Transgenic Crop Plants (PDFDrive)

Transgenic Crop Plants
Chittaranjan Kole Charles H. Michler

l l
Albert G. Abbott Timothy C. Hall

l
Editors
Transgenic Crop Plants

Volume 2: Utilization and Biosafety
Editors
Prof. Chittaranjan Kole Prof. Albert G. Abbott
Department of Genetics & Biochemistry Department of Genetics & Biochemistry
Clemson University Clemson University
Clemson, SC 29634, USA Clemson, SC 29634, USA
ckole@clemson.edu aalbert@clemson.edu
Prof. Charles H. Michler Prof. Timothy C. Hall

Director Institute of Developmental &
Hardwood Tree Improvement and Molecular Biology
Regeneration Center at Purdue University Department of Biology
NSF I/UCRC Center for Tree Genetics Texas A&M University
West Lafayette, IN, USA College Station, TX, USA
michler@purdue.edu tim@idmb.tamu.edu
ISBN: 978-3-642-04811-1 e-ISBN: 978-3-642-04812-8

DOI 10.1007/978-3-642-04812-8
Springer Heidelberg Dordrecht London New York
Library of Congress Control Number: 2009939124
# Springer-Verlag Berlin Heidelberg 2010

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,
reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication
or parts thereof is permitted only under the provisions of the German Copyright Law of September 9,
1965, in its current version, and permission for use must always be obtained from Springer. Violations
are liable to prosecution under the German Copyright Law.
The use of general descriptive names, registered names, trademarks, etc. in this publication does not
imply, even in the absence of a specific statement, that such names are exempt from the relevant protec-
tive laws and regulations and therefore free for general use.
Cover design: WMXDesign GmbH, Heidelberg, Germany
Printed on acid-free paper
Springer is part of Springer Science+Business Media (www.springer.com)

Preface
Transgenic Plants – known also as Biotech Plants, Genetically Engineered Plants,

or Genetically Modified Plants – have emerged amazingly fast as a boon for science
and society. They have already played and will continue to play a significant role in
agriculture, medicine, ecology, and environment. The increasing demands for food,
feed, fuel, fiber, furniture, perfumes, minerals, vitamins, antibiotics, narcotics, and
many health-related drugs and chemicals necessitate the development and cultiva-
tion of transgenic plants with augmented or suppressed trait(s). From a single
transgenic plant (Flavr Savr tomato with a longer shelf-life) introduced for com-
mercialization in 1994, we have now 13 transgenic crops covering 800 million ha in
25 countries of six continents. Interestingly, the 13.3 million farmers growing
transgenic crops globally include 12.3 million (90%) small and resource poor
farmers from 12 developing countries. Increasing popularity of transgenic plants
is well evidenced from an annual increase of about 10% measured in hectares but
actually of 15% in “trait hectares.” Considering the urgent requirement of trans-
genic plants and wide acceptance by the farmers, research works of transgenic
plants are now being conducted on 57 crops in 63 countries. Transgenic plants have
been developed in over 100 plant species and they are going to cover the fields,
orchards, plantations, forests, and even the seas in the near future. These plants have
been tailored with incorporation of useful alien genes for several desirable traits
including many with “stacked traits” and also with silencing of genes controlling
some undesirable traits.
Development, applications and socio-political implications of transgenic plants
are immensely important fields now in education, research, and industries. Plant
transgenics has deservedly been included in the course curricula in most, if not all,
leading universities and academic institutes all over the world, and therefore
reference books on transgenic plants with a class-room approach are essential for
teaching, research, and extension. There are some elegant reviews on the transgenic
plants or plant groups (including a 10-volume series “Compendium of Transgenic
Crop Plants” edited by two of the present team of editors C. Kole and T.C. Hall
published by Wiley-Blackwell in 2008) and on many individual tools and
v
vi Preface
techniques of genetic transformation in plants. All these reviews could surely serve
well the purpose for individual crop plants or particular methodologies. Since
transgenic plant development and utilization is studied, taught, and practiced by
students, teachers, and scientists of over a dozen disciplines under basic science,
agriculture, medicine, and humanities at public and private sectors, introductory
reference books with lucid deliberations on the concepts, tools, and strategies to
develop and utilize transgenic plants and their global impacts could be highly useful
for a broad section of readers.
Deployment of transgenic crop plants are discussed, debated, regulated, and
sponsored by people of diverse layers of the society, including social activists,
policy makers, and staff of regulatory and funding agencies. They also require lucid
deliberations on the deployment, regulations, and legal implications of practicing
plant transgenics. More importantly, depiction of the positive and realistic picture
of the transgenic plants should and could facilitate mitigation of the negative
propaganda against transgenic plants and thereby reinforce moral and financial
support from all individuals and platforms of the society. Global population is
increasing annually by 70 millions and is estimated to grow to eight billion by 2025.
This huge populace, particularly its large section from the developing countries,
will suffer due to hunger, malnutrition, and chemical pollution unless we produce
more and more transgenic plants, particularly with stacked traits. Compulsion to
meet the requirements of this growing population on earth and the proven innocu-
ous nature of transgenic plants tested and testified for the last 13 years could
substantiate the imperative necessity of embracing transgenics.
Traditional and molecular breeding practiced over the last century has provided
enormous number of improved varieties in economic crops and trees including
wheat and rice varieties that fostered the “green revolution.” However, these crop
improvement tools depend solely on the desirable genes available naturally, crea-
table by mutation in a particular economic species, or their shuffling for desired
recombinations. Transgenic breeding has opened a novel avenue to incorporate
useful alien genes from not only other cross-incompatible species and genera of the
plant kingdom, but also from members of the prokaryotes including bacteria, fungi,
and viruses, and even from higher animals including mice and humans. An array of
plant genetic engineering achievements starting from the development of insect
resistance cotton by transforming the cry genes from the bacteria Bacillus
thuringiensis to the present-day molecular pharming that enables the expression
of interferon- gene from human in tobacco evidence for this pan-specific gene
transfer.
Human and animal safety is another general concern related to transgenic food
or feed. However, there is no reliable scientific documentation of these health
hazards even after 13 years of cultivation of transgenic plants and consumption of
about 1 trillion meals containing transgenic ingredients. Utilization of transgenic
plants has reduced the pesticide applications by 359,000 tons that would otherwise
affect human and animal health besides causing air, water, and soil pollution and
also mitigated the chance of consumption of dead microbes and insects along with
foods or feeds.
Preface vii
Gene flow from transgenic crop species to their cross-compatible wild relatives
is a genuine concern and therefore required testing of a transgenic crop plant before
deployment followed by comprehensive survey of the area for presence of inter-
fertile wild and weedy plants before introduction of a transgenic crop are being
seriously conducted.
Addition of novel genotypes with transsgenes in the germplasms is increasing
the biological diversity rather than depleting it. Using the genetically engineered
plants has also eliminated greenhouse gas emission of 10 million metric tons
through fuel savings. In fact, 1.8 billion liters of diesel have been saved because
of reduced tillage and plowing owing only to herbicide-resistant transgenic crops.
Many transgenics are now being used for soil reclamation. Above all, cultivation of
transgenic crops has returned $44 billion of net income to the farmers. Perhaps,
these are the reasons that 25 Nobel Laureates and 3,000-plus eminent scientists
appreciated the merits and safety and also endorsed transgenic crops as a powerful
and safe way to improve agriculture and environment besides the safety of geneti-
cally modified foods. Many international and national organizations have also
endorsed health and environmental safety of transgenic plants; these include
Royal Society (UK), National Academy of Sciences (USA), World Health Organi-
zation, Food and Agriculture Organization (UN), European Commission, French
Academy of Medicine and American medical Association, to name a few.
Production, contributions, and socio-political implications of biotech plants are
naturally important disciplines now in education, research, and industries and
therefore introductory reference books are required for students, scientists, indus-
tries, and also for social activists and policy makers. The two book volumes on
“Transgenic Crop Plants” will hopefully fill this gap. These two book volumes have
several unique features that deserve mention. The outlines of the chapters for these
two books are formulated to address the requirements of a broad section of readers.
Students and scholars of all levels will obtain a lot of valuable reading material
required for their courses and researches. Scientists will get information on con-
cepts, strategies, and clues useful for their researches. Seed companies and indus-
tries will get information on potential resources of plant materials and expertise for
their own R&D activities. In brief, the contents of this series have been designed
to fulfill the demands of students, teachers, scientists, and industry people, for small
to large libraries. Students, faculties, or scientists involved in various subjects will
be benefited from this series; biotechnology, bioinformatics, molecular biology,
molecular genetics, plant breeding, biochemistry, ecology, environmental science,
bioengineering, chemical engineering, genetic engineering, biomedical engineer-
ing, pharmaceutical science, agronomy, horticulture, forestry, entomology, pathol-
ogy, nematology, virology, just to name a few.
It had been our proud privilege to edit the 23 chapters of these two books those
were contributed by 71 scientists from 14 countries and the list of authors include
one of the pioneers of plant transgenics, Prof. Timothy C. Hall (one of the editors
also); some senior scientists who have themselves edited books on plant trans-
genics; and many scientists who have written elegant reviews on invitation for
quality books and leading journals. We believe these two books will hopefully
viii Preface
serve the purposes of the broad audience who are studying, teaching, practicing,
supporting, funding, and also those who are debating for or against plant trans-
genics. The first volume dedicated to “Principles and Development” elucidates the
basic concepts, tools, strategies, and methodologies of genetic engineering, while
the second volume on “Applications and Safety” enumerates the utilization of
transgenic crop plants for various purposes of agriculture, industry, ecology, and
environment, and also genomics research. This volume also deliberates compre-
hensively on the legal and regulatory aspects; complies to the major concerns; and
finally justifies the compulsion of practicing plant transgenics.
Glimpses on the contents of this volume (Volume 2: Transgenic Crop Plants:
Applications and Safety) will perhaps substantiate its usefulness. This volume
enumerates the application of transgenic technologies in crop plants for particular
objectives in the first ten chapters. Biotic stress resistant, specifically insect resis-
tant, transgenics have been developed and commercialized in several crops. An
example with Bt-expressing cotton and maize alone, with current market share of
about $3.26 billion substantiates their success and popularity (Chap.1). Abiotic
stresses, particularly drought, salinity, and temperature extremes, have always been
difficult to manipulate. Still success stories are pouring in recently from works
mainly in cereals and vegetables (Chap.2). Herbicide-resistant transgenic plants
(in cotton and canola) were first deregulated in 1995 and in 2008 more than 80%
of the transgenic plants grown globally possess a transgenic trait for herbicide
resistance. Chapter 3 details the present and emerging herbicide-resistant transgenic
plants. Although the first transgenic trait was developmental, shelf-life in tomato
to be precise, transgenics research for these traits are yet to make significant
commercial headway but started producing encouraging results (Chaps.4 and 5).
Deployment of transgenic plants for biofuel, pharmaceuticals, and other biopro-
ducts has been enunciated in three chapters (Chaps.6, 7, and 9). Transgenic plants
have been labeled as a culprit for potential threats to ecology and environment by a
few groups of social activists. Chapter 8 addresses these weird concerns with
suitable examples of utilization of transgenic plants for phytoremediation, biomo-
nitoring, and the production of bioplastics and biopolymers for amelioration of
ecology and environment. Plant genomics has emerged fast within the last three
decades and facilitated fine-scale view of the plant genes and genomes. Transgenic
plants have provided enormous resources for functional genomics studies and
expected to play their roles as more plants systems and genes are targeted (Chap.
10). Scientists practicing transgenics are no less aware of the potential risks of
genetic engineering than the few people with antagonistic views. Neither are the
regulatory agencies at institutional, state, national, and international level regulatory
agencies unaware of the steps to be involved for inspection, monitoring, and
approval of transgenic plants for commercial use. Chapter 11 delineates all these
aspects with examples from US and other continents and countries. Any original
innovation or effort deserves recognition and also an incentive. The scope of
patenting and intellectual property rights for materials owned and generated and
methodologies implemented have been appreciated and enforced legally. These
aspects related to transgenic crop plants have been discussed in Chap.12.
Preface ix
The concluding chapter (Chap.13) briefs the contributions and concerns with the
compliances and compulsion of practicing plant transgenics for science and society.
We thank all the 41 scientists from nine countries for their elegant and lucid
contributions to this volume and also for their sustained support through revision,
updating and fine-tuning their chapters. We also acknowledge for the recent
statistics that have been accessed from the web sites of Monsanto Company on
“Conversations about Plant Biotechnology” and “International Service for the
Acquisition of Agri-Biotech Applications on ISAAA Brief 39-2008: Executive
Summary” and used them in this preface and elsewhere in the volume.
We enjoyed a lot of our Clemson–Purdue–Texas A&M triangular interaction,
constant consultations, and dialogs while editing this book, and also our working
with the editorial staff of Springer, particularly Dr. Sabine Schwarz who had been
supportive since inception till publication of this book.
We will look forward to suggestions from all corners for future improvement of
the content and approach of this book volume.
Chittaranjan Kole, Clemson, SC

Charles H. Michler, West Lafayette, IN
Albert G. Abbott, Clemson, SC
Timothy C. Hall, College Station, TX
Contents
1 Transgenic Crop Plants for Resistance to Biotic Stress . . . . . . . . . . . . . . . 1

N. Ferry and A.M.R. Gatehouse
2 Transgenic Plants for Abiotic Stress Resistance . . . . . . . . . . . . . . . . . . . . . . 67

Margaret C. Jewell, Bradley C. Campbell, and Ian D. Godwin
3 Transgenic Crops for Herbicide Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . 133

Stephen O. Duke and Antonio L. Cerdeira
4 Understanding and Manipulation of the Flowering

Network and the Perfection of Seed Quality . . . . . . . . . . . . . . . . . . . . . . . . . 167
Stephen L. Goldman, Sairam Rudrabhatla, Michael G. Muszynski,
Paul Scott, Diaa Al-Abed, and Shobha D. Potlakayala
5 Biotechnological Interventions to Improve Plant

Developmental Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Avtar K. Handa, Alka Srivastava, Zhiping Deng, Joel Gaffe,
Ajay Arora, Martı́n-Ernesto Tiznado-Hernández, Ravinder K. Goyal,
Anish Malladi, Pradeep S. Negi, and Autar K. Mattoo
6 Transgenics for Biofuel Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

Anjanabha Bhattacharya, Pawan Kumar, and Rippy Singh
7 Plant Produced Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

Jared Q. Gerlach, Michelle Kilcoyne, Peter McKeown,
Charles Spillane, and Lokesh Joshi
8 Biotech Crops for Ecology and Environment . . . . . . . . . . . . . . . . . . . . . . . . 301

Saikat Kumar Basu, François Eudes, and Igor Kovalchuk
xi
xii Contents
9 Algal Biotechnology: An Emerging Resource with Diverse

Application and Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Cunningham Stephen and Joshi Lokesh
10 Transgenic Crops and Functional Genomics . . . . . . . . . . . . . . . . . . . . . . . . . 359

Narayana M. Upadhyaya, Andy Pereira, and John M. Watson
11 Deployment: Regulations and Steps for Commercialization . . . . . . . . 391

Kelly D. Chenault Chamberlin
12 Patent and Intellectual Property Rights Issues . . . . . . . . . . . . . . . . . . . . . . . 411

Jim M. Dunwell
13 Transgenic Crop Plants: Contributions, Concerns,

and Compulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435
Brian R. Shmaefsky
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479
Contributors
Diaa Al-Abed Edenspace Systems Corporation, Manhattan, KS 66502, USA,

dalabed@edenspace.com
Ajay Arora Division of Plant Physiology, Indian Agricultural Research

Institute, New Delhi 110012, India
Saikat Kumar Basu Department of Biological Sciences, University of

Lethbridge, 4401 University Drive, Lethbridge, AB, Canada T1K 3M4; Biopro-
ducts and Bioprocesses, Agriculture and Agri-Food Canada, 5403 1st Avenue
South, Lethbridge, AB, Canada T1J 4B1
Anjanabha Bhattacharya National Environmental Sound Production Agricul-

ture Laboratory, University of Georgia, Tifton, GA 31794, USA, anjan@uga.edu
Bradley C. Campbell School of Land, Crop and Food Sciences, The University
of Queensland, Brisbane, QLD 4072, Australia
Antonio L. Cerdeira Brazilian Department of Agriculture, Agricultural

Research Service, EMBRAPA/Environment, C.P. 69, Jaguariuna-SP-13820000,
Brazil
Kelly D. Chenault Chamberlin USDA-ARS, Wheat, Peanut, and Other Field

Crops Unit, 1301 N. Western, Stillwater, OK 74075, USA, kelly.Chamberlint@ars.
usda.gov
Zhiping Deng Department of Plant Biology, Carnegie Institution of Washington,

Stanford, CA 94305, USA
Stephen O. Duke Agricultural Research Service, United States Department of

Agriculture, P. O. Box 8048, University, MS 38677, USA, sduke@olemiss.edu
xiii
xiv Contributors
Jim M Dunwell University of Reading, Whiteknights, Reading RG6 6AS, UK,

j.dunwell@reading.ac.uk
François Eudes Bioproducts and Bioprocesses, Agriculture and Agri-Food

Canada, 5403 1st Avenue South, Lethbridge, AB, Canada T1J 4B1
N. Ferry School of Biology, Institute for Research on Environment and Sustain-

ability, Devonshire Building, Newcastle University, Newcastle upon Tyne, NE1
7RU, UK, natalie.ferry@ncl.ac.uk
Joel Gaffe Laboratoire Adaptation et Pathogénie des Microorganismes, LAPM,

UMR 5163 CNRS-UJF, Institut Jean Roget BP 170, 38042 Grenoble cedex 9,
France
A.M.R. Gatehouse School of Biology, Institute for Research on Environment

and Sustainability, Devonshire Building, Newcastle University, Newcastle upon
Tyne, NE1 7RU, UK
Jared Q. Gerlach Glycoscience and Glycotechnology Group and the Martin

Ryan Institute, National Centre for Biomedical Engineering Science, National
University of Ireland, Galway, Ireland
Ian D. Godwin School of Land, Crop and Food Sciences, The University of
Queensland, Brisbane, QLD 4072, Australia, i.godwin@uq.edu.au
Stephen L. Goldman Department of Environmental Sciences, The University of

Toledo, Toledo, OH 43606, USA, sgoldma@utoledo.edu
Ravinder K. Goyal Department of Biochemistry and Microbiology, University

of Victoria, Victoria, BC, Canada V8W 3P6
Avtar K. Handa Department of Horticulture and Landscape Architecture, Pur-

due University, West Lafayette, IN 47907-2010, USA, ahanda@purdue.edu
Margaret C. Jewell School of Land, Crop and Food Sciences, The University of
Queensland, Brisbane, QLD 4072, Australia
Lokesh Joshi Glycoscience and Glycotechnology Group and the Martin Ryan
Institute, National Centre for Biomedical Engineering Science, National University
of Ireland, Galway, Ireland, lokesh.joshi@nuigalway.ie
Michelle Kilcoyne Glycoscience and Glycotechnology Group and the Martin

Ryan Institute, National Centre for Biomedical Engineering Science, National
University of Ireland, Galway, Ireland
Contributors xv
Igor Kovalchuk Department of Biological Sciences, University of Lethbridge,

4401 University Drive, Lethbridge, AB, Canada T1K 3M4, igor.kovalchuk@uleth.ca
Pawan Kumar National Environmental Sound Production Agriculture Labora-

tory, University of Georgia, Tifton, GA 31794, USA
Joshi Lokesh Glycoscience and Glycotechnology Group and the Martin Ryan
Institute, National Centre for Biomedical Engineering Science, National University
of Ireland, Galway, Ireland, lokesk.joshi@nuigalway.ie
Anish Malladi Department of Horticulture, University of Georgia, Athens, GA

30602, USA
Autar K. Mattoo Sustainable Agricultural Systems Laboratory, The Henry A.

Wallace Beltsville Agric Research Center, Beltsville, MD 20705-2350, USA
Peter McKeown Genetics and Biotechnology Laboratory, Department of

Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland
Michael G. Muszynski Department of Genetics, Development and Cell Biology,

Iowa State University, Ames, IA 50011, USA, mgmuszyn@iastate.edu
Pradeep S. Negi Department of Horticulture and Landscape Architecture,

Purdue University, West Lafayette, IN 47907-2010, USA
Andy Pereira Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA

24061, USA
Shobha D. Potlakayala Penn State Milton S. Hershey College of Medicine,

Hershey, PA 17033, USA, p_shobhadevi@yahoo.com
Sairam Rudrabhatla Environmental Engineering, College of Science,

Engineering and Technology, Penn State University, Middletown, PA 17057,
USA, svr11@psu.edu
Paul Scott Department of Genetics, Development and Cell Biology, Iowa State
University, Ames, IA 50011, USA, mgmuszyn@iastate.edu; Department of Agron-
omy, USDA-ARS, Iowa State University, Ames, IA 50011, USA, paul.scott@
ars.usda.gov
Brian R. Shmaefsky Lone Star College – Kingwood, HSB 202V, 20,000

Kingwood Drive, Kingwood, TX 77339-3801, USA, Brian.R.Shmaefsky@
lonestar.edu
xvi Contributors
Rippy Singh National Environmental Sound Production Agriculture Laboratory,

University of Georgia, Tifton, GA 31794, USA
Charles Spillane Genetics and Biotechnology Laboratory, Department of

Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland
Alka Srivastava Department of Horticulture and Landscape Architecture, Pur-

due University, West Lafayette, IN 47907-2010, USA
Cunningham Stephen Glycoscience and Glycotechnology Group and the

Martin Ryan Institute, National Centre for Biomedical Engineering Science,
National University of Ireland, Galway, Ireland
Martı́n-Ernesto Tiznado-Hernández Fisiologı́a y Biologı́a Molecular de

Plantas, Coordinación de Tecnologı́a de Alimentos de Origen Vegetal, Centro de
Investigación en Alimentación y Desarrollo, A.C., Hermosillo, Sonora, México
Narayana M. Upadhyaya CSIRO Plant Industry, GPO Box 1600, Canberra,

ACT 260, Australia, Narayana.upadhyaya@csiro.au
John M. Watson CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 260,
Australia
Abbreviations
1-FFT Fructan:fructan 1-fructosyltransferase

1-MCP 1-Methylcyclopropene
1-SST Sucrose:sucrose 1-fructosyltransferase
2,4-D 2,4-Dichlorophenoxyacetic acid
2D-PAGE Two-dimensional polyacrylamide gel electrophoresis
4C3H 4-Coumarate 3-hydroxylase
4CL 4-Hydroxycinnamoyl CoA ligase
6G-FFT Fructan:fructan 6G-fructosyltransferase
6-SFT Sucrose:fructan 6-fructosyltransferase
AAFC Agriculture and Agri-Food Canada
ABA Abscisic acid
ABRE ABA responsive element
Ac Activator gene
ACC 1-Aminocyclopropane-1-carboxylate
AChE Acetylcholinesterase
ACP Acyl-carrier protein
ADP Adenosine di-phosphate
ae1 amylose extender gene
AHK2/3 Arabidopsis histidine kinase
AL-PCD Apoptotic-like programmed cell death
ALS Acetolactate synthase
AMGT Agrobacterium-mediated gene transfer
AMPA Aminomethylphosphonic acid
ANVISA National Agency for Health and Surveillance of the Ministry
of Health
AOS Allene oxide synthase
AOX Altenative oxidase
ap apetalla gene
AP1 APETALA1 gene
AP2 APETALA2/ Apetela2 gene
xvii
xviii Abbreviations
APHIS Animal and Plant Health Inspection Service

APX Ascorbate peroxidase
ARF Auxin response factor
arsC Arsenate reductase
Asc Ascorbate
at antherless gene
AtCKX1 Arabidopsis thaliana cytokinin oxidase gene
Avr Avirulence
AZ Abscission zone
BA Benzyladenine
BADH Betaine aldehyde dehydrogenase
BAP 6-Benzylaminopurine
BAR Bialaphos resistance gene
BBC British Broadcasting Corporation
BC Biotech crop
BGI-RIS Beijing Genomics Institute- Rice Information System
bla Beta-lactamase
BMR Brown midrib
BRS Biotechnology Regulatory Service
Bt Bacillus thuriengensis
bZIP Basic leucine zipper
CAD Cinnamoyl alcohol dehydrogenase
CAL CAULIFLOWER gene
CAMBIA Center for the Application of Molecular Biology to International
Agriculture
CaMV Cauliflower mosaic virus
CAT Chloramphenicol acetyltransferase/catalase
CBER Centre for Biologics Evaluation and Research
CBF CRT binding factor
cbLCV Cabbage leaf curl virus
CBS Columbia Broadcasting System
CCR Cinnamoyl CoA reductase
CDC Centers for Disease Control
CDF Cycling DOF Factor
cDNA Complementary-DNA
CEL Cellulase
CEPA Canadian Environmental Protection Act
CFIA Canadian Food Inspection Agency
CFSAN Centre for Food Safety and Applied Nutrition
CGH-1 Cardenolide 16’-O-glucohydrolase
CGIAR Consultative Group on International Agricultural Research
cGMP Current GMP
CHO Chinese hamster ovary
CHO Choline dehydrogenase
Abbreviations xix
chs chalcone synthase gene

CIGB Cuban Centre for Biotechnology and Genetic Engineering
CMO Choline monooxygenase
CMS Cytoplasmic male sterility
CMS Cellular membrane stability
CNN Cable News Network
CNR Colorless non-ripening gene
CO Constans gene
ConA Concanavalin A
CONABIA National Advisory Commission on Agricultural Biotechnology
conz1 constans of Zea mays1 gene
COR Cold responsive
COR Cold responsive gene
CP Chloroplast
CpTI Cowpea trypsin inhibitor
CRE Cytokinin response
CRIIGEN Comité de Recherche et d’Information Indépendantes sur le Génie
CRT C-Repeat
Cry Crystal
CTNBio National Technical Commission on Biosafety
CVM Centre for Vetinary Medicine
CV-N Cyanovirin
D2GT2A Diacylglycerol acyltransferase 2A
dab delayed abscission gene
DDB Damaged DNA binding protein
DDT Dichlorodiphenyltrichloroethane
DEFRA Department of Environment, Food and Rural Affairs
DET Detiolated gene
DGDG Digalactosyldiacylglycerol
DHAsc Dehydroascorbate
dlf1 delayed flowering1 gene
DNMA Directorate of Agricultural Markets
DRE Dehydration responsive element
DREB DRE binding protein
driPTGS Direct repeat-induced PTGS
Ds Dissociation gene
DsE Enhancer trap Ds
DsG Gene trap Ds
DSL Domestic Substance List
dzr1 delta zein regulator1 gene
EA Environmental assessment
EBV Epstein-barr virus
EC European Commission
EDB Ethylene dibromide
xx Abbreviations
EFSA European Food Safety Authority

EFSA European Food Standards Agency
EIN Ethylene-insensitive gene
EIS Environmental Impact Statement
EMEA European Agency for Evaluation of Medicinal Products
EMS Ethylmethane sulfonate
En Enhancer transposon
EPA Eicosapentaenoic
EPA Environmental Protection Agency
EPSP Enolpyruvyl-shikimate-3-phosphate
EPSPS 5-Enolpyruvyl-shikimate-3-phosphate synthase
ER Endoplasmic reticulum
ERE Ethylene responsive element
ERS Economic Research Service
EST Expressed sequence tag
ETC Electron transport chain
ETH Ethylene
EU European Union
F Florigenic signal
F1 Fraction 1 anti-phagocytic capsular envelope protein
FAD1 Flavin adenine dinucleotide
FAD3 Omega-3 fatty acid desaturase
FAO Food and Agriculture Organization of the United Nations
FD FLOWERING LOCUS D gene
FDA Food and Drug Administration
FFDCA Federal Food, Drug and Cosmetic Act
FIFRA Federal Insecticide, Fungicide and Rodenticide Act
fl2 floury-2 gene
FONSI Finding of no significant impact
FPPA Federal Plant Pest Act
Fr Fertility restorer gene
FST Flanking sequence tag
FT Flowering Locus T/Flowering Transition gene
FT-ICR-MS Fourier-transform ion cyclotron mass spectrometry
Fuc Fucose
FucT Fucosyltransferase
FUL FRUITFUL gene
Fx Fucoxantine
G3P Glycerol-3-phosphate
GA/ GA3 Gibberellic acid
Gal Galactose
GalNAc N-Acetylgalactosamine
GalT Galactosyltransferase
GAT Glyphosate N-acetyltransferase
Abbreviations xxi
GC Glutathione synthetase
GCase Glucosyl-N-acylspingosineglycohydrolase
GCS g-Glutamylcysteine synthase
GDP Gross domestic product
GE Genetic engineering/Genetically engineered
GFLV Grapevine fanleaf virus
GFP Green fluorescent protein
GI Gigantea gene
gigz1 gigantea of Zea mays1 gene
GlcNAc N-Acetylglucosamine
GlyBet Glycine betaine
GM Genetically modified
GMHT Genetically modified herbicide tolerant
GMO Genetically modified organism
GMP Genetically modified plant
GMP Good manufacturing practise
GMPO Genetically modified plant organism
GMS Genic male sterility
GNA Galanthus nivalis agglutinin
GOI Gene of interest
GOX Glyphosate oxidoreductase
GPAT Glycerol-3-phosphate acyltransferase
GPX Glutathione peroxidase
GR Glutathione reductase/ Glyphosate resistant
GRC Glyphosate resistant Crop
GS1 Glutamine synthase gene 1
GSH Glutathione/ Glutamate synthase
GSSG Glutathione disulfide
GST Glutathione S-transferase
GTN Glycerol trinitrate
GUS ß-Glucuronidase
HBcAg Hepatitis B core antigen
HBsAg Hepatitis B surface antigen
HBV Hepatitis B virus
HCMV Human cytomegalovirus
hEPO Human erythropoietin
hGM-CSF Human granulocyte-macrophage colony-stimulating factor
HIV Human immunodeficiency virus
HMX Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine
HR Hypersensitive response/ Herbicide resistant
HRC Herbicide resistant crop
HS Heat shock
HSP Heat shock protein
HSV Herpes simplex virus
xxii Abbreviations
HT/Ht Herbicide tolerant/tolerance

I Inhibitor transposon
i.p. Intraperitoneal
iAc Immobile Ac transposon
IBA Indole-3-butyric acid
ICTSD International Center for Trade and Sustainable Development
id1 indeterminate1 gene
ida inflorescence deficient in abscission gene
IDD ID-domain
IFN Interferon
IgA Immunoglobulin A
IL-12 Interleukin-12
IMI Imidazolinone
IPM Integrated pest management
IPP Isopentyl diphosphate
IPR Intellectual Property Rights
IPT Isopentenyl transferase/ Isopentyl transferase
IR Insect resistant/resistance
IRGSP International Rice Genome Sequencing Project
ISAAA International Service of AgriBiotech Applications
ISIS Institute for Science and International Security
ISR Induced systemic resistance
JA Jasmonic acid
LB Left border of T-DNA
LD Long-day
LEA Late embryogenesis abundant
Leu Leucine
LFY Leafy gene
LOG Lonely guy gene
LOX1/2/3 Lipoxygenase gene
lpa1 Lysophosphatidic acid receptor
LPS Lipopolysaccharide
LRR Leucine rich repeats
LTB Heat-labile enterotoxin, subunit B
Lys Lysine
MAb Monoclonal antibody
MAPK Mitogen-activated protein kinase
MGDG Monogalactosyldiacylglycerol
MHBsAg Middle HBsAg
Mi Meloidogyne incognita resistance gene
MIP Major intrinsic protein
miRNA Micro-RNA
MPSS Massively parallel signature sequencing
mRNA Messenger-RNA
Abbreviations xxiii
MS Mass spectrometry
MS Murashige and Skoog (medium)
MST Members of the Landless Rural Workers Movement
MT Metallothionein
MTP Metal-tolerance protein
MTT Multi-tasking transgenics
Mu Mutator transposon
MuIL-12 Murine IL-12
MV Methyl viologen
MVL Microcystis viridis lectin
NAA a-Napthalene acetic acid
NAM Napthaleneacetamide
NAS National Academy of Sciences
NAS Nicotinamine synthase
NBS Nucleotide binding site
NCBI National Center for Biotechnology Information
NDV Newcastle disease virus
NEPA National Environmental Policy Act
Neu5Ac 5-N-Acetyl-D-neuraminic acid
NIH National Institute of Health
NIL Near-isogenic line
NMR Nuclear magnetic resonance
NO Nitric oxide
NOI Notice of Intent
NoV Norovirus
NR Nitrate reductase
NST Nac secondary wall thickening promoter factor
NUE Nitrogen use efficiency
o2 opaque-2 gene
OECD Organization for Economic Cooperation and Development
OFB Office of Food Biotechnology
OMT O-Methyl transferase
ORF Open reading frame
Ori Origin of replication
OSTP Office of Science and Technology Policy
PAHs Polycyclic aromatic hydrocarbon
PAL Phenyalanine ammonia lyase
PAMPs Pathogen associated molecular patterns
PAT Phosphinothricin-acetyltransferance
PC Phytochelatin
PCB Polychlorinated biphenyl
PCD Programmed cell death
PCR Polymerase chain reaction
PCS Phytochlelatin synthase
xxiv Abbreviations
PEG Polyethylene glycol

PETN Pentaerythritol tetranitrate
PG Polygalacturonase
PG Phosphatidylglycerol
PHB Polyhydroxybutyrate
pi pistillata gene
PiP Plant Incorporated Protectant
PIP Plasma membrane intrinsic protein
PL Pectate lyase
PLC Phospholipase C
PLD Phospholipase D
PLE Phospholipid cleaving enzyme
PME Pectin methylesterase
PMP Plant-made pharmaceutical
PNT Plant with novel trait
PPO Polyphenol oxidase
PR Pathogenesis-related
Pro Proline
PS Phytosiderophores
PSI Photosystem 1
PSII Photosystem 2
PTGS Post-transcriptional gene silencing
Put Putrescine
PVP Plant Variety Protection
PVX Potato virus X
PyMSP4/5 Murine P. yoelii merozoite surface protein 4/5
QPM Quality protein maize
QTL Quantitative trait loci
RAP-DB Rice Annotation Project-Database
rasiRNA Repeat-associated siRNA
RB Non-toxin B-chain from ricin
RB Right border of T-DNA
rDNA Recombinant-DNA
RDX Hexahydro-1,3,5-trinitro-1,3,5 triazine
Rf Restorer of fertility gene
RFLP Restriction fragment length polymorphism
R-gene Resistance-gene
rGSII Recombinant Griffonia simplicifolia lectin II
rhCVFVIII Recombinant human clotting factor VIII
rhEPO Recombinant human erythropoietin
rhIF Recombinant human intrinsic factor
RHS Royal Horticultural Society
RID1 Rice Indeterminate1 gene
RIL Recombinant inbred line
Abbreviations xxv
rin ripening inhibitor gene

Rip Ribosome inactivating protein
RNAi RNA-interference
ROIs Reactive oxygen intermediates
ROS Reactive oxygen species
RT-PCR Reverse transcriptase-PCR
RWC Relative water content
s.c. Subcutaneously
SA Salicylic acid
SA Splice acceptor
SAGE Serial analyses of gene expression
SAGPyA Secretariat of Agriculture, Livestock, Fisheries and Food
SAM S-Adenosylmethionine
SAM Shoot apical meristem
SAR Systemic acquired resistance
scFv Single chain variable fragment
scN Soyacystatin N
SD Short-day
SE Substantial equivalence/ equivalent
SENASA National Agri-food Health and Quality Service
Ser Serine
SFI1 Segestria florentina venom peptide
sh2 shrunken2 gene
siRNA Short/Small interfering RNA
SIV Simian immunodeficiency virus
SL Selenocysteine lyase
SMT Seleno-cysteine methyl transferase
soc suppessor of overexpression of constans gene
SOC1 Suppressor of Overexpression of Constans1 gene
SOD Superoxide dismutase
SOliD Supported Oligo Ligation Detection
Spd Spermidine
Spm Spermine
Spm Suppressor-Mutator transposon
SQDG Sulfoquinovosyldiacylglycerol
ssRNA Single-stranded RNA
STP Signal transduction pathway
su1 sugary1 gene
SVN Scytovirin
TA Transcriptional activator
TAC Tiller angle control gene
TAGI The Arabidopsis Genome Initiative
tasiRNA Transacting siRNA
tb1 teosinte branched1 gene
xxvi Abbreviations
TCE 2.4,6-Trichloroethylene
TCOH Chloral and trichoethanol
TCP 2,4,6-Tricholorophenol
T-DNA Transferred-DNA
TDZ Thidiazuron
TET Transiently expressed transposase
TETRYL N-Methyl-N, 2, 4, 6-tetranitroaniline
TF Transcription factor
TFL Terminal Flower gene
Thr Threonine
ti Trypsin inhibitor allele
TILLING Targeting induced local lesions in genomes
TIP Tonoplast intrinsic protein
TMV Tobacco mosaic virus
TNT Trinitrotoluene
Trp Tryptophan
TRV Tobacco rattle virus
TSCA Toxic Substances Control Act
UAS Upstream activator sequence
uf uniflora gene
UN United Nations
UNCTAD United Nations Conference on Trade and Development
US United States
USAID US Agency for International Development
USDA United States Department of Agriculture
USPTO United States Patent and Trademark Office
UV Ultraviolet
VB Vector backbone
Vgt1 Vegetative to generative transition1 gene
Vgt2 Vegetative to generative transition2 gene
VIGS Virus-induced gene silencing
VIP Vegetative Insecticidal Protein
VLP Virus-like particle
VRO Variety Registration Office
WHO World Health Organisation
WT Wild type
WUE Water use efficiency
XTH Xyloglucan endotransglucosylase/hydrolase
Xyl Xylose
XylT Xylosyltransferase
YCF1 Yeast vacuolar glutathione Cd transporter
YFP Yellow fluorescent protein
ZCN Zea CENTRORADIALIS gene
zfl1 Zea FLO/LFY1 gene
Abbreviations xxvii
zfl2 Zea FLO/LFY2 gene

ZFN Zinc-finger nuclease
ZMM4/5 Zea mays FULL1-like gene
ZmRap2 Zea mays related to AP2 gene
g-GCS g-Glutamyl cysteine synthetase
o3 Omega 3
Chapter 1
Transgenic Crop Plants for Resistance
to Biotic Stress
N. Ferry and A.M.R. Gatehouse
1.1 Introduction
We couldn’t feed today’s world with yesterday’s agriculture and we won’t be able to feed
tomorrow’s world with today’s. – Lord Robert May, President of the Royal Society, March
2002
The human population is everexpanding; conservative estimates predict that the

population will reach ten billion by 2050 (United Nations Population Division), and
the ability to provide enough food is becoming increasingly difficult (Chrispeels
and Sadava 2003). The planet has a finite quantity of land available to agriculture
and the need for increasing global food production has led to increasing exploitation
of previously uncultivated land for agriculture; as a result wilderness, wetland,
forest and other pristine environments have been, and are being, encroached upon
(Ferry and Gatehouse 2009). The minimization of losses to biotic stress caused by
agricultural pests would go some way to optimizing the yield on land currently
under cultivation. For nearly 50 years, mainstream science has told us that this
would be impossible without chemical pesticides (Pimental 1997). The global
pesticide market is in excess of $30 billion per year (Levine 2007); despite this,
approximately 40% of all crops are lost directly to pest damage (Fig. 1.1).
These figures are simplified rough estimates; in reality crop losses to biotic stress
are extremely difficult to quantify and vary by crop, year, and region.
N. Ferry (*)
School of Biology, Institute for Research on Environment and Sustainability, Devonshire
Building, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK
e-mail: natalie.ferry@ncl.ac.uk
C. Kole et al. (eds.), Transgenic Crop Plants, 1

DOI 10.1007/978-3-642-04812-8_1, # Springer-Verlag Berlin Heidelberg 2010
2 N. Ferry and A.M.R. Gatehouse
Fig. 1.1 The world agricultural cake
1.2 Biotic Stress due to Insect Pests
1.2.1 Crop Losses due to Insect Pests
[A]nimals annually consume an amount of produce that sets calculation at defiance; and,
indeed, if an approximation could be made to the quantity thus destroyed, the world would
remain skeptical of the result obtained, considering it too marvelous to be received as truth.
– John Curtis, 1860
Arthropods are the most widespread and diverse group of animals, with an esti-
mated 4–6 million species worldwide (Novotny et al. 2002). While only a small
percentage of arthropods are classified as pests, they cause major devastation of
crops, destroying around 14% of the world annual crop production, contributing to
20% of losses of stored grains and causing around US$100 billion of damage each
year (Nicholson 2007).
Herbivorous insects and mites are a major threat to food production for human
consumption. Larval forms of lepidopterans are considered the most destructive
insects, with about 40% of all insecticides directed against heliothine species
(Brooks and Hines 1999). However, many species within the orders Acrina, Cole-
optera, Diptera, Hemiptera, Orthoptera and Thysanoptera are also considered
agricultural pests with significant economic impact (Fig. 1.2).
Insect pests may cause direct damage by feeding on crop plants in the field
or by infesting stored products and so competing with humans for plants as a
food resource. Some cause indirect damage, especially the sap-feeding (sucking)
insects by transmitting viral diseases or secondary microbial infections of crop
plants.
1 Transgenic Crop Plants for Resistance to Biotic Stress 3
Fig. 1.2 Phylloxera, a sap-sucking pest of grape, almost devastated the European wine industry in
the nineteenth century
1.2.1.1 The Phylloxera Plague
In the late nineteenth century, a phylloxera epidemic destroyed most of the

vineyards for wine grapes in Europe, most notably in France. Grape phylloxera
(Daktulosphaira vitifoliae, family Phylloxeridae) is a pest of commercial grape-
vines worldwide, originally native to eastern North America. These minute, pale
yellow sap-sucking insects feed on the roots of grapevines. In Vitis vinifera, the
resulting deformations and secondary fungal infections can damage roots, gradually
cutting off the flow of nutrients and water to the vine. Phylloxera was inadvertently
introduced to Europe in the 1860s. The European wine grape V. vinifera was highly
susceptible to the pest and the epidemic devastated most of the European wine-
growing industry. Some estimates hold that between two-thirds and nine-tenths of
all European vineyards were destroyed. Native American grapes Vitis labrusca are
naturally Phylloxera-resistant. The grafting of European grape vines onto resistant
grape rootstock is the preferred method to cope with the pest problem even today
(http://www.calwineries.com). Thus, phylloxera provides a clear example of how a
single insect pest can nearly devastate a whole industry.
Innumerable examples exist of insect pests that are highly injurious to agri-
cultural production. The most notable for their destructive capacity being the
Fig. 1.3 Monument to the

cotton boll weevil Source:
Wikimedia Commons
migratory locust (Locusta migratoria), Colorado potato beetle (Leptinotarsa

decemlineata), boll weevil (Anthonomus grandis), Japanese beetle (Popillia japon-
ica), and aphids, which are among the most destructive pests on earth as vectors of
plant viruses (many species in ten families of the Aphidoidea), and the western corn
rootworm (Diabrotica virgifera virgifera), also called the billion dollar bug because
of its economic impact in the US alone.
Curiously, one of these pests, the cotton boll weevil, responsible for near-
destruction of the cotton industry in North America, is also ultimately responsible
for subsequent diversification of agriculture in many regions, thus warranting a
monument in the town of Enterprise, Alabama, in profound appreciation of its role
in bringing to an end the state’s dependence on a poverty crop (Fig. 1.3).
The global challenge facing agriculture is to secure large and high-quality crop
yields and to make agricultural production environmentally sustainable. Control of
insect pests would go some way towards achieving this goal.
1.2.2 Insecticides
Insecticides have been, and still are, a highly effective method to control pests
quickly when they threaten to destroy crops. The chemical nature of the
insecticides used has evolved over time. In early farming practices, inorganic
chemicals were used for insect control; however, with the advances in synthetic
organic chemistry that followed the two world wars the synthetic insecticides
were born. In the 1940s, the neurotoxic organochlorine, DDT, was the pesticide
of choice, but following its indiscriminate use it was reported to bio-accumu-
late in the food chain where it affected the fertility of higher organisms – such
as birds. Rachel Carson first highlighted this in the book Silent Spring pub-
lished in 1962; while her presumptions have since been proven to be wrong,
the book was nevertheless an important signature event in the birth of the
environmental movement. This pesticide was subsequently replaced by the
comparatively safer organophosphate and carbamate-based pesticides (both
acetylcholinesterase inhibitors) and many of these were replaced in turn by
the even safer pyrethroid-based pesticides (axonic poisons). Synthetic pyre-
throids continue to be used today despite the fact that they are broad-spectrum
pesticides.
The major limiting factor on the insecticide strategy is the occurrence of
resistance in insect populations. In fact, resistance to insecticides has now been
reported in more than 500 species (Nicholson 2007). Furthermore, resistance has
evolved to every major class of chemical. The underlying causes of insecticide
resistance are manyfold. Owing to wide usage and narrow target range, arthropods
have been put under a high degree of selection pressure (Feyereisen 1995). Insecti-
cide resistance may be characterized by:
(a) Metabolic detoxification (upregulation of esterases, glutathione-S-transferases,
and monoxygenases)
(b) Decreased target site sensitivity (via mutation of the target receptor)
(c) Sequestration or lowered insecticide availability
In addition, cross-resistance to different classes of chemicals has occurred
because of the fact that many insecticides target a limited number of sites in the
insect nervous system (Raymond-Delpech et al. 2005). The five target sites in
insects comprise: nicotinic acetylcholine receptors (e.g., imidacloprid), voltage-
gated sodium channels (e.g., DDT, pyrethroids), g-aminobutyric acid receptors
(e.g., fipronil), glutamate receptors (e.g., avermectins), and acetylcholinesterase
(AChE) (e.g., organophosphates and carbamates). The world insecticide market is
dominated by compounds that inhibit the enzyme AChE. Together, AChE inhibi-
tors and insecticides acting on the voltage-gated sodium channel, in particular the
pyrethroids, account for approximately 70% of the world market (Nauen et al.
2001).
Unfortunately, as insecticide target sites are conserved between invertebrates
and vertebrates, insecticides have undesirable nontarget effects and unaccept-
able ecological impacts. Insecticides are implicated in the poisoning of nontar-
get insects, other arthropods, marine life, birds, and humans (Fletcher et al.
2000). The poisoning of nontarget organisms has obvious implications for
biodiversity.
1.2.3 Integrated Pest Management and Organic Agriculture
In parallel to the development of modern insecticides, the specific microbial toxins

produced by the soil-dwelling bacterium Bacillus thuringiensis (Bt) are increas-
ingly being adopted as biopesticides. In fact, microbial sprays of Bt are used in
organic agriculture. This shift is due in part to a demand for increased safety both
for humans and for the environment.
Organic agriculture is a form of agriculture that relies on crop rotation, green
manure, compost, biological pest control, and mechanical cultivation to maintain
soil productivity and control pests, excluding or strictly limiting the use of synthetic
fertilizers and synthetic pesticides, plant growth regulators, and genetically modi-
fied organisms (Directorate General for Agriculture and Rural Development of the
European Commission).
Integrated pest management (IPM) has also been proposed as a sustainable
control system for insects. Several control systems are combined, including the
judicious application of chemicals and biopesticides, use of trap crops, biological
control, rotation, good husbandry and cultural control to manage all the pests of a
particular crop (Gatehouse and Gatehouse 1999). Increasing crop varietal resistance
is critical to both IPM and organic agriculture.
It is unfortunate that ultimately neither organic agriculture nor IPM will be able
to feed the world. In order to feed an increasing world population, more food must
be produced in the future and on either the same amount, or less land (Ferry and
Gatehouse 2009). Neither of these farming methods will be as productive as will be
necessary to meet increased demands (Amman 2009).
1.2.4 Transgenic (Genetically Modified) Crops
Genetically modified (GM) maize and cotton varieties that express insecticidal
proteins derived from B. thuringiensis (Bt) have become an important component
in agriculture worldwide. At present, 20.3 million hectares of land is planted with
insect-protected transgenic Bt cotton and maize (James 2007), with economic
benefits from Bt cotton estimated at US$9.6 billion and maize US$3.6 billion
(James 2007). Significantly, Phipps and Park (2002) showed that on a global
basis GM technology has reduced pesticide use. These authors estimated that
pesticide use was reduced by a total of 22.3 million kg of formulated product in
2000 alone.
1.2.4.1 Bacillus thuringiensis Toxins
B. thuringiensis (Bt) is a soil-dwelling bacterium of major agronomic and scientific

interest. Whilst the subspecies of this bacterium colonize and kill a large variety of
Table 1.1 Insecticidal properties of Bt toxins

Insect order Cry protein
Lepidoptera Cry1A, Cry1B, Cry1C, Cry1E, Cry1F, Cry1I, Cry1J, Cry1K, Cry2A, Cry9A,
Cry9I, Cry15A
Coleoptera Cry1I, Cry3A, Cry3B, Cry3C, Cry7A, Cry8A, Cry8B, Cry8C, Cry14A, Cry23A
Diptera Cry2A, Cry4A, Cry10A, Cry11A, Cry11B, Cry16A, Cry19A, Cry20A, Cry21A
Hymenoptera Cry22A
Nematodes Cry5A, Cry6A, Cry6B, Cry12A, Cry13A, Cry14A
Liver fluke Cry5A
host insects, each strain tends to be highly specific. Toxins for insects in the orders
Lepidoptera (butterflies and moths), Diptera (flies and mosquitoes), Coleoptera
(beetles and weevils), and Hymenoptera (wasps and bees) (Table 1.1) have been
identified (de Maagd et al. 2001), but interestingly none with activity towards
Homoptera (sap suckers) have, as yet, been identified, although a few with activity
against nematodes have been isolated (Gatehouse et al. 2002). Further, there is little
evidence of effective Bt toxins against many of the major storage insect pests.
Bt toxins (also referred to as d-endotoxins; Cry proteins) exert their pathological
effects by forming lytic pores in the cell membrane of the insect gut. On ingestion,
they are solubilized and proteolytically cleaved in the midgut to remove the
C-terminal region, thus generating an “activated” 65–70 kDa toxin. The active
toxin molecule binds to a specific high-affinity receptor in the insect midgut epithelial
cells. Following binding, the pore-forming domain, consisting of a-helices, inserts
into the membrane; this results in cell death by colloid osmotic lysis, followed by
death of the insect (de Maagd et al. 2001). A number of putative receptors in the
insect gut have been identified and include aminopeptidase N proteins (Knight et al.
1994; Sangadala et al. 1994; Gill et al. 1995; Luo et al. 1997), cadherin-like proteins
(Vadlamudi et al. 1995; Nagamatsu et al. 1998; Gahan et al. 2001) and glycolipids
(Denolf 1996).
Transgenic plants expressing Bt toxins were first reported in 1987 (Vaeck et al.
1987) and following this initial study numerous crop species have been transformed
with genes encoding a range of different Cry proteins targeted towards different
pest species. Since bacterial cry genes (genes encoding Bt toxins) are rich in A/T
content compared to plant genes, both the full-length and truncated versions of
these cry genes have had to undergo considerable modification of codon usage and
removal of polyadenylation sites before successful expression in plants (de Maagd
et al. 1999). Crops expressing Bt toxins were first commercialized in the mid-1990s,
with the introduction of Bt potato and cotton. Currently, 20.3 million hectares of
land is planted with Bt cotton and maize (James 2007).
Bt Maize
Lepidopteran pests such as European corn borer Ostrinia nubilalis, fall armyworm
Spodoptera frugiperda, and corn earworm Helicoverpa zea perennially cause leaf
and ear damage to corn. The Bt concept was particularly attractive for maize, since
it made it possible to combat European corn borer larvae hidden inside the stem of
the plant for the first time. Bt maize has now been grown on a large scale for over a
decade, particularly in the US. In 2007, insect-resistant Bt maize was grown on 21%
of the total maize cultivation area, and Bt maize with a combination of insect and
herbicide resistance was grown on a further 28% (James 2007). Various Bt maize
varieties are also authorized in the EU. In 2007, there was notable cultivation of Bt
maize primarily in Spain, where it was grown on around 75,000 hectares (Ortego
et al. 2009).
Transgenic corn hybrids expressing the insecticidal protein Cry1Ab from
B. thuringiensis (Bt) var. kurstaki were originally developed to control European
corn borer, and offer the potential for reducing losses by fall armyworm and corn
earworm. Several events of transgenic Bt corn have been developed with different
modes of toxin expression (Ostlie et al. 1997). Amongst the most promising events
were Bt11 expressing the cry1Ab gene from B. thuringiensis subsp. kurstaki
(Novartis Seeds) and MON810 expressing a truncated form of the cry1Ab gene
from B. thuringiensis subsp. kurstaki HD-1 (Monsanto Co.). In both events, the
endotoxins are expressed in vegetative and reproductive structures throughout the
season (Armstrong et al. 1995; Williams and Davis 1997). Crops containing either
of these events are collectively referred to as having “YieldGard technology.”
Furthermore, a modified cry9C gene from B. thuringiensis subsp. tolworthi strain
BTS02618A is expressed in maize (tradename StarLink – marketed by Aventis
CropScience). StarLink corn has only been approved in the US for livestock
feed use.
In recent years, there has been increasing focus on another maize pest, this
time a Coleopteran (beetle); the western corn rootworm. Western corn root-
worm is one of the most devastating corn rootworm species in North America.
Its larvae are root pests and can destroy significant percentages of corn if left
untreated. In the US, current estimates show that 30 million acres (120,000 km)
of corn (out of 80 million grown) are infested with corn rootworms. The
United States Department of Agriculture (USDA) estimates that corn root-
worms cause US$1 billion in lost revenue each year. To make matters worse,
this pest is extending its geographical range all of the time – including
spreading throughout Europe. Bt maize which is resistant to the western corn
rootworm has been authorized in the US since 2003 and has been grown on a
large scale since. YieldGard Rootworm uses event MON 863 and expresses the
Cry3Bb1 protein from B. thuringiensis (subsp. kumamotoensis) in the plant,
protecting the plant against root feeding from the western and northern corn
rootworm larvae. Products containing both YieldGard Corn Borer (MON810)
and YieldGard Rootworm (MON 863) are marketed under the trade name
YieldGard Plus (http://www.agbios.com). Corn rootworm-resistant maize is
also produced by expression of the cry34Ab1 and cry35Ab1 genes from
B. thuringiensis strain PS149B1 (DOW AgroSciences LLC and Pioneer
Hi-Bred International, Inc.).
Bt Cotton
Cotton fibers used in textiles around the world come from the seed hairs of
Gossypium hirsutum. Cotton develops in closed, green capsules known as bolls
that burst open when ripe, revealing the white, fluffy fibers. But cotton is more than
just a fiber for textiles. It is also an important source of raw materials used in animal
feed and for various processed food ingredients, including cottonseed oil, protein-
rich cottonseed meal (mostly used as animal feed) and even leftover fibers can be
used as food additives.
Lepidopteran, particularly heliothine, pests can have an enormously damaging
effect on a cotton crop and controlling these insects in conventional farming
involves treatment with a number of insecticide sprays. In 1996, Bollgard1 cotton
(Monsanto) was the first Bt cotton to be marketed in the US. Bollgard cotton
produces the Cry1Ac toxin from B. thuringiensis (subsp. kurstaki), which has
excellent activity on tobacco budworm and pink bollworm. These two insects are
extremely important as both are difficult and expensive to control with traditional
insecticides and the damage caused by them directly impacts on the harvestable
plant organ, the cotton bolls themselves.
Bollgard II1 was introduced in 2003, representing the next generation of Bt
cottons. Bollgard II contains Cry1Ac plus a second gene from the Bt bacteria which
encodes the production of Cry 2Ab (also subsp. kurstaki). WideStrike (a Trademark
of DowAgrosciences) was registered for use in 2004, and like Bollgard II, it
expresses two Bt toxins but this time Cry1Ac and Cry1F were used in combination.
Both Bollgard II and WideStrike have better activity on a wider range of caterpillar
pests than the original Bollgard technology. GM Bt cotton has become widespread,
covering a total of 15 million hectares in 2007, or 43% of the world’s cotton. Most
GM cotton is grown in the US and China, but it can also be found in India, South
Africa, Australia, Argentina, Mexico, and Columbia (http://www.agbios.com).
Currently, 20% of the cotton grown commercially in China expresses Cry1Ac in
combination with a plant protease inhibitor, cowpea trypsin inhibitor (CpTI) (He
et al. 2009).
Bt Potato
Potato (Solanum tuberosum L.) is a major world food crop. Potato is exceeded only
by wheat, rice, and maize in terms of world production for human consumption
(Ross 1986). Many commercial potato varieties are highly susceptible to damage by
the Colorado potato beetle. In 1999, 93% of the 1.1 million potato acres grown in
the US were treated with a total of 2.6 million pounds of insecticide (http://www.
usda.gov/). To date, few traditionally bred varieties have been produced with
resistance to this major pest. Unfortunately, many of the pesticides currently used
are broad-spectrum pesticides, killing not only the target pest but most of its natural
enemies as well. The Cry3A d-endotoxin from B. thuringiensis Berliner subsp.
tenebrionis is toxic to coleopterans, particularly chrysomelids (Krieg et al. 1983;
Bauer 1990; MacIntosh et al. 1990). It is insecticidal against the Colorado potato
beetle, L. decemlineata (Ferro and Gerlernter 1989). In 1995, Bt.Cry3A (NewLeafTM)
potato became the first Bt-crop to be commercialized, although they are currently
withdrawn from the US market.
1.2.4.2 Evolution of Resistance in Pest Populations
Perhaps one of the most important issues surrounding the cultivation of Bt crops
relates to the evolution of target pest resistance, which could limit the life span of the
technology. In the case of Bt toxins, this is a major concern for the organic farming
community, since the potential for insect populations to evolve resistance to Bt will
not only limit the effectiveness of Bt-expressing crops but also Bt-based biopesti-
cides. Bt resistance in insect pests has been reported to develop within 4–5 genera-
tions in the laboratory (Stone et al. 1989). To date, the mechanism of resistance to
Cry toxins in insects has been most commonly ascribed to the loss or inactivation of
specific toxin-binding sites on midgut cell membranes (Ferré and Van Rie 2002).
Other resistance mechanisms that have been proposed include a defect in the toxin
activation by midgut proteases (Oppert et al. 1994; Sayyed et al. 2001), or an
increased repair and/or replacement rate of Cry-damaged midgut cells by stem
cells (Forcada et al. 1999). Studies have also revealed evidence for novel resistance
mechanisms based on active defensive responses (Rahman et al. 2004; Ma et al.
2005). When one considers the ability of insects to evolve resistance to chemical
pesticides (French-Constant 2004), the development of field resistance is inevitable
and in fact was recently reported to have already occurred (Tabashnik and Carrière
2009). Analysis of monitoring data shows that some field populations of H. zea have
evolved resistance to Cry1Ac, the toxin produced by first-generation Bt cotton
(Tabashnik et al. 2008). Nonetheless, resistance of H. zea to Cry1Ac has not caused
widespread crop failures in the field for several reasons (Tabashnik et al. 2008).
First, the documented resistance is spatially limited. Second, from the outset,
insecticide sprays have been used to improve the control of H. zea on Bt cotton
because Cry1Ac alone is not sufficiently effective to manage this pest. Finally, GM
cotton producing two Bt toxins (Cry2Ab and Cry1Ac) was planted on more than one
million ha in the US in 2006; control of H. zea by Cry2Ab would limit problems
associated with resistance to Cry1Ac (Jackson et al. 2004).
Considerable effort has been devoted to delaying the onset of evolution of
resistance, e.g., the use of refugia has been required/recommended in most regions
growing Bt-crops depending upon the country in question (Tabashnik and Carrière
2009). Gene-stacking and integrated pest management should be combined to
control this problem.
1.2.4.3 Unexpected Benefits
Interestingly, the expression of Bt has resulted in improved crop quality as a

consequence of decreased levels of Fusarium infestation and fumonisin mycotoxin
production as a direct result of reduced levels of insect pest damage. This benefit is
particularly important in food crops such as maize (Glaser and Matten 2003).
1.2.4.4 A Global Technology
Of the global total of 12 million biotech farmers in 2007, over 90% were small and
resource-poor farmers from developing countries; the balance of one million were
large farmers from both industrialized countries such as Canada and developing
countries such as Argentina. Of the 11 million small farmers, most were Bt cotton
farmers; 7.1 million in China (Bt cotton), 3.8 million in India (Bt cotton), and the
balance of 100,000 in the Philippines (GM maize), South Africa (GM cotton, maize
and soybeans often grown by subsistence women farmers) and the other eight
developing countries which grew GM crops in 2007. This modest uptake by
subsistence farmers contributes towards the Millennium Development Goals of
reducing poverty by 50% by 2015 and is a very important development (James
2007).
1.2.5 Other Sources of Insecticidal Molecules
The concept of employing genes encoding Bt toxins to produce insect-resistant

transgenic plants arises from the successful use of Bt-based biopesticides.
A number of other strategies for protecting crops from insect pests actually exploit
endogenous resistance mechanisms (Harborne 1998; Gatehouse 2002a, b). Genes
encoding such defensive proteins are obvious candidates for enhancing crop resis-
tance to insect pests.
1.2.5.1 Enzyme Inhibitors
Interfering with digestion, and thus affecting the nutritional status of the insect, is a
strategy widely employed by plants for defense, and has been extensively investi-
gated as a means of producing insect-resistant crops (Gatehouse 2002a, b). Insect-
digestive proteases tend to fall into four mechanistic classes (serine, cysteine,
aspartic or metallo proteases – depending on the enzyme-active site residue).
Numerous studies since the 1970s have confirmed the insecticidal properties of a
broad range of protease inhibitors from both plant and animal sources (Jouanin et al.
1998; Gatehouse 2002a, b). Proof of concept for exploiting such molecules for crop
protection was first demonstrated with expression of a serine protease inhibitor
from cowpea (CpTI), which was shown to significantly reduce insect growth and
survival (Hilder et al. 1987). These studies were subsequently extended to include a
greater range of target pests (Gatehouse et al. 1994; Graham et al. 1995; Xu et al.
1996), and a broader range of inhibitors and plant species, including economically
important pest species, particularly lepidopterans (Broadway 1997; De Leo et al.

2001).
Since many economically important coleopteran pests predominantly utilize
cysteine proteases for protein digestion, inhibitors for this class of enzyme (cysta-
tins) have also been investigated as a means for controlling pests from this order.
Oryzacystatin, a cysteine protease inhibitor isolated from rice seeds, is effective
towards both coleopteran insects and nematodes when expressed in transgenic
plants (Leple et al. 1995; Urwin et al. 1995; Pannetier et al. 1997). Similarly, the
cysteine/aspartic protease inhibitor equistatin, from sea anemone, is also toxic to
several economically important coleopteran pests, including the Colorado potato
beetle (Outchkourov et al. 2003). More recent studies have included the stacking of
different families of inhibitors to increase the spectrum of activity (Abdeen et al.
2005).
A major limitation, however, to this strategy for the control of insect pests arises
from the ability of some lepidopteran and coleopteran species to respond and adapt
to ingestion of protease inhibitors by either overexpressing native gut proteases, or
producing novel proteases that are insensitive to inhibition (Bown et al. 1997;
Jongsma and Bolter 1997). Thus, detailed knowledge about the enzyme–inhibitor
interactions, both at the molecular and biochemical levels, together with detailed
knowledge on the response of insects to exposure to such proteins is essential to
effectively exploit this strategy. The concept of inhibiting protein digestion as a
means of controlling insect pests has been extended to the inhibition of carbohy-
drate digestion. For example, inhibitors of a-amylase have been expressed in
transgenic plants and shown to confer resistance to bruchid beetles (Shade et al.
1994; Schroeder et al. 1995).
1.2.5.2 Lectins
Lectins, found throughout the plant and animal kingdoms, form a large and diverse
group of proteins identified by a common property of specific binding to carbohy-
drate residues, either as free sugars, or more commonly, as part of oligo- or
polysaccharides. Many physiological roles have been attributed to plant lectins,
including defense against pests and pathogens (Chrispeels and Raikhel 1991;
Peumans and Vandamme 1995).
Although some lectins are toxic to mammals, and are thus not suitable candi-
dates for transfer to crops for enhanced levels of protection, this is by no means
universal. Many lectins are not toxic to mammals, yet are effective against insects
from several different orders (Gatehouse et al. 1995), including homopteran pests
such as hoppers and aphids (Powell et al. 1995; Sauvion et al. 1996; Gatehouse et al.
1997). This finding has generated significant interest, not least since no Bts effective
against this pest order have been identified to date. One such lectin is the snowdrop
lectin (Galanthus nivalis agglutinin; GNA). Both constitutive and phloem-specific
(Rss1 promoter) expression of GNA in rice is an effective means of significantly
reducing survival of rice brown planthopper (Nilaparvata lugens), and green
leafhopper (Nephotettix virescens) both serious economic pests of rice (Rao et al.
1998; Foissac et al. 2000; Tinjuangjun et al. 2000). GNA has been expressed in
combination with other genes encoding insecticidal proteins, including the cry
genes (Maqbool et al. 2001). Although lectins such as GNA, and ConA are not as
effective against aphids as they are against hoppers, they nonetheless have signifi-
cant effects on aphid fecundity when expressed in potato (Down et al. 1996;
Gatehouse et al. 1997, 1999) and wheat (Stoger et al. 1999).
The precise mode of action of lectins in insects is not fully understood although
binding to gut epithelial cells appears to be a prerequisite for toxicity. In the case of
rice brown planthopper, GNA not only binds to the luminal surface of the midgut
epithelial cells, but also accumulates in the fat bodies, ovarioles and throughout the
haemolymph, suggesting that the lectin is able to cross the midgut epithelial barrier
and pass into the insect’s circulatory system, resulting in a systemic toxic effect
(Powell et al. 1998; Du et al. 2000).
As with protease inhibitors, the levels of protection conferred by the expression
of lectins in transgenic plants are generally not high enough to be considered
commercially viable. However, the absence of genes with proven high insecticidal
activity against homopteran pests may well mean that transgenic crops with partial
resistance may still find acceptance in agriculture, especially if expressed with other
genes that confer partial resistance, or if introduced into partially resistant genetic
backgrounds.
1.2.5.3 A Brief Aside; Plant Parasitic Nematodes
Plant parasitic nematodes include several groups causing severe crop losses. The
most common genera are: Aphelenchoides (foliar nematodes), Meloidogyne (root-
knot nematodes), Heterodera, Globodera (cyst nematodes) such as the potato cyst
nematode, Nacobbus, Pratylenchus (lesion nematodes), Ditylenchus, Xiphinema,
Longidorus, and Trichodorus. Several phytoparasitic nematode species cause his-
tological damages to roots, including the formation of visible galls (Meloidogyne).
Some nematode species transmit plant viruses through their feeding activity on
roots. One of them is Xiphinema index, vector of GFLV (grapevine fanleaf virus),
an important disease of grapes. Bt toxins, lectins (Burrows et al. 1999) and protease
inhibitors have shown some promise for control, particularly the expression of
cystatins (Cowgill et al. 2002). For a recent review of the topic, the reader is
referred to Fuller et al. (2008).
1.2.5.4 Other Sources of Insecticidal Molecules
Generating insecticidal transgenic crops harboring genes from nonconventional

sources is an extremely active area, with amongst others, foreign genes from plants
(e.g., enzymes inhibitors and novel lectins) and animal sources including insects
(e.g., biotin-binding proteins, neurohormones, venoms and enzyme inhibitors)

being a major focus (Ferry et al. 2006).
The development of second-generation transgenic plants with greater durable
resistance might result from the expression of multiple insecticidal genes such as
the Vip (vegetative insecticidal proteins) produced by B. thuringiensis during its
vegetative growth. The benefit of such an approach is a broader insect target range
than conventional Bt proteins and the proposed expectation to control current Bt
resistant pests due to the low levels of homology between the domains of the two
proteins classes (Christou et al. 2006).
With Bt toxins as the classical reference, toxins from other insect pathogens
provide a potential repository of novel insecticidal compounds. Photorhabdus spp.
are bacterial symbionts of entomopathogenic nematodes, which are lethal to a wide
range of insects (Chattopadhyay et al. 2004). Photorhabdus toxin expression in
Arabidopsis caused significant insect mortality (see for review Ferry et al. 2006).
Thus, toxins from other insect pathogens are also opening up new routes to pest
control using transgenic-based strategies.
Interesting recent developments include the use of novel proteins from insect
biological control agents and insect hormones to generate transgenic crops. A
teratocyte secretory protein from a hymenopteran endoparasitoid (a parasitic
wasp often used in biocontrol programs) has been expressed in transgenic tobacco
and shown to increase resistance to lepidopteran pests (Maiti et al. 2003). Similar
protection has also been achieved with insect peptide hormones (Tortiglione et al.
2003). Interestingly, they replaced the tomato systemin peptide region of prosyste-
min (a plant signaling molecule) with the insect peptide and showed that this
resulted in the production of biologically active insecticidal peptides.
Reliance on the expression of a single gene product for pest control is a relatively
short-term strategy that parallels the use of exogenously applied chemical pesti-
cides. Thus, pyramiding (stacking) of genes encoding different Bt toxins has been
developed as a method for preventing the onset of evolution of pest resistance, and
for conferring greater levels of pest control (Boulter et al. 1990; Maqbool et al.
2001; Zhao et al. 2003). This strategy has now been adopted in commercially
available crops (see above). For example, corn lines have recently been developed
(Moellenbeck et al. 2001; Ostlie 2001) coexpressing two d-endotoxins from Bt for
resistance to corn rootworm. Hybrid proteins have also been developed to enhance
and extend the activity of Bt toxins. The use of a single Bt toxin in a crop is limited
in that many insects attack a single crop and toxins generally show very high
specificity towards a single pest species. Therefore, toxins have been engineered
to modify their receptor recognition and pore formation. Each toxin consists of
three domains. Domain I is involved in membrane insertion and pore formation.
Domains II and III are both involved in receptor recognition and binding. Addi-
tionally, a role for domain III in pore function has been found. This approach has
proved successful in both enhancing activity (Karlova et al. 2005) and extending
host range (Singh et al. 2004). Such hybrid/fusion proteins offer an alternative/
complementary strategy to address potential limitations in conventional transgenic
insect pest control.
1.2.5.5 Transgenic Plants Expressing Fusion Proteins
The concept of “gene stacking” has recently been extended to the development
and use of fusion proteins. Such proteins not only provide a means of increasing
durability, but also provide a “vehicle” for more effective targeting of insecti-
cidal molecules, including peptides. It thus offers an alternative/complementary
strategy to address potential limitations in conventional transgenic insect pest
control. For example, recognition of binding sites in the insect gut is an important
factor determining the toxicity of Bt. Enhancing toxin-binding capabilities should
thus extend host range and delay resistance in pest populations. Bt is believed to
bind primarily to aminopeptidase N or cadherin membrane proteins, while the
generation of a fusion protein with the nontoxic B-chain from ricin (RB) was
shown to extend the binding of Bt to include specific glycoproteins. Transgenic
plants expressing the Bt fused RB demonstrated that the addition of the RB-
binding domain provided a wider repertoire of receptor sites within target species
and significantly enhanced the levels of toxicity of Bt. For example, survival of
the armyworm Spodoptera littoralis, a species of insect not sensitive to Bt, was
reduced by ~90% when feeding on transgenic maize expressing the fusion,
compared to plants expressing either Bt Cry1Ac alone, or the RB-binding
domain. Expression of the fusion protein resulted in the insect becoming suscep-
tible to Bt (Mehlo et al. 2005). This strategy has shown great potential beyond
just extending the toxicity of Bt. Zhu-Salzman et al. (2003) have generated fusion
proteins with anchor regions to other insecticidal proteins to the insect gut
epithelium. Using the legume lectin rGSII, they proposed a system to combat
the ability of certain insect species to activate protease inhibitor-insensitive
proteolytic enzymes. The soybean cysteine protease inhibitor soyacystatin N
(scN) was covalently linked to the GlcNAc-specific legume lectin using a natu-
rally occurring linker region from the potato multicystatin. In this instance, the
fusion protein not only has a novel binding ability that is proposed to initiate a
concentration effect by localizing the inhibitor at the anterior of the gut, but the
fused lectin moiety additionally offers a degree of protection to the insecticidal
moiety by blocking the access of scN-insensitive proteases, thereby preventing
proteolytic destruction of the cystatin.
Not only do fusion proteins have potential for use in transgenic crops, but also to
improve the efficacy of biopesticide-based sprays. Neuropeptides potentially offer a
high degree of biological activity, and thus provide an attractive alternative pest
management strategy. There are major drawbacks to their use, particularly as
topical sprays. They are unlikely to be rapidly absorbed through the insect cuticle
to their site of action, and are prone to proteolysis and rapid degradation in the
environment. Should they survive the application process and are then taken up by
the insect, they are then unlikely to survive the conditions of the insect gut or be
delivered to the correct targets within the insect. The discovery that snowdrop lectin
(GNA) remains stable and active within the insect gut after ingestion, and that it is
able to cross the gut epithelium, provided an opportunity for its use as a “carrier
molecule” to deliver other peptides to the circulatory system of target insect
species. This strategy effectively delivered the insect neuropeptide hormone, alla-
tostatin, to the haemolymph of the tomato moth Lacanobia oleracea (Fitches et al.
2002). Subsequent expression of the fusion protein in potato further provided proof
of concept for the efficacy of fusion proteins as a means of delivery. The results
demonstrated significant reduction in mean larval weight when compared to the
controls. GNA can be used to deliver insecticidal peptides isolated from the venom
of the spider Segestria florentina (SFI1) to the haemolymph of L. oleracea (Fitches
et al. 2004). Neither the GNA nor the SFI1 moieties alone were acutely toxic;
however, the SFI1/GNA fusion was insecticidal to first stage larvae, causing 100%
mortality after 6 days. This spider venom neurotoxin is believed to irreversibly
block the presynaptic neuromuscular junctures. Such venom toxins show high
degrees of specificity and thus lend themselves to environmentally benign pest
management strategies.
1.2.6 Manipulation of Plant Endogenous Defenses
Alternative strategies for protecting crops from insect pests, that are not dependent
on the expression of single or stacked genes, seek to exploit the induced endoge-
nous resistance mechanisms exhibited by plants to most insect herbivores. For
further information, the reader is referred to Chap. 10 of this volume. Such induced
defenses are exemplified by the wounding response, first identified as the local and
systemic synthesis of proteinase inhibitors (PIs), which block insect digestion in
response to plant damage (Gatehouse 2002a, b). Many transgenic strategies have
attempted to exploit the potential overexpression of plant PIs to protect crops from
pest damage (Jouanin et al. 1998) but these have relied on the transfer of a single PI
gene, and many insects have been able to adapt to this.
More recent research has shown that induced defenses also involve the plant’s
ability to produce toxic or repellent secondary metabolites as direct defenses,
and volatile molecules, which play an important role in indirect defense (Kessler
and Baldwin 2002). Insect herbivores activate induced defenses both locally and
systemically via signaling pathways involving systemin, jasmonate, oligogalacturo-
nic acid and hydrogen peroxide (Fig. 1.4).
Ecologists have long understood that plants exhibit multi-mechanistic resistance
towards herbivores, but the molecular mechanisms underpinning these complicated
responses have remained elusive (Baldwin et al. 2001). However, recent studies
investigating the plant’s herbivore-induced transcriptome, using microarrays and
differential display technologies, have provided novel insights into plant–insect
interactions. The jasmonic acid cascade plays a central role in transcript accumula-
tion in plants exposed to herbivory (Hermsmeier et al. 2001). A single microarray-
based study revealed that the model plant Arabidopsis undergoes changes in levels
of over 700 mRNAs during the defense response (Schenk et al. 2000). In contrast,
only 100 mRNAs were upregulated by spider mite (Tetranicus urticae) infestation
in lima bean (Phaseolus lunatus), although a further 200 mRNAs were upregulated
Herbivory
physical forces
local peptide hormone release eg.

prosystemin
insect dervived elicitors ethylene
systemin
plant-plant pest deterrent/
interactions receptor binding attraction of
natural
ABA enemies
linolenic acid
SA
octadecanoid pathway Voltiles e.g.
-green leaf
methyl
volatiles
jasmonate
-C10, C15
jasmonic acid
terpenoids
-indole
auxin, SA (-)
ethylene, ABA (+)
polygalactouronidase
oligalacturonic acid plant-plant
interactions
vascular bundle (systemic
NAPDH induction of
oxidase
Pls)
Cell wall
H202 early
genes -
late signalling
genes
Insecticidal (defence)
compounds
e.g. Protease mesophyll
Inhbitors cells
Fig. 1.4 The generalized plant-wounding response. Generalized overview of the plant wounding
response, and signalling molecules which can modulate it, showing the pathways necessary for
both local and systemic induction of insecticidal proteins. (Adapted from: Ferry et al. 2004)
in an indirect response mediated by feeding-induced volatile signal molecules

(Arimura et al. 2000).
Deciphering of the signals regulating herbivore-responsive gene expression will
afford many opportunities to manipulate the response. Signaling molecules such as
salicylic acid, jasmonic acid and ethylene do not activate defenses independently by
linear cascades, but rather establish complex interactions that determine specific
responses. Knowledge of these interactions can be exploited in the rational design
of transgenic plants with increased insect resistance (Rojo et al. 2003; De Vos et al.
2005; Giri et al. 2006).
1.2.6.1 A Special Case: Sap-Feeding Insects
While most herbivorous insects cause extensive damage to plant tissues when
feeding, many insects of the order Homoptera feed from the contents of vascular
tissues by inserting a stylet between the overlying cells, thus limiting cell damage
and minimizing induction of a wounding response. In contrast to wounding, plant
responses following attack by these insects have been shown to be typical of pathogen
attack, with examples of gene-for-gene interactions being known (Walling 2000;
Moran et al. 2002). However, these pathogen-induced pathways can induce expres-
sion of many of the genes upregulated by wounding because of pathway cross-talk.
Moran and Thompson (2001) demonstrated that phloem feeding by the green
peach aphid (Myzus persicae) on Arabidopsis induced expression of genes asso-
ciated with salicylic acid (SA) responses to pathogens, as well as a gene involved in
the jasmonic acid-mediated response pathway. These results suggest stimulation of
response pathways involved in both pathogen and herbivore responses. Microarray
data has identified genes involved in oxidative stress, calcium-dependent signaling,
pathogenesis-related responses, and signaling as key components of the induced
response (Moran et al. 2002). It may be that transgenic strategies that activate such
signaling cascades could enhance plant resistance to these problematic pests.
1.2.6.2 Indirect Defense (Volatile Production)
The role of plant volatiles in indirect defense has been described as “top-down”
defense (Baldwin et al. 2001). Some volatiles appear to be common to many
different plant species, including C6 aldehydes, alcohols and esters (green leaf
volatiles), C10 and C15 terpenoids, and indole, whereas others are specific to a
particular plant species. Many volatiles are preformed and act in herbivore deter-
rence; furthermore, the wounding response also includes the formation of volatile
compounds. Top-down control of herbivore populations is achieved by attracting
predators and parasitoids to the feeding herbivore, mediated by these volatile
organic compounds (VOCs). For example, genes involved in the biosythesis of
the maize VOC bouquet are upregulated by insect feeding (Frey et al. 2000; Shen
et al. 2000). In addition, herbivore oviposition has been shown to induce VOC
emissions, which attract egg parasitoids (Hilker and Meiners 2002). Herbivore-
induced VOCs can also elicit production of defence-related transcripts in plants
near the individual under attack (Arimura et al. 2000; Dicke et al. 2003). Exposure
to herbivore-induced volatiles in lima bean results in transcription of genes
involved in ethylene biosynthesis (Arimura et al. 2000).
Manipulation of volatile biosynthesis can affect insect resistance. Transgenic
potatoes in which production of hydroperoxide lyase (the enzyme involved in green
leaf volatile biosynthesis) was reduced were found to support improved aphid
performance and fecundity, suggesting toxicity of these volatiles to M. persicae
(Vancanneyt et al. 2001). In a review of the topic Degenhardt et al. (2003) discuss
the potential of modifying terpene emission with the aim of making crops more
attractive to herbivore natural enemies.
1.2.6.3 Detoxification and Insect Modulation of the Wounding Response
However, insect pests are able to feed on plants despite their defenses, both
constitutive and inducible. Many insects are able to detoxify potentially toxic
secondary metabolites, using cytochrome P-450 monoxygenases and glutathione-
S-transfereases. These enzymes are induced by exposure to toxic plant secondary
compounds, for example, xanthotoxin (a furanocoumarin) induces P-450 expres-

sion in corn earworm (Li et al. 2000). More recently, Li et al. (2002) have shown
that corn earworm uses signaling molecules from its plant host, jasmonate and
salicylate, to activate four of its cytochrome P450 genes, thus making the induction
of detoxifying enzymes rapid and specific. Recent strategies based on RNAi
technology have shown that it is possible to overcome these insect responses
(discussed later in this chapter).
1.2.7 RNAi
Disrupting gene function by the use of RNAi is a well-established technique in

insect genetics based on delivery by injection into insect cells or tissues. The
observation that RNAi could also be effective in reducing gene expression,
measured by mRNA level, when fed to insects (Turner et al. 2006) has led to two
recent articles in which transgenic plants producing double-stranded RNAs
(dsRNAs) which are shown to exhibit partial resistance to insect pests. Transgenic
maize producing dsRNA directed against V-type ATPase of corn rootworm showed
suppression of mRNA in the insect and reduction in feeding damage compared to
controls (Baum et al. 2007). Similarly, transgenic tobacco and Arabidopsis expres-
sing dsRNA directed against a detoxification enzyme (Cytochrome P450 gene
CYP6AE14) for the breakdown of gossypol (a defensive metabolite) in cotton
bollworm caused the insect to become more sensitive to gossypol in the diet
(Mao et al. 2007). This approach holds great promise for future development. It
is also proving effective for nematode control (Bakhetia et al. 2005). For a recent
review on RNAi-mediated crop protection against insect pests the reader is referred
to Price and Gatehouse (2008).
Advances in our understanding of induced responses in plants and their regula-
tion, has refocused attention on potential exploitation of endogenous resistance
mechanisms for crop protection. While plant resistance is an integral component of
organic and IPM strategies, it does not afford similar levels of protection as those
provided by the use of “direct” protective methods such as the expression of Bt
toxins. The goal of the plant breeder, and now the biotechnologist, is to engineer
durable multi-mechanistic resistance to insect pests in crops, and increased know-
ledge of induced defense mechanisms and their molecular control is likely to play
an important role in realizing this aim.
1.2.8 Environmental Impact of IR Crops
Almost from the beginning of the production of transgenic crops there have been
concerns over their use and introduction into the environment. There is interna-
tional agreement that GM crops should be evaluated for their safety, including their
environmental impact (Dale 2002). During the past 15–20 years, there have been
extensive research programs of risk assessment, with several areas of major concern
identified.
1.2.8.1 Impact on Nontarget Organisms
Assessing the consequences of pest control on nontarget organisms is an important

precursor to their becoming adopted in agriculture. The expression of transgenes
that confer enhanced levels of resistance to insect pests is of particular significance
since it is aimed at manipulating the biology of organisms in a different trophic
level to that of the plant. Potential risks to beneficial nontarget arthropods exist.
Those groups most at risk include: nontarget Lepidoptera, beneficial insects (polli-
nators, natural enemies) and soil organisms.
Exposure of nontarget Lepidoptera to insecticidal transgene products may occur
through both direct consumption of transgenic plant tissues including via consump-
tion of transgenic pollen; many nontarget Lepidoptera are rare butterflies having
great conservation value. The case of the Monarch butterfly (Danaus plexippus), a
conservation flagship species in the US, highlighted the need for ecological impact
research. In a letter to Nature, Losey et al. (1999) claimed that both survival and
consumption rates of Monarch larvae fed milkweed leaves (natural host) dusted
with Bt pollen were significantly reduced, and that this would have profound
implications for the conservation of this species. However, a series of ecologically
based studies rigorously evaluated the impact of pollen from such crops on Mon-
archs and demonstrated that the commercial wide-scale growing of Bt-maize did
not pose a significant risk to the Monarch population (Hellmich et al. 2001;
Gatehouse et al. 2002). In fact, the initial experiments did not quantify the dose
of pollen used, or indeed, if this was a realistic level likely to be encountered in the
field; nevertheless, this work highlighted the importance of studying nontarget
effects. In a separate field study Wraight et al. (2000) showed that Papilio polyxenes
(black swallowtail) larvae were unaffected by pollen from Bt expressing maize
event Mon810 at 0.5, 1, 2, 4 and 7 m from the transgenic field edge, highlighting the
need for a case-by-case study of organisms considered to be at risk. In addition to
the potential direct impacts of Bt toxins on susceptible target insects, as in the case
of the Morarch butterfly, some Lepidoptera have been shown to have a reduced
sensitivity to the lepidopteran-specific Bt toxins. For example, S. littoralis can
survive on maize expressing Cry1Ab (Hilbeck et al. 1998) and thus present a
route of exposure to the next trophic level. In the case of Bt Cry3Aa or Cry3Bb
expressing potatoes or maize, some Lepidoptera may represent nontarget secondary
pests, and whilst not directly affected by the transgene product themselves may
again present a route of exposure to the next trophic level, as do other nontarget
herbivores. Organisms such as those belonging to the orders Homoptera, Hemi-
ptera, Thysanoptera, and Tetranychidae are not targeted by Bt toxins expressed in
transgenic plants; however, they do utilize the Bt crop (Groot and Dicke 2002). The
direct effect that this may have on these insects is dependent on the presence of Bt
receptors in the first instance, and it is so far unclear whether such receptors are
present in nontarget organisms (de Maagd et al. 2001). In addition, the fate of the
toxin ingested by nontarget herbivores is unclear, since if it retains toxicity then this
may have implications at the next trophic level.
The impacts of insect-resistant transgenic crops at higher trophic levels have also
been considered, where there are concerns over the risks to beneficial arthropod
biodiversity (Schuler et al. 1999; Bell et al. 2001); in particular, predators and
parasitoids, which play an important role in suppressing insect pest populations
both in the field and under specialized cultivation systems (glasshouses). Natural
enemies may ingest transgene products via feeding on herbivorous insects that have
themselves ingested the toxin from the plant; such tritrophic interactions will be
influenced by the susceptibility of the herbivore to the plant protection product. If,
as in the case with Bt toxins, the prey item is susceptible to the toxin, then the
predator will not come into contact with the toxin as the pest will effectively be
controlled, and in target insects the toxin is bound to receptors in the midgut
epithelium that are structurally rearranged and may lose their entomotoxicity (de
Maagd et al. 2001). In nontarget insects (and resistant insects), the toxins do not
bind and may thus retain biological activity. However, the overwhelming weight of
evidence from independent laboratory and field studies show that Bt toxins have a
limited ability to affect the next trophic level (reviewed in Sanvido et al. 2007;
Ferry and Gatehouse 2009; Romeis et al. 2008).
Pollinators represent another group of nontarget organisms highlighted as at risk
from Bt toxins in GM crops. The current generation of transgenic crops produce Bt
toxin in the pollen as well as in the vegetative tissues. Several studies have been
conducted to determine toxicity of Bt toxins to pollinators (Vandenberg 1990; Sims
1995, 1997; Arpaia 1997; Malone and Pham-Delegue 2001); generally, they all
conclude that neither the adults nor the larvae of bees were affected by Bt toxins. For
a comprehensive review of the impact of transgenic crops on pollinators, the reader
is referred to two recent reviews (Malone et al. 2008; Malone and Burgess 2009).
Finally, nontarget species may come into contact with Bt toxins via the environ-
ment. Several studies have shown that Bt toxins released from transgenic plants
bind to soil particles (Palm et al. 1996; Crecchio and Stotzky 1998; Saxena et al.
1999). Soil-dwelling and epigeic insects such as Collembola and Carabidae may
thus be exposed to the toxins. Several studies (Saxena and Stotzky 2001; Ferry et al.
2007) show no differences in mortality or body mass of bacteria, fungi, protozoa,
nematodes and earthworms or carabid beetles exposed to Bt.
Exposure to the transgene products, however, does not necessarily imply a
negative impact. Most studies to date have demonstrated that crops transformed
for enhanced pest resistance have no deleterious effects on beneficial insects
(reviewed in Ferry et al. 2003; Romeis et al. 2008).
1.2.9 Conclusions
Ultimately one must consider the impact of transgenic crops and specifically Bt
toxins in comparison to other pest control strategies such as conventional crop
protection using insecticides. While pesticides have no doubt brought vast yield
improvements, they have well-documented undesirable nontarget effects (Devine
and Furlong 2007). It is worth remembering that whilst potential risks do exist to
the environment from the cultivation of GM crops, their potential to decrease
reliance on external inputs (less insecticide sprays) and to increase the availability
of genetic resources available to breeders is great (Ferry and Gatehouse 2009).
1.3 Biotic Stress due to Weeds
1.3.1 Crop Losses due to Weeds
Weeds can compete with productive crops or pasture, or convert productive land
into unusable scrub. Weeds are also often poisonous, distasteful, produce burrs, or
thorns that interfere with the use and management of desirable plants by contam-
inating harvests or excluding livestock. Weeds tend to thrive at the expense of the
more refined edible or ornamental crops. They provide competition for space,
nutrients, water and light, although how seriously they will affect a crop depends
on a number of factors. Some crops have greater resistance to competition than
others, for example smaller, slower-growing seedlings are more likely to be over-
whelmed than those that are larger and more vigorous. Weeds also differ in their
competitive abilities, and this can vary according to specific conditions and the time
of year. Tall-growing vigorous weeds such as fat hen (Chenopodium album) can
have the most pronounced effects on adjacent crops. Chickweed (Stellaria media),
a low-growing plant, can happily coexist with a tall crop during the summer, but
plants that have overwintered will grow rapidly in early spring and may swamp
crops.
Simply put, weeds are any plant growing in an area where it is not wanted. Of
over 250,000 plant species in the world, only a few hundred are troublesome weeds.
Although no single characteristic clearly defines a weed, two attributes are common
in the worst weeds: competitiveness and persistence. The world’s worst weeds are
shown in Table 1.2 (Chrispeels and Sadava 2003).
1.3.2 Weed Management: Prevention, Control, and Eradication
As weeds are persistent, once they have established in a field they are extremely
difficult to eradicate. One of the most successful attempts in the eradication of a
weed is provided by witchweed (Striga sp.) in the US. Witchweed is a parasitic
plant; its seeds germinate in response to a chemical, strigol, produced by the roots of
the host plant. The germinated seedling attaches to the root through special haus-
toria, as this occurs underground infestation can go unnoticed, consequently serious
Table 1.2 World’s worst weeds (from: Chrispeels and Sadava 2003)
Plant family Weed examples Crop
Grass (Poaceae) Bermuda grass (Cynodon dactylon) Wheat
Barnyard grass (Echinochloa crus-galli)
Johnson grass (Sorghum halepense)
Sedge (Cyperaceae) Purple nutsedge (Cyperus rotundus) None
Yellow nutsedge (Cyperus esculentus)
Smallflower umberella sedge (Cyperus
difformis)
Sunflower (Asteraceae) Canada thistle (Cirsium arvense) Sunflower
Common cocklebur (Xanthium strumarium)
Common dandelion (Taraxacum officinale)
Buckwheat (Polygonaceae) Wild buckwheat (Polygonum convolvulus) Buckwheat
Curly dock (Rumex crispus)
Red sorrel (Rumex acetosella)
Pigweed (Amaranthaceae) Smooth pigweed (Amaranthus hybridus) Grain
amaranth
Spiny amaranth (Amaranthus sinosus)
Redroot pigweed (Amaranthus retroflexus)
Mustard (Brassicaceae) Shepherd’s purse (Capsella bursa-pastoris) Canola
Hoary cress (Brassica draba)
Wild mustard (Brassica kaber)
Legume (Fabaceae) Black medic (Medicago lupulina) Soybean
Sensitive plant (Mimosa pudica)
Kudzu (Pueraria lobata)
Morning glory Field bindweed (Convolvulvus arvensis) Sweet potato
(Convolvulaceae)
Field dodder (Cuscuta campestris)
Swamp morning glory (Ipomoea aquatica)
Spurge (Euphorbiaceae) Leafy spurge (Euphorbia esula) Cassava
Garden spurge (Euphorbia hirta)
Spotted spurge (Euphorbia maculata)
Goosefoot (Chenopodiaceae) Common Lamb’s quarters (Chenopodium Sugarbeet
album)
Russian thistle (Salsola iberica)
Nettleleaf goosefoot (Chenopodium murale)
Mallow (Malvaceae) Venice mallow (Hibiscus trionum) Cotton
Velvetleaf (Abutilon theophrasti)
Arrowleaf sida (Sida rhombifolia)
Nightshade (Solanaceae) Black nightshade (Solanum nigrum) Potato
Jimsonweed (Datura stramonium)
Cutleaf groundcherry (Physalis angulata)
infestation leading to yield loss occurs before the parasitic plant even appears
through the soil. Once the witchweed comes through the soil it becomes photosyn-
thetically active, but still dependent on its host for water. Such parasitic weeds are
extremely difficult to control and yield losses of approximately 50% can be
expected under drought conditions. In the US, only quarantine of infected areas
reduced witchweed infection from 150,000 hectares to a couple of 1,000 hectares,
but this quarantine lasted for nearly 50 years (Chrispeels and Sadava 2003)!
For this reason, preventative weed management is critical. This involves keeping
weed seeds and vegetative propagules from getting to the field in the first place and
is mainly achieved through seed cleaning and the purchase of certified clean seed.
Good management practice and the cleaning of farm equipment moved between
different areas are critical in preventative control. Ultimately weed control is
achieved by mechanical, biological and predominately chemical means.
1.3.3 Herbicides
The economic importance of weeds is emphasized by the fact that the herbicides
comprise as large a share of the world agrochemical market as all other pesticides
combined, farmers spend an annual US$10 billion to control weeds in the US alone
(Chrispeels and Sadava 2003). Herbicides have evolved over time, and herbicide
use has seen a move towards more biodegradable chemicals that only need to be
applied at a very low concentration of active ingredient. Thus, the new generation
of herbicides are relatively environmentally benign. Many herbicides exploit the
differences in plant physiology between the crop species and its weeds (usually the
differences between monocots and dicots); they may be systemic or act on contact
(Table 1.3). Major crops such as wheat, rice, and maize; and legumes such as
soybean, common bean, and peanut come from the same plant families as their
weeds, thus creating a control problem.
The mode of action of common herbicides is varied (Naylor 2002). For example,
inhibition of photosynthesis and light-dependent membrane destruction (acting on
photosystems II and I, respectively) are the mode of action of the foliar-acting
nonselective herbicides like atrazine, paraquat and diquat. Hormone herbicides,
such as 2,4-dichlorophenoxyacetic acid (2,4-D) induce abnormal plant growth by
interring with auxin regulation. The sulfonyl ureas, imidazolines and the environ-
mentally benign, but nonselective glyphosate (acting on 5-enolpyruvylshikimate-
3-phosphate synthase) inhibit amino acid synthesis. Others include inhibitors of
lipid synthesis, inhibition of cell division and pigment synthesis. The advantages of
herbicides are clear – they control multiple weed species, control perennial weeds,
cause no injury to the crop plant, and can readily be applied to large areas.
Table 1.3 Herbicide modes of action

Herbicide type
Contact herbicides destroy only that plant tissue in contact with the chemical spray. Generally,
these are the fastest-acting herbicides. They are ineffective on perennial plants that are able to
regrow from roots or tubers
Systemic herbicides are foliar-applied and are translocated through the plant and destroy a greater
amount of the plant tissue. Modern herbicides such as glyphosate are designed to leave no
harmful residue in the soil
Soil-borne herbicides are applied to the soil and are taken up by the roots of the target plant
Pre-emergent herbicides are applied to the soil and prevent germination or early growth of weed
seeds
Nonchemical weed control includes: cultural weed control practices such as

intercropping and rotation; mechanical removal of weeds; and biological control,
all of which are labor intensive and unpredictable. Chemical treatment is by far the
most effective method, but it is not without problems.
1.3.3.1 Resistance to Herbicides
A common consequence of repeated use of herbicide application is the evolution of

resistance in weed populations because of strong selection pressure. The first
example of a problematic herbicide resistant weed was reported in the 1970s
(Chrispeels and Sadava 2003) when common groundsel resistant to triazine was
identified in the US. The mechanism of herbicide resistance is usually a change in
the target site with a reduction in herbicide affinity – this may be achieved through a
single point mutation and alteration of a single amino acid. By 2001, over 150 weed
species had resistance to at least one herbicide. Some biotypes of weeds have
resistance to herbicides with different modes of action.
Rigid Ryegrass
When a weed evolves resistance to a particular herbicide, the farmer is forced to use
an alternative control strategy. This often involves the use of another herbicide with
a different mode of action. Subsequent selection pressure may lead to a weed
population acquiring multiple herbicide resistance. In nearly all the cases so far,
resistance to only one or two chemical families has been reported. Rigid ryegrass is
a notable exception. In the most severe cases, biotypes exist that are resistant to nine
chemical families with five different modes of action! Resistance is conferred by
multiple mechanisms; both altered sites of action and the ability to metabolize
particular herbicides, via nonspecific monoxygenases.
1.3.4 Transgenic Crops for Weed Control
The adoption of transgenic herbicide-resistant (HR) crops has made remarkable

changes to global agriculture within the last decade. Currently, an estimated 114.3
million hectares of transgenic crops are planted throughout a variety of agroeco-
systems in 23 developing and industrial countries. Approximately, 90% of the land
area with transgenic crops includes a trait for glyphosate resistance (GR) (Owen
2008).
While there are a number of herbicide-tolerant genetically modified crops that
have been developed for several herbicides with different modes of phytotoxic
action, the primary influence in world agriculture are glyphosate-resistant crops
(GRCs) (Duke 2005; Duke and Powles 2008). More specifically, of the 23 countries
that grow plant glyphosate-resistant crops, the US, Canada, Argentina and Brazil
are the countries that account for most hectares (Owen 2008). In these countries, the
principle GRCs planted include maize (Zea mays), soybean (Glycine max), cotton
(G. hirsutum), and canola (Brassica napus). For further information, the reader is
referred to Chap. 3 of this volume.
1.3.4.1 Round-up1 Ready Crops
Glyphosate (Round-up1) is a highly effective broad-spectrum herbicide that

inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, a branch point
enzyme in aromatic amino acid biosynthesis. A naturally occurring EPSP synthase
gene (cp4) was identified from Agrobacterium sp. strain CP4, whose protein
product provided glyphosate tolerance (Fig. 1.5) in plants (Padgette et al. 1995).
COO–
SHKP COO–
+ P O CH2
phosphoenol-
OH pyruvate
P O
(PEP)
GLYPHOSATE 5-enolpyruvylshikimate-3-phosphate synthase

OH (EPSP synthase)
shkG
H3PO4
COO– COO–
H3PO4
CH2 CH2
shkH
chorismate synthase
P O O COO– O COO–
OH OH
5-enolpyruvylshikimate-3-phosphate chorismate (CHA)
(EPSP )
Fig. 1.5 Glyphosate; mode of action. Glyphosate kills plants by interfering with the synthesis of
the amino acids phenylalanine, tyrosine and tryptophan. It does this by inhibiting the enzyme
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which catalyzes the reaction of shiki-
mate-3-phosphate (SHKP) and phosphoenolpyruvate to form 5-enolpyruvyl-shikimate-3-phos-
phate (EPSP). EPSP is subsequently dephosphorylated to chorismate, an essential precursor in
plants for the aromaticamino acids: phenylalanine, tyrosine and tryptophan. These amino acids
are used as building blocks in peptides, and to produce secondary metabolites such as folates,
ubiquinones and naphthoquinone
Furthermore, glyphosate detoxification pathways are known in microbes involving

glyphosate oxidoreductase (gox) genes (Jacob et al. 1988). These two genes confer
glyphosate resistance in selected crops.
Crops engineered for resistance to this broad-spectrum herbicide allow control
of multiple weeds without injury to the main crop. The economic benefits to farmers
of glyphosate-resistant crops are estimated at US$17.5 billion, mainly accrued from
reduced sprays and lower inputs of labor. In addition, herbicide-tolerant crops
reduce the amounts of active ingredient required for weed control; they also
promote the usage of no/low till farming and thus lower fuel consumption with
direct benefits to both soil structure and carbon emissions (James 2007).
1.3.4.2 Other Transgenic Herbicide-Tolerant Crops
Traits for resistance to three other classes of herbicides have been developed, but
have not reached the same level of popularity as glyphosate resistance. Resistance
to oxynil herbicides conferred by the BXN nitrilase from Klebsiella pneumoniae
(subsp. ozaenae) (Stalker et al. 1988) was the first trait engineered in cotton
(developed by Calgene, Davis, CA [now Monsanto]). Because glyphosate is less
expensive and controls more weed species, interest in using the oxynil herbicides
has waned and 2004 was the final year of BXN1 cotton sales. BXN canola was
commercialized by Rhone-Poulenc Canada (now Bayer Crop Science, Monheim,
Germany) and subsequently discontinued. Phosphinothricin acetyltransferase (PAT
or BAR) detoxifies phosphinothricin- or bialaphos-based herbicides (glufosinate).
The pat gene is native to Streptomyces viridichromogenes and bar is from
S. hygroscopicus where they act in both the biosynthesis and detoxification of the
tripeptide bialaphos (De Block et al. 1987). Like glyphosate, phosphinothricin
herbicides control a broad spectrum of weed species and break down rapidly in
the soil so that the problems with residual activity and environmental impact are
greatly reduced. Bayer CropScience markets this trait as LibertyLink1 in several
species. The pat and bar genes are also popular plant transformation markers in the
research community. Finally, BASF (Ludwigshafen, Germany) markets nontrans-
genic CLEARFIELD1 imidazolinone-resistant canola, wheat, sunflower, corn,
lentils, and rice, while DuPont (Wilmington, DE) markets nontransgenic STS1
soybeans with tolerance to sulfonylurea herbicides. A sulfonylurea-tolerant flax
variety called CDC Triffid, developed by the University of Saskatchewan (Canada),
was grown commercially in Canada in 2000 but is no longer offered. All these crops
contain mutagenized versions of the acetohydroxyacid synthase, also called acet-
olactate synthase (ALS), which are not inhibited by imidazolinone and/or sulfonyl-
urea herbicides (Devine and Preston 2000). Herbicides that inhibit ALS are
considered low or very low use-rate herbicides with a good spectrum of weed
control and are likely to remain an important part of weed resistance management
programs (Castle et al. 2006).
1.3.5 HT Canola
There are two GM traits for herbicide resistance in canola: glyphosate and glufo-
sinate resistance. Resistance to glyphosate herbicides is conferred by the genes
5-enol-pyruvylshikimate-3-phosphate synthase from Agrobacterium sp. CP4 (CP4
epsps) (event GT73, Monsanto) that is targeted to the chloroplast, and a glyphosate
oxidoreductasegene from Achromobacter sp. strain LBAA (gox v247) (both
Roundup Ready1). Resistance to phosphinothricin herbicides is conferred by
phosphinothricin-acetyltransferance (pat gene), and marketed as LibertyLink1
by AgrEvo (now Bayer CropScience).
Herbicide-resistant canola (oilseed rape) allows post-emergence application of a
single herbicide with a wide spectrum of activity. For effective control, it has to be
applied before the weeds reach 10 cm height. This extends the potential time period
for spraying (Benbrook 2004). The timing is more flexible and the application of a
single herbicide simplifies weed control (Firbank and Forcella 2000). Since many
herbicides allow post-emergence applications, HT crops do not generally provide a
new option. However, post-emergence spraying with non-GMHT oilseed rape is
confined to a short 3–5 week period after crop emergence (Pallutt and Hommel
1998). Thus, GMHT canola offers greater flexibility (Owen 1999).
With HT oilseed rape, the intention is to reduce the amount of active ingredient
of herbicide used and to rely preferably on one broad-spectrum herbicide only
(http://www.canola-councildemo.org). The intent is also to reduce the number of
spraying rounds, which helps reduce soil compaction and erosion (Madsen et al.
1999). During the first years of cultivating GMHT oilseed rape, most farmers had
reduced active ingredient rates and applications (Champion et al. 2003). GMHT
oilseed rape was usually sprayed only once, with an average active ingredient
amount that was either lower (Champion et al. 2003) or not significantly different
from conventional oilseed rape (Schütte et al. 2004). However, in Canada (where
HT canola is widely cultivated), application of herbicide was seen to actually
increase (Canola Council of Canada 2001). In fact, after years of continued
GMHT oilseed rape cultivation, secondary adverse effects on application rates
were reported. This was due to weeds becoming herbicide tolerant on- and off-
site (Devos et al. 2004; Hayes et al. 2004). HT oilseed rape volunteers occurred in
subsequent rotations (Devos et al. 2004), and multiple tolerances of volunteers to
herbicides were recorded because of seed impurities and seed banks after out-
crossing events between fields (Hall et al. 2000). Outcrossing to weedy relatives
also occurred (Daniels et al. 2005); these weedy relatives, however, have not yet
been proved to be fit enough to persist on cultivated fields (Hails and Morley 2005).
Nevertheless, improved weed suppression with HT oilseed rape in many agro-
nomic respects has been demonstrated (Westwood 1997; Bohan et al. 2005), and
ultimately the technology remains popular with farmers because of its simplicity
and reduced costs (Canola Council of Canada 2001; Graef et al. 2007). Genes have
always moved between the natural and agroecosystem, and to date – despite the
formation of hybrids between HT canola and wild relatives in Canada (Gressel
2008) – the technology has proven safe and effective (Cerdeira and Duke 2006;
Darmency et al. 2007; James 2007; Gressel 2008).
1.3.6 HT Maize
C. album, Amaranthus retroflexus, Abutilon theophrasti and several annual grass

species cause economic losses in maize, if left uncontrolled. The option to use
glyphosate in these systems is extremely valuable because it is a broad-spectrum
herbicide that provides excellent control of a wide range of weed species. Resis-
tance to glyphosate-herbicides in maize is conferred by the epsps gene in event
GA21 (the GA21 trait for glyphosate-resistant maize relies on a modified maize
epsps gene) (Roundup Ready), developed by DeKalb (now Monsanto) in 1998.
This is now being largely replaced by event NK603, Roundup Ready corn 2, with
two epsps expression cassettes under the transcriptional regulatory control of the
rice (Oryza sativa L.) actin 1 (P-Ract1) and the enhanced cauliflower mosaic virus
35S (P-e35S) promoters to impart fully constitutive expression. Maize has also
been developed with resistance to phosphinothricin herbicides via transformation
with the pat gene in events T14 and T25, developed by Aventis (now Bayer
CropScience), and marketed as LibertyLink.
Maize is an open-pollinated, wind-facilitated species and gene flow via pollen is
well recognized (Haslberger 2001). Thus, the movement of GM traits is a signifi-
cant consideration in maize production (Luan et al. 2001; Ma et al. 2004). Gener-
ally, the introgression of GR traits in seed maize can be managed successfully
(1% outcross) by establishing isolation distances of 200 m between fields (Ma
et al. 2004). However, in typical maize production regions of the US, these isolation
distances are not possible and GR trait introgression into non-GR fields is prevalent.
The occurrence of the GR transgene in non-GM maize can have significant
economic consequences, if the grower of the non-GM maize has a contract to
provide a GM-free product. Furthermore, incidences of GM gene introgression in
local landraces of maize in Oaxaca, Mexico have been reported (Quist 2001). The
implications for transgene occurrence reflect concerns for the maintenance of the
genetic resource of the landrace maize. However, the initial report of transgene
introgression was followed by a second report that suggested that no transgenes
existed in these landraces of maize (Ortiz-Garcia et al. 2005). Regardless, given the
adoption of GR maize, the discovery of transgene introgression into landrace maize
is likely in the longer term.
1.3.7 HT Cotton
Cotton is a slow-growing plant, and only a limited selection of herbicides can be

used for weed control. These two factors sometimes make weed control difficult.
Cotton is a semitropical, perennial plant, although it is grown as an annual crop.
Its growth is especially slow in the northern cotton belt in the US, where it is often
planted early into cool soils. Post-emergence broadleaf control is accomplished
with herbicides that can injure cotton but that are applied in a directed spray that
misses most of the cotton plant; thus, weed control in cotton is particularly difficult.
Cotton with resistance to glyphosate herbicides has been developed based on the
expression of the CP4 epsps in events MON1445/1698, Monsanto, Roundup Ready1.
Resistance to phosphinothricin herbicides is based on the expression of the bar
gene in LLCotton25, Bayer CropScience LibertyLink1. Herbicide-tolerant cotton
expanded from around 2% of the cotton acreage in 1996 to 26% in 1998, and
reached 46% in 2000 (James 2001). In 2008, adoption of GM cotton in the US with
either or both herbicide tolerance and Bt reached 86% (James 2007).
There are limited reports that cotton demonstrates introgression at low frequen-
cies (Ellstand et al. 1999). Pollen movement in cotton is dependent on insects.
Cotton is predominantly self-pollinated and natural outcrossing is typically quite
low (Xanthopoulos and Kechagia 2000). Thus, there is a very low probability that
the GR transgene would move into non-GR cotton cultivars via pollen flow. While
gene flow via GR cotton seed can occur, the consequences of this are not thought to
be important.
1.3.8 HT Soybean
Soybean engineered for resistance to glyphosate herbicides has been developed by

Monsanto with the CP4 epsps gene, event GTS-40-3-2 and is marketed as Roundup
Ready1.
Glyphosate-resistant soybeans were one of the earliest transgenic crops brought
to market and they have experienced rapid adoption to the point that over 85% of
US soybeans and 56% of soybeans globally are now glyphosate-resistant (James
2007). Soybeans are an autogamous (self-fertilizing) species with limited opportu-
nity for pollen-directed gene flow (Palmer et al. 2001). Spontaneous gene flow in
cultivated soybeans ranges from 0.02 to 5% depending on distance and is facilitated
by thrips (Thrips tabaci) and honeybees (Apis mellifera). While the movement of
the GR transgene has been observed in soybeans, there are extremely limited
opportunities for this occurrence and pollen-mediated gene flow in GR soybeans
is essentially a nonissue (Abud et al. 2004; Owen 2005). However, gene flow by
seed is highly probable and represents a significant economic problem (Swoboda
2002; Owen 2005).
1.3.9 Other GMHT Crops
In terms of commercial crops, glyphosate-resistant alfalfa had been developed and

was launched in 2006 (James 2007).
However, of special interest genetically engineered herbicide-resistant crops are

being tested as a solution to the parasitic plants Orobanche spp. (broomrapes) and
Striga spp. (witchweeds), which parasitize the roots of important crops, heavily
reducing yields. Joel et al. (1997) showed that the use of three target-site resistances
decimated Orobanche, demonstrating the potential of transgenic herbicide-resistant
crops in the control of parasitic weeds. This is potentially of great importance as
every year parasitic plant damage to crops accounts for an estimated US$7 billion in
yield loss in sub-Saharan Africa, and affects the welfare and livelihood of over 100
million people. For this technology to have a great impact in Africa, crops adapted
to African agroecologies need to be available to farmers.
1.3.10 Changes in Agronomic Practice
The adoption of GM Crops, and specifically glyphosate-resistant crops, has resulted

in significant changes in agronomic practices. Most obviously, these changes
include the increase glyphosate use at the cost of other herbicides and the manner
and frequency in which glyphosate is used. In addition, the amount of tillage that is
conducted for crop production has significantly changed (Young 2006; Service
2007a, b; Foresman and Glasgow 2008). This reduction in tillage has an important
benefit of reducing the use of petroleum-based fuels as well as an implicit gain in
time use efficiency by growers. A significant reduction in pesticide use has been
attributed to the adoption of herbicide-tolerant (HT) crops (Sankula 2006). Further-
more, the benefits ascribed to herbicide-tolerant crops have dramatically changed
the crop cultivars selected by growers and have hastened the development of new
transgenic crops for commercial distribution worldwide (Duke 2005; Dill et al.
2008).
Despite wide scale commercial plantings of GM soybean, canola, cotton and
maize, several herbicide-resistant crops have been developed but have not been
commercially introduced. Notably, sugarbeet (Beta vulgaris) cultivars that are
resistant to glyphosate were deregulated in 1999, but not commercially offered
until 2008 because of concerns about the acceptability of sugar refined from a GM
crop (Duke 2005; Gianessi 2005). The development of GM rice (O. sativa) cultivars
modified to be resistant to glufosinate herbicide proceeded from 1998 to 2001, but
were withdrawn and commercial development terminated because of concerns
about market acceptance of the GM rice (Gealy and Dilday 1997; Gealy et al.
2007). Similarly, wheat (Triticum aestivum) glyphosate-resistant cultivars were
under development but the program was terminated in 2004 (Dill 2005). While
the GR-based wheat production systems demonstrated excellent opportunities for
improved weed management, concerns about the acceptance of the flour made from
GR wheat cultivars as an export commodity in GM-adverse countries resulted in the
decision to halt further development (Stokstad 2004; Howatt et al. 2006).
In addition to concerns centered on consumer acceptability, several concerns
over potential environmental impact have been raised. These are addressed below.
1.3.11 Weeds, Gene Flow, Invasiveness, and Biodiversity Impacts
There have been significant concerns regarding the potential of GM crops, particu-
larly glyphosate-resistant crops, to become major agricultural problems (by inva-
sion, volunteerism) or to create “superweeds” by cross-pollination (Ellstrand 2003).
1.3.11.1 Gene Flow
While GM crops do have the potential to cross-pollinate other crops and wild
relatives, there are four basic elements determining the likelihood and conse-
quences of gene flow: first, the distance of pollen movement from the GM crop;
second, the synchrony of flowering between crop and pollen recipient; third, sexual
compatibility between crop and recipient; and fourth, ecology of the recipient
species (Dale et al. 2002). Research has shown that pollination declines sharply
with distance from the pollen source (Lutman 1999) and one could reduce the
chances of GM pollen reaching other crops through the use of isolation or buffer
zones, although pollen may travel further if plants are insect-pollinated. Ellstrand et al.
(1999) reviewed the sexual compatibility of crops with weeds and feral species. For
example, oilseed rape (canola), barley, wheat and beans can hybridize with weeds in
some countries; however, in the UK, for example, the probability of hybridization
with weeds is considered minimal for wheat, low for oilseed rape and barley,
and high for sugarbeet. Although sugarbeet can readily hybridize, in the case of
herbicide-tolerant varieties of sugarbeet the crop would be harvested before flower-
ing and hence shed no pollen. Indeed, methods have been developed to block
expression in the pollen of transgenic plants, including engineering of the chloro-
plast genome (Heifetz 2000) and transgene mitigation strategies (Gressel 2008).
Transgene Mitigation Strategies
Transgenic crops may interbreed with nearby weeds, increasing their competitive-
ness, and may themselves become a “volunteer” weed in the following crop. The
desired transgene can be coupled in tandem with genes that would render hybrid
offspring or volunteer weeds less able to compete with crops, weeds and wild
species. Genes that prevent seed shatter or secondary dormancy, or that dwarf the
recipient could all be useful for mitigation and may have value to the crop. Many
such genes have been isolated in the past few years (Gressel 1999). Examples
include: apomixis (asexual reproduction) as a fail-safe so that the seed is actually of
vegetative origin and not from sexual pollination (Zemetra et al. 1998); chromo-
some-specific cytogenetic fail-safes (the risk of transgenic traits spreading into
weeds can be reduced by orders of magnitude by using cytogenetic mapping to
locate transgenes and releasing only those transgenic lines in which it is on a genome
incompatible with local weeds (Gressel and Rotteveel 2000)); plastome-specific
cytogenetic fail-safes (it is possible to introduce some traits into the chloroplast or
mitochondrial genomes;thus, in species where these genomes are completely

maternally inherited, the transgenes cannot move into other species (Daniell et al.
1998)); seed dormancy (the transgenic abolition of secondary dormancy is neutral
to the crop but deleterious to weeds (Gressel 1999)); seed ripening and shattering
(uniform ripening and anti-shattering genes are harmful to weeds but neutral for
some crops (Schaller et al. 1998)), and dwarfing (dwarfing is disadvantageous for
weeds, because they can no longer compete with the crop for light (Gressel 1999)).
However, these strategies are not as of yet fully developed and in use, with the
exception of plastid transformation.
1.3.11.2 Invasiveness
The potential exists for GM crops to become invasive. There has been a great deal
of concern that such crops could persist in the wild and disperse from their
cultivated habitat. However, recent studies (and experience from the field) have
indicated that the ability of GMHT crops to invade and persist was actually no
better than that of their conventional counterparts (Crawley et al. 2001). Finally,
GM crops persisting in fields after harvest thus becoming a weed in a different crop
may be dealt with in two ways; simple treatment with an appropriate herbicide or
mitigation technologies that prevent the transgene being carried over to the next
generation (Gressel 2008).
1.3.11.3 A Dose of Reality
In order to put these concerns into perspective, one must understand that flow from
the agroecosystem to natural ecosystems has always occurred. Gene flow is a
continuing process and is the source of biological diversity (Thies and Devare
2007). There has always been gene flow from commercial crops to relatives living
in near proximity (Ferry and Gatehouse 2009). In reality, the vast majority of the
major cultivated crops have no wild or weedy relatives outside of their centers of
origin (Gressel 2008); however, some crops are grown in areas where gene flow
may occur and appropriate measures must be taken in these areas.
1.3.12 Coexistence of GM and Non-GM Crops
A pervasive problem that exists with the production of GR crops is their coexis-
tence with non-GM crops (Byrne and Fromherz 2003). The issue of coexistence
includes three possibilities: (1) introgression of the trait via pollen (pollen drift), (2)
containment of plant products during the production year (grain segregation), and
(3) volunteer GRC plants in following years (Owen 2005).
While GR crops and non-GM crops can coexist, growers must go to great lengths
to accomplish segregation (Anonymous 2007). Grain segregation, while difficult to
maintain, can be accomplished and will effectively minimize the impact of GR

crops on non-GM crops. Given that the mixing of grain is equivalent to loss of
intellectual property (IP), this can be costly to growers; thus, appropriate tactics to
isolate GR crops from non-GM crops must be in place (Owen 2000). Controlling
volunteer GR crops is also relatively easy, depending on the rotational crop, but
does require diligence on the part of the grower (Owen 2005). The introgression of
the HT trait via pollen movement is another possibility and management of this
problem is considerably more difficult, particularly in open-pollinated crops such as
maize (Luan et al. 2001; Palmer et al. 2001; Westgate et al. 2003; Abud et al. 2004).
A number of factors affect the success of maize pollen movement and subsequent
pollination, and generally, the greater the distance between the pollen source and
the donor, the less likely is the introgression of the GR trait (Luan et al. 2001;
Westgate et al. 2003). However, given the tolerance levels established for some GM
traits in non-GM crops, the isolation distances required to mitigate the risks of gene
flow are too large to be realistic (Matus-Cadiz et al. 2004). Other open-pollinated
crops have also undergone a great deal of scrutiny (Charles 2007; Fisher 2007;
Harriman 2007; Weise 2007). It is suggested that the issues of the coexistence of
GRCs with non-GM crops will continue to be a concern as long as there are
economic differences between the crop cultivar types (Hurburgh 2000; Ginder
2001; Hurburgh 2003). Nevertheless, coexistence guidelines and buffer zone (iso-
lation zone) regulations are in existence (Boller et al. 2004).
1.3.13 Stacked Traits
Stacked products are a very important feature and future trend for GM crops, which
meets the multiple needs of farmers and consumers; these are now increasingly
deployed by ten countries – US, Canada, the Philippines, Australia, Mexico, South
Africa, Honduras, Chile, Colombia, and Argentina (James 2007). In fact, 37% of all
GM crops in the US in 2007 were stacked products containing two or three traits
that delivered multiple benefits. Currently, there are a number of GM crops that
have stacked herbicide resistance and Bt events (Owen 2006).
In some events, the pat gene is used as a marker for the Bt – the pat gene also
confers resistance for glufosinate herbicide. Interestingly, the Bt trait is combined
with a GR hybrid, the resultant GM cultivar is resistant to both glyphosate and
glufosinate. Monsanto has announced new transgenic maize cultivars that will
combine several Bt events plus two herbicide resistance events.
The Bt event functions ecologically differently compared to herbicide-resistant
transgenes; the transgene for herbicide resistance can be considered benign to the
weed population and has no impact until the herbicide is applied (Owen 2009). In
contrast, Bt exerts continuous selection pressure on the target insect whether the
insect population is at economic threshold or not. Furthermore, given the potential for
the introgression of transgenes into near-relatives, Bt could potentially improve the
fitness of compatible weed species (Snow and Pedro 1997). For example, the fitness
of canola was increased by the inclusion of Bt (Steward et al. 1997); thus, gene flow
was kept in check. However, there is no reason to believe that stacked traits will
have any greater impact on nontargets than the crops with single traits, and for Bt
non-target impacts have been shown to be minimal (Ferry and Gatehouse 2009).
1.3.14 Conclusions
Herbicide-resistant crops, specifically GR crops, have been globally adopted as the

basis for the production of maize, soybean, cotton and canola (Dill et al. 2008).
Their adoption provides economic benefits to agriculture and has major positive
impacts on the environment; specifically, conservation tillage which can reduce soil
erosion (Duke and Powles 2008; Shipitalo et al. 2008). However, growers must
adopt measures to proactively sustain the technology (Mueller et al. 2005; Johnson
and Gibson 2006; Sammons et al. 2007; Christoffoleti et al. 2008) and to steward
GR crops to avoid the evolution of GR weeds (Duke and Powles 2008; Owen 2009).
While there are tactics that are capable of mitigating some of the other concerns,
including the risks of transgene introgression into near-relative plants (Snow and
Pedro 1997; Gepts and Papa 2003), an improved process to assess the environmen-
tal risk of GR crop technologies and communicating those risks to the lay public
should be in place (Owen 2009).
1.4 Biotic Stress due to Plant Pathogens
1.4.1 Crop Losses due to Plant Pathogens
Plants are challenged constantly by many different potential pathogens (Table 1.4).
There are hundreds of thousands of viral, bacterial, and fungal species in the world
and thousands of these are pathogens that infect plants (Chrispeels and Sadava 2003).
Any one pathogen can severely depress the yield of a given crop. Pathogens may
reduce yield by causing tissue lesions; by reducing leaf, root, or seed growth; or by
clogging up vascular tissue and causing wilt. Even in the absence of obvious symp-
toms, pathogens can still be a major metabolic drain that reduces productivity. They
may also cause pre- or postharvest (stored products) damage to the harvested product.
Table 1.4 Organisms that cause infectious disease in plants

Types of plant pathogens
Fungi
Ascomycetes e.g., Fusarium, Verticillium
Basidiomycetes e.g., Rhizoctonia, Puccinia (rust)
Oomycetes e.g., Phytopthora (blight)
Bacteria e.g., Xanthomonas, Pseudomonas
Phytoplasmas and Spiroplasmas
Viruses Viroids and virus-like organisms
Nematodes, protozoa, and parasitic plants are also considered plant
pathogens
1.4.1.1 Current Disease Control Measures
Disease control in commercial crops is both economically important and environ-

mentally controversial. Disease management includes: quarantine, cultural prac-
tices such as modification of the plant environment (e.g., soil management) to
reduce pest numbers, host plant resistance, biological control and chemical control.
Chemical control can be highly effective. The first fungicide was discovered in the
1880s when Bordeaux mixture (a mixture of copper sulfate and lime) was found to
suppress grape downy mildew. Numerous compounds with antifungal or antibac-
terial activity have since been discovered, most often applied as sprays, dusts or
seed coatings. Many older compounds are broad spectrum and toxic with the newer
chemistries acting systemically with a narrower target range. Such chemicals are
expensive and normally reserved for use on high-value fruit and vegetable crops
(Ferry and Gatehouse 2009). They are expensive to manufacture and a great deal of
investment must be put into human and ecological safety testing before a product
can be released (Chrispeels and Sadava 2003). As with other chemical control
strategies, pathogens can evolve resistance to these compounds.
Thus, plant breeders have often relied on genetic disease resistance traits to
manage pathogens of particular crops. In classical plant breeding, this has relied on
crosses between elite crops and wild relatives (that are more genetically diverse) to
introduce new disease resistance traits into the crops. Extensive backcrossing of the
elite line is then required to eliminate the undesirable traits in the wild relative and
thus makes traditional breeding a time-consuming process with a time of about
15 years required before a new resistant variety is available for release to growers.
Nevertheless, such traditional plant breeding has had significant successes. For
example, the development of F1 hybrid crops (derived from crossing two inbred
lines) resulted in vastly improved yields (Chrispeels and Sadava 2003). Despite this
enormous success, the F1 hybrids also serve as a warning as to the dangers of large-
scale monoculture and the need for effective and durable disease resistance. In
1970, the highly successful but genetically uniform F1 maize crops in the US were
left devastated by a disease, southern corn leaf blight (Bipolaris maydis Nisik),
which caused damage to 710 million bushels of maize (Ullstrup 1972).
In extreme cases, crop disease can change political as well as agricultural
landscapes!
1.4.1.2 The Irish Potato Famine
The Irish Potato Famine (the Great Hunger) started in 1845, lasted until 1849–1852
and led to the death of approximately one million people through starvation and
disease; a further million are thought to have emigrated as a result of the famine.
Some estimate that the population of Ireland was reduced by 25% (Kinealy 1995).
The cause of the famine was a potato disease commonly known as late blight caused
by the oomycete, Phytophthora infestans. Although blight ravaged potato crops
throughout Europe, the impact and human cost in Ireland was high as a third of the
population was entirely dependent on the potato for food; it was also exacerbated by
a host of political, social and economic factors. The famine was a watershed in the
history of Ireland. Its effects permanently changed the island’s demographic,
political and cultural landscape. For both the native Irish and the resulting diaspora,
the famine became a rallying point for nationalist movements. The fallout of the
famine continued for decades afterwards and Ireland’s population still has not
recovered to prefamine levels.
1.4.1.3 The Bengal Famine of 1943
The Bengal famine of 1943 occurred in British administered Bengal. It is estimated

that around four million people died from starvation and malnutrition during the
period. During the Second World War, the British had just suffered defeat in nearby
Burma and British authorities feared a subsequent Japanese invasion of India
by way of Bengal, so emergency measures were introduced to stockpile food for
British soldiers and prevent access to supplies by the Japanese in case of an
invasion. However, in the rice-growing season of 1942 weather conditions were
exactly right to encourage an epidemic of the rice disease brown spot following a
cyclone and flooding; brown spot in rice is caused by the fungus Helminthosporium
oryzae. When food shortages became apparent, the Bengal government reacted to
the crisis incompetently refusing to stop the export of food from Bengal to allied
soldiers and failing to provide adequate famine relief in Bengal itself. Winston
Churchill was Prime Minister at the time and while his involvement in the disaster,
and indeed his knowledge of it remains unclear, it has been suggested that in
response to an urgent request by the Secretary of State for India to release food
stocks for India, Churchill responded with a telegram asking if food was so scarce,
“why Gandhi hadn’t died yet” (Mishra 2007). Needless to say, whether this is
completely true or is in fact an unfortunate misquote used out of context, the British
Administration in India declined in popularity and there is little doubt that this
incident fuelled feelings for Indian Independence.
Given the vast array of diseases that threaten crops, and the scale of disaster that
can ensue, it is a wonder that crops be produced at all?
1.4.2 Plant Defense Against Pathogens
Plants have evolved mechanisms to resist pathogen invasion that consist of different
defense layers. Firstly, waxy coatings on epidermal cells provide a physical barrier;
secondly, plants contain large amounts of preformed secondary metabolites that
have antimicrobial activity (constitutive defenses including glucosides, saponins,
alkaloids, antifungal proteins, antifeedants and enzyme inhibitors), these are effec-
tive in many cases – but pathogens have evolved enzymes capable of detoxifying
these compounds. Often induced defenses are the plants’ last line of defense against
pathogens and are sufficient to (partially) ward off invading microorganisms.
Essentially, plants have two major types of induced disease resistance, basal defense
and resistance (R) gene-mediated defense. All plants have basal defense, this
is a general immune response to pathogens and other environmental stresses.
R-gene-mediated defense is more specific and is only found in certain plant species.
R-gene-mediated defense involves recognition of a specific pathogen effector by a
plant ligand receptor. These pathogen effectors can suppress plant basal defense,
making any plant without the R-gene defense susceptible. The ligand-effector
recognition can result in a dramatic immune response such as cell death. In tomato,
Solanum lycopersicum, the Mi gene, a member of a large family of R-genes, mediate
resistance to potato aphids, whiteflies, and root-knot nematodes (Kaloshian and
Walling 2005). Both types of plant defenses (R and basal) involve signaling via
three major plant hormones: salicylic acid, jasmonic acid, and ethylene (ETH). In
some instances, defense responses are induced distal to the site of infection and this
is referred to as systemic acquired resistance (SAR).
At least three nonspecific induced defense pathways are described which are
triggered by these specific signaling molecules:
(a) The salicylic acid (SA)-dependent pathway is induced by necrosis inducing
pathogens and triggers systemic acquired resistance (SAR)
(b) A second pathway is triggered by nonpathogenic rhizobacteria, it is dependent
on jasmonic acid (JA) and ethylene (ETH) and constitutes induced systemic
resistance (ISR)
(c) JA and ETH regulate a third pathway that is effective against a different set of
pathogens and not affected by ISR
Most of the inducible defense-related genes are regulated by these signaling
pathways (Delaney et al. 1994; Sticher et al. 1997; Van Loon 1997; Reymond and
Farmer 1998; Knoester et al. 1998; Ananieva and Ananiev 1999).
Defense gene regulation has been extensively studied and severa1 rapid pro-
cesses characteristic of the hypersensitive response (HR) appear to involve prima-
rily activation of preexisting components rather than changes in gene expression.
One of these rapid processes is the striking release of reactive oxygen species.
1.4.2.1 Oxidative Burst
The oxidative burst, a rapid production of reactive oxygen species (ROS), is a well-
documented early plant response to biotic stress (e.g., Apel and Hirt 2004). ROS
comprise radicals and other nonradical but reactive species derived from oxygen.
Among them, the superoxide anion (O 2 ) and hydrogen peroxide (H2O2) exert
various effects on cells, directly or in cooperation with other molecules. In excess,
ROS pose a threat to important biomolecules and cell membranes. One of the
consequences of ROS activity is oxidative damage of membrane integrity due to
lipid peroxidation processes which may result in the generation of highly cytotoxic
compounds. Glutathione-S-transferases (GSTs), induced upon pathogen attack,
may detoxify lipid peroxides by conjugating them with glutathione (GSH). These
enzymes can also catalyze the GSH-dependent reduction/inactivation of H2O2,

forming glutathione disulfide (GSSG) and increasing GSH synthesis by feedback
induction (Marrs 1996). On the other hand, numerous studies indicate an essential
role of ROS in plant defense responses to biotic stress. In addition to direct
antimicrobial activity and contribution to the strengthening of barriers against
pathogens, recent reports point to H2O2 and O 2 as signal transduction agents
activating defense pathways and as key mediators in cell death during hypersensi-
tive response (HR) (Grant and Loake 2000). To maintain a balance between
negative and beneficial functions of ROS, their levels are strictly controlled by a
complex and flexible network of antioxidant systems (Mittler et al. 2004). The
major enzymatic ROS-scavenging components of this network are superoxide
dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). Superoxide
dismutases dismute O 2 to H2O2, an excess of which may be subsequently detox-
ified by CATs and/or APX. Ascorbate peroxidases, in contrast to CATs localized in
peroxisomes, are present in almost all cellular compartments; they exhibit a high
affinity to H2O2 and are considered to be responsible for the fine modulation of ROS
level (Mittler 2002). Moreover, APX, in cooperation with two main low-molecular
antioxidants, ascorbate (Asc) and glutathione (GSH), and enzymes for their regen-
eration, monodehydroascorbate reductase, dehydroascorbate reductase and gluta-
thione reductase (GR), constitute the ascorbate–glutathione (Asc–GSH) cycle,
believed to be the central part of the antioxidant network (Noctor and Foyer
1998). Ascorbate, present at high concentrations in all cellular compartments and
capable of direct scavenging of O 2 and hydroxyl radicals, is considered to be the
most powerful cell antioxidant (Noctor and Foyer 1998). Ascorbate and glutathione
are the major cellular redox buffers, which together with their oxidized forms,
dehydroascorbate (DHAsc) and glutathione disulfide (GSSG), enable cells to main-
tain a redox balance. Changes in the levels or redox state of ascorbate and
glutathione pools as well as in H2O2 homeostasis, and thereby in cellular/compart-
ment redox state, are considered to be pivotal signaling events influencing gene
expression and modulating the plant defense response (Pastori and Foyer 2002;
Foyer and Noctor 2005).
Numerous genes are induced during the plant defense response and presumably
these function in host plant pathogen defense. Induced defenses include the activa-
tion of phytoalexins (including terpenoids, glycosteroids and alkaloids) and PR
proteins (including chitinase, b-1,3 glucanase and other antimicrobial proteins) and
the production of reactive oxygen species (ROS) leading to a hypersensitive
response (HR).
1.4.3 Utilizing Defense Mechanisms
Most conventional breeding strategies have relied on identifying major resistance

genes (often in wild or ancestral plants) while fewer have concentrated on breeding
for polygenic resistance. New opportunities and strategies to enhance disease
resistance are afforded by genetic engineering of plants – these may follow the
single gene model, or may involve manipulation of defense-activating mechanisms.
1.4.4 Transgenic Disease Resistance
Many transgenic-based approaches exist for conferring enhanced levels of disease

resistance, as exemplified by Fig. 1.6, which provides a simplified model.
1.4.4.1 Expression of Single Genes
R-Genes
A very effective defense mechanism in plants is gene-for-gene resistance, this

induced resistance mechanism is based on the interaction between plant-derived
Pathogen
ty
ici
gen (3) Pathogen mimicry
o
th
Pa tors
c
fa
(1b) Induced (2a) Detection of
defense pathogen
(1a) Constitutive
defense
(2b) Defense
regulation
Host cell Nucleus
Transgenic Strategies. 1a. Constitutive expression of antimicrobial factors,

1b. Pathogen induced expression of one or more genes, 2a. Altering
recognition of the pathogen (e.g. R-genes) and 2b. altering downstream
regulators, 3. Priming recognition of pathogens (genetic vaccination).
Fig. 1.6 Simplified model of transgenic strategies to enhance plant defense

resistance (R) gene products and an avirulence (avr)-gene product (elicitor) pro-
duced by the pathogen. The interaction is generally very specific and results in the
triggering of strong resistance responses including hypersensitive response (HR)
and localized cell death at the point of infection. There is no common structure
between avirulence gene products, except that most are secreted proteins. Because
there would be no evolutionary advantage to a pathogen keeping a protein that only
serves to be recognized by the plant, it is believed that the products of avr genes,
sometimes known as effector proteins, play an important role in virulence in
genetically susceptible hosts. The guard hypothesis model proposes that the R
proteins interact, or guard, a protein known as the guardee which is the target of
the Avr protein. When it detects interference with the guardee protein, it activates
resistance. R-gene specificity (recognizing certain avr gene products) is believed to
be conferred by the leucine-rich repeats (LLRs). LRRs are multiple, serial repeats
of a motif of approximately 24 amino acids in length, with leucines or other
hydrophobic residues at regular intervals. LRRs are involved in protein–protein
interactions, and the greatest variation amongst resistance genes occurs in the LRR
domain. LRR swapping experiments between resistance genes in flax rust resulted
in the specificity of the resistance gene for the avirulence gene changing (Bergelson
et al. 2001). Gene-for-gene resistance thus provides obvious targets to engineer
disease resistance in transgenic plants. However, pathogens can often breakdown
R-gene-mediated resistance if the corresponding avr gene mutates becoming inac-
tivated. Pathogen adaptation is also seen in conventionally bred crops. The use of
R-genes is limited by them conferring resistance to only a single pathogen or race
of pathogen; however, they can provide effective (even broad spectrum) resistance
if transformed by genetic engineering into new species or new genera of plant
(Oldroyd and Staskawicz 1998). For example, Rxo1, an R-gene derived from
maize, a nonhost of the rice bacterial pathogen Xanthomonas oryzae pv. oryzicola,
was successfully transformed into rice and shown to confer resistance against this
pathogen (Zhao et al. 2005). Interspecies differences do radically influence R-gene
function making it preferable to use R-genes from related species (Ayliffe and
Lagudah 2004); however, the transgenic approach circumvents the tedious and
time-consuming backcrossing required to introduce a single trait via traditional
crop breeding.
Constitutive Expression of Inducible Antimicrobial Factors
Several examples of transgenic strategies to enhance plant constitutive defenses are

described below. This is an extension of the single gene paradigm that has worked
so well for insect-resistant transgenic plants.
Expression of Chitinases
Chitinases are glycosyl hydrolases catalyzing the degradation of chitin, an insoluble
linear b-1,4-linked polymer of N-acetylglucosamine. They are produced by a wide
range of organisms from microbes to insects and mammals, including plants

(Kasprzewska 2003). Plants produce chitinases as defense against chitin-containing
fungal pathogens by inhibiting spore germination, germ tube elongation and
degrading hyphal tips (Khan et al. 2008).
Transgenic plants expressing chitinase ChiC genes have already been produced
in several species. In carrot, the tobacco class I ChiC gene has shown resistance to
Botrytis cinerea (Punja and Raharjo 1996). Transgenic tobacco transformed with
bean class I ChiC exhibited enhanced resistance to Rhizoctonia solani (Broglie
et al. 1991) and Alternaria alternata (Lan et al. 2000). The rice chitinase gene
(RCC2) exhibited increased resistance to Sphaerotheca humuli in transgenic straw-
berry (Asao et al. 1997). Peanut (Arachis hypogae) plants transformed with tobacco
ChiC gene were resistant to a fungal pathogen (Cercospora arachidicola), the
causal organism of leafspot disease (Rohini and Rao 2001). Potato has been
transformed with the Streptomyces griseus ChiC gene and resistance to Alternaria
solani (causal agent of early blight) demonstrated (Kahn et al. 2008) and cotransfer
and expression of ChiC, glucanase, and bialaphos resistance (bar) genes in creeping
bent grass conferred resistance to the fungal pathogens Sclerotinia homoeocarpa
and R. solani (Wang et al. 2003).
Expression of Defensins
Defensins are the best characterized cysteine-rich antimicrobial proteins in plants
(Broekaert et al. 1995). All known members of this family have four disulphide
bridges and are folded in a globular structure that includes three b-strands and an
a-helix (Segura et al. 1998). Inhibition of fungal growth by defensins seems to
occur by permeabilization of the plasma membrane through binding to a putative
receptor (De Samblanx et al. 1997). Genes encoding plant defensins are develop-
mentally regulated, with predominant expression in the outer cell layers, and are
induced in response to pathogen infection and stress (Segura et al. 1998). Enhanced
tolerance to the fungus Alternaria longipes is observed in transgenic tobacco
overexpressing a radish defensin (Rs-AFP2) (Chiang and Hadwinger 1991). In
support of this role, Manners et al. (1998) show that the promoter of the plant
defensin gene PDF1.2 from Arabidopsis is systemically activated by fungal patho-
gens and responds to methyl jasmonate, but not to salicylic acid.
Expression of Oxalate Oxidase and H2O2 -Generating Enzymes

Rapid generation of superoxide and accumulation of H2O2 is a characteristic early
feature of the hypersensitive response following perception of pathogen avirulence
signals. In germinating barley and wheat seeds as well as in barley leaves
challenged with powdery mildew, oxalate oxidase activity has been identified as
a generator of H2O2 (Lane et al. 1993; Zhou et al. 1998). Oxalate oxidase utilizes
oxalic acid and oxygen as substrates producing H2O2 and CO2. For certain necro-
trophic fungi, such as Sclerotinia sclerotiorum, oxalic acid is a major pathoge-
nicity determinant, significantly altering environmental pH (Cessna et al. 2000).
Pathogen-inducible oxalate oxidase both acts as a generator of H2O2, killing the

invading pathogen, and simultaneously detoxifies the acid, which is phytotoxic at
high concentrations. Sunflower plants expressing a wheat oxalate oxidase accumu-
late enhanced levels of salicylic acid and PR1 even in the absence of pathogen
infection and display improved tolerance to Sclerotinia infection (Hu et al. 2003).
This effect is most likely due to the production of H2O2 from endogenous oxalic
acid. Amine oxidases are a class of enzymes mainly found in the plant apoplast that
act on a variety of amine substrates, including mono-, di- and polyamines and
release the corresponding aldehyde as well as NH3 and H2O2. Amines are present in
the plant apoplast and accumulate in response to environmental stress (Bolwell
et al. 1999). Thus, in plants several systems are available that can produce H2O2
following pathogen attack. The importance of H2O2 in plant defense has clearly
been shown by several groups who have reported increased pathogen resistance in
transgenic plants by introducing either H2O2-generating systems or by inhibiting
H2O2-degrading systems. The expression of a fungal glucose oxidase resulted in
enhanced resistance to P. infestans and Erwinia carotovora in potato (Wu et al.
1995). Similarly, the prevention of H2O2 breakdown resulted in higher levels of
H2O2 and increased disease resistance (Chamnongpol et al. 1998). Whether this
improved pathogen tolerance is due to the direct antimicrobial effect of H2O2, or
due to the fact that the plant defense system is induced by the increased levels of
H2O2, was, in either case, not investigated.
Inducible Expression of Single Antimicrobial Factors
Stilbene Synthase
Production of stilbene, a phytoalexin, is a well-characterized defense reaction in
grape vine. Stilbene plays an important role in resistance to fungal and bacterial
infection in plants. It strongly inhibits the growth of fungi and sprout of spores.
Stilbene synthase gene (Vst1) is responsible for the synthesis of resveratrol (trans-
3,40 5-trihydroxystilbene) when plants are challenged by fungal diseases. Thus,
introduction of stilbene synthase genes into transgenic plants can be used to induce
synthesis of phytoalexins. Vst1 has been transferred into many plants to enhance
fungal resistance including common spring wheat using biolistic transformation,
where plantlets with mild resistance to powdery mildew were identified (Leckband
and Lörz 1998). Other crops transformed to produce stilbene include: tomato
(Thomzik et al. 1997); apple (Szankowski et al. 2003); and Vst1 overexpressed in
its native grape (Fan et al. 2008).
1.4.4.2 Activation of Expression of Multiple Genes
Polygenic Resistance
There are several types of disease resistance in terms of the effects of genes on the
plant and pathogen. However, in terms of the number of genes involved, there are
two general types of resistance. The first (as described above for R-genes) is called
major-gene or single-gene resistance. This type of resistance is well defined and
more easily measured; it is sometimes called qualitative resistance because plants
are either resistant or susceptible, without intermediate levels. The second type,
called polygenic resistance, involves several or many genes. This type of resistance
is harder to define; exactly which genes are involved may be unknown. It usually is
effective against all races of a pathogen. This type is often called quantitative,
because there are intermediate levels ranging from resistant to susceptible. It is also
harder to measure than major-gene or single-gene resistance. Often polygenic
resistance does not give a plant as high a level of resistance as major gene
resistance. Polygenic durable resistance to multiple diseases of a crop would be
highly desirable. In nature, most host plant resistance is based on multiple genes
and a diverse set of resistant factors. This diverse, polygenic resistance system helps
to prevent plant-feeding microorganisms from overcoming the resistance in the host
plant. At present, polygenic resistance may be bred into a crop via quantitative trait
loci (QTL) analysis, and the use of molecular markers in molecular breeding
approaches; promising transgenic strategies (as discussed below) activate poly-
genic resistance via elicitor-induced resistance and activation of multiple genes.
Detection of Pathogens; Expression of Elicitors and Defense Activators
Most plant disease resistance (R) proteins contain a series of leucine-rich repeats
(LRRs), a nucleotide binding site (NBS), and a putative amino-terminal signaling
domain, termed NBS–LRR proteins. The LRRs of a wide variety of proteins from
many organisms serve as protein interaction platforms, and as regulatory modules
of protein activation. Genetically, the LRRs of plant R proteins are determinants of
response specificity, and their action can lead to plant cell death in the form of the
hypersensitive response (HR). It is thought that this halts pathogen growth. In the
absence of specific recognition, a basal defense response also occurs, which is
apparently driven by pathogen-associated molecular patterns (PAMPS), such as
flagellin and lipopolysaccharides (LPS) elicitors of defense responses (Belkhadir
et al. 2004). The basal defense response overlaps significantly with R-protein-
mediated defense, but is temporally slower and of lower amplitude. However,
Takakura et al. 2008 show that expression of a bacterial flagellin gene (N1141) in
transgenic rice triggers disease resistance and enhances resistance against blast
(Magnaporthe grisea). Furthermore, a number of transgenic plants expressing
NBS–LRR proteins under the control of the CaMV 35S promoter have been
described (Mindrinos et al. 1994; Ellis et al. 1999; Stokes et al. 2002). For example,
Grant et al. (2003) show that an Arabidopsis mutant, adr1 (activated disease
resistance), contains both elevated levels of SA and ROIs, accumulates a number
of defense-related gene transcripts and exhibits resistance against a number of
microbial pathogens. ADR1 encodes a distinct NBS–LRR protein which possesses
N-terminal kinase subdomains. Furthermore, transient expression of ADR1 is suffi-
cient to engage defense-related gene expression and establish disease resistance
in the absence of significant seed yield penalty. ADR1 plants showed striking
resistance against both P. parasitica (Noco2) and another biotrophic pathogen,
E. cichoracearum (UED1).
Pathogen Phytosensing
A related strategy for crop defense involves engineered phytosensors indicating the
presence of key plant pathogens to provide an important first line of defense
(Mazarei et al. 2008).
Plant defense mechanisms are highly regulated on the transcriptional level, and
can be induced by chemical elicitors produced by pathogens. These elicitors have
been shown to cause changes in gene expression, which initiate a whole plant
response from a localized encounter with a pathogenic organism (Metraux et al.
2002). This is controlled by signal transduction pathways, inducible promoters and
cis-regulatory elements corresponding to key genes involved in HR, SAR, ISR, and
pathogen specific responses, any of which could be useful in building phytosensors.
Cis-acting elements are conserved among plant species, which enables them to
be used efficiently as synthetic inducible promoters. Employing synthetic promo-
ters with potential inducible elements to engineer plants that can sense the presence
of plant pathogens at the molecular level provides novel technologies for monitor-
ing and increasing resistance to diseases (Gurr and Rushton 2005). Identified
inducible promoters and cis-acting elements could be utilized in plant sentinels,
or “phytosensors,” by fusing these to reporter genes to produce plants with altered
phenotypes in response to the presence of pathogens. Mazarei et al. (2008) have
employed cis-acting elements from promoter regions of pathogen-inducible genes
as well as those responsive to the plant defense signal molecules salicylic acid,
jasmonic acid, and ethylene. Synthetic promoters were constructed by combining
various regulatory elements supplemented with the enhancer elements from the
cauliflower mosaic virus CaMV 35S promoter to increase basal level of GUS
expression. Histochemical and fluorometric GUS expression analyses showed that
both transgenic Arabidopsis and tobacco plants responded to elicitor and phytohor-
mone treatments with increased GUS expression when compared to untreated
plants. Pathogen-inducible phytosensor studies analyzed the sensitivity of the
synthetic promoters against virus infection. Transgenic tobacco plants infected
with alfalfa mosaic virus showed an increase in GUS expression when compared
to mock-inoculated control plants. The end goal of such studies is to engineer
transgenic plants for the purpose of early pathogen detection.
1.4.4.3 Regulation of Inducible Defenses
The overexpression of regulatory genes provides another tool to activate plant

defenses in response to pathogen attack. For example, transduction of the SA signal
requires the function of NPR1 (also known as NIM1), a regulatory protein that was
identified in Arabidopsis through genetic screens for SAR-compromized mutants

(Cao et al. 1994; Delaney et al. 1995; Shah et al. 1997). Mutant npr1 plants
accumulate normal levels of SA after pathogen infection but are impaired in their
ability to express PR genes and to mount a SAR response. The NPR1 gene encodes
a protein with a BTB/BOZ domain and an ankyrin-repeat domain (Cao et al. 1997).
Upon induction of SAR, NPR1 is translocated to the nucleus (Kinkema et al. 2000)
where it interacts with members of the TGA/OBF subclass of basic domain/leucine
zipper (bZIP) transcription factors (Zhang et al. 1999; Després et al. 2000; Zhou
et al. 2000; Subramaniam et al. 2001; Fan and Dong 2002) that are involved in the
SA-dependent activation of PR genes (Lebel et al. 1998; Niggeweg et al. 2000).
Physical interaction between NPR1 and TGA transcription factors has been shown
to be required for the binding activity of these factors to promoter elements that
play a crucial role in the SA-mediated activation of PR genes (Després et al. 2000;
Fan and Dong 2002).
In separate studies NPR1 overexpression and enhanced resistance are correlated
with either elevated or earlier expression of PR transcripts (Cao et al. 1998). Genes
with high sequence similarity to NPR1 are found in Arabidopsis, tobacco, tomato,
rice and maize (Campbell et al. 2003). Overexpression of NPR1 in rice has been
shown to enhance resistance to bacterial blight (X.o.o) (Chern et al. 2001).
1.4.5 Pathogen Mimicry and Virus Resistance
Numerous reports concern transgenic resistance to plant viruses (Fuchs and

Gonsalves 2007) in which RNA-mediated gene silencing is the predominant strat-
egy (viral RNA is degraded and viral DNA is deactivated by methylation). Most of
these strategies can be categorized as pathogen mimicry. Transgenes constitutively
expressed to provide RNA-mediated virus resistance fall into three main types:
1. Sense or antisense viral sequences
2. Inverted repeats/hairpin RNA of viral sequences, and
3. Sequences of engineered microRNAs targeted against the virus
RNA-mediated resistance against viruses was first reported by Lindbo et al.
(1993). Since the discovery of what is now generally called RNA silencing in
plants, the same specific RNA degradation mechanism has been identified in nearly
all eukaryotes (Baulcombe 2004). In plants, genetic and molecular analyses have
revealed at least three natural pathways for RNA silencing: cytoplasmic short
interfering RNA (siRNA) silencing; endogenous mRNA silencing by microRNA
(miRNA); and the silencing associated with DNA methylation and suppression of
transcription (Baulcombe 2004). All these pathways involve the cleavage of double-
stranded RNA molecules into short 21–26 nuclotide RNAs, known as siRNAs and
miRNAs (Baulcombe 2004). To date, it has primarily been the cytoplasmic siRNA
silencing pathway that has been exploited by genetic engineering to confer resis-
tance to plant viruses.
The best documented examples of commercial transgenic resistance involve type

(1) resistance, although the mechanism of resistance was not initially known. In the
1990s, the papaya industry in Hawaii suffered a 50% reduction in production because
of papaya ringspot virus; the solution came through the expression of viral coat protein
in transgenic plants (thought to be immune priming, analogous to vaccination).
1.4.5.1 Papaya Ringspot Virus
Papaya ringspot is a destructive disease (a potyvirus) characterized by yellowing and

stunting of the crown of papaya trees, a mottling of the foliage, damage to younger
leaves, water-soaked streaking of the petioles (stalks), and small darkened rings
on the surface of fruit. Papaya ringspot virus is transmitted from infected papaya
trees to healthy trees by the feeding action of various species of aphids, especially
the green peach aphid and melon aphid. The virus is transmitted in a nonpersistent
manner, meaning that the virus does not multiply within the aphid but is instead
carried on its mouth parts and is transmitted from plant to plant while feeding.
In 1995, American researchers developed a transgenic papaya resistant to the
virus, by expressing a copy of a viral coat protein in the plant (Ling et al. 1991).
It was field-tested in Hawaii where it was shown to be effective against the virus.
The virus-resistant papaya is now widely used by commercial papaya producers in
Hawaii. The presence of small RNA species, presumably siRNA, corresponding to
regions of the viral coat protein gene was later shown to be present in transgenic
lines resistant to PRSV; thus it is posttranscriptional gene silencing involved in the
establishment of resistance (Krubphachaya et al. 2007). Pathogen-derived resis-
tance has also been shown to be effective against maize streak virus (Shepherd et al.
2007), potato leaf roll virus (Vazquez Rovere et al. 2001) and tomato yellow leaf
curl (Yang et al. 2004).
An important drawback of RNA-based approaches to enhanced resistance is the
high level of sequence specificity required for RNA degradation. Viruses contain-
ing >10% nucleotide divergence are insensitive to RNA degradation. Another
drawback is the size of the transgene, commonly >300 bp, required to trigger
efficient RNA silencing. However, Parizotto et al. (2004) has provided experimen-
tal evidence using genetically engineered microRNA (miRNA) to show that a 21
nucleotide sequence complementary to GFP mRNA was sufficient to trigger com-
plete GFP silencing in transgenic plants. In the future, similar miRNA-derived
strategies could provide resistance to a large number of plant viruses. However, the
major technical limitation for technologies based on RNA silencing is that many
important plant crop species are difficult or impossible to transform, precluding the
constitutive expression of constructs directing production of double-stranded RNA.
Moreover, public concerns over the potential ecological impact of virus-resistant
transgenic plants have so far significantly limited their use (Fermin et al. 2004).
For DNA viruses (particularly, Gemini viruses), transgenic resistance involves
both RNA-mediated resistance and mutated viral proteins exerting negative effects
on viral replication (Vanderschuren et al. 2007).
1.4.6 Conclusions
While insect-resistant and herbicide-tolerant transgenic plants are the most widely
grown GM crops, very few strategies using genetically engineered plants for
disease resistance have had a similar impact (Collinge et al. 2008). Given the effort
put into biotechnological strategies for disease resistance over the last two decades,
why have so few crops expressing these traits become commercialized crops?
The development of molecular markers, which are particularly useful in screen-
ing for disease resistance and facilitate conventional breeding, provide a higher
commercial incentive for seed companies because of strong public opposition to
GM crops. Often, transgenic disease resistant plants have only partial resistance,
or resistance to rapid breakdown (single genes), again giving a low economic
incentive to developers. At present, clear field results show that transgenic virus
resistance is effective; however, there are no signs that commercial bacterial- or
fungal-resistant crops will be introduced onto the market at any point soon.
1.5 Transgenic Crops for Resistance to Biotic Stress:

Conclusions
Approximately 10.3 million farmers in 22 countries grew transgenic (genetically

modified) crops in 2006. Yet this technology remains one of the most controversial
agricultural issues of current times. Many consumer and environmental lobby
groups believe that GM crops will bring very little benefit to growers and to the
general public, and that they will have a deleterious effect on the environment. The
human population is currently 6.1 billion and it is predicted to increase to 9–10
billion in the next 50 years (Fig. 1.7). This is at a time when food and fuel are
competing for land (Fig. 1.8) and climate change threatens to compromise current
resources. Population growth, changing diets, higher transport costs and a drive
towards biofuels are forcing food prices up (Fig. 1.9). The UN’s Food and Agricul-
ture Organization (FAO) stated that the food crisis had thrown an additional 75
million people into hunger and poverty in 2007 alone.
World Population Growth (billion people)
1950 1975 2000 2025 2050
2.5bn 4.1bn 6.1bn 8.0bn 9.2bn
Fig. 1.7 World population growth

Fig. 1.8 Demand for biofuels Demand for Biofuels

Rise in use of course grains 2005-7 (FAO/OECD).
Total: Biofuel use:

80m tonnes 47m tonnes
Price Rises in a Single Year (Mar 2007-Mar 2008)

Source: Bloomberg, FAO/ Jackson Son & Co
130%
87%
74%
31%
Corn Rice Soya Wheat
Fig. 1.9 Price rises of major food crops (2007–2008)
It is, and will continue to be, a priority for agriculture to produce more crops on
less land. The minimization of losses to biotic stress caused by agricultural pests
would go some way to optimizing the yield on land currently under cultivation.
Traditionally, agricultural production has kept pace, even outstripped human
population growth; however, we currently face a set of unique challenges. One of
the greatest dangers to agriculture is its vulnerability to global climate change. The
expected impacts are for more frequent and severe drought and flooding, and
shorter growing seasons. The performance of crops under stress will depend on
their inherent genetic capacity and on the whole agroecosystem in which they are
managed. It is for this reason that any efforts to increase the resilience of agriculture
to climate change must involve the adoption of stress-resistant plants as well as
more prudent management of crops, animals and the natural resources that sustain
their production. Currently, we may be at the limit of the existing genetic resources
available in our major crops (Gressel 2008). Thus, new genetic resources must be
found and only new technologies will enable this.
References
Abdeen A, Virgos A, Olivella E, Villanueva J, Aviles X, Gabarra R, Prat S (2005) Multiple insect
resistance in transgenic tomato plants over-expressing two families of plant proteinase inhibi-
tors. Plant Mol Biol 57(2):189–202
Amman K (2009) Biodiversity and genetically modified crops. In: Ferry N, Gatehouse AMR (eds)
Environmental impact of genetically modified/novel crops. CAB International, Oxford, UK,
pp 240–265
Ananieva KI, Ananiev ED (1999) Effect of methyl ester of jasmonic acid and benzylaminopurine
on growth and protein profile of excised cotyledons of Cucurbita pepo L. (zucchini). Biol Plant
42:549–557
Anonymous (2007) Can biotech and organic crops coexist? Council for Biotechnology Informa-
tion. http://www.whybiotech.com
Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal trans-
duction. Annu Rev Plant Biol 55:373–399
Arimura G, Tashiro K, Kuhara S, Nishioka T, Ozawa R, Takabayashi J (2000) Gene responses in
bean leaves induced by herbivory and by herbivore-induced volatiles. Biochem Biophys Res
Commun 277(2):305–310
Armstrong CL, Parker GB, Pershing JC, Brown SM, Sanders PR, Duncan DR, Stone T, Dean DA,
DeBoer DL, Hart J, Howe AR, Morrish FM, Pajeau ME, Petersen WL, Reich BJ, Rodriguez R,
Santino CG, Sato SJ, Schuler W, Sims SR, Stehling S, Tarochione LJ, Fromm ME (1995) Field
evaluation of European corn borer control in progeny of 173 transgenic corn evens expressing
an insecticidal protein from Bacillus thuringiensis. Crop Sci 35:550–557
Arpaia S (1997) Ecological impact of Bt-transgenic plants: 1. Assessing possible effects of
CryIIIB toxin on honey bee (Apis mellifera) colonies. J Genet Breed 50:315–319
Asao H, Nishizawa Y, Arai S, Sato T, Hirai M, Yoshid K, Shinmyo A, Hibi T (1997) Enhanced
resistance against a fungal pathogen Sphaerotheca humuli in transgenic strawberry expressing
a rice chitinase gene. Plant Biotechnol 14:145–149
Ayliffe MA, Lagudah ES (2004) Molecular genetics of disease resistance in cereals. Ann Bot
94:765–773
Bakhetia M, Urwin PE, McPherson MJ, Atkinson HJ (2005) RNA interference and control of plant
parasitic nematodes. Trends Plant Sci 10:362–367
Baldwin IT, Halitschke R, Kessler A, Schittko U (2001) Merging molecular and ecological
approaches in plant-insect interactions. Curr Opin Plant Biol 4(4):351–358
Bauer LS (1990) Response of the cottonwood leaf beetle (Coleoptera: Chrysomelidae) to Bacillus
thuringiensis var. San Diego. Environ Entomol 19:428–431
Baulcombe D (2004) RNA silencing in plants. Nature 431:356–363
Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P, Ilagan O, Johnson S, Plaetinck G,
Munyikwa T, Pleau M, Vaughn T, Roberts J (2007) Control of coleopteran insect pests through
RNA interference. Nat Biotechnol 25:1322–1326
Belkhadir Y, Subramaniam R, Dangl JL (2004) Plant disease resistance protein signaling: NBS–
LRR proteins and their partners. Curr Opin Plant Biol 7:391–399
Bell HA, Fitches EC, Marris GC, Bell J, Edwards JP, Gatehouse JA, Gatehouse AMR (2001)
Transgenic crop enhances beneficial biocontrol agent performance. Transgenic Res 10:35–42
Benbrook CM (2004) Genetically engineered crops and pesticide use in the United States: the first
nine years, 7th edn. BioTech InfoNet, Idaho, USA
Bergelson J, Kreitman M, Stahl EA, Tian D (2001) Evolutionary dynamics of plant R-genes.
Science 292:2281–2285
Bohan DA, Boffey WH, Brooks DR, Clark SJ, Dewar AM, Firbank L, Haughton AJ, Hawes C,
Heard MS, May MJ, Osborne JL, Perry JN, Rothery P, Roy DB, Scott RJ, Squire GR, Woiwod
IP, Champion GT (2005) Effects on weed and invertebrate abundance and diversity of
herbicide-tolerant winter-sown oilseed rape. Proceedings of the Royal Society of London
272:463–474
Boller EF, Häni F, Poehling HM (2004) Ecological infrastructures: ideabook on functional
biodiversity at the farm level. IOBCwprs Commission on Integrated Production Guidelines
and Endorsement. www.iobc.ch
Bolwell GP, Blee KA, Butt VS, Davies DR, Gardner SL, Gerrish C, Minibayeva F, Rowntree EG,
Wojtaszek P (1999) Recent advances in understanding the origin of the apoplastic oxidative
burst in plant cells. Free Radic Res 31:S137–S145
Boulter D, Edwards GA, Gatehouse AMR, Gatehouse JA, Hilder VA (1990) Additive protective
effects of different plant-derived insect resistance genes in transgenic tobacco plants. Crop Prot
9:351–354
Bown DP, Wilkinson HS, Gatehouse JA (1997) Differentially regulated inhibitor-sensitive and
insensitive protease genes from the phytophagous insect pest, Helicoverpa armigara, are
members of complex multigene families. Insect Biochem Mol Biol 27(7):625–638
Broadway RM (1997) Dietary regulation of serine proteinases that are resistant to serine proteinase
inhibitors. J Insect Physiol 43(9):855–874
Broekaert WF, Terras FR, Cammue BP, Osborn RW (1995) Plant defensins: novel antimicrobial
peptides as components of the host defense system. Plant Physiol 108:1353–1358
Broglie K, Chet I, Holliday M, Cressman R, Biddle P, Knowlton S, Mauvais CJ, Broglie R (1991)
Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science
254:1194–1197
Brooks EM, Hines ER (1999) Viral biopesticides for heliothine control - fact or fiction. Today’s
Life Sci Jan/Feb:38–44
Burrows PR, Barker ADP, Newell CA, Hamilton WDO (1999) Plant-derived enzyme inhibitors and
lectins for resistance against plant-parasitic nematodes in transgenic crops. Pestic Sci 52:176–183
Byrne PF, Fromherz S (2003) Can GM and non-GM crops coexist? Setting a precedent in Boulder
County, Colorado, USA. Food Agr Environ 1:258–261
Calwineries. http://www.calwineries.com. Accessed 1 Oct 2008
Campbell EJ, Schenk PM, Kazan K, Penninckx IAMA, Anderson JP, Maclean DJ, Cammue BPA,
Ebert PR, Manners JM (2003) Pathogen-responsive expression of a putative ATP-binding
cassette transporter gene conferring resistance to the diterpenoid sclareol is regulated by
multiple defense signaling pathways in Arabidopsis. Plant Physiol 133:1272–1284
Canola Council of Canada (2001) Canola Council of Canada, 2001. An agronomic and economic
assessment of transgenic canola. The Growers manual. p 26. http://www.canola-council.org/
production/gmo2.html
Cao H, Bowling SA, Gordon AS, Dong X (1994) Characterization of an Arabidopsis mutant that is
nonresponsive to inducers of systemic acquired resistance. Plant Cell 6:1583–1592
Cao H, Glazebrook J, Clarke JD, Volko S, Dong X (1997) The Arabidopsis NPR1 gene that controls
systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell 88:57–63
Cao H, Li X, Dong X (1998) Generation of broad-spectrum disease resistance by overexpression
of an essential regulatory gene in systemic acquired resistance. Proceedings of the National
Academy of Sciences 95:6531–6536
Castle LA, Wu G, McElroy D (2006) Agricultural input traits: past, present and future. Curr Opin
Biotechnol 17(2):105–112
Cerdeira AL, Duke SO (2006) The current status and environmental impacts of glyphosate-
resistant crops; a review. J Environ Qual 35:1633–1658
Cessna SG, Sears VE, Dickman MB, Low PS (2000) Oxalic acid, a pathogenicity factor for
Sclerotinia sclerotiorum, suppresses the oxidative burst of the host plant. Plant Cell 12:2191–2199
Chamnongpol S, Willekens H, Moeder W, Langebartels C, Sandermann H, van Montagu M, Inze
D, van Camp W (1998) Defense activation and enhanced pathogen tolerance induced by H2O2
in transgenic tobacco. Proceedings of the National Academy of Sciences 95:5818–5823
Champion GT, May MJ, Bennett S, Brooks DR, Clark SJ, Daniels RE, Firbank LG, Haughton AJ,
Hawes C, Heard MS, Perry JN, Randle Z, Rossall MJ, Rothery P, Skellern MP, Scott RJ,
Squire GR, Thomas MR (2003) Crop management and agronomic context of the farm scale
evaluations of genetically modified herbicide-tolerant crops. Philos Trans R Soc Lond B
358:1801–1818
Charles D (2007) U.S. courts say transgenic crops need tighter scrutiny. Science 315:1069
Chattopadhyay A, Bhatnagar NB, Bhatnagar R (2004) Bacterial insecticidal toxins. Crit Rev
Microbiol 30:33–54
Chern MS, Fitzgerald HA, Yadav RC, Canlas PE, Dong X, Ronald PC (2001) Evidence for a
disease-resistance pathway in rice similar to the NPR1-mediated signaling pathway in Arabi-
dopsis. Plant J 27:101–113
Chiang CC, Hadwinger LA (1991) The Fusarium solani-induced expression of a pea gene family
encoding high cysteine content proteins. Mol Plant Microbe Interact 4:324–331
Chrispeels M, Sadava D (2003) Plants, genes and crop biotechnology. ASPB/Jones and Bartlett,
Boston, MA 562 p
Chrispeels MJ, Raikhel NV (1991) Lectins, lectin genes, and their role in plant defense. Plant Cell
3(1):1–9
Christoffoleti PJ, Galli AJB, Carvalho SJP, Moreira MS, Nicolai M, Foloni LL, Martins BAB,
Ribeiro DN (2008) Glyphosate sustainability in South American cropping systems. Pest Manag
Sci 64:422–427
Christou P, Capell T, Kohli A, Gatehouse JA, Gatehouse AMR (2006) Recent developments and
future prospects in insect pest control in transgenic crops. Trends Plant Sci 11:302–308
Collinge DB, SØgaard Lund O, Thordal-Christensen H (2008) What are the prospects for geneti-
cally engineered, disease resistant plants? Eur J Plant Pathol 121:217–231
Cowgill SE, Bardgett RD, Kiezebrink DT, Atkinson HJ (2002) The effect of transgenic nematode
resistance on non-target organisms in the potato rhizosphere. J Appl Ecol 39:915–923
Crawley MJ, Brown SL, Hails RS, Kohn DD, Rees M (2001) Biotechnology – Transgenic crops in
natural habitats. Nature 409:682–683
Crecchio C, Stotzky G (1998) Insecticidal activity and biodegradation of the toxin from Bacillus
thuringiensis subspecies kurstaki bound to humic acids from soil. Soil Biol Biochem 30:463–470
Dale PJ (2002) The environmental impact of genetically modified (GM) crops: a review. J Agr Sci
138:245–248
Dale PJ, Clarke B, Fontes EMG (2002) Potential for the environmental impact of transgenic crops.
Nat Biotechnol 20:567–574
Daniell H, Datta R, Varma S, Gray S, Lee SB (1998) Containment of herbicide resistance through
genetic engineering of the chloroplast genome. Nat Biotechnol 16:345–348
Daniels R, Boffey C, Mogg R, Bond J, Clarke R (2005) The potential for dispersal of herbicide
tolerance genes from genetically-modified, herbicide-tolerant oilseed rape crops to wild
relatives. Final Report to DEFRA. http://www.defra.gov.uk/environment/gm/research/pdf/
epg_1-5-151.pdf
Darmency H, Vigouroux Y, Gestat De Garambe T, Richard-Molard M, Muchembled C (2007)
Transgene escape in sugar beet production fields: data from six years farm scale monitoring.
Environ Biosafety Res 6:197–206
De Block M, Botterman J, Vandewiele M, Dockx J, Thoen C, Gossele V, Movva NR, Thompson
C, Van Montagu M, Leemans J (1987) Engineering herbicide resistance in plants by expression
of a detoxifying enzyme. EMBO J 6:2513–2518
De Leo F, Bonade-Bottino M, Ceci LR, Gallerani R, Jouanin L (2001) Effects of a mustard trypsin
inhibitor expressed in different plants on three lepidopteran pests. Insect Biochem Mol
31(6–7):593–602
de Maagd RA, Bosch D, Stiekema W (1999) Bacillus thuringiensis toxin-mediated insect resis-
tance in plants. Trends Plant Sci 4(1):9–13
de Maagd RA, Bravo A, Crickmore N (2001) How Bacillus thuringiensis has evolved specific
toxins to colonize the insect world. Trends Genet 17:193–1999
De Samblanx GW, Goderis IJ, Thevissen K, Raemaekers R, Fant F, Borremans F, Acland DP,
Osborn RW, Patel S, Broekaert WF (1997) Mutational analysis of a plant defensin from radish
(Raphanus sativus L.) reveals two adjacent sites important for antifungal activity. J Biol Chem
272:1171–1179
De Vos M, Van Oosten VR, Van Poecke RMP, Van Pelt JA, Pozo MJ, Mueller MJ, Buchala AJ,
Metraux JP, Van Loon LC, Dicke M, Pieterse CMJ (2005) Signal signature and transcriptome
changes of Arabidopsis during pathogen and insect attack. Mol Plant Microbe Interact
18(9):923–937
Degenhardt J, Gershenzon J, Baldwin IT, Kessler A (2003) Attracting friends to feast on foes:
engineering terpene emission to make crop plants more attractive to herbivore enemies. Curr
Opin Biotechnol 14(2):169–176
Delaney TP, Uknes S, Vernooij B, Friedrich L, Weymann K, Negrotto D, Gaffney T, Gut-Rella M,
Kessmann H, Ward E, Ryals J (1994) A central role of salicylic acid in plant disease resistance.
Science 266:1247–1250
Delaney TP, Friedrich L, Ryals JA (1995) Arabidopsis signal transduction mutant defective in
chemically and biologically induced disease resistance. Proceedings of the National Academy of
Sciences 92:6602–6606
Denolf P (1996) Isolation, cloning and characterisation of Bacillus thuringiensis delta-endotoxin
receptors in Lepidoptera. PhD, University of Gent, Belgium
Després C, DeLong C, Glaze S, Liu E, Fobert PR (2000) The Arabidopsis NPR1/NIM1 protein
enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription
factors. Plant Cell 12:279–290
Devine GJ, Furlong MJ (2007) Insecticide use: contexts and ecological consequences. Agr Hum
Val 24:281–306
Devine MD, Preston C (2000) The molecular basis of herbicide resistance. In: Cobb AH,
Kirkwood RC (eds) Herbicides and their mechanisms of action. Sheffield Academic Press,
Sheffield, UK, pp 72–104
Devos Y, Reheul D, De Schrijver A, Cors F, Moens W (2004) Management of herbicide-tolerant
oilseed rape in Europe: a case study on minimizing vertical gene flow. Environ Biosafety Res
3:135–148
Dicke M, Agrawal AA, Bruin J (2003) Plants talk, but are they deaf? Trends Plant Sci 8(9):
403–405
Dill GM (2005) Glyphosate-resistant crops; history, status and future. Pest Manag Sci 61:219–224
Dill GM, CaJacob CA, Padgette SR (2008) Glyphosate-resistant crops: adoption, use and future
considerations. Pest Manag Sci 64:326–331
Directorate General for Agriculture and Rural Development of the European Commission. What is
organic farming? http://ec.europa.eu/agriculture/index_en.htm
Down RE, Gatehouse AMR, Hamilton WDO, Gatehouse JA (1996) Snowdrop lectin inhibits
development and decreases fecundity of the glasshouse potato aphid (Aulacorthum solani)
when administered in vitro and via transgenic plants both in laboratory and glasshouse trials.
J Insect Physiol 42(11–12):1035–1045
Du JP, Foissac X, Carss A, Gatehouse AMR, Gatehouse JA (2000) Ferritin acts as the most
abundant binding protein for snowdrop lectin in the midgut of rice brown planthoppers
(Nilaparvata lugens). Insect Biochem Mol Biol 30(4):297–305
Duke SO, Powles SB (2008) Glyphosate: a once-in-a-century herbicide. Pest Manag Sci 64:
319–325
Duke SO (2005) Taking stock of herbicide-resistant crops ten years after introduction. Pest Manag
Sci 61:211–218
Ellis JG, Lawrence GJ, Luck JE, Dodds PN (1999) Identification of regions in alleles of the flax
rust resistance gene L that determine differences in gene-for-gene specificity. Plant Cell
11:495–506
Ellstand NC, Prentice HC, Hancock JF (1999) Gene flow and introgression from domesticated
plants into their wild relatives. Annu Rev Ecol Syst 30:539–563
Ellstrand NC (2003) Current knowledge of gene flow in plants. Philos Trans R Soc Lond B Biol
Sci 358:1163–1170
Ellstrand NC, Prentice HC, Hancock JF (1999) Gene flow and introgression from domesticated
plants into their wild relatives. Annu Rev Ecol Syst 30:539–563
Fan C, Pu N, Wang X, Wang Y, Fang L, Xu W, Zhang J (2008) Agrobacterium – mediated genetic
transformation of grapevine (Vitis vinifera L.) with a novel stilbene synthase gene from
Chinese wild Vitis pseudoreticulata. Plant Cell Tissue Organ Cult 92:197–206
Fan W, Dong X (2002) In vivo interaction between NPR1 and transcription factor TGA2 leads to
salicylic acid–mediated gene activation in Arabidopsis. Plant Cell 14:1377–1389
Fermin G, Inglessis V, Garboza C, Rangle S, Dagert M, Gonsalves D (2004) Engineered resistance
against Papaya ringspot virus in Venezuelan transgenic papaya. Plant Dis 88:516–522
Ferré J, Van Rie J (2002) Biochemistry and genetics of insect resistance to Bacillus thuringiensis.
Annu Rev Entomol 47:501–533
Ferro DN, Gerlernter WD (1989) Toxicity of a new strain of Bacillus thuringiensis to Colorado
potato beetle (Coleoptera: Chrysomelidae). J Econ Entomol 82:750–755
Ferry N, Gatehouse AMR (eds) (2009) Environmental impact of genetically modified/novel crops.
CAB International, Oxford, UK
Ferry N, Edwards MG, Gatehouse JA, Capell T, Christou P, Gatehouse AMR (2006) Transgenic
plants for insect pest control: a forward looking scientific perspective. Transgenic Res 15:13–19
Ferry N, Edwards MG, Mulligan EA, Emami K, Petrova A, Frantescu M, Davison GM, Gatehouse
AMR (2003) Engineering resistance to insect pests. In: Christou P, Klee H (eds) Handbook of
plant biotechnology. Wiley, New York, NY, pp 373–394
Ferry N, Mulligan EA, Majerus MEN, Gatehouse AMR (2007) Bitrophic and tritrophic effects of
Bt Cry3A transgenic potato on beneficial, non-target, beetles. Transgenic Res 16:795–812
Feyereisen R (1995) Molecular biology of inseticide resistance. Toxicol Lett 82(83):83–90
French-Constant RH (2004) The genetics and genomics of insecticide resistance. Trends Genet
20:163–170
Firbank LG, Forcella F (2000) Genetically modified crops and farmland biodiversity. Science
289:1481–1482
Fisher L (2007) Growers continue to grow and use roundup ready alfalfa but Monsanto Company
is disappointed with preliminary injunction affecting purchase and planting. http://www.
monsanto.com. Accessed 15 Oct 2008
Fitches E, Audsley N, Gatehouse JA, Edwards JP (2002) Fusion proteins containing neuropeptides
as novel insect control agents: snowdrop lectin delivers fused allatostatin to insect haemo-
lymph following oral ingestion. Insect Biochem Mol Biol 32(12):1653–1661
Fitches E, Edwards MG, Mee C, Grishin E, Gatehouse AMR, Edwards JP, Gatehouse JA (2004)
Fusion proteins containing insect-specific toxins as pest control agents: snowdrop lectin
delivers fused insecticidal spider venom toxin to insect haemolymph following oral ingestion.
J Insect Physiol 50(1):61–71
Fletcher MR, Hunter K, Barnett EA, Sharp EA (2000) Pesticide poisoning of animals 1998:
investigations of suspected incidents in the United Kingdom. A Report of the Environment
Panel of the Advisory Committee on Pesticides. London, UK, MAFF, (PB4786), p 54
Foissac X, Loc NT, Christou P, Gatehouse AMR, Gatehouse JA (2000) Resistance to green
leafhopper (Nephotettix virescens) and brown planthopper (Nilaparvata lugens) in transgenic
rice expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA). J Insect Physiol
46(4):573–583
Forcada C, Alcacer E, Garcera MD, Tato A, Martinez R (1999) Resistance to Bacillus thurin-
giensis Cry1Ac toxin in three strains of Heliothis virescens: proteolytic and SEM study of the
larval midgut. Arch Insect Biochem Physiol 42:51–63
Foresman C, Glasgow L (2008) US grower perceptions and experiences with glyphosate-resistant
weeds. Pest Manag Sci 64:388–391
Foyer CH, Noctor G (2005) Redox homeostasis and antioxidant signaling: a metabolic interface
between stress perception and physiological responses. Plant Cell 17:1866–1875
Frey M, Stettner C, Pare PW, Schmelz EA, Tumlinson JH, Gierl A (2000) An herbivore
elicitor activates the gene for indole emission in maize. Proceedings of the National Academy
of Sciences 97(26):14801–14806
Fuchs M, Gonsalves D (2007) Safety of virus-resistant transgenic plants two decades after their
introduction: lessons from realistic field risk assessment studies. Annu Rev Phytopathol
45:173–202
Fuller VL, Lilley CJ, Urwin PE (2008) Nematode resistance. New Phytol 180(1):27–44
Gahan LJ, Gould F, Heckel DG (2001) Identification of a gene associated with Bt resistance in
Heliothis virescens. Science 293(5531):857–860
Gatehouse JA, Gatehouse AMR (1999) Genetic engineering of plants for insect resistance.
In: Rechcigl JE, Reichcigl NA (eds) Biological and biotechnological control of insect pests.
CRC Press LLC, USA, pp 211–241
Gatehouse AMR, Davison GM, Stewart JN, Galehouse LN, Kumar A, Geoghegan IE, Birch ANE,
Gatehouse JA (1999) Concanavalin A inhibits development of tomato moth (Lacanobia
oleracea) and peach-potato aphid (Myzus persicae) when expressed in transgenic potato plants.
Mol Breed 5(2):153–165
Gatehouse JA (2002a) Plant resistance towards insect herbivores: a dynamic interaction. Tansley
Review No 140. New Phytol 156:145–169
Gatehouse AMR, Davison GM, Newell CA, Merryweather A, Hamilton WDO, Burgess EPJ,
Gilbert RJC, Gatehouse JA (1997) Transgenic potato plants with enhanced resistance to the
tomato moth, Lacanobia oleracea: growth room trials. Mol Breed 3(1):49–63
Gatehouse AMR, Ferry N, Raemaekers RJM (2002) The case of the Monarch butterfly; a verdict is
returned. Trends Genet 18:249–251
Gatehouse AMR, Hilder VA, Powell KS, Wang M, Davison GM, Gatehouse LN, Down RE,
Edmonds HS, Boulter D, Newell CA, Merryweather A, Hamilton WDO, Gatehouse JA (1994)
Insect-resistant transgenic plants – choosing the gene to do the job. Biochem Soc Trans
22(4):944–949
Gatehouse AMR, Powell K, Peumans W, Damme EV, Gatehouse JA (1995) Insecticidal properties
of plant lectins: their potential in plant protection. In: Pusztai A, Bardocz S (eds) Lectins
biomedical perspectives. Taylor & Francis, London, UK, pp 35–57
Gatehouse JA (2002b) Plant resistance towards insect herbivores: a dynamic interaction. New
Phytol 156(2):145–169
Gealy DR, Dilday RH (1997) Biology of red rice (Oryza sativa L.) accessions and their suscepti-
bility to glufosinate and other herbicides. Weed Sci Soc Am 37:34
Gealy DR, Bradford KJ, Hall L, Hellmich R, Raybold A, Wolt J, Zilberman D (2007) Implications
of gene flow in the scale-up and commercial use of biotechnology-derived crops: economic and
policy considerations. CAST Issue Paper. CAST, Ames, IA, p 24
Gepts P, Papa R (2003) Possible effects of (trans)gene flow from crops on the genetic diversity
from landraces and wild relatives. Environ Biosafety Res 2:89–103
Gianessi LP (2005) Economic and herbicide use impacts of glyphosate-resistant crops. Pest Manag
Sci 61:241–245
Gill SS, Cowles EA, Francis V (1995) Identification, isolation, and cloning of a Bacillus thur-
ingiensis CryIAc toxin-binding protein from the midgut of the Lepidopteran insect Heliothis
virescens. J Biol Chem 270(45):27277–27282
Ginder RG (2001) Channelling, identity preservation and the value chain: lessons from the recent
problems with StarLink corn. Iowa State University, Ames, IA
Giri AP, Wunsch H, Mitra S, Zavala JA, Muck A, Svatos A, Baldwin IT (2006) Molecular
interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and
its natural host Nicotiana attenuata. VII. Changes in the plant’s proteome. Plant Physiol
142:1621–1641
Glaser JA, Matten SR (2003) Sustainability of insect resistance management strategies for
transgenic Bt corn. Biotechnol Adv 22:45–69
Graef F, Stachow U, Werner A, Schütte G (2007) Agricultural practice changes with cultivating
genetically modified herbicide-tolerant oilseed rape. Agr Syst 94(2):111–118
Graham J, McNicol RJ, Greig K (1995) Towards genetic based insect resistance in strawberry
using the Cowpea trypsin inhibitor gene. Ann Appl Biol 127(1):163–173
Grant JJ, Loake GJ (2000) Role of reactive oxygen intermediates and cognate redox signaling in
disease resistance. Plant Physiol 124:21–29
Grant JJ, Chini A, Basu D, Loake GJ (2003) Targeted activation tagging of the NBS–LRR gene,
ADR1, conveys resistance to virulent pathogens. Mol Plant Microbe Interact 16:669–80
Gressel J (1999) Tandem constructs: preventing the rise of superweeds. Trends Biotechnol
17:361–366
Gressel J, Rotteveel AW (2000) Genetic and ecological risks from biotechnologically-derived
herbicide-resistant crops: decision trees for risk assessment. Plant Breed Rev 18:251–303
Gressel J (2008) Genetic glass ceilings. Transgenics for crop biodiversity. John Hopkins Univ
Press, Baltimore, MD
Groot AT, Dicke M (2002) Insect-resistant transgenic plants in a multi-trophic context. Plant J
31:387–406
Gurr SJ, Rushton PJ (2005) Engineering plants with increased disease resistance: how are we
going to express it? Trends Biotechnol 10:390–396
Hails RS, Morley K (2005) Genes invading new populations: a risk assessment perspective. Trends
Ecol Evol 20:245–252
Hall K, Topinka J, Huffman L, Davis A (2000) Pollen flow between herbicide-resistant Brassica
napus is the cause of multiple-resistant B. napus volunteers. Weed Sci 48:688–694
Harborne J (1998) Introduction to ecological chemistry. Academic, London, UK
Harriman P (2007) Roundup ready concerns land alfalfa seed in court. http://64.26.159.139/static/
news/NEWSID_8242.php
Haslberger A (2001) GMO contamination of seeds. Nat Biotechnol 19:613
Hayes KR, Gregg PC, Gupta VVSR, Jessop R, Lonsdale WM, Sindel B, Stanley J, Williams CK
(2004) Identifying hazards in complex ecological systems. Part 3: Hierarchical Holographic
Model for herbicide tolerant oilseed rape. Environ Biosafety Res 3:109–128
He KJ, Wang ZY, Zhang YJ (2009) Monitoring Bt resistance in the field: China as a case study.
In: Ferry N, Gatehouse AMR (eds) Environmental impact of genetically modified/novel crops.
CAB International, Oxford, UK, pp 344–360
Heifetz PB (2000) Genetic engineering of the chloroplast. Biochimie 82:655–666
Hellmich RL, Siegfried BD, Sears MK, Stanley-Horn DE, Daniels MJ, Mattila HR, Spencer T
(2001) Monarch larvae sensitivity to Bacillus thuringiensis-purified proteins and pollen.
Proceedings of the National Academy of Sciences 98:11925–11930
Hermsmeier D, Schittko U, Baldwin IT (2001) Molecular interactions between the specialist
herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata.
I. Large-scale changes in the accumulation of growth- and defence-related plant mRNAs. Plant
Physiol 125(2):683–700
Hilbeck A, Baumgartner M, Fried PM, Bigler F (1998) Effects of transgenic Bacillus thuringiensis
corn-fed prey on mortality and development of immature Chrysoperla carnea (Neuroptera:
Chrysopidae). Environ Entomol 27:480–487
Hilder VA, Gatehouse AMR, Sheerman SE, Barker RF, Boulter D (1987) A novel mechanism of
insect resistance engineered into tobacco. Nature 330(6144):160–163
Hilker M, Meiners T (2002) Induction of plant responses to oviposition and feeding by herbivo-
rous arthropods: a comparison. Entomol Exp Appl 104(1):181–192
Howatt KA, EndresGJ HendricksonPE, AberleEZ LukachJR, JenksBM RivelandNR, ValentiSA

RystedtCM (2006) Evaluation of glyphosate-resistant hard red spring wheat (Triticum aesti-
vum). Weed Technol 20:706–716
http://www.agbios.com
http://www.canola-councildemo.org. Accessed 14 July 2008
Hu X, Bidney DL, Yalpani N, Duvick JP, Crasta O, Folkerts O, Lu G (2003) Overexpression of a
hydrogen peroxide-generating oxalate oxidase gene evokes defense responses in sunflower.
Plant Physiol 133:170–181
Hurburgh CR (2000) The GMO controversy and grain handling for 2000. Iowa State University,
Ames, IA
Hurburgh CR (2003) Constraints for isolation and traceability of grains. Iowa State University,
Ames, IA
Jackson RE, Bradley JR Jr, Van Duyn JW (2004) Performance of feral and Cry1Ac-selected
Helicoverpa zea (Lepidoptera: Noctuidae) strains on transgenic cottons expressing either one
or two Bacillus thuringiensis ssp. kurstaki proteins under greenhouse conditions. J Entomol Sci
39:46–55
Jacob GS, Garbow JR, Hallas LE, Kimack NM, Kishore GM, Schaeffer J (1988) Metabolism of
glyphosate in Pseudomonas sp. strain LBr. Appl Environ Microbiol 54:2953–2958
James C (2001) Global review of commercialized transgenic crops. ISAAA Briefs 24, ISAAA
James C (2007) Global status of commercialized biotech/GM crops. ISAAA Briefs 37, ISAAA
Joel JD, Kleifeld U, Losner-Goshen D, Herzlinger G, Gressel J (1995) Transgeniccrops against
parasites. Nature 374:220–221
Johnson WG, Gibson KD (2006) Glyphosate-resistant weeds and resistance management strate-
gies: an Indiana grower perspective. Weed Technol 20:768–772
Jongsma MA, Bolter C (1997) The adaptation of insects to plant protease inhibitors. J Insect
Physiol 43(10):885–895
Jouanin L, Bonade-Bottino M, Girard C, Morrot G, Giband M (1998) Transgenic plants for insect
resistance. Plant Sci 131(1):1–11
Kahn RS, Sjahril R, Nakamura I, Mii M (2008) Production of transgenic potato exhibiting enhanced
resistance to fungal infections and herbicide applications. Plant Biotechnol Rep 2:13–20
Kaloshian I, Walling LL (2005) Hemipterans as plant pathogens. Annu Rev Plant Biol 43:491–521
Karlova R, Weemen-Hendriks M, Naimov S, Ceron J, Dukiandjiev S, de Maagd RA (2005)
Bacillus thuringiensis delta-endotoxin Cry1Ac domain III enhances activity against Heliothis
virescens in some, but not all Cry1-Cry1Achybrids. J Invertebr Pathol 88(2):169–172
Kasprzewska A (2003) Plant chitinases – regulation and function. Cell Mol Biol Lett 8:809–824
Kessler A, Baldwin IT (2002) Plant responses to insect herbivory: the emerging molecular
analysis. Annu Rev Plant Biol 53:299–328
Khan RS, Sjahril R, Nakamura I, Mii M (2008) Production of transgenic potato exhibiting enhanced
resistance to fungal infections and herbicide applications. Plant Biotechnol Rep 2:13–20
Kinealy C (1995) This great calamity: the Irish Famine 1845–1852. Gill and MacMillan, New
York, NY
Kinkema M, Fan W, Dong X (2000) Nuclear localization of NPR1 is required for activation of PR
gene expression. Plant Cell 12:2339–2350
Knight PJK, Crickmore N, Ellar DJ (1994) The receptor for Bacillus thuringiensis Cryla(C)
delta-endotoxin in the brush-border membrane of the Lepidopteran Manduca sexta is
aminopeptidase-N. Mol Microbiol 11(3):429–436
Knoester M, van Loon LC, van den Heuvel J, Hennig J, BolJF LHJM (1998) Ethylene-insensitive
tobaccolacks nonhost resistance against soil-borne fungi. Proceedings of the National Academy
of Sciences 95:1933–1937
Krieg A, Hugner AM, Lagenbruch GA, Schnetter W (1983) Bacillus thuringiensis var. tenebrio-
nis: Ein neuer gegenuber Larven von Coleopteran wirksamer Pathotyp. Z Angew Entomol
96:500–508
Krubphachaya P, Juricek M, Kertbundit S (2007) Induction of RNA-mediated resistance to papaya

ringspot virus type W. J Biochem Mol Biol 40(3):404–411
Lan HY, Tian YC, Wang CH, Liu GZ, Zhang LH, Wang LL, Chen ZH (2000) Studies of
transgenic tobacco plants expressing beta-1, 3-glucanase and chitinase genes and their poten-
tial for fungal resistance. Yi Chuan Xue Bao 27:70–77
Lane BG, Dunwell JM, Ray JA, Schmitt MR, Cuming AC (1993) Germin, a protein of early plant
development, is an oxalate oxidase. J Biol Chem 268:12239–12242
Lebel E, Heifetz P, Thorne L, Uknes S, Ryals J, Ward E (1998) Functional analysis of regulatory
sequences controlling PR-1 gene expression in Arabidopsis. Plant J 16:223–233
Leckband G, Lörz H (1998) Transformation and expression of a stilbene synthase gene of
Vitis vinifera L. in barley and wheat for increased fungal resistance. Theor Appl Genet
96:1432–2242
Leple JC, Bonadebottino M, Augustin S, Pilate G, Letan VD, Delplanque A, Cornu D, Jouanin L
(1995) Toxicity to Chrysomela tremulae (Coleoptera, Chrysomelidae) of transgenic poplars
expressing a cysteine proteinase-inhibitor. Mol Breed 1(4):319–328
Levine MJ (2007) Pesticides: a toxic time bomb in our midst. Praeger, USA, pp 213–214
Li XC, Berenbaum MR, Schuler MA (2000) Molecular cloning and expression of CYP6B8: a
xanthotoxin-inducible cytochrome P450 cDNA from Helicoverpa zea. Insect Biochem Mol
Biol 30(1):75–84
Li XC, Schuler MA, Berenbaum MR (2002) Jasmonate and salicylate induce expression of
herbivore cytochrome P450 genes. Nature 419(6908):712–715
Lindbo JA, Silva-Rosales L, Proebsting WM, Dougherty WG (1993) Induction of a highly specific
antiviral state in transgenic plants: implications for regulation of gene expression and virus
resistance. Plant Cell 5:1749–1759
Ling K, Namba S, Gonsalves C, Slightom JL, Gonsalves D (1991) Protection against detrimental
effects of potyvirus infection in transgenic tobacco plants expressing the papaya ringspot virus
coat protein gene. Biotechnology 9:752–758
Losey JE, Rayor LS, Carter ME (1999) Transgenic pollen harms monarch larvae. Nature 399:
214–214
Luan V, Figueroa SJ, Baltazar MB, Gomez MR, Townsend LR, Schoper JB (2001) Maize pollen
longevity and distance isolation requirements for effective pollen control. Crop Sci 41:1551–1557
Luo K, Sangadala S, Masson L, Mazza A, Brousseau R, Adang MJ (1997) The Heliothis virescens
170 kDa aminopeptidase functions as “receptor A” by mediating specific Bacillus thuringiensis
Cry1A delta-endotoxin binding and pore formation. Insect Biochem Mol Biol 27(8–9):735–743
Lutman P (ed) (1999) Gene flow and agriculture: relevance for transgenic crops (BCPC Sympo-
sium Proceedings No 72, Keele Proceedings). British Crop Protection Council, London, UK
Ma BL, Subedi KD, Reid LM (2004) Extent of cross-fertilization in maize by pollen from
neighboring transgenic hybrids. Crop Sci 44:1273–1282
Ma G, Roberts H, Sarjan M, Featherstone N, Lahnstein J, Akhurst R, Schmidt O (2005) Is the
mature endotoxin Cry1Ac from Bacillus thuringiensis inactivated by a coagulation reaction in
the gut lumen of resistant Helicoverpa armigera larvae? Insect Biochem Mol Biol 35:729–739
MacIntosh SC, Stone TB, Sims SR, Hunst PL, Green-plate JT, Marrone PG, Perlak FJ, Fischhoff
DA, Fuchs RL (1990) Specificity and efficacy of purified Bacillus thuringiensis proteins
against agronomically important insects. J Invertebr Pathol 56:258–266
Madsen KH, Blacklow WM, Jensen JE, Streibig JC (1999) Simulation of herbicide use in a crop
rotation with transgenic herbicide-tolerant oilseed rape. Weed Res 39:95–106
Maiti IB, Dey N, Pattanaik S, Dahlman DL, Rana RL, Webb BA (2003) Antibiosis-type insect
resistance in transgenic plants expressing a teratocyte secretory protein (TSP14) gene from a
hymenopteran endoparasite (Microplitis croceipes). Plant Biotechnol J 1:209–219
Malone LA, Pham-Delegue M-H (2001) Effects of transgene products on honey bees (Apis
mellifera) and bumblebees (Bombus sp.). Apidologie 32:287–304
Malone LA, Burgess EPJ (2009) Impact of genetically modified crops on pollinators. In: Ferry N,
Gatehouse AMR (eds) Environmental impact of genetically modified/novel crops. CAB
International, Oxford, UK, pp 199–225
Malone LA, Gatehouse AMR, Barratt BIP (2008) Beyond Bt: alternative strategies for insect-
resistant crops. In: Romeis J, Shelton T, Kennedy G (eds) Integration of insect-resistant
genetically modified crops within integrated pest management programs. Series on progress
in biological control. Springer, Berlin
Manners JM, Penninckx IAMA, Vermaere K, Kazan K, Brown RL, Morgan A, Maclean DJ, Curtis
MD, Cammue BPA, Broekaert WF (1998) The promoter of the plant defensin gene PDF1.2
from Arabidopsis is systemically activated by fungal pathogens and responds to methyl
jasmonate but not to salicylic acid. Plant Mol Biol 38:1071–1080
Mao YB, Cai WJ, Wang JW, Hong GJ, Tao XY, Wang LJ, Huang YP, Chen XY (2007) Silencing a
cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance
to gossypol. Nat Biotechnol 25:1307–1313
Maqbool SB, Riazuddin S, Loc NT, Gatehouse AMR, Gatehouse JA, Christou P (2001) Expres-
sion of multiple insecticidal genes confers broad resistance against a range of different rice
pests. Mol Breed 7(1):85–93
Marrs KA (1996) The functions and regulation of glutathione S-tranferases in plants. Annu Rev
Plant Physiol Plant Mol Biol 47:127–158
Matus-Cadiz PH, Horak MJ, Blomquist LK (2004) Gene flow in wheat at the field scale. Crop Sci
44:718–727
Mazarei M, Teplova I, Hajimorad MR, Stewart CN (2008) Pathogen phytosensing: plants to report
plant pathogens. Sensors 8:2628–2641
Mehlo L, Gahakwa D, Nghia PT, Loc NT, Capell T, Gatehouse JA, Gatehouse AMR, Christou P
(2005) An alternative strategy for sustainable pest resistance in genetically enhanced crops.
Proceedings of the National Academy of Sciences 102(22):7812–7816
Metraux JP, Nawrath C, Genoud T (2002) Systemic acquired resistance. Euphytica 124:
237–243
Mindrinos M, Katagiri F, Yu GL, Ausubel FM (1994) The Arabidopsis thaliana disease resistance
gene rps2 encodes a protein containing a nucleotide binding site and leucine rich repeats. Cell
78:1089–1099
Mittler R, Vanderawera S, Gollery M, Van Breusegen F, (2004) Reactive oxygen gene network of
patients. Trends in plant Science 9:490–498
Mishra P (2007) Exit wounds. The New Yorker, 13 Aug 2007
Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410
Moellenbeck DJ, Peters ML, Bing JW, Rouse JR, Higgins LS, Sims L, Nevshemal T, Marshall L,
Ellis RT, Bystrak PG (2001) Insecticidal proteins from Bacillus thuringiensis protect from corn
rootworms. Nat Biotechnol 19:668–672
Moran PJ, Thompson GA (2001) Molecular responses to aphid feeding in Arabidopsis in relation
to plant defence pathways. Plant Physiol 125(2):1074–1085
Moran PJ, Cheng YF, Cassell JL, Thompson GA (2002) Gene expression profiling of
Arabidopsis thaliana in compatible plant-aphid interactions. Arch Insect Biochem Physiol
51(4):182–203
Mueller TC, Mitchell PD, Young BG, Culpepper AS (2005) Proactive versus reactive manage-
ment of glyphosate-resistant or -tolerant weeds. Weed Technol 19:924–933
Nagamatsu Y, Toda S, Koike T, Miyoshi Y, Shigematsu S, Kogure M (1998) Cloning, sequencing,
and expression of the Bombyx mori receptor for Bacillus thuringiensis insecticidal CryIA(a)
toxin. Biosci Biotechnol Biochem 62(4):727–734
Nauen R, Ebbinghaus-Kintscher U, Elbert A, Jeschke P, Tietjen K (2001) Acetycholine receptors
as sites for developing neonicotinoid insecticides. In: Ishaaya I (ed) Biochemical sites impor-
tant in insecticide action and resistance. Springer, Berlin, pp 77–105
Naylor REL (2002) Weed management handbook. Osney Mead, Blackwell Science Ltd,
Oxford, UK
Nicholson GM (2007) Fighting the global pest problem: preface to the special toxicon issue on
insecticidal toxins and their potential for insect pest control. Toxicon 49:413–422
Niggeweg R, Thurow C, Kegler C, Gatz C (2000) Tobacco transcription factor TGA2.2 is the main
component of as-1-binding factor ASF-1 and is involved in salicylic acid- and auxin-inducible
expression of as-1-containing target promoters. J Biol Chem 275:19897–19905
Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control.
Annu Rev Plant Physiol Plant Mol Biol 49:249–279
Novotny V, Basset Y, Miller SE, Weiblen GD, Bremer B, Cizek L, Drozd P (2002) Low host
specificity of herbivorous insects in a tropical forest. Nature 416:841–844
Oldroyd GED, Staskawicz BJ (1998) Genetically engineered broad-spectrum disease resistance in
tomato. Proceedings of the National Academy of Sciences 95:10300–10305
Oppert B, Kramer KJ, Johnson DE, MacIntosh SC, McGaughey WH (1994) Altered protoxin
activation by midgut enzymes from a Bacillus thuringiensis resistant strain of Plodia inter-
punctella. Biochem Biophys Res Commun 198:940–947
Ortego F, Pons X, Albajes R, Castañera P (2009) European commercial GM plantings and field
trials. In: Ferry N, Gatehouse AMR (eds) Environmental impact of genetically modified/novel
crops. CAB International, Oxford, UK, pp 327–344
Ortiz-Garcia S, Ezcurra E, Schoel B, Acevedo F, Soberon J, Snow AA (2005) Absence of
detectable transgenes in local landraces of maize in Oaxaca, Mexico (2003–2004). Proceedings
of the National Academy of Sciences 102:12338–12343
Ostlie K (2001) Crafting crop resistance to corn rootworms. Nat Biotechnol 19:624–625
Ostlie KR, Hutchison WD, Hellmich RL (1997) Bt corn and European corn borer (NCR Publ 602).
University of Minnesota, Saint Paul, MN
Outchkourov NS, Rogelj B, Strukelj B, Jongsma MA (2003) Expression of sea anemone equistatin
in potato. Effects of plant proteases on heterologous protein production. Plant Physiol 133
(1):379–390
Owen M (1999) Weed management update for the next millenium. http://www.weeds.iastate.edu/
mgmt/qtr99-1/weedupdate.htm
Owen MDK (2008) Weed species shifts in glyphosate-resistant crops. Pest Manag Sci 64:
377–387
Owen MDK (2000) Current use of transgenic herbicide-resistant soybean and corn in the USA.
Crop Prot 19:765–771
Owen MDK (2005) Maize and soybeans – controllable volunteerism without ferality. In: Gressel J
(ed) Crop ferality and volunteerism. CRC Press, Boca Raton, FL, pp 149–165
Owen MDK (2006) Weed management update – who cares? In: Proceedings Integrated Crop
Management Conference, Iowa State University, Ames, IA, USA, pp 149–153
Owen MDK (1999) Weed management update for the next millennium. In: Proceedings Integrated
Crop Management Conference, vol 11, Iowa State University, Ames, IA, USA, pp 305–310
Owen MDK (2009) Herbicide tolerant genetically modified crops: resistance management. In:
Ferry N, Gatehouse AMR (eds) Environmental impact of genetically modified/novel crops.
CAB International, Oxford, UK, pp 115–165
Padgette R, Kolacz KH, Delannay X, Re DB, LaVallee BJ, Tinius CN, Rhodes WK, Otero YI,
Barry GF, Eichholtz DA, Peschke VM, Nida DL, Taylor NB, Kishore GM (1995) Develop-
ment, identification, and characterization of a glyphosate-tolerant soybean line. Crop Sci
35:1451–1461
Pallutt B, Hommel B (1998) Konzept und erste Ergebnisse zur Bewertung von Glufosinat-
tolerantem Raps und Mais im Rahmen einer 4-feldrigen Fruchtfolge. Z PflKrankh PflSchutz
Sonderheft 16:427–433
Palm CJ, Schaller DL, Donegan KK, Seidler RJ (1996) Persistence in soil of transgenic plant
produced Bacillus thuringiensis var. kurstaki d-endotoxin. Can J Microbiol 42:1258–1262
Palmer RG, Gai J, Sun H, Burton JW (2001) Production and evaluation of hybrid soybean.
In: Janick J (ed) Plant breeding reviews. Wiley, New York, NY, pp 263–308
Pannetier C, Giband M, Couzi P, LeTan V, Mazier M, Tourneur J, Hau B (1997) Introduction

of new traits into cotton through genetic engineering: insect resistance as example. Euphytica
96(1):163–166
Parizotto EA, Dunoyer P, Rahm N, Himber C, Voinnet O (2004) In vivo investigation of the
transcription, Processing, endonucleolytic activity, and functional relevance of the spatial
distribution of a plant miRNA. Genes Dev 18:2237–2242
Pastori GM, Foyer CH (2002) Common components, networks, and pathways of cross-tolerance
to stress. The central role of “redox” and abscisic acid-mediated controls. Plant Physiol
129:460–468
Peumans WJ, Vandamme EJM (1995) Lectins as plant defense proteins. Plant Physiol 109(2):
347–352
Phipps RH, Park JR (2002) Environmental benefits of genetically modified crops: global and
European perspectives on their ability to reduce pesticide use. J Anim Feed Sci 11:1–8
Pimental D (1997) Water resources: agriculture, the environment, and society. Bioscience
47(2):97–106
Powell KS, Gatehouse AMR, Hilder VA, Gatehouse JA (1995) Antifeedant effects of plant-lectins
and an enzyme on the adult stage of the rice brown planthopper, Nilaparvata lugens. Entomol
Exp Appl 75(1):51–59
Powell KS, Spence J, Bharathi M, Gatehouse JA, Gatehouse AMR (1998) Immunohistochemical
and developmental studies to elucidate the mechanism of action of the snowdrop lectin on the
rice brown planthopper, Nilaparvata lugens (Stal). J Insect Physiol 44(7–8):529–539
Price DR, Gatehouse JA (2008) RNAi-mediated crop protection against insects. Trends Biotech-
nol 26(7):393–400
Punja ZK, Raharjo SH (1996) Response of transgenic cucumber and carrot plants expressing
different chitinase enzymes to inoculation with fungal pathogens. Plant Dis 80:999–1005
Quist DA (2001) Transgenic DNA introgressed into traditional maize landraces in Oaxaca,
Mexico. Nature 414:541–543
Rahman MM, Roberts HL, Sarjan M, Asgari S, Schmidt O (2004) Induction and transmission of
Bacillus thuringiensis tolerance in the flour moth Ephestia kuehniella. Proceedings of the
National Academy of Sciences 101:2696–2699
Rao KV, Rathore KS, Hodges TK, Fu X, Stoger E, Sudhakar D, Williams S, Christou P, Bharathi
M, Bown DP, Powell KS, Spence J, Gatehouse AMR, Gatehouse JA (1998) Expression of
snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper.
Plant J 15(4):469–477
Raymond-Delpech V, Matsuda K, Sattelle BM, Rauh JJ, Sattelle DB (2005) Ion channels:
molecular targets of neuroactive insecticides. Invert Neurosci 5:119–133
Reymond P, Farmer EE (1998) Jasmonate and salicylate as global signals for defense gene
expression. Curr Opin Plant Biol 1:404–411
Rohini VK, Rao KS (2001) Transformation of peanut (Arachis hypogaea L.) with tobacco
chitinase gene: variable response of transformants to leaf spot disease. Plant Sci 160:889–898
Rojo E, Solano R, Sanchez-Serrano JJ (2003) Interactions between signaling compounds involved
in plant defence. J Plant Growth Regul 22(1):82–98
Romeis J, Bartsch D, BiglerF CMP, Gielkens MMC, Hartley SE, Hellmich RL, Huesing JE,
Jepson PC, Layton R, Quemada H, Raybould A, Rose RI, Schiemann J, Sears MK, Shelton
AM, Sweet J, Vaituzis Z, Wolt JD (2008) Assessment of risk of insect-resistant transgenic
crops to nontarget arthropods. Nat Biotechnol 26:203–208
Ross H (1986) Potato breeding – problems and perspectives: advances in plant breeding, J Plant
Breed (Supl 13). Paul Parey, Berlin 132 p
Sammons RD, Heering DC, DinicolaN GH, Elmore GA (2007) Sustainability and stewardship of
glyphosate and glyphosate-resistant crops. Weed Technol 21:347–354
Sangadala S, Walters FS, English L, Adang MJA (1994) Mixture of Manduca sexta aminopepti-
dase and phosphatase enhances Bacillus thuringiensis insecticidal Cryia(C) toxin binding and
(Rb+-K+)-Rb-86 efflux in vitro. J Biol Chem 269(13):10088–10092
Sankula S (2006) Quantification of the impacts on US agriculture of biotechnology-derived

crops planted in 2005. Executive Summary, National Center for Food and Agricultural Policy.
http://www.ncfap.org. Accessed 15 Oct 2008
Sanvido O, Romeis J, Bigler F (2007) Ecological impacts of genetically modified crops: ten years
of field research and commercial cultivation. Green Gene Technol 107:235–278
Sauvion N, Rahbe Y, Peumans WJ, Van Damme EJM, Gatehouse JA, Gatehouse AMR (1996)
Effects of GNA and other mannose binding lectins on development and fecundity of the peach-
potato aphid Myzus persicae. Entomol Exp Appl 79(3):285–293
Saxena D, Stotzky G (2001) Bacillus thuringiensis (Bt) toxin released from root exudates and
biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and
fungi in soil. Soil Biol Biochem 33:1225–1230
Saxena D, Flores S, Stotzky G (1999) Insecticidal toxin in root exudates from Bacillus thurin-
giensis corn. Nature 402:480
Sayyed A, Gatsi R, Kouskoura T, Wright DJ, Crickmore N (2001) Susceptibility of a field-derived,
Bacillus thuringiensis-resistant strain of diamondback moth to in vitro-activated Cry1Ac toxin.
Appl Environ Microbiol 67:4372–4373
Schaller H, Bouvier-Navé P, Benveniste P (1998) Overexpression of an Arabidopsis cDNA
encoding a sterol-C241-methyltransferase in tobacco modifies the ratio of 24-methyl choles-
terol to sitosterol and is associated with growth reduction. Plant Physiol 118:461–469
Schenk PM, Kazan K, Wilson I, Anderson JP, Richmond T, Somerville SC, Manners JM (2000)
Coordinated plant defence responses in Arabidopsis revealed by microarray analysis. Proceed-
ings of the National Academy of Sciences 97(21):11655–11660
Schroeder HE, Gollasch S, Moore A, Tabe LM, Craig S, Hardie DC, Chrispeels MJ, Spencer D,
Higgins TJV (1995) Bean alpha-amylase inhibitor confers resistance to the pea weevil (Bru-
chus pisorum) in transgenic peas (Pisum sativum L). Plant Physiol 107:1233–1239
Schuler TH, Potting RPJ, Denholm I, Poppy GM (1999) Parasitoid behaviour and Bacillus
thuringiensis plants. Nature 400:825–826
Schütte G, Stachow U, Werner A (2004) Agronomic and environmental aspects of the cultivation
of transgenic herbicide resistant plants. 11/04. Umweltbundesamt, Berlin
Segura A, Moreno M, Molina A, Garcia-Olmedo F (1998) Novel defensin subfamily from spinach
(Spinacia oleracea). FEBS Lett 435:159–162
Service RF (2007a) Glyphosate – the conservationist’s friend? Science 316:1116–1117
Service RF (2007b) A growing threat down on the farm. Science 316:1114–1117
Shade RE, Schroeder HE, Pueyo JJ, Tabe LM, Murdock LL, Higgins TJV, Chrispeels MJ (1994)
Transgenic pea seeds expressing the alpha-amylase inhibitor of the common bean are resistant
to bruchid beetles. Biotechnology 12(8):793–796
Shah J, Tsui F, Klessig DF (1997) Characterization of a salicylic acid-insensitive mutant (sai1) of
Arabidopsis thaliana, identified in a selective screen utilizing the SA-inducible expression
of the tms2 gene. Mol Plant Microbe Interact 10:69–78
Shen BZ, Zheng ZW, Dooner HK (2000) A maize sesquiterpene cyclase gene induced by insect
herbivory and volicitin: characterization of wild-type and mutant alleles. Proceedings of the
National Academy of Sciences 97(26):14807–14812
Shepherd DN, Mangwende T, Martin DP, Bezuidenhout M, Carolissen KFJ, CH MAL, Rybicki
EP, Thomson JA (2007) Maize streak virus-resistant transgenic maize: a first for Africa. Plant
Biotechnol 5:759–767
Shipitalo MJ, Malone RW, Owens LB (2008) Impact of glyphosate-tolerant sobyean and
glufosinate-tolerant corn production on herbicide losses in surface runoff. J Environ Qual
37:401–408
Sims SR (1995) Bacillus thuringiensis var. kurstaki [Cry1A(c)] protein expressed in transgenic
cotton: effects on beneficial and other non-target insects. Southwest Entomol 20:493–500
Sims SR (1997) Host activity spectrum of the CryIIA Bacillus thuringiensis subsp. kurstaki protein:
effects on Lepidoptera, Diptera, and non-target arthropods. Southwest Entomol 22:395–404
Singh PK, Kumar M, Chaturvedi CP, Yadav D, Tuli R (2004) Development of a hybrid delta-
endotoxin and its expression in tobacco and cotton for control of apolyphagous pest Spodop-
tera litura. Transgenic Res 13(5):397–410
Snow AA, Pedro MP (1997) Commercialization of transgenic plants: potential ecological risks.
Bioscience 47:86–96
Stalker DM, McBride KE, Malyj LD (1988) Herbicide resistance in transgenic plants expressing a
bacterial detoxification gene. Science 242:419–423
Steward CN Jr, All JN, Raymer PL, Ramachandran S (1997) Increased fitness of transgenic
insecticidal rapeseed under insect selection pressure. Mol Ecol 6:773–779
Sticher L, Mauch-Mani B, Métraux JP (1997) Systemic acquired resistance. Annu Rev Phyto-
pathol 35:235–270
Stoger E, Williams S, Christou P, Down RE, Gatehouse JA (1999) Expression of the insecticidal
lectin from snowdrop (Galanthus nivalis agglutinin; GNA) in transgenic wheat plants: effects
on predation by the grain aphid Sitobion avenae. Mol Breed 5(1):65–73
Stokes TL, Kunkel BN, Richards EJ (2002) Epigentic variation in Arabidopsis disease resistance.
Genes Dev 16:171–182
Stokstad E (2004) Monsanto pulls the plug on genetically modified wheat. Science 304
(5674):1088–1089
Stone TB, Sims SR, Marrone PG (1989) Selection of tobacco budworm for resistance to a
genetically engineered Pseudomonas fluorescens containing the d-endotoxin of Bacillus thur-
ingiensis subsp. Kurstaki. J Invertebr Pathol 53:228–234
Subramaniam R, Desveaux D, Spickler C, Michnick SW, Brisson N (2001) Direct visualization of
protein interactions in plant cells. Nat Biotechnol 19:769–772
Szankowski I, Briviba K, Fleshhut J, Schonherr J, Jacobsen HJ, Kiesecker H (2003) Transforma-
tion of apple (Malus domestica Borkh.) with the stilbene synthase gene from grapevine (Vitis
vinifera L.) and a PGIP gene from kiwi (Actinidia deliciosa). Plant Cell Rep 22:141–149
Tabashnik BE, Carrière Y (2009) Insect resistance to genetically modified crops. In: Ferry N,
Gatehouse AMR (eds) Environmental impact of genetically modified/novel crops. CAB
International, Oxford, UK, pp 74–101
Tabashnik BE, Gassmann AJ, Crowder DW, Carriére Y (2008) Insect resistance to Bt crops:
evidence versus theory. Nat Biotechnol 26:199–202
Takakura Y, Che FS, Ishida Y, Tsutsumi F, Kurotani KI, Usami S, Isogai A, Imaseki H (2008)
Expression of a bacterial flagellin gene triggers plant immune responses and confers disease
resistance in transgenic rice plants. Mol Plant Pathol 9:525–529
The United States Department of Agriculture. http://www.usda.gov/wps/portal/usdahome
Thies JE, Devare MH (2007) An ecological assessment of transgenic crops. J Dev Stud 43:97–129
Thomzik JE, Stenzel K, Stöcker R, Schreier PH, Hain R, Stahl DJ (1997) Synthesis of a grapevine
phytoalexin in transgenic tomatoes (Lycopersicon esculentum Mill.) conditions resistance
against Phytophtora infestans. Physiol Mol Plant Pathol 51:265–278
Tinjuangjun P, Loc NT, Gatehouse AMR, Gatehouse JA, Christou P (2000) Enhanced insect
resistance in Thai rice varieties generated by particle bombardment. Mol Breed 6(4):391–399
Tortiglione C, Fogliano V, Ferracane R, Fanti P, Pennacchio F, Monti LM, Rao R (2003) An insect
peptide engineered into the tomato prosystemin gene is released in transgenic tobacco plants
and exerts biological activity. Plant Mol Biol 53(6):891–902
Turner CT, Davy MW, MacDiarmid RM, Plummer KM, Birch NP, Newcomb RD (2006) RNA
interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-
stranded RNA feeding. Insect Mol Biol 15:383–391
Ullstrup AJ (1972) The impacts of the southern corn leaf blight epidemics of 1970–1971. Annu
Rev Phytopathol 10:37–50
United Nations Population Division. http://www.un.org/esa/population/unpop.htm
Urwin PE, Atkinson HJ, Waller DA, McPherson MJ (1995) Engineered oryzacystatin-I expressed
in transgenic hairy roots confers resistance to Globodera pallida. Plant J 8(1):121–131
Vadlamudi RK, Weber E, Ji IH, Ji TH, Bulla LA (1995) Cloning and expression of a receptor for
an insecticidal toxin of Bacillus thuringiensis. J Biol Chem 270(10):5490–5494
Vaeck M, Reynaerts A, Hofte H, Jansens S, Debeuckeleer M, Dean C, Zabeau M, Vanmontagu M,
Leemans J (1987) Transgenic plants protected from insect attack. Nature 328(6125):33–37
Van Loon LC (1997) Induced resistance in plants and the role of pathogenesis related proteins. Eur
J Plant Pathol 103:753–765
Vancanneyt G, Sanz C, Farmaki T, Paneque M, Ortego F, Castanera P, Sanchez-Serrano JJ (2001)
Hydroperoxide lyase depletion in transgenic potato plants leads to an increase in aphid
performance. Proceedings of the National Academy of Sciences 98(14):8139–8144
Vandenberg JD (1990) Safety of four entomopathogens for cages adult honey bees (Hymenoptera:
Apidae). J Econ Entomol 83:755–759
Vanderschuren H, Stupak M, Futterer J, Gruissem W, Zhang P (2007) Engineering resistance to
Gemini viruses – review and perspectives. Plant Biotechnol 5:207–220
Vazquez Rovere C, Asurmendi S, Hopp HE (2001) Transgenic resistance in potato plants expres-
sing potato leaf roll virus (PLRV) replicase gene sequences is RNA-mediated and suggests the
involvement of post-transcriptional gene silencing. Arch Virol 146:1337–53
Walling LL (2000) The myriad plant responses to herbivores. J Plant Growth Regul 19(2):195–216
Wang YX, Kausch AP, Chandlee JM, Luo H, Ruemmele BA, Browning M, Jackson N, Goldsmith
MR (2003) Co-transfer and expression of chitinase, glucanase, and bar genes in creeping
bentgrass for conferring fungal disease resistance. Plant Sci 165:497–506
Weise E (2007) Effect of genetically engineered alfalfa cultivate a debate, USA Today
Westgate ME, Lizaso J, Batchelor W (2003) Quantitative relationship between pollen shed density
and grain yield in maize. Crop Sci 43:934–942
Westwood J (1997) Growers endorse herbicide resistant crops, recognize need for responsible use,
ISB News No 3
Williams WP, Davis FM (1997) Maize germplasm with resistance to south-western corn borer and
fall armyworm. In: Mihm JA (ed) Insect resistant maize: recent advances and utilization.
Proceedings International Symposium, 27 Nov–3 Dec 1994. CIMMYT, Mexico, pp 226–229
Wraight CL, Zangerl AR, Carroll MJ, Berenbaum MR (2000) Absence of toxicity of Bacillus
thuringiensis pollen to black swallowtails under field conditions. Proceedings of the National
Academy of Sciences 14:7700–7703
Wu G, Shortt BJ, Lawrence EB, Levine EB, Fitzsimmons KC, Shah DM (1995) Disease resistance
conferred by expression of a gene encoding H2O2-generating glucose oxidase in transgenic
potato plants. Plant Cell 7:1357–1368
Xanthopoulos FP, Kechagia UE (2000) Natural crossing in cotton (Gossypium hirsutum L.). Aust J
Agr Res 51:979–983
Xu DP, Xue QZ, McElroy D, Mawal Y, Hilder VA, Wu R (1996) Constitutive expression of a
cowpea trypsin inhibitor gene, CpTi, in transgenic rice plants confers resistance to two major
rice insect pests. Mol Breed 2(2):167–173
Yang Y, Sherwood TA, Patte CP, Hiebert E, Polston JE (2004) Use of tomato yellow leaf curl
virus (TYLCV) rep gene sequences to engineer TYLCV resistance in tomato. Phytopathology
94:490
Young BG (2006) Changes in herbicide use patterns and production practices resulting from
glyphosate-resistant crops. Weed Technol 20:301–307
Zemetra RS, Hansen J, Mallory-Smith CA (1998) Potential for gene transfer between wheat
(Triticum aestivum) and jointed goatgrass (Aegilops cylindrica). Weed Sci 46:313–317
Zhang Y, Fan W, Kinkema M, Li X, Dong X (1999) Interaction of NPR1 with basic leucine zipper
protein transcription factors that bind sequences required for salicylic acid induction of the
PR-1 gene. Proceedings of the National Academy of Sciences 96:6523–6528
Zhao B, Lin X, Poland J, Trick H, Leach J, Hulbert S (2005) A maize resistance gene functions
against bacterial streak disease in rice. Proceedings of the National Academy of Sciences
102:15383–15388
Zhao J-Z, Cao J, Li Y, Collins HL, Roush R, Earle ED, Shelton AM (2003) Transgenic plants
expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nat Biotechnol
21:1493–1497
Zhou F, Zhang Z, Gregersen PL, Mikkelsen JD, de Neegaard E, Collinge DB, Thordal-Christensen
H (1998) Molecular characterization of the oxalate oxidase involved in the response of barley
to the powdery mildew fungus. Plant Physiol 117:33–41
Zhou JM, TrifaY SH, Pontier D, Lam E, Shah J, Klessig DF (2000) NPR1 differentially interacts
with members of the TGA/OBF family of transcription factors that bind an element of the PR-1
gene required for induction by salicylic acid. Mol Plant Microbe Interact 13:191–202
Zhu-Salzman K, Ahn J-E, Salzman RA, Koiwa H, Shade RE, Balfe S (2003) Fusion of a soybean
cysteine protease inhibitor and a legume lectin enhances anti-insect activity synergistically.
Agr Forest Entomol 5:317–323
Chapter 2
Transgenic Plants for Abiotic Stress Resistance
Margaret C. Jewell, Bradley C. Campbell, and Ian D. Godwin
2.1 Introduction
Modern agricultural crop production relies on the growth of a few of the world’s
plant species selected for their superior qualities and suitability as food, animal
feed, fiber or industrial end uses. Centuries of selection and, more recently, scien-
tific breeding for adaptation to biotic and abiotic stresses have been necessary to
improve yield, yield stability, and product quality in agricultural species.
Nevertheless, abiotic stresses remain the greatest constraint to crop production.
Worldwide, it has been estimated that approximately 70% of yield reduction is the
direct result of abiotic stresses (Acquaah 2007). The ever increasing pressure put on
agricultural land by burgeoning human populations has resulted in land degrada-
tion, a cultivation shift to more marginal areas and soil types, and heavier require-
ments for agricultural productivity per unit area. Additionally, climate change has
exacerbated the frequency and severity of many abiotic stresses, particularly
drought and high temperatures, with significant yield reductions reported in major
cereal species such as wheat, maize, and barley (Lobell and Field 2007). In many
parts of the world, rainfall has become less predictable, more intense, and, due to
increasing temperatures, subject to higher evapotranspiration. For example, in the
major grain growing areas of eastern Africa, the predominant rainy season is
starting later and ending earlier (Segele and Lamb 2005) with longer dry spells in
between (Seleshi and Camberlin 2006).
Agricultural practices to improve crop productivity per unit area have, in many
cases, accelerated the rate of land degradation, with particularly marked effects in
irrigated areas. Irrigation has led to salinity across large tracts of agricultural land,
I. D. Godwin (*)
School of Land, Crop and Food Sciences, The University of Queensland, Brisbane, Qld 4072,
Australia
e-mail: i.godwin@uq.edu.au

68 M.C. Jewell et al.
with cases, such as in India, where it has reportedly led to the loss of seven million
hectares from cultivation (Martinez-Beltran and Manzur 2005). In Australia, almost
eight million hectares of land are under threat of dryland salinity (Munns et al.
2002). Higher yields are also only sustainable with higher nutrient use, and the
heavy demand for fertilizers has caused rising costs for farmers worldwide. The
environmental and economic consequences of increased nutrient use have been
widely reported. For sustainability of crop production, there is a need to reduce the
environmental footprint of food and fiber production, and nutrient use efficient
crops are highly sought after.
Transgenic approaches are one of the many tools available for modern plant
improvement programs. Gene discovery and functional genomics projects have
revealed multitudinous mechanisms and gene families, which confer improved
productivity and adaptation to abiotic stresses. These gene families can be manipu-
lated into novel combinations, expressed ectopically, or transferred to species in
which they do not naturally occur or vary. Hence, the ability to transform the major
crop species with genes from any biological source (plant, animal, microbial) is an
extremely powerful tool for molecular plant breeding. Transgenic plants can be
used as sources of new cultivars (or their germ plasm as new sources of variation in
breeding programs) and they are also extremely useful as proof-of-concept tools to
dissect and characterize the activity and interplay of gene networks for abiotic
stress resistance.
In this chapter, we will outline the major yield-limiting abiotic stresses faced by
crop plants: drought, salinity, cold, nutrient deficiency, and metal toxicity. Within
each section, we will then cite specific examples of transgenic crop approaches to
overcome these stresses and also discuss a number of conserved plant stress
response mechanisms, which have been demonstrated to confer better adaptation
to a number of different abiotic stresses.
2.2 Water Scarcity and Agriculture
Drought is the most significant environmental stress on world agricultural produc-

tion (Tuberosa and Salvi 2006; Cattivelli et al. 2008) and enormous effort is being
made by plant scientists to improve crop yields in the face of decreasing water
availability. During the twentieth century, the world’s population tripled from
approximately 1.65 to 5.98 billion and population projections of 8.91 and 9.75
billion are expected to occur by 2050 and 2150, respectively. Developing countries
in Africa and Asia account for approximately 80% of this growth and, with an
estimated 800 million people in these countries already undernourished, the Food
and Agriculture Organization (FAO) of the United Nations predicts that a 60%
increase in world food production over the next two decades is required in order to
sustain these populations.
Agriculture accounts for approximately 70% of global water use and irrigation
accounts for up to 90% of total water withdrawals in arid nations (World Water
2 Transgenic Plants for Abiotic Stress Resistance 69
Council 2008; FAO 2009a). Approximately, 40% of all crops produced in develop-
ing countries are grown on irrigated arable land, which accounts for only 20% of the
total arable land in these nations (FAO 2009c). The water withdrawal requirement
for irrigation is expected to increase by 14% in developing countries by 2030 and
strategies to decrease this demand by developing crops that require less irrigation
will, therefore, play a vital role in maintaining world food supply.
The area of plant drought tolerance research and improvement encompasses an
enormous range of environmental, genetic, metabolic, and physiological considera-
tions and an exhaustive discussion of all available avenues for developing drought-
resistant crop varieties is beyond the scope of this chapter. Rather, this section
attempts to provide an overview of some of the genetic mechanisms that have been
manipulated in order to develop transgenic crops with improved drought tolerance
and focuses on research that has involved long-term and field-based drought stress
treatments performed on agronomic and horticultural crop species at yield deter-
mining plant life stages.
2.2.1 Improving Drought Tolerance in Agricultural Crops
All plants require water to complete their life cycle, with most plant cells consisting
of at least 70% water on a fresh weight basis. When insufficient water is available,
plant water status is disrupted, which causes imbalances in osmotic and ionic
homeostasis, loss of cell turgidity, and damage to functional and structural cellular
proteins and membranes. Consequently, water-stressed plants wilt, lose photosyn-
thetic capacity, and are unable to sequester assimilates into the appropriate plant
organs. Severe drought conditions result in reduced yield and plant death.
The overall aim of genetically improving crops for drought resistance is to
develop plants able to obtain water and use it to produce sufficient yields for
human needs under drought conditions. While advances have been made in devel-
oping crops that are genetically improved with traits such as herbicide and pesticide
resistance, attempts to improve plant drought resistance have been hindered by the
complexity of plant drought resistance mechanisms at the whole plant, cellular,
metabolic, and genetic levels. Interactions between these mechanisms and the
complex nature of drought itself, adds another layer of intricacy to this problem.
2.2.2 Complexity of Drought and Plant Responses

to Drought Stress
Drought (nonavailability of water for crop growth) and water deficit (insufficient
plant water status) are variable, complex, and recurring features in most parts of the
world. Even in areas with very high precipitation, many crops are likely to experi-
ence a certain level of water deficit at some stage during the growing cycle.
Elucidation of plant drought resistance and response mechanisms has been
compounded by the variable levels and forms of drought. Drought can be spatially
and temporally variable; terminal, short-term, or sporadic; severe, moderate, or
minor; and can occur at rates ranging from very sudden to gradual. In addition, the
effects of drought and water deficit on crop productivity vary for different crops,
macro- and microenvironments across a single field, plant life stages, and the plant
material to be harvested. Furthermore, the effects of drought on crop productivity
are often compounded by associated stresses such as heat, salt, and nutrient stress.
2.2.3 Plant Drought Resistance and Response
Plant drought resistance mechanisms can be broadly grouped into avoidance or

tolerance mechanisms. Drought avoidance mechanisms are associated with physi-
ological whole-plant mechanisms such as canopy resistance and leaf area reduction
(which decrease radiation adsorption and transpiration), stomatal closure and cutic-
ular wax formation (which reduce water loss), and adjustments to sink-source
allocations through altering root depth and density, root hair development, and
root hydraulic conductance (Beard and Sifers 1997; Rivero et al. 2007).
Drought tolerance mechanisms are generally those that occur at the cellular and
metabolic level. These mechanisms are primarily involved in turgor maintenance,
protoplasmic resistance, and dormancy (Beard and Sifers 1997). Plants respond to
water-limiting conditions by altering the expression of a complex array of genes
(Fig. 2.1) and, although elucidation of biochemical pathways associated with many
of these genes has been the focus of an enormous amount of research over the last
two to three decades, the mechanisms by which these genes and their products
interact remains relatively poorly understood.
Abiotic stress leads to a series of morphological, physiological, biochemical,
and molecular changes that adversely affect plant growth and productivity (Wang
et al. 2001). Drought, salinity, extreme temperatures, and oxidative stress are
often interconnected, and may induce similar cellular damage. For example,
drought and/or salinization are manifested primarily as osmotic stress, resulting
in the disruption of homeostasis and ion distribution in the cell (Serrano et al.
1999; Zhu 2001). Oxidative stress, which frequently accompanies high tempera-
ture, salinity, or drought stress, may cause denaturation of functional and struc-
tural proteins (Smirnoff 1998). As a consequence, these diverse environmental
stresses often activate similar cell signaling pathways (Knight 2000; Shinozaki
and Yamaguchi-Shinozaki 2000; Zhu 2001, 2002) and cellular responses, such as
the production of stress proteins, upregulation of antioxidants and accumulation of
compatible solutes (Vierling and Kimpel 1992; Zhu et al. 1997; Cushman and
Bohnert 2000; Wang et al. 2003b).
Oxidative stress
Environmental
Osmotic and
Drought
Stimulus Disrupted osmotic/ionic
Cold
homeostatis
Salinity
Damaged proteins and
Heat
membranes
Pollution
Signal sensing,
Osmosensors
transduction
perception,
phospholipid cleaving enzymes

second messengers
Ca2+ sensors
MAP kinases
calcium-dependent protien kinases
Transcriptional
control
Transcription
factors
Chaperone
Detoxification
Stress responsive
functions
mechanisms
Gene
activation
Water and ion

Osmoprotection
movement
resistance
tolerance/
Stress
Re-established homeostasis
membrane/protein protection
Fig. 2.1 Plant responses to abiotic stress

2.2.4 The Genetic Basis of Drought Tolerance
Expression studies have shown that drought-specific genes can be grouped into
three major categories: (1) Genes involved in signal transduction pathways (STPs)
and transcriptional control; (2) Genes with membrane and protein protection func-
tions; and (3) Genes assisting with water and ion uptake and transport (Vierling 1991;
Ingram and Bartels 1996; Smirnoff 1998; Shinozaki and Yamaguchi-Shinozaki
2000).
Plants adapt to drought conditions by tightly regulating specific sets of these
genes in response to drought stress signals, which vary depending on factors such as
the severity of drought conditions, other environmental factors, and the plant
species (Wang et al. 2003b). To date, successes in genetic improvement of drought
resistance have involved manipulation of a single or a few genes involved in
signaling/regulatory pathways or that encode enzymes involved in these pathways
(such as osmolytes/compatible solutes, antioxidants, molecular chaperones/osmo-
protectants, and water and ion transporters; Wang et al. 2003b). The disadvantage
of this is that there are numerous interacting genes involved, and efforts to improve
crop drought tolerance through manipulation of one or a few of them is often
associated with other, often undesirable, pleiotropic and phenotypic alterations
(Wang et al. 2003b). These complex considerations, when coupled with the com-
plexity of drought and the plant–environment interactions occurring at all levels of
plant response to water deficit, illustrate that the task plant researchers are faced
with in engineering drought resistant crops is dauntingly multi-faceted and
extremely difficult.
2.2.5 Engineering Improved Drought Avoidance in Crops
Most transformation studies to improve plant drought resistance have produced

transformants that display a variety of both tolerance and avoidance traits. An
exception was demonstrated by Rivero et al. (2007) who manipulated a leaf
senescence gene. Leaf senescence is an avoidance strategy and is accelerated in
drought-sensitive plants to decrease canopy size. In crop plants, accelerated senes-
cence is often associated with reduced yield and is thought to be the result of an
inappropriately activated cell death program. Therefore, suppression of drought-
induced leaf senescence in tobacco plants was investigated as a tool to enhance
drought resistance. Transgenic plants were developed by expressing isopentyl
transferase (IPT), a key enzyme in the biosynthesis of cytokinin (a leaf senescence
inhibitor) under the control of the senescence-associated receptor protein kinase
promoter (PSARK). The SARK gene, which is induced during late maturation and
drought and decreased during senescence development, encodes a maturation/-
sensescence-dependent protein kinase. Although transgenic plants wilted under a
15-day glasshouse drought stress treatment, senescence did not occur and a reduced
photosynthetic capacity was maintained. Following rewatering, transgenic plants

recovered full leaf turgor and resumed growth and maximum photosynthetic
capacity, while the control plants were unable to recover from the drought stress.
Water use efficiency (WUE) of the transgenic plants was also markedly higher than
wild-type (WT) plants, and was two to three times higher after rewatering than
before the drought treatment. An experiment to assess whether the transgenic plants
could produce significant yields under water-limiting conditions determined that
biomass and seed yield were not as affected in transgenic plants than in WT
controls, although this result was not significant (Rivero et al. 2007).
Other drought avoidance/whole-plant traits that have been investigated include
stay-green and cuticular biosynthesis. Stay-green is a variable and quantitative
trait, which generally refers to delayed senescence. It has not yet been used
to successfully produce transgenic plants with increased drought resistance in
the field. Cuticular biosynthesis was investigated by transgenic expression of
AtMYB41, which encodes an R2R3-MYB transcription factor (TF) in Arabidopsis.
AtMYB41 is expressed at high levels in response to drought, abscisic acid (ABA;
Sect. 2.2.6.1), and salt treatments, and was demonstrated to have a role in cell
expansion and cuticle deposition. The transformation of Arabidopsis with AtMYB41
was associated with undesirable pleiotropic phenotypes including dwarfism,
enhanced sensitivity to desiccation, and enhanced permeability of leaf surfaces
(Cominelli et al. 2008).
2.2.6 Improving Plant Drought Tolerance
2.2.6.1 Absicisic Acid and Transcriptional Regulation
The plant hormone ABA regulates the plant’s adaptive response to environmental
stresses such as drought, salinity, and chilling via diverse physiological and devel-
opmental processes. ABA has functional roles ranging from seed maturation
processes to lateral root development (McCourt and Creelman 2008; Wasilewska
et al. 2008). Under abiotic stress, ABA induces stomatal closure, reduces water loss
via transpiration, and induces gene expression (Chandler and Robertson 1994).
Gene expression and biochemical studies into ABA synthesis in Arabidopsis and
some other model plants have largely elucidated the basic ABA biosynthetic
pathway (Schwartz et al. 2003) and many of the key enzymes involved in ABA
synthesis have been investigated transgenically in relation to improving plant
drought tolerance. For example, transgenic Arabidopsis plants constitutively over-
expressing the zeaxanthin epoxidase gene, AtZEP, which encodes an enzyme
required for an initial step in ABA synthesis from isopentyl diphosphate (IPP)
and b-carotene (Schwartz et al. 2003) showed increased tolerance to drought and
salinity stress. The increased drought stress tolerance was attributed to increased
leaf and lateral root development, longer primary roots, higher fresh weight, and
increased survival compared with control plants following drought treatment.
Furthermore, compared with WT plants, the rate of water loss was lower, the levels
of ABA were higher, the expression of stress responsive genes such as Rd29a was
much higher, and stomatal aperture was smaller under salt and/or drought stress.
Overall, the increased stress resistance was attributed to increased ABA levels
in response to osmotic stress, which resulted in enhanced expression of ABA-
responsive stress-related genes (Park et al. 2008).
Many of the drought stress response pathways that have been identified to date
appear to be under transcriptional regulation and ABA plays a key role in this
process (Fig. 2.2). Transcriptional regulation involves interaction between TFs and
specific cis-acting elements located within or near the promoter region upstream of
expressed genes. Figure 2.2 shows links between responses to low temperature and
dehydration stress at the transcriptional level. It can be seen that ABA is involved in
both types of abiotic stress.
Many transcriptional responses to drought stress have been well characterized
and are classified as being ABA-dependent, ABA-independent, or both. ABA is
Low Dehydration
Temperature
ABA independent ABA dependent
CBF1, 2, 3 /
DREB1a, b, c
Zn MYC/
DREB2 CBF4 bZIP
finger MYB
DREB2
AREB/ABF
CRT/DRE ABRE MYCR/MYBR

(Rd29a, Cor15a) (Rd29a, Rd29b) (Rd22, AtADH1)
Fig. 2.2 Plant transcriptional processes induced by dehydration and low temperature stress.
Displayed are transcription factors (rounded rectangles) both ABA-dependent (shaded) and
ABA-independent (unshaded), posttranscriptional modification such as phosphorylation (elipses),
transcription factor binding sites and representative promotors (rectangles), possible regulation
points (dotted arrows), and possible cross-talk (bidirectional arrows)
often used in stress and stress acclimation studies because it is produced in response
to stress. ABA induces expression of many genes that are also induced by drought,
cold, and salinity when applied exogenously (Shinozaki and Yamaguchi-Shinozaki
1996). There are two types of ABA-dependent transcription. The “direct” pathway
involves cis-acting ABA-responsive elements (ABREs), which are directly acti-
vated by binding with TFs such as basic-domain leucine zipper (bZIP)-type DNA-
binding proteins (Shinozaki and Yamaguchi-Shinozaki 1996; Kobayashi et al.
2008). Alternatively, the “indirect” ABA-dependent transcription pathway involves
other cis-acting elements, such as MYC and MYB. These elements are activated
through binding with ABA- or drought-inducible TFs, such as basic helix–loop–
helix (bHLH)-related protein AtMYC2 and an MYB-related protein, AtMYB2
(Abe et al. 2003). An example of the indirect pathway can be seen in the expression
of rd22 from Arabidopsis (Shinozaki and Yamaguchi-Shinozaki 1996).
Some genes are induced by drought stress but are not expressed in response to
exogenous ABA applications and these genes are the product of ABA-independent
STPs. One such gene is rd29a (also known as lti78 and cor78). Yamaguchi-
Shinozaki and Shinozaki (Yamaguchi-Shinozaki and Shinozaki 1994) identified a
dehydration-responsive element (DRE) in the promoter region of rd29a and the
DRE-binding (DREB) protein transcription pathway has since been explored for its
important roles in drought, cold, and salinity stress (Shinozaki and Yamaguchi-
Shinozaki 1996; Qin et al. 2007). Several C-repeat (CRT) binding factor (CBF)/
DREB proteins have now been identified from the promoter regions of other stress-
inducible Arabidopsis genes, such as cor15a, kin1, cor6.6 and cor47/rd17, and the
CBF/DREB pathway has been shown to be conserved across species (Benedict
et al. 2006; Pasquali et al. 2008). CBF/DREB1 and DREB2, belong to the ethylene-
responsive element/apetela 2 (ERE/AP2) TF family; their expression is induced by
cold or drought stress and both activate expression of genes possessing a CRT/DRE
cis-element (Stockinger et al. 1997; Liu et al. 1998). Likewise, DREB2A positively
regulates expression of many abiotic stress-related genes possessing DRE
sequences in their 5’-upstream regions. DREB2A overexpression in Arabidopsis
confers significant drought tolerance in transgenic plants (Sakuma et al. 2006a, b).
DREB genes have been used in transformation of several crops, including wheat
and rice, in attempts to increase drought tolerance (Chen et al. 2008; Kobayashi
et al. 2008).
Although DREs are cis-acting elements that were first thought to activate
ABA-independent stress-responsive gene expression, some are also implicated in
ABA-dependent expression (Shinozaki and Yamaguchi-Shinozaki 2000). CBF4 is
an apparent homolog of the CBF/DREB1 proteins that is thought to be a critical
regulator of gene expression in drought stress signal transduction. The action of
CBF4 is thought to be through its binding with CRT/DRE elements in promoter
regions of drought- and cold-inducible genes (Haake et al. 2002). CBF4 gene
expression has been shown to be upregulated in response to drought and ABA;
however, constitutive expression of CBF4 was found to result in expression of
cold- and drought-induced genes under nonstress conditions and this was associated
with retarded growth, shorter petioles, darker green leaves, and delayed time to
flowering in Arabidopsis seedlings (Haake et al. 2002). Another study showed that
CBF4 expression was induced by salt, but not by drought, cold, or ABA (Sakuma
et al. 2002). Similar observations, and observations of higher levels of soluble
sugars and proline, have been recorded during many CBF overexpression studies,
which suggest that the use of constitutively expressed CBF/DREB genes may not be
applicable to the development of crops with improved drought tolerance. It is
thought that the use of stress-inducible promoters that have low expression levels
under non-stress conditions could be used in conjunction with CBF genes to
alleviate the retarded growth observed in CBF overexpression studies (Zhang
et al. 2004).
Many studies have illustrated the potential of manipulating CBF/DREB genes
to confer improved drought tolerance. For example, overexpression of CBF1/
DREB1B from Arabidopsis was able to improve tolerance to water-deficit stress in
tomato. Furthermore, when driven by three copies of an ABA-responsive complex
(ABRC1) from the barley HAV22 gene, transgenic tomato plants expressing CBF1
exhibited enhanced tolerance to chilling, water deficit, and salt stress, and main-
tained normal growth and yield under normal growing conditions when compared
with control plants (Lee et al. 2003a). Other studies have also found that expression
of CBF/DREB genes under stress-inducible promoters result in transgenic plants
that do not express detectable levels of these genes under non-stress conditions,
minimizing growth retardation and other adverse effects (Al-Abed et al. 2007).
The CRT/DRE motif also acts as one of the binding sites for the ERF family of
TFs (Trujillo et al. 2008). A novel ERF from sugarcane, SodERF3, was found to
enhance salt and drought tolerance when overexpressed in tobacco plants. Under
drought treatment, transgenic plants were significantly taller than controls and
were able to flower under an extended growth period. Furthermore, the absence
of observable differences in height, number of leaves, leaf area, leaf weight, and
stalk weight between transgenic and control plants illustrates that this gene has
potential for engineering drought stress tolerance in plants (Trujillo et al. 2008).
Other TFs involved in mediation of ABA-dependent and ABA-independent
signal transduction and gene expression include NAC, WRKY, RING finger, and
zinc-finger TFs (Seki et al. 2003; Zhang et al. 2004; Chen et al. 2006). Nelson et al.
(2007) showed that constitutive expression of a TF from the nuclear factor (NF-Y)
family, AtNF-YB1, which belongs to the CCAAT-binding TF family, improved
performance of Arabidopsis under drought conditions. Consequently, an orthologous
maize TF gene, ZmNF-YB2, was constitutively expressed in maize. Transgenic lines
were exposed to both glasshouse-based and field-based drought stress treatments.
Transgenic lines exhibited less wilting and faster recovery and re-established
growth more rapidly than WT (on average) under glasshouse-based drought treat-
ment. Transgenic lines subjected to field-based drought stress at the late vegetative
stage exhibited superior health, higher chlorophyll indices and photosynthetic rates,
lower leaf temperatures, higher stomatal conductance, and less yield reduction
than WT plants. Furthermore, under favorable conditions, transgenic plants were
greener, flowered 1–3 days earlier, and had slightly compressed internodes. Most
importantly, the stress adaptation response contributed to a yield advantage in
transgenic maize grown within drought environments, suggesting that ZmNF-YB2

has a realistic application for use in commercial agriculture under severe water-
limiting conditions (Nelson et al. 2007).
Another TF that has been manipulated in order to increase plant drought
tolerance is the HARDY (HRD) gene, which has been linked to increased transpira-
tion efficiency related to stomatal adjustment. HRD is an AP2/ERF-like TF isolated
from hrd-dominant (hrd-D) Arabidopsis mutants, which displayed vigorous rooting
and dark green leaves that were smaller and thicker than WT plants. Karaba et al.
(2007) isolated the HRD gene and constitutively expressed it in Arabidopsis under
the control of the cauliflower mosaic virus (CaMV) 35S promoter. The thicker
leaves, higher root density, and increased root strength were associated with
abundant chloroplasts, increased secondary and tertiary root initiation and prolifer-
ation, and extra corticle cell layers and more compact, stele bearing vascular tissue,
respectively. Furthermore, the mutants survived longer periods of drought stress
and could reach full maturity under high levels of salt stress. HRD was also
constitutively expressed in rice and transgenic plants displayed no reduction in
growth, seed yield, or germination, but had significantly increased leaf canopy with
more tillers under normal greenhouse conditions compared with WT controls.
Under drought stress, the transgenic plants were of deeper green color (attributable
to increased number of bundle sheath cells), displayed distinctive drought tolerance
and lower stomatal conductance, had higher net carbon assimilation and photosyn-
thetic rates, and possessed higher root biomass (Karaba et al. 2007).
Recently, a novel drought-tolerant gene, HDG11, which encodes a protein from
the homeodomain (HD)-START TF family (also known as the Class IV HD-leucine
zipper TF family) was identified in Arabidopsis and was found to confer drought
resistance via enhanced root growth and decreased stomatal density when con-
stitutively overexpressed in transgenic tobacco (Yu et al. 2008). The constitutive
expression of the gene was not associated with retarded growth or any other
observable deleterious phenotypic effects and, the gene was also shown to trans-
activate a number of other genes involved in the drought stress response including
ERECTA (Sect. 2.2.6.2.1; Yu et al. 2008). SNAC1 from rice has also been shown to
have trans-activation activity. NAC TFs comprise a large gene family with proteins
exhibiting a highly conserved N-terminal DNA-binding domain and a diversified
C-terminal domain. NAC was derived from the names of the first three described
proteins containing the DNA-binding domain, namely, NAM (no apical meristem),
ATAF1-2, and CUC2 (cup-shaped cotyledon; Souer et al. 1996; Aida et al. 1997).
NAC is a plant-specific TF family with diverse roles in development and stress
regulation. When constitutively overexpressed in rice, SNAC1 was found to signifi-
cantly improve plant resistance to severe drought stress during reproductive and
vegetative growth and was not associated with any negative phenotypic effects or
yield penalty (Hu et al. 2006). Transgenic plants were more sensitive to ABA and
closed more stomata than WT plants but maintained continual photosynthetic
activity. There was no difference between root morphologies of transgenic and
WT plants indicating that the improved drought resistance was not related to
increased root-water uptake.
The WRKY superfamily of plant TFs has a conserved sequence (WRKYGQK)

at their N-terminal ends (Wu et al. 2008b). Transgenic rice seedlings, expressing
OsWRKY11 under the control of a rice heat shock protein (HSP) promoter, HSP101,
were shown to survive longer and lose less water under a short, severe drought
treatment, than WT plants (Wu et al. 2008b). A TFIIIA-type zinc-finger protein
gene, ZFP252, was also found to confer improved drought stress resistance in rice.
Young transgenic rice plants overexpressing ZFP252 survived longer, displayed
less relative electrolyte leakage, and accumulated more compatible osmolytes than
WT plants or plants with ZFP252 knocked out during a 14-day period of drought
stress (Xu et al. 2008a). A salt- and drought-induced RING-finger protein, SDIR1,
was found to confer enhanced drought tolerance to tobacco and rice (Zhang et al.
2008b). Arabidopsis E3 ligase SDIR1 is a positive regulator in ABA signal trans-
duction. Tobacco and rice plants constitutively overexpressing the SDIR1 gene
displayed less leaf wilting and rolling, longer survival, and improved recovery
under drought conditions than control plants. The mechanism of drought tolerance
was thought to be due to decreased stomatal aperture, which increased transpiration
efficiency of transgenic plants.
Some genes have been shown to suppress expression of drought-response
transcription pathways. For example, Jiang et al. (2008) recently characterized
SAZ, an Arabidopsis gene from the SUPERMAN (SUP) family of plant-specific
zinc-finger genes, which encode proteins containing single C2H2-type zinc-finger
motif with a conserved short amino acid sequence and a class II ERF-associated
amphiphilic repression (EAR) motif-like TF domain at the carboxy-terminal region.
SAZ was found to be rapidly downregulated in response to drought and other
abiotic stresses and SAZ gene knockouts resulted in elevated expression of the
ABA-responsive genes rd29B and rab18 under stressed and unstressed condi-
tions. This shows that gene knockouts and gene silencing may also be applicable
to the development of crops with improved drought resistance.
2.2.6.2 Signal Sensing, Perception, and Transduction
Prior to transcriptional activation of genes, drought stress signals are received

and messages conveyed to the appropriate components of the downstream path-
way (Xiong and Ishitani 2006). In general, STPs involve perception of stress by
specific receptor molecules, which vary in identity, structure, perception, signal
relay mechanism, and location within the cell (Xiong and Ishitani 2006). Plant
stress STPs often involve secondary messengers, which may modify signals (often
via reversible protein phosphorylation) prior to conveying them from receptor
molecules to the activators of the appropriate gene expression pathway (Xiong
and Ishitani 2006). Other molecules may also be involved in stress STPs and the
functions of these include recruitment and assembly of signaling complexes,
targeting of signaling molecules, and regulation of signaling molecule lifespan
(Xiong and Ishitani 2006).
The major molecules involved in drought stress signal sensing, perception,

and transduction include receptor molecules/osmosensors, phospholipid-cleaving
enzymes (PLEs), reactive oxygen species (ROS), mitogen-activated protein kinases
(MAPK), and Ca2+ sensors.
Receptor Molecules/Osmosensors
Receptor molecules/osmosensors are the initial stress signal perceivers and

they convey the signal to the appropriate molecule to initiate STPs. On the
basis of analyses of plants and other species, receptor molecules are thought to
include receptor-like kinases, two-component receptors, receptor tyrosine kinases,
G-protein-coupled receptors, iontropic channel-related receptors, histidine kinases,
and nuclear hormone receptors. Receptor molecules that have been identified to
date in plants include: ROP10, a small G protein from the ROP family of Rho
GTPases, that negatively regulates ABA response in Arabidopsis (Zheng et al.
2002); ATHK1, a putative homolog of the yeast SLN1, which is a functional
histidine kinase feeding into the HOG MAPK pathway (Urao et al. 1999; Reiser
et al. 2003); NtC7, a receptor-like membrane protein from tobacco (Tamura et al.
2003); and Cre1, a putative cytokinin sensor and histidine kinase from Arabidopsis
(Reiser et al. 2003).
The ERECTA gene from Arabidopsis is a putative leucine-rich repeat receptor-
like kinase (LRR/RLK). It was the first gene to be shown to act on the coordina-
tion between transpiration and photosynthesis (Masle et al. 2005). ERECTA was
analyzed by its transgenic expression in null-mutants and was shown to have roles
in lowering stomatal conductance, controlling leaf photosynthesis and organogene-
sis, modulation of cell expansion, cell division, cell–cell contact, cell–cell and
tissue–tissue signaling, cell proliferation, and inflorescence differentiation. Owing
to the range of traits attributed to ERECTA expression, ERECTA is thought to act as
a master gene in transpiration regulation (Masle et al. 2005). No known studies
have yet involved transgenic expression of ERECTA in economic crops; however,
initial studies suggest that this gene may be useful in the design of crops with
improved transpiration efficiencies, reduced stomatal limitations, and increased
yield potentials.
Phospholipid-Cleaving Enzymes
PLEs degrade phospholipid membranes, catalyzing the release of lipid and lipid-
derived secondary messengers (Chapman 1998; Sang et al. 2001). Phospholipases
C (PLC) and D (PLD) are both involved in ABA-mediated signal transduction and
drought stress tolerance perception in plants. Phosphatidic acid (PtdOH), a product
of the PLC and PLD pathways, is also important in the signaling process (Bartels
et al. 2007; Wang et al. 2008a).
Wang et al. (2008a) successfully produced maize plants constitutively over-

expressing ZmPLC1, a phospholipase catalyzing the hydrolysis of 4,5-bisphosphate
to form diacylglycerol (DAG) and inositol 1,4,5-trisphosphate, the products of
which are the second messengers Ca2+ and PtdOH, respectively. This pathway is
important in a wide variety of abiotic stress-responsive processes. Transgenic maize
plants carrying the ZmPLC1 gene were shown to have increased photosynthetic
activity, reduced anthesis to silking interval (ASI; an indicator of maize yield
potential), better recovery, and higher grain yield than WT plants when subjected
to 21 days of drought stress at the ten-leaf stage. Because there was no significant
difference between stomatal conductances of WT and transgenic plants, the higher
photosynthetic rate was attributed to better photochemical activity rather than the
improved guard cell signaling. This has also been demonstrated in other studies
(Staxen et al. 1999; Hunt et al. 2003; Mills et al. 2004). Sang et al. (2001) showed
that overexpression of PLD results in enhanced sensitivity of transgenic tobacco
and plays a key role in controlling stomatal movements and plant response to water
stress.
Reactive Oxygen Species
ROS are generated in plants as photoreaction and cellular oxidation byproducts

under normal conditions and can cause cellular damage under water deficit when
they accumulate to toxic levels. Some of these species also have important roles in
early stress response through activation of cellular defense mechanisms and miti-
gation of cellular damage. While plant mechanisms must be in place to detoxify
high levels of ROS that occur under drought, low levels of these beneficial ROS
must also be maintained. Those ROS known to have important signaling roles in
plant stress STPs include nitric oxide (NO) and hydrogen peroxide (H2O2).
Mitogen-Activated Protein Kinases
MAPKs are enzymes that catalyze reversible phosphorylations, important for

relaying signals. They function via cascades, which involve sequential phosphory-
lation of a kinase by its upstream kinase (Xiong and Ishitani 2006). Recently, the
MKK2 pathway was identified in Arabidopsis as having involvement in cold and
osmotic stress signal transduction. An example of a MAPK having specific involve-
ment in drought and salt stress is the p44MMK4 kinase from alfalfa (Medicago
sativa; Jonak et al. 1996). Phosphatases involved in the sequential phosphorylation
of MAPKs and other protein kinases are also important for stress signaling. For
example, the ABI1 and AB12 proteins from Arabidopsis have been shown to act in
a negative regulatory feedback loop of the ABA signaling pathway (Merlot et al.
2001). Some MAPK and MAPKK proteins have also been shown to activate the
Rd29a stress pathway in Arabidopsis (Hua et al. 2006). Other protein kinases
involved in stress signaling include calcium-dependent protein kinases (CDPKs),
kinases from the SNF1 family of protein kinases, and serine-threonine-type protein
kinases (Xiong and Ishitani 2006; Bartels et al. 2007).
Ca2+ Sensors
Ca2+ sensors are important for coupling extracellular signaling to intercellular

responses and comprise calmodulin (CaM) and CaM-related proteins (Sneddon
and Fromm 1998; Sneddon and Fromm 2001), calcineurin B-like proteins (CBL;
also known as SCaBP/SOS3-like calcium-binding proteins; Kudla et al. 1999), and
CDPKs (Harmon et al. 2000). Ca2+ sensors that have been attributed with roles in
drought tolerance in plants include the CBL1 gene (Kudla et al. 1999) and the
AtCAMBP25 protein (Perruc et al. 2004) from Arabidopsis.
2.2.6.3 Stress-Responsive Mechanisms
The outcome of stress signal perception, transduction, and transcriptional up- or

downregulation of genes is the production of molecules with various plant protec-
tion, repair, and stabilization functions. These molecules can be broadly grouped
into five functional groups: (1) detoxification; (2) chaperoning; (3) late embryogen-
esis abundant (LEA) protein functions; (4) osmoprotection; and (5) water and ion
movement.
Detoxification
To prevent stress injury, cellular ROS need to remain at nontoxic levels under
drought stress. Antioxidants involved in plant strategies to degrade ROS include:
(1) enzymes such as catalase, superoxide dismutase (SOD), ascorbate peroxidase
(APX), and glutathione reductase; and (2) nonenzymes such as ascorbate, glutathi-
one, carotenoids, and anthocyanins (Wang et al. 2003b). Some proteins, osmolytes,
and amphiphilic molecules also have antioxidative functionality (Bowler et al.
1992; Noctor and Foyer 1998).
Chaperoning
Chaperone functions involve specific stress-associated proteins, which are respon-

sible for protein synthesis, targeting, maturation and degradation, and function in
protein and membrane stabilization, and protein renaturation. HSPs, which can be
divided into five conserved families, have been shown to have particularly impor-
tant stress-related chaperone functions in plants (Hendrick and Hartl 1993; Boston
et al. 1996; Hartl 1996; Waters et al. 1996; Torok et al. 2001). HSPs, which are
induced by heat, have been implicated in plant cell protection mechanisms under
drought stress. Protein denaturation occurs under drought stress because decreased
cellular volume increases the likelihood of degradative molecular interactions (Cho

and Hong 2006). HSPs maintain or repair companion protein structure and target
incorrectly aggregated and non-native proteins for degradation and removal from
cells (Cho and Hong 2006). One such protein, NtHSP70-1, was constitutively
overexpressed in tobacco to ascertain its role in plant drought response and toler-
ance (Cho and Hong 2006). The drought tolerance of transgenic seedlings was
increased and their optimum water content was maintained after progressive
drought stress (Cho and Hong 2006). Few other studies have involved transforming
plants with HSPs; however, HSP24 from Trichederma harzianum was found to
confer significantly higher resistance to salt, drought, and heat stress when consti-
tutively expressed in Saccharomyces cerevisiae (Liming et al. 2008).
Late Embryogenesis Abundant Protein Functions
LEA proteins are produced in response to dehydration stress and function in water
status stabilization, protection of cytosolic structures, ion sequestration, protein
renaturation, transport of nuclear targeted proteins, prevention of membrane leak-
age, and membrane and protein stabilization. LEA and LEA-type genes are found
universally in plants. They accumulate in seeds during the late stages of embryo-
genesis and are associated with the acquisition of desiccation tolerance under
drought, heat, cold, salt, and ABA stress (Sivamani et al. 2000; Bartels et al.
2007). They are also present in the biomass tissue of resurrection plants and are
upregulated in many desiccation-sensitive plants in response to drought stress
(Bartels et al. 2007). LEA proteins are divided into groups based on conserved
sequence motifs (Zhang et al. 2000; Wise 2003). Five of these groups have been
characterized at the molecular and structural level (Table 2.1); however, recent
research indicates that additional groups of LEA and LEA-like proteins are still
being identified (Park et al. 2003; Wang et al. 2006; March et al. 2007). Common
Table 2.1 The five groups of LEA proteins

LEA group Description
Group 1 Contain a 20-amino acid motif and are represented by the wheat Em protein, for
which gene homologs have been identified in a wide range of plant species
Group 2 The most extensively studied group. They contain a lysine-rich 15-amino acid
(dehydrins) motif (K-segment; EKKGIMDKIKEKLPG), which is predicted to form an
amphipathic a-helix, a tract of contiguous serine residues and a conserved
motif containing the consensus sequence DEYGNP in the N-terminal section
of the protein
Group 3 Contain a characteristic repeat motif of 11 amino acids, which have been
predicted to form an amphipathic a-helix with possibilities for intra- and
inter-molecular interactions
Group 4 Have a conserved N-terminus, which is proposed to form a-helices and a diverse
C-terminal region with a random coil structure
Group 5 Contain more hydrophobic residues than the other groups, are insoluble after
boiling, and are likely to adopt a globular structure
Source: Bartels and Salamini (2001), Ramanjulu and Bartels (2002)
features of LEA proteins generally include hydrophilicity (Garay-Arroyo et al. 2000;

Park et al. 2003), heat stability (Close and Gallagher-Ludeman 1989; Ceccardi et al.
1994; Houde et al. 1995; Thomashow 1998, 1999), and transcriptionally regulated
and ABA-responsive gene expression (Close and Gallagher-Ludeman 1989). It is
generally assumed that they play a role in water-deficit tolerance and the possible
functions of LEA proteins include binding and replacement of water (Dure 1993), ion
sequestration (Bray 1993), maintenance of protein and membrane structure (Baker
et al. 1988), molecular chaperones (Close 1996), membrane stabilization (Koag et al.
2003), and nuclear transport of specific molecules (Goday et al. 1994). One class of
LEAs, the dehydrins, which have detergent and chaperone-like properties, stabilize
membranes, proteins, and cellular compartments (Close 1996).
LEA genes have been manipulated in many plants in order to increase drought
resistance. For example, a wheat dehydrin, DHN-5, was ectopically overexpressed
in Arabidopsis and transgenic plants displayed superior growth, seed germination
rate, water retention, ion accumulation, more negative water potential, and higher
proline contents than WT plants under salt and/or drought stress (Brini et al. 2007a).
The barley (Hordeum vulgare L.) group 3 LEA gene, HVA1 was constitutively
overexpressed in rice plants to increase drought tolerance. Transgenic plants dis-
played significantly increased tolerance to water deficit and salinity, which was
associated with higher growth rates, delayed onset of stress damage symptoms, and
improved recovery following stress removal (Xu et al. 1996). A more recent study
involving the overexpression of this gene in rice showed that transgenic plants had
significantly higher relative water content (RWC), improved turgor, less reduction
in shoot and root growth, and improved cell membrane stability under prolonged
drought conditions. It was found that HVA1 did not function as an osmolyte and that
membrane protection was the mechanism, which inferred drought resistance in rice
plants (Chandra Babu et al. 2004). HVA1 was also expressed in Basmati rice under
control of either a constitutive rice promoter or a stress-inducible promoter. Trans-
genic plants exhibited increased stress tolerance in terms of cell integrity and
growth, and it was found that inducible expression of HVA1 resulted in transgenic
plants that were able to grow normally under nonstress conditions (Rohila et al.
2002). Transgenic wheat plants expressing HVA1 displayed more root fresh and dry
weights, and shoot dry weight than WT plants under water-deficit conditions
(Sivamani et al. 2000). Similarly, HVA1 overexpressing transgenic mulberry,
Morus indica, exhibited improved cellular membrane stability, photosynthetic
yield, less photo-oxidative damage, and superior WUE than WT plants under salt
and drought stress (Lal et al. 2008). The discussion of expression of HVA1 in
mulberry will be continued in Sect. 2.3.5.
Other group 3 LEA genes that have been manipulated in order to improve plant
drought tolerance include a Brassica napus group 3 LEA gene, which conferred
improved salt and drought tolerance when constitutively expressed in Chinese
cabbage (Park et al. 2005a), and TaLEA3 from wheat, which increased RWC,
leaf water potential, and relative average growth rate of transgenic plants compared
to WT plants under drought stress when constitutively overexpressed in the peren-
nial grass Leymus chinensis (Wang et al. 2008b). Two group 4 LEA proteins,
BhLEA1 and BhLEA2 from the resurrection plant Boea hygrometrica, conferred
improved drought tolerance in transgenic tobacco. This was associated with plant
cell protection and increased membrane and protein stability during dehydration
(Liu et al.). A novel LEA gene from Tamarix androssowii also conferred increased
drought tolerance when expressed in transgenic tobacco (Wang et al. 2006).
Osmoprotection
Osmoprotection involves the upregulation of compatible solutes (osmolytes) that

function primarily to maintain cell turgor, but are also involved in antioxidation and
chaperoning through direct stabilization of membranes and/or proteins (Yancey
et al. 1982; Bohnert and Jensen 1996; Lee et al. 1997; Hare et al. 1998; McNeil et al.
1999; Diamant et al. 2001). Compatible solutes are low molecular weight, highly
soluble compounds that are usually nontoxic at high cellular concentrations. The
three major groups of compatible solutes are amino acids (such as proline), quater-
nary amines (glycine betaine (GlyBet), polyamines, and dimethylsulfonioproprio-
nate), and polyol/sugars (such as mannitol, galactinol, and trehalose; Wang et al.
2003b). Many genes involved in the synthesis of these osmoprotectants have been
explored for their potential in engineering plant abiotic stress tolerance (Vinocur
and Altman 2005).
GlyBet and trehalose act as osmoprotectants by stabilizing quaternary structures
of proteins and highly ordered states of membranes. Mannitol serves as a free-
radical scavenger. Proline serves as a storage sink for carbon and nitrogen and a
free-radical scavenger. It also stabilizes subcellular structures (membranes and
proteins), and buffers cellular redox potential under stress. Many crops lack the
ability to synthesize the special osmoprotectants that are naturally accumulated by
stress tolerant organisms. It is believed that osmoregulation would be the best
strategy for abiotic stress tolerance, especially if osmoregulatory genes could be
triggered in response to drought, salinity, and high temperature. Therefore, a widely
adopted strategy to develop stress-tolerant crops has been to engineer or over-
express certain osmolytes in plants (Bhatnagar-Mathur et al. 2008).
GlyBet is a compatible solute that has been extensively studied for its role in
drought stress response and increasing the levels of GlyBet in plants via genetic
engineering has enhanced the drought tolerance of many model plants (Sakamoto
and Alia 1998; Sakamoto and Murata 2000; Mohanty et al. 2002). A two-step
enzymatic process accomplishes production of GlyBet in plants. The first step
involves conversion of choline to betaine aldehyde by choline monoxygenase
(CMO), a stromal enzyme with a Rieske-type (2Fe-2S) center (Brouquisse et al.
1989), and the second step involves betaine aldehyde dehydrogenase (BADH), a
nuclear-encoded chloroplast stromal enzyme, which converts betaine aldehyde to
GlyBet (Weigel et al. 1986). Quan et al. (2004) reported one of the first attempts
to increase the GlyBet expression levels of maize by overexpressing the betA
gene, which encodes choline dehydrogenase (CHO), another key enzyme in the
choline–betaine aldehyde reaction (Zhang et al. 2008a). The study showed that
transgenic maize plants were more drought tolerant than WT plants at three
different life stages, including the ten-leaf-flowering stage, and also that yields of
transgenic plants were less affected by drought stress than WT.
Tobacco lacks GlyBet; however, it possess some BADH activity and the transfer
of CMO is, therefore, a means of installing the GlyBet pathway in tobacco.
Furthermore, because conversion of choline to GlyBet occurs in the chloroplast,
it is also possible to use chloroplast genetic engineering to transfer CMO into
GlyBet non-accumulators (Zhang et al. 2008a). Zhang et al. (2008a) transformed
tobacco with a gene for CMO from beetroot via chloroplast genetic engineering and
found that the transgenic plants accumulated GlyBet in leaves, roots, and seeds, and
exhibited improved tolerance to toxic choline levels and salt and drought stress.
GlyBet accumulation in the chloroplasts may be more effective than in other
organelles, such as the nucleus, for abiotic stress protection because of protection
and stabilization of chloroplast proteins, membrane, and photosynthesis systems
under stress conditions (Zhang et al. 2008a).
Lv et al. (2007) found that transgenic cotton plants constitutively overexpressing
betA had increased RWCs, increased photosynthesis, better osmotic adjustment,
decreased percentage of ion leakage, decreased lipid membrane peroxidation, and
increased yield in response to drought stress at the seedling, squaring, and anthesis
stages.
Water and Ion Movement
Water and ions move through plants via transcellular and intracellular pathways.
Aquaporins (major intrinsic proteins; MIPs), which are either tonoplast- (TIP) or
plasma membrane- (PIP) localized, facilitate water, glycerol, small molecule, and
gas transfer through membranes and, therefore, have a role in water homeostasis
(Bartels et al. 2007). Active transport of solutes into the cell and cellular organelles,
particularly the vacuole, is another means of cell turgor maintenance as increased
solute potential facilitates the passive movement of water into cells and cellular
compartments (Li et al. 2008).
Successful attempts made in engineering plants expressing genes for enzymes
involved in proton pumps that generate energy for tonoplast transport of solutes
into vacuoles include the overexpression of the Arabidopsis H+-pyrophosphatase
(H+-PPase; AVP1) in Arabidopsis (Gaxiola et al. 2001), upregulation of AVP1 in
tomato (Park et al. 2005c), heterologous expression of the Thellungiella halophila
vacuolar-H+-PPase (V-H+-PPase; TsVP) in tobacco (Gao et al. 2006), and over-
expression of the wheat Na–H+ antiporter, TNHX1, and H+-PPase, TVP1, in
Arabidopsis (Brini et al. 2007b; Li et al. 2008). In all the cases, the transgenic
plants displayed superior drought and/or salinity resistance compared with WT
plants with resistance being attributed to mechanisms such as increased vacuolar
H+ to drive secondary uptake of ions into the vacuole and more enhanced develop-
ment and robustness of root systems (Gaxiola et al. 2001; Li et al. 2005a; Park et al.
2005c; Gao et al. 2006; Brini et al. 2007a, b). Recently, Li et al. (2008) reported that
heterologous expression of the potassium-dependent TsVP gene from the halophyte

T. halophyta in maize under the control of the maize ubiquitin promoter could infer
drought tolerance. Under drought stress, transgenic plants had a higher percentage
of seed germination, better-developed root systems, more biomass, increased solute
accumulation, less cell membrane damage, less growth retardation, shorter ASI,
and much higher grain yields than WT plants.
Attempts have also been made to improve drought tolerance of plants by altering
the expression of aquaporins (Aharon et al. 2003; Porcel et al. 2005; Yu et al. 2005;
Peng et al. 2006; Jang et al. 2007; Cui et al. 2008; Miyazawa et al. 2008; Zhang et al.
2008c). Aquaporins facilitate transport of water and other small solutes and ions
across membranes via the apoplastic route (Aharon et al. 2003; Cui et al. 2008;
Jang et al. 2007; Peng et al. 2006; Zhang et al. 2008c). Research into the role of
aquaporins in plant drought tolerance has shown that various aquaporins function
differently depending on the severity and type of stress. For example, some aqua-
porins, such as the Arabidopsis Rd28, and rice RWC3, are upregulated under drought
stress and others, such as NtQP1 and AtPIP1, remain unchanged under drought
stress (Cui et al. 2008). Additionally, some aquaporins genes, such as AtPIP1b have
been shown to diminish the drought tolerance capability of some plants, while
others, such as the Vicia faba PIP1, Panax ginseng PgTIP1, Brassica napus
BnPIP1, and Brassica juncea BjPIP1, have been shown to improve drought toler-
ance (Aharon et al. 2003; Yu et al. 2005; Peng et al. 2006; Cui et al. 2008; Zhang
et al. 2008c). There is also evidence that overexpression of aquaporins in some plants
causes them to respond differently to different stresses. For example, Jang et al.
(2007) found that Arabidopsis and tobacco plants overexpressing Arabiopsis PIP’s
displayed enhanced water flow and improved germination under cold stress, but
exhibited rapid water loss, retarded seedling growth, and inferior germination under
drought conditions. It is therefore thought that different aquaporin isoforms are
associated with different physiological processes and that plants respond to drought
conditions either by increasing aquaporin expression, which facilitates water move-
ment (especially into the tonoplast in order to maintain cell-turgor) or downregulat-
ing aquaporin expression to avoid excessive water loss (Aharon et al. 2003; Peng
et al. 2006). Overexpression of aquaporins has also been implicated in conferring
heavy metal tolerance to transgenic plants by alleviation of metal ion-induced water
deficit and oxidative damage caused by metal ions (Zhang et al. 2008c).
2.3 Engineering Salt Tolerance in Plants
2.3.1 Impacts of Salinity on Agricultural Production
The damaging effects of salt accumulation in agricultural soils have severely

affected agricultural productivity in large swathes of arable land throughout the
world. Salt-affected land accounts for more than 6% of the world’s total land area
(FAO 2009c) and is distributed largely amongst coastal salt marshes or inland
desert sands. These have primarily arisen naturally through mineral weathering
(which leads to the release of soluble salts such as chlorides of calcium, magnesium
and sodium, and, to a lesser extent, sulfates and carbonates) or wind and rain
deposition of oceanic water (Szabolcs 1989; Munns and Tester 2008; FAO 2009b).
Secondary salinization occurs when irrigation and tree clearing of agricultural
land cause water tables to rise and concentrate salts in the root zone (Rengasamy
2006). Approximately 20% of the world’s irrigated land, from which one-third of
the world’s food supply is produced, is presently affected by salinity (Ghassemi
et al. 1995). With the expected increase in world population, the loss of arable land
due to salinity presents a serious challenge to food sustainability and productivity.
Removal of salts from the root zone (reclamation) is perhaps the most effective
way to ameliorate the detrimental effects of salinity; however, this is a slow and
expensive process. The use of plant breeding and genetic engineering technologies
to alter the salt tolerance of crops will, therefore, play an important role in main-
taining global food production in the future.
2.3.2 Improving Salinity Tolerance of Agricultural Crops
Plants have evolved a complex adaptive capacity to perceive and respond to salt
stress. The existence of salt-tolerant flora (halophytes) and differences in salt
tolerance between genotypes within the salt-sensitive plant species (glycophytes)
give rise to the belief that salt tolerance has a genetic basis (Yamaguchi and
Blumwald 2005).
As for drought, efforts to improve the salt tolerance of crops have met with
limited success because of the physiological and genetic complexity of the trait.
Salinity tolerance is a multi-genic trait, with quantitative trait loci (QTL) identified
in barley, wheat, soybean, citrus, rice, and tomato (Flowers and Flowers 2005;
Jenks et al. 2007). Genetic approaches currently being used to improve salinity
tolerance include the exploitation of functional genomics, bioinformatics, and
natural genetic variations, either through direct selection in stressful environments
or through the mapping of QTLs and subsequent marker-assisted selection
(Yamaguchi and Blumwald 2005), or the generation of transgenic plants (Vij and
Tyagi 2007).
2.3.3 Physiological Effects of Salinity on Plants and Salinity

Tolerance Mechanisms
Salinity imposes a variety of stresses on plant tissues. Two of these are osmotic
stress, which results from the relatively high soil solute concentrations, and ion
cytotoxicity. The decreased rate of leaf growth that occurs after an increase in soil
salinity is primarily due to the osmotic effect of the salt around the roots, which
inhibits plant water uptake and causes leaf cells to lose water. However, this loss of
cell volume and turgor is transient and reductions in cell elongation and also cell
division lead to slower leaf appearance and smaller final size over the longer term
(Bartels and Sunkar 2005; Munns and Tester 2008).
Under prolonged salinity stress, inhibition of lateral shoot development becomes
apparent within weeks and, within months, there are effects on reproductive
development, such as early flowering and reduced floret number. Concomitantly,
older leaves may die while the production of younger leaves continues. The cellular
and metabolic processes involved are similar to those occurring in drought-affected
plants and are responses to the osmotic effect of salt (Yeo et al. 1991; Munns and
Tester 2008).
Ion cytotoxicity occurs when salt accumulates to toxic concentrations in fully
expanded leaves (which, unlike younger leaves, are unable to dilute high salt
concentrations), causing leaf death. Replacement of K+ by Na+ in biochemical
reactions leads to conformational changes and loss of protein function, as Na+ and
Cl– ions penetrate hydration shells and interfere with noncovalent interactions
between amino acids. If the rate of leaf death generated by ion cytotoxicity is
greater than the rate at which new leaves are produced, the photosynthetic capacity
of the plant will no longer be able to supply the carbohydrate requirement of young
leaves, which further reduces their growth rate (Munns and Tester 2008).
Halophytes, though taxonomically widespread, are relatively rare amongst the
flowering plants and virtually all crop plants are glycophytes (Flowers and Flowers
2005). However, there is considerable variability in the tolerance of glycophytes to
salt. Munns and Tester (2008), categorize salinity tolerance under three broad
categories: (1) Tolerance to osmotic stress, which immediately reduces cell expan-
sion in root tips and young leaves, and causes stomatal closure; (2) Na+ exclusion
from leaf blades, which ensures that Na+ remains at nontoxic concentrations within
leaves; and (3) Tissue tolerance to Na+ or Cl–, which requires compartmentalization
of Na+ and Cl– at the cellular and intracellular level to avoid accumulation of toxic
concentrations within the cytoplasm.
2.3.4 Salt Tolerance Using Transgenic Approaches
2.3.4.1 Osmoprotectants
Osmoprotectants were discussed previously (Sect. 2.2.6.3.4) in relation to their use

in developing drought-tolerant crops and the transfer of GlyBet intermediates have
improved the drought and salt tolerance of transgenic plants in many cases.
Mohanty et al. (2002) demonstrated that Agrobacterium-mediated transformation
of an elite indica rice cultivar to increase GlyBet synthesis through the incorpora-
tion of the codA gene, which encodes choline oxidase, was an effective way to
improve salinity tolerance. Challenge studies performed with R1 plants by exposure

to salt stress for one week, followed by a recovery period, revealed that in some
cases more than 50% of the transgenic plants could survive salt stress and set seed
whereas WT plants failed to recover. A more recent example of enhanced GlyBet
synthesis experiments involved transformation of maize with the BADH gene,
introduced by the pollen-tube pathway (Wu et al. 2008a). Transgenic lines were
examined for tolerance to NaCl by induced salt stress and, after 15 days of
treatment, most transgenic seedlings survived and grew well, whereas WT seed-
lings wilted and showed loss of chlorophyll.
The amino acid proline is known to occur widely in higher plants and normally
accumulates in large quantities in response to environmental stresses (Kavi Kishore
et al. 2005; Ashraf and Foolad 2007). In plants, the precursor for proline biosynthe-
sis is l-glutamic acid. Two enzymes, pyrroline-5-carboxylate synthetase (P5CS) and
pyrroline-5-carboxylate reductase (P5CR), play major roles in the proline biosyn-
thetic pathway (Delauney and Verma 1993; Ashraf and Foolad 2007). Su and Wu
(2004) established that the rate of growth of transgenic rice plants expressing
mothbean D1-pyrroline-5-carboxylate synthetase (p5cs) cDNA under either a con-
stitutive or stress-inducible promoter led to the accumulation of p5cs mRNA
and proline in third-generation (R2) transgenic rice seedlings. Significantly higher
salinity and water-deficit stress tolerance of R2 seedlings were attributed to faster
growth of shoots and roots in comparison with non-transformed plants. Stress-
inducible expression of the p5cs transgene showed significant advantages over
constitutive expression in increasing the biomass production of transgenic rice
grown in soil under stress conditions. The osmoprotectant role of proline has been
verified in other plants such as potato, where salt tolerance, measured by comparing
tuber yield of transgenic lines cultivated in a greenhouse and watered with saline
water to that of plants watered with normal tap water, had a less significant effect on
tuber yield of transgenic plants than WT (Hmida-Sayari et al. 2005).
Polyamines, including spermidine (Spd, a triamine), spermine (Spm, a tetra-
mine), and their obligate precursor putrescine (Put, a diamine), are aliphatic amines
widely present in living organisms. The polyamine biosynthetic pathway is
depicted in Fig. 2.3. Recently, it has been demonstrated that plant polyamines are
involved in the acquisition of tolerance to such stresses as high and low tempera-
tures, salinity, hyperosmosis, hypoxia, and atmospheric pollutants (Liu et al. 2007).
Furthermore, genetic transformation of several plant species with polyamine bio-
synthetic genes encoding arginine decarboxylase (ADC), ornithine decarboxylase
(ODC), S-adenosylmethionine decarboxylase (SAMDC), or Spd synthase (SPDS)
led to improved environmental stress tolerance (Liu et al. 2007). He et al. (2008)
tested transgenic apple engineered with (SPDS)-overexpressing transgenic Euro-
pean pear (Pyrus communis L. “Ballad”) for changes in enzymatic and nonenzy-
matic antioxidant capacity in response to NaCl or mannitol stress. Their research
revealed that transgenic plants accumulated more Spd than WT. The transgenic line
contained higher antioxidant enzyme activities (less malondialdehyde and H2O2)
than the WT, implying that it suffered from less injury and enhanced enzymatic and
nonenzymatic antioxidant capacity (He et al. 2008).
Polyamine Biosynthetic Pathway

NH2
O O
N CH3
N
S OH OH
N H2N
N O
NH2 NH2
Ornithine
HO OH Ornithine
–CO2
decarboxylase
S-adensoyl-L-methionine (AdoMet)
–CO2 AdoMet H2N

decarboxylase NH2
Putrecine
NH2 NH2
Spermidine
N CH3 N CH3
N N synthase
+
S S
N N N N
O O
NH2
H2N
N NH2
HO OH HO OH H
Methylthio-Adenosine Decarboxylated AdoMet (dcAdoMet) Spermidine
Spermine
synthase
H2N N
H N NH2
H
Spermine
Fig. 2.3 Polyamine biosynthesis
Mannitol is a primary photosynthetic product that is associated with excep-

tional salt tolerance. In celery, mannitol metabolism is clearly altered by salt
stress, with several lines of evidence indicating a connection between mannitol
and salt tolerance, including increases in the capacity for mannitol biosynthesis
and accumulation and decreases in catabolism (Williamson et al. 2002). Sickler
et al. (2007) showed that Arabidopsis plants transformed with celery’s mannose-
6-phosphate reductase (M6PR) gene produced mannitol and grew normally in the
absence of stress. However, in the presence of salt stress, daily analysis of the
increase in growth (fresh and dry weight, leaf number, leaf area per plant, and
specific leaf weight) over a 12-day period showed less effect of salt on transfor-
mants than WT plants. The daily energy use efficiency for photochemistry by
photosystem 2 (PSII) was also measured and demonstrated that, unlike trans-
formed plants, which were not affected, WT plants treated with 100 mM NaCl
displayed a reduction in PSII yield after 6 days with a 50% loss in yield after
12 days. Similarly, under atmospheric levels of CO2, growth with 200 mM
NaCl caused an increase in sub-stomatal levels of CO2 in WT plants but not in
transformants.
Trehalose is a disaccharide sugar widely distributed in bacteria, fungi, insects,
plants and invertebrate animals. In microbes and yeast, trehalose is produced from
glucose by trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phos-
phatase (TPP), and functions in sugar storage, metabolic regulation, and protection
against abiotic stress (Strom and Kaasen 1993; Wiemken 1990). Trehalose acts as a
compatible solute, protecting membranes and proteins and conferring desiccation
tolerance on cells in the absence of water (Crowe et al. 1984). Ge et al. (2008)
illustrated the protective role of trehalose in higher plants. Expression analysis
demonstrated that OsTPP1 isolated and cloned from rice, was initiated and tran-
siently upregulated after salt, osmotic, and ABA treatments but slowly upregulated
under cold stress. OsTPP1 overexpression in rice enhanced salt and cold stress
tolerance. Tolerance of transgenic plants to abiotic stress was examined by observ-
ing 2-week-old seedlings exposed to salt. Following one week of exposure, seed-
lings exhibited salt-induced damage symptoms such as wilted leaves. However,
after prolonged salt treatment, transgenic lines were more vigorous and displayed
increased leaf greenness and viability over control plants.
Generally, two broad themes have emerged from the results of attempts to
engineer overexpression of osmoprotectants. The first is that metabolic limitations
have been encountered in generating absolute levels of target osmolytes, especially
when compared with salt-tolerant halophytes and the second is that the degree to
which transformed plants are able to tolerate salinity stress is not necessarily
correlative with the levels of osmoprotectants attained.
2.3.4.2 Transporter Genes
Mechanisms that confer salt tolerance vary with the plant species; however, the
ability to maintain low cytosolic Na+ is thought to be one of the key determinants of
plant salt tolerance (Tester and Davenport 2003). Salt “inclusion” and “exclusion”
are recognized as different mechanisms by which higher plants tolerate salinity.
The functional removal of Na+ from the cytoplasm of plant cells and the mainte-
nance of low cytosolic Na+ concentrations under salinity conditions (Blumwald
et al. 2000) is accomplished by either pumping Na+ out of cells (plasma membrane
antiporter) or into vacuoles (vacuolar antiporter) in exchange for H+. Na+/H+
antiporter activity is driven by the electrochemical gradient of protons (H+) gener-
ated by the H+ pumps (H+-ATPase) in the plasma membrane or the tonoplast
(Chinnusamy and Zhu 2003; Tester and Davenport 2003). In Arabidopsis, active
exclusion of Na+ is mediated by the plasma membrane-localized Na+/H+ antiporter,
AtSOS1 (Shi et al. 2003). In contrast, the sequestration of excess Na+ into the
vacuole is mediated by the vacuolar membrane-localized Na+/H+ antiporter,
AtNHX1 (Gaxiola et al. 1999; Shi et al. 2008). In a similar way, overexpression
of the S. cerevisiae HAL1 gene (Gaxiola et al. 1992) conferred salt tolerance in
yeast by increasing intracellular K+ and decreasing Na+ levels.
The successful use of transporter genes has been demonstrated in several plants.
He et al. (2005) created transgenic cotton plants expressing AtNHX1 and found that
transgenic plants generated more biomass and produced more fibers under salt
stress in a greenhouse. It was suggested that the increased fiber yield was due to
superior photosynthetic performance and higher nitrogen assimilation rates
observed in the transgenic plants compared with WT. Interestingly, the researchers
demonstrated that field-grown irrigated AtNHX1-expressing cotton plants produced
higher fiber yields (fiber plus seeds) than WT, with an average increase of more
than 25% per line. Furthermore, the fibers produced by transgenic plants were
generally more uniform, stronger, and longer than those of WT. Similarly, Chen
et al. (2007) engineered maize plants overexpressing the rice OsNHX1 gene.
Transformants accumulated more biomass under greenhouse-based salt stress.
Higher Na+ and K+ content was observed in transgenic leaves than in WT when
treated with 100–200 mM NaCl, while the osmotic potential and the proline content
in transgenic leaves was lower than in WT. Salt stress field trials revealed that
the transgenic maize plants produced higher grain yields than WT plants at the
vegetative growth stage.
Biochemical studies suggest that Na+/H+ exchangers in the plasma membrane of
plant cells contribute to cellular sodium homeostasis by transporting Na+ ions out of
the cell (Qiu et al. 2002). SOS1 encodes a plasma membrane Na+/H+ exchanger in
Arabidopsis (Qiu et al. 2002) and the important role of the plasma membrane
Na+/H+ exchangers for plant salt tolerance was supported by the finding that over-
expression of SOS1 improved plant salt tolerance (Shi et al. 2003). Zhao et al.
(2006) demonstrated that expressing the plasma membrane Na+/H+ antiporter
SOD2 from yeast (Schizosaccharomyces pombe) in transgenic rice also increased
salt tolerance. Transgenic plants accumulated more K+, Ca2+, and Mg2+ and less
Na+ in their shoots compared with non-transformed controls. Moreover, measure-
ments on isolated plasma membrane vesicles derived from the SOD2 transgenic
rice plant roots showed that the vesicles had enhanced P-ATPase hydrolytic activity
as well as being able to maintain higher levels of photosynthesis and root proton
exportation capacity. Martinez-Atienza et al. (2007) identified an AtSOS1 homolog,
OsSOS1, in rice, which demonstrated a capacity for Na+/H+ exchange in plasma
membrane vesicles of yeast (S. cerevisiae) cells and reduced their net cellular Na+
content. OsSOS1 was also shown to suppress the salt sensitivity of an sos1-1 mutant
of Arabidopsis.
In relation to the introduction of genes that modulate cation transport systems,
many researchers have sought to employ the overexpression of the S. cerevisiae
HAL1 gene, which has conferred salt tolerance in yeast by facilitating intracellular
K+ accumulation and decreasing intracellular Na+ (Gaxiola et al. 1992; Rios et al.
1997). Rus et al. (2001) established ectopic expression of HAL1 in transgenic
tomato plants, and showed that transformants were able to minimize the reduction
in fruit production caused by salt stress. Maintenance of fruit production by
transgenic plants was correlated with enhanced growth under salt stress of calli
derived from the plants. The HAL1 transgene enhanced water and K+ contents in
leaf calli and leaves in the presence of salt, which indicates that, similar to the
yeast gene, plant HAL1 functions by facilitating K+/Na+ selectivity under salt
stress. Ellul et al. (2003), utilizing an optimized Agrobacterium-mediated gene
transfer protocol, developed HAL1-expressing watermelon (Citrullus lanatus). Salt
tolerance of transgenic plants was confirmed in a semi-hydroponic system on the
basis of the higher growth performance of transgenic lines compared to control
plants. The halotolerance observed supports the potential usefulness of the HAL1
gene as a molecular tool for genetic engineering salt-stress protection in other
crop species.
2.3.4.3 Detoxifying Genes
The mechanisms of plant detoxification of ROS under drought stress were intro-
duced in Sects. 2.2.6.2 and 2.2.6.2.3. As an antioxidant enzyme, glutathione
peroxidase (GPX) reduces hydroperoxides in the presence of glutathione to protect
cells from oxidative damage, including lipid peroxidation (Maiorino et al. 1995).
Gaber et al. (2006) generated transgenic Arabidopsis plants overexpressing GPX-
2 genes in cytosol (AcGPX2) and chloroplasts (ApGPX2). The activities toward
a-linolenic acid hydroperoxide in ApGPX2- and AcGPX2-expressing plants were
6.5–11.5 and 8.2–16.3 nmol min–1 mg protein–1, respectively, while no activity was
detected in the WT plants. Both transgenic lines showed enhanced tolerance to
oxidative damage caused by the treatment with H2O2, Fe ions, or methylviologen
(MV) and environmental stress conditions, such as chilling with high light intensity,
high salinity or drought. The degree of tolerance of the transgenic plants to all
types of stress was correlated with the levels of lipid peroxide suppressed by the
overexpression of the GPX-2 genes.
SOD is the first enzyme in the enzymatic antioxidative pathway and halophytic
plants, such as mangroves, reported to have a high level of SOD activity. SOD
plays a major role in defending mangrove species against severe abiotic stresses.
Prashanth et al. (2008) further characterized the Sod1 cDNA (a cDNA encoding a
cytosolic copper/zinc SOD from the mangrove plant Avicennia marina) by trans-
forming it into rice. Transgenic plants were more tolerant to MV-mediated oxida-
tive stress in comparison to WT and withstood salinity stress of 150 mM of NaCl for
a period of 8 days while WT plants wilted at the end of the hydroponic stress
treatment. Pot-grown transgenic plants tolerated salinity stress better than the WT
when irrigated with saline water.
In plant cells, APXs are directly involved in catalyzing the reduction of H2O2 to
water, which is facilitated by specific electron donation by ascorbic acid. APXs are
ubiquitous in plant cells and are localized in chloroplasts (Takahiro et al. 1995),
peroxisomes (Shi et al. 2001), and cytosol (Caldwell et al. 1998). Xu et al. (2008b)
transformed Arabidopsis plants with a pAPX gene from barley (HvAPX1). The
transgenic line was found to be more tolerant to salt stress than the WT. There were
no significant differences in Na+, K+, Ca2+, and Mg2+ contents and the ratio of K+ to
Na+ between pAPX3 and WT plants, which indicated that salt tolerance in trans-
genic plants was not due to the maintenance and re-establishment of cellular ion
homeostasis. However, the degree of H2O2 and lipid peroxidation (measured as the
levels of malondialdehyde) accumulation under salt stress was higher in the WT
than in transgenic plants. The mechanism of salt tolerance in transgenic plants was
explained by a reduction of oxidative stress injury.
Apart from catalase and various peroxidases and peroxiredoxins (Dietz 2003),
four enzymes, APX, dehydroascorbate reductase, monodehydroascorbate reductase
and glutathione reductase (GR), are involved in the ascorbate-glutathione cycle, a
pathway that allows the scavenging of superoxide radicals and H2O2 (Asada 1999).
Most of the ascorbate-glutathione cycle enzymes are located in the stroma, cytosol,
mitochondria, and peroxisomes (Jimenez et al. 1998). APX and GR, the first and
last enzymes in this cycle, respectively, are responsible for H2O2 detoxification in
green leaves (Foyer et al. 1994). GR has a central role in maintaining the reduced
glutathione (GSH) pool during stress (Pastori et al. 2000).
Lee and Jo (2004) introduced BcGR1, a Chinese cabbage gene that encodes
cytosolic GR into tobacco plants via Agrobacterium-mediated transformation.
Homozygous lines containing BcGR1 were generated and tested for their acquisi-
tion of increased tolerance to oxidative stress. When ten-day old transgenic tobacco
seedlings were treated with 5 to 20 mM MV, they showed significantly increased
tolerance compared to WT seedlings. The most drastic difference was observed at a
concentration of 10 mM MV. In addition, when leaf discs were subjected to MV, the
transgenic plants were less damaged than the WT with regard to their electrical
conductivity and chlorophyll content.
2.3.5 Late Embryogenesis Abundant (LEA) Proteins
The LEA proteins were introduced in Sect. 2.2.6.3.3 in relation to their use in
improving plant drought tolerance. These proteins have also been used in engineer-
ing salt-tolerant crops. Park et al. (2005b) introduced a B. napus LEA protein
gene, ME-leaN4 (Wakui and Takahata 2002) into lettuce (Lactuca sativa L.)
using Agrobacterium-mediated transformation. Transgenic lettuce demonstrated
enhanced growth ability compared with WT plants under salt- and water-deficit
stress. After 10-day growth under hydroponic 100 mM NaCl conditions, average
plant length and fresh weight of transgenic lettuce were higher than those of WT
and the increased tolerance was also reflected by delayed leaf wilting caused by
water-deficit stress.
Brini et al. (2007a) analyzed the effect of ectopic expression of dehydrin (Dhn-5;
Table 2.1) cDNA in Arabidopsis under salt and osmotic stress. When compared to
WT plants, the Dhn-5-expressing transgenic plants exhibited stronger growth under
high concentrations of NaCl or water deprivation, and showed a faster recovery
from mannitol treatment. Leaf area and seed germination rate decreased much more
in WT than in transgenic plants subjected to salt stress. Moreover, the water
potential was more negative in transgenic than in WT plants and the transgenic
lines had higher proline contents and lower water loss rates under water stress. Na+
and K+ also accumulated to a greater extent in the leaves of the transgenic plants.
Lal et al. (2008) reported the effects of overexpression of the HVA1 gene in
mulberry under a constitutive promoter. HVA1 is a group 3 LEA (Table 2.1)
isolated from barley aleurone layers and has been found to be inducible by ABA.
Transgenic plants were subjected to simulated salinity and drought stress conditions
to study the role of HVA1 in conferring tolerance. Using leaf discs as explants,
growth performance under salt-stress and water-deficit conditions were carried out
from 8- to 10-month-old transgenic plants. Leaf discs of uniform size were cut
and used for simulated salt and water-deficit stress treatments for different dura-
tions. After the stress treatments, leaf discs were analyzed for proline content,
photosynthetic yield, RWC, and cellular membrane stability (CMS). Transgenic

plants showed better CMS, photosynthetic yield, less photooxidative damage, and
better WUE as compared with the nontransgenic plants under both salinity and
drought stress. Under salinity stress, transgenic plants showed a manyfold increase
in proline concentration over WT plants and, under water-deficit conditions, proline
accumulated only in the nontransgenic plants. Results also indicated that the
production of HVA1 proteins enhanced the performance of transgenic mulberry
by protecting plasma and chloroplast membrane stability under abiotic stress.
2.3.6 Transcription Factors
The importance of TFs in plant stress response was discussed in Sect. 2.2.5. In
addition to binding to cis-acting elements in the promoters of environmental stress-
responsive genes, TFs can activate and repress gene expression through interactions
with other TFs, thus playing a central role in plant response to environmental
stresses (Chen and Zhu 2004). It is generally accepted that activation or ectopic
expression of a specific TF can result in expression of many functional genes
related to stresses.
CBF/DREBs are key regulatory factors that function primarily in freezing
tolerance by activating a network of target genes (Fowler and Thomashow 2002;
Maruyama et al. 2004). Oh et al. (2007) isolated a barley gene, HvCBF4, whose
expression is induced by low temperature stress. Transgenic overexpression of
HvCBF4 in rice resulted in an increase in tolerance to drought, high-salinity,
and low temperature stresses without stunting growth. Interestingly, under low
temperature conditions, the maximum photochemical efficiency of PSII in the
dark-adapted state in HvCBF4 plants was higher by 20 and 10% than that in non-
transgenic and CBF3/DREB1A-expressing plants, respectively. Using the 60K Rice
Whole Genome microarray, 15 rice genes were identified that were activated by
HvCBF4. When compared with 12 target rice genes of CBF3/DREB1A, 5 genes
were common to both HvCBF4 and CBF3/DREB1A, and 10 and 7 genes were
specific to HvCBF4 and CBF3/DREB1A, respectively. Results suggested that CBF/
DREBs of barley acted differently from those of Arabidopsis in transgenic rice.
The NAC family of TFs (Sect. 2.2.6.1) has applicability for generating salt-
tolerant crops. Hu et al. (2008) characterized a stress-responsive NAC gene
(SNAC2) isolated from upland rice for its role in stress tolerance. Northern blot
and SNAC2 promoter activity analyses demonstrated that SNAC2 expression was
induced by drought, salinity, cold, wounding and ABA treatment. The SNAC2 gene
was overexpressed in japonica rice to test the effect on improving stress tolerance.
To test salinity tolerance, germinated positive transgenic and WT seeds were
transplanted on Murashige and Skoog (MS) medium containing 150 mM NaCl
and the normal MS medium without NaCl as a control. Under saline conditions,
transgenic seedlings grew faster and their shoots were significantly longer than WT
after 20 days. However, there was no difference in root length or root numbers
between transgenic and WT seedlings grown under saline conditions and no dif-
ference in growth performance was observed between transgenic and WT seedlings
in the normal MS medium. Hu et al. (2008) also evaluated germination ability of
transgenic lines harboring SNAC2 under salt-stress conditions. After 4 days of
germination on the medium containing 150 mM NaCl, only 40% of WT seeds
were poorly germinated, whereas more than 70% of transgenic seeds germinated
efficiently. In the MS medium, more than 90% of both transgenic and WT seeds
germinated well and there was no significant difference in germination rates,
suggesting that overexpression of SNAC2 does not affect seed germination under
normal conditions. The significantly higher germination rate of transgenic seeds
than that of WT under saline conditions further supported the improved salt
tolerance of SNAC2-overexpressing plants. DNA chip profiling analysis of the
transgenic plants revealed many upregulated genes related to stress response and
adaptation such as peroxidase, ornithine aminotransferase, heavy metal-associated
protein, sodium/hydrogen exchanger, HSP, GDSL-like lipase, and phenylalanine
ammonia lyase. This data suggests that SNAC2 is a novel stress-responsive NAC
TF that possesses potential utility in improving stress tolerance of rice.
The TFIIIA-type zinc-finger proteins, first discovered in Xenopus, represent an
important class of eukaryotic TFs (Miller et al. 1985). More than 40 TFIIIA-type
zinc-finger protein genes have been identified from various plants, including petu-
nia, soybean, Arabidopsis, and rice (Kim et al. 2001; Sugano et al. 2003; Mittler
et al. 2006; Huang and Zhang 2007) and these genes have been shown to be induced
by various abiotic stresses. Xu et al. (2008a) have recently reported the functional
analysis of ZFP252 (a salt and drought stress responsive TFIIIA-type zinc-finger
protein gene from rice), using gain- and loss-of-function strategies. They discov-
ered that overexpression of ZFP252 in rice increased the amount of free proline and
soluble sugars, elevated the expression of stress defense genes, and enhanced rice
tolerance to salt and drought stresses compared with ZFP252 antisense and non-
transgenic plants. Their findings suggest that ZFP252 plays an important role in rice
response to salt and drought stresses and is useful in engineering crop plants with
enhanced drought and salt tolerance (Xu et al. 2008a).
2.3.6.1 Signal Transduction Genes
Plant salt-stress-response genes are involved in many plant cellular processes,

including physiological metabolism, cell defense, energy production and transpor-
tation, ion transfer and balance, and cell growth and division. These genes function
through certain coordination mechanisms to maintain the normal growth of plants
under salt stress. As discussed previously, components of the STP may also be
shared by various stress factors such as drought, salt, and cold (Shinozaki and
Yamaguchi-Shinozaki 1999).
Signal molecules, H2O2 and NO, are involved in the ABA-induced stomatal
closure and gene expression and activities of antioxidant enzymes (Zhang et al.
2006, 2007a). ABA-induced H2O2 production mediates NO generation, which in
turn activates MAPK and results in upregulation of the expression and activities of
antioxidant enzymes (Zhang et al. 2007a). The importance of ABA in plant
environmental stress responses was discussed in Sect. 2.2.6.1. The oxidative cleav-
age of cis-epoxycarotenoids by 9-cis-epoxycartenoid dioxygenase (NCED) is the
key regulatory step of ABA biosynthesis in higher plants. Overexpression of
SgNCED1 in transgenic tobacco plants resulted in 51–77% more accumulation of
ABA in leaves (Zhang et al. 2008d). Transgenic tobacco plants were shown to have
decreased stomatal conductance and transpiration and photosynthetic rates and
increased activities of SOD, catalase, and APX activities. H2O2 and NO in leaves
were also induced in the transgenic plants. Compared with WT, the transgenic
plants displayed improved growth under 0.1 M mannitol-induced drought stress and
0.1 M NaCl induced salinity stress. It was suggested that the ABA-induced H2O2
and NO generation upregulates stomatal closure and antioxidant enzymes and,
therefore, increases drought and salinity tolerance in transgenic plants.
Salt stress is known to trigger a rapid and transient increase of free calcium
concentration in plant cells (Knight 2000; Pauly et al. 2000). As such, Ca2+
signaling processes are one of the earliest events in salt signaling and may play
an essential role in the ion homeostasis and salt tolerance in plants ( Zhu 2003;
Reddy and Reddy 2004). CBLs represent a unique family of calcium sensors in
plants and function as a positive regulator in the salt-stress-response pathway.
Extensive studies have progressed toward understanding of Arabidopsis CBLs,
yet knowledge of their functions in other plant species is still quite limited. Wang
et al. (2007a) have reported the cloning and functional characterization of ZmCBL4,
a novel CBL gene from maize. ZmCBL4 encodes a putative homolog of the
Arabidopsis CBL4/SOS3 protein. Under normal conditions, ZmCBL4 was shown
to be expressed differentially at a low level in various organs of maize plants and its
expression was regulated by NaCl, LiCl, ABA and PEG treatments. Expression of
35S::ZmCBL4 not only complemented the salt hypersensitivity in an Arabidopsis
sos3 mutant, but also enhanced the salt tolerance in Arabidopsis WT plants at the
germination and seedling stages. ZmCBL4-expressing Arabidopsis lines accumu-
lated less Na+ and Li+ as compared with WT plants. Wang et al. (2007a) concluded
that the maize CBL gene functions in salt-stress-elicited calcium signaling and thus
in maize salinity tolerance.
2.4 Engineering Cold Tolerance in Plants
2.4.1 Impacts of Cold Stress on Agricultural Production
Agricultural borders for crop species are defined geographically by occurrences of

low temperatures and frost, which cause severe yield losses in marginal areas.
Approximately two-thirds of the world’s landmass is annually subjected to tem-
peratures below freezing and half to temperatures below –20 C.
Most crops of tropical origin, as well as many of subtropical origin, are sensitive
to chilling temperatures. Amongst the major world food crops, maize and rice are
sensitive to chilling temperatures and yield loss or crop failure of these species can
occur at temperatures below 10 C. Many other crops, such as soybean, cotton,
tomato, and banana, are injured at temperatures below 10–15 C (Lynch 1990). The
temperature below which chill injury can occur varies with species and regions of
origin and ranges from 0 to 4 C for temperate fruits, 8 C for subtropical fruits, and
about 12 C for tropical fruits (Lyons 1973).
Cold acclimation, also known as cold hardening, describes an increase in
tolerance over time to cold temperatures and cellular desiccation in response to
conditions such as cold temperature, short photoperiods, and mild drought and
results from changes in gene expression and physiology (Xin and Browse 2000;
Kalberer et al. 2006). Most temperate plants can cold-acclimate and acquire
tolerance to extracellular ice formation in their vegetative tissues. Winter-habit
plants such as winter wheat, barley, oat, rye, and oilseed rape have a vernalization
requirement, which allows them to survive freezing stress as seedlings during
winter. However, after vernalization and at the end of the vegetative phase, the
cold acclimation ability of winter cereals gradually decreases, making them sensi-
tive to freezing injuries (Fowler et al. 1996; Chinnusamy et al. 2007). Therefore, it
is not surprising that the impacts of cold stress on plant life have been comprehen-
sively studied. Many attempts have been made to improve cold resistance of
important crop plants; however, progress in achieving frost hardiness of plants
either by classical breeding or by gene transfer is difficult because of the fact that
cold resistance is not a quality conferred by the product of one gene, but, as for most
abiotic stress tolerance mechanisms, is quantitative in nature (Mahajan and Tuteja
2005).
2.4.2 Physiological Effects of Cold Stress on Plants and Cold

Tolerance Mechanisms
The symptoms of chilling-induced stress injury in cold-sensitive plants are variable

and generally manifest within 48 to 72 h of stress exposure. Observed phenotypic
symptoms in response to chilling stress include reduced leaf expansion, wilting,
chlorosis, and necrosis (Mahajan and Tuteja 2005). Chilling also severely inhibits
plant reproductive development, with species such as rice displaying sterility when
exposed to chilling temperatures during anthesis (Jiang et al. 2002). The extent of
plant damage caused by exposure to low temperature depends on factors such as the
developmental stage, the duration and severity of the frost, the rates of cooling (and
rewarming), and whether ice formation takes place intra- or extra-cellularly (Beck
et al. 2004).
Chilling stress (<20 C) is a direct result of low temperature effects on cellular
macromolecules, which leads to slowing of metabolism, solidification of cell
membranes, and loss of membrane functions. This kind of damage results primarily
from loss of function of biomembranes associated with decreased fluidity and
inactivation or deceleration of membrane-bound ion pumps. Absorbance of light
energy, which occurs independently of temperature, results in oxidative stress
if metabolism cannot keep pace with the excitation of the photosynthetic compo-
nents. Freezing stress (<0 C), which causes extracellular ice-crystal formation,
freeze-induced dehydration, and concentration of cell sap, has major mechanical
impacts on cell walls and plasma membranes and leads to cell rupture (Margesin
et al. 2007).
Generally, freezing results in loss of membrane integrity and solute leakage. The
integrity of intracellular organelles is also disrupted under freezing stress and leads
to loss of compartmentalization and reduction and impaired photosynthesis, protein
assembly, and general metabolic processes. The primary environmental factors
responsible for triggering increased tolerance against freezing are collectively
known as “cold acclimation” (Mahajan and Tuteja 2005).
Frost resistance can be achieved by two main mechanisms: (1) avoidance of ice
formation in tissues; or (2) tolerance of apoplastic extracellular ice. An individual
plant may employ both mechanisms of frost resistance in different tissues (Sakai
and Larcher 1987; Margesin et al. 2007). A key function of cold acclimation is to
stabilize membranes against freezing injury through mechanisms such as adjust-
ment of lipid composition and accumulation of protective sugars, hydrophilic and
LEA proteins, and antioxidants (Thomashow 1999).
2.4.3 Cold Tolerance Using Transgenic Approaches
2.4.3.1 ROS Detoxifying Substances
The mechanisms of ROS accumulation and detoxification were discussed in

Sects. 2.2.6.2 and 2.3.4.3. The decrease in the amount of unsaturated fatty acids
during lipid peroxidation elevates membrane viscosity, promotes lipid transition
from a liquid crystalline phase to a gel phase, raises proton permeability of mem-
branes, diminishes membrane electric conductance, and causes inactivation of
membrane-localized enzymes. The adaptation of plant cells to low temperature is
based on their ability to maintain saturation of fatty acids in membrane lipids, thus
modifying membrane fluidity (Szalontai et al. 2003).
Demin et al. (2008) examined the role of D12-acyl-lipid desaturase in plant
resistance to hypothermia-induced oxidative stress. The study focused on modula-
tion of free-radical processes occurring at low temperature in leaf cells of potato
plants transformed with the gene for D12-acyl-lipid desaturase from cyanobacte-
rium. Plants were grown in vitro on MS agar medium containing 2% sucrose.
During hypothermia (–9 C), the rate of O2– generation and H2O2 concentration
decreased significantly. In addition, the contents of both primary products (conju-
gated dienes and trienes) and secondary products (malonic dialdehyde) of lipid
peroxidation were lower in transformed plant leaves than in leaves of WT plants. It

was hypothesized that insertion of D12-acyl-lipid desaturase into the plant genome
stabilizes the composition and physical properties of biomembranes by promoting
polyunsaturation of fatty acids, which averts the accelerated generation of O2– and
suppresses lipid peroxidation during hypothermia.
Evidence suggests that ROS are the cause of photosystem 1 (PSI) inactivation in
chilling-sensitive plants during cold stress in the light, and that H2O2 accumulation
is a major factor leading to the decline in PSI activity (Sonoike 1996). Cotton
is considered to be a chilling-sensitive species; its photosynthetic performance
and ability to rapidly recover photosynthetic activity following chilling in the
light diminish considerably with time of exposure (Koniger and Winter 1993).
Kornyeyev et al. (2003) compared the photosynthetic performance between leaf
discs of WT cotton and transgenic cotton overexpressing chloroplast stroma-based
APX+ plants during exposure to 10 C and 500 mmol photons m–2 s–1. They showed
that APX+ leaves did not exhibit as large an increase in cellular H2O2 as WT leaves
shortly after the imposition of the chilling treatment. In addition, APX+ leaves
exhibited slightly but significantly less PSI and PSII photoinhibition.
The electron transport chain (ETC) of plant mitochondria has also been
researched in relation to production of freeze-tolerant transgenic plants. Plant
ETCs have unique features compared with other eukaryotes, including the ubiqui-
tous presence of a terminal alternative oxidase (AOX) that competes for electrons
with the standard cytochrome (Cyt) pathway (Finnegan et al. 2004). The mainte-
nance of ETC redox balance is critical because electron input in excess of ETC
capacity can be responsible for the production of ROS, particularly O2– and H2O2. It
has been proposed that AOX may be an important factor in allowing plants to
tolerate chilling-induced ROS damage to membrane phospholipids (Purvis and
Shewfelt 1993). Evidence suggests that the AOX pathway of plant mitochondria
uncouples respiration from mitochondrial ATP production and may ameliorate
plant performance under stressful environmental conditions, such as cold tempera-
tures, by preventing excess accumulation of ROS (Wagner et al. 1998). Fiorani
et al. (2005) tested this model in whole tissues by growing AtAOX1a-transformed
Arabidopsis plants at 12 C. Twenty-one days after sowing, antisense mutants
showed (on average) a 27% reduction in leaf area and 25% smaller rosettes versus
30% increased leaf area and 33% larger rosette size for overexpressing lines
compared with WT plants. These results demonstrate that AOX activity plays a
role in shoot acclimation to low temperature in Arabidopsis.
2.4.3.2 Membrane Modifications
Chilling-resistant plants have a greater abundance of unsaturated fatty acids than

chilling-sensitive plants and it has been shown that the proportion of unsaturated
fatty acids increases during acclimation to cold temperature (Palta et al. 1993). This
modification allows membranes to remain fluid by lowering the temperature
at which the membrane lipids experience a gradual phase change from fluid to
semi-crystalline. Thus, desaturation of fatty acids provides protection against

damage from chilling temperatures (Khodakovskaya et al. 2006). Khodakovskaya
et al. (2006) developed a cold-inducible genetic construct cloned using a chloroplast-
specific omega-3-fatty acid desaturase gene (FAD7) under the control of a cold-
inducible promoter (cor15a) from Arabidopsis and expressed it in young tobacco
plants. When seedlings were exposed to low-temperature (0.5, 2, or 3.5 C)
for up to 44 days, survival within independent cor15a-FAD7 transgenic lines
(40.2%–96%) was far superior to the WT (6.7%–10.2%). In addition, the major
trienoic fatty acid species remained stable in cold-induced cor15a-FAD7 plants
under prolonged cold storage while the levels of hexadecatrienoic acid (16:3)
and octadecatrienoic acid (18:3) declined in WT plants under the same condi-
tions (79 and 20.7%, respectively). Electron microscopy showed that chloroplast
membrane ultrastructure in cor15a-FAD7 transgenic plants was unaffected by
prolonged exposure to cold temperatures. In contrast, WT plants experienced a
loss of granal stacking and disorganization of the thylakoid membrane under the
same conditions.
In higher plants, the most abundant lipids of thylakoid membranes are glycoli-
pids (monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG),
sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG)). PG is the
only phospholipid in thylakoid membranes. The chilling resistance of higher plants
is apparently closely correlated with the level of cis-unsaturated fatty acids in PG
from chloroplast membranes (Nishida and Murata 1996). Chilling-resistant plants
contain a large proportion of cis-unsaturated fatty acids at the sn-1 position of
PG and there are few cis-unsaturated fatty acids in chilling-sensitive plants. The
sn-2 position is occupied mainly by saturated and trans-unsaturated fatty acids
(Bertrams and Heinz 1981) and hence the content of cis-unsaturated fatty acids at
the sn-1 position of PG determines plant chilling resistance. The dominant factor
that determines the level of cis-unsaturated fatty acids in PG is the substrate
selectivity of glycerol-3-phosphate (G3P) acyltransferase (GPAT: EC2.3.1.15) in
chloroplasts, which catalyzes the first step of glycerolipid biosynthesis by transfer-
ring the acyl group of acyl-(acyl carrier protein; ACP) to the sn-1 position of G3P to
yield 1-acylglycerol-3-phosphate (lysophosphatidate; LPA; (Roughan and Slack
1982)). GPAT from chilling-resistant plants prefers oleoyl-ACP (18:1-ACP) to
palmitoyl-ACP (16:0-ACP) as a substrate resulting in a high level of cis-unsaturated
fatty acids in PG. The enzymes from chilling-sensitive plants hardly distinguish
18:1-ACP from 16:0-ACP resulting in a low level of cis-unsaturated fatty acids at
the sn-1 position of PG (Weber et al. 1991). In this way, fatty acids remain
saturated, which renders the plants sensitive to chilling stress. Sui et al. (2007)
isolated a tomato GPAT gene (LeGPAT), which despite the chilling sensitivity
of tomato exhibited selectivity to 18:1 over 16:0. Overexpression of LeGPAT
increased total activity of LeGPAT and cis-unsaturated fatty acids in the thylakoid
membrane. Chilling treatment (4 C for 4 h) induced less ion leakage from the
transgenic plants than from the WT. The photosynthetic rate and the maximal
photochemical efficiency of PSII (Fv/Fm) in transgenic plants decreased more
slowly during chilling stress and recovered faster than WT plants under optimal
conditions. These results indicate that overexpression of LeGPAT increased the

levels of PG cis-unsaturated fatty acids in thylakoid membranes, which was benefi-
cial for the recovery of chilling-induced PSI photoinhibition in tomato.
2.4.3.3 LEA and Chaperoning Modifications
Several proteins are expressed in plants upon exposure to low temperature. These
are either located in the cytosol or secreted to the apoplast. They have various
putative functions, including cryoprotection, altered lipid metabolism, protein
protection, desiccation tolerance, and sugar metabolism (Hiilovaara-Teijo and
Palva 1999; Margesin et al. 2007). During cold acclimation, several stress proteins
that may function as chaperones and membrane stabilizers during freeze dehydra-
tion are expressed in the cytosol (Puhakainen et al. 2004). Of the many low
temperature-responsive genes characterized to date, several have been predicted
to encode proteins with the characteristics of the dehydrin class of LEA proteins
(Table 2.1). Houde et al. (2004) reported an improvement of the selection procedure
and the transfer of the wheat Wcor410a acidic dehydrin gene to strawberry. The
WCOR410 protein was expressed in transgenic strawberry at a level comparable
with that in cold-acclimated wheat. Freezing tests showed that cold-acclimated
transgenic strawberry leaves had a 5 C improvement in freezing tolerance over WT
leaves or transformed leaves not expressing the WCOR410 protein. However, no
difference in freezing tolerance was found between the different plants under
non-acclimated conditions, suggesting that the WCOR410 protein needs to be
activated by another factor induced during cold acclimation. The data demonstrated
that WCOR410 protein prevents membrane injury and greatly improves freezing
tolerance in leaves of transgenic strawberry.
HSPs (Sect. 2.2.6.1) accumulate in response to low temperature. The HS
response in plants has been extensively investigated (Waters et al. 1996). Plants
synthesize predominantly small (15–30 kDa) HSPs (sHSPs) during the heat-shock
response, and it has been suggested that the accumulation of sHSPs is correlated
with thermotolerance (Vierling 1991). Guo et al. (2007), characterized a sweet
pepper cDNA clone, CaHSP26 encoding the chloroplast (CP)-sHSP, with regard to
its sequence, response to various temperatures, and function in transgenic tobacco
plants. Expression of the CaHSP26 gene showed that the mRNA accumulation of
CaHSP26 was induced by heat stress. Higher transcript levels were observed when
sweet pepper leaves were treated at 42 C for 3 h. However, the expression of the
CaHSP26 gene was not induced by chilling stress (4 C) in the absence of HS and
the transcripts were detected at 48 h at 4 C after HS while not at 25 C. The
photochemical efficiency of PSII (Fv/Fm) and the oxidizable P700 in transgenic
tobacco overexpressing CaHSP26 were higher than that in WT tobacco during
chilling stress under low irradiance. These results suggest that the CP sHSP protein
plays an important role in the protection of PSII and PSI during chilling stress under
low irradiance.
2.4.3.4 Osmoprotectants/Compatible Solutes
Long-term acclimation to the cold and winter survival in herbaceous plants is

strongly correlated with the recovery of photosynthesis at low temperature and
the maintenance of soluble carbohydrate reserves (Stitt and Hurry 2002; Strand
et al. 2003), namely, through upregulation of sucrose biosynthesis. Strand et al.
(2003) tested this hypothesis by comparing the acclimation responses of WT
Arabidopsis with transgenic plants overexpressing sucrose phosphate synthase
(over-sps) or with antisense repression of either cytosolic fructose-1,6-bisphospha-
tase (antifbp) or sucrose phosphate synthase (antisps). Plants overexpressing
sucrose phosphate synthase showed improved photosynthesis and increased flux
of fixed carbon into sucrose when shifted to 5 C, whereas both antisense lines
showed reduced flux into soluble sugars relative to WT plants. The improved
photosynthetic performance by the overexpressing sps plants was associated with
an increase in freezing tolerance relative to WT (–9.1 and –7.2 C, respectively). In
contrast, both antisense lines showed impaired development of freezing tolerance
(–5.2 and –5.8 C for antifbp and antisps, respectively). Similarly, metabolic engi-
neering for the biosynthesis of fructans is a potential strategy for increasing water-
stress tolerance. Fructans are a class of water-soluble fructose polymers based
on sucrose, which accumulate in many bacterial and plant species serving as an
important storage carbohydrate and protecting plants against water deficit caused
by low matrix potential, salinity, or low temperatures (Spollen and Nelson 1994). In
plants, fructans are synthesized in vacuoles from sucrose by the action of two or
more different fructosyltransferases, including sucrose:sucrose 1-fructosyltransferase
(1-SST), sucrose:fructan 6-fructosyltransferase (6-SFT), fructan:fructan 1-fructo-
syltransferase (1-FFT), and fructan:fructan 6G-fructosyltransferase (6G-FFT; Vijn
and Smeekens 1999). Kawakami et al. (2008) used rice, which is highly sensitive to
chilling temperatures and is not able to synthesize fructans, to study the effect of
fructan biosynthesis against water stress. Two wheat fructan-synthesizing enzymes,
1-SST, encoded by wft2, or 6-SFT, encoded by wft1, were introduced into rice
plants. The transgenic seedlings with wft2 accumulated significantly higher con-
centrations of oligo- and polysaccharides than nontransgenic rice seedlings, and
exhibited enhanced chilling tolerance (11-day exposure to 5 C). The oligo- and
polysaccharide concentrations of seedlings expressing wft1 were visibly lower than
those of lines expressing wft2, and no correlation between oligo- and polysaccha-
ride concentrations and chilling tolerance was detected in wft1-expressing rice
lines. The results suggest that transgenic rice lines expressing wheat-derived fruc-
tosyltransferase genes accumulated large amounts of fructans in mature leaf blades
and exhibited enhanced chilling tolerance at the seedling stage.
2.4.3.5 Transcription Factors
Cold acclimation in plants is a complex process involving changes in the expression

of numerous cold-responsive (COR) genes (Chinnusamy et al. 2006). This results in
modification of plant cell structural, biochemical, and photosynthetic properties

that facilitate an increase in the plant’s freezing stress tolerance. Regulatory factors
influencing expression of COR genes and/or freezing tolerance have been identified
over the last decade (Chinnusamy et al. 2006). The cold-induced CBF transcrip-
tional regulatory factor CBF1-3 controls the cold-responsive expression of a major
regulon of COR genes that increases plant freezing tolerance (van Buskirk and
Thomashow 2006). Ectopic expression of CBF transgenes under warm conditions
activates a suite of COR genes without cold stimulus, increasing plant freezing
tolerance and inducing biochemical and structural alterations normally observed
during exposure to cold. Pino et al. (2008) studied the effect of ectopic AtCBF
overexpression on physiological alterations that occur during cold exposure in
frost-sensitive (Solanum tuberosum) and frost-tolerant (S. commersonii) potato.
Relative to WT plants, ectopic AtCBF1 overexpression enhanced expression of
COR genes without a cold stimulus in both species and imparted a significant
increase in freezing tolerance gain in both species (2 C in S. tuberosum and up to
4 C in S. commersonii). Transgenic S. commersonii displayed improved cold
acclimation potential, whereas transgenic S. tuberosum was still incapable of cold
acclimation. During cold treatment, leaves of WT S. commersonii showed signifi-
cant thickening resulting from palisade cell lengthening and intercellular space
enlargement, whereas those of S. tuberosum did not. Ectopic AtCBF1 activity
induced these same leaf alterations in the absence of cold in both species. In
transgenic S. commersonii, AtCBF1 activity also mimicked cold treatment by
increasing proline and total sugar contents in the absence of cold. Relative to WT,
transgenic S. commersonii leaves were darker green, had higher chlorophyll and
lower anthocyanin levels, greater stomatal numbers, and displayed greater photo-
synthetic capacity, suggesting higher productivity potential. These results suggest
that an endogenous CBF pathway is involved in many of the structural, biochemical,
and physiological alterations associated with cold acclimation in potato.
The cuticle is one of the most important barriers for all terrestrial plants and
mitigates damage to above-ground biomass caused by low humidity and other
biotic and abiotic stresses (Jenks and Ashworth 1999). The important physiological
and ecological functions of the cuticle (which include control of transpiration and
leaching and facilitation of foliar penetration of pesticides) are related to the
presence of cuticular waxes that are embedded in or deposited on the cutin matrix
(Kunst and Samuels 2003). Increased accumulation of cuticular waxes in leaves has
been achieved through the overexpression of TF genes such as WIN1/SHN1 in
Arabidopsis (Broun et al. 2004) and WXP1 in alfalfa (Zhang et al. 2005). Elevated
leaf cuticular wax deposition led to significant improvement in drought tolerance of
the transgenic plants (Broun et al. 2004; Zhang et al. 2005). Zhang et al. (2007b)
reports the functional characterization of two putative ERF TF genes WXP1 and its
paralog WXP2 from Medicago truncatula. Transgenic expression of WXP1 and
WXP2 in Arabidopsis led to significantly increased cuticular wax deposition on
leaves of 4- and 6-week-old transgenic plants. Differences in the accumulation of
various wax components, as well as their chain length distributions, were found in
the WXP1 and WXP2 plants. Analysis of fresh weight loss from detached leaves
revealed that the transgenic leaves retained more water than controls. Both WXP1
and WXP2 transgenic plants showed significantly enhanced whole-plant drought
tolerance. Analysis of freezing tolerance at the whole-plant level and measurement
of electrolyte leakage from detached leaves revealed that the WXP1 plants had
increased freezing tolerance while the WXP2 plants were more sensitive to low
temperature when compared to the control. Transgenic expression of WXP1 had no
obvious effects on plant growth and development; however, the expression of
WXP2 led to slower plant growth. These results indicate that WXP1 is a useful
candidate gene for improving plant drought and freezing tolerance by genetic
transformation.
2.5 Nutrient Deficiency and Nutrient Use Efficiency
In all, plants require 17 essential elements, 14 of which are taken up in inorganic

forms by the roots. The absence or paucity of any one of these essential elements
will commonly lead to plant death or inability to complete its life cycle. In the
presence of nutrient deficiencies, even at asymptomatic levels, crop performance,
yield, and quality are frequently compromised. Hence, fertilizer and other soil
ameliorations are essential to ensuring food security and the sustainability of agri-
culture, and these add a major economic and potentially environmental burden to
crop production. The ability to improve and enhance the efficiency of nutrient
uptake and utilization in major crop plants has become a major objective in modern
plant improvement. Interestingly, different species have evolved differential mech-
anisms for improving their ability to scavenge essential elements at low concentra-
tions in the soil. In this section, we will discuss the ways in which biotechnological
intervention can be applied to improve the ability of crop plants to survive and
produce yield in nutrient-poor environments. The discussion will focus on the most
limiting nutrient, nitrogen, with some discussion of phosphorus and iron, which are
particularly susceptible to soil pH variation.
2.5.1 Nitrogen
Nitrogen (N) is quantitatively the most important nutrient that plants obtain
from the soil (Paungfoo-Lonhienne et al. 2008), with an estimated 1011 tons of
N fertilizer applied annually worldwide (Lea and Azevedo 2006). The total cost of
this agricultural input exceeds US $50 billion. In general, however, plants are only
able to acquire 30–40% of this applied N, with significant losses occurring through
leaching, denitrification, and the volatilizationz of ammonium to the atmosphere
(Lea and Azevedo 2006).
Nitrogen assimilation is the process by which inorganic N forms (typically
nitrate) are reduced to ammonium, and then converted into organic forms (amino
acids) for transportation and use by the plant. Nitrate is the main form of N taken up
by plants and is reduced in the plant through the combined action of the enzymes,
nitrate reductase (NR) and nitrite reductase. It has long been believed that this step
is the major control point in nitrate assimilation and it has been shown that
expressing a constitutive NR as opposed to a fully regulated NR demonstrates
accumulation of high concentrations of asparagine and glutamine (Good et al. 2004;
Lea and Azevedo 2006).
Inorganic N is then converted into organic amino acids by the GS-GOGAT
cycle, and these amino acids are used to transport N around the plant. The major N
transport amino acids are glutamate, glutamine, aspartate, and asparagine, with
differences in importance among different phyla and also environmental condi-
tions. Ammonium is incorporated into glutamine through the enzyme glutamine
synthetase (GS). This places the ammonium molecule initially into the amide
position of the glutamine molecule from where the N is relocated to the a-amino
position of glutamate through the action of glutamate synthase (GOGAT) (Glass
et al. 2002; Good et al. 2004; Lea and Azevedo 2006). The GS-GOGAT has been
considered a primary target for the manipulation of nitrogen use efficiency (NUE).
NUE is usually described as unit biomass per unit of N present in the plant or
the yield of N in the plant per unit of available N in the soil (Bushoven and Hull
2001; Lea and Azevedo 2006). NUE is dependent on physiological traits such as
uptake and assimilation of N (Good et al. 2004). While there are other processes
that use N in plants, it is these two traits that control NUE. The ability to uptake and
transport N throughout the plant is, along with photosynthetic efficiency and WUE,
one of the major determinants of plant survival, growth, and yield. In addition, the
mobilization of organic N into fruits and seeds is a major determinant of yield and
product quality, especially in cereal and legume grains, where the protein content
determines the nutritional and end-use qualities.
N nutrients are powerful signaling molecules within the plant (Vidal and
Gutierrez 2008). Nitrate controls the expression of thousands of genes involved in
many plant processes, with some genes responding to nanomolar concentrations
(Wang et al. 2003a, 2007b). This includes the nitrate transporter molecules (for an
introduction to transporter genes, see the description in Sect. 2.3.4.2). The nitrate
transporters, NRT1.2 and NRT2.1, are not only involved with N uptake, but they
are also sensor molecules, and have been shown to increase N uptake rate at low
rhizosphere concentration.
It is known that root branching occurs at higher frequency in regions of the
rhizosphere with local N-enrichment (Scott Russell 1977). The transporter NRT1.1
is involved in signaling, which leads to colonization of nitrate-rich patches in the
soil by promoting localized root proliferation in concert with ANR1, a MADS-box
TF gene not yet fully functionally characterized (Remans et al. 2006). Both these
genes have regulatory sequences which direct reporter gene expression in root
primordia and root tips.
Amino acid synthesis and transport genes have been manipulated to alter the
NUE in numerous plants. For example, the overexpression of a bacterial asparagine
synthetase gene in the leaves of lettuce was shown to affect N status (Giannino
et al. 2008). Early vegetative growth rate of these transgenic lettuce plants was
approximately 1.3-fold higher for the first 35 days post-germination, accompanied
by higher leaf number and leaf area. However, these plants also attained reproduc-
tive phase earlier, which was not advantageous for a leaf vegetable. Similar effects
have been reported in transgenic oilseed rape (Seiffert et al. 2004) and Lotus
(Vincent et al. 1997). The transgenics had approximately twofold more aspartate
and asparagine than WT, and interestingly also had elevated levels of glutamine
with no effect on glutamate concentration. Leaf assays also revealed a lower level
of free nitrate in the transgenic lettuce lines, a phenomenon previously reported in
lettuce overexpressing a nitrate reductase gene (Curtis et al. 1999). The authors
suggested that these plants may be suitable for breeding new quick-harvest lettuce
varieties with lower N fertilizer requirement, or enhanced NUE.
2.5.2 Phosphorus
Worldwide, it is estimated that 5.7 billion hectares of land lack sufficient quantities
of plant-available Phosphorus (P) (Batjes 1997). P deprivation in plants has been
widely studied, as P becomes limiting to plant productivity with falling soil pH, and
tends to bind very tightly with soil. In mildly acid soils with pH < 6, soil P rapidly
becomes immobile and forms insoluble compounds, commonly with Fe and Al.
Even in soils with abundant P, usually only about 1% of the soil P is actually in a
readily available, soluble form, and over 90% is generally bound tightly to soil
particles in organic and inorganic forms, which require mineralization before they
become plant available.
P uptake and transporter genes are generally regarded as high or low affinity,
with the high affinity genes becoming more important for scavenging nutrient from
the soil as P becomes limiting. Many Australian soils are particularly P-deficient
and, as a result, many native Australian plants have evolved different mechanisms
to overcome this with high-affinity P transporters, association with vesicular-
arbuscular mycorrhiza, the secretion of organic acids into the rhizosphere, and
the formation of proteoid roots (Lambers et al. 2006).
Attempts to overexpress high affinity P transporters have met with mixed
success. Overexpression of high-affinity transporter genes in barley did not have
any effect on P uptake or plant productivity and growth (Rae et al. 2004), despite
having demonstrated that these genes increased P uptake in transgenic rice callus
cultures. However, the overexpression of homologous high-affinity P transporter
genes in rice enabled the plants to accumulate twice as much P as WT rice, and the
resulting phenotype produced more tillers (Seo et al. 2008). Further research has
also demonstrated that many plant phosphate transporters have complex interac-
tions with vesicular arbuscular mycorrhizae (Glassop et al. 2007).
Plants have been demonstrated to alter the rhizosphere with specific exudates,
commonly organic acids or enzymes, to improve the availability of nutrients such
as phosphate. The exudation into the rhizosphere of acid phoaphatase has led to
improved biomass and P accumulation in Arabidopis (Xiao et al. 2007) and rice
(Park et al. 2007). Similar results have been achieved with the ectopic expression of
phytase in root tissues of potato (Hong et al. 2008).
2.5.3 Iron
Iron is not only an essential plant nutrient, but its deficiency, known as anemia, also
represents the world’s most frequent and debilitating human mineral nutrient
deficiency (Wintergerst et al. 2007). Hence, improving the ability of plants to
uptake, translocate and store iron in bioavailable forms is not only a plant health
and productivity issue, it is a major human and animal nutritional target.
Plants have the ability to sense low-iron conditions, which induces a coordinated
response. The predominant form of iron in aerobic soils is Fe(III), and plants must
possess strategies to utilize this form. Most plants use what has become widely
known as Strategy I, whereby they acidify the rhizosphere with the induction of a
specific proton pump which solubilizes more Fe, and then, by the production of
Fe chelate reductase, convert Fe(III) to Fe(II), which is then transported into the cell
by a membrane-bound Fe(II) transporter.
Grasses and cereals, however, utilize Strategy II, which involves the production
and secretion of a specialized group of chemicals, known as phytosiderophores (PS)
into the rhizosphere. PS are a form of mugineic acids, which form strong chelates
with Fe(III), and help to solubilize them for plant uptake by specialized protein
forms, known as the Yellow Stripe proteins (von Wiren et al. 1994; Curie et al.
2001), which are actually Fe(III) transporter genes.
Interestingly, rice is a special case and appears to be able to utlilize both
strategies of iron uptake. Unusually, among the grasses rice has an efficient Fe(II)
uptake system and does not produce much PS (Mori et al. 1991). Rice has been
engineered to produce larger quantities of PS, and the transgenic lines were
more tolerant to Fe-deficient soils (Suzuki et al. 2008). Other successful approaches
to improve Fe uptake of transgenic rice in alkaline soils have included the
overexpression of Fe chelate reductase genes (Ishimaru et al. 2007) and barley
nicotianamine amino transferase genes (Takahashi et al. 2001).
2.6 Engineering Metal Toxicity Tolerance in Plants
2.6.1 Heavy Metal Contaminated Soil
Soils naturally consist of varying high levels of heavy metals; however, concentra-
tions in soils are greatly increased where humans have extracted them and used
them for industrial purposes (Greger 1999; Harmsen 2002; UN-Oceans 2008).
Heavy metals, especially those that are present from weathering of parent rock, are
usually bound or immobile in soils and their removal via leaching or plant accu-
mulation is slow and inefficient. As a consequence, metals accumulate in soils
(Haygarth and Jones 1992). Heavy metals usually exist as cations under biological
conditions and form complexes with soil sediments and colloids, which are made
up of negatively charged organic substances and inorganic clay particles. Forma-
tion of this complex is a slow process and, because unbound metals are bioavailable
and may deleteriously affect agricultural products or leach into groundwater, areas
where human activities have led to elevated soil metal content are a socioeconomic
as well as an environmental concern (Greger 1999; Harmsen 2002).
Common heavy metal soil pollutants include arsenic, cadmium, chromium,
copper, nickel, lead, and mercury (UN-Oceans 2008). Cd and Hg are particularly
concerning because they are widespread and have no known function in human
metabolism (Nordberg et al. 2002; UN-Oceans 2008).
2.6.2 Engineering Heavy Metal Tolerance in Plants
Few known studies have focused on engineering heavy metal tolerance in plants.
It is assumed that this is due to the complex interactions of stress factors, which
mean that many studies focusing on improving other types of abiotic stress, such as
osmotic and low temperature stress, include analysis of improved tolerance to one
or a few heavy metals. For example, Zhang et al. (2008c) isolated an aquaporin
gene BjPIP1 from the heavy metal hyperaccumulator Indian mustard, which is
upregulated in leaves under drought, salt, low temperature, and heavy metal stress.
Constitutive expression of BjPIP1 in tobacco decreased water loss rate, transpira-
tion rate, and stomatal conductance of transgenic plants compared to WT under
osmotic stress. On exposure to Cd, transgenic plants displayed enhanced Cd
resistance of root growth and lowered transpiration rates and stomatal conductance,
increased activities of antioxidative enzymes, lower levels of electrolyte leakage,
and lower malondialdehyde content. The study suggested that the increased heavy
metal resistance was conferred by maintaining reasonable water status in transgenic
plants. Other proteins that have been implicated in conferring heavy metal tolerance
include LEA proteins and cation-efflux transport proteins (Zhang et al. 2007c).
When Koh et al. (2006) introduced a yeast Cd factor (YCF1), which sequesters
glutathione chelates of heavy metals and xenobiotics into vacuoles, into Arabidop-
sis in order to improve heavy metal tolerance, transgenic plants were found to have
improved salt tolerance as well (Zhang et al. 2007c). While the use of genetic
engineering to develop crops with improved heavy metal tolerance remains rela-
tively unexplored, a related field is the use of genetic engineering to develop plants
with increased ability to uptake and accumulate metals in order to remove the
soil contamination in a process known as phytoremediation. Many of the plant
characteristics required to confer metal accumulation ability also confer heavy
metal tolerance and, as such, plants engineered with enhanced phytormediation
capabilities inevitably possess enhanced heavy metal tolerance as well. Unlike

engineering plant heavy metal tolerance, this field has been the focus of many
studies over the last decade. Therefore, the remainder of this chapter will briefly
discuss the use of transgenic technology to improve the phytoremediation capabil-
ities of plants.
2.6.3 Soil Heavy Metal Management and Remediation Options
There are many management and remediation options available to prevent entry
of soil heavy metals into the human food chain via agricultural crops. These include:
prevention, agronomic management, and physical remediation. These options,
while contributing to reductions in human exposure to some heavy metals, can be
unrealistic, labor intensive, and prohibitively expensive (McLaughlin et al. 1999;
Ensley 2000; Glass 2000; Clarkson 2002; Madden et al. 2002; Prasad 2002;
Robinson et al. 2003; Song et al. 2003).
2.6.3.1 Phytoremediation: Engineering Crops to Increase Heavy

Metal Uptake
Phytoremediation is a biological technology, which utilizes the physiology of

plants to extract and detoxify soil and water pollutants (Kramer and Chardonnens
2000; Rosselli et al. 2003). The use of plants for contaminated land reclamation is
based on the existence of metal hyperaccumulator plants, which are capable of
growing on metal contaminated soils and accumulating extremely high levels of
heavy metals in their harvestable tissue (Robinson et al. 2003). The major metal
tolerance mechanisms identified in heavy metal accumulator plants include: (1) cell
wall metal ion binding; (2) inhibition of metal ion transport across plasma mem-
branes; (3) active metal ion efflux from cells; (4) chelation and detoxification of
metal ions; and (5) compartmentalisation of metal ions in organelles (Bargagli
1998).
The several types of phytoremediation include: phytostabilization (the use of
plants to immobilize metals in the environment to prevent them from leaching into
groundwater (Prasad 2002)), phytodegradation (the use of plants to degrade con-
taminants in the root zone), phytoextraction, and phytovolatization. Phytoextrac-
tion and phytovolatilisation are particularly important for metal phytoremediation.
Phytoextractor plants remove metal ions from soils and store them in above-ground
tissue using the photosynthetically driven processes of soil-ion extraction and
translocation. Normal harvesting processes enable easy removal of contaminants
and their responsible disposal. Phytovolatilization is similar to phytoextraction but,
rather than be stored in biomass, extracted elements are converted into less toxic,
volatile forms via plant metabolic pathways and released into the atmosphere
(Kramer and Chardonnens 2000; Robinson et al. 2003).
2.6.4 Phytovolatilization of Soils Contaminated with Hg
Relatively low exposure to Hg and mercurial compounds can cause severe detri-
mental physiological effects in all biological organisms. Atmospheric Hg increased
from approximately 2 106 kg to 4 106 kg over the twentieth century because
of Hg use in the chemical, medical, paper, mining, and defense industries (Bizily
et al. 2000). Environmental Hg is present as Hg(II), elemental Hg (Hg(0)), and
organomercurial compounds (R-Hg+) such as CH3–Hg+. Toxicity symptoms
between these forms differ; CH3–Hg+ is the most dangerous because it is lethal at
doses two to three orders of magnitude lower than Hg(0) or Hg(II), diffuses
passively through biological membranes, is highly reactive with biological com-
pounds, has a long retention time in the body, and is biomagnified through the food
chain (Rugh et al. 2000).
There are no known naturally occurring plants that are able to detoxify or
hyperaccumulate Hg (Pilon-Smits and Pilon 2002). Therefore, a bacterial Hg
volatilization pathway has been used in the development of Hg phytoremediators
(Pilon-Smits and Pilon 2002). Bacterial colonies that metabolically convert Hg(II)
and R-Hg+ compounds into the less toxic Hg(0) have been discovered inhabiting
Hg-contaminated sites. The volatilization pathway is conferred by the presence of
the mer-operon, which encodes a set of genes involved in the detection, mobiliza-
tion, and enzymatic detoxification of R-Hg+ compounds via two main reactions,
which are catalyzed by organomercurial lyase (MerB) and mercuric ion reductase
(MerA) (Rugh et al. 1998). A modified form of the MerA gene, merA9, was trans-
ferred into Arabidopsis and tobacco and transgenic plants were found to be resistant
to 50 mm Hg(II) (Rugh et al. 2000). Expression of the same gene in the forest
species, Liriodendron tulipifera (yellow poplar) resulted in transgenic plants, which
thrived in solution containing 50 mm Hg(II) and volatized Hg(0) at a rate tenfold
higher than WT controls (Kramer and Chardonnens 2000). MerA has also been
expressed in the eastern cottonwood (Populus deltoids) with similar success. The
transformation of Arabidopsis with constructs of both the merA and merB genes
resulted in transgenic plants able to tolerate media containing 40-fold higher CH3–
Hg+ levels than WT plants and were able to volatilize Hg at a rate severalfold higher
than WTs (Bizily et al. 2000). Tobacco was also transformed with MerA and MerB
via the chloroplast genome and transgenic plants grew well with root Hg concen-
trations up to 2,000 mg g–1, accumulated R-Hg+ compounds and inorganic mercur-
ials to levels surpassing soil concentrations and displayed a 100-fold increase in the
efficiency of shoot Hg accumulation over WT plants (Hussein et al. 2007). MerA
and MerB have also been expressed simultaneously in transgenic poplars and
eastern cottonwood trees (Lyyra et al. 2007; Young et al. 2007). Transgenic poplars
were tolerant to 50 mM HgCl2 and 2 mM CH3HgCl in culture; however, a high
level of variation in Hg-tolerance was observed in transgenic plants (Young et al.
2007). Transgenic eastern cottonwood trees were highly resistant to R-Hg+ com-
pounds and detoxified them two to three times more rapidly than controls (Lyyra
et al. 2007).
2.6.5 Phytoextraction of Soils Contaminated with Cd
Plants able to tolerate high Cd levels do so via exclusion (reducing metal uptake by
cell wall binding) or intracellular compartmentalization (binding of metals with
detoxifying ligands within the cell and the sequestration of these complexes in
organelles where the metals cannot interact with the plants metabolic processes
(Robinson et al. 1994)). The two major heavy metal detoxifying ligands in plant
cells are the metallothioneins (MTs) and the phytochelatins (PCs; Cobbett and
Goldsborough 2000). Class I and II MTs are low molecular weight metal-binding
products of mRNA translation. PCs are represented by the class III MTs and are
products of enzymatic reactions (Cobbett and Goldsborough 2002).
2.6.5.1 Metallothioneins
It has been difficult to analyze plant metallothionein (MT) proteins because they are
unstable under aerobic conditions; however, isolated and cloned mammalian MT
genes have been assessed for their ability to increase metal tolerance when
expressed in transgenic organisms. Table 2.2 lists the findings of some of these
studies. These results, obtained in laboratory trials, demonstrate that expression of
mammalian MTs in plants can provide protection against toxic effects of heavy
metal ions such as Zn2+, Cd2+, and Hg(II); but it has not yet been possible to achieve
similar results in the field (Kramer and Chardonnens 2000).
2.6.5.2 Phytochelatins
Phytochelatins (PCs) were first discovered in plants and are structurally related to
GSH, which is thought to act as the substrate for PC synthesis by PC synthase
(PCS). Various methods have been employed to enhance the effectiveness of PC-
assisted metal detoxification. Indian mustard was engineered to express the E. coli
GSH synthesis gene, gsh2, and transgenic plants displayed increased GSH and PC
levels and a 25% increase in shoot Cd concentration (Zhu et al. 1999). The enzyme
that catalyzes synthesis of the GSH precursor, g-glutamylcysteine synthase (GCS),
was also expressed in B. juncea and shoot Cd concentrations were increased by
40–90% in the transformed plants (Kramer and Chardonnens 2000).
A variety of PC genes have been isolated from different species and over-
expressed in endogenous or exogenous species to determine their potential for
Cd phytoremediation. The most extensively studied PCS gene is AtPCS1 from
Arabidopsis, which has been found to play roles in increased tolerance and/or
accumulation of Cd and As (Gasic and Korban 2007b). However, depending on
the expression level, transfer of PCS genes have also been associated with
decreased Cd accumulation and Cd hypersensitivity (Lee et al. 2003b, c; Li et al.
Table 2.2 Genetic transfer of MT genes for improved phytoremediation capabilities of plants
Transformed Transgene(s) Findings References
species
Eschericia Human MT genes Increased metal adsorption Kramer and
coli Chardonnens
(2000)
Ralstonia Mouse MTs genes Enhanced Cd immobilizing ability Valls et al.
eutrophia when expressed on cell surface (2000)
Tobacco Mammalian MT Root-shoot Cd transport is reduced Kramer and
gene Chardonnens
(2000)
Brassica Yeast MT gene, Increased leaf Cd accumulating Kramer and
oleracea CUP1 ability Chardonnens
(2000)
Tobacco Construct containing Up to 90% increased Cd Macek et al.
CUP1 and an accumulation in harvestable (2002)
additional metal- parts without any visible
binding domain difference in growth
characteristics
Sunflower CUP1 Enhanced Cd tolerance when Watanabe et al.
expressed at the callus stage (2005)
Tobacco Arabidopsis MT Expression strongly induced by Tonkovska et al.
gene, MT2aI Cu2+, Zn2+, and Cd2+ (2003)
suggesting the promoter has
specificity for heavy metal
stress
Tobacco Construct encoding a 45–75% increase in Cd Pavlikova et al.
poyhistidine accumulation in harvestable (2004a, b)
cluster with a plant parts and increased
yeast MT resistance to Cd-induced stress
Arabidopsis Garlic MT gene Stronger Cd tolerance and higher Zhang et al.
Cd accumulation (2006)
Arabidopsis Brassica juncea MT Increased tolerance to Cu2+ and Zhigang et al.
gene, BjMT2, Cd2+ based on shoot growth and (2006)
chlorophyll content and
decreased root growth in the
absence of heavy metal
exposure
Tobacco Silene vulgaris L. Signigicantly increased Cd Gorinova et al.
MT gene accumulation in roots and (2007)
leaves
Arabidopsis Brassica rapa MT Chloroplast target of gene resulted Sun et al. (2007)
gene in detoxification of Cd and
H2O2
2004, 2005b; Gasic and Korban 2007a). Other studies have shown that AtPCS1 can
lead to increased Cd tolerance and accumulation in roots but not translocation of Cd
into harvestable tissue (Pomponi et al. 2006). PCS genes from Cyndon dactylon
(CdPCS1) and wheat (TaPCS1) have also been transformed into tobacco and have
led to increases in leaf accumulation of Cd and other heavy metals (Li et al. 2006;
Martinez et al. 2006). Tobacco transformed with TaPCS1 was grown in the field to
assess its Cd-accumulating ability and it was found that transgenic plants could
accumulate more heavy metals and 100 times more biomass on contaminated soils
than the hyperaccumulator Thlaspi caerlescens (Martinez et al. 2006). Another
approach was employed by Song et al. (2003) and was aimed at increasing
the efficiency of metal transport across the tonoplast. In this study, YCF1 from
S. cerevisiae was overexpressed in Arabidopsis and transformed plants displayed
increased tolerance to Cd and Pb and improved vacuolar Cd sequestration. Many
studies have also shown that dual- or multiple-gene expression using constructs
with combinations of PC synthesis genes such as GCS, GSH synthase, ATP sul-
furylase, and serine acetyltranferase may be the most promising route toward the
development of a useful Cd phytoremediator and a phytoremediator useful for
removal of mixtures of heavy metals from soils (Bennett et al. 2003; Wawrzynski
et al. 2006; Guo et al. 2008; Reisinger et al. 2008).
2.7 Conclusion and Future Directions
This review has briefly summarized some of the stress resistance mechanisms that
have been targeted for manipulation in the endeavor to develop crop plants with
improved resistance to drought, salinity, cold, nutrient deficiencies, and metal
toxicities. In the majority of cases, there are many knowledge gaps that need to
be filled prior to development and release of these crops. Water-limiting environ-
ments are the most widespread form of stress, and also the least amenable to rapid
advances in resistance. Reasons for this include the incomplete knowledge regard-
ing plant drought responses, lack of field trials and drought stress treatments which
are truly reflective of climatic conditions, the lack of site and crop specificity of
drought tolerance studies, and the lack of integration of disciplines.
The last century of breeding effort and crop physiology studies have led to
increases in the economic yield of most major crop species and have elucidated
many traits that are associated with plant adaptability to drought-prone environ-
ments (such as small plant size, reduced leaf area, early maturity, and prolonged
stomatal closure). However, many attempts to improve drought tolerance through
breeding have been associated with reduced yield potential (Cattivelli et al. 2008).
Nevertheless, enormous advances should have been make in the understanding of
the physiological and molecular responses of plants to water deficit through breed-
ing and physiological studies, and scientists in these areas will continue to have a
fundamental role in the development of transgenic drought-resistant crops. One of
the greatest limitations in drought stress tolerance breeding has been the fact that
drought takes many varied forms. Depending on the crop and the season, water
stress may be experienced in early vegetative stages, during transition to flowering
and even post-anthesis. In some cases, this may be a terminal stress or, more
commonly, a recurring event broken by sporadic rainfall precipitating a recovery
of the whole plant, sometimes with reduced yield potential as a result of conserva-
tive plant cell survival responses.
It is entirely possible that the development of transgenic crops with improved

abiotic stress tolerance may lead to unforeseen outcomes, both positive and nega-
tive. The development of “super varieties” that offer promising solutions to the
problems of water deficit or salinity stress, may also give rise to problems asso-
ciated with high-input agriculture in monoculture situations. Additionally, this
review has shown that there are a plethora of genetic components that may be
manipulated in order to confer improved abiotic stress tolerance of crops and that
there is, therefore, unlikely to be a single gene that will result in a suitable “drought-
tolerant” or “salt-tolerant” variety for all conditions. Consequently, it may be
beneficial to trial novel technologies such as overexpression of multiple genes
known to be involved in the adaptation to stress and introgression of these genes
in random combinations into many cultivars of the one crop prior to field release.
This may increase the stability of these genes and allow specific environments to
“naturally select” the transgenes that are most suitable for their particular climatic
conditions in the same way that increased natural genetic diversity does so in
natural populations.
Salt tolerance is a physiologically complex trait and halophytes and less tolerant
plants show a wide range of adaptations to salt stress. Attempts to enhance
tolerance have involved conventional breeding programs, the use of in vitro selec-
tion, physiological trait pooling, interspecific hybridization, the use of halophytes
as alternative crops, the use of marker-aided selection, and the use of transgenic
plants. The assessment of salt tolerance in transgenic experiments as described
above has mostly been carried out using a limited number of seedlings or mature
plants in laboratory experiments. In most cases, experiments were carried out in
greenhouse conditions where the plants were not exposed to conditions that prevail
in high-salinity soils (e.g., alkaline soil pH, high diurnal temperatures, low humid-
ity, presence of sodic salts, and elevated concentrations of selenium and/or boron
(Yamaguchi and Blumwald 2005)). Therefore, the salt tolerance of plants needs to
be evaluated under field conditions and, more importantly, salt tolerance needs
to be evaluated as a function of yield. The evaluation of field performance under
salt stress is difficult because of the variability of salt levels in field conditions
(Daniells et al. 2001) and the potential for interactions with other environmental
factors, including soil fertility, temperature, light intensity and water loss due to
transpiration. Evaluating tolerance is further complicated because of variation in
sensitivity to salt during plant life cycles. According to Flowers and Flowers
(2005), conventional breeding programs have rarely delivered enhanced salt toler-
ance, while wide crossing to achieve salt tolerance generally reduces yield to
unacceptably low levels (Flowers and Flowers 2005). There has been success
using physiological criteria as the basis of selection for rice (Dedolph and Hettel
1997) and such an approach has been advocated for wheat (Munns et al. 2002).
According to Flowers and Flowers (2005) recent analysis has shown that, while
it is possible to produce a wide range of transgenic plants where some aspect of a
trait relating to salt tolerance is altered, none or few transgenic plants have been
tested in the field and few claims for success meet minimal criteria required to
demonstrate enhanced tolerance (Flowers 2004).
While adaptation to stress under natural conditions has some ecological advan-
tages, the metabolic and energy costs may sometimes mask and limit its benefit to
agriculture and result in yield penalty. An ideal genetically modified crop should
possess a highly regulated stress-response capability that does not affect crop
performance when stress is absent. In this respect, conventional breeding and
selection techniques will continue to make a contribution (Wang et al. 2001). As
a result of this, transgenic approaches to plant improvement are best regarded as a
means of widening genetic variation. Transgenic plants will nevertheless continue
to be extremely useful tools in basic plant science research, and will lead to
improved understanding of the gene networks and molecular physiology of plant
responses to abiotic stresses.
References
Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Arabidopsis AtMYC2

(bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling.
Plant Cell 15:63–78
Acquaah G (2007) Principles of plant genetics and breeding. Blackwell, Oxford, UK
Aharon R, Shahak Y, Wininger S, Bendov R, Kapulnik Y, Galili G (2003) Overexpression of a
plasma membrane aquaporin in transgenic tobacco improves plant vigor under favorable
growth conditions but not under drought or salt stress. Plant Cell 15:439–447
Aida M, Ishida T, Fukaki H, Fujisawa H, Tasaka M (1997) Genes involved in organ separation in
Arabidopsis: An analysis of the cup-shaped cotyledon mutant. Plant Cell 9:841–857
Al-Abed D, Madasamy P, Talla R, Goldman S, Rudrabhatla S (2007) Genetic engineering of maize
with the Arabidopsis DREB1A/CBF3 gene using split-seed explants. Crop Sci 47:2390–2402
Asada K (1999) The water-water cycle in chloroplasts: scavenging of active oxygens and dissipa-
tion of excess photons. Annu Rev Plant Biol 50:601–639
Ashraf M, Foolad MR (2007) Roles of glycine betaine and proline in improving plant abiotic stress
resistance. Environ Exp Bot 59:206–216
Baker J, Steele C, Dure L (1998) Sequence and characterisation of 9 Lea proteins and the genes
from cotton. Plant Mol Biol 11:277–291
Bargagli R (1998) Trace elements in terrestrial plants: an ecophysiological approach to biomoni-
toring and biorecovery. Springer, Berlin
Bartels D, Salamini F (2001) Desiccation tolerance in the resurrection plant Craterostigma
plantagineum. A contribution to the study of drought tolerance at the molecular level. Plant
Physiol 127:1346–1353
Bartels D, Sunkar R (2005) Drought and salt tolerance in plants. Crit Rev Plant Sci 24:23–58
Bartels D, Phillips J, Chandler J (2007) Desiccation tolerance: gene expression, pathways, and
regulation of gene expression. In: Jenks MA, Wood AJ (eds) Plant Desiccation Tolerance.
Blackwell, Ames, IA, pp 115–137
Batjes NH (1997) A world dataset of derived soil properties by FAO-UNESCO soil unit for global
modelling. Soil Use Manag 13:9–16
Beard JB, Sifers SI (1997) Genetic diversity in dehydration avoidance and drought resisitance
within the Cynodon and Zoysia species. Int Turfgrass Soc Res J 8:603–610
Beck EH, Heim R, Hansen J (2004) Plant resistance to cold stress: mechanisms and environmental
signals triggering frost hardening and dehardening. J Biosci 29:449–459
Benedict C, Skinner JS, Meng R, Chang Y, Bhalerao R, Huner NPA, Finn CE, Chen THH,
Hurry V (2006) The CBF1-dependent low temperature signalling pathway, regulon and
increase in freeze tolerance are conserved in Populus spp. Plant Cell Environ 29:1259–1272
Bennett LE, Burkhead JL, Hale KL, Terry N, Pilon M, Pilon-Smits EAH (2003) Analysis of
transgenic Indian mustard plants for phytoremediation of metal-contaminated mine tailings.
J Environ Qual 32:432–440
Bertrams M and Heinz E (1981) Positional specificity and fatty acid selectivity of purified
sn-glycerol-3-phosphate acyltransferase from chloroplasts. Plant Physiol 68:653–657
Bhatnagar-Mathur P, Vadez V, Sharma KK (2008) Transgenic approaches for abiotic stress
tolerance in plants: retrospect and prospects. Plant Cell Rep 27:411–424
Bizily SP, Rugh CL, Meagher RB (2000) Phytodetoxification of hazardous organomercurials by
genetically engineered plants. Nat Biotechnol 18:216–217
Blumwald E, Aharon GS, Apse MP (2000) Sodium transport in plant cells. Biochem Biophys Acta
Biomembr 1465:140–151
Bohnert HJ, Jensen RG (1996) Strategies for engineering water-stress tolerance in plants. Trends
Boston RS, Viitanen PV, Vierling E (1996) Molecular chaperones and protein folding in plants.
Plant Mol Biol 32:191–222
Bowler C, Van Montagu M, Inze D (1992) Superoxide dismutase and stress tolerance. Annu Rev
Plant Physiol Plant Mol Biol 43:83–116
Bray EA (1993) Molecular responses to water deficit. Plant Physiol 103:1035–1040
Brini F, Hanin M, Lumbreras V, Amara I, Khoudi H, Hassairi A, Pages M, Masmoudi K (2007a)
Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in
Arabidopsis thaliana. Plant Cell Rep 26:2017–2026
Brini F, Hanin M, Mezghani I, Berkowitz GA, Masmoudi K (2007b) Overexpression of wheat Na
+/H+ antiporter TNHX1 and H+-pyrophosphatase TVP1 improve salt- and drought-stress
tolerance in Arabidopsis thaliana plants. J Exp Bot 58:301–308
Broun P, Poindexter P, Osborne E, Jiang CZ, Riechmann JL (2004) WIN1, a transcrip-
tional activator of epidermal wax accumulation in Arabidopsis. Proc Natl Acad Sci USA
101:4706–4711
Brouquisse R, Weigel P, Rhodes D, Yocum CF, Hanson AD (1989) Evidence for a ferredoxin-
dependent choline mono-oxygenase from spinach chloroplast stroma. Plant Physiol
90:322–329
Bushoven JT, Hull RJ (2001) Nitrogen use efficiency is linked to nitrate reductase activity and
biomass partitioning between roots and shoots of perennial ryegrass and creeping bentgrass. Int
Turfgrass Soc Res J 9:245–252
Caldwell CR, Turano FJ, McMahon MB (1998) Identification of two cytosolic ascorbate peroxi-
dase cDNAs from soybean leaves and characterization of their products by functional expres-
sion in E. coli. Planta 204:120–126
Cattivelli L, Rizza F, Badeck F-W, Mazzucotelli E, Mastrangelo AM, Francia E, Mare C,
Tondelli A, Stanca AM (2008) Drought tolerance improvement in crop plants: an integrated
view from breeding to genomics. Field Crops Res 105:1–14
Ceccardi TL, Meyer NC, Close TJ (1994) Purification of a maize dehydrin. Protein Expr Purif
5:266–269
Chandler PM, Robertson M (1994) Gene expression regulated by abscisic acid and its relation to
stress tolerance. Annu Rev Plant Physiol Plant Mol Biol 45:113–141
Chandra Babu R, Zhang J, Blum A, David Ho TH, Wu R, Nguyen HT (2004) HVA1, a LEA gene
from barley confers dehydration tolerance in transgenic rice (Oryza sativa L.) via cell mem-
brane protection. Plant Sci 166:855–862
Chapman KD (1998) Phospholipase activity during plant growth and development and in response
to environmental stress. Trends Plant Sci 3:419–426
Chen WJ, Zhu T (2004) Networks of transcription factors with roles in environmental stress
response. Trends Plant Sci 9:591–596
Chen C-W, Yang Y-W, Lur H-S, Tsai Y-G, Chang M-C (2006) A novel function of abscisic acid
in the regulation of rice (Oryza sativa L.) root growth and development. Plant Cell Physiol
47:1–13
Chen M, Chen QJ, Niu XG, Zhang R, Lin HQ, Xu CY, Wang XC, Wang GY, Chen J (2007)
Expression of OsNHX1 gene in maize confers salt tolerance and promotes plant growth in the
field. Plant Soil Environ 53:490–498
Chen JQ, Meng XP, Zhang Y, Xia M, Wang XP (2008) Over-expression of OsDREB genes lead to
enhanced drought tolerance in rice. Biotechnol Lett 30:2191–2198
Chinnusamy V, Zhu JK (2003) Plant salt tolerance. In: Hirt H, Shinozaki K (eds) Plant responses
to abiotic stress. Springer, Berlin, pp 241–270
Chinnusamy V, Zhu J, Zhu JK (2006) Gene regulation during cold acclimation in plants. Physiol
Plant 126:52–61
Chinnusamy V, Zhu J, Zhu JK (2007) Cold stress regulation of gene expression in plants. Trends
Plant Sci 12:444–451
Cho EK, Hong CB (2006) Over-expression of tobacco NtHSP70–1 contributes to drought-stress
tolerance in plants. Plant Cell Rep 25:349–358
Clarkson TW (2002) Mercury. In: Sarkar B (ed) Heavy metals in the environment. Marcel Dekker,
New York, pp 457–501
Close TJ (1996) Dehydrins: emergence of a biochemical role of a family of plant dehydration
proteins. Physiol Plant 97:795–803
Close KR, Gallagher-Ludeman LA (1989) Structure-activity relationships of auxin-like plant
growth regulators and genetic influences on the culture induction response in maize (Zea
mays L.). Plant Sci 61:245–252
Cobbett CS, Goldsborough PB (2000) Mechanisms of metal resistance: phytochelatins and
metallothioneins. In: Ensley BD (ed) Phytoremediation of toxic metals: using plants to clean
up the environment. John Wiley, New York, pp 247–269
Cobbett CS, Goldsborough PB (2002) Phytochelatins and metallothioneins: roles in heavy metal
detoxification and homeostasis. Annu Rev Plant Biol 53:159–182
Cominelli E, Sala T, Calvi D, Gusmaroli G, Tonelli C (2008) Over-expression of the Arabidopsis
AtMYB41 gene alters cell expansion and leaf surface permeability. Plant J 53:53–64
Crowe JH, Crowe LM, Chapman D (1984) Preservation of membranes in anhydrobiotic organ-
isms: the role of trehalose. Science 223:701–703
Cui X-H, Hao Fu-Shun, Chen H, Chen J, Wang X-C (2008) Expression of the Vicia faba VfPIP1
gene in Arabidopsis thaliana plants improves their drought resistance. J Plant Res
121:207–214
Curie C, Panaviene Z, Loulergue C, Dellaporta SL, Briat JF, Walker EL (2001) Maize
yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake. Nature
409:346–349
Curtis IS, Power JB, de Laat AMM, Caboche M, Davey MR (1999) Expression of a chimeric
nitrate reductase gene in transgenic lettuce reduces nitrate in leaves. Plant Cell Rep
18:889–896
Cushman JC, Bohnert HJ (2000) Genomic approaches to plant stress tolerance. Curr Opin Plant
Biol 3:117–124
Daniells IG, Holland JF, Young RR, Alston CL, Bernardi AL (2001) Relationship between yield of
grain sorghum (Sorghum bicolor) and soil salinity under field conditions. Aust J Exp Agric
41:211–217
Dedolph C, Hettel G (eds) (1997) Rice varieties boost yield and improve saline soils. IRRI,
Manila, The Philippines
Delauney AJ, Verma DPS (1993) Proline biosynthesis and osmoregulation in plants. Plant J
4:215–223
Demin IN, Deryabin AN, Sinkevich MS, Trunova TI (2008) Insertion of cyanobacterial desA gene
coding for D12-acyl-lipid desaturase increases potato plant resistance to oxidative stress
induced by hypothermia. Russ J Plant Physiol 55:639–648
Diamant S, Eliahu N, Rosenthal D, Goloubinoff P (2001) Chemical chaperones regulate

molecular chaperones in vitro and in cells under combined salt and heat stresses. J Biol
Chem 276:39586–39591
Dietz KJ (2003) Plant peroxiredoxins. Annu Rev Plant Biol 54:93–107
Dure III L (1993) A repeating 11-mer amino acid motif and plant desiccation. Plant J 3:363–369
Ellul P, Rios G, Atares A, Roig LA, Serrano R, Moreno V (2003) The expression of the
Saccharomyces cerevisiae HAL1 gene increases salt tolerance in transgenic watermelon
[Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Theor Appl Genet 107:462–469
Ensley BD (2000) Rationale for use of phytoremediation. In: Raskin I, Ensley BD (eds) Phytor-
emediation of toxic metals: using plants to clean up the environment. John Wiley, New York,
pp 3–11
FAO (2009a) Aquastat. www.fao.org/nr/water/aquastat/data/query/index.html
FAO (2009b) FAO land and plant nutrition management service. www.fao.org/ag/agl/agll/spush/
FAO (2009c) Food and Agriculture Organisation of the United Nations. www.fao.org/askfao/
topicsList.do?mainAreaId=20263
Finnegan PM, Soole KL, Umbach AL (2004) Alternative mitochondrial electron transport proteins in
higher plants. In: Day DA, Millar AH, Whelan J (eds) Plant mitochondria: from gene to function.
Advances in photosynthesis and respiration. Kluwer, Dordrecht, The Netherlands, pp 163–230
Fiorani F, Umbach AL, Siedow JN (2005) The alternative oxidase of plant mitochondria is
involved in the acclimation of shoot growth at low temperature. A study of Arabidopsis
AOX1a transgenic plants. Plant Physiol 139:1795–1805
Flowers TJ (2004) Improving crop salt tolerance. J Exp Bot 55:307–319
Flowers TJ, Flowers SA (2005) Why does salinity pose such a difficult problem for plant breeders?
Agr Water Manag 78:15–24
Fowler S, Thomashow MF (2002) Arabidopsis transcriptome profiling indicates that multiple
regulatory pathways are activated during cold acclimation in addition to the CBF cold response
pathway. Plant Cell 14:1675–1690
Fowler DB, Limin AE, Wang SY, Ward RW (1996) Relationship between low-temperature
tolerance and vernalization response in wheat and rye. Can J Plant Sci 76:37–42
Foyer CH, Descourvieres P, Kunert KJ (1994) Protection against oxygen radicals – an important
defence-mechanism studied in transgenic plants. Plant Cell Environ 17:207–523
Gaber A, Yoshimura K, Yamamoto T, Yabuta Y, Takeda T, Miyasaka H, Nakano Y, Shigeoka S
(2006) Glutathione peroxidase-like protein of Synechocystis PCC 6803 confers tolerance to
oxidative and environmental stresses in transgenic Arabidopsis. Physiol Plant 128:251–262
Gao F, Gao Q, Duan X, Yue G, Yang A, Zhang J (2006) Cloning of an H+-PPase gene from
Thellungiella halophila and its heterologous expression to improve tobacco salt tolerance.
J Exp Bot 57:3259–3270
Garay-Arroyo A, Colmenero-Flores JM, Garciarrubio A, Covarrubias AA (2000) Highly hydro-
philic proteins in prokaryotes and eukaryotes are common during conditions of water deficit.
J Biol Chem 275:5668–5674
Gasic K, Korban SS (2007a) Expression of Arabidopsis phytochelatin synthase in Indian mustard
(Brassica juncea) plants enhances tolerance for Cd and Zn. Planta 225:1277–1285
Gasic K, Korban SS (2007b) Transgenic Indian mustard (Brassica juncea) plants expressing an
Arabidopsis phytochelatin synthase (AtPCS1) exhibit enhanced As and Cd tolerance. Plant
Mol Biol 64:361–369
Gaxiola R, De Larrinoa F, Villalba JM, Serrano R (1992) A novel and conserved salt-induced
protein is an important determinant of salt tolerance in yeast. EMBO J 11:3157–3164
Gaxiola RA, Rao R, Sherman A, Grisafi P, Alper SL, Fink GR (1999) The Arabidopsis thaliana
proton transporters, AtNhx1 and Avp1, can function in cation detoxification in yeast. Proc Natl
Acad Sci USA 96:1480–1485
Gaxiola RA, Li J, Undurraga S, Dang LM, Allen GJ, Alper SL, Fink GR (2001) Drought- and salt-
tolerant plants result from overexpression of the AVP1 H+-pump. Proc Natl Acad Sci USA
98:11444–11449
Ge LF, Chao DY, Shi M, Zhu MZ, Gao JP, Lin HX (2008) Overexpression of the trehalose-
6-phosphate phosphatase gene OsTPP1 confers stress tolerance in rice and results in the
activation of stress responsive genes. Planta 228:191–201
Ghassemi F, Jakeman AJ, Nix HA (1995) Salinisation of land and water resources. Human causes,
extent management & case studies. University of New South Wales, Sydney, Australia
Giannino D, Nicolodi C, Testone G, Frugis G, Pace E, Santamaria P, Guardasole M, Mariotti D
(2008) The overexpression of asparagine synthetase A from E. coli affects the nitrogen status in
leaves of lettuce (Lactuca sativa L.) and enhances vegetative growth. Euphytica 162:11–22
Glass DJ (2000) Economic potential of phytoremediation. In: Raskin I, Ensley BD (eds) Phytor-
emediation of toxic metals: using plants to clean up the environment. John Wiley, New York,
pp 15–31
Glass ADM, Britto DT, Kaiser BN, Kinghorn JR, Kronzucker HJ, Kumar A, Okamoto M,
Rawat S, Siddiqi MY, Unkles SE, Vidmar JJ (2002) The regulation of nitrate and ammonium
transport systems in plants. Oxford Univeristy Press, Oxford, UK, pp 855–864
Glassop D, Godwin RM, Smith SE, Smith FW (2007) Rice phosphate transporters associated
with phosphate uptake in rice roots colonised with arbuscular mycorrhizal fungi. Can J Bot
85:644–651
Goday A, Jensen AB, Culianez-Macia FA, Mar Alba M, Figueras M, Serratosa J, Torrent M,
Pages M (1994) The maize abscisic acid-responsive protein Rab17 is located in the nucleus
and interacts with nuclear localization signals. Plant Cell 6:351–360
Good AG, Shrawat AK, Muench DG (2004) Can less yield more? Is reducing nutrient input into
the environment compatible with maintaining crop production? Trends Plant Sci 9:597–605
Gorinova N, Nedkovska M, Todorovska E, Simova-Stoilova L, Stoyanova Z, Georgieva K,
Demirevska-Kepova K, Atanassov A, Herzig R (2007) Improved phytoaccumulation of cad-
mium by genetically modified tobacco plants (Nicotiana tabacum L.). Physiological and
biochemical response of the transformants to cadmium toxicity. Environ Pollut 145:161–170
Greger M (1999) Metal availability and bioconcentration in plants. In: Prasad MNV, Hagemeyer J
(eds) Heavy metal stress in plants: from molecules to ecosystems. Springer, Berlin, pp 1–27
Guo SJ, Zhou HY, Zhang XS, Li XG, Meng QW (2007) Overexpression of CaHSP26 in transgenic
tobacco alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance.
J Plant Physiol 164:126–136
Guo J, Dai X, Xu W, Ma M (2008) Overexpressing GSH1 and AsPCS1 simultaneously increases
the tolerance and accumulation of cadmium and arsenic in Arabidopsis thaliana. Chemosphere
72:1020–1026
Haake V, Cook D, Riechmann JL, Pineda O, Thomashow MF, Zhang JZ (2002) Transcription
factor CBF4 is a regulator of drought adaptation in Arabidopsis. Plant Physiol 130:639–648
Hare PD, Cress WA, Van Staden J (1998) Dissecting the roles of osmolyte accumulation during
stress. Plant Cell Environ 21:535–553
Harmon AC, Gribskov M, Harper JF (2000) CDPKs – a kinase for every Ca2+ signal? Trends Plant
Sci 5:154–159
Harmsen K (2002) Long-term behaviour of heavy metals in agricultural soils: a simple analytical
model. In: Adriano DC (ed) Biogeochemistry of trace metals. Lewis Publ, Boca Raton, FL,
pp 217–247
Hartl FU (1996) Molecular chaperones in cellular protein folding. Nature 381:571–580
Haygarth PM, Jones KC (1992) Atmospheric deposition of metals to agricultural surfaces. In:
Adriano DC (ed) Biogeochemisty of trace metals. Lewis Publ, Boca Raton, FL, pp 249–276
He C, Yan J, Shen G, Fu L, Holaday AS, Auld D, Blumwald E, Zhang H (2005) Expression of
an Arabidopsis vacuolar sodium/proton antiporter gene in cotton improves photosynthetic
performance under salt conditions and increases fiber yield in the field. Plant Cell Physiol
46:1848–1854
He L, Ban Y, Inoue H, Matsuda N, Liu J, Moriguchi T (2008) Enhancement of spermidine content
and antioxidant capacity in transgenic pear shoots overexpressing apple spermidine synthase in
response to salinity and hyperosmosis. Phytochemistry 69:2133–2141
Hendrick JP, Hartl FU (1993) Molecular chaperone functions of heat-shock proteins. Annu Rev
Biochem 62:349–384
Hiilovaara-Teijo M, Palva ET (1999) Molecular responses in cold adapted plants. In: Margesin R,
Schinner F (eds) Cold adapted organisms ecology, physiology. Enzymology and molecular
biology. Springer, Berlin, pp 349–384
Hmida-Sayari A, Gargouri-Bouzid R, Bidani A, Jaoua L, Savoure A, Jaoua S (2005) Overexpres-
sion of D1-pyrroline-5-carboxylate synthetase increases proline production and confers salt
tolerance in transgenic potato plants. Plant Sci 169:746–752
Hong YF, Liu CY, Cheng KJ, Hour AL, Chan MT, Tseng TH, Chen KY, Shaw JF, Yu SM
(2008) The sweet potato sporamin promoter confers high-level phytase expression and
improves organic phosphorus acquisition and tuber yield of transgenic potato. Plant Mol
Biol 67:347–361
Houde M, Daniel C, Lachapelle M, Allard F, Laliberte S, Sarhan F (1995) Immunolocalization of
freezing-tolerance-associated proteins in the cytoplasm and nucleoplasm of wheat crown
tissues. Plant J 8:583–593
Houde M, Sylvain D, N’Dong D, Sarhan F (2004) Overexpression of the acidic dehydrin
WCOR410 improves freezing tolerance in transgenic strawberry leaves. Plant Biotechnol J
2:381–387
Hu H, Dai M, Yao J, Xiao B, Li X, Zhang Q, Xiong L (2006) Overexpressing a NAM, ATAF, and
CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice. Proc
Natl Acad Sci USA 103:12987–12992
Hu H, You J, Fang Y, Zhu X, Qi Z, Xiong L (2008) Characterization of transcription factor gene
SNAC2 conferring cold and salt tolerance in rice. Plant Mol Biol 67:169–181
Hua ZM, Yang X, Fromm ME (2006) Activation of the NaCl- and drought-induced RD29A and
RD29B promoters by constitutively active Arabidopsis MAPKK or MAPK proteins. Plant Cell
Environ 29:1761–1770
Huang J, Zhang HS (2007) The plant TFIIIA-type zinc finger proteins and their roles in abiotic
stress tolerance. Hereditas (Beijing) 29:915–922
Hunt L, Mills LN, Pical C, Leckie CP, Aitken FL, Kopka J, Mueller-Roeber B, McAinsh MR,
Hetherington AM, Gray JE (2003) Phospholipase C is required for the control of stomatal
aperture by ABA. Plant J 34:47–55
Hussein HS, Ruiz ON, Terry N, Daniell H (2007) Phytoremediation of mercury and organomer-
curials in chloroplast transgenic plants: Enhanced root uptake, translocation to shoots, and
volatilization. Environ Sci Technol 41:8439–8446
Ingram J, Bartels D (1996) The molecular basis of dehydration tolerance in plants. Annu Rev Plant
Physiol Plant Mol Biol 47:377–403
Ishimaru Y, Kim S, Tsukamoto T, Oki H, Kobayashi T, Watanabe S, Matsuhashi S, Takahashi M,
Nakanishi H, Mori S, Nishizawa NK (2007) Mutational reconstructed ferric chelate reductase
confers enhanced tolerance in rice to iron deficiency in calcareous soil. Proc Natl Acad Sci
USA 104:7373–7378
Jang JY, Lee SH, Rhee JY, Chung GC, Ahn SJ, Kang H (2007) Transgenic Arabidopsis and
tobacco plants overexpressing an aquaporin respond differently to various abiotic stresses. Mol
Jenks MA, Ashworth EN (1999) Plant epicuticular waxes: function, production, and genetics. In:
Janick J (ed) Horticultural review. John Wiley, New York, USA, pp 1–68
Jenks MA, Hasegawa I, Mohan PM, Jain S (eds) (2007) Advances in molecular breeding toward
drought and salt tolerant crops. Springer, Dordrecht, The Netherlands
Jiang QW, Kiyoharu O, Ryozo I (2002) Two novel mitogen-activated protein signalling compo-
nents, OsMEK1 and OsMAP1, are involved in a moderate low-temperature signaling pathway
in rice. Plant Physiol 129:1880–1891
Jiang CJ, Aono M, Tamaoki M, Maeda S, Sugano S, Mori M, Takatsuji H (2008) SAZ, a new
SUPERMAN-like protein, negatively regulates a subset of ABA-responsive genes in Arabi-
dopsis. Molecular Genetics and Genomics 279:183–192
Jimenez A, Hernandez JA, Pastori G, Del Rio LA, Sevilla F (1998) Role of the ascorbate-
glutathione cycle of mitochondria and peroxisomes in the senescence of pea leaves. Plant
Physiol 118:1327–1335
Jonak C, Kiegerl S, Ligterink W, Barker PJ, Huskisson NS, Hirt H (1996) Stress signaling in
plants: a mitogen-activated protein kinase pathway is activated by cold and drought. Proc Natl
Acad Sci USA 93:11274–11279
Kalberer SR, Wisniewski M, Arora R (2006) Deacclimation and reacclimation of cold-hardy
plants: current understanding and emerging concepts. Plant Sci 171:3–16
Karaba A, Dixit S, Greco R, Aharoni A, Trijatmiko KR, Marsch-Martinez N, Krishnan A,
Nataraja KN, Udayakumar M, Pereira A (2007) Improvement of water use efficiency in rice
by expression of HARDY, an Arabidopsis drought and salt tolerance gene. Proc Natl Acad Sci
USA 104:15270–15275
Kavi Kishore PB, Sangam S, Amrutha RN, Laxmi PS, Naidu KR, Rao KRSS, Rao S, Reddy KJ,
Theriappan P, Sreenivasulu N (2005) Regulation of proline biosynthesis, degradation, uptake
and transport in higher plants: its implications in plant growth and abiotic stress tolerance. Curr
Sci 88:424–438
Kawakami A, Sato Y, Yoshida M (2008) Genetic engineering of rice capable of synthesizing
fructans and enhancing chilling tolerance. J Exp Bot 59:793–802
Khodakovskaya M, McAvoy R, Peters J, Wu H, Li Y (2006) Enhanced cold tolerance in transgenic
tobacco expressing a chloroplast o-3 fatty acid desaturase gene under the control of a cold-
inducible promoter. Planta 223:1090–1100
Kim JC, Lee SH, Cheong YH, Yoo CM, Lee SI, Chun HJ, Yun DJ, Hong JC, Lee SY, Lim CO,
Cho MJ (2001) A novel cold-inducible zinc finger protein from soybean, SCOF-1, enhances
cold tolerance in transgenic plants. Plant J 25:247–259
Knight H (2000) Calcium signaling during abiotic stress in plants. Int Rev Cytol 195:269–324
Koag MC, Fenton RD, Wilkens S, Close TJ (2003) The binding of maize DHN1 to lipid vesicles.
Gain of structure and lipid specificity. Plant Physiology 131:309–316
Kobayashi F, Ishibashi M, Takumi S (2008) Transcriptional activation of Cor/Lea genes and
increase in abiotic stress tolerance through expression of a wheat DREB2 homolog in trans-
genic tobacco. Transgen Res 17:755–767
Koh EJ, Song WY, Lee Y, Kim KH, Kim K, Chung N, Lee KW, Hong SW, Lee H (2006)
Expression of yeast cadmium factor 1 (YCF1) confers salt tolerance to Arabidopsis thaliana.
Plant Sci 170:534–541
Koniger M, Winter K (1993) Reduction of photosynthesis in sun leaves of Gossypium hirsutum L.
under conditions of high light intensities and suboptimal leaf temperatures. Agronomie 13:659
Kornyeyev D, Logan BA, Allen RD, Holaday AS (2003) Effect of chloroplastic overproduction of
ascorbate peroxidase on photosynthesis and photoprotection in cotton leaves subjected to low
temperature photoinhibition. Plant Sci 165:1033–1041
Kramer U, Chardonnens AN (2000) The use of transgenic plants in the bioremediation of soils
contaminated with trace elements. Appl Microbiol Biotechnol 55:661–672
Kudla J, Xu Q, Harter K, Gruissem W, Luan S (1999) Genes for calcineurin B-like proteins
in Arabidopsis are differentially regulated by stress signals. Proc Natl Acad Sci USA
96:4718–4723
Kunst L, Samuels AL (2003) Biosynthesis and secretion of plant cuticular wax. Prog Lipid Res
42:51–80
Lal S, Gulyani V, Khurana P (2008) Overexpression of HVA1 gene from barley generates tolerance
to salinity and water stress in transgenic mulberry (Morus indica). Transgen Res 17:651–663
Lambers H, Shane MW, Cramer MD, Pearse SJ, Veneklaas EJ (2006) Root structure and
functioning for efficient acquisition of phosphorus: matching morphological and physiological
traits. Ann Bot 98:693–713
Lea PJ, Azevedo RA (2006) Nitrogen use efficiency. Ann Appl Biol 149:243–247
Lee H, Jo J (2004) Increased tolerance to methyl viologen by transgenic tobacco plants that over-
express the cytosolic glutathione reductase gene from Brassica campestris. J Plant Biol
47:111–116
Lee GJ, Roseman AM, Saibil HR, Vierling E (1997) A small heat shock protein stably binds heat-
denatured model substrates and can maintain a substrate in a folding-competent state. EMBO J
16:659–671
Lee J-T, Prasad V, Yang PT, Wu JF, David Ho TH, Charng YY, Chan MT (2003a) Expression of
Arabidopsis CBF1 regulated by an ABA/stress inducible promoter in transgenic tomato
confers stress tolerance without affecting yield. Plant Cell Environ 26:1181–1190
Lee S, Moon JS, Ko TS, Petros D, Goldsbrough PB, Korban SS (2003b) Overexpression of
Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress.
Lee S, Petros D, Moon JS, Ko TS, Goldsbrough PB, Korban SS (2003c) Higher levels of ectopic
expression of Arabidopsis phytochelatin synthase do not lead to increased cadmium tolerance
and accumulation. Plant Physiol Biochem 41:903–910
Li Y, Dhankher OP, Carreira L, Lee D, Chen A, Schroeder JI, Balish RS, Meagher RB (2004)
Overexpression of phytochelatin synthase in Arabidopsis leads to enhanced arsenic tolerance
and cadmium hypersensitivity. Plant Cell Physiol 45:1787–1797
Li J, Yang H, Peer WA, Richter G, Blakeslee J, Bandyopadhyay A, Titapiwantakun B, Undurraga S,
Khodakovskaya M, Richards EL, Krizek B, Murphy AS, Gilroy S, Gaxiola R (2005a) Plant
science: Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ development. Science
310:121–125
Li Y, Dhankher OP, Carreira L, Balish RS, Meagher RB (2005b) Arsenic and mercury tolerance
and cadmium sensitivity in Arabidopsis plants expressing bacterial g-glutamylcysteine synthe-
tase. Environ Toxicol Chem 24:1376–1386
Li J, Guo J, Xu W, Ma M (2006) Enhanced cadmium accumulation in transgenic tobacco
expressing the phytochelatin synthase gene of Cynodon dactylon L. J Integr Plant Biol
48:928–937
Li B, Wei A, Song C, Li N, Zhang J (2008) Heterologous expression of the TsVP gene improves
the drought resistance of maize. Plant Biotechnol J 6:146–159
Liming Y, Qian Y, Pigang L, Sen L (2008) Expression of the HSP24 gene from Trichoderma
harzianum in Saccharomyces cerevisiae. J Therm Biol 33:1–6
Liu Q, Sakuma Y, Abe H, Kasuga M, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1998) Two
transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain,
separate two cellular signal transduction pathways in drought- and low temperature-responsive
gene expression, respectively, in Arabidopsis. Plant Cell 12:165–178
Liu JH, Kitashiba H, Wang J, Ban Y, Moriguchi T (2007) Polyamines and their ability to provide
environmental stress tolerance to plants. Plant Biotechnol 24:117–126
Lobell DB, Field CB (2007) Global scale climate – crop yield relationships and the impacts of
recent warming. Environ Res Lett 2:014002
Lv S, Yang A, Zhang K, Wang L, Zhang J (2007) Increase of glycinebetaine synthesis improves
drought tolerance in cotton. Mol Breed 20:233–248
Lynch DV (1990) Chilling injury in plants: the relevance of membrane lipids. In: Katterman F (ed)
Environmental injury to plants. Academic, New York, pp 17–34
Lyons JM (1973) Chilling injury in plants. Annu Rev Plant Physiol 24:445–466
Lyyra S, Meagher RB, Kim T, Heaton A, Montello P, Balish RS, Merkle SA (2007) Coupling two
mercury resistance genes in Eastern cottonwood enhances the processing of organomercury.
Plant Biotechnol J 5:254–262
Macek T, MackovaÌ M, PavliÌkovaÌ D, SzaÌkovaÌ J, Truksa M, Cundy AS, Kotrba P, Yancey N,
Scouten WH (2002) Accumulation of cadmium by transgenic tobacco. Acta Biotechnol
22:101–106
Madden EF, Sexton MM, Smith DR, Fowler BA (2002) Lead. In: Sarkar B (ed) Heavy metals in
the environment. Marcel Dekker, New York, pp 409–455
Mahajan S, Tuteja N (2005) Cold, salinity and drought stresses: an overview. Arch Biochem
Biophys 444:139–158
Maiorino FM, Brigelius-Flohe R, Aumann KD, Roveri A, Schomburg D, Flohe L (1995) Diversity
of glutathione peroxidases. Methods Enzymol 252:38–48
March TJ, Able JA, Schultz CJ, Able AJ (2007) A novel late embryogenesis abundant protein and
peroxidase associated with black point in barley grains. Proteomics 7:3800–3808
Margesin R, Neuner G, Storey KB (2007) Cold-loving microbes, plants, and animals – fundamen-
tal and applied aspects. Naturwissenschaften 94:77–99
Martinez M, Bernal P, Almela C, Velez D, Garcia-Agustin P, Serrano R, Navarro-Avino J (2006)
An engineered plant that accumulates higher levels of heavy metals than Thlaspi caerulescens,
with yields of 100 times more biomass in mine soils. Chemosphere 64:478–485
Martinez-Atienza J, Jiang X, Garciadeblas B, Mendoza I, Zhu JK, Pardo JM, Quintero FJ (2007)
Conservation of the salt overly sensitive pathway in rice. Plant Physiol 143:1001–1012
Martinez-Beltran J, Manzur CL (2005) Overview of salinity problems in the world and FAO
strategies to address the problem. In: Proceedings of International Salinity Forum, Riverside,
CA, USA, pp 311–313
Maruyama K, Sakuma Y, Kasuga M, Ito Y, Seki M, Goda H, Shimada Y, Yoshida S, Shinozaki K,
Yamaguchi-Shinozaki K (2004) Identification of cold-inducible downstream genes of the
Arabidopsis DREB1A/CBF3 transcriptional factor using two microarray systems. Plant J
38:982–993
Masle J, Gilmore SR, Farquhar GD (2005) The ERECTA gene regulates plant transpiration
efficiency in Arabidopsis. Nature 436:866–870
McCourt P, Creelman R (2008) The ABA receptors – we report you decide. Curr Opin Plant Biol
11:474–478
McLaughlin MJ, Parker DR, Clarke JM (1999) Metals and micronutrients – food safety issues.
Field Crops Res 60:143–163
McNeil SD, Nuccio ML, Hanson AD (1999) Betaines and related osmoprotectants. Targets for
metabolic engineering of stress resistance. Plant Physiol 120:945–949
Merlot S, Gosti F, Guerrier D, Vavasseur A, Giraudat J (2001) The ABI1 and ABI2 protein
phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling
pathway. Plant J 25:295–303
Miller J, McLachlan AD, Klug A (1985) Repetitive zinc-binding domains in the protein transcrip-
tion factor IIIA from Xenopus oocytes. EMBO J 4:1609–1614
Mills LN, Hunt L, Leckie CP, Aitken FL, Wentworth M, McAinsh MR, Gray JE, Hetherington AM
(2004) The effects of manipulating phospholipase C on guard cell ABA-signalling. J Exp Bot
55:199–204
Mittler R, Kim Y, Song L, Coutu J, Coutu A, Ciftci-Yilmaz S, Lee H, Stevenson B, Zhu JK (2006)
Gain- and loss-of-function mutations in Zat10 enhance the tolerance of plants to abiotic stress.
FEBS Lett 580:6537–6542
Miyazawa S-I, Yoshimura S, Shinzaki Y, Maeshima M, Miyake C (2008) Deactivation of
aquaporins decreases internal conductance to CO2 diffusion in tobacco leaves grown under
long-term drought. Funct Plant Biol 35:556–564
Mohanty A, Kathuria H, Ferjani A, Sakamoto A, Mohanty P, Murata N, Tyagi AK (2002)
Transgenics of an elite indica rice variety Pusa Basmati 1 harbouring the coda gene are highly
tolerant to salt stress. Theor Appl Genet 106:51–57
Mori S, Nishizawa N, Hayashi H, Chino M, Yoshimura E, Ishihara J (1991) Why are you rice
plants highly susceptible to iron-deficiency? Plant Soil 130:143–156
Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol 59:651–681
Munns R, Husain S, Rivelli AR, James RA, Condon AG, Lindsay MP, Lagudah ES, Schachtman DP,
Hare RA (2002) Avenues for increasing salt tolerance of crops, and the role of physiologically
based selection traits. Plant Soil 247:93–105
Nelson DE, Repetti PP, Adams TR, Creelman RA, Wu J, Warner DC, Anstrom DC, Bensen RJ,
Castiglioni PP, Donnarummo MG, Hinchey BS, Kumimoto RW, Maszle DR, Canales RD,
Krolikowski KA, Dotson SB, Gutterson N, Ratcliffe OJ, Heard JE (2007) Plant nuclear factor
Y (NF-Y) B subunits confer drought tolerance and lead to improved corn yields on water-
limited acres. Proc Natl Acad Sci USA 104:16450–16455
Nishida I, Murata N (1996) Chilling sensitivity in plants and cyanobacteria: the crucial contribu-
tion of membrane lipids. Annu Rev Plant Physiol Plant Mol Biol 47:541–568
Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control.
Annu Rev Plant Biol 49:249–279
Nordberg GFB, Sandstrom G, Becking F, Goyer RA (2002) Essentiality and toxicity of metals. In:
Sarkar B (ed) Heavy metals in the environment. Marcel Dekker, New York, pp 1–33
Oh SJ, Kwon CW, Choi DW, Song SI, Kim JK (2007) Expression of barley HvCBF4 enhances
tolerance to abiotic stress in transgenic rice. Plant Biotechnol J 5:646–656
Palta JP, Whitaker BD, Weiss LS (1993) Plasma membrane lipids associated with genetic
variability in freezing tolerance and cold acclimation of Solanum species. Plant Physiol
103:793–803
Park J-A, Cho SK, Kim JE, Chung HS, Hong J-P, Hwang B, Hong CB, Kim WT (2003) Isolation
of cDNAs differentially expressed in response to drought stress and characterization of the Ca-
LEAL1 gene encoding a new family of atypical LEA-like protein homologue in hot pepper
(Capsicum annuum L. cv. Pukang). Plant Sci 165:471–481
Park B-J, Liu Z, Kanno A, Kameya T (2005a) Genetic improvement of Chinese cabbage for
salt and drought tolerance by constitutive expression of a B. napus LEA gene. Plant Sci
169:553–558
Park BJ, Liu Z, Kanno A, Kameya T (2005b) Increased tolerance to salt- and water-deficit stress in
transgenic lettuce (Lactuca sativa L.) by constitutive expression of LEA. Plant Growth Regul
45:165–171
Park S, Li J, Pittman JK, Berkowitz GA, Yang H, Undurraga S, Morris J, Hirschi KD, Gaxiola RA
(2005c) Up-regulation of a H+-pyrophosphatase (H+-PPase) as a strategy to engineer drought-
resistant crop plants. Proc Natl Acad Sci USA 102:18830–18835
Park MR, Baek SH, de los Reyes BG, Yun SJ (2007) Overexpression of a high-affinity phosphate
transporter gene from tobacco (NtPT1) enhances phosphate uptake and accumulation in
transgenic rice plants. Plant Soil 292:259–269
Park H-Y, Seok H-Y, Park B-K, Kim S-H, Goh C-H, B-h L, Lee C-H, Moon Y-H (2008)
Overexpression of Arabidopsis ZEP enhances tolerance to osmotic stress. Biochem Biophys
Res Commun 375:80–85
Pasquali G, Biricolti S, Locatelli F, Baldoni E, Mattana M (2008) Osmyb4 expression
improves adaptive responses to drought and cold stress in transgenic apples. Plant Cell Rep
27:1677–1686
Pastori GM, Mullineaux PM, Foyer CH (2000) Post-transcriptional regulation prevents accumu-
lation of glutathione reductase protein and activity in the bundle sheath cells of maize. Plant
Physiol 122:667–675
Pauly N, Knight MR, Thuleau P, Van Der Luit AH, Moreau M, Trewavas AJ, Ranjeva R,
Mazars C (2000) Control of free calcium in plant cell nuclei. Nature 405:754–755
Paungfoo-Lonhienne C, Lonhienne TGA, Rentsch D, Robinson N, Christie M, Webb RI,
Gamage HK, Carroll BJ, Schenk PM, Schmidt S (2008) Plants can use protein as a nitrogen
source without assistance from other organisms. Proc Natl Acad Sci USA 105:4524–4529
Pavlikova D, Macek T, MackovaÌ M, Sura M, Szakova J, Tlustos P (2004a) The evaluation of
cadmium, zinc and nickel accumulation ability of transgenic tobacco bearing different trans-
genes. Plant, Soil and Environ 50:513–517
Pavlikova D, Macek T, MackovaÌ M, Szakova J, Balik J (2004b) Cadmium tolerance and
accumulation in transgenic tobacco plants with a yeast metallothionein combined with a
polyhistidine tail. International Biodeterioration and Biodegradation 54:233–237
Peng LX, Gu LK, Zheng CC, Li DQ, Shu HR (2006) Expression of MaMAPK gene in seedlings of
Malus L. under water stress. Acta Biochim Biophys Sin 38:281–286
Perruc E, Charpenteau M, Ramirez BC, Jauneau A, Galaud JP, Ranjeva R, Ranty B (2004) A novel
calmodulin-binding protein functions as a negative regulator of osmotic stress tolerance in
Arabidopsis thaliana seedlings. Plant J 38:410–420
Pilon-Smits E, Pilon M (2002) Phytoremediation of metals using transgenic plants. Crit Rev Plant
Sci 21:439–456
Pino MT, Skinner JS, Jeknic Z, Hayes PM, Soeldner AH, Thomashow MF, Chen THH (2008)
Ectopic AtCBF1 over-expression enhances freezing tolerance and induces cold acclimation-
associated physiological modifications in potato. Plant Cell Environ 31:393–406
Pomponi M, Censi V, Di Girolamo V, De Paolis A, Di Toppi LS, Aromolo R, Costantino P,

Cardarelli M (2006) Overexpression of Arabidopsis phytochelatin synthase in tobacco plants
enhances Cd2+ tolerance and accumulation but not translocation to the shoot. Planta 223:180–190
Porcel R, Gomez M, Kaldenhoff R, Ruiz-Lozano JM (2005) Impairment of NtAQP1 gene
expression in tobacco plants does not affect root colonisation pattern by arbuscular mycorrhizal
fungi but decreases their symbiotic efficiency under drought. Mycorrhiza 15:417–423
Prasad MNV (2002) Phytoremediation of metal-polluted ecosystems: hype for commercialisation.
Russ J Plant Physiol 50:686–700
Prashanth SR, Sadhasivam V, Parida A (2008) Over expression of cytosolic copper/zinc superox-
ide dismutase from a mangrove plant Avicennia marina in indica Rice var Pusa Basmati-1
confers abiotic stress tolerance. Transgen Res 17:281–291
Puhakainen T, Hess MW, Mäkelä P, Svensson J, Heino P, Palva ET (2004) Overexpression of
multiple dehydrin genes enhances tolerance to freezing stress in Arabidopsis. Plant Mol Biol
54:743–753
Purvis AC, Shewfelt RL (1993) Does the alternative pathway ameliorate chilling injury in
sensitive plant tissues? Physiol Plant 88:712–718
Qin QL, Liu JG, Zhang Z, Peng RH, Xiong AS, Yao QH, Chen JM (2007) Isolation, optimization,
and functional analysis of the cDNA encoding transcription factor OsDREB1B in Oryza Sativa
L. Mol Breed 19:329–340
Qiu QS, Guo Y, Dietrich MA, Schumaker KS, Zhu JK (2002) Regulation of SOS1, a plasma
membrane Na+/H+ exchanger in Arabidopsis thaliana, by SOS2 and SOS3. Proc Natl Acad Sci
USA 99:8436–8441
Quan R, Shang M, Zhang H, Zhao Y, Zhang J (2004) Engineering of enhanced glycine betaine
synthesis improves drought tolerance in maize. Plant Biotechnol J 2:477–486
Rae AL, Jarmey JM, Mudge SR, Smith FW (2004) Over-expression of a high-affinity phosphate
transporter in transgenic barley plants does not enhance phosphate uptake rates. Funct Plant
Biol 31:141–148
Ramanjulu S, Bartels D (2002) Drought- and desiccation-induced modulation of gene expression
in plants. Plant Cell Environ 25:141–151
Reddy VS, Reddy ASN (2004) Proteomics of calcium-signaling components in plants. Phyto-
chemistry 65:1745–1776
Reiser V, Raitt D, Saito H (2003) Yeast osmosensor Sln1 and plant cytokinin receptor Cre1
respond to changes in turgor pressure. Yeast 20:S169
Reisinger S, Schiavon M, Terry N, Pilon-Smits EAH (2008) Heavy metal tolerance and accumu-
lation in Indian mustard (Brassica juncea L.) expressing bacterial g-glutamylcysteine synthe-
tase or glutathione synthetase. Int J Phytoremediation 10:440–454
Remans T, Nacry P, Pervent M, Filleur S, Diatloff E, Mounier E, Tillard P, Forde BG, Gojon A
(2006) The Arabidopsis NRT1.1 transporter participates in the signaling pathway triggering
root colonization of nitrate-rich patches. Proc Natl Acad Sci USA 103:19206–19211
Rengasamy P (2006) World salinization with emphasis on Australia. J Exp Bot 57:1017–1023
Rios G, Ferrando A, Serrano R (1997) Mechanisms of salt tolerance conferred by overexpression
of the HAL1 gene in Saccharomyces cerevisiae. Yeast 13:515–528
Rivero RM, Kojima M, Gepstein A, Sakakibara H, Mittler R, Gepstein S, Blumwald E (2007)
Delayed leaf senescence induces extreme drought tolerance in a flowering plant. Proc Natl
Acad Sci USA 104:19631–19636
Robinson NJ, Urwin PE, Robinson PJ, Jackson PJ (1994) Gene expression in relation to meatl
toxicity and tolerance. In: Basra AS (ed) Stress-Induced Gene Expression in Plants. Harwoon,
Switzerland, pp 209–248
Robinson BSG, MIlls T, Clothier B, van der Velde M, Laplane R, Fung L, Deurer M, Hurst S,
Thayalakumaran T, van der Dijssel C (2003) Phytoremediation: using plants as biopumps to
improve degraded environments. Aust J Soil Res 41:599–612
Rohila JS, Jain RK, Wu R (2002) Genetic improvement of Basmati rice for salt and drought
tolerance by regulated expression of a barley Hva1 cDNA. Plant Sci 163:525–532
Rosselli W, Keller C, Boschi K (2003) Phytoextraction capacity of trees growing on a metal
contaminated soil. Plant Soil 256:265–272
Roughan PG, Slack CR (1982) Cellular organization of glycerolipid metabolism. Annu Rev Plant
Physiol 33:97–132
Rugh CL, Senecoff JF, Meagher RB, Merkle SA (1998) Development of transgenic yellow poplar
for mercury phytoremediation. Nat Biotechnol 16:925–928
Rugh CL, Bizily SP, Meagher RB (2000) Phytoreduction of environmental mercury pollution. In:
Raskin I, Ensley BD (eds) Phytoremediation of toxic metals: using plants to clean up the
environment. John Wiley, New York, pp 151–169
Rus AM, Estan MT, Gisbert C, Garcia-Sogo B, Serrano R, Caro M, Moreno V, Bolarin MC (2001)
Expressing the yeast HAL1 gene in tomato increases fruit yield and enhances K+/Na+ selectiv-
ity under salt stress. Plant Cell Environ 24:875–880
Sakai A, Larcher W (1987) Frost survival of plants. Responses and adaptation to freezing stress.
Springer-Verlag, Berlin
Sakamoto A, Alia MN (1998) Metabolic engineering of rice leading to biosynthesis of glycine-
betaine and tolerance to salt and cold. Plant Mol Biol 38:1011–1019
Sakamoto A, Murata N (2000) Genetic engineering of glycinebetaine synthesis in plants: current
status and implications for enhancement of stress tolerance. J Exp Bot 51:81–88
Sakuma Y, Liu Q, Dubouzet JG, Abe H, Shinozaki K, Yamaguchi-Shinozaki K (2002) DNA-
binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors
involved in dehydration- and cold-inducible gene expression. Biochem Biophys Res Commun
290:998–1009
Sakuma Y, Maruyama K, Qin F, Osakabe Y, Shinozaki K, Yamaguchi-Shinozaki K (2006a) Dual
function of an Arabidopsis transcription factor DREB2A in in water-stress-responsive and
heat-stress-responsive gene expression. Proc Natl Acad Sci USA 103:18822–18827
Sakuma Y, Maruyama K, Qin F, Osakabe Y, Shinozaki K, Yamaguchi-Shinozaki K (2006b)
Functional analysis of an Arabidopsis transcription factor DREB2A drought-responsive gene
expression. Plant Cell 18:1292–1309
Sang Y, Zheng S, Li W, Huang B, Wang X (2001) Regulation of plant water loss by manipulating
the expression of phospholipase D. Plant J 28:135–144
Schwartz SH, Qin X, Zeevaart JAD (2003) Elucidation of the indirect pathway of abscisic acid
biosynthesis by mutants, genes, and enzymes. Plant Physiol 131:1591–1601
Scott Russell R (1977) Plant root systems: their function and interaction with the soil. McGraw-
Hill, London, UK
Segele ZT, Lamb PJ (2005) Characterization and variability of Kiremt rainy season over Ethiopia.
Meteorol Atmos Phys 89:153–180
Seiffert B, Zhou ZW, Wallbraun M, Lohaus G, Mollers C (2004) Expression of a bacterial
asparagine synthetase gene in oilseed rape (Brassica napus) and its effect on traits related to
nitrogen efficiency. Physiol Plant 121:656–665
Seki M, Kamei A, Yamaguchi-Shinozaki K, Shinozaki K (2003) Molecular responses to drought,
salinity and frost: common and different paths for plant protection. Curr Opin Biotechnol
14:194–199
Seleshi Y, Camberlin P (2006) Recent changes in dry spell and extreme rainfall events in Ethiopia.
Theor Appl Clim 83:181–191
Seo HM, Jung Y, Song S, Kim Y, Kwon T, Kim DH, Jeung SJ, Yi YB, Yi G, Nam MH, Nam J
(2008) Increased expression of OsPT1, a high-affinity phosphate transporter, enhances phos-
phate acquisition in rice. Biotechnol Lett 30:1833–1838
Serrano R, Mulet JM, Rios G, Marquez JA, De Larrinoa IF, Leube MP, Mendizabal I, Pascual-
Ahuir A, Proft M, Ros R, Montesinos C (1999) A glimpse of the mechanisms of ion
homeostasis during salt stress. J Exp Bot 50:1023–1036
Shi WM, Muramoto Y, Ueda A, Takabe T (2001) Cloning of peroxisomal ascorbate peroxidase
gene from barley and enhanced thermotolerance by overexpressing in Arabidopsis thaliana.
Gene 273:23–27
Shi H, Lee BH, Wu SJ, Zhu JK (2003) Overexpression of a plasma membrane Na+/H+ antiporter
gene improves salt tolerance in Arabidopsis thaliana. Nat Biotechnol 21:81–85
Shi LY, Li HQ, Pan XP, Wu GJ, Li MR (2008) Improvement of Torenia fournieri salinity
tolerance by expression of Arabidopsis AtNHX5. Funct Plant Biol 35:185–192
Shinozaki K, Yamaguchi-Shinozaki K (1996) Molecular responses to drought and cold stress. Curr
Opin Biotechnol 7:161–167
Shinozaki K, Yamaguchi-Shinozaki K (1999) Molecular responses to drought stress. In: Shinozaki
K, Yamaguchi-Shinozaki K (eds) Molecular responses to cold, drought. Heat and salt stress in
higher plants. R. G. Landes, Austin, pp 11–28
Shinozaki K, Yamaguchi-Shinozaki K (2000) Molecular responses to dehydration and low
temperature: differences and cross talk between two stress signalling pathways. Curr Opin
Plant Biol 3:217–233
Sickler CM, Edwards GE, Kiirats O, Gao Z, Loescher W (2007) Response of mannitol-producing
Arabidopsis thaliana to abiotic stress. Funct Plant Biol 34:382–391
Sivamani E, Bahieldin A, Wraith JM, Al-Niemi T, Dyer WE, Ho T-HD QuR (2000) Improved
biomass productivity and water use efficiency under water deficit conditions in transgenic
wheat constitutively expressing the barley HVA1 gene. Plant Sci 155:1–9
Smirnoff N (1998) Plant resistance to environmental stress. Curr Opin Biotechnol 9:214–219
Sneddon WA, Fromm H (1998) Calmodulin, calmodulin-regulated proteins and plant responses to
the environment. Trends Plant Sci 3:299–304
Sneddon WA, Fromm H (2001) Calmodulin as a versatile calcium signal transducer in plants. New
Phytol 151:35–66
Song W-Y, Sohn EJ, Martinoia E, Lee YJ, Yang Y-Y, Jasinski M, Farestier C, Hwang I, Lee Y
(2003) Engineering tolerance and accumulation of lead and cadmium in transgenic plants. Nat
Sonoike K (1996) Photoinhibition of photosystem I: its physiological significance in the chilling
sensitivity of plants. Plant Cell Physiol 37:239–247
Souer E, Van Houwelingen A, Kloos D, Mol J, Koes R (1996) The no apical Meristem gene of
petunia is required for pattern formation in embryos and flowers and is expressed at meristem
and primordia boundaries. Cell 85:159–170
Spollen WG, Nelson CJ (1994) Response of fructan to water deficit in growing leaves of tall
fescue. Plant Physiol 106:329–336
Staxen I, Pical C, Montgomery LT, Gray JE, Hetherington AM, McAinsh MR (1999) Abscisic
acid induces oscillations in guard-cell cytosolic free calcium that involve phosphoinositide-
specific phospholipase C. Proc Natl Acad Sci USA 96:1779–1784
Stitt M, Hurry V (2002) A plant for all seasons: alterations in photosynthetic carbon metabolism
during cold acclimation in Arabidopsis. Curr Opin Plant Biol 5:199–206
Stockinger EJ, Gilmour SJ, Thomashow MF (1997) Arabidopsis thaliana CBF1 encodes an AP2
domain-containing transcription activator that binds to the C-repeat/DRE, a cis-acting DNA
regulatory element that stimulates transcription in response to low temperature and water
deficit. Proc Natl Acad Sci USA 94:1035–1040
Strand A, Foyer CH, Gustafsson P, Gardestrom P, Hurry V (2003) Altering flux through the
sucrose biosynthesis pathway in transgenic Arabidopsis thaliana modifies photosynthetic
acclimation at low temperatures and the development of freezing tolerance. Plant Cell Environ
26:523–535
Strom AR, Kaasen I (1993) Trehalose metabolism in Escherichia coli: stress protection and stress
regulation of gene expression. Mol Microbiol 8:205–210
Su J, Wu R (2004) Stress-inducible synthesis of proline in transgenic rice confers faster growth
under stress conditions than that with constitutive synthesis. Plant Sci 166:941–948
Sugano S, Kaminaka H, Rybka Z, Catala R, Salinas J, Matsui K, Ohme-Takagi M, Takatsuji H
(2003) Stress-responsive zinc finger gene ZPT2–3 plays a role in drought tolerance in petunia.
Plant J 36:830–841
Sui N, Li M, Zhao SJ, Li F, Liang H, Meng QW (2007) Overexpression of glycerol-3-phosphate
acyltransferase gene improves chilling tolerance in tomato. Planta 226:1097–1108
Sun HK, Hyun SL, Won YS, Kwan SC, Hur Y (2007) Chloroplast-targeted BrMT1 (Brassica rapa
type-1 metallothionein) enhances resistance to cadmium and ROS in transgenic Arabidopsis
plants. J Plant Biol 50:1–7
Suzuki M, Morikawa KC, Nakanishi H, Takahashi M, Saigusa M, Mori S, Nishizawa NK (2008)

Transgenic rice lines that include barley genes have increased tolerance to low iron availability
in a calcareous paddy soil. Soil Sci Plant Nutr 54:77–85
Szabolcs I (1989) Salt-affected soils. CRC, Boca Raton, FL
Szalontai B, Kota Z, Nonaka H, Murata N (2003) Structural consequences of genetically engi-
neered saturation of the fatty acids of phosphatidylglycerol in tobacco thylakoid membranes.
An FTIR study. Biochemistry 42:4292–4299
Takahashi M, Nakanishi H, Kawasaki S, Nishizawa NK, Mori S (2001) Enhanced tolerance of rice
to low iron availability in alkaline soils using barley nicotianamine aminotransferase genes.
Takahiro I, Sakai K, Takeda T, Shigeoka S (1995) Cloning and expression of cDNA encoding a
new type of ascorbate peroxidase from spinach. FEBS Lett 367:28–32
Tamura T, Hara K, Yamaguchi Y, Koizumi N, Sano H (2003) Osmotic stress tolerance of
transgenic tobacco expressing a gene encoding a membrane-located receptor-like protein
from tobacco plants. Plant Physiol 131:454–462
Tester M, Davenport R (2003) Na+ tolerance and Na+ transport in higher plants. Ann Bot
91:503–527
Thomashow MF (1998) Role of cold-responsive genes in plant freezing tolerance. Plant Physiol
118:1–7
Thomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechan-
isms. Annu Rev Plant Biol 50:571–599
Tonkovska G, Atanassov A, Atanassov I (2003) The promoter region of Arabidopsis metallothio-
nein MT2a gene is strongly induced by treatment with CuII, ZnII and Cd II ions in transgenic
Nicotiana benthamiana plants. Biotechnology and Biotechnological Equipment 17:134–139
Torok Z, Goloubinoff P, Horvath I, Tsvetkova NM, Glatz A, Balogh G, Varvasovszki V, Los DA,
Vierling E, Crowe JH, Vigh L (2001) Synechocystis HSP17 is an amphitropic protein that
stabilizes heat-stressed membranes and binds denatured proteins for subsequent chaperone-
mediated refolding. Proc Natl Acad Sci USA 98:3098–3103
Trujillo LE, Sotolongo M, Menendez C, Ochogavia ME, Coll Y, Hernandez I, Borras-Hidalgo O,
Thomma BPHJ, Vera P, Hernandez L (2008) SodERF3, a novel sugarcane ethylene responsive
factor (ERF), enhances salt and drought tolerance when overexpressed in tobacco plants. Plant
Cell Physiol 49:512–525
Tuberosa R, Salvi S (2006) Genomics-based approaches to improve drought tolerance of crops.
Trends Plant Sci 11:405–412
UN-Oceans (2008) Atlas of the oceans. http://www.oceansatlas.org/
Urao T, Katagiri T, Mizoguchi T, Yamaguchi-Shinozaki K, Hayashida N, Shinozaki K (1999) A
transmembrane hybrid-type histidine kinase in Arabidopsis functions as an osmosensor. Plant
Cell 11:1743–1754
Valls M, Atrian S, de Lorenzo V, Fernandez LA (2000) Engineering a mouse metallothionein on
the cell surface of Ralstonia eutrophia CH34 for immobilisation of heavy metals in soil. Nat
van Buskirk HA, Thomashow MF (2006) Arabidopsis transcription factors regulating cold accli-
mation. Physiol Plant 126:72–80
Vidal EA, Gutierrez RA (2008) A systems view of nitrogen nutrient and metabolite responses in
Arabidopsis. Curr Opin Plant Biol 11:521–529
Vierling E (1991) The roles of heat shock proteins in plants. Annu Rev Plant Physiol Plant Mol
Biol 42:579–620
Vierling E, Kimpel JA (1992) Plant responses to environmental stress. Curr Opin Biotechnol
3:164–170
Vij S, Tyagi AK (2007) Emerging trends in the functional genomics of the abiotic stress response
in crop plants: review article. Plant Biotechnol J 5:361–380
Vijn I, Smeekens S (1999) Fructan: more than a reserve carbohydrate? Plant Physiol 120:351–359
Vincent R, Fraisier V, Chaillou S, Limami MA, Deleens E, Phillipson B, Douat C, Boutin JP, Hirel
B (1997) Overexpression of a soybean gene encoding cytosolic glutamine synthetase in shoots
of transgenic Lotus corniculatus L plants triggers changes in ammonium assimilation and plant
development. Planta 201:424–433
Vinocur B, Altman A (2005) Recent advances in engineering plant tolerance to abiotic stress:
achievements and limitations. Curr Opin Biotechnol 16:123–132
von Wiren N, Mori S, Marschner H, Romheld V (1994) Iron inefficiency in maize mutant ys1 (Zea
mays L. cv. Yellow Stripe) is caused by a defect in uptake of iron phytosiderophores. Plant
Physiol 106:71–77
Wagner AM, Wagner MJ, Moore AL (1998) In vivo ubiquinone reduction levels during thermo-
genesis in araceae. Plant Physiol 117:1501–1506
Wakui K, Takahata Y (2002) Isolation and expression of Lea gene in desiccation-tolerant
microspore-derived embryos in Brassica spp. Physiol Plant 116:223–230
Wang WX, Vinocur B, Shoseyov O, Altman A (2001) Biotechnology of plant osmotic stress
tolerance: physiological and molecular considerations. Acta Hortic 560:285–292
Wang RC, Okamoto M, Xing XJ, Crawford NM (2003a) Microarray analysis of the nitrate response
in Arabidopsis roots and shoots reveals over 1, 000 rapidly responding genes and new linkages
to glucose, trehalose-6-phosphate, iron, and sulfate metabolism. Plant Physiol 132:556–567
Wang W, Vinocur B, Altman A (2003b) Plant responses to drought, salinity and extreme
temperatures: towards genetic engineering for stress tolerance. Planta 218:1–14
Wang Y, Jiang J, Zhao X, Liu G, Yang C, Zhan L (2006) A novel LEA gene from Tamarix
androssowii confers drought tolerance in transgenic tobacco. Plant Sci 171:655–662
Wang M, Gu D, Liu T, Wang Z, Guo X, Hou W, Bai Y, Chen X, Wang G (2007a) Overexpression
of a putative maize calcineurin B-like protein in Arabidopsis confers salt tolerance. Plant Mol
Biol 65:733–746
Wang RC, Xing XJ, Crawford N (2007b) Nitrite acts as a transcriptome signal at micromolar
concentrations in Arabidopsis roots. Plant Physiol 145:1735–1745
Wang CR, Yang AF, Yue GD, Gao Q, Yin HY, Zhang JR (2008a) Enhanced expression of phospho-
lipase C 1 (ZmPLC1) improves drought tolerance in transgenic maize. Planta 227:1127–1140
Wang L, Li X, Chen S, Liu G (2008b) Enhanced drought tolerance in transgenic Leymus chinensis
plants with constitutively expressed wheat TaLEA 3. Biotechnol Lett 31:1–7
Wasilewska A, Vlad F, Sirichandra C, Redko Y, Jammes F, Valon C, Frey NFD, Leung J (2008)
An update on abscisic acid signaling in plants and more. Mol Plant 1:198–217
Watanabe M, Shinmachi F, Noguchi A, Hasegawa I (2005) Introduction of yeast metallothionein
gene (CUP1) into plant and evaluation of heavy metal tolerance of transgenic plant at the callus
stage. Soil Sci Plant Nutr 51:129–133
Waters ER, Lee GJ, Vierling E (1996) Evolution, structure and function of the small heat shock
proteins in plants. J Exp Bot 47:325–338
Wawrzynski A, Kopera E, Wawrzynska A, Kaminska J, Bal W, Sirko A (2006) Effects of
simultaneous expression of heterologous genes involved in phytochelatin biosynthesis on
thiol content and cadmium accumulation in tobacco plants. J Exp Bot 57:2173–2182
Weber S, Wolter FP, Buck F, Frentzen M, Heinz E (1991) Purification and cDNA sequencing of an
oleate-selective acyl-ACP: sn-glycerol-3-phosphate acyltransferase from pea chloroplasts.
Plant Mol Biol 17:1067–1076
Weigel P, Weretilnyk EA, Hanson AD (1986) Betaine aldehyde oxidation by spinach chloroplasts.
Wiemken A (1990) Trehalose in yeast, stress protectant rather than reserve carbohydrate. Int J Gen
Mol Microbiol 58:209–217
Williamson JD, Jennings DB, Guo WW, Pharr DM, Ehrenshaft M (2002) Sugar alcohols, salt
stress, and fungal resistance: polyols – multifunctional plant protection? J Am Soc Hort Sci
127:467–473
Wintergerst ES, Maggini S, Hornig DH (2007) Contribution of selected vitamins and trace
elements to immune function. Ann Nutr Metab 51:301–323
Wise MJ (2003) LEAping to conclusions: a computational reanalysis of late embryongenesis

abundant proteins and their possible roles. BMC Bioinformatics 4:52
World Water Council (2008) Water crisis. In: Waord Water Council (ed) Water at a Glance. World
Water Council, Marseilles
Wu W, Su Q, Xia X, Wang Y, Luan Y, An L (2008a) The Suaeda liaotungensis kitag betaine
aldehyde dehydrogenase gene improves salt tolerance of transgenic maize mediated with
minimum linear length of DNA fragment. Euphytica 159:17–25
Wu X, Shiroto Y, Kishitani S, Ito Y, Toriyama K (2008b) Enhanced heat and drought tolerance in
transgenic rice seedlings overexpressing OsWRKY11 under the control of HSP101 promoter.
Plant Cell Rep 28:21–30
Xiao B, Huang Y, Tang N, Xiong L (2007) Over-expression of a LEA gene in rice improves
drought resistance under the Weld conditions. Theor Appl Genet 115:35–46
Xin Z, Browse J (2000) Cold comfort farm: the acclimation of plants to freezing temperatures.
Plant Cell Environ 23:893–902
Xiong L, Ishitani M (2006) Stress signal transduction: components, pathways, and network
integration. In: Rai AK, Takabe T (eds) Abiotic stress tolerance in plants: toward the improve-
ment of global environment and food. Springer, Dordercht, The Netherlands, pp 3–29
Xu D, Duan X, Wang B, Hong B, Ho T-HD WuR (1996) Expression of a late embryogenesis
abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in
transgenic rice. Plant Physiol 110:249–257
Xu DQ, Huang J, Guo SQ, Yang X, Bao YM, Tang HJ, Zhang HS (2008a) Overexpression of a
TFIIIA-type zinc finger protein gene ZFP252 enhances drought and salt tolerance in rice
(Oryza sativa L.). FEBS Lett 582:1037–1043
Xu WF, Shi WM, Ueda A, Takabe T (2008b) Mechanisms of salt tolerance in transgenic
Arabidopsis thaliana carrying a peroxisomal ascorbate peroxidase gene from Barley. 1 project
supported by a grant of CAS (Chinese Academy of Sciences) Research program on soil
biosystems and agro-product safety (No. CXTD-Z2005-4) and the knowledge innovation
project of CAS (No. KZCX3-SW-439). Pedosphere 18:486–495
Yamaguchi T, Blumwald E (2005) Developing salt-tolerant crop plants: challenges and opportu-
nities. Trends Plant Sci 10:615–620
Yamaguchi-Shinozaki K, Shinozaki K (1994) A novel cis-acting element in an Arabidopsis gene is
involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 6:251–264
Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN (1982) Living with water stress:
evolution of osmolyte systems. Science 217:1214–1222
Yeo AR, Lee KS, Izard P, Boursier PJ, Flowers TJ (1991) Short- and long-term effects of salinity
on leaf growth in rice (Oryza sativa L.). J Exp Bot 42:881–889
Young IC, Eun WN, Hyo SL, Mu SH, Jae SL, Kwan SC (2007) Mercury-tolerant transgenic
poplars expressing two bacterial mercury-metabolizing genes. J Plant Biol 50:658–662
Yu Q, Hu Y, Li J, Wu Q, Lin Z (2005) Sense and antisense expression of plasma membrane
aquaporin BnPIP1 from Brassica napus in tobacco and its effects on plant drought resistance.
Plant Sci 169:647–656
Yu H, Chen X, Hong YY, Wang Y, Xu P, Ke SD, Liu HY, Zhu JK, Oliver DJ, Xiang CB (2008)
Activated expression of an Arabidopsis HD-START protein confers drought tolerance with
improved root system and reduced stomatal density. Plant Cell 20:1134–1151
Zhang L, Ohta A, Takagi M, Imai R (2000) Expression of plant group 2 and group 3 lea genes in
Saccharomyces cerevisiae revealed functional divergence among LEA Proteins. J Biochem
127:611–616
Zhang JZ, Creelman RA, Zhu J-K (2004) From laboratory to field. Using information from
Arabidopsis to engineer salt, cold, and drought tolerance in crops. Plant Physiol 135:615–621
Zhang JY, Broeckling CD, Blancaflor EB, Sledge MK, Sumner LW, Wang ZY (2005) Over-
expression of WXP1, a putative Medicago truncatula AP2 domain-containing transcription
factor gene, increases cuticular wax accumulation and enhances drought tolerance in trans-
genic alfalfa (Medicago sativa). Plant J 42:689–707
Zhang A, Jiang M, Zhang J, Tan M, Hu X (2006) Mitogen-activated protein kinase is involved in

abscisic acid-induced antioxidant defense and acts downstream of reactive oxygen species
production in leaves of maize plants. Plant Physiol 141:475–487
Zhang A, Jiang M, Zhang J, Ding H, Xu S, Hu X, Tan M (2007a) Nitric oxide induced by hydrogen
peroxide mediates abscisic acid-induced activation of the mitogen-activated protein kinase
cascade involved in antioxidant defense in maize leaves. New Phytol 175:36–50
Zhang JY, Broeckling CD, Sumner LW, Wang ZY (2007b) Heterologous expression of two
Medicago truncatula putative ERF transcription factor genes, WXP1 and WXP2, in Arabidopsis
led to increased leaf wax accumulation and improved drought tolerance, but differential
response in freezing tolerance. Plant Mol Biol 64:265–278
Zhang YX, Xu J, Wang X, Chai TY (2007c) Research advances in drought resistance and heavy
metals tolerance of transgenic plant. Chin J Appl Ecol 18:1631–1639
Zhang YY, Li Y, Gao T, Zhu H, D-j W, H-w Z, Y-s N, Liu L-j Wu, Y-r Chu C-c, H-s G, Xie Q
(2008a) Arabidopsis SDIR1 enhances drought tolerance in crop plants. Biosci Biotechnol
Biochem 72:2251–2254
Zhang J, Tan W, Yang XH, Zhang HX (2008b) Plastid-expressed choline monooxygenase gene
improves salt and drought tolerance through accumulation of glycine betaine in tobacco. Plant
Cell Rep 27:1113–1124
Zhang Y, Wang Z, Chai T, Wen Z, Zhang H (2008c) Indian mustard aquaporin improves drought
and heavy-metal resistance in tobacco. Mol Biotechnol 40:1–13
Zhang Y, Yang J, Lu S, Cai J, Guo Z (2008d) Overexpressing SgNCED1 in tobacco increases ABA
level, antioxidant enzyme activities, and stress tolerance. J Plant Growth Regul 27:151–158
Zhao F, Guo S, Zhang H, Zhao Y (2006) Expression of yeast SOD2 in transgenic rice results in
increased salt tolerance. Plant Sci 170:216–224
Zheng ZL, Nafisi M, Tam A, Li HM, Crowell DN, Chary SN, Schroeder JI, Shen J, Yang Z (2002)
Plasma membrane associated ROP10 small GTPase is a specific negative regulator of abscisic
acid responses in Arabidopsis. Plant Cell 14:2787–2797
Zhigang A, Cuijie L, Yuangang Z, Yejie D, Wachter A, Gromes R, Rausch T (2006) Expression of
BjMT2, a metallothionein 2 from Brassica juncea, increases copper and cadmium tolerance in
Escherichia coli and Arabidopsis thaliana, but inhibits root elongation in Arabidopsis thaliana
seedlings. J Exp Bot 57:3575–3582
Zhu JK (2001) Cell signaling under salt, water and cold stresses. Curr Opin Plant Biol 4:401–406
Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol
53:247–273
Zhu JK (2003) Regulation of ion homeostasis under salt stress. Curr Opin Plant Biol 6:441–445
Zhu JK, Hasegawa PM, Bressan RA (1997) Molecular aspects of osmotic stress in plants. Crit Rev
Zhu YL, Pilon-Smits E, Jouanin L, Terry N (1999) Overexpression of glutathione synthetase in
Indian mustard enhances cadmium accumulation and tolerance. Plant Physiol 119:73–79
Chapter 3
Transgenic Crops for Herbicide Resistance
Stephen O. Duke and Antonio L. Cerdeira
3.1 Introduction
A year after the introduction of the first commercial transgenic crop (Flavr Savr™
tomato with a longer shelf life) in 1994, transgenic, herbicide-resistant crops
(HRCs) were introduced (Table 3.1) with the introduction of bromoxynil- (3,5-
dibromo-4-hydroxybenzonitrile) resistant cotton and glufosinate- [2-amino-4-
(hydroxymethylphosphinyl)butanoic acid] resistant canola. Bromoxynil resistance
had little market penetration during the years when it was available. The next year,
1996, marked the introduction of the first glyphosate- [N-(phosphonomethyl) gly-
cine] resistant (GR) crop (soybean). Other GR and glufosinate-resistant crops were
introduced in the subsequent years. GR crops now represent well over 80% of all
transgenic crops grown worldwide (James 2008). Accordingly, this chapter will
deal primarily with GR crops. Several reviews (e.g., Duke 2005; Duke and Cerdeira
2005; Cerdeira and Duke 2006) and two books (McClean and Evans 1995; Duke
1996) are available on the topic of HRCs, but this rapidly evolving topic requires
timely updates.
Before the advent of transgenic crops, there was both controversy and optimism
about their potential impact on farming, human health, and the environment (e.g.,
Goldberg et al. 1990; Duke et al. 1991). We have now (early 2009) had 14 years of
experience with HRCs over vast areas in many parts of the world, providing a
wealth of information on the utilization of this technology, as well as the impacts of
HRCs on the environment. This review will deal, in part, with these topics as they
apply to HRCs
S.O. Duke (*)

Agricultural Research Service, United States Department of Agriculture, University of Mississippi,
MS 38677, USA
e-mail: sduke@olemiss.edu

DOI 10.1007/978-3-642-04812-8_3, # Springer‐Verlag Berlin Heidelberg 2010
134 S.O. Duke and A.L. Cerdeira
Table 3.1 Transgenic herbicide-resistant crops that have been

deregulated (approved for sale) in North America
Herbicide Crop Year approved
Bromoxynila Cotton 1995
Canola 2000
Glufosinate Canola 1995
Maize 1997
Cotton 2004
Riceb 2006
Glyphosate Soybean 1996
Canola 1996
Cotton 1997
Maize 1998
Sugarbeetc 1999
Alfalfad 2005
a
Removed from market
b
Deregulated, but not commercialized
c
Removed from market, but reintroduced in 2008
d
Returned to regulated status in 2007 by court
3.2 Present HRCS and Their Impact
3.2.1 Commercially Available HRCs
In 2008, after 14 years of HRCs, there are only nine different HRCs being grown in
the US (Table 3.1), and only a few of these are grown in other countries. From 1995
until 2000, one or two new HRCs were introduced to the market every year, after
which the number of introductions has dwindled, with only a single new HRC in
occasional subsequent years.
The adoption rate of GR soybean was rapid in the US (Fig. 3.1), currently
representing more than 90% of the area planted in soybean. The adoption rate of
GR soybean in Argentina was even more rapid, reaching almost 90% adoption
within 4 years of introduction (Penna and Lema 2003). Adoption of this HRC has
also been rapid in other parts of South America.
Both cotton and maize have varieties that are either stand-alone GR varieties or
varieties that combine GR and transgenic Bt (Bacillus thuriengensis toxin) traits for
insect resistance. In both the crops, there are also stand-alone Bt toxin varieties. To
generate the data in Fig. 3.1, adoption rates of the two types of GR varieties must be
added. GR cotton adoption was initially similar to that of soybean, but it has
stabilized at about 70% (Fig. 3.1), partly because of the adoption of glufosinate-
resistant cotton in places where it fits the weed problems better than GR cotton. The
economics for GR maize was not quite as good as with existing weed management
methods when it was first introduced, but its adoption in the US is now rising
rapidly and has almost caught up with that of cotton (Fig. 3.1). Relatively little GR
canola is grown in the US, but about 90% of the canola grown in Canada was
GR canola in 2006 (Dill et al. 2008). Of the canola grown in the US, 62% was GR
3 Transgenic Crops for Herbicide Resistance 135
100
Soybean
80
Cotton
Maize
% of cultivated land
60
40
20
0
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008
Year
Fig. 3.1 US adoption rate of glyphosate-resistant crops. Data from: http://www.ers.usda.gov/Data/

BiotechCrops
and 31% was glufosinate-resistant in 2005 (Sankula 2006). After a false start in
1999, GR sugar beet was reintroduced in 2008, with an unprecedented adoption rate
of about 60% for the initial year of availability and an anticipated 95% adoption rate
in 2009 (Thomas Schwarz, Beet Sugar Development Foundation, pers. comm). The
adoption rate was limited by the availability of transgenic seed. GR alfalfa was
introduced and well accepted by farmers in 2005, but deregulation was challenged
in court by organic alfalfa growers in 2007, resulting in the removal of the product
from the market.
Glufosinate-resistant crops are also available (Table 3.1), but they have garnered
a much smaller fraction of the HRC market. Their biggest market penetration is
with canola in the US. Glufosinate-resistant cotton has been adopted at high rates in
the US state of Texas. Partly owing to the evolution of GR weeds, glufosinate-
resistant crop adoption is increasing. Crops with both GR and glufosinate resistance
are being made available.
The economics for the biotechnology industry with HRCs is also good. HRCs
offer profits from both a “technology fee” added to seed costs and for the purchase
of the herbicide. No other type of transgenic trait offers this opportunity for dual
profits from the seed and a chemical upon which the value of the gene is dependent.
There has been some consideration of linking expression of transgenic traits to a
chemical inducer of transgene expression (e.g., Jepson et al. 1998), but farmers
would be unlikely to pay much for such a chemical, and the cost of applying the
inducer would probably skew the economics away from such a strategy, unless the
value of the trait was large.
3.2.2 Herbicide Resistance in HRCs
Plants can be made resistant to herbicides or other phytotoxins by a number of

mechanisms. The molecular target site of the herbicide can be modified so that it no
longer binds it and is thereby resistant. One or more herbicide-inactivating or
herbicide-degrading enzymes can be introduced to or increased in a plant. The
plant can be altered to have a mechanism that prevents the herbicide from reaching
the molecular target site (increased sequestration, or decreased uptake or transloca-
tion). All three mechanisms have been described in weeds that have evolved
resistance to herbicides. Metabolic inactivation or degradation is the principle
mechanism in most cases of natural crop resistance to selective herbicides.
The first two approaches have been useful in producing commercial HRCs.
Glyphosate’s molecular target site is an enzyme of the aromatic amino acid path-
way (the shikimate pathway). This enzyme, 5-enolpyruvylshikimate-3-phosphate
synthase (EPSPS), is highly sensitive to glyphosate (Duke 1988), and there are
apparently no other good inhibitors of the enzyme. All plant EPSPS is sensitive to
glyphosate, making it a nonselective herbicide that can be used to kill almost all
weed species. Some fungi and bacteria also have EPSPS, and there are bacterial
versions of the enzyme that are very resistant to glyphosate. A gene encoding
Agrobacterium sp. EPSPS, the CP4 gene, has been used as a transgene for almost
all GR crops (Cerderia and Duke 2007). In some maize varieties, a maize EPSPS
gene modified by site-directed mutagenesis has been used as the transgene in GA21
varieties (Dill 2005). In canola, a gene from the soil bacterium Ochrobactrum
antropi that encodes a glyphosate-degrading enzyme (glyphosate oxidoreductase,
GOX) has been used with the CP4 EPSPS. This enzyme catalyzes the degradation
of glyphosate to aminomethylphosphonic acid (AMPA) and glyoxylate, both rela-
tively innocuous compounds.
The CP4 EPSPS alone makes soybean approximately 50-fold less sensitive to
glyphosate (Nandula et al. 2007) (Fig. 3.2). CP4 and GOX genes together provide
about the same level of resistance to canola (Nandula et al. 2007). Despite a
generally high resistance factor, there can be problems if the transgene promoter
is not sufficiently active in reproductive tissue, as appeared to be case in some of the
first GR cotton varieties (Pline et al. 2002a,b; Pline-Srnic 2005; Yasuor et al. 2006;
Dill et al. 2008). This problem was solved by using a promoter that is stronger in
reproductive tissues. A similar, but lesser problem, has been reported in some maize
varieties (Thomas et al. 2004).
Under some environmental circumstances, glyphosate can cause leaf damage in
GR soybean (e.g., Reddy and Zablotowicz 2003), although this is a rare occurrence
from which the crop eventually recovers. The degradation product of glyphosate,
AMPA, is a weak phytotoxin (Hoagland 1980). Reddy et al. (2004) correlated
120
Shoot dry weight (% of nontreated control)
100
80
60
40
20 Non-GR HBKC 5025

GR Asgrow 4603
0
0.001 0.01 0.1 1 10 100
Glyphosate (kg ae/ha)
Fig. 3.2 Response of glyphosate-resistant (GR) Asgrow 4603RR and nonGR HBKC 5025 soy-
bean in the one- to two-trifoliolate leaf (22-day-old, 45-cm tall) growth stage to glyphosate-
potassium 3 weeks after treatment. Mean values of nine replications are plotted. From Nandula
et al. (2007)
mild phytotoxicity in glyphosate-resistant (GR) soybeans to high levels of AMPA

formed from high-glyphosate-dose applications. Applications of AMPA alone that
resulted in the same endogenous AMPA concentrations caused the same level of
phytotoxicity.
Papers have been published supporting the view that glyphosate impairs growth
of GR crops under some conditions because of effects on micronutrient status (e.g.,
Jolley et al. 2004; Bott et al. 2008). Glyphosate chelates divalent metal cations
(Duke 1988), which could limit their availability when the glyphosate levels are
very high and the divalent metal cation concentration is very low. Conversely,
binding of Ca2+, Mn2+, or Fe2+ can reduce the efficacy of glyphosate (e.g., Schonherr
and Schreiber 2004; Bernards et al. 2005). The pH can strongly influence how
tightly each cation species is chelated to glyphosate. There have been exceedingly
few reports of nutrient deficiencies in the huge areas in which GR crops are grown.
Considering the very disproportionate abundance between the number of molecules
of glyphosate that would reach the soil and the number of divalent cations available
for binding, it seems implausible that glyphosate would have a significant effect on
the availability of essential minerals in GR crops. However, if a plant had a
borderline mineral deficiency, chelation in vivo might cause a problem. The proper
experiments have not been conducted to determine if this possibility could cause a
significant problem in the field. Ozturk et al. (2007) reported that glyphosate
inhibits ferric reductase in plant roots, suggesting that this effect would cause
iron deficiency in GR crops. However, this work was carried out with glyphosate-
sensitive sunflower and the assay was an in vivo assay performed hours after
treatment with high concentrations of glyphosate so that there was no way to
determine if the effects were direct or indirect. These studies should be repeated
with GR crops to answer this question.
Root-associated microbes are involved in plant mineral nutrition (e.g., Jakobsen
et al. 2002). Some of these microbes are glyphosate-sensitive (e.g., Moorman et al.
1992). Some of the glyphosate applied to foliage is exuded by roots (e.g., Coupland
and Caseley 1979; Laitinen et al. 2007). Thus, mineral nutrition could be altered by
adverse effects on root-associated microflora. However, considering that nutrient
deficiency problems have not been a commonly reported problem with GR crops
after more than 10 years of adoption over vast areas, the potential mineral nutrition
problems reported by only very few laboratories are of questionable impact in the
field.
Glyphosate is toxic to Bradyrhizobium japonicum (Moorman et al. 1992).
Several studies have indicated that there is potential for reduced nitrogen fixation
in GR soybeans, but yield reductions due to such an effect have not been docu-
mented in the field when glyphosate is used at the label rate (Zablotowicz and
Reddy 2004, 2007).
The no longer used bromoxynil-resistant crops owed their resistance to a trans-
gene of microbial origin (Klebsiella ozaenae) that converts the benzonitrile to a
nonphytotoxic benzoic acid derivative (Stalker et al. 1996). This gave the crops a
more than tenfold level of resistance. Bromoxynil is an older category of selective
herbicide that inhibits photosynthesis by binding the D1 protein of photosystem II
(Devine et al. 1993).
Glufosinate is a synthetic version of the natural product, phosphinothricin. It is
not accepted by organic farmers, partly because it is chemically synthesized as a
racemic mixture, and the D enantiomer of the racemic mixture is not a natural
compound. It acts by inhibition of glutamine synthetase, thereby causing accumu-
lation of toxic levels of ammonium ion and indirectly stopping photosynthesis
(Lydon and Duke 1999). It is considered a broad-spectrum herbicide, but is not
quite as effective in some situations as glyphosate. One of the microbes that
produce phosphinothricin, Streptomyces hygroscopicus, has an enzyme that inacti-
vates phosphinothricin and glufosinate by acylating it. The gene (bar) encoding this
enzyme, phosphinothricin N-acetyltransferase (PAT), is used as a transgene for
glufosinate-resistant crops (Vasil 1996). In addition to the HRC use, the bar gene
has been used extensively as a selectable marker.
3.2.3 Impacts on Weed Management
Nothing has had more impact on weed management in such a short time period as
GR crops, except perhaps the introduction of synthetic, selective herbicides. Sev-
eral factors have contributed to the strong acceptance of GR technology. A strong
argument can be made that glyphosate is the most effective and useful herbicide
available (Duke and Powles 2008b). It is a slow-acting, highly translocated, foliarly
applied product that kills weeds by inhibiting a molecular target site that is ubiqui-
tous to all plants, EPSPS (Duke et al. 2003a). Its slow action allows translocation to
meristematic tissues, ensuring that all growing points, including subterranean ones,
are killed. It has very good environmental safety characteristics (see Sect. 3.2.5) in
that it is highly nontoxic, does not easily move to ground or surface water, and has a
relatively short half-life in soil. Before GR crops, it could only be used in places
without crops or with methods that avoided crop contact. GR crops opened the door
to widespread utilization of this exceptional herbicide directly on field crops.
In addition to the generally profound economic advantages to using GR crop/
glyphosate technology (Gianessi 2005, 2008; Clewis and Wilcut 2007), this tech-
nology simplifies weed management (Bonny 2008). The farmer can often rely on
only glyphosate applied once or twice during a growing season for weed control,
rather than using a complicated strategy of both soil-incorporated and foliarly
applied herbicides, involving multiple herbicides with different molecular target
sites. Since glyphosate is used only as a postemergence herbicide, the farmer can
wait to see what kind of weed problem emergences before spraying. Tillage can
often be reduced or eliminated, creating both environmental (see below) and
economic benefits. The benefits of this technology are not farm size dependent. A
small farmer who farms only on weekends might derive more benefits, as this
technology is more forgiving than traditional methods of weed management. Thus,
one does not have to hire pest management consultants for prescription recommen-
dations in order to obtain excellent weed control at a reasonable cost. This technol-
ogy is also useful to farmers who grow multiple GRCs, in that they can apply one
herbicide to multiple crops. Prior to GRCs, a particular herbicide could rarely be
used on more than one crop.
Initially, weed management with GR crop technology was excellent, as indi-
cated by the rapid adoption. But, the specter of the evolution of GR weeds is
jeopardizing reliance on this weed control method (Powles 2008a). Although
Bradshaw et al. (1997) gave reasons why the evolution of GR resistance was
implausible, the first report of an evolved GR weed occurred the same year as
their paper (Heap 1997). Since then, the number of cases of evolved GR resistance
has grown at a steady pace all over the world (Table 3.2, Fig. 3.3), and about half of
the cases have occurred in GR crops, where the selection pressure is intense
(Fig. 3.3). In most of the other cases, resistance evolved in vineyards or orchards
in which glyphosate was sprayed several times a year for several consecutive years.
The levels of evolved resistance of weeds to glyphosate are much lower than
that of GR crops, usually in the range of two- to tenfold. The most common
mechanism of resistance in these evolved biotypes is reduced translocation (e.g.,
Lolium spp., Conyza spp.) (Preston and Wakelin 2008), although mutations in
EPSPS that provide marginal resistance have also occurred in Eleucine indica
(Baerson et al. 2002) and some populations of Lolium spp. (Wakelin and Preston
2006; Perez-Jones et al. 2007). In at least some evolved GR Conyza, increased
numbers of EPSPS transcripts may also contribute to resistance (Dinelli et al. 2008).
Table 3.2 Occurrence of evolved GR weeds by species, year, and country

Species Year Location
Amaranthus palmeri 2005 USA
Amaranthus rudis 2005 USA
Ambrosia artemisifolia 2004 USA
Ambrosia trifida 2004 USA.
Conyza bonariensis 2003 South Africa
2004 Spain
2005 Brazil
2006 Columbia
2007 USA
Conyza canadensis 2000 USA
2005 Brazil
2006 China, Spain
2007 Czech Republic
Digitaria insularia 2006 Paraguay
2008 Brazil
Echinochloa colona 2007 Australia
Eleusine indica 1997 Malaysia
2006 Colombia
Euphorbia heterophylla 2006 Brazil
Lolium multiflorum 2001 Chile
2003 Brazil
2004 USA
2006 Spain
2007 Argentina
Lolium rigidum 1996 Australia
1998 USA
2001 South Africa
2005 France
2006 Spain
Parthenium hysterophorus 2005 Columbia
Plantago lanceolata 2003 South Africa
Sorghum halepense 2005 Argentina
2007 USA
Urochloa panicoides 2008 Australia
Source: From the International Survey of Herbicide-Resistant Weeds: http://
www.weedscience.org/In.asp and Vila-Aiub et al. (2008)
Nature abhors a vacuum. In addition to the evolution of GR weeds, when farmers

rely on glyphosate year after year, other species of weeds can fill the ecological
niches vacated by the species that are easily managed with glyphosate. The process
of weed species shifts in GR crop fields has been documented (e.g., Reddy 2004;
Owen 2008). Some of these species have a low level of natural (not evolved)
resistance, and others can avoid glyphosate by germinating later in the growing
season or having a broad germination pattern throughout the season. One potential
mechanism of some species that are naturally resistant is enhanced conversion of
glyphosate to AMPA (Reddy et al. 2008).
The advent of evolved GR weeds and shifts to naturally GR or glyphosate-
avoiding weeds has made the GR crop/glyphosate weed management package less
18
16
14
Glyphosate-resistant species
12 Evolved glyphosate-resistant species
10
2
Evolved in HR crops
0
1996 1998 2000 2002 2004 2006 2008
Year
Fig. 3.3 Incidence of species that have evolved resistance to glyphosate by year
effective in some locales. Application rates and number of applications of glypho-

sate in GR crops have increased. Herbicides, in addition to glyphosate, are being
used with GR crops. New herbicide-resistant crops are being developed to have
resistance to glyphosate, plus resistance to another herbicide or class of herbicides
that might be effective on weeds that are not controlled by glyphosate. Other
options for coping with GR weeds and weed shifts are resistance management
strategies that involve approaches such as alternation of herbicides, utilization of
alternative/or residual herbicides in conjunction with glyphosate, and use of culti-
vation (e.g., Gustafson 2008; Neve 2008; Werth et al. 2008).
The effects of HRCs on weed management is in a constant state of flux because
of weed dynamics, economics, and changes in the availability of both old and new
weed management tools. GR crops have been a valuable asset for weed manage-
ment and will probably continue to be, even as GR weeds evolve and weed species
shift. The effective longevity of this technology will depend, in part, on how wisely
it is used (Powles 2008a).
3.2.4 Impacts on Food and Feed
One of the concerns of those opposing transgenic crops is that the transgene will
alter the quality and/or safety of the consumable part of the plant. This could occur
through the protein from the transgene being toxic, through a metabolic product of
the enzyme encoded by the transgene being toxic, through pleiotropic effects of the
transgene, through alteration of expression of nontransgenic genes by the position
of the transgene in the genome or through more indirect effects. For regulatory
approval, transgenic crops are scrutinized to a far greater level than conventional
crops using analytical, nutritional, and toxicological methods (Atherton 2002;
Malarkey 2003; König et al. 2004), although some have proposed that even more
extensive tests be done by metabolomic, proteomic, and transcriptomic analysis to
detect potential unintended effects of the transgene and its insertion on food safety
and quality (Kuiper et al. 2002; Cellini et al. 2004).
In a safety evaluation for the CP4 EPSPS enzyme introduced into soybean
to provide glyphosate resistance, Harrison et al. (1996) found the protein to be:
(1) nontoxic to mice when consumed at doses thousands of times higher than
potential human exposure; (2) readily degraded by digestive fluids; and (3) not
structurally or functionally related to any known protein allergens or toxins, based
on amino acid sequence homology searches.
The potential allergenic properties of the protein products of transgenes must be
determined before approval. These data are provided to regulatory agencies, but
publications on this topic are scarce. However, there are a few published studies
showing no allergenic properties of transgene products associated with HRCs. Sten
et al. (2004) in a study with soybean-sensitized patients, found that the allergenicity
of 10 GR and eight nonGR soybean cultivars were not different. Chang et al. (2003)
found no significant allergenicity to rats of the CP4 EPSPS gene product conferring
glyphosate resistance. On the basis of both an analysis of published literature and
experimental studies, Herouet et al. (2005) concluded that there is a reasonable
certainty of no harm resulting from the inclusion of the gene for glufosinate
resistance in human food or in animal feed.
Most of the tests have simply examined HRCs for equivalence in food quality to
nonHRCs. Health Canada’s review of the information, presented in support of the
food use of refined oil from glufosinate-resistant canola line HCN92, concluded
that such refined oil does not raise concerns related to safety. Health Canada is of
the opinion that refined oil from canola line HCN92 is as safe and nutritious as
refined oil from the current commercial varieties (http://www.hc-sc.gc.ca/food-
aliment/mh-dm/ofb-bba/nfi-ani/e_nf7web00.html). The nutritional properties of
glufosinate-resistant sugar beets and maize grains were found to be essentially
equivalent to nontransgenic cultivars in feeding studies with swine and ruminants
(Daenicke et al. 2000; Bohme et al. 2001); similar results have been produced with
glufosinate-resistant rice in swine feeding studies (Cromwell et al. 2005).
Studies with GR maize line GA21 evaluated the compositional and nutritional
safety of maize line GA21 compared to that of conventional maize (Sidhu et al.
2000). Compositional analyzes were conducted to measure proximate, fiber, amino
acid, fatty acid, and mineral contents of grain and proximate, fiber, and mineral
contents of forage collected from 16 field sites over two growing seasons. No
significant differences were found. Similarly, Tutel’ian et al. (2001) found no
compositional differences between conventional maize and maize line GA21. The
nutritional safety of maize line GA21 was also evaluated by Sidhu et al. (2000) in a
poultry feeding study. Results from the poultry feeding study showed that there
were no differences in growth, feed efficiency, adjusted feed efficiency, and fat pad
weights among chickens fed with GA21 grain or with parental control grain. These
data taken together demonstrate that GR GA21 maize is as safe and nutritious as
conventional maize for food and feed use.
The occurrence of mycotoxins, specifically aflatoxin, contamination is of con-
cern with maize cultivation in warm climates subject to preharvest moisture stress.
Studies by Reddy et al. (2007) indicated increased incidence of aflatoxin contami-
nation in GR compared to conventional maize in 1 year out of four. But, in 1 year
out of four, fumonisin levels were higher in nonGR maize than in GR maize. The
observed effects were thought to be due to differences in how the crops were grown,
rather than due to glyphosate or the transgene.
Several other studies have found no substantial difference in the nutrient content
of GR and nontransgenic crops. These studies include maize (Ridley et al. 2002;
Autran et al. 2003), soybean (Padgette et al. 1996), wheat (Obert et al. 2004), and
cotton (Nida et al. 1996). In the Autran et al. (2003) study, the characteristics of
glyphosate- and glufosinate-resistant maize in different foods (e.g., beer, hominy,
oil, grits) were compared and found not to be substantially different from the
respective, nontransgenic parental lines.
Lappe et al. (1999) reported reductions of isoflavone levels on GR soybean
varieties in the absence of glyphosate (i.e., a pleiotropic effect of the CP4 gene).
However, this study was not done by comparing isogenic lines. Padgette et al.
(1996) found no effects of the transgene on isoflavone content of soybean. Glypho-
sate targets the shikimate pathway (Duke et al. 2003a), and the estrogenic isofla-
vones of soybeans are products of this pathway. Glyphosate resistance from the
CP4 EPSPS gene is not always complete (Pline et al. 2002a,b), and glyphosate
preferentially translocates to metabolic sinks such as seeds (Duke 1988). Therefore,
we reasoned that at relatively high and late applications of glyphosate to GR
soybeans, a reduction of the content of these compounds could occur. In a well-
replicated field study at two sites, hundreds of kilometers apart, no significant
effects of glyphosate on isoflavones were found at the highest and latest legal
application rates (Duke et al. 2003b).
Table 3.3 summarizes most of the published results of animal feeding studies
with GR crops. All the studies support the view that food from GR crops is
substantially equivalent to nontransgenic crops. In addition to these studies, no
evidence of the CP4 gene or its protein product could be detected in pork from
swine fed with GR soybean meal (Jennings et al. 2003). No effects on GR soybeans
could be found on the immune system of mice (Teshima et al. 2000).
HRCs, being highly resistant to the herbicide to which they have been made
resistant, could also contain levels of the herbicide or its metabolite(s) that exceed
the legal tolerance levels. Surprisingly, little has been published on herbicide residues
in HRC foods. Most of what we know is from studies with nonHRC crops. However,
herbicide residue data must be supplied for regulatory approval of HRCs.
Glyphosate acid and its salts are moderately toxic compounds in EPA toxicity
class II. Glyphosate (either the anion or the isopropylamine salt) is practically
Table 3.3 Some results of animal feeding studies with glyphosate-resistant crops
Crop Animal Result Reference
Maize Rat No effect Hammond et al. (2004)
No effect Healy et al. (2008)
Maize Swine No effect Hyun et al. (2004)
Maize Cattle No effect Erickson et al. (2003)
Maize Dairy cattle No effect Donkin et al. (2003)
No effect Ipharraguerre et al. (2003)
No effect Grant et al. (2003)
Maize Poultry No effect Sidhu et al. 2000
Soybean Rat No effect Zhu et al. 2004
No effect Hammond et al. (1996)
No effect Appenzeller et al. (2008)
Soybean Mice No effect Brake and Evenson (2004)
Soybean Swine No effect Cromwell et al. (2002)
Soybean Dairy cattle No effect Hammond et al. (1996)
Soybean Catfish No effect Hammond et al. 1996
Soybean Poultry No effect Hammond et al. (1996)
No effect Taylor et al. (2007)
Canola Rainbow trout No effect Brown et al. (2003)
Canola Poultry No effect Taylor et al. (2004)
Alfalfa cattle No effect Combs and Hartnell (2008)
Sugarbeet Sheep No effect Hartnell et al. (2005)
nontoxic by ingestion, with a reported acute oral LD50 of >5,000 mg kg1 in the rat
(Vencill 2002). The trimethylsulfonium salt of glyphosate is more toxic, with an
oral LD50 of about 705 mg kg1. It is not a restricted use pesticide and is a best-
selling weed killer for home use. Animals do not contain the herbicide molecular
target site (EPSPS) of glyphosate.
Occasional reports of severe effects of ingestion of formulated glyphosate occur
(e.g., Stella and Ryan 2004); however, the glyphosate molecule itself is considered
one of the most toxicologically benign herbicides available. Williams et al. (2000)
extensively reviewed the toxicology literature on glyphosate and its metabolites and
concluded that under present and expected conditions of use, glyphosate does not
pose a significant health risk to humans.
In a testing program to detect whether GR soybeans had been sprayed with
glyphosate or not, Lorenzatti et al. (2004) found glyphosate and AMPA in green,
immature seeds. Both glyphosate and its degradation product, AMPA, were found
in mature, harvested seeds of different GR soybean varieties grown in widely
separated geographical regions (Duke et al. 2003b). Even though the glyphosate
applications were at legal, but at relatively high rates and late timing, the residues
were within the established tolerance levels. We were surprised to find higher
AMPA than glyphosate levels since at that time plants were thought to degrade
glyphosate very little, if at all (Duke 1988; Duke et al. 2003a). Subsequently, we
found that some legume plant species readily convert glyphosate to AMPA (Reddy
et al. 2008). In a study conducted earlier, but published later (Arregui et al. 2004),
similar levels of glyphosate were found in harvested seed of GR soybeans, but these
scientists found much lower AMPA levels. We have found no publications on

glyphosate residues in GR crops other than soybean.
Glufosinate is not a restricted use pesticide and is sold for home weed control in
the USA. Its acute oral toxicity in rats is an LD50 of ca. 2.2 g kg1. Glufosinate
chemically resembles glutamine, a molecule used to transmit nerve impulses in the
brain. Ebert et al. (1990) concluded in an extensive review that glufosinate is safe
under conditions of recommended use. Similarly, Hack et al. (1994) also concluded
from their studies that glufosinate is unlikely to cause health effects of either users
or consumers when used as directed, although the herbicide target site, glutamine
synthetase, is also found in animals.
In a study to determine if glufosinate applied to glufosinate-resistant maize and
canola could lead to an increase in herbicide residues or to the formation of new
metabolites, Ruhland et al. (2004) found that L-glufosinate was in the form of
known metabolites and the parent compound in both maize and canola. The highest
content was in the leaves, and the lowest in the grains. No levels were found above
the established tolerance levels.
A last, but understudied, aspect of food quality and HRCs is their influence on
contamination of food with poisonous weed seeds. Weed seeds can be the sources
of toxic compounds (e.g., Powell et al. 1990). HRCs are generally more weed-free
than conventional crops, resulting in less foreign matter, including weed seeds, in
the harvested product (Canola Council of Canada 2001; Shaw and Bray 2003).
Therefore, there is less likelihood of significant contamination of harvested food
with toxic weed seeds in HRCs than with conventional crops.
3.2.5 Environmental Impacts
There are numerous possible environmental effects of HRCs. These can be either
positive or negative. They can be associated with the transgene or with the herbi-
cide to which the transgene is linked. But, ultimately, potential environmental
impacts of HRCs must be compared with the impacts of the technologies that
they replace. All the published studies and analyses of this type have found that
the environmental benefits of substituting HRCs for conventional crops are usually
substantial (e.g., Wauchope et al. 2002; Nelson and Bullock 2003; Bennett et al.
2004; Amman 2005; Brimner et al. 2005; Brookes and Barfoot 2006; Cerdeira and
Duke 2007; Cerdeira et al. 2007; Kleter et al. 2007, 2008; Devos et al. 2008;
Gardner and Nelson 2008; Shiptalo et al. 2008). Of course, the potential benefits
vary with the HRC, the geographic location of use, the way the farmer uses the
HRC, and different components of environmental impact. Since nature is not static,
the environmental impact will change with time as farmers using HRC technology
adjust their methods to deal with changing weed and other problems.
Whether HRCs have increased or decreased herbicide use in terms of the amount
of material used per hectare of crop can be argued either way, depending on many
factors. However, in terms of environmental impact this factor is not useful, as the
relative toxicity and environmental fate of glyphosate or glufosinate compared with

the herbicides that they replace in HRCs can be large. Glyphosate and glufosinate
have relatively low mammalian toxicity (Ebert et al. 1990; Giesy et al. 2000;
Williams et al. 2000), compared to many of the herbicides that they can replace
when used with transgenic crops. Glyphosate, in particular, has lower acute toxicity
than aspirin or many other commonly ingested compounds.
In a study, using acute mammalian toxicity data, Gardner and Nelson (2008)
compared the number of LD50 doses per unit area that were decreased by GR crops
in the United States. Depending on the crop and the location, they calculated that
conventional weed management with other synthetic herbicides could result in as
much as 3,000 more LC50 doses per hectare with maize, more than 375 with cotton,
and more than 90 with soybean. An example of some of their data with cotton is
provided in Fig. 3.4. They concluded that GR technology has a positive environ-
mental effect. In a European study, Devos et al. (2008) found that use of GR maize
and glufosinate-resistant maize would lower the pesticide occupational and envi-
ronmental risk or weed management. The main benefits were due to the lower
potential of these chemicals to contaminate groundwater and their lower acute
toxicity. Glyphosate gave slightly greater reductions than glufosinate. Their calcu-
lations were done with the assumption that other herbicides would not be used with
glyphosate nor glufosinate. However, glyphosate is increasingly used with other
Fig. 3.4 Increase in LD50 doses per hectare of herbicides needed if GR cotton were not available
in the US. From Gardner and Nelson (2008)
herbicides in GR crops because of evolved glyphosate resistance and shifts to

naturally resistant weed species.
Neither glyphosate nor glufosinate are problematic pesticides in the context
of surface and groundwater contamination of the herbicides that they replace
(reviewed by Duke and Cerdeira 2005; Cerdeira and Duke 2006; Borggaard and
Gimsing 2008). Glyphosate does not move well in soil because of its strong
sorption to soil minerals (e.g., Mamy and Barriuso 2005). Furthermore, in most
soils, it degrades more rapidly than the herbicides that it replaces (e.g., Mamy et al.
2005). In addition to microbial degradation, both glyphosate and AMPA can
undergo nonbiological degradation in soil (Barrett and McBride 2005), although
the relative contribution of the two types of processes is unknown.
Concern over AMPA contamination of groundwater has recently been expressed
(Mamy et al. 2005, 2008). AMPA is more persistent than glyphosate in the
environment, and it more readily leaches in soil (Mamy et al. 2008; Simonsen
et al. 2008). Both glyphosate and AMPA are leached from soil containing high
phosphate levels than soil with low levels (Simonsen et al. 2008). The relative
toxicities of AMPA versus contaminants from conventional herbicide use have not
been adequately analyzed.
Fuel utilization associated with weed management has been reduced by GR
crops because of reduced tillage and fewer trips across the field to spray herbicides
(reviewed by Cerdeira and Duke 2006). Bennett et al. (2004) estimated that there
would be a 50% fossil fuel savings in growing sugar beet in Europe by switching to
GR crops. Brookes and Barfoot (2006) estimated that GR crop use in 2005
worldwide reduced carbon emissions approximately the same as the removal of
four million family automobiles from the road.
Effects of the herbicides used with HRCs on nontarget organisms are a concern.
Obviously, since glyphosate is a herbicide, glyphosate drift to nontarget plants can
be harmful (e.g., Bellaloui et al. 2006). This is not a new problem, as there have
been problems with herbicide drift since the advent of herbicides that are sprayed.
Concentrations reaching nontarget plants are generally only a small fraction of the
recommended dose for weed control. At very low doses that one might expect with
drift, herbicides can often stimulate growth, activate host defense systems against
pathogens, or enhance nitrogen utilization, depending on the herbicide (reviewed
by Duke et al. 2006). The phenomenon of a stimulatory effect at subtoxic concen-
tration of a toxin is termed hormesis. Glyphosate clearly enhances growth of plants
at very subtoxic application rates (e.g., Schabenberger et al. 1999; Wagner et al.
2003; Cedergreen et al. 2007; Velini et al. 2008). But, at least in barley, the effect is
not sustained over time (Cedergreen 2008). The mechanism(s) of herbicide-caused
hormesis is poorly understood and may well differ between herbicides with differ-
ent molecular target sites.
Perhaps, the greatest damage done by conventional agriculture, other than taking
land out of its natural state, has been caused by tillage. The primary reason for
tillage has been weed management. Glyphosate use in GR crops has resulted in
significantly reduced tillage, especially in soybean and cotton (Fig. 3.5) (American
Soybean Association 2001; Penna and Lema 2003; Dill et al. 2008; Locke et al. 2008;
a
60
50
Cropped Area
% Soybean
40
30
20
10
0
Non
GR
Non
GR
Non
GR
Non
GR
Non
GR
GR
GR
GR
GR
GR
b
90
80
70
%Cotton Cropped
60
50
Area
40
30
20
10
0
Non
GR
Non
GR
Non
GR
Non
GR
Non
GR
GR
GR
GR
GR
GR
2002 2003 2004 2005 2006
Conventional Conservation Till No-Till
Fig. 3.5 Comparison of US tillage practices in glyphosate-resistant (GR) and nonGR soybean
(a) cotton (b) from 2002 through 2006 as a percentage of hectares planted. From Dill et al.
(2008)
Powles 2008b). Even in some nonGR crops, reductions in the price of glyphosate
and the increases in the cost of diesel fuel have favored conservation tillage rather
than traditional tillage (Nail et al. 2006). Reduced tillage sometimes reduces soil
compaction (Koch et al. 2003; Shukla et al. 2003). To our knowledge, this aspect of
the influence of HRCs has not been studied. However, as both evolved and naturally
resistant GR weeds increase, some farmers of GRCs are returning to occasional tillage
for more complete weed management.
There is concern about the potential of HRCs to create new weed problems,
either themselves becoming a weed or the HR transgene escaping to relatives, either
feral crops or related species, to create new weed problems. Gene flow to native
populations of species with which the HRC can crossbreed could result in unwanted
agricultural and/or environmental effects.
The HR transgene confers no advantage where the matching herbicide is not
sprayed, so the HR crop is no more likely to invade a natural habitat than the nonHR
crop. None of the crops that have been made herbicide resistant with transgenes are
crops that become weeds outside of agricultural fields. However, HRCs are some-
times problems in agricultural fields in which the same herbicide is used in
subsequent years with a different HRC. This has already been a problem with GR
crops (e.g., York et al. 2004; Soltani et al. 2006; Beckie and Owen 2007), requiring
the use of herbicides other than glyphosate to control the “volunteer” GR crop.
Gene flow to nontransgenic crops of the same species has been a commercial and
political problem, but not an environmental threat. Organic farmers cannot retain
the organic status of their crops if transgene presence is above a set limit, nor can
crops be sold to markets that require the product to be nontransgenic if transgenic
occurrence is above the level set the regulatory jurisdiction. For crops like soybean,
outcrossing is not a problem, but considerable outcrossing can occur with maize,
rice, sugar beet, and canola. Substantial gene flow can occur between HR and
nonHR canola (e.g., Hall et al. 2000; Reiger et al. 2002; Mallory-Smith and Zapiola
2008). Outcrossing may account for the contamination of nontransgenic rice with
the glufosinate-resistant gene (Vermij 2006), even though glufosinate-resistant rice
has only been grown experimentally. To our knowledge, no herbicide-resistant
transgenes have been found to be a problem in nonherbicide-resistant maize,
although there is significant potential for this to happen (Allnutt et al. 2008).
There has been considerable controversy about whether Mexican maize landraces
are contaminated with transgenes (reviewed by Cerdeira and Duke 2006). No gene
flow from transgenic to nontransgenic cotton has been reported in cultivated fields,
but it should occur because of insect pollination. Gene flow from GR alfalfa
to organic alfalfa was the ostensible reason for its re-regulation. Flow of a GR
transgene from bentgras being evaluated for commercial use to nontrangenic bent
grass occurred rapidly, and mitigation efforts have not been effective (Mallory-
Smith and Zapiola 2008; Zapiola et al. 2008).
A much bigger concern is the potential effect of gene flow from HRCs to weedy
relatives. The only environmental aspects of transgenic crops that are not also
associated with nontransgenic crops are those associated with the transgenes. HR
transgenes offer no advantage in natural ecosystems where the herbicide is not
used, but when coupled with transgenes imparting traits that would improve fitness
in a natural ecosystem (e.g., insect or drought resistance), the HR trait would
improve the likelihood of introgression of the gene into the unintended recipient
species (see below). Once a transgene escapes to another species, it is unlikely that
it could be eliminated in the population by human efforts. Indeed, removal of a GR
transgene from a nontransgenic population of bentgrass has not been accomplished
with the mitigation methods used (Zapiola et al. 2008).
Gene flow or introgression involves the movement of a gene or genes into a
sexually compatible species. This type of gene flow is also termed vertical gene
flow. The first generation is generally not very fit (e.g., Scheffler and Dale 1994),
but subsequent backcrosses to the noncrop species will eventually fully introgress
the transgene into this species. Yearly spraying of the herbicide to which the
transgene confers resistance should facilitate the process by selecting only crosses
with the transgene and removing competing weeds to give the unfit F1 and usually
the F2 generations a significant survival advantage.
Transmission of HR transgenes could make weedy relatives of the HRC much
more problematic for the farmer. This has not happened with soybean, cotton, and
maize, presumably because there are few or no weedy species with which they are
sexually compatible in the places in which they are grown.
Canola outcrosses with several weedy related Brassica species, including related
Brassica crops (e.g., cabbage, cauliflower, broccoli, etc.). These crosses are gener-
ally quite unfit, but introgression of a GR transgene from canola to its weedy
relative, bird rape (Brassia rapa) has occurred in field situation, and the intro-
gressed gene appears to be stable in the population, even in the absence of spraying
glyphosate (Warwick et al. 2008). A potential exists for a transgene to move from
through compatible relative (a bridge species) to a third Brassica species to increase
the means by which a GR gene could spread (Brown and Brown 1996).
Work by Darmency et al. (2007) indicates that gene flow from sugar beets to
weedy beets is sufficient to cause problems for the farmers growing HR beets within
a few years if herbicides are not rotated. However, the degree of outcrossing that
they measured varied considerably between lines of transgenic beets, years, and
field locations.
Rice outcrosses readily with feral rice (e.g., red rice), as well as weedy related
species. Nontransgenic, imidazolinone (IMI) herbicide-resistant rice created by
selection in cell culture (Tan et al. 2005) was commercialized in the USA in
2002. Outcrossing to produce IMI-resistant weeds occurs (e.g., Shivrain et al.
2007). The incidence of IMI-resistant weeds in rice is growing in locations where
IMI-resistant rice is grown (Valverde 2007), although whether this is due to evolved
resistance or gene flow is unclear in most cases, because IMI resistance evolves
quickly in some species when exposed to this herbicide class (Heap 2008). We can
expect that gene flow from rice to feral rices and to sexually compatible species will
occur with any transgene, including those imparting herbicide resistance.
Keeping HRCs out of areas in which sexually compatible species exist is a
daunting task. For example, transgenic, GR and glufosinate-resistant canola are not
approved as crops in Japan, but the seed can be imported for processing. Saji et al.
(2005) found these HRCs growing along routes to processing plants in Japan.
There is considerable literature on strategies and technologies for mitigating or
eliminating vertical gene flow (reviewed by Gressel 2002; Cerdeira and Duke
2006). Much of this literature deals with simply reducing movement of the genes
to plants other than the transgenic crop (e.g., Devos et al. 2005). However, if the
gene would pose a potential problem in a natural environment, a fail-safe approach
would be preferable. Strategies coupling one or more technologies such as genetic
use restriction technology genes to make transmission of a functional transgene
impossible (Oliver et al. 1998) or linking the HR transgene to one that would confer
unfitness to a wild plant (e.g., genes that prevent shattering or dormancy; Al-Ahmad
et al. 2004; Gressel and Al-Ahmad 2004) have the potential for fulfilling this need.
In the latter approach, the HR gene(s) should be in the same construct as the
unfitness gene(s) to keep the genes from segregating.
Lastly, some have voiced concern that there could be gene flow from HRCs and
other transgenic crops to totally unrelated organisms (horizontal gene transfer),
especially soil or gut microbes (e.g., Bertolla and Simonet 1999; Giovannetti 2003),
from degraded HRCs in the field or through ingestion of food composed of GRCs.
Almost all of the transgenes currently used for HRCs are from soil microbes. These
genes are much more likely to be transferred to unrelated microbes from the natural,
microbial sources than from HRCs (Kim et al. 2005). Levy-Booth et al. (2008)
found that degradation of the cp4-epsps transgene from GR soybean leaf material in
the soil was rapid, but could still be detected in soil after 30 days. Degradation of
the transgene and a natural soybean gene in soil were similar. Dale et al. (2002)
concluded that there is no compelling argument that there would be any more
likelihood of such gene transfer transgenic crops than from nontransgenic crops.
There has so far been no credible evidence of gene transfer from transgenic crops to
microbes (Dunfield and Germida 2004), although some have criticized detection
methods (e.g., Nielsen and Townsend 2004). Nevertheless, after 14 years of grow-
ing these crops on huge areas, there have been no reports of transgenes being
transferred to microbes of any type.
3.2.6 Herbicide-Resistant Crops and Crop Disease
There are some untended benefits of GR crops and perhaps glufosinate-resistant

crops. Both glyphosate and glufosinate are fungitoxic (reviewed by Duke et al.
2007). Thus, when used at full application rates, these herbicides may, under some
circumstances, be providing sufficient protection from plant pathogens to prevent
crop damage or to preclude spraying with a fungicide. Perhaps, the most carefully
studied example is that of glyphosate effects of Asian rust in GR soybeans (Feng
et al. 2005, 2008), in which glyphosate applications to GR soybeans reduced rust
infection and damage, both as a preventative and a curative (Fig. 3.6) treatment.
However, in many field situations, the optimal timing of glyphosate application for
effective weed management is unlikely to also be most effective for rust control
(Bradley and Sweets 2008). The effects of glyphosate on reducing this disease will
a b
Water 1DAI Surf 1DAI
% ASR incidence (N=15)
60 Fungicide A 1DAI 60
Water 1DAI
Glyp1x 1DAI Glyp.5x 3DAI
Glyp1x 3DAI
40 40 Glyp1x 3DAI
Glyp1x 6DAI
Glyp2x 3DAI
20 20
0 0
0 10 20 30 40 0 10 20 30 40
Days After Inoculation Days After Inoculation
Fig. 3.6 (a) Curative activity for Asian rust in glyphosate-resistant soybeans sprayed with
glyphosate (Glyp, 1 at 0.84 kg AE ha1) at 1, 3 or 6 days after inoculation (DAI), water and
fungicide A (carbendazim + flusilazole) at 1 DAI. (b) Curative activity as a function of glyphosate
dose (0.5, 1 or 2) from spray application at 3 DAI, surfactant (1 g L1 MON 0818) or water
alone at 1 DAI. From Feng et al. (2008)
Table 3.4 Reports of glyphosate interactions and lack of interactions with plant disease in
glyphosate-resistant crops
Crop Disease Effect References
Soybean Phakopsora pachyrhizi Reduces Feng et al. (2005, 2008)
Fusarium spp Increases Kremer et al. (2005)
S. sclerotiorum No effect Lee et al. (2003)
Increases Nelson et al. (2002)
F. solani Increases Sanogo et al. (2001), Nijiti et al. (2003)
Cotton Rhizoctonia solani Reduces Pankey et al. (2005)
Wheat Puccinia triticina Reduces Feng et al. (2008), Anderson
and Kolmer (2005)
Sugarbeet Rhizoctonia solani Increases Larson et al. (2005)
Fusarium oxysporum Increases Larson et al. (2005)
probably increase as glyphosate use increases, both in application rates and number
of applications, because of the evolution of GR weeds and weed species shifts to
more naturally GR weeds.
On the other hand, glyphosate treatment is known to predispose nontransgenic
plants to plant disease by more than one mechanism associated with the mechanism
of action of glyphosate as a herbicide (reviewed by Duke et al. 2007), although
these mechanisms should not exist in GR crops. There are many reported cases of
exacerbation of plant disease symptoms with glyphosate, but these studies have
generally been done with glyphosate-susceptible plants (reviewed by Duke et al.
2007). Nevertheless, there are a few reports of this phenomenon in GR crops
(Table 3.4). Likewise, there are reports of reduced crop disease with glyphosate
(Table 3.4). Thus, the interaction of glyphosate, fungal plant diseases, and GR crops
is variable, depending on the crop, the disease, and perhaps the timing of herbicide
application and infection. Also, differences in cultural practices between HRCs and
nontransgenic crops can influence plant disease (e.g., Lee et al. 2005). Glyphosate
effects on root exudates and soil moisture can influence root-borne soybean dis-
eases (Kremer et al. 2005; Means and Kremer 2007; Means et al. 2007). Larson
et al. (2005) found the that several factors, including the disease isolate, whether or
not the crop was sprayed with glyphosate, and the variety of GR sugar beet
determined whether there was increased disease with the GR crop. In a recent
review, Powell and Swanton (2008) concluded that there was insufficient informa-
tion from realistic field studies to determine whether glyphosate influences
Fusarium spp. diseases in GR crops.
3.3 Coming Herbicide-Resistant Crops
3.3.1 Transgenes for Herbicide Resistance
Many transgenes have been reported and/or patented for the production of HRCs.
Only a few of them are listed in Table 3.5. Technology is at a stage at which
resistance to any herbicide can be imparted by transgene technology. Yet, only
Table 3.5 Some of the transgenes that have been used for making crops resistant to herbicides
or classes of herbicides
Herbicide or herbicide class Gene source and gene product References
2,4-D Microbial degradation enzyme Llewellyn and Last
(1996), Bisht et al.
(2004)
Asulam Resistant microbial dihydropteroate Surov et al. (1998)
synthase
Dalapon Microbial degradation enzyme Buchanan-Wollaston
et al. (1992)
Dicamba Microbial degradation enzyme Behrens et al. (2007),
Herman et al.
(2005)
Hydroxyphenylpyruvate Microbial, herbicide-resistant HPPD Matringe et al. (2005)
dioxidase (HPPD)
inhibitors
Paraquat Chloroplast superoxide dismutase Sen Gupta et al. (1993)
Phenmedipham Microbial degradation enzyme Streber et al. (1994)
Phytoene desaturase (PDS) Genes from microbes and the aquatic Sandmann et al.
inhibitors weed Hydrilla encoding resistant PDS (1996), Arias et al.
(2005)
Protoporphyrinogen oxidase Resistant microbial PPO and resistant Li and Nicholl (2005)
(PPO) inhibitors Arabidopsis thaliana PPO
three types of HRCs have been marketed; crops resistant to bromoxynil, glufosi-
nate, and glyphosate, utilizing only five transgenes. Considering the huge economic
impact of the GR crops, one would expect that other HRCs would have been
marketed. Considerable effort and expense was put into development of HRCs
that resist both hydroxyphenylpyruvate dioxygenase- and protoporphyrinogen
oxidase-inhibiting herbicides (Li and Nicholl 2005; Matringe et al. 2005), yet the
decision was apparently made to postpone or terminate plans to commercialize
these HRCs. Devine (2005) attributed the few HRCs making it to the market place
to the high cost of developing and getting regulatory approval of HRCs.
Further complications are international trade issues and the impact on the
existing registration of the herbicide to which they are made resistant. Realistically,
none of the herbicides on the market to which crops can be made resistant offer the
advantages of glyphosate (Duke and Powles 2008b) or perhaps even glufosinate,
both relatively safe, broad-spectrum herbicides.
In addition to the few number of herbicides used (only two), the number of crops
represented among all the currently available HRCs is few (Table 3.1). This is
partly because the cost of obtaining approval of HRCs is considered too great for
minor crops (Devine 2005). However, this does not explain the absence of GR
wheat and rice and glufosinate-resistant wheat, where the market could be very
large, worldwide. GR wheat was almost marketed in North America, but concern
over acceptance of wheat products from this source in Europe prevented it being
marketed. A similar concern slowed the acceptance of GR sugar beets until 2008.
The horrendous problems with outcrossing of rice with weedy feral and other
species of may be related to GR rice being unavailable.
Table 3.6 Recently deregulated HRCs (http://www.aphis.usda.gov/brs/not_reg.html) that are not

yet on the market and HRCs that have recent approval for field-testing in the USA (http://www.isb.
vt.edu/cfdocs/fieldtests1.cfm)
Crop Herbicide(s) Company Resistance gene(s)
Petition for deregulation
Soybeana Glyphosate and ALS Pioneer Glyphosate acetyltransferase and a modified
inhibitors soybean ALS
Cotton Glyphosate and Bayer Crop Maize EPSPS modified by site-directed
glufosinate Sci. mutagenesis and bar gene
Maize Glyphosate and ALS Pioneer Glyphosate acetyltransferase and a modified
inhibitors maize ALS
Approval for field-testing
Alfalfa Glyphosate/ Pioneer Glyphosate acetyltransferase and a modified
sulfonylureas plant ALS
Soybean Glyphosate/dicamba Monsanto CP4 EPSPS and a demethylase
Glufosinate/dicamba Monsanto CP4 EPSPS and a demethylase
Glyphosate Pioneer Glyphosate acetyltransferase
Glyphosate/isoxazole MS Technologies
Not available
Glufosinate MS Technologies
Not avaiable
Maize Glyphosate Pioneer Glyphosate acetyltransferase
Bentgrass Glufosinate HybriGene, Phosphinothrichin acetyl transferase
LLC
a
Deregulation approved
Many types of HRCs have gotten to the point of being field-tested, but few have
proceeded to be deregulated (approved for commercial use). Table 3.6 lists pro-
ducts for which deregulation has been applied, as well as recently approved
applications for field-testing in the USA. This information on the USDA/APHIS
website provides the best indication of what new products are in the offing.
Considering that many of these crops have glyphosate resistance, we consider
these products separately.
3.3.2 Glyphosate-Resistant Crops
Considering the enormous success of GR crops and the fact that glyphosate is now a
generic herbicide, other companies have discovered or created new glyphosate-
resistant transgenes and, in at least one case, are going forward with the commer-
cialization process. This crop uses an artificially evolved glyphosate-resistant gene
(Castle et al. 2004; Siehl et al. 2005). A gene from the soil bacterium Bacillus
licheniformis, which encoded a weak glyphosate N-acetyltransferase (GAT) was
put through 11 iterations of gene shuffling to increase its activity by almost
four orders of magnitude. Properties of the resultant GAT are described by Siehl
et al. (2005, 2007). Plants made resistant to glyphosate with this transgene were
ca. 100-fold more resistant to glyphosate than to nontransgenic lines (Green et al.
2008). Other glyphosate-inactivating enzymes are apparently encoded by genes of
soil microbes, because there are other routes of degradation or inactivation. For
example, a C-P lyase that converts glyphosate to inorganic phosphate and sarcosine
is found in several bacteria, including Arthrobacter spp., Rhizobium spp., and
Pseudomonas spp. (Kishore and Jacob 1987; Liu et al. 1991; Dick and Quinn
1995). Not all bacterial C-P lyases will degrade glyphosate (White and Metcalf
2004). A good C-P lyase transgene for use in GR crops has apparently not yet been
developed. A microbial transgene-encoded EPSPS with some properties that might
be superior to that used in commercialized GR crops is available (Vande Berg et al.
2008). Thus, transgenes for glyphosate resistance are available for companies that
do not have GR crops.
3.3.3 Other Herbicide-Resistant Crops
Crops with both the GAT gene and a gene for resistance to acetolactate synthase
inhibitors are in the final stage of development (Green et al. 2008), including testing
for food safety (Table 3.6) (McNaughton et al. 2007). Monsanto is developing a
dicamba (3,6-dichloro-2-methoxybenzoic acid) resistance trait (Table 3.6) that
detoxifies this herbicide by demethylating it (Herman et al. 2005; Behrens et al.
2007). Dow Agroscience has developed HRCs with resistance to broadleaf-killing
auxinic herbicides like 2,4-D [(2,4-dichlorophenoxy)acetic acid] and grass-killing
aryloxyphenoxypropionate herbicides such as diclofop (()-2-[4-(2,4-dichlorophe-
noxy) phenoxy]propanoic acid). These crops are apparently being field-tested, but
there is no specific information on the APHIS website as to what genes are being
used. Thus, we have left them out of Table 3.6. A recent abstract mentions that a
transgene encoding an a-ketoglutarate-dependent dioxygenase is used to confer
resistance to these two herbicide classes (Simpson et al. 2008). A patent had been
filed for such a unique bacterial (Ralstonia eutropha)-derived transgene by Dow
Agroscience in 2005 (Wright et al. 2005).
3.4 The Future of HRCs
The future of HRCs will be influenced by the development of other approaches to

weed management. Availability of acceptable new technologies that are economi-
cally competitive with HRCs may eventually stem their rapidly increasing adop-
tion. There are other strategies for using transgenes for weed management (Gressel
2002; Duke 2003, 2006) including enhancing crop competitiveness and allelopathy,
and enhancement of weed biocontrol agents. There is little likelihood of these
approaches having a significant effect in the next decade, but ultimately they
could have substantive impacts.
To some extent, regulatory requirements regarding environmental impacts of

weed management methods will affect this scenario. The increasing costs of
meeting the requirements for deregulation of transgenic crops and the conflicting
regulatory processes in different parts of the world have hampered the adoption of
HRCs. Eventual harmonization of this process could benefit this technology.
There are many other factors that could have an impact on the future of HRCs,
such as the amount of biofuel crops that will be grown (Baylis 2008). Since these
crops are not meant for human consumption, and, in some cases, could not be used
for human or animal consumption, the regulatory requirements for deregulation
of HRC biofuel crops may be simplified and less costly to fulfill. Indeed, GR
sugarcane is being developed in Brazil (http://monsanto.mediaroom.com/index.
php?s = 43&item = 656). Still, the issue of herbicide resistance management and
gene flow will be as important for these crops as for those meant for human and
livestock consumption.
Agriculture has clearly found HRCs to be of great value, but their utility is being
eroded by the evolution of GR weeds and weed species shifts in GRCs. Both of
these processes are the result of over reliance on this highly effective technology.
Some of the new HRCs that are to be introduced will help cope with this situation,
but we see overreliance on glyphosate continuing to undermine its effectiveness.
The unique properties of glyphosate as a herbicide (Duke et al. 2003a; Duke and
Powles 2008b), almost necessitate that its utility be preserved for future harvests
(Duke and Powles 2008a; Powles 2008a). It will be shame to see the evolution of
GR weeds continue unabated, as the strategies for preventing it are well known.
Finally, the future of HRCs has been and may continue to be affected by public
opinion. For example, transgenic crops have made meager inroads in Europe for
this reason. Even though Europeans grow almost no transgenic crops, they buy
substantial amounts of transgenic crops grown elsewhere for animal feed. At the
time of this writing, this situation does not appear to be changing significantly. In
countries such as Australia and those of South America, resistance to transgenic
crops has waned considerably and adoption is increasing rapidly. We expect
available HRCs to be almost universal outside of Europe and perhaps a few small
areas of resistance within a few more years.
References
Al-Ahmad H, Galili S, Gressel J (2004) Tandem constructs to mitigate transgene persistence:

Tobacco as a model. Mol Ecol 13:697–710
Allnutt TR, Dwyer M, McMillan J, Henry C, Langrell S (2008) Sampling and modeling for
quantification of adventitious genetically modified presence in maize. J Agri Food Chem
56:3232–3237
American Soybean Association (2001) Conservation tillage study, American Soybean Associa-
tion, St. Louis, MO, USA. http://www.soygrowers.com/ctstudy/ctstudy_files/frame.htm.
Accessed 24 Dec 2008
Amman K (2005) Effects of biotechnology on biodiversity: herbicide-tolerant and insect-resistant
GM crops. Trends Biotechnol 23:388–394
Anderson JA, Kolmer JA (2005) Rust control in glyphosate tolerant wheat following application of
the herbicide glyphosate. Plant Dis 89:1136–1142
Appenzeller LM, Munley SM, Hoban D, Sykes GP, Malley LA, Delaney B (2008) Subchronic
feeding study of herbicide-tolerant soybean DP-356O43–5 in Sprague-Dawley rats. Food
Chem Toxicol 46:2201–2213
Arias RS, Netherland MD, Atul P, Dayan FD (2005) Biology and molecular evolution of
resistance to phytoene desaturase inhibitors in Hydrilla verticillata and its potential use of
produce herbicide-resistant crops. Pest Manag Sci 61:258–268
Arregui MC, Lenardon A, Sanchez D, Maitre MI, Scotta R, Enrique S (2004) Monitoring
glyphosate residues in transgenic glyphosate-resistant soybean. Pest Manag Sci 60:163–166
Atherton KT (2002) Safety assessment of genetically modified crops. Toxicology 181–182:421–426
Autran JC, Benetrix F, Bloc D, Burghart P, Chaurand M, Combe N, Melcion JP (2003) Composition
and technological value of genetically modified and conventional maize. Sci Aliments 23:223–247
Baerson SR, Rodriquez DJ, Tran M, Feng Y, Biest NA, Dill GM (2002) Glyphosate-resistant
goosegrass. Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-
phosphate synthase. Plant Physiol 129:1265–1275
Barrett KA, McBride MB (2005) Oxidative degradation of glyphosate and aminomethylpho-
sphonate by manganese oxide. Environ Sci Technol 39:9223–9228
Baylis A (2008) Biofuels: What impact on crop protection and seeds now? Outlooks on Pest
Manag 19:270–274
Beckie HJ, Owen MDK (2007) Herbicide-resistant crops as weeds in North America. CAB Rev 2,
No. 044, pp 22, http://www.cabi.org/cabreviews/default.aspx?LoadModule¼Review&
ReviewID¼33801&site¼167&page¼1178
Behrens MR, Mutlu N, Chakraborty S, Dumitru R, Jiang WZ, LaVallee BJ, Herman PL, Clemente
TE, Weeks DP (2007) Dicamba resistance: Enlarging and preserving biotechnology-based
weed management strategies. Science 316:1185–1188
Bellaloui N, Reddy K, Zablotowicz RM, Mengistu A (2006) Simulated glyphosate drift influences
nitrate assimilation and nitrogen fixation in non-glyphosate-resistant soybean. J Agri Food
Chem 54:3357–3364
Bennett R, Phipps R, Strange A, Grey P (2004) Environmental and human health impacts of
growing genetically modified herbicide-tolerant sugar beet: a life-cycle assessment. Plant
Biotechnol J 2:273–278
Bernards ML, Thelen KD, Muthukumaran RB, Penner D, McCracken JL (2005) Glyphosate
interaction witth manganese in tank mixtures and its effect on glyphosate absorption and
translocation. Weed Sci 53:787–794
Bertolla F, Simonet P (1999) Horizontal gene transfers in the environment: natural transformation
as a putative process for gene transfers between transgenic plants and microorganisms. Res
Bisht NC, Burma PK, Pental D (2004) Development of 2, 4-D-resistant transgenics in Indian
oilseed mustard (Brassica juncea). Curr Sci 87:367–370
Bohme H, Aulrich K, Daenicke R, Flachowsky G (2001) Genetically modified feeds in animal
nutrition. 2nd communication: Glufosinate tolerant sugar beets (roots and silage) and maize
grains for ruminants and pigs. Arch Anim Nutrit 54:197–207
Bonny S (2008) Genetically modified glyphosate-tolerant soybean in the USA: adoption factors,
impacts and prospects: a review. Agron Sustain Dev 28:21–32
Borggaard OK, Gimsing AL (2008) Fate of glyphosate in soil and the possibility of leaching to
ground and surface waters: a review. Pest Manag Sci 64:441–456
Bott S, Tesfamariam T, Candan H, Cakamak I, Römheld V, Neumann G (2008) Glyphosate-
induced impairment of plant growth and micronutient status in glyphosate-resistant soybean
(Glycine max L.). Plant Soil 312:185–194
Bradley KW, Sweets LE (2008) Influence of glyphosate and fungicide coapplications on weed
control, spray penetration, soybean response, and yield in glyphosate-resistant soybean.
Agron J 100:1360–1365
Bradshaw LD, Padgette SR, Kimball SL, Wells BH (1997) Perspectives on glyphosate resistance.
Weed Technol 11:189–198
Brake BG, Evenson DP (2004) A generational study of glyphosate-tolerant soybeans on mouse
fetal, postnatal, pubertal and adult testicular development. Food Chem Toxicol 42:29–36
Brimner TA, Gallivan GJ, Stephenson GR (2005) Influence of herbicide-resistant canola on the
environmental impact of weed management. Pest Manag Sci 61:47–52
Brookes G, Barfoot P (2006) Global impact of biotech crops: Socio-economic and environmental
effects in the first ten years of commercial use. AgBioForum 9:139–151
Brown J, Brown A (1996) Gene transfer between canola (Brassica napus L. and B. campestris L.)
and related species. Ann Appl Biol 129:555–560
Brown PB, Wilson KA, Jonker Y, Nickson TE (2003) Glyphosate tolerant canola meal is
equivalent to the parental line in diets fed to rainbow trout. J Agri Food Chem 51:4268–4272
Buchanan-Wollaston V, Snape A, Cannon F (1992) A plant selective marker gene based on the
detoxification of the herbicide dalapon. Plant Cell Rep 11:627–631
Canola Council of Canada (2001) An agronomic and economic assessment of transgenic canola.
http://www.canola-council.org/
Castle LA, Siehl DL, Gorton R, Patten PA, Chen YH, Cho BS, H-J DN, Wong J, Liu D, Lassner
MW (2004) Discovery and directed evolution of a glyphosate tolerance gene. Science
304:1151–1154
Cedergreen N (2008) Is the growth stimulation by low doses of glyphosate sustained over time?
Environ Poll 156:1099–1104
Cedergreen N, Streibig JC, Kudsk P, Mathiassen SK, Duke SO (2007) The occurrence of hormesis
in plants and algae. Dose-Response 5:150–162
Cellini F, Chesson A, Colquhoun I, Constable A, Davies HV, Engel KH, Gatehouse AMR,
Kaerenlampi S, Kok EJ, Leguay JJ, Lehesranta S, Noteborn HPJM, Pedersen J, Smith M
(2004) Unintended effects and their detection in genetically modified crops. Food Chem
Toxicol 42:1089–1125
Cerdeira AL, Duke SO (2006) The current status and environmental impacts of glyphosate-
resistant crops: a review. J Environ Qual 35:1633–1658
Cerdeira AL, Duke SO (2007) Environmental impacts of transgenic herbicide-resistant crops.
CAB Rev: Perspectives in Agri Vet Sci Nutri Nat Res 2:#033, pp, 14, http://www.cabi.org/
cabreviews/default.aspx?LoadModule¼Review&ReviewID¼31919167&page¼1178
Cerdeira AL, Gazziero DLP, Duke SO, Matallo MB, Spadoto CA (2007) Review of potential
environmental impacts of transgenic glyphosate-resistant soybean in Brazil. J Environ Sci
Health, Part B: Pest Food Contam Agri Wastes 42:539–549
Chang HS, Kim NH, Park MJ, Lim SK, Kim SC, Kim JY, Kim JA, Oh HY, Lee CH, Huh K,
Jeong TC, Nam DH (2003) The 5-enolpyruvylshikimate-3-phosphate synthase of glyphosate-
tolerant soybean expressed in Escherichia coli shows no severe allergenicity. Mol Cell 15:20–26
Clewis SB, Wilcut JW (2007) Economic assessment of weed management in strip- and conven-
tional-tillage nontransgenic and transgenic cotton. Weed Technol 21:45–52
Combs DK, Hartnell GF (2008) Alfalfa containing the glyphosate-tolerant trait has no effect on
feed intake, milk composition, or milk production of dairy cattle. J Dairy Sci 91:673–678
Coupland D, Caseley JC (1979) Presence of 14C activity in root exudates and guttation fluid from
Agropyron repens treated with 14C-labeled glyphosate. New Phytol 83:17–22
Cromwell GL, Lindemann MD, Randolph JH, Parker GR, Coffey RD, Laurent KM, Armstrong
CL, Mikel WB, Stanisiewski EP, Hartnell GF (2002) Soybean meal from Roundup Ready or
conventional soybeans in diets for growing-finishing swine. J Anim Sci 80:708–715
Cromwell GL, Henry BJ, Scott AL, Gerngross MF, Dusek DL, Fletcher DW (2005) Glufosinate
herbicide-tolerant (LibertyLink) rice vs. conventional rice in diets for growing-finishing swine.
J Anim Sci 83:1068–1074
Daenicke R, Aulrich K, Flachowsky G (2000) Investigations on the nutritional value of sugar
beets and sugar beet leaf silage of isogenic and transgenic plants for muttons. VDLUFA-
Schriftenreihe 55:84–86
Dale PJ, Clarke B, Fontes EMG (2002) Potential for the environmental impact of transgenic crops.
Darmency H, Vigouroux Y, De Garambe T, Gestat R-MM, Muchembled C (2007) Transgene
escape in sugar beet production fields: data from six years farm scale monitoring. Environ
Biosaf Res 6:197–206
Devine MD (2005) Why are there not more herbicide-tolerant crops? Pest Manag Sci 61:312–317
Devine MD, Duke SO, Fedtke K (1993) Physiology of herbicide action. PTR Prentiss-Hall,
Englewood Cliffs, NJ, USA, p 441
Devos Y, Reheul D, De Schrijver A, Cors F, Moens W (2005) Management of herbicide-tolerant
oilseed rape in Europe: a case study on minimizing vertical gene flow. Environ Biosaf Res
3:135–148
Devos Y, Cougnon M, Vergucht S, Bulcke R, Haesaert G, Steurbaut W, Reheul D (2008)
Environmental impact of herbicide regimes used with genetically modified herbicide-resistant
maize. Transgen Res 17:1059–1077
Dick RE, Quinn JP (1995) Glyphosate-degrading isolates from environmental samples: occur-
rence and pathways of degradation. Appl Microbiol Biotechnol 43:545–550
Dill GM (2005) Glyphosate-resistant crops: history, status and future. Pest Manag Sci 61:219–224
Dill GM, CaJacob CA, Padgette SR (2008) Glyphosate-resistant crops: adoption, use and future
considerations. Pest Manag Sci 64:326–331
Dinelli G, Marotti I, Bonetti A, Catizone P, Urbano JM, Barnes J (2008) Physiological and
molecular bases of glyphosate resistance in Conyza bonariensis biotypes from Spain. Weed
Res 48:257–265
Donkin SS, Velez JC, Totten AK, Stanisiewski EP, Hartnell GF (2003) Effects of feeding silage
and grain from glyphosate-tolerant or insect-protected corn hybrids on feed intake, ruminal
digestion, and milk production in dairy cattle. J Dairy Sci 86:1780–1788
Duke SO (1988) Glyphosate. In: Kearney PC, Kaufman DD (eds) Herbicides – chemistry, degrada-
tion and mode of action, vol III. Marcel Dekker, New York, USA, pp 1–70
Duke SO (ed) (1996) Herbicide-resistant crops. CRC Press, USA, p 420
Duke SO (2003) Weeding with transgenes. Trends Biotechnol 21:192–195
Duke SO (2005) Taking stock of herbicide-resistant crops ten years after introduction. Pest Manag
Sci 61:211–218
Duke SO (2006) The use of transgenes for weed management. J Plant Dis Protect 20:3–10 Spl Iss
Duke SO, Cerdeira AL (2005) Potential environmental impacts of herbicide-resistant crops. In:
Collection of biosafety reviews, vol 2. International Centre for Genetic Engineering and
Biotechnology, Trieste, Italy, pp. 66–143
Duke SO, Powles SB (2008a) Editorial: glyphosate-resistant weeds and crops. Pest Manag Sci
64:317–318
Duke SO, Powles SB (2008b) Glyphosate: a once in a century herbicide. Pest Manag Sci
64:319–325
Duke SO, Holt JS, Hess FD, Christy AL (1991) Herbicide-resistant crops. Comments from CAST,
No 1999-1, Council for Agricultural Science and Technology, Ames, IA, USA, p. 24
Duke SO, Baerson SR, Rimando, AM (2003a) Herbicides: glyphosate. In: Plimmer JR, Gammon
DW, Ragsdale NN (eds) Encyclopedia of agrochemicals. John Wiley, New York, USA
http://www.mrw.interscience.wiley.com/eoa/articles/agr119/frame.html
Duke SO, Rimando AM, Pace PF, Reddy KN, Smeda RJ (2003b) Isoflavone, glyphosate, and
aminomethylphosphonic acid levels in seeds of glyphosate-treated, glyphosate-resistant
soybean. J Agri Food Chem 51:340–344
Duke SO, Cedergreen N, Velini ED, Belz RG (2006) Hormesis: Is it an important factor in
herbicide use and allelopathy. Outlooks Pest Manag 17:29–33
Duke SO, Wedge DE, Cerdeira AL, Matallo MB (2007) Interactions of synthetic herbicides with
plant disease and microbial herbicides. In: Vurro M, Gressel J (eds) Novel Biotechnologies for
Biocontrol Agent Enhancement and Management. Springer, Dordrecht, The Netherlands,
pp 277–296
Dunfield KE, Germida JJ (2004) Impact of genetically modified crops on soil- and plant-associated
microbial communities. J Environ Qual 33:806–815
Ebert E, Leist KH, Mayer D (1990) Summary of safety evaluation toxicity studies of glufosinate
ammonium. Food Chem Toxicol 28:339–349
Erickson GE, Robbins ND, Simon JJ, Berger LL, Klopfenstein TJ, Stanisiewski EP, Hartnell GF
(2003) Effect of feeding glyphosate-tolerant (Roundup-Ready events GA21 or nk603) corn
compared with reference hybrids on feedlot steer performance and carcass characteristics.
J Anim Sci 81:2600–2608
Feng PCC, Baley GJ, Clinton WP, Bunkers GJ, Alibhai MF, Paulitz TC, Kidwell KK (2005)
Glyphosate inhibits rust diseases in glyphosate-resistant wheat and soybean. Proc Natl Acad
Sci USA 102:17290–17295
Feng PCC, Clark C, Andrade GC, Balbi MC, Caldwell P (2008) The control of Asian rust by
glyphosate-resistant soybeans. Pest Manag Sci 64:353–359
Gardner JG, Nelson GC (2008) Herbicides, glyphosate resistance and acute mammalian toxicity:
simulating an environmental effect of glyphosate-resistant weeds in the USA. Pest Manag Sci
64:470–478
Gianessi LP (2005) Economic and herbicide use impacts of glyphosate-resistant crops. Pest Manag
Sci 61:241–245
Gianessi LP (2008) Economic impacts of glyphosate-resistant crops. Pest Manag Sci 64:346–352
Giesy JP, Dobson S, Solomon KR (2000) Ecotoxicological risk assessment for Roundup herbicide.
Rev Environ Contam Toxicol 167:35–120
Giovannetti M (2003) The ecological risks of transgenic plants. Rivista Biol 96:207–223
Goldberg RJ, Rissler J, Shand H, Hassebrook C (1990) Biotechnology’s Bitter Harvest: Herbicide-
tolerant crops and the threat to sustainable agriculture. Environental Defense Fund, New York,
USA 73 p
Grant RJ, Fanning KC, Kleinschmit D, Stanisiewski EP, Hartnell GF (2003) Influence of glypho-
sate-tolerant (event nk603) and corn rootworm protected (event MON863) corn silage and
grain on feed consumption and milk production in Holstein cattle. J Dairy Sci 86:1707–1715
Green JM, Hazel CB, Forney DR, Pugh LM (2008) New multiple-herbicide crop resistance and
formulation technology to augment the utility of glyphosate. Pest Manag Sci 64:332–339
Gressel J (2002) Molecular biology of weed control. Taylor & Francis, London, UK, p 504
Gressel J, Al-Ahmad H (2004) Methods and transgenic plants for mitigating introgression of
genetically engineered genetic traits from crop plants into related species. US Patent Appl Publ
US Der No 889 737, abandoned App US 2004–774 388 20040210
Gustafson DI (2008) Sustainable use of glyphosate in North American cropping systems. Pest
Manag Sci 64:409–416
Hack R, Ebert E, Ehling G, Leist KH (1994) Glufosinate ammonium-some aspects of its mode of
action in mammals. Food Chem Toxicol 32:461–470
Hall L, Topinka K, Huffman J, Davis L, Good A (2000) Pollen flow between herbicide-resistant
B. napus volunteers. Weed Sci 48:688–694
Hammond BG, Vicini JL, Hartnell GF, Naylor MW, Knight CD, Robinson EH, Fuchs RL,
Padgette SR (1996) The feeding value of soybeans fed to rats, chickens, catfish and dairy
cattle is not altered by genetic incorporation of glyphosate tolerance. J Nutri 126:717–727
Hammond B, Dudek R, Lemen J, Nemeth M (2004) Results of a 13 week safety assurance study
with rats fed grain from glyphosate tolerant corn. Food Chem Toxicol 42:1003–1014
Harrison LA, Bailey MR, Naylor MW, Ream JE, Hammond BG, Nida DL, Burnette BL, Nickson
TE, Mitsky TA, Taylor TA, Fuchs RL, Padgette SR (1996) The expressed protein in glypho-
sate-tolerant soybean, 5-enolpyruvylshikimate-3-phosphase synthase from Agrobacterium sp.
strain CP4 is rapidly digested in vitro and is not toxic to acutely gavaged mice. J Nutr
126:728–740
Hartnell GF, Hvelplund T, Weisbjerg MR (2005) Nutrient digestibility in sheep fed diets contain-
ing Roundup Ready or conventional fodder beet, sugar beet, and beet pulp. J Anim Sci
83:400–407
Healy C, Hammond B, Kirkpatrick J (2008) Results of a 13-week safety assurance study with rats
fed grain from corn rootworm-protected, glyphosate -tolerant MON 88017 corn. Food Chem
Toxicol 46:2517–2524
Heap IM (1997) The occurrence of herbicide-resistant weeds worldwide. Pest Sci 51:235–243
Heap IM (2008) International survey of herbicide resistant weeds. http://www.weedscience.org/
In.asp
Herman PL, Behrens M, Chakraborty S, Chrastil BM, Barycki J, Weeks DP (2005) A three-
component dicamba O-demethylase from Pseudomonas maltophilia, Strain DI-6: gene isola-
tion, characterization, and heterologous expression. J Biol Chem 280:24759–24767
Herouet C, Esdaile DJ, Mallyon BA, Debruyne E, Schulz A, Currier T, Hendrickx K, van der Klis
R-J, Rouan D (2005) Safety evaluation of the phosphinothricin acetyltransferase proteins
encoded by the pat and bar sequences that confer tolerance to glufosinate-ammonium herbicide
in transgenic plants. Regul Toxicol Pharmacol 41:134–149
Hoagland RE (1980) Effects of glyphosate on metabolism of phenolic compounds: VI. Effects of
glyphosine and glyphosate metabolites on phenylalanine ammonia-lyase activity, growth, and
protein, chlorophyll, and anthocyanin levels in soybean (Glycine max) seedlings. Weed Sci
28:393–400
Hyun Y, Bressner GE, Ellis M, Lewis AJ, Fischer R, Stanisiewski EP, Hartnell GF (2004) Perfor-
mance of growing-finishing pigs fed diets containing Roundup Ready corn (event nk603), a
nontransgenic genetically similar corn, or conventional corn lines. J Anim Sci 82:571–580
Ipharraguerre IR, Younker RS, Clark JH, Stanisiewski EP, Hartnell GF (2003) Performance of
lactating dairy cows fed corn as whole plant silage and grain produced from a glyphosate-
tolerant hybrid (event NK603). J Dairy Sci 86:1734–1741
Jakobsen I, Smith SE, Smith FA (2002) Function and diversity of arbuscular mycorrhizae in
carbon and mineral nutrition. In: van der Heijden MGA, Sanders IR (eds) Mycorrhizal
Ecology. Springer, Berlin, pp 75–92
James C (2008) International service for the acquisition of agri-biotech applications. http://www.
isaaa.org/resources/publications/briefs/37/pptslides/Brief37slides.pdf
Jennings JC, Kolwyck DC, Kays SB, Whetsell AJ, Surber JB, Cromwell GL (2003) Determining
whether transgenic and endogenous plant DNA and transgenic protein are detectable in muscle
from swine fed Roundup Ready soybean meal. J Anim Sci 81:1447–1455
Jepson I, Martinez A, Sweetman JP (1998) Chemical-inducible gene expression systems for plants
– a review. Pest Sci 54:360–367
Jolley VD, Hansen NC, Shiffer AK (2004) Nutritional and management related interactions with
iron-deficiency stress response mechanisms. Soil Sci Plant Nutr 50:973–981
Kim YT, Kim SE, Park KD, Kang TH, Lee YM, Lee SH, Moon JS, Kim SU (2005) Investigation
of possible gene transfer from leaf tissue of transgenic potato to soil bacteria. J Microbiol
Kishore GM, Jacob GS (1987) Degradation of glyphosate by Pseudomonas sp. PG2982 via a
sarcosine intermediate. J Biol Chem 262:12164–12168
Kleter GA, Bhula R, Bodnaruk K, Carazo E, Felso AS, Harris CA, Katayama A, Kuiper HA,
Racke KD, Rubin B, Shevah Y, Stephenson GR, Tanaka K, Unsworth J, Wauchope RD, Wong
S-S (2007) Altered pesticide use on transgenic crops and the associated general impact from an
environmental perspective. Pest Manag Sci 64:479–488
Kleter GA, Harris C, Stephenson G, Unsworth J (2008) Comparison of herbicide regimes and the
associated potential effects of glypyosate-resistant crops versus what they replace in Europe.
Pest Manag Sci 64:479–488
Koch HJ, Pringas C, Scherer J (2003) Conservation tillage for sustainable sugarbeet production in
Germany – environmental and phytopathological aspects. Zuckerindustrie 128:810–813
König A, Cockburn A, Crevel RWR, Debruyne E, Grafstroem R, Hammerling U, Kimber I,
Knudsen I, Kuiper HA, Peijnenburg AACM, Penninks AH, Poulsen M, Schauzu M, Wal JM
(2004) Assessment of the safety of foods derived from genetically modified (GM) crops. Food
Chem Toxicol 42:1047–1088
Kremer RJ, Means NE, Kim S (2005) Glyphosate affects soybean root exudation and rhizosphere
microorganisms. Int J Environ Anal Chem 85:1165–1174
Kuiper HA, Noteborn HPJM, Kok EJ, Kleter GA (2002) Safety aspects of novel food. Food Res Int
35:267–271
Laitinen P, Rämö S, Siimes K (2007) Glyphosate translocation from plants to soil - does this
constitute a significant proportion of residues in soil? Plant Soil 300:51–60
Lappe MA, Bailey EB, Childress C, Setchell KDR (1999) Alterations in clinically impor-
tant phytoestrogens in genetically modified, herbicide-tolerant soybeans. J Med Food
1:241–245
Larson RL, Hill AL, Fenwick A, Kniss AR, Hanson LE, Miller SD (2005) Influence of glyphosate
on Rhizoctonia and Fusarium root rot in sugar beet. Pest Manag Sci 62:1182–1192
Lee CD, Penner D, Hammerschmidt R (2003) Influence of formulated glyphosate and activator
adjuvants on Schlerotinia schlerotiorum in glyhphosate-resistant and susceptible Glycine max.
Weed Sci 48:710–715
Lee CD, Renner KA, Penner D, Hammerschmidt R, Kelly JD (2005) Glyphosate-resistant soybean
management system effect on Scleroinia stem rot. Weed Technol 19:580–588
Levy-Booth DJ, Campbell RG, Gulden RH, Hart M, Powell JR, Klironomos JN, Pauls KP,
Swanton CJ, Trevors JT, Dunfield KE (2008) Real-time polymerase chain reaction monitoring
of recombinant DNA entry into soil from decomposing Roundup Ready leaf biomass. J Agri
Food Chem 56:6339–6347
Li X, Nicholl D (2005) Development of PPO inhibitor-resistant cultures and crops. Pest Manag Sci
61:277–285
Liu C-M, McLean PA, Sookdeo CC, Cannon FC (1991) Degradation of the herbicide glyphosate
by members of the family Rhizobiaceae. Appl Environ Microbiol 57:1799–1804
Llewellyn D, Last D (1996) Genetic engineering of crops for tolerance to 2, 4-D. In: Duke SO (ed)
Herbicide-Resistant Crops. CRC Press, Boca Raton, Florida, USA, pp 159–174
Locke MA, Zablotowicz RM, Reddy KN (2008) Integrating soil conservation practices and
glyphosate-resistant crops: impacts on soil. Pest Manag Sci 64:457–469
Lorenzatti E, Maitre MI, Argelia L, Lajmanovich R, Peltzer P, Anglada M (2004) Pesticide
residues in immature soybeans of Argentina croplands. Fresenius Environ Bull 13:675–678
Lydon J, Duke SO (1999) Inhibitors of glutamine biosynthesis. In: Singh BK (ed) Plant Amino
Acids: Biochemistry and Biotechnology. Marcel Dekker, New York, USA, pp 445–464
Malarkey T (2003) Human heath concerns with GM crops. Mut Res 544:217–221
Mallory-Smith C, Zapiola M (2008) Gene flow from glyphosate-resistant crops. Pest Manag Sci
64:428–440
Mamy L, Barriuso E (2005) Glyphosate adsorption in soils compared to herbicides replaced with
the introduction of glyphosate resistant crops. Chemosphere 61:844–855
Mamy L, Barriuso E, Gabrielle B (2005) Environmental fate of herbicides trifluralin, metazachlor,
metamitron and sulcotrione compared with that of glyphosate, a substitute broad spectrum
herbicide for different glyphosate-resistant crops. Pest Manag Sci 61:905–916
Mamy L, Gabrielle B, Barriuso E (2008) Measurement and modeling of glyphosate fate compared
with that of herbicides replaced as a result of the introduction of glyphosate-resistant oilseed
rape. Pest Manag Sci 64:262–275
Matringe M, Sailland A, Pelissier B, Roland A, Zind O (2005) p-Hydroxyphenylpyruvate dioxy-
genase inhibitor-resistant plants. Pest Manag Sci 61:269–276
McClean GD, Evans G (eds) (1995) Herbicide-resistant crops and pastures in australian farming
systems. Bureau of Resource Sciences, Australia, p 294
McNaughton J, Roberts M, Smith B, Rice D, Hinds M, Schmidt J, Locke M, Brink K, Rood T,
Layton R, Lamb I, Delaney B (2007) Comparison of broiler performance when fed diets
containing event DP-356Ø43–5 (Optimum GAT), nontransgenic near-isoline control or com-
mercial soybean meal, hulls, and oil. Poult Sci 86:2569–2581
Means NE, Kremer RJ (2007) Influence of soil moisture on root colonization of glyphosate-treated
soybean by Fusarium species. Commun Soil Sci Plant Anal 38:1713–1720
Means NE, Kremer RJ, Ramsier C (2007) Effects of glyphosate and foliar amendments on activity
of microorganisms in the soybean rhizosphere. J Environ Sci Health, Part B: Pest Food Contam
Agri Wastes 42:125–132
Moorman TB, Becerril JM, Lydon JM, Duke SO (1992) Production of hydroxybenzoic acids by
Bradyrhizobium japonicum strains after treatment with glyphosate. J Agri Food Chem
40:289–293
Nail EL, Young DL, Schillinger WF (2006) Diesel and glyphosate price changes benefit the
economics of conservation tillage versus traditional tillage. Soil Till Res 94:321–327
Nandula VK, Reddy KN, Rimando AM, Duke SO, Poston DH (2007) Glyphosate-resistant and
-susceptible soybean (Glycine max) and canola (Brassica napus) dose response and
metabolism relationships with glyphosate. J Agri Food Chem 55:3540–3545
Nelson DS, Bullock GC (2003) Simulating a relative environmental effect of glyphosate-resistant
soybeans. Ecol Econ 45:273–278
Nelson KA, Renner KA, Hammerschmidt R (2002) Cultivar and herbicide selection affects
soybean development and incidence of Sclerotinia stem rot. Agron J 94:1270–1281
Neve P (2008) Simulation modeling to understand the evolution and management of glyphosate
resistance in weeds. Pest Manag Sci 64:392–401
Nida DL, Patzer S, Harvey P, Stipanovic R, Wood R, Fuchs RL (1996) Glyphosate-tolerant cotton:
The composition of the cottonseed is equivalent to that of conventional cottonseed. J Agri Food
Chem 44:1967–1974
Nielsen KM, Townsend JP (2004) Monitoring and modeling horizontal gene transfer. Nat Bio-
technol 22:1110–1114
Nijiti VN, Myers O, Schroeder D, Lightfoot DA (2003) Roundup Ready soybean: glyphosate
effects on Fusarium solani root colonization and sudden death syndrome. Agron J
95:1140–1145
Obert JC, Ridley WP, Schneider RW, Riordan SG, Nemeth MA, Trujillo WA, Breeze ML, Sorbet
R, Astwood JD (2004) The composition of grain and forage from glyphosate tolerant wheat
MON 71800 is equivalent to that of conventional wheat (Triticum aestivum L.). J Agri Food
Chem 52:1375–1384
Oliver MJ, Quisenberry JE, Trolinder NLG, Keim DL (1998) Regeneration of genetically mod-
ified whole plant from plant cell transfected with DNA sequence comprising regulatory
regions and genes for phenotype-regulating protein, recombinase, and genetic repressor. US
Patent 283 604
Owen MDK (2008) Weed species shifts in glyphosate-resistant crops. Pest Manag Sci 64:377–387
Ozturk L, Yazici A, Eker S, Gokmen O, Römheld V, Cakmak I (2007) Glyphosate inhibition of
ferric reductase activity in iron deficient sunflower roots. New Phytol 177:899–906
Padgette SR, Taylor NB, Nida DL, Bailey MR, MacDonald J, Holden LR, Fuchs RL (1996)
The composition of glyphosate-tolerant soybean seeds is equivalent to that of conventional
soybeans. J Agri Food Chem 126:702–716
Pankey JH, Griffin JL, Colyer PD, Schneider W, Miller DK (2005) Preemergence herbicide
and glyphosate effects on seedling disease in glyphosate-resistant cotton. Weed Technol
19:312–318
Penna JA, Lema D (2003) Adoption of herbicide tolerant soybeans in Argentina: an economic
analysis. In: Kalaitzandonakes N (ed) Economic and environmental impacts of agrotechnol-
ogy. Kluwer-Plenum Publ, New York, USA, pp 203–220
Perez-Jones A, Park K-W, Polge N, Colquhoun J, Mallory-Smith CA (2007) Investigating the
mechanisms of glyphosate resistance in Lolium multiflorum. Planta 226:395–404
Pline WA, Wilcut JW, Duke SO, Edmisten KL, Wells R (2002a) Tolerance and accumulation of
shikimic acid in response to glyphosate applications in glyphosate-resistant and conventional
cotton (Gossypium hirsutum L.). J Agri Food Chem 50:506–512
Pline WA, Viator R, Wilcut JW, Edmisten KL, Thomas J, Wells R (2002b) Reproductive
abnormalities in glyphosate-resistant cotton caused by lower CP4-EPSPS levels in the male
reproductive tissue. Weed Sci 50:438–447
Pline-Srnic W (2005) Technical performance of some commercial glyphosate-resistant crops. Pest

Powell JR, Swanton CJ (2008) A critique of studies evaluating glyphosate effects on diseases
associated with Fusarium spp. Weed Res 48:307–318
Powell RG, Plattner RD, Suffness M (1990) Occurrence of sesbanimide in seeds of toxic Sesbania
species. Weed Sci 38:148–152
Powles SB (2008a) Evolved glyphosate-resistant weeds around the world: lessons to be learnt. Pest
Powles SB (2008b) Evolution in action: glyphosate-resistant weeds threaten world crops. Outlooks
Pest Manag 16:256–259
Preston C, Wakelin AM (2008) Resistance to glyphosate from altered translocation patterns. Pest
Reddy KN (2004) Weed control and species shift in bromoxynil- and glyphosate-resistant cotton
(Gossypium hirsutum) rotation systems. Weed Technol 18:131–139
Reddy KN, Zablotowicz RM (2003) Glyphosate-resistant soybean response to various salts of
glyphosate and glyphosate accumulation in soybean nodules. Weed Sci 51:496–502
Reddy KN, Duke SO, Rimando AM (2004) Aminomethylphosphonic acid, a metabolite of
glyphosate, causes injury in glyphosate-treated, glyphosate-resistant soybean. J Agri Food
Chem 52:5139–5143
Reddy KN, Abbas HK, Zablotowicz RM, Abel CA, Koger CH (2007) Mycotoxin occurrence and
Aspergillus flavus soil propagules in a corn and cotton glyphosate-resistant cropping systems.
Food Additives Contaminants 24:1367–1373
Reddy KN, Rimando AM, Duke SO, Nandula VK (2008) Aminomethylphosphonic acid accumu-
lation in plant species treated with glyphosate. J Agri Food Chem 56:2125–2130
Reiger MA, Lamond M, Preston C, Powles SB, Roush RT (2002) Pollen-mediated movement of
herbicide resistance between commercial canola fields. Science 296:2386–2388
Ridley WP, Sidhu RS, Pyla PD, Nemeth MA, Breeze ML, Astwood JD (2002) Comparison of the
nutritional profile of glyphosate-tolerant corn event NK603 with that of conventional corn (Zea
mays L.). J Agri Food Chem 50:7235–7243
Ruhland M, Engelhardt G, Pawlizki K (2004) Distribution and metabolism of D/L, L- and
D-glufosinate in transgenic, glufosinate-tolerant crops of maize (Zea mays L. spp. mays) and
oilseed rape (Brassica napus L. var. napus) (dagger). Pest Manag Sci 60:691–696
Saji H, Nakajim N, Aono M, Tamaoki M, Kubo A, Wakiyama S, Hatase Y, Nagatsu M (2005)
Monitoring the escape of transgenic oilseed rape around Japanese ports and roadsides. Environ
Biosaf Res 4:217–222
Sandmann G, Misawa N, Böger P (1996) Steps toward genetic engineering of crops resistant
against bleaching herbicides. In: Duke SO (ed) Herbicide-resistant Crops. CRC Press, USA,
pp 189–200
Sankula S (2006) Quantification of the impacts on U.S. agriculture of biotechnology-derived crops
planted in 2005 national center for food and agricultural policy. Washington, USA 110 p
Sanogo S, Yang XB, Lundeen P (2001) Field response of glyphosate-tolerant soybean to herbi-
cides and sudden death syndrome. Plant Dis 85:773–779
Schabenberger O, Kells JJ, Penner D (1999) Statistical tests for hormesis and effective dosage in
herbicide dose-response. Agron J 91:713–721
Scheffler JS, Dale PJ (1994) Opportunities for gene transfer from transgenic oilseed rape (Brassica
napus) to related species. Transgen Res 3:263–278
Schonherr J, Schreiber L (2004) Interactions of calcium ions with weakly acidic active ingredients
slow cuticular penetration: a case study with glyphosate. J Agri Food Chem 52:6546–6551
Sen Gupta A, Heinen JL, Holaday AS, Burke JJ, Allen RD (1993) Increased resistance to oxidative
stress in transgenic plants that overexpress chloroplast Cu/Zn superoxide dismutase. Proc Natl
Shaw DR, Bray CS (2003) Foreign material and seed moisture in glyphosate-resistant and
conventional soybean systems. Weed Technol 17:389–393
Shiptalo MJ, Malone RW, Owens LB (2008) Impact of glyphosate-tolerant soybean and glufosinate-
tolerant corn production on herbicides losses in surface runoff. J Environ Qual 37:401–408
Shivrain VK, Burgos NR, Anders MM, Rajguru SN, Moore J, Sales MA (2007) Gene flow
between Clearfield rice and red rice. Crop Protect 26:349–356
Shukla MK, Lal R, Ebinger M (2003) Tillage effects on physical and hydrobiological properties of
a Typic Argiaquoll in central Ohio. Soil Sci 168:802–811
Sidhu RS, Hammond BG, Fuchs RL, Mutz JN, Holden LR, George B, Olson T (2000) Glyphosate-
tolerant corn: the composition and feeding value of grain from glyphosate-tolerant corn is
equivalent to that of conventional corn (Zea mays L.). J Agri Food Chem 48:2305–2312
Siehl DL, Castle LA, Gorton R, Chen YH, Bertain S, Cho H-J, Keenan R, Liu D, Lassner MW
(2005) Evolution of a microbial acetyltransferase for modification of glyphosate: a novel
tolerance strategy. Pest Manag Sci 61:235–240
Siehl DL, Castle LA, Gorton R, Keenan RJ (2007) The molecular basis of glyphosate resistance by
an optimized microbial acetyltransferase. J Biol Chem 282:11446–11455
Simonsen L, Fomsgaard IS, Svensmark B, Spliid NH (2008) Fate and availability of glyphosate
and AMPA in agricultural soil. J Environ Sci Health, Part B: Pesticides Food Contaminants
Agri Wastes 43:365–375
Simpson DM, Wright TR, Chambers RS, Peterson MA, Cui C, Robinson AE, Richburg JS, Ruen
DC, Ferguson S, Maddy BE, Schreder EF (2008) Dow AgroSciences herbicide tolerance traits
in corn and soybean. Proc N Central Weed Sci Soc, Champaign, IL, USA
Soltani N, Shropshire C, Sikkema PH (2006) Control of volunteer glyphosate-tolerant maize (Zea
mays) in glyphosate-tolerant soybean (Glycine max). Crop Protect 25:178–181
Stalker DM, Kiser JA, Baldwin G, Coulombe B, Houck CM (1996) Cotton weed control using the
BXN™ system. In: Duke SO (ed) Herbicide-resistant crops. CRC Press, Boca Raton, FL,
pp 93–105
Stella J, Ryan M (2004) Glyphosate herbicide formulation: A potentially lethal ingestion. Emerg
Med Australas 16:235–239
Sten E, Skov PS, Anderson SV, Torp AM, Olesen A, Bindlsley-Jensen U, Poulsen LK, Bindsley-
Jensen C (2004) A comparative study of the allergenic potency of wild-type and glyphosate-
tolerant gene-modified soybean cultivars. Acta Pathologica Microbiologica Immunologica
Scandinavica 112:21–28
Streber WR, Kutschka U, Thomas F, Pohlenz H-D (1994) Expression of a bacterial gene in trans-
genic plants confers resistance to the herbicide phenmedipham. Plant Mol Biol 25:977–987
Surov T, Aviv D, Aly R, Joel DM, Goldman-Guez T, Gressel J (1998) Generation of transgenic
asumlam-resistant potatoes to facilitate eradications of parasitic broomrapes (Orobanche spp.)
with the sul gene as the selectable marker. Theor Appl Genet 96:132–137
Tan S, Evans RR, Dahmer ML, Singh BK, Shaner DL (2005) Imidazolinone-tolerant crops:
history, current status and future. Pest Manag Sci 61:246–257
Taylor ML, Stanisiewski EP, Riordan SG, Nemeth MA, George B, Hartnell GF (2004) Compari-
son of broiler performance when fed diets containing Roundup Ready (event RT73), nontrans-
genic control, or commercial canola meal. Poultry Sci 83:456–461
Taylor M, Hartnell G, Lucas D, Davis S, Nemeth M (2007) Comparison of broiler perfor-
mance and carcass parameters when fed diets containing soybean meal produced from
glyphosate -tolerant (MON 89788), control, or conventional reference soybeans. Poult Sci
86:2608–2614
Teshima R, Akiyama H, Okunuki H, Sakushima J, Goda Y, Onodera H, Sawada J, Toyoda M
(2000) Effect of GM and non-GM soybeans on the immune system of BN rats and B10A mice.
Shokuhin Eiseigaku Zasshi 41:188–193
Thomas WE, Pline-Srnic WA, Thomas JF, Edmisten KL, Wells R, Wilcut JW (2004) Glyphosate
negatively affects pollen viability but not pollination and seed set in glyposate-resistant corn.
Weed Sci 52:725–734
Tutel’ian VA, Aksiuk IN, Sorokina EI, Aleshko-Ozhevskii IP, Gapparov MM, Zhminchenko VM,
Kodentsova VM, Nikol’skaia GV (2001) Medical and biological assessment of genetically
modified corn line MON 810 resistant to European corn borer and line GA 21 resistant to
glyphosate: a chemical study. Voprosy Pitaniia 70:25–27
Valverde BE (2007) Status and management of grass-weed herbicide resistance in Latin America.
Weed Technol 21:310–323
Vande Berg BJ, Hammer PE, Chun BL, Schouten LC, Carr B, Guo R, Peters C, Hinson TK,
Beilinson V, Shekita A, Deter R, Chen Z, Samoylov V, Bryant CT, Stauffer ME, Eberle T,
Moellenbeck DJ, Carozzi NB, Koziel MG, Duck NB (2008) Characterization and plant
expression of glyphosate-tolerant enolpyruvylshikimate phosphate synthase. Pest Manag Sci
64:340–345
Vasil IK (1996) Phosphinothricin-resistant crops. In: Duke SO (ed) Herbicide-resistant crops.
CRC Press, Boca Raton, FL, pp 85–91
Velini ED, Alves E, Godoy MC, Meschede DK, Souza RT, Duke SO (2008) Glyphosate applied at
low doses can stimulate plant growth. Pest Manag Sci 64:489–496
Vencill WK (ed) (2002) Herbicide Handbook, 8th edn. Weed Science Society of America,
USA, p 493
Vermij P (2006) Liberty Link rice raises specter of tightened regulations. Nat Biotechnol
24:1301–1302
Vila-Aiub MM, Vidal RA, Balbi MC, Gundel PE, Trucco F, Ghersa CM (2008) Glyphosate-
resistant weeds of South American cropping systems. Pest Manag Sci 64:366–371
Wagner R, Kogan M, Parada AM (2003) Phytotoxic activity of root absorbed glyphosate in corn
seedlings (Zea mays L.). Weed Biol Manag 3:228–232
Wakelin AM, Preston C (2006) A target-site mutation is present in a glyphosate-resistant Lolium
rigidum population. Weed Res 46:703–705
Warwick SI, Legere A, Simard M-J, James T (2008) Do escaped transgenes persist in nature? the
case of an herbicide resistant transgene in a weedy Brassica rapa population. Mol Ecol
17:1387–1395
Wauchope RD, Estes TL, Allen R, Baker JL, Hornsby AG, Jones RL, Richards RP, Gustosfson DI
(2002) Predicted impact of transgenic, herbicide tolerant corn on drinking water quality in
vulnerable watersheds of the mid-western USA. Pest Manag Sci 58:146–160
Werth JA, Preston C, Taylor IN, Charles GW, Robets GN, Baker J (2008) Managing the risk of
glyphosate ressistance in Australian glyphosate-resistant cotton production systems. Pest
White AK, Metcalf WW (2004) Two C-P lyase operons in Pseudomonas stutzeri and their roles in
the oxidation of phosphonates, phosphite, and hypophosphite. J Bacteriol 186:4730–4739
Williams GM, Kroes R, Munro IC (2000) Safety evaluation and risk assessment of the herbicide
Roundup and its active ingredient, glyphosate, for humans. Regul Toxicol Pharmacol 31:117–165
Wright TR, Lira JM, Merlo DJ, Hopkins N (2005) A bacterial gene for an aryloxyalkanoate
dioxygenase conferring resistance to phenoxy auxin and aryloxyphenoxypropionate herbi-
cides. Patent Application WO2005US1437 20050502
Yasuor H, Abu-Abied M, Belausov E, Madmony A, Sadot E, Riov J, Rubin B (2006) Glyphosate-
induced anther indehiscence in cotton is partially temperature dependent and involves cytoskele-
ton and secondary wall modifications and auxin accumulation. Plant Physiol 141:1306–1315
York AC, Steward AM, Vidrine PR, Culpepper AS (2004) Control of volunteer glyphosate-
resistant cotton in glyphosate-resistant soybean. Weed Technol 18:532–539
Zablotowicz RM, Reddy KN (2004) Impact of glyphosate on the Bradyrhizobium japonicum sym-
biosis with glyphosate-resistant transgenic soybean: a mini review. J Envron Qual 33:825–831
Zablotowicz RM, Reddy KN (2007) Nitrogenase activity, nitrogen content, and yield responses to
glyphosate in glyphosate-resistant soybean. Crop Prot 26:370–376
Zapiola ML, Campbell CK, Butler MD, Mallory-Smith C (2008) Escape and establishment of
transgenic glyphosate-resistant creeping bentgrass Agrostis stolonifera in Oregon, USA:
a 4-year study. J Appl Ecol 45:486–494
Zhu Y, Li D, Wang F, Yin J, Jin H (2004) Nutritional assessment and fate of DNA of soybean meal
from Roundup Ready or conventional soybeans using rats. Arch Anim Nutr 58:295–310
Chapter 4
Understanding and Manipulation of the
Flowering Network and the Perfection
of Seed Quality
Stephen L. Goldman, Sairam Rudrabhatla, Michael G. Muszynski, Paul Scott,

Diaa Al-Abed, and Shobha D Potlakayala
4.1 Introduction
In spite of an impressive 2,263 metric tons reported for cereal production in 2004,
sustainable output has not kept pace with global population increases. By 2020, the
world’s population is expected to exceed 8 billion and the projected minimum
annual 1.3% output increase needed to feed the population is not likely to be met.
Complicating the problem further is that the projected expansions are not expected
to be distributed evenly. Among the Asian Countries, India could become the most
densely populated country in the world.
Although India is second to the US in arable acreage, food is already being
imported. As reported by Saritha Rai in the New York Times (2006), India has
imported 2.2 million tons of wheat. This has been attributed to global climate
change resulting in decreases in rainfall in the arid/subhumid zones of the country.
Temperature increases and significant alterations in rainfall pattern are expected to
acerbate in the absence of global enforceable environmental laws designed to
mitigate sustainability while doing no harm to the biosphere.
Similar problems are expected to plague the Philippines and Indonesia. The
world’s governments can no longer look at agricultural production statistics on a
country-by-country basis as though the resolution of hunger is confined within
national borders. Although the largest population increases are expected in Asia,
the highest percent rise falls in Africa. The number of people is expected to multiply
at an annual rate of three percent accompanied by significant demographic changes
as people move from rural areas to the city, thus decreasing farm output. By 2020,
Africa alone is projected to require 60 million additional metric tons from import
suppliers.
S. Rudrabhatla (*)
Environmental Engineering, College of Science, Engineering and Technology, Pennsylvania State
University, Middletown, PA 17057, USA
e-mail: svr11@psu.edu

168 S.L. Goldman et al.
Where are these food sources to be found? India is already importing cereal and
its demand will increase over the next two decades. Moreover, China, the world’s
most populous country cannot sustain its growing population, since it has only 7%
of the globe’s arable land. Already America is experiencing the tension resulting
from an insufficient amount of corn where the interests of energy production
compete with the interests of the cereal’s use as food, forage, and commercial
additive. This tension is predictable as energy demand has resulted in historic per
bushel prices exceeding US$7.00, with significant increases in food cost. Placing
more land in production has its drawbacks, especially if increased cultivation
results in global loss with respect to biodiversity. In this connection, threats to
deceases in biodiversity with respect to land-use change have already resulted in
successful legal challenges.
Given the aforementioned facts, it is likely that the most immediate relief to
projected global food shortages will come not only as a function of increased seed
quality (either through standard breeding or transgenic production), but also as a
function of being able to manipulate flower development. Indeed, new plants will
have to be developed whose time-to-flower cycle is altered, thus mitigating chal-
lenges to both pollen production and fertilization associated with increased temper-
ature and diminished water supplies. The ability to alter development by changing
flowering time, life cycle length or, for that matter, where a plant is cultivated
should result not only in significant increases in the availability of land usage per se,
but also in production. If these alterations are likewise accompanied by improve-
ments in seed quality as measured by protein or fat content, for example, positive
movement is made towards the requisite goal of feeding people globally. All
aspects of flowering must be dissected and understood if its mechanism(s) is(are)
to be manipulated toward this end, and it is to this understanding that this review is
dedicated.
4.2 The Regulation of Flowering
4.2.1 The Floral Transition
The production of grain crops depends on the successful union of gametes at an

optimal time to ensure plentiful generation of seed for harvest. The production and
union of fertile gametes occur at flowering, which is a critical event in the life
cycle of all higher plants. In order to flower, plants must first transition from
vegetative to reproductive growth. During vegetative development, the shoot
apical meristem (SAM), a population of totipotent cells at the growing point of
the plant, gives rise to leaves and other above-ground organs. To switch to
reproductive growth, the SAM ceases leaf production and becomes committed to
the production of reproductive structures, such as an inflorescence bearing flowers
(Fig. 4.1). The period when the SAM is reprogrammed is called the floral
4 Understanding and Manipulation of the Flowering Network 169
Fig. 4.1 The floral transition in maize, when the vegetative shoot apical meristem (left) ceases leaf
production and adopts a reproductive fate (right). Scale bar is 200 mM
transition and the timing of the transition largely determines when a plant flowers.
Flowering time is often measured by counting the number of leaves produced prior
to the transition of the SAM and, thus, is a direct measure of the timing of the
transition. Accordingly, late flowering varieties produce more leaves than early
flowering varieties, as the transition is delayed and the SAM remains in the
vegetative stage of growth for a longer period of time. The timing of the floral
transition has been manipulated by plant breeders for centuries. Elite varieties
have been selected to balance the extent of vegetative growth with the duration of
grain fill to maximize yield in their adapted geographic locations. Identifying key
regulators of the floral transition and understanding their functions will allow for
the development of varieties improved for productivity and yield within a rapidly
changing environment.
Higher plants have developed sophisticated genetic mechanisms to ensure that
flowering coincides with an optimal time for reproductive success (Blazquez and
Weigel 2000; Hayama and Coupland 2003; Izawa et al. 2003; Komeda 2004;
Putterill et al. 2004; Simpson et al. 2004). Previous physiological studies revealed
that a combination of various environmental inputs and endogenous cues affect the
timing of the transition. For many species, day length is the predominant input,
while other species are day-neutral and rely almost entirely on endogenous signals.
These early studies also established several relevant points regarding the floral
transition (Bernier and Perilleux 2005). First, the inductive flowering signal
originates in leaves, whether the transition is triggered by external or endogenous
cues. Second, the inductive signal, often referred to as florigen (F), is mobile and is
transmitted from the leaves through the phloem to the shoot apex. Lastly, the
SAM, the target of the inductive signal in the shoot apex, must be competent to
perceive the signal in order to transition. Thus, we know that leaves and the shoot
apex are key tissues involved in the initiation, propagation and perception of floral
signaling.
4.2.2 The Floral Transition Network
At present, the molecular mechanisms that regulate the floral transition have been
primarily elaborated in the model plant Arabidopsis thaliana using the wealth of
flowering time mutants available in that species. Proper timing of the transition is
controlled by signaling through a complex network of floral inductive and repres-
sive activities. These activities function through four main regulatory pathways:
autonomous, photoperiod, gibberellin and vernalization (Koornneef et al. 1998;
Mouradov et al. 2002; Simpson and Dean 2002). The four regulatory pathways
converge on the following key floral integrators; Leafy (LFY), Suppressor of Over-
expression of Constans (SOC1), Flowering Locus T (FT) and Flowering Locus D
(FD) (Lee et al. 2000; Samach et al. 2000; Abe et al. 2005; Michaels et al. 2005;
Parcy 2005; Corbesier et al. 2007). From these integrators, the flowering signals are
transmitted to early-acting floral meristem identity MADS-box genes Apetala1
(AP1), Cauliflower (CAL) and Fruitful (FUL) that promote formation of inflores-
cence meristems which ultimately produce flowers (Fig. 4.2; Yanofsky 1995;
Ferrándiz et al. 2000). Less is known about what controls the floral transition in
the cereals, but a growing body of evidence suggests some of the floral regulators
and their signaling modules are conserved in Oryza sativa (rice), Triticum aestivum
(wheat) and Zea mays (maize) (Table 4.1; Danyluk et al. 2003; Hayama et al. 2003;
Izawa et al. 2003; Izawa 2007; Yan et al. 2003, 2006; Muszynski et al. 2006; Adam
et al. 2007; Chengxia and Dubcovsky 2008; Danilevskaya et al. 2008). The genes
known or presumed to regulate flowering time in the major cereal crops are listed in
Table 4.1. The core photoperiod regulatory module in Arabidopsis – GIGANTEA
(GI), CONSTANS (CO) and FT – has been shown to be conserved in rice; although,
under long-day photoperiods, rice FT expression is suppressed, leading to suppres-
sion of flowering, which is the opposite regulation of FT in Arabidopsis (Fig. 4.2;
Yano et al. 2000; Hayama et al. 2002; Kojima et al. 2002). Signaling downstream of
FT to the floral meristem identity genes also appears conserved among Arabidopsis,
rice, wheat and maize (Lim et al. 2000; Lee et al. 2003).
Although a number of studies show that diverse plant species share parts of the
regulatory pathways defined in Arabidopsis, whether all the components of these
regulatory pathways exist in other plants is not apparent. Furthermore, for those
components that are conserved, how they integrate within the larger network is
unknown. Further analysis of floral regulatory mechanisms in several species is
required to define common pathways as well as uncover unique regulatory elements
that may have evolved to accommodate particular environmental conditions and
species-specific physiologies. For example, although both Arabidopsis and rice
predominantly rely on photoperiod to regulate the timing of the transition flowering
is promoted in Arabidopsis by long days and in rice by short days. Unlike rice,
many temperate cereals, such as wheat, are long-day plants that also require
vernalization, a period of prolonged cold temperature, to promote flowering. Con-
versely, although domesticated from teosinte, an obligate short-day species, tem-
perate maize is largely photoperiod insensitive and relies primarily on endogenous
Photoperiod Response Autonomous

Arabidopsis Rice Maize
(LD) (SD) (DN)
circadian
clock
RID1 id1
LEAF GI OsGI
CO Hd1 (F)
FT Hd3a ZCN8(?)
FD + FT OsDlf1(?) + Hd3a dlf1 + ZCN8(?)
LFY x zfl1, zfl2(?)
APEX
AP1/CAL/FUL OsMADS14/18(?) ZMM4, ZMM15
Fig. 4.2 The floral transition networks in Arabidopsis (left), rice (center) and maize (right). Under
inductive photoperiods (long day for Arabidopsis and short day for rice), the circadian clock drives
expression of key leaf-expressed flowering time genes (GI and CO or OsGI and Hd1). These
activate the downstream mobile floral stimulus FT or Hd3a which moves from the leaves through
the phloem to the shoot apex. In the shoot apex, the mobile floral stimulus interacts with the floral
inductive gene FD or OsDlf1(?) to activate the floral meristem identity MADS-box genes AP1/
CAL/FUL or OsMADS14/18(?). In Arabidopsis, LFY integrates inductive signals from the leaf to
the floral meristem identity genes in a parallel pathway. In rice, the LFY ortholog does not appear
to participate in floral inductive signaling; but RID1, an id1 ortholog, has been shown to control
initiation of floral induction. In temperate maize, which is relatively day-neutral, an autonomous
signal induces expression of id1 in leaves that regulates a florigenic factor (F). Signals downstream
of F (perhaps ZCN8, a possible maize FT ortholog) are transmitted to the shoot apex and regulate
dlf1 expression or DLF1 activity. Interaction of DLF1 and ZCN8 presumably activates down-
stream targets x and the ZMM4 and ZMM15. Redundant inductive signaling through an id1-
independent alternate pathway converges downstream of dlf1. The position of genes marked by
(?) is speculative, hypothetical interactions are marked by dotted lines and the alternate pathway in
maize is colored blue
cues to flower. The molecular nature of the endogenous signaling pathways is not
known but some parts of the maize flowering network are shared with other cereals
and Arabidopsis (Table 4.1). The succeeding section describes what is known about
the floral transition regulatory network in maize and how the network components
relate to components from other species.
4.2.3 Flowering in Maize
Growth of maize (Zea mays) is largely determined by the activity of the SAM
(Fig. 4.1). During the first 3–4 weeks after germination, the SAM produces vegeta-
tive organs, such as leaves and stem tissue. After about 4–5 weeks of growth, the
Table 4.1 Genes regulating flowering time in the major cereal crops; maize, rice and wheat
Gene/locus Activitya Protein Similarity to Reference
homology Arabidopsisb
Maize
Constans of Zea mays1 UNK B-box zinc finger CONSTANS (CO) Miller et al.
(conz1) (2008)
Delayed flowering1 P Basic-leucine FLORAL LOCUS D Muszynski et al.
(dlf1) zipper (bZIP) (FD) (2006)
Gigantea of Zea mays1 UNK Nuclear protein GIGANTEA (GI) Miller et al.
(gigz1A and B) (2008)
Indeterminate1 (id1) P C2H2 zinc finger None Colasanti and
Sundaresan
(2000)
Phytochrome B (phyb) R Phytochrome PHYTOCHROME B Sheehan et al.
(2007)
Zea FLORICAULA/ P DNA-binding LEAFY (LFY) Bomblies et al.
LEAFY (zfl1 and protein (2003)
zfl2)
Zea mays UNK Phosphatidyl- FLORAL LOCUS T Danilevskaya
CENTRORADIALIS ethanolamine (FT) and et al. (2008)
(ZCN) binding TERMINAL
protein FLOWER (TFL)
Zea mays MADS-box4 P MIKC-type APETALA1 (AP1), Danilevskaya
(ZMM4) MADS-box CAULIFLOWER et al. (2008)
protein (CAL) and
FRUITFUL
(FUL)
ZmRap2.7 R AP2/ethylene- TARGET OF EAT1 Salvi et al. (2007)
responsive (TOE1)
element-
binding
protein
Rice
Early heading date1 P B-type response None Doi et al. (2004)
(Ehd1) regulator
GRAIN number, plant R Zinc-finger CCT None
height and heading domain
date7 (Ghd7) protein
Heading date1 (Hd1) P B-box zinc finger CO Yano et al.
(2000)
Heading date3a (Hd3a) P Phosphatidyl- FT Kojima et al.
ethanolamine (2002)
binding
protein
Heading date6 (Hd6) R a-subunit protein CK2 Takahashi et al.
kinase (2001)
OsGI P Nuclear protein GI Hayama et al.
(2002)
OsMADS14 P MIKC-type AP1/CAL/FUL Jeon et al. (2000)
MADS-box
protein
OsMADS51 P Type I MADS- None
box protein
(continued )
Table 4.1 (continued)

Gene/locus Activitya Protein Similarity to Reference
homology Arabidopsisb
OsSOC1 (OsMADS50) P MIKC-type SOC1 Lee et al. (2003)
MADS-box
protein
Rice FLO-LFY (RFL) P DNA-binding LFY Rao et al. (2008)
protein
Rice Id1 (RID1) P C2H2 zinc finger none Wu et al.
(2008a,b)
Photoperiodic R Heme oxygenase HY1 Izawa et al.
sensitivity5 (se5) (2000)
Wheat
TaFDL2 P Basic-leucine FD Chengxia and
zipper (bZIP) Dubcovsky
(2008)
TaFT (VRN3) P Phosphatidyl- FT Yan et al. (2006)
ethanolamine
binding
protein
TaVRN1 P MIKC-type AP1/CAL/FUL Yan et al. (2003)
MADS-box
protein
TaVRN2 R Zinc-finger CCT None Yan et al. (2004)
domain
protein
TaVRT2 R MIKC-type SHORT Kane et al.
MADS-box VEGETATIVE (2007)
protein STAGE3 (SVP3)
a
Gene activity has been shown to either promote (P) or repress (R) flowering or has not yet been
determined (UNK)
b
Similarity to Arabidopsis flowering time genes
SAM switches from vegetative over to reproductive growth during the period of the
floral transition. This period is distinguished in the SAM by the cessation of leaf
initiation and its rapid increase in size (Shaver 1983; Irish and Nelson 1991). The
SAM continues to increase in size as it progresses through the floral transition,
which terminates with the SAM acquiring inflorescence identity to become the
tassel primordium (Fig. 4.1). In maize, the tassel is the apical inflorescence, which
bears the male flowers that produce pollen. A similar transition occurs later in time,
leading to the conversion of several axillary meristems into ear primordia. In maize,
the ear is the axillary inflorescence, which bears the female flowers that exsert silks
(elongated pistils).
Flowering in temperate maize is largely controlled by endogenous signals,
possibly involving plant size or leaf number (Irish and Nelson 1991). Much of
our understanding regarding the molecular regulation of flowering in maize comes
from the analysis of genetic variants with altered flowering time. Three single gene
late-flowering mutants are described in maize; the recessive indeterminate1 (id1)
and delayed flowering1 (dlf1) mutations, and the dominant Leafy (Lfy) mutation.
All three mutations delay the floral transition and therefore we expect functional id1
and dlf1 to promote flowering, while normal Lfy function is difficult to deduce
(Shaver 1983; Neuffer et al. 1997; McSteen et al. 2000). The id1 mutants flower
extremely late, producing many more leaves and flowering many weeks later than
normal. Additionally, id1 mutants often have aberrant morphology, such as the
absence of ears (Galinat and Naylor 1951). The id1 gene has been cloned and
encodes a unique type of Zn-finger transcription factor, defined by the ID domain
(IDD). id1 is specifically expressed in immature leaf tissue and has been suggested
to control a florigenic signal (Colasanti et al. 1998; Colasanti and Sundaresan
2000). Subsequent analysis showed that the ID1 protein localizes to nuclei and
binds DNA suggesting it regulates the transcription of downstream flowering-time
genes (Kozaki et al. 2004). Furthermore, ID1 protein accumulates soon after
germination and high protein levels are detected in discrete regions of immature
leaves up to the time of the floral transition, after which time levels decline (Wong
and Colasanti 2007). id1 is unique to grasses and a putative homolog was not
found in Arabidopsis suggesting id1 participates in a monocot-specific pathway
(Colasanti et al. 2006). Recently, a rice ortholog of id1, RID1, was identified that
was proposed to control the initiation of floral induction and appears to regulate
multiple inductive pathways (Wu et al. 2008a,b). Plants lacking RID1 activity never
flower and remain in a perpetual vegetative state of growth. The other recessive
late-flowering mutation, dlf1, has a more moderate late-flowering phenotype, with
mutant plants producing a modest number of additional leaves and flowering
10–14 days later than normal (Neuffer et al. 1997; Muszynski et al. 2006). The
dlf1 gene was cloned recently and encodes a basic leucine-zipper (bZIP) homo-
logous protein that resembles the Arabidopsis floral integrator FD (Muszynski et al.
2006). Similar to FD, dlf1 is expressed in shoot apices, peaking in expression near
the time of the floral transition. dlf1 was shown to function downstream of id1,
thereby defining an id1-dlf1 floral inductive module (Fig. 4.2). To date, an id1-dlf1
homologous flowering module has not been described in rice or other grass spe-
cies, although an apparent dlf1 ortholog was identified in wheat (Chengxia and
Dubcovsky 2008). Leafy (unrelated to the Arabidopsis LEAFY gene or similar
homologous genes) is a unique, dominant, late-flowering mutation, which increases
the number of leaves specifically between the uppermost ear and tassel (Shaver
1983). Additionally, the Lfy mutation disrupts the coordination of inflorescence
maturation, such that the ear matures before the tassel, resulting in mutant plants
exserting silks prior to shedding pollen (M.G. Muszynski, unpub.). A mutation with
a similar phenotype has not been identified in other grasses. Little is known about
the morphological and developmental aspects of this dominant mutation and it has
yet to be molecularly isolated. Although relatively uncharacterized from a genetic
perspective, it has been used to a modest degree in maize breeding programs in
Canada to increase leaf biomass as a means to improve yield (Costa et al. 2002;
Subedi and Ma 2005; Subedi et al. 2006).
Additional information regarding the molecular control of the floral transition in
maize comes from the study of flowering time variants which display a quantitative
mode of inheritance. Two quantitative trait loci (QTL) mediating early flowering
were identified on chromosome 8 and named vegetative to generative transition1

(Vgt1) and Vgt2 (Vladutu et al. 1999; Salvi et al. 2002). Positional cloning and
association mapping recently pinpointed the molecular position of Vgt1 to an
intergenic region ~70 kb upstream of an APETALA2 (AP2)-like transcription factor,
designated ZmRap2.7 (Salvi et al. 2007). Variation in flowering time in various
inbred lines was associated with sequence changes in this putative cis-element that
presumably controls the expression of ZmRap2.7. Transgenic analysis showed that
overexpression of ZmRap2.7 cDNA caused late flowering, whereas downregulation
in antisense maize plants caused early flowering, demonstrating ZmRAP2.7 is a
repressor of the floral transition. The similarity of ZmRAP2.7 to members of a
family of Arabidopsis AP2-like genes that also have a negative effect on flowering
suggests a potential orthologous role for ZmRap2.7 in the regulation of the floral
transition (Aukerman and Sakai 2003). How ZmRAP2.7 functions within the maize
flowering network is currently unknown.
Homology-based investigations have also informed our understanding of flow-
ering in maize. A recent study has identified maize homologs of GI (gigantea of
Zea mays1, gigz1A and B) and CO (constans of Zea mays1, conz1) that display
different diurnal expression patterns in response to varied day lengths, suggesting
temperate maize does sense and respond to photoperiod (Miller et al. 2008).
Whether these homologs affect the floral transition was not determined. Associa-
tions with flowering time and inflorescence architecture were correlated with
differences in gene copy number of duplicate maize genes homologous to FLOR-
ICAULA of Antirrhinum majus and LEAFY of Arabidopsis (Bomblies and Doebley
2006). It was also shown that mutation of both paralogous Zea FLO/LFY1 (zfl1)
and zfl2 genes leads to very mild late flowering and altered inflorescence patterning
(Bomblies et al. 2003). Additional maize floral regulatory candidates have been
identified based on their sequence homology to FT, which was recently shown to
be part of the mobile floral stimulus in several species (Jaeger and Wigge 2007;
Lin et al. 2007; Mathieu et al. 2007; Tamaki et al. 2007). Maize has at least 25 FT-
like and related paralogos TERMINAL FLOWER (TFL)-like genes, designated
ZCN (for Zea mays CENTRORADIALIS) that could encode candidates for a
conserved florigenic protein (Danilevskaya et al. 2008). Determining which family
member(s) participate in florigenic signaling will require functional analysis of
each gene but ZCN8 stands out as a likely candidate based on its expression
pattern and ability to interact with the DLF1 protein in yeast two-hybrid assays
(Danilevskaya et al. 2008). Comprehensive phylogenetic analyses have determined
that for the meristem identity AP1/CAL/FUL MADS-box gene clade, only FUL-
like homologs exist within maize and other noneudicot species (Litt and Irish
2003; Malcomber et al. 2006). The grass FUL-like genes fall into three subgroups;
FUL1 and FUL2 are sister clades that are both sister to the FUL3 clade. A recent
study has identified two maize FUL1-like genes, ZMM4 and ZMM5, which are
initially expressed near the time of the floral transition (Danilevskaya et al. 2008).
Overexpression of ZMM4 promotes early flowering and is able to suppress the
late-flowering phenotype of id1 and dlf1 mutants, suggesting ZMM4 has floral
inductive activity.
Collectively, these studies have enabled the configuration of a maize floral

transition regulatory gene network (Fig. 4.2). Unlike Arabidopsis and rice, where
signaling is photoperiod-responsive and linked to the circadian clock, signaling in
maize is principally autonomous and not connected to day length. In the maize
network, id1 sits at the top of an inductive signaling hierarchy initiated in leaves by
unknown autonomous inputs. id1 is proposed to regulate the production or trans-
mission of a florigenic signal (F) that moves from the leaves to the shoot apex
(Fig. 4.2). The relationship between F and ZCN8, a possible maize FT ortholog, is
not known. Signals downstream of F are transmitted to the shoot apex and regulate
dlf1 expression or DLF1 protein activity. The model network predicts interaction of
DLF1 and ZCN8, which presumably activates several targets downstream of dlf1,
including an early target (x) which feedback regulates dlf1 expression and one or
more ZMM MADS-box floral identity genes (Muszynski et al. 2006). Redundant
inductive signaling through an id1-independent alternate pathway converges down-
stream of dlf1 to activate both x and the ZMM MADS genes. The positions of
ZmRAP2.7 and the zfl genes are not known, although the zfl genes make attractive
candidates for participating in the alternate inductive pathway.
Modern maize inbreds are exquisitely adapted to a wide range of geographical
and environmental conditions, with selection for specific maturities to flower at
particular latitudes. In fact, most inbreds are selected to flower as late as possible,
but early enough to assure that yield is maximized. Inbreds developed for one area
of adaptation are rarely used in another because of limitations of flowering, grain fill
or kernel maturation. Thus, breeding between inbreds with different maturities is
uncommon, leading to a reduction in germplasm diversity and impeding the transfer
of superior alleles to new lines. A comprehensive understanding of the genetic
determinants regulating flowering would enable breeders to manipulate maturity
through molecular breeding or transgenic methods and in this way increase the
diversity of germplasm utilized in a selection program. This is especially significant
at a time when climate change may dramatically alter an environmental component
of a particular geography, thus rendering elite lines maladapted to these new
conditions.
4.3 In Vitro Flowering: An Alternative Route to Rapid

Flower Production
Plants differ with respect to the length of their life cycle and the timing of the floral
transition, to say nothing of robustness with respect to gamete and seed production.
Seeds likewise differ with respect to ease of germination. When these limitations
are factored against challenges arising from both biotic and abiotic stressors, the
accessibility of year round flower production has broad economic implications.
This is especially true for secondary plant products whose biosynthesis is restricted
either to the inflorescence or to the flower. Recently concentrated efforts have been
directed towards producing fertile flowers in vitro whose seed may be harvested
directly from the culture dish. The ideal in vitro flowering protocol couples a
reduction in flowering and seed maturation, in effect shortening the entire life
cycle, thus increasing the number of segregating populations that can be produced
annually. Hence, the ability to manipulate in vitro flower production will have an
incalculable impact on the introgression of transgenes into robust breeding lines to
say nothing of plant breeding per se.
In vitro flowers have already been produced in culture using a number of
different hormone regimes being sensitive to particular cytokinins in a concentra-
tion-dependent fashion. In vitro flowers in plant species as diverse as legumes, pearl
millet, ornamentals and bamboo (Abou-Alaiwi 2007) result following sequentially
the formation of in vitro shoots that lead to flowers. While flowers have been
produced, the technical problems associated with the development of a medium
are complex. Specifically, from a population of explants, flowers may be produced
through an intervening shoot or through the induction of vegetative shoots or
infertile shoots where the inflorescence develops aberrantly leading either to the
absence or the production of defective gametes. Most elusive of all to date has been
the expression of direct flowering where the vegetative development is completely
inhibited (Lin et al. 2005; Rudrabhatla and Goldman 2009, US Patent # 7, 547,
548). For the purpose of this discussion, a true in vitro flower is one that is fertile
and therefore capable of developing seeds that are indistinguishable from those
harvested from traditional crosses.
4.3.1 In vitro Flowering in Grasses
4.3.1.1 Pearl Millet
Devi et al. (2000) reported the production of in vitro flowers from cultured apical
meristems of Pennisetum glaucum (L.) R. Br. that is hormone- and concentration-
dependent. If the apices were cultured with low levels of 2,4-dichlorophenoxyacetic
acid (2,4-D) and the concentration of benzyladenine (BA) varied, the shoot apical
meristem enlarged and adventitious shoots developed. At higher concentration of
BA, somatic embryos were also observed. In the absence of 2,4-D, shoot tips
produced many leaves and in vitro flowers but only upon the inclusion of BA in
the medium. Notably, the plants developed from in vitro shoots are fertile. The
varied developmental profiles reported are indistinguishable among the four geno-
types tested and determined solely as a function of media composition with
inclusion of BA being an obligate requirement for in vitro flower development.
4.3.1.2 Bamboos
As a general rule, in vitro flowers have been derived from cultured shoots following
a number of manipulations that require the addition of specific concentrations of
cytokinin. Working with Dendroclamus strictus Nees, a bamboo, Singh et al. (2000)
b 40
R 23
Hi-II
In Vitro flower induction
B 73
LH 198 LH227
Frequency (%)
20
0
1 2 3 4 5 6
Media
produced flowers from cultured shoots on a modified Murashige and Skoog medium
containing thidiazuron (TDZ). While shoots were robustly produced over a wide
concentration of cytokinin (0.01–1.0 mg/l), in vitro flower production was restricted
only to medium containing TDZ between 0.5 and 1.0 mg/l. The formation of anthers
did not necessarily specify the development of normal flowers. Not only were there
problems with pollen dehiscence but also with pollen development. In fact, only 20%
of the grains were normal, with the remaining 80% being empty.
Lin et al. (2003) described in vitro flowering in another bamboo species,
Bambusa edulis, albeit with significant shoot formation. Unlike Singh et al.
(2000) the shoots were initiated from in vitro derived spikelets. Specifically, these
were cultured on Murashige and Skoog (MS) medium containing 9.3 mM kinetin
and kinetin þ 13.57 mM 2,4-D. The callus-derived somatic embryos were then ger-
minated on media supplemented with 0.5 uM TDZ plus 3% sucrose. After several
subcultures, shoots were recovered. The shoots then were transferred to media
containing different cytokinins and the effects determined by testing with a number
of different concentrations. Coincident with this transfer, cytokinin is not only
sufficient but necessary for the induction of in vitro flowers.
Following the production of proliferating intervening shoots, culture on different
cytokinins altered the developmental fate of the cells of the axillary meristems.
When BA is substituted for TDZ, the number of flowering shoots is profoundly
reduced; an effect further complicated as a function of exogenous hormone con-
centration. Hence, the sensory apparatus of cytokinin signaling can discriminate not
only differences among cytokinin molecules but also in concentration leading to
profound alterations in morphogenesis. This is in keeping with the earlier observa-
tion of Singh et al. (2001) who demonstrated that differences among endogenous
concentrations can have profound and antagonistic effects on root and shoot
meristem development. Finally, auxins can also effect in vitro development.
Although not sufficient in themselves to specify flower formation, the addition of
auxin in the media modifies morphogenetic fate in bamboo. If NAA is included in
the media during shoot proliferation at high auxin to cytokinin ratios, the number as
well as the percentages of reproductive shoots was significantly reduced and,
accordingly, in vitro flowering decreased.
4.3.1.3 Maize
Maize in vitro flower induction originating from split-seed was observed after
3–4 weeks in medium containing BA and kinetin (Fig. 4.3 a, b; Al-Abed 2007;
<
Fig. 4.3 (a) In vitro flowers originating from maize split seed explants. Close images of in vitro
induced ears (a) and (b) and tassels (b) and (c). (b) Comparisons of maize hybrids and inbred lines
for the induction of in vitro flowers on various concentrations of BAP and Kinetin. R 23, Hi-II, B73
and LH 198 LH 227 are different maize hybrid and inbred lines. Numbers 1–6 correspond to
different concentration combinations of BAP and kinetin. All percentage data were transformed
using arc-sin transformation before statistical analysis
Al-Abed et al. 2006). Although both ears and tassels were formed, only the ears
were fertile. The induction frequency rose as a function of cytokinin concentra-
tion with an optimum of 17.6 mM 6-benzylaminopurine (BAP) and 22 mM
kinetin. Four genotypes were tested to determine genotype independence, and
no significant differences were observed in the frequency of in vitro flowering
among the varieties. Interestingly, although the incidence of inflorescence pro-
duction among the varieties B73, Hi-II and LH198 LH227 proved to be
cytokinin concentration dependent, there were no significant differences for
in vitro flower induction frequency among the four genotypes when the concen-
tration of BAP was stabilized at 17.6 mM and kinetin varied between 4.6 and
18.4 mM. Similarly, when the concentration of BAP was stabilized at 22 mM and
kinetin concentration varied as before no differences in induction were observed.
Of equal interest, TDZ could not successfully substitute for either BA or kinetin;
an incorporation of TDZ in the culture media resulted in no in vitro inflores-
cences formed.
When the media contained BAP alone there was a significant difference in
flower induction frequency among the four micromolar concentrations tested (8.8,
13.2, 17.6 and 22 mM). The number of flowers increased as BAP concentrations
increased up to 22 mM. Shoots from line R23 were induced to flower, although the
number of ears induced was higher than the number of tassels. On the other hand,
there was no significant difference between the number of ears and tassels induced
from B73, Hi-II and LH198 LH227. With further increases in BAP concentration
(26.4 mM and higher), the shoots were stunted and additional rounds of multiple
shoot production began.
From these results, it may be concluded that flower induction frequency is
correlated with incremental changes of BAP and kinetin concentration. Moreover,
prolonged culture in cytokinin-supplemented media affects the development of the
ear and subsequent silk formation. If shoot-derived ears were kept on 17.6 mM BAP
þ 9.2 mM kinetin for more than 4 weeks, none of the ears formed silks. In contrast,
if the shoots were cultured on 17.6 mM BAP þ 9.2 mM kinetin and then transferred
to MS lacking hormones, the silks developed with the ears growing larger and
more robust.
4.3.2 In Vitro Flowering in Dicots
4.3.2.1 Tomato
Dielen et al. (2001) investigated the floral transition in the tomato mutant uniflora
(uf). Under winter growth conditions, homozygous plants exhibit a prolonged
vegetative phase and flower production is suppressed. Floral transition can be
induced on a medium containing sucrose and a variety of cytokinins, in addition
to nitrogenous nutrients. The inclusion of gibberellic acid (GA3) was found to
inhibit transition.
Sheeja and Mandal (2003) produced in vitro tomato flowers through an inter-
vening callus on medium containing 2 mg/l BAP. Flowers, however, required
continuous light (2.2 mM2/s) in order to open. The number of flowers produced
was low, averaging approximately 11 per explant. The flowers were fertile and
tomatoes set 160 days after pollination. Flower production capacity was limited
with respect to variety. Of the seven varieties tested on MS þ 2 mg/l BAP, only one
variety, KS118, was able to produce fully formed flowers from callus developed
from leaf explants.
4.3.2.2 Roses
In vitro flowering was induced in six vegetatively produced rose cultivars in MS-
modified media containing different phytohormone combinations. All contained
the auxin a-naphthaleneacetic acid (NAA) at 0.1 mg/l and a cytokinin which was
varied. Media containing 0.5 mg/l TDZ or 0.5 mg/l zeatin induced in vitro floral bud
production at frequencies exceeding 40%. The phytohormone response of each rose
cultivar was not uniform.
4.3.2.3 Ginseng
Lin et al. (2005) reported the direct formation of Panax gensing inflorescences. In
this connection, plantlets derived from somatic embryos produced clusters of
dormant buds when cultured on B5 medium containing BA (1 mg/l) and
GA3(1 mg/l). The buds were subcultured again on the same medium and approxi-
mately 15% of the buds cultured produced inflorescences directly without a vege-
tative phase. In addition to direct production of flowers, a mixture of plant organs
developed on the explant including vegetative shoots and indirect flowers in
addition to dormant buds. If TDZ was substituted for BA, the number of flowers
produced without an intervening shoot increased as did the number of dormant
buds. These observations confirm the importance of cytokinin as a prerequisite for
flower development (Chang and Hsing 1980; Lim et al. 1997). While no gross
structural or morphological differences could be detected among flowers produced
either by direct or indirect flowering, Lin et al. (2005) reported neither the produc-
tion of functional gametes nor seed. Hence, the utility of a season-independent,
direct in vitro flowering system remains a goal.
4.3.2.4 Soybean
To date, in vitro flowers have also been obtained from dicots through culturing an
intervening shoot as shown by Franklin et al. (2000) using the legume Pisum
sativum. Following germination on hormone-free MS media, cotyledonary node
and shoot tip explants were transferred to an MS medium containing 2mg/l BAP.
The resulting elongated shoots were removed 15 days post-implantation and incu-
bated on medium containing either auxin alone (indole-3-butyric acid, IAA; or
NAA) or in combination with (GA3). Following culture on this medium roots and
mature pods were formed within 25 days.
The number of shoots per cotyledonary node or shoot tip is BAP concentration
dependent. The inclusion of BAP, either below 0.5 mg/l or above 5.0 mg/l, results
in the complete absence of shoots derived from cotyledonary node explants. Indeed,
shoot formation is maximized between 1 and 2.0 mg/l. Consistent with this obser-
vation is the fact that the number of shoots per shoot tip explant is also BAP
concentration dependent, albeit over a much narrower range. While BAP results in
shoots regardless of the explant, a component of shoot development is fine-tuned
based on the tissue that is the donor cell source.
The in vitro flowers formed on these shoots, however, are dependent on the
subsequent inclusion of specific auxins at highly defined concentrations. While
indole-3-butyric acid (IBA) can induce flowering over a wide range of cytokinin
concentrations (0.1–2.0 mg/l with or without GA3), substitution with NAA cannot.
Not only is GA3 required but the range over which it is effective is restricted to
0.1–0.5 mg/l.
In contrast, Rudrabhatla and Goldman (2009), (patent pending) report that
culture of soybean cotyledons or radicles on MSB5 medium supplemented with
specific cytokinin combinations produced in vitro flowers either directly or indi-
rectly through an intervening shoot. For example, after 7–15 days in MSB5 medium
containing TDZ and BAP, the bulging of cotyledons is prominent and small
greenish protuberances appear at the proximal end of the cotyledon (Fig. 4.4a).
The cotyledons are transferred to a modified MS basal medium containing B5
vitamins but lacking hormones. The small greenish protuberances morph into
bud-like structures (Fig. 4.4b). Both shoot buds and/or flower buds form at the
proximal end of the cotyledon. The formation of soybean in vitro shoots and/or
flowers is not only defined by the cytokinin molecule per se but is also concentra-
tion dependent. As will become apparent, soybean tissues have the capacity to
discriminate differences among classes of cytokinins on the same explant and in
doing so alter the developmental fate of that fixed cell population.
If soybean cotyledons are challenged with BAP alone, only multiple shoots were
observed (Barwale et al. 1986), while a low frequency of flower buds were observed
with TDZ alone. The highest frequency of flower buds was observed in the
combination treatment of TDZ (2.0 mg/l) and BAP (1.0 mg/l) (Fig. 4.4c). A
decrease in the frequency of flower buds (8.75%) was observed when the con-
centration of TDZ was kept constant and the concentration of BAP increased to
2.0 mg/l (Fig. 4.4d). Further increase in BAP (3.0 mg/l) resulted in the complete
suppression of flower buds with a concomitant increase in the production of multiple
shoot buds. Hence, using the same cell population and manipulating the growth
regulators, the meristem identity of the tissue was changed from flower buds to shoots.
All the flower buds were clumped or grouped together, even in those cultures
producing multiple shoots along with flowers suggesting the flower buds arise from
a specific group of cells. Among all the combinations tested, 2.0 mg/l TDZ along
Fig. 4.4 Effect of hormones on in vitro flowering and viable seed set in soybean. Cotyledons
producing direct flowers (a) and multiple shoots (b) on MSB5. Direct pod formation from
cotyledons without vegetative phase (c and d)
with 1.0 mg/l BAP induced 70–75 flower buds. As soybean is a self-pollinated
species, most of the in vitro flowers produced pods and set seed. The in vitro pods
fully matured within 35 days after anthesis, and had well-developed seeds. Those
in vitro developed seeds, when cultured on MSB5 basal medium, readily germi-
nated and formed fully fertile plants that were easily hardened and grown in the
greenhouse.
This developmental course can be predictably varied by changing the concen-
tration of BAP and TDZ in the media. In this connection, cotyledons incubated on
MSB5 containing 3 mg/l BAP and 1 mg/l TDZ suppressed direct flower formation
leading directly to the development of in vitro soybean shoots. From these shoots,
fertile flowers developed and seeds indistinguishable from those grown in the field
or greenhouse were recovered. The elimination of TDZ from this media likewise
produced shoots, which eventually gave rise to fertile flowers at a low frequency
(Rudrabhatla and Goldman 2009, patent pending).
Similar results have also been obtained using radicle explants as summarized
in Fig. 4.5a–c. Three-day-old germinated seeds were collected, the seed coats
Fig. 4.5 In vitro flowering from radical explants in soybean. Soybean in vitro flowering from
radical explants through in vitro developed shoots (a), flower (b) and pod (c)
removed and segments of the radicle and plumule excised. These were placed on
MSB5 supplemented with TDZ (2 mg/l) and BAP (1 mg/l) and directly formed
clustered fertile in vitro flowers (Fig. 4.5a). If the concentration of BAP was raised
to 3 mg/l and the concentration of TDZ remained either constant at 1 mg/l or was
raised incrementally to 2 mg/l, direct flower formation was suppressed and shoots
formed. Notably, these shoots too developed normal fertile flowers.
Taken as a whole, the phenomenon of in vitro flowering supports the hypothesis
that cytokinins are essential not only for meristem maintenance and integrity but
also as a determinant of cell fate. This conclusion is based on the fact that attempts
to induce in vitro flowering through manipulation of cytokinin has resulted in the
formation of shoots lacking flowers, shoots producing sterile flowers, shoots pro-
ducing fertile flowers, direct sterile in vitro flowers where the intervening shoot is
absent and direct fertile in vitro flowers. Each of these outcomes may be attributed
to tension that results when cytokinin is supplied exogenously thus challenging the
plant’s ability to maintain phytohormone homeostasis in the meristem.
The role of cytokinin and its obligate requirement for meristem stability, integrity,
and function is now well established (Werner et al. 2001, 2003; Riefler et al. 2006;
Kurokawa et al. 2007). Transgenic tobacco plants overexpressing any one of the
cytokinin oxidase genes, AtCKX1, AtCKX2, AtCKX3 or AtCKX4, produce plants
with significantly reduced cytokinin concentration. Such plants are not only stunted
but show significant size reduction in the shoot apical meristem (Werner et al.
2001). In subsequent experiments using Arabidopsis, Werner et al. (2003) demon-
strated that overexpression of any of the six members of the AtCKX oxidase/
hydrogenase gene family has profound effects on the aerial portions of the plant
impacting both shoot and floral development. The effect on development is likely a
function of perturbations in differential cytokinin activity that is normally asso-
ciated with growth zones, which are reduced because of the expression of the
AtCKX genes that shrink the size of the SAM.
Key to the production of in vitro flowers and the route by which they are
produced is likely to be, in part, a function of challenges to cytokinin homeostasis
in the meristem. T-DNA insertion mutants into the cytokinin receptor genes
Arabidopsis histidine kinase genes (AHK2, AHK3) and cytokinin response (CRE/
AHK4) result in profound developmental changes (Riefler et al. 2006). The effect
of these mutated genes is to reduce signaling and lead not only to increases in
endogenous cytokinin concentration but to do so in a defined manner leading to
the accumulation of specific metabolites. The ahk2 and ahk3 double mutants
affect leaf development reducing cell number and total chlorophyll content while
showing reduced sensitivity to cytokinin-dependent inhibition of dark-induced
chlorophyll loss. The effect of the triple mutants on flowering remains a matter
for discussion. While Higuchi et al. (2004) and Nishimura et al. (2004) claimed
complete plant sterility, Riefler et al. (2006) demonstrated that some plants may
be rescued. The said effect of disturbing homeostasis on flower development is
unmistakable and consistent with the observation that mutants express anywhere
from 16- to 19-fold increases in zeatin metabolites. While reduced signaling
increases cytokinin accumulation, it remains unclear as to whether the changes
seen in the development result from increased biosynthesis or decreased
degradation.
A decreased signaling results in increased endogenous cytokinin activity dimi-
nishing homeostasis and resulting in disturbances in the development. Indeed, the
source of the change in endogenous cytokinin concentration is unimportant.
Recently, Kurokawa et al. (2007) have detailed the expression of lonely guy
(LOG) gene in rice. Plants overexpressing LOG produce plants with diminished
panicles, reductions in the number of floral organs and a flattened meristem
resulting in anomalies in organ development. The wild-type gene is definitive for
the final step of cytokinin biosynthesis in the shoot tip.
The differential response to cytokinin on in vitro flowering described here
substantiates the fact that the uptake of exogenous cytokinin disturbs the delicate
internal homeostatic balance needed if plant development is to proceed normally.
The parameters regulating in vitro flowering are complex and result by signaling
appropriate developmental pathways and repressing others. While cytokinin sig-
naling is initiated through a number of histidine kinase receptors followed by
phosphor relay, it remains unknown how increases in hormone concentration effect
differential gene expression as a result of perturbations in homeostasis. This said,

while the precise genes that are either expressed or repressed are unknown at this
time, their definition is made possible. Our results clearly indicate that the unique
hormonal combination applied at appropriate stage of in vitro culture seems to be a
key factor that defines whether floral meristems are formed or not. Hence, it should
be possible to determine if any, of the sentinel cloned genes at which all floral
pathways converge are expressed, as well as those that are unique. Indeed, the
collapsing of the vegetative phase and the appearance of flower buds beginning the
3rd or 4th week after transfer to flower induction medium raises questions as to
whether the four flower induction pathways previously described for Arabidopsis
finds a parallel in soybean or, alternatively, whether the in vitro flowering system
expresses a regulatory pathway previously unknown. Identification of the genes
regulating in vitro production of soybean flowers may offer the possibility of
transferring genes that are absent to complete the pathway or identifying those
genes that are uniquely expressed in response to the specific combination of the
floral induction medium.
4.4 Genes for Improving Grain Quality in Corn and Soybeans
4.4.1 Key Issues
Grain crops form the basis of the global food supply, either through direct con-
sumption or in animal feed to produce meat. A number of trends lead to an
increasing global demand for grain. First, the global population is increasing. The
current population is 6.5 billion and this is expected to increase to 9 billion by 2050.
Second, global meat consumption is increasing in developing countries. In the past
25 years, meat consumption in developing countries has doubled (Speedy 2003).
Meat production by nonruminant animals often depends on grain, and conversion of
grain to meat is relatively inefficient. Thus, an increase in meat consumption creates
a larger demand for grain. Third, increasing amounts of grains are used to produce
renewable biofuels such as ethanol and biodiesel. This increasing demand is
compounded by environmental factors such as reductions in fresh water and arable
land. It is clear that crop yields will need to be increased substantially to keep pace
with the demand for grain.
In addition to increasing crop yields, it is important to ensure that our grain
supplies are used efficiently. It may be possible to use less grain for a given purpose
if that grain is particularly well suited to that purpose. For example, oil crops that
contain more oil require consumption of less grain to meet the demand for oil. Thus,
part of the demand for grain can be met by producing better grain. The definition of
grain quality depends on the end use of the grain. This chapter will examine the
major uses of grain and the genes that have been important in improving grain for
these purposes.
4.4.2 Protein
4.4.2.1 Amino Acid Balance
Meeting protein nutrition needs is one of the greatest challenges because plants tend
to be low in protein and this protein has poor nutritional quality. Animal protein
sources have much higher nutritional value. The nutritional quality of a protein
source is defined, in part, by its amino acid balance. Of the 20 common natural
amino acids, nonruminant animals can produce 10 amino acids from other sources
and the remaining 10 are required in the diet. While the exact amino acid require-
ment depends on the animal species, developmental stage and other factors, in
general, monocot grains tend to be deficient in lysine and tryptophan while dicot
grains tend to be deficient in the sulfur-containing amino acids, cysteine and
methionine. These deficiencies are largely a consequence of the amino acid balance
of the seed storage proteins that accumulate to high levels in the seed and support
the seedling until it is capable of photoautotrophic growth. The major seed storage
proteins of cereals tend to be prolamins, which are characterized by low levels of
lysine and tryptophan, while the main seed storage proteins in legumes tend to be
globulins, which have low levels of sulfur-containing amino acids. These deficien-
cies lead to poor utilization of plant protein in diets, so improving the balance of
amino acids is an important grain quality objective.
Several approaches have been used to improve the amino acid balance of grain.
Because the seed storage proteins have such a large impact on determining the
amino acid balance, one approach to solving this problem is to alter seed protein
deposition. In cereals, several genes involved in deposition of the prolamin family
of seed storage proteins called zeins have been found to impact amino acid balance.
Recessive mutant alleles of the opaque 2 (o2) gene of maize cause an increase in the
lysine and tryptophan content of the grain (Mertz et al. 1964). The o2 gene encodes
a basic leucine zipper (bZIP) transcription factor (Hartings et al. 1989) that binds to
promoter elements of some zeins (Schmidt et al. 1990). Unfortunately, the soft, low-
density kernels produced on these mutant plants are not well suited to agronomic
production. Development of nutritionally improved maize with acceptable agro-
nomic properties has required an extensive breeding effort to overcome the unfa-
vorable effects of the mutation. The resulting varieties are called Quality Protein
Maize (QPM) and carry the o2 gene as well as a number of unidentified “modifier
loci” that condition desirable agronomic properties (Prasanna et al. 2001).
Mutation in the floury-2 (fl2) gene also improves the lysine and methionine
content of maize (Mertz et al. 1965). This mutation encodes a zein with an altered
signal sequence that interferes with zein deposition (Coleman et al. 1995).
The delta zein regulator (dzr1) gene conditions elevated methionine levels in
maize and was originally identified in a screening program aimed at identifying
high lysine genotypes (Phillips et al. 1981). This gene regulates the deposition of a
methionine-rich seed storage protein, the 10 kDa delta zein by altering the mRNA
stability of the gene (Cruz-Alvarez et al. 1991) and has been used to produce a
series of high methionine inbred lines (Olsen et al. 2003).
Transgenic approaches to improving the amino acid balance have taken advan-
tage of information gained from the naturally occurring mutants to achieve effects
similar to those of the natural mutations. Downregulation of zein transcription
results in improved amino acid balance (Segal et al. 2003; Huang et al. 2004) as
would be predicted from studies of o2, and enhancing the stability of the methionine-
rich delta zein message improves methionine content (Lai and Messings 2002) as
would be predicted from studies of dzr1.
A different transgenic approach involves modifying genes encoding enzymes
involved in metabolism of amino acids of interest. Introduction of feedback-
insensitive versions of an enzyme in the lysine biosynthetic pathway results in
increased levels of total and free lysine when used in combination with a transgene
designed to downregulate the zein storage proteins (Huang et al. 2005). Down-
regulation of degradative enzymes is also effective is increasing free lysine levels
(Houmard et al. 2007). An approach that involved both expression of a feedback-
insensitive biosynthetic enzyme and downregulation of a degradative enzyme with
a single transgene was effective in increasing the free lysine levels 40-fold over
nontransgenic controls.
Approaches to improving the amino acid balance of dicot grain have been
recently reviewed (Krishnan 2005). Expression of methionine-rich proteins from
other species has been widely used to address this problem. For example, the 2S
albumin from Brazil nut is a methionine-rich seed storage protein that has been
shown to increase methionine content when introduced into the seeds of transgenic
plants (Altenbach et al. 1989). Analysis of transgenic soybeans containing this
protein indicated that the allergenic properties of this protein were present in the
transgenic grain (Nordlee et al. 1996) and this approach has not been pursued
further. Maize methionine-rich zein genes have also been expressed in transgenic
soybeans (Dinkins et al. 2001; Kim and Krishnan 2004). While the transgene-
encoded proteins accumulated in soybean seeds, the change to methionine content
was modest.
4.4.2.2 Antigenicity
Allergenicity limits the utility of plant proteins in diets. Genetic approaches have
been used to produce varieties with lacking certain antigens. In soybean, for
example, the P34 protein is a major allergen. A transgene was introduced that
uses gene silencing to prevent accumulation of this protein (Herman et al. 2003). In
addition, several null alleles that fail to accumulate this protein were identified
through screening of a germplasm of the Glycine genus (Joseph et al. 2006). These
resources may allow the development of varieties with reduced antigenicity.
4.4.2.3 Digestibility
The digestibility of soybeans is reduced by the presence of trypsin inhibitors in the

seeds. A null allele for the Kunitz trypsin inhibitor allele has been identified and
designated ti (Orf and Hymowitz 1979). A line containing this mutation was shown
to have improved protein efficiency ratio when compared to a nonmutant isoline.
This difference was attributed to improved digestibility due to reduced trypsin
inhibitor activity.
4.4.2.4 Oil
The fatty acid composition of vegetable oil has a large impact on its functional
properties and its dietary impact on health. The specific composition with the
highest value depends on the end use of the oil. Given the wide range of uses of
vegetable oils, many types of modifications have added value. The fatty acid
biosynthetic pathway has been manipulated by mutagenesis or genetic engineering
to produce oils with many potentially valuable compositions (reviewed in Fehr
2007). Manipulation of fatty acid levels is complicated by the fact that most steps in
the biosynthetic pathway of these compounds are encoded by families of genes.
4.4.2.5 Oxidative Stability
Unsaturated fatty acids, such as linolenic acid, are susceptible to oxidation at the
high temperatures used in frying. This oxidation results in undesirable flavors and
odors, and therefore low-linolenic acid oils are desirable. Breeders have used EMS
(ethyl methyl sulfonate) mutagenesis to produce lines with low linolenic acid
(Hammond and Fehr 1983; Wilcox et al. 1984). Reduced linolenic acid levels in
these lines have been attributed to lesions in the members of the omega-3 fatty acid
desturase (FAD3) gene family (Byrum et al. 1997).
4.4.2.6 Health Benefits
High levels of saturated fatty acids are correlated with coronary heart disease. In
soybean oil, palmitic acid is the most abundant saturated fatty acid. Alleles of
several genetic loci have been used by breeders to reduce palmitic acid levels in
soybean, but most have not been molecularly characterized. The exception is
FATB, which encodes a 16:0 thioesterase.
4.4.2.7 Flavor
Lipoxygenases are enzymes that catalyze the oxidation of polyunsaturated fatty

acids (reviewed in Feussner and Wasternack 2002). These reactions produce a
number of aromatic compounds that contribute to undesirable beany flavors in
bean products. Three major isoforms of soybean lipoxygenase are designated
LOX1, LOX2 and LOX3. Soybeans lines containing null alleles of these isoforms
have been identified (Kitamura et al. 1983; Davies and Nielsen 1986). Characteri-
zation of soybeans containing LOX null alleles has been shown to accumulate
reduced levels of compounds responsible for undesirable flavors (Davies and
Nielsen 1987; Hildebrand et al. 1990; Moreira et al. 1993; Nishiba et al. 1995)
and that tofu and soymilk made from LOX null soybeans had a less beany fla-
vor than similar products made from normal beans (Davies and Nielsen 1987;
Torres-Penaranda et al. 1998).
4.4.3 Starch
Starch accumulates in maize kernels as insoluble granules. When heated in the

presence of water, starch gelatinizes. This behavior makes starch useful for many
purposes in food as an adhesive and thickening agent and industrial applications
such as making paper, paint and cosmetics. With such a broad range of applications,
nearly any modification to the physical properties of starch will make it better suited
to one of its end use.
4.4.3.1 Industrial Applications
The physical properties of starch are determined by its chemical structure. Starch is
a polymer of glucose, but it can be fractionated into two polymers with different
degrees of branching. Amylose is a largely linear polymer, while amylopectin
contains a higher degree of branches. The ratio of amylose to amylopectin has a
large impact on the physical properties of starch. Several genes control this ratio,
and mutations in these genes are used in varieties that produce different industrial
starches. It was shown that the waxy (wx1) gene lacks a glucosyl transferase activity
(Nelson and Rines 1962), later determined to be due to the granule-bound starch
synthase. Mutants of wx1 do not accumulate amylose (Sprague et al. 1943) and are
particularly useful as adhesives.
The amylase extender (ae1) gene of maize accumulates high levels of amylose,
and encodes starch branching enzyme IIb (Stinard et al. 1993). The high amylose
starches derived from this mutant forms strong gels well suited to confections and
thickening agents.
4.4.3.2 Food Quality
In addition to mutants that change starch structure, mutants that change the amount
of starch produced are valuable. Sweet corn that is used directly for human
consumption is based on mutations that reduce the level of starch in the kernel.
This reduction in starch is accompanied by a concomitant increase in the sugar
content of the developing kernels, resulting in the sweetness and flavor desired by
consumers. The sugary-1 (su1) gene encodes a starch debranching enzyme that is
essential for the production of normal starch granules (James et al. 1995). Recently,
the shrunken-2 (sh2) mutation has been employed to develop “supersweet” types of
sweet corn (Tracy 1997). The sh2 gene encodes an ADP-glucose pyrophosphory-
lase (Bhave et al. 1990).
4.4.4 Other Components
Plants store phosphorus in the form of a phosphorylated sugar called phytic acid.
Nonruminant animals including humans cannot efficiently digest phytate, leading
to a deficiency in phosphorous in grain-based diets. In addition, phytate is an
efficient chelator of several minerals, such as iron and zinc that are limiting in
some diets, reducing their biological availability. In addition, undigested phosphate
in animal manure contaminates ground water and contributes to eutrophication of
streams and lakes. Thus, reduction of phytic acid is desirable for both nutritional
and environmental reasons.
Mutants of maize (Raboy et al. 2000) and soybean (Wilcox et al. 2000; Hitz et al.
2002) with reduced content of phytic acid identified, however, pleiotropic effects
on seed weight in maize (Raboy et al. 2000) and seedling emergence in soybean
(Oltmans et al. 2005) are a barrier to the development of commercial varieties with
reduced phytate. A transgenic approach involving embryo-specific downregulation
of the maize lysophosphatidic acid receptor (lpa1) gene may successfully overcome
these problems (Shi et al. 2007).
4.5 Concluding Remarks and Future Prospects
Boosting global production of quality grain to feed our planet’s increasing popula-
tion demands advanced understanding of plant biology and creative strategies to
manipulate the inherent development and physiology of our food crops. One
strategy is to couple altering the timing of the floral transition with in vitro flower-
ing to change the length of the crop plant’s life cycle, thereby, allowing faster
breeding, quicker adaptation to new environments and increased production of
higher-quality grain. To be successful, a more complete understanding of the
mechanistic interactions that regulate flowering in diverse crop plants is required.
For example, the nature of the primary endogenous flowering stimulus in maize is
unknown and represents a serious gap in our understanding of how the maize floral
transition network functions. Identifying the primary floral stimulus is essential to
predictably manipulate flowering in this grain crop. Further, how treatment with
different hormone regimes impacts floral network function is not understood.
Dissecting how hormone and floral network signaling intersect is an important
area for future research. Increased investment in both basic and applied plant
biology research will enhance our understanding of these key developmental

processes and provide the components necessary to enable the modulation of a
crop’s life cycle. In conjunction with improvements to grain quality, progress can
be made towards the goal of feeding people globally.
References
Abe M, Kobayashi Y, Yamamoto S, Daimon Y, Yamaguchi A, Ikeda Y, Ichinoki H, Notaguchi M,

Goto K, Araki T (2005) FD, a bZIP protein mediating signals from the floral pathway integrator
FT at the shoot apex. Science 309:1052–1056
Abou-Alaiwi (2007) Advances in biotechnology for the enhancement of medicinal plants. PhD
Dissertation. University of Toledo, OH, USA
Adam H, Jouannic S, Orieux Y, Morcillo F, Richaud F, Duval Y, Tregear JW (2007) Functional
characterization of MADS box genes involved in the determination of oil palm flower
structure. J Exp Bot 58:1245–1259
Al-Abed D (2007) Genetic engineering of maize (Zea Mays L.) using novel explants “split seed.”
PhD Dissertation. University of Toledo, OH, USA
Al-Abed D, Rudrabhatla S, Talla R, Goldman S (2006) Split-seed: a new tool for maize research-
ers. Planta 223:1355–1360
Altenbach SB, Pearson KW, Meeker G, Staraci LC, Sun SM (1989) Enhancement of the methio-
nine content of seed proteins by the expression of a chimeric gene encoding a methionine-rich
protein in transgenic plants. Plant Mol Biol 13:513–522
Aukerman MJ, Sakai H (2003) Regulation of flowering time and floral organ identity by a
microRNA and its APETALA2-like target genes. Plant Cell 15:2730–2741
Barwale UB, Kerns HR, Widholm JM (1986) Plant regeneration from callus cultures of several
soybean genotypes via embryogenesis and organogenesis. Planta 67:473–481
Bernier G, Perilleux A (2005) A physiological overview of the genetics of flowering time control.
Plant Biotechnol J 3:3–16
Bhave MR, Lawrence S, Barton C, Hannah LC (1990) Identification and molecular characteriza-
tion of Shrunken-2 cDNA clones of maize. Plant Cell 2:581–588
Blazquez MA, Weigel D (2000) Integration of floral inductive signals in Arabidopsis. Nature
404:889–892
Bomblies K, Doebley JF (2006) Pleiotropic effects of the duplicate maize FLORICAULA/LEAFY
genes zfl1 and zfl2 on traits under selection during maize domestication. Genetics 172:519–531
Bomblies K, Wang RL, Ambrose BA, Schmidt RJ, Meeley RB, Doebley J (2003) Duplicate
FLORICAULA/LEAFY homologs zfl1 and zfl2 control inflorescence architecture and flower
patterning in maize. Development 130:2385–2395
Byrum JR, Kinney AJ, Stecca KL, Grace DJ, Diers BW (1997) Alteration of the omega-3 fatty
acid desaturase gene is associated with reduced linolenic acid in the A5 soybean genotype.
Theor Appl Genet 94:356–359
Chang WC, Hsing YI (1980) In vitro flowering of embryoids derived from mature root callus of
ginseng (Panax ginseng). Nature 284:341–342
Chengxia L, Dubcovsky J (2008) Wheat FT protein regulates VRN1 transcription through inter-
actions with FDL2. Plant J 55:543–554
Colasanti J, Sundaresan V (2000) “Florigen” enters the molecular age: long-distance signals that
cause plants to flower. Trends Biochem Sci 25:236–240
Colasanti J, Yuan Z, Sundaresan V (1998) The indeterminate gene encodes a zinc finger protein
and regulates a leaf-generated signal required for the transition to flowering in maize. Cell
93:593–603
Colasanti J, Tremblay R, Wong AYM, Coneva V, Kozaki A, Mable BK (2006) The maize
INDETERMINATE1 flowering time regulator defines a highly conserved zinc finger protein
family in higher plants. BMC Genom 7:1–15
Coleman CE, Lopes MA, Gillikin JW, Boston RS, Larkins BA (1995) A defective signal peptide in
the maize high-lysine mutant floury 2. Proc Natl Acad Sci USA 92:6828–6831
Corbesier L, Vincent C, Jang S, Fornara F, Fan Q, Searle I, Giakountis A, Farrona S, Gissot L,
Turnbull C, Coupland G (2007) FT protein movement contributes to long-distance signaling in
floral induction of Arabidopsis. Science 316:1030–1033
Costa C, Dywer LM, Stewart DW, Smith DL (2002) Nitrogen effect on kernel yield and yield
components of Leafy and non-Leafy maize genotypes. Crop Sci 42:1556–1563
Cruz-Alvarez M, Kirihara JA, Messing DJ (1991) Post-transcriptional regulation of methionine
content in maize kernels. Mol Gen Genet 225:331–339
Danilevskaya O, Meng X, Hou Z, Ananiev E, Simmons C (2008) A genomic and expression
compendium of the expanded PEBP gene family from maize. Plant Physiol 146:250–264
Danyluk J, Kane NA, Breton G, Limin AE, Fowler DB, Sarhan F (2003) TaVRT-1, a putative
transcription factor associated with vegetative to reproductive transition in cereals. Plant
Physiol 132:1849–1860
Davies CS, Nielsen NC (1986) Genetic analysis of a null-allele for lipoxygenase-2 in soybean.
Crop Sci 26:460–463
Davies CS, Nielsen NC (1987) Flavor improvement of soybean preparations by genetic removal of
lipoxygenase-2. J Am Oil Chem Soc 64:1428–1433
Devi P, Zhong H, Sticklen MB (2000) In vitro morphogenesis of pearl millet (Pennisetum glaucum
(L.) R.Br.): efficient production of multiple shoots and inflorescences from shoot apices. Plant
Cell Rep 19:546–550
Diaa Al-Abed, Parani Madaswamy, Talla, Reddy, Goldman, Stephen and Rudrabhatla, Sairam.
Genetic Engineering of Maize with the Arabidopsis DREB1A/CBF3 Gene Using Split-seed
Explants. Crop Science, Vol 47. No. 6, November-December, 2007
Dielen V, Lecouvet V, Dupont S, Kinet JM (2001) In vitro control of floral transition in tomato
(Lycopersicon esculentum Mill.), the model for autonomously flowering plants, using the late
flowering uniflora mutant. J Exp Bot 52:715–723
Dinkins RD, Reddy MSS, Meurer CA, Yan B, Trick H, Thibaud-Nissen F, Finer JJ, Parrott WA,
Collins GB (2001) Increased sulfur amino acids in soybean plants overexpressing the maize 15
kDa zein protein. In Vitro Cell Dev Biol-Plant 37:742–747
Doi K, Izawa T, Fuse T, et al (2004) Ehd1, a B-type response regulator in rice, confers short-day
promotion of flowering and controls FT-like gene expression independently of Hd1. Genes &
Development 18:926–936
Fehr WR (2007) Breeding for modified fatty acid composition in soybean. Crop Sci 47(S3): S72–S87
Ferrándiz C, Liljegren S, Yanofsky M (2000) FRUITFULL negatively regulates the SHATTER-
PROOF genes during Arabidopsis fruit development. Science 289:436–438
Feussner I, Wasternack C (2002) The lipoxygenase Pathway. Annu Rev Plant Biol 53:
275–297
Franklin G, Pius PK, Ignacimuthu S (2000) Differential morphogenetic response of cotyledonary
explants of Vigna mungo. Biol Planta 43:157–160
Frizzi A, Huang S, Gilbertson LA, Armstrong TA, Luethy MH, Malvar TM (2008) Modifying
lysine biosynthesis and catabolism in corn with a single bifunctional expression/silencing
transgene cassette. Plant Biotechnol J 6:13–21
Galinat WC, Naylor AW (1951) Relation of photoperiod to inflorescence proliferation in Zea mays
L. Am J Bot 38:38–47
Hammond EG, Fehr WR (1983) Registration of A5 germplasm line of soybean (reg No GP44).
Crop Sci 23:192
Hartings H, Maddaloni M, Lazzaroni N, Di Fonzo N, Motto M, Salamini F, Thompson R (1989)
The O2 gene which regulates zein deposition in maize endosperm encodes a protein with
structural homologies to transcriptional activators. EMBO J 8:2795–2801
Hayama R, Coupland G (2003) Shedding light on the circadian clock and the photoperiodic control
of flowering. Curr Opin Plant Biol 6:13–19
Hayama R, Izawa T, Shimamoto K (2002) Isolation of rice genes possibly involved in the
photoperiodic control of flowering by a fluorescent differential display method. Plant Cell
Physiol 43:494–504
Hayama H, Ito A, Moriguchi T, Kashimura Y (2003) Identification of a new expansin gene closely
associated with peach fruit softening. Postharvest Biol Technol 29:1–10
Herman EM, Helm RM, Jung R, Kinney AJ (2003) Genetic modification removes an immunodo-
minant allergen from soybean. Plant Physiol 132:36–43
Higuchi M, Pischke MS, Mahonen AP, Miyawaki K, Hashimoto Y, Seki M, Kobayashi M,
Shinozaki K, Kato T, Tabata S (2004) In planta functions of the Arabidopsis cytokinin receptor
family. Proc Natl Acad Sci USA 101:8821–8826
Hildebrand DF, Hamilton-Kemp TR, Loughrin JH, Ali K, Andersen RA (1990) Lipoxygenase
3 reduces hexanal production from soybean seed homogenates. J Agric Food Chem 38:
1934–1936
Hitz WD, Carlson TJ, Kerr PS, Sebastian SA (2002) Biochemical and molecular characterization
of a mutation that confers a decreased raffinosaccharide and phytic acid phenotype on soybean
seeds. Plant Physiol 128:650–660
Houmard NM, Mainville JL, Bonin CP, Huang S, Luethy MH, Malvar TM (2007) High-lysine
corn generated by endosperm-specific suppression of lysine catabolism using RNAi. Plant
Huang SS, Adams WR, Zhou Q, Malloy KP, Voyles DA, Anthony J, Kriz AL, Luethy MH (2004)
Improving nutritional quality of maize proteins by expressing sense and antisense zein genes.
J Agric Food Chem 52:1958–1964
Huang S, Kruger DE, Frizzi A, D’Ordine RL, Florida CA, Adams WR, Brown WE, Luethy MH
(2005) High-lysine corn produced by the combination of enhanced lysine biosynthesis and
reduced zein accumulation. Plant Biotechnol J 3:555–569
Irish EE, Nelson TM (1991) Vegetative to floral conversion occurs in multiple steps in maize tassel
development. Development 112:891–898
Izawa T (2007) Daylength measurements by rice plants in photoperiodic short-day flowering. Int
Rev Cytol 256:191–222
Izawa T, Takahashi Y, Yano M (2003) Comparative biology comes into bloom: genomic and
genetic comparison of flowering pathways in rice and Arabidopsis. Curr Opin Plant Biol
6:113–120
IzawaT TY, Yano M (2003) Comparative biology comes into bloom: genomic and gen-
etic comparison of flowering pathways in rice and Arabidopsis. Curr Opin Plant Biol
6:113–120
Izawa T, Oikawa T, Tokutomi S, Okuno K, Shimamoto K (2000) Phytochromes confer the
photoperiodic control of flowering in rice (a short-day plant). The Plant Journal 22:391–399
Jaeger KE, Wigge PA (2007) FT protein acts as a long-range signal in Arabidopsis. Curr Biol
17:1050–1054
James MG, Robertson DS, Myers AM (1995) Characterization of the maize gene sugary1, a
determinant of starch composition in kernels. Plant Cell 7:417–429
Jeon JS, Lee S, Jung KH, Yang WS, Yi GH, Oh BG, An GH (2000) Production of transgenic rice
plants showing reduced heading date and plant height by ectopic expression of rice MADS-box
genes. Mol Breed 6:581–592
Joseph LM, Hymowitz T, Schmidt MA, Herman EM (2006) Evaluation of glycine germplasm for
nulls of the immunodominant allergen P34/Gly m Bd 30k. Crop Sci 46:1755–1763
Kane NA, Agharbaoui Z, Diallo AO, Adam Hl, Tominaga Y, Ouellet F et al (2007) TaVRT2
represses transcription of the wheat vernalization gene TaVRN1. Plant J 51:670–680
Kim WS, Krishnan HB (2004) Expression of an 11-kDa methionine-rich delta-zein in transgenic
soybean results in the formation of two types of novel protein bodies in transitional
cells situated between the vascular tissue and storage parenchyma cells. Plant Biotechnol J
2:199–210
Kitamura K, Davies CS, Kaizuma N, Nielsen NC (1983) Crop Sci 23:924–927
Kojima S, Takahashi Y, Kobayashi Y, Monna L, Sasaki T, Araki T, Yano M (2002) Hd3a, a rice
ortholog of the Arabidopsis FT gene, promotes transition to flowering downstream of Hd1
under short-day conditions. Plant Cell Physiol 43:1096–1105
Komeda Y (2004) Genetic regulation of time to flower in Arabidopsis thaliana. Annu Rev Plant
Biol 55:521–535
Koornneef M, Alonso-Blanco C, Blankestijn-de Vries H, Hanhart CJ, Peeters AJ (1998) Genetic
interactions among late-flowering mutants of Arabidopsis. Genetics 148:885–892
Kozaki A, Hake S, Colasanti JJ (2004) The maize ID1 flowering time regulators is a zinc finger
protein with novel DNA binding properties. Nucl Acids Res 32:1710–1720
Krishnan HB (2005) Engineering soybean for enhanced sulfur amino acid content. Crop Sci
45:454–461
Kurokawa K, Itoh T, Kuwahara T, Oshima K, Toh H, Toyoda A, Takami H, Morita H,
Sharma VK, Srivastava TP (2007) Comparative metagenomics revealed commonly enriched
gene sets in human gut microbiomes. DNA Res 14:169–181
Lai J, Messing J (2002) Increasing maize seed methionine by mRNA stability. Plant J 30:395–402
Lee H, Suh S, Park E, Cho E, Ahn JH, Kim S, Lee JS, Kwon YM, Lee I (2000) The AGAMOUS-
LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis. Gen Dev
14:2366–2376
Lee S, Kim J, Son JS, Nam J, Jeong DH (2003) Systematic reverse genetic screening of T-DNA
tagged genes in rice for functional genomic analyses: MADS-box genes as a test case. Plant
Lim HT, Lee HS, Eriksson T (1997) Regeneration of Panax ginseng C.A. Meyer by organogenesis
and nuclear DNA analysis of regenerants. Plant Cell Tiss Org Cult 49:179–187
Lim J, Moon YH, An G, Jang SK (2000) Two rice MADS domain proteins interact with
OsMADS1. Plant Mol Biol 44:513–527
Lin CS, Lin CC, Chung WC (2003) In vitro flowering of Bambusa edulis and subsequent plantlet
survival. Plant Cell Tiss Org Cult 72:71–78
Lin SI, Wang JG, Poon SY, Su CL, Wang SS, Chiou TJ (2005) Differential regulation of
FLOWERING LOCUS C expression by vernalization in cabbage and Arabidopsis. Plant
Physiol 137:1037–1048
Lin MK, Belanger H, Lee YL, Varkonyl-Gasic E, Taoka KI, Miura E, Xoconostie-Cázares B,
Gendler K, Joprgensen RA, Phinney B, Lough TJ, Lucas WJ (2007) FLOWERING LOCUS T
protein may act as the long-distance florigenic signal in the cucurbits. Plant Cell 19:1488–1506
Litt A, Irish VR (2003) Duplication and diversification in the APETALA1/FRUITFULL floral
homeotic gene lineage: implications for the evolution of floral development. Genetics
165:821–833
Malcomber ST, Preston JC, Reinheimer R, Kossuth J, Kellogg EA (2006) Developmental gene
evolution and the origin of grass inflorescence diversity. Adv Bot Res 44:425–481
Mathieu J, Warthmann N, Kuttner F, Schmid M (2007) Export of FT protein from phloem
companion cells is sufficient for floral induction in Arabidopsis. Curr Biol 17:1055–1060
McSteen P, Laudencia-Chingcuanco D, Colasanti J (2000) A floret by any other name: control of
meristem identity in maize. Trends Plant Sci 5:61–66
Mertz E, Bates L, Nelson OE Jr (1964) Mutant gene that changes protein composition and
increases lysine content of maize endosperm. Science 16:279–280
Mertz ET, Veron O, Bates LS, Nelson OE (1965) Growth of rats fed opaque-2 maize. Science
148:1741–1742
Michaels SD, Himelblau E, Kim SY, Schomburg FM, Amasino RM (2005) Integration of flower-
ing signals in winter-annual Arabidopsis. Plant Physiol 137:149–156
Miller TA, Muslin E, Dorweiler JE (2008) A maize CONSTANS-like gene, conz1, exhibits
distinct diurnal expression patterns in varied photoperiods. Planta 227:1377–1388
Moreira MA, Tavares SR, Ramos V, Barrose EG (1993) Hexanal production and TBA number are
reduced in soybean [Glycine max (L.) Merr.] seeds lacking lipoxygenase isozymes 2 and 3.
J Agric Food Chem 41:103–106
Mouradov A, Cremer F, Coupland G (2002) Control of flowering time: Interacting pathways as a
basis for diversity. Plant Cell 14:S111–S130
Muszynski MG, Dam T, Li B, Shirbroun DM, Hou Z, Bruggemann E, Archibald R, Ananiev
EV, Danilevskaya ON (2006) delayed flowering 1 encodes a basic leucine zipper protein
that mediates floral inductive signals at the shoot apex in maize. Plant Physiol 142:
1523–1536
Nelson OE Jr, Rines HW (1962) The enzymatic deficiency in the waxy mutant of maize. Biochem
Biophys Res Commun 9:297–300
Neuffer M, Coe E, Wessler S (1997) Mutants of maize. Cold Spring Harbor Lab Press, Cold Spring
Harbor, NY
Nishiba Y, Furuta S, Hajika M, Igita K, Suda I (1995) Hexanal accumulation and DETBA value in
homogenate of soybean seeds lacking two or three lipoxygenase isoenzymes. J Agric Food
Chem 43:738–741
Nishimura C, Ohashi Y, Sato S, Kato T, Tabata S, Ueguchi C (2004) Histidine kinase homologs
that act as cytokinin receptors possess overlapping functions in the regulation of shoot and root
growth in Arabidopsis. Plant Cell 16:1365–1377
Nordlee JA, Taylor SL, Townsend JA, Thomas LA, Bush RK (1996) Identification of a Brazil-nut
allergen in transgenic soybeans. New Engl J Med 334:688–692
Olsen MS, Krone TL, Phillips RL (2003) BSSS53 as a donor source for increased whole-kernel
methionine in maize: selection and evaluation of high-methionine inbreds and hybrids. Crop
Sci 43:1634–1642
Oltmans SE, Fehr WR, Welke GA, Raboy V, Peterson KL (2005) Agronomic and seed traits of
soybean lines with low-phytate phosphorus. Crop Sci 45:593–598
Orf JH, Hymowitz T (1979) Inheritance of the absence of the Kunitz trypsin inhibitor in seed
protein of soybeans. Crop Sci 19:107–109
Parcy F (2005) Flowering: a time for integration. Int J Dev Biol 49:585–593
Phillips RL, Morris PR, Wold F, Gengenbach BG (1981) Seedling screening for lysine-plus-
threonine resistant maize. Crop Sci 21:601–607
Prasanna BM, Vasal SK, Kassahun B, Singh NN (2001) Quality protein maize. Curr Sci
81:1308–1319
Putterill J, Laurie R, Macknight R (2004) It’s time to flower: the genetic control of flowering time.
Bioessays 26:363–373
Raboy V, Gerbasi PF, Young KA, Stoneberg SD, Pickett SG, Bauman AT, Murthy PPN, Sheridan
WF, Ertl DS (2000) Origin and seed phenotype of maize low phytic acid 1–1 and low phytic
acid 2–1. Plant Physiol 124:355–368
Rao NN, Prasad K, Kumar PR, Vijayraghavan U (2008) Distinct regulatory role for RFL, the rice
LFY homolog, in determining flowering time and plant architecture. Proc Natl Acad Sci USA
105:3646–3651
Riefler M, Novak O, Strnad M, Schmulling T (2006) Arabidopsis cytokinin receptor mutants
reveal functions in shoot growth, leaf senescence, seed size, germination, root development,
and cytokinin metabolism. Plant Cell 18:40–54
Rudrabhatla SV, Goldman SL (2009) Method for producing direct in vitro flowering and variable
seed from cotyledon, radical, and leaf explants, and plants produced therefrom. United States
Patent Number 7,547,548
Salvi ND, George L, Eapen S (2002) Micropropagation and field evaluation of micropropagated
plants of turmeric. Plant Cell Tiss Org Cult 68:143–151
Salvi SG, Sponza G, Morgante M, Tomes D, Niu X, Fengler KA, Meeley R, Ananiev EV,
Svitashev S, Bruggemann E (2007) Conserved noncoding genomic sequences associated
with a flowering time quantitative trait locus in maize. Proc Natl Acad Sci USA 104:
11376–11381
Samach A, Onouchi H, Gold SE, Ditta GS, Schwarz-Sommer Z, Yanofsky MF, Coupland G
(2000) Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis.
Science 288:1613–1618
Schmidt R, Burr F, Aukerman M, Burr B (1990) Maize regulatory gene Opaque-2 encodes
a protein with a “leucine-zipper” motif that binds to zein DNA. Proc Natl Acad Sci USA
87:46–50
Segal G, Song RT, Messing J (2003) A new opaque variant of maize by a single dominant RNA-
interference-inducing transgene. Genetics 165:387–397
Shaver DL (1983) Genetics and breeding of maize with extra leaves above the ear. 38th Proc Annu
Corn and Sorghum Res Conf, Chicago, IL, USA, Am Seed Trade Assoc, Washington DC,
USA, pp 161–180
Sheehan MJ, Kennedy LM, Costich DE, Brutnell TP (2007) Subfunctionalization of PhyB1
and PhyB2 in the control of seedling and mature plant traits in maize. The Plant Journal
49:338–353
Sheeja TE, Mandal AB (2003) In vitro flowering and fruiting in tomato (Lycopersicum esculentum
Mill.). Asia Pacif J Mol Biol Biotechnol 11(1):37–42
Shi J, Wang H, Schellin K, Li B, Faller M, Stoop JM, Meeley RB, Ertl DS, Ranch JP, Glassman K
(2007) Embryo-specific silencing of a transporter reduces phytic acid content of maize and
soybean seeds. Nat Biotechnol 25:930–937
Simpson GG, Dean C (2002) Arabidopsis, the rosetta stone of flowering time? Science 296:
285–289
Simpson GG, Quesada V, Henderson IR, Dijkwel PP, Macknight R (2004) RNA processing and
Arabidopsis flowering time control. Biochem Soc Trans 32:565–566
Singh M, Jaiswal U, Jaiswal VS (2000) Thidiazuron-induced in vitro flowering in Dendrocalamus
strictus Nees. Curr Sci 79(11):1529–1530
Singh M, Jaiswal U, Jaiswal VS (2001) Thidiazuron-induced shoot multiplication and plant
regeneration in bamboo (Dendrocalamus strictus Nees.). J Plant Biochem Biotechnol
10:133–137
Speedy AW (2003) Global production and consumption of animal source foods. J Nutr 133:
4048–4053
Sprague GF, Brimhall B, Hixon RM (1943) Some effects of the waxy gene in corn on properties of
the endosperm starch. J Am Soc Agron 35:817–822
Stinard PS, Robertson DS, Schnable PS (1993) Genetic isolation, cloning, and analysis of a
mutator-induced, dominant antimorph of the maize amylose extender1 locus. Plant Cell
5:1555–1566
Subedi KD, Ma BL (2005) Seed priming does not improve corn yield in a humid temperate
environment. Agron J 97:211–218
Subedi KD, Ma BL, Smith DL (2006) Response of leafy and non-leafy maize hybrid to plant
population densities and fertilizer nitrogen levels. Crop Sci 46:1860–1869
Takahashi Y, Shomura A, Sasaki T, Yano M (2001) Hd6, a rice quantitative trait locus involved in
photoperiod sensitivity, encodes the subunit of protein kinase CK2. Proc Natl Acad Sci USA
98:7922–7927
Tamaki S, Matsuo S, Wong HL, Yokoi S, Shimamoto K (2007) Hd3a protein is a mobile flowering
signal in rice. Science 316:1033–1036
Torres-Penaranda AV, Reitmeier CA, Wilson LA, Fehr WR, Narvel JM (1998) Sensory char-
acteristics of soymilk and Tofu made from lipoxygenase-free and normal soybeans. J Food Sci
63:1084–1087
Tracy WF (1997) History, genetics, and breeding of supersweet (shrunken2) sweet corn. Plant
Breed Rev 14:189–236
Vladutu C, McLaughlin J, Phillips RL (1999) Fine mapping and characterization of linked
quantitative trait loci involved in the transition of the maize apical meristem from vegetative
to generative structures. Genetics 153:993–1007
Werner T, Motyka V, Strnad M, Schmülling T (2001) Regulation of plant growth by cytokinin.

Proc Natl Acad Sci USA 98:10487–10492
Werner T, Motyka V, Laucou V, Smets R, Van Onckelen H, Schmülling T (2003) Cytokinin-
deficient transgenic Arabidopsis plants show multiple developmental alterations indicating
opposite functions of cytokinins in regulating shoot and root meristem activity. Plant Cell
15:2532–2550
Wilcox J, Cavins J, Nielsen N (1984) Genetic alteration of soybean oil composition by a chemical
mutagen. J Am Oil Chem Soc 61:97–100
Wilcox JR, Premachandra GS, Young KA, Raboy V (2000) Isolation of high seed inorganic P,
low-phytate soybean mutants. Crop Sci 40:1601–1605
Wong AYM, Colasanti J (2007) Maize floral regulator protein INDETERMINATE1 is
localized to developing leaves and is not altered by light or the sink/source transition. J Exp
Bot 58:403–414
Wu C, You C, Li C, Long T, Chen G, Byrne ME, Zhang Q (2008a) RID1, encoding a Cys2/
His2-type zinc finger transcription factor, acts as a master switch from vegetative to floral
development in rice. Proc Nal Acad Sci USA 105:12915–12920
Wu K, Zhang L, Zhou C, Yu CW, Chaikam V (2008b) HDA6 is required for jasmonate response,
senescence and flowering in Arabidopsis. J Exp Bot 59:225–234
Yan L, Loukoianov A, Tranquilli G, Helguera M, Fahima T, Dubcovsky J (2003) Positional
cloning of the wheat vernalisation gene VRN1. Proc Natl Acad Sci USA 100:6263–6268
Yan L, Fu D, Li C, Blechl A, Tranquilli G, Bonafede M (2006) The wheat and barley vernalization
gene VRN3 is an orthologue of FT. Proc Natl Acad Sci USA 103:19581–19586
Yan LL, Loukoianov A, Blechl A, Tranquilli G, Ramakrishna W, San Miguel P, Bennetzen JL,
Echenique V, Dubcovsky J (2004) The wheat VRN2 gene is a flowering repressor down-
regulated by vernalization. Science 303:1640–1644
Yano M, Katayose Y, Ashikari M, Yamanouchi U, Monna L, Fuse T, Baba T, Yamamoto K,
Umehara Y, Nagamura Y (2000) Hd1, a major photoperiod sensitivity quantitative trait locus
in rice, is closely related to the Arabidopsis flowering time gene CONSTANS. Plant Cell
12:2473–2484
Yanofsky MF (1995) Floral meristems to floral organs: genes controlling early events in Arabi-
dopsis flower development. Annu Rev Plant Physiol Plant Mol Biol 46:167–188
Chapter 5
Biotechnological Interventions to Improve Plant
Developmental Traits
Avtar K. Handa, Alka Srivastava, Zhiping Deng, Joel Gaffe, Ajay Arora,
Martı́n-Ernesto Tiznado-Hernández, Ravinder K. Goyal, Anish Malladi,
Pradeep S. Negi, and Autar K. Mattoo
5.1 Introduction
Unprecedented progress during the last three decades in our understanding of the
principles of a living cell, particularly the identification of genes and signaling path-
ways involved in cell differentiation and organ development, has brought us a broader
insight into plant biological processes. Technological advancements are revealing
new and fundamental knowledge at the molecular and cellular levels, knowledge that
is critical towards achieving the goal of precision-based crop improvement. Modern-
day genetic engineering has emerged as a promising precision-based technology for
boosting up food production in the world and introducing desirable traits such as
nutritional enhancement and disease and pest resistance, both important components
of agricultural sustainability (Chrispeels et al. 2002; Fatima et al. 2008; Negi and
Handa 2008). Achieving results that benefit the world will depend on the success of
applying new knowledge to real-world field scenarios. The challenge, therefore, is
also to simultaneously obtain knowledge on agroecosystem structure and function to
understand how manipulation and control of specific gene expression will translate
into directing processes at the ecological scale (Mattoo and Teasdale 2009).
Developmental traits are coordinated at various levels in a plant and involve
organ-to-organ communications via long-distance signaling processes that integrate
transcription, hormonal action and environmental cues. Thus, plant architecture,
root–soil–microbe interactions, flowering, fruit (and seed) development, and fruit
ripening (and seed germination) are highly regulated genetic programs that are also
impacted by processes such as organ abscission, organ senescence (and ripening),
and programmed cell death (PCD). We note that belowground processes provide
the anchor for a healthy and robust plant (Mattoo and Teasdale 2009) but in this
A.K. Handa (*)

Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette,
IN 47907-2010, USA
e-mail: ahanda@purdue.edu

200 A.K. Handa et al.
chapter we have focused on the aboveground plant processes, bringing together

information on the genes and biotechnological applications that can potentially
affect production and quality of crop plants.
5.2 Shoot Branching
Shoot branching (tillering in monocots) involves the initiation and development of

axillary buds of a leaf, which form new branches or flowers. It is one of the most
important traits of plant architecture, affecting plants’ ability to intercept light
energy for photosynthesis and adaptation to grow in different environmental con-
ditions. Both branching number and branch angle are traits that control crop yield.
Branching is regulated by endogenous plant hormones and environmental sig-
nals. Plant hormones such as auxin, cytokinin, and strigolactones or their precur-
sors, have major roles in controling branching. Auxin is actively transported
basipetally down the shoot to inhibit shoot branching (Ongaro and Leyser 2008),
while cytokinin and strigolactones are transported acropetally to promote and
inhibit bud growth, respectively. Modification of genes involved in the biosynthe-
sis, transport, or perception of these three hormones is expected to affect branching.
For example, three rice mutants, d3 (Ishikawa et al. 2005), htd1 (Zou et al. 2006)
and dwarf10 (Arite et al. 2007), which are either deficient in strigolactone percep-
tion or biosynthesis, display increased branch numbers. D3, a leucine-rich repeat
F-box protein, is required for strigolactone perception, while HTD1 and
DWARF10, which encode different members of carotenoid cleavage dioxygenases,
are required for strigolactone biosynthesis.
Domestication of major cereal crops involved modification of shoot branching,
allowing for denser planting and less shelf-shading. Cultivated varieties usually
have fewer branches compared to their wild ancestors (Doust 2007). Some of the
genes affecting branching number or angle have been cloned from cereal crops
(Table 5.1). For example, teosinte, wild ancestor of maize, displays many tillers
with slender ears, while maize shows strong apical dominance and typically deve-
lops a few tillers with thicker ears (Fig. 5.1). The apical dominance in maize is
mainly controlled by teosinte branched1 (tb1) (Doebley et al. 1997). This gene was
cloned by transposon tagging and shown to repress growth of axillary buds and
promote female inflorescences. Overexpression of a rice TB1 gene under the
constitutive rice actin promoter reduced lateral branching, whereas a rice line with
nonfunctional TB1 exhibited enhanced lateral branching (Takeda et al. 2003).
Similarly, overexpression of a maize TB1 gene in wheat suppressed tiller develop-
ment (Lewis et al. 2008), while a loss of function of the Arabidopsis TB1 homolog,
Branched1, in the T-DNA knockout lines or RNAi lines (Chap. 1-6), resulted in
increased branching (Aguilar-Martı́nez et al. 2007). These results indicate that TB1-
mediated control of branching is conserved between monocots and dicots. Since
branching is also affected by environmental and growth conditions, it remains to be
seen whether fine-tuning of expression levels of branching-related genes via bio-
engineering (Chap. 1-7) will improve crop yield.
5
Table 5.1 Genes with potential to modify plant branching

Gene Function Effect Phenotypes Reference
TEOSINTE TCP transcription Inhibits shoot Overexpression in rice and maize Lewis et al. (2008), Takeda et al.
BRANCHED1 factor branching decreases branching, loss of function (2003), Aguilar-Martı́nez et al.
(TB1) in Arabidopsis increases lateral (2007)
branching
PROSTRATE Zinc finger Increases branching Overexpression in rice causes prostrate Jin et al. (2008), Tan et al. (2008)
GROWTH1 transcription factor and tiller angles growth
(PROG1)
MONOCULM1 GRAS family protein, Promotes bud Overexpression in rice increases tiller Li et al. (2003)
(MOC1) putative transcription initiation and number, and loss of function in rice
factor development reduces tillering capacity
Tiller Angle Unknown function Increases tiller angle Overexpression in rice causes wider Yu et al. (2007)
Control 1 tiller angle
(TAC1)
LAZY1 Unknown function Promotes erect growth Loss of function in rice promotes Li et al. (2007a), Yoshihara and Iino
(LA1) prostrate growth (wider angle) (2007)
Biotechnological Interventions to Improve Plant Developmental Traits
201
Fig. 5.1 Mature plants and

inflorescence of teosinte
branched 1 mutant and wild-
type maize. a, b, the mutant;
c, d, an inbred maize line.
Modified from Doebley et al.
(1997)
5.3 Male Sterility
Male sterility in higher plants is characterized by a failure to produce functional

anthers, pollen or male gametes. It is a useful trait, both for studying the male
developmental pathway and for exploiting heterosis to obtain high-yielding
crop plants. Kaul (1988) classified male sterile types as structural, sporogenous
5 Biotechnological Interventions to Improve Plant Developmental Traits 203
or functional. Structural male sterile plants may show absence of stamens with
anthers or presence of anthers that are devoid of sporogenous tissues. Sporo-
genous male sterile plants show normal anther development but the development of
pollen is impaired because of irregularities in the division of sporogenous cells.
Plants that show anthers with viable pollen development but are still incapable of
affecting fertilization because of anther dehiscence failure, abnormal exine mor-
phology or failure of pollen tube germination, are classified as functional male
sterile types. Additionally, Taylor and Jorgensen (1992) have proposed the term
“conditional male fertility” for the type of male sterility in which viable pollen is
unable to germinate and shows pollen tube growth in self-crosses of the chalcone
synthase-deficient progeny. At the genetic level, three types of plant male steri-
lities have been identified, namely, genic male sterility (GMS), cytoplasmic male
sterility (CMS) and gene-cytoplasmic male sterility.
5.3.1 Genic Male Sterility
GMS is caused by spontaneous or induced mutations in nuclear genes. It is

ordinarily governed by ms, a single recessive gene. GMS is inherited in Mendelian
fashion and has been reported in approximately 175 species of angiosperms,
including crop plants such as barley, maize, pea, tomato, rice and pepper. These
plants can be propagated when pollinated by wild type, male fertile plants. Several
male sterile plants exhibit structurally malformed male sexual organs. For exam-
ple, genes such as apetalla-3 (ap-3), pistillata (pi) and antherless (at) in Arabi-
dopsis affect the development of male sexual organs resulting in sterility
(Chaudhury 1993). Mutations also affect genes participating in the differentiation
and function of such anther cell types as the stomium, tapetum, endothecium and
the archesporial and sporogenous layers of the anther primordium. Arabidopsis
mutant ms2 shows defective tapetal layer development resulting in pollen abortion
shortly after release from the tetrads (Aarts et al. 1993). Defective meiosis in
Arabidopsis (msW and msY; Dawson et al. 1993) and tomato mutants (ms3, ms15
and ms29; Rick 1948) also resulted in male sterile plants. Furthermore, mutations
in genes controlling metabolic pathways may also result in sterile/nonviable
pollens. Induced petunia white anther (wha) mutant is characterized by the
inability of the pollen grains to germinate in vitro. This phenotype is caused by
a lesion in chs (chalcone synthase) gene that encodes the first enzyme in the
flavonoid biosynthesis pathway (Napoli et al. 1999). Such examples of spontane-
ous or induced GMS are limited in their use for plant breeding purposes by the
availability of a fertility restoration gene. Genetic engineering approaches to
introduce a dominant male sterile gene into plant cells (Chap. 1–3) have overcome
such barriers and resulted in male sterile transgenic lines in many crop plants.
These approaches have been discussed in greater detail under biotechnological
advances in Sect. 5.3.3.
5.3.2 Cytoplasmic and Gene-Cytoplasmic Male Sterility
CMS, in most cases, is determined by mutation in mitochondrial genes (Hanson and

Bentolila 2004; Pelletier and Budar 2007). CMS has been reported in approxi-
mately 150 species, comprising both wild and cultivated plants (Raghavan 1997).
It is maternally inherited, easier to maintain in crop plants and therefore of much
value to plant breeders. When the vegetative plant part is the harvested product,
CMS alone can be used to obtain higher-yielding hybrid lines. However, when seed
or fruit is the desired product, the heterotic hybrid lines should be self-pollinating
and able to set seeds. A fertility restorer (Fr) gene must be introduced in such
hybrids. Restorer genes, also known as Restorer of fertility (Rf) or Fr, are nuclear
genes that mask the male sterility effect of CMS, such that the plant is rendered
male fertile in their presence (Schnable and Wise 1998). Rf and CMS together form
a binary system (genic-cytoplasmic or nucleo-cytoplasmic) that regulates the mode
of reproduction of individuals in populations (Pelletier and Budar 2007). It is
possible to remove the effect of Rf genes through interspecific crosses or somatic
hybridization such that the nuclear genome of one plant species is present in the
male sterility-inducing cytoplasmic background of another plant species (Chase
2007). Male sterility arising as a result of interspecific nuclear-cytoplasmic combi-
nation is known as alloplasmic male sterility (Hanson and Bentolila 2004). The
absence of Rf gene from the alloplasmic nuclear-cytoplasmic combination unmasks
the male sterility-inducing effect of the mitochondrial genes. However, in some
cases, hybridization itself has proven mutagenic causing novel cytoplasmic genome
configuration that arises because of disturbances in cytoplasmic genome replication
and organization (Hanson and Conde 1985).
CMS systems have been extensively used to generate high-yielding hybrid
varieties in crop plants. In maize, several types of male sterility-inducing cyto-
plasms have been identified of which CMS-T, -S and -C are the most widespread
and well characterized. These CMS systems are differentiated by their reaction to
restorers; CMS-T is restored by Rf1 and Rf2, CMS-S is restored by Rf3, and CMS-C
is restored by Rf4 (Sofi et al. 2007). Many CMS systems have been found in
Brassica crops including the well-studied ogura (ogu), polima (pol) and napus
(nap) CMS systems. The pol and nap CMS systems are restored by Rfp and Rfn
genes, respectively. Both Rfp and Rfn map to the same chromosomal position and
they are allelic (Li et al. 1998). In rice, CMS-BT (Boro type), CMS-WA (wild
abortive) and CMS-HL (Honglian) are well established: Rf1a and Rf1b that confer
fertility to CMS-BT and Rf5 and Rf6(t) that confer fertility to CMS-HL are all
located on chromosome 10 (Fuji et al. 2008). The restorer genes, Rf3 and Rf4, which
confer fertility in CMS-WA are located on chromosomes 1 and 10, respectively.
CMS systems and their restorer genes in other crop plants are listed in Table 5.2.
Many male sterility-conferring cytoplasmic genes have now been identified as
open reading frames (ORFs, Table 5.2). For example, the male sterility-inducing
CMS systems T-urf13 in maize, pvs-orf239 in bean and orf138, orf224 in Brassica
are ORFs formed as a result of complex mitochondrial DNA (mtDNA)
Table 5.2 Plant cytoplasmic male sterility systems

Plant CMS system Mt ORFa/gene Restorer locus Reference
Radish Ogura orf 138 and orfB Rfo Brown et al. (2003)
Kosena orf125 Rf K Koizuka et al. (2003)
Rice CMS-BT Orf 79/B-atp6 Rf1a, Rf1b Wang et al. (2006a, b)
CMS-WA Unknown Rf3, Rf4 Li et al. (2007a)
CMS-HL orf H79/atp6 Rf5, Rf6 (t) Zhang et al. (2007a)
Petunia Pcf Rf1 Bentolila et al. (2002)
Maize CMS –T T-urf13 Rf2, Rf1, Rf8, Rf* Wise et al. (1999)
orf355-orf77,atp6,
CMS-S Cob Rf3 Wen and Chase (1999)
CMS-C atp6-C Rf4, Rf5, Rf-I Dewey et al. (1991)
Brassica pol orf224/atp6 Rfp Singh et al. (1996)
nap orf222 Rfn(Mmt) Homme et al. (1997)
Sunflower PET1 orfB,orf552 Rf1 Horn et al. (2003)
Sorghum A1 Rf1 Klein et al. (2005)
A3 Orf107 Rf3, Rf4 Kuhlman et al. (2006)
a
Mitochondrial open reading frames associated with CMS
rearrangement (Hanson and Bentolila 2004). These ORFs often comprise a mix of
conventional mitochondrial genes and sequences of unknown origins. They are
co-transcribed with essential mitochondrial genes and encode proteins with at least
two shared features: lower molecular size (<30 kDa) and at least one hydrophobic
domain.
Studies comparing the respiratory activities of CMS and fertile lines have shown
differences in the activity level of the respiratory complexes (Conley and Hanson
1995; Sabar et al. 2000). ATP synthase as well as cytochrome oxidase are asso-
ciated with the inner membrane of the mitochondria (Hanson and Bentolila 2004).
The presence of ATP synthase subunits in the CMS-associated chimeric genes
raises the possibility of an impaired ATP synthase activity being the cause of
disrupted pollen development that results in male sterility. Premature PCD, in
which mitochondria play a central role, has also been suggested to be a possible
cause of CMS. According to this suggestion, PCD destroys the tapetal cells that
provide nourishment to the developing pollen inside the anthers and thereby
causing male sterility (Balk and Leaver 2001; Ku et al. 2003; Rogers 2006).
Fr genes encode members of pentatricopeptide repeat protein (PPR) family. PPR
genes have been found in the restorer loci of Petunia, Brassica, Raphanus and rice
(Hanson and Bentolila 2004). These proteins, targeted to chloroplast and mitochon-
dria, are involved in organelle biogenesis in plants (Lurin et al. 2004). Studies
involving nuclear gene mutants with compromized plastid functions have revealed
PPR proteins to be involved in RNA processing, intron splicing and translation
(Stern et al. 2004; Shikanai 2006). These studies indicate probable functions for
mitochondria-targeted PPR proteins including those encoded by restorer genes.
In most fertility-restored CMS lines, the protein product for the CMS determining
locus does not accumulate. This loss of protein accumulation is accompanied by
lower accumulation of CMS-associated transcripts or truncated transcripts.
Whether the decreased transcript level or transcript truncation results in the lack
of protein accumulation or is a by-product of failed translation or imperfect editing

of CMS-associated transcript in the presence of a restorer gene is not known
(Hanson and Bentolila 2004; Newton et al. 2004; Linke and Borner 2005; Chase
2007).
5.3.3 Bioengineering Male Sterility for Hybrid Plants
Mariani et al. (1990) successfully engineered the first GMS plant, thereby paving
the way for the development of more transgenic male sterile crop plants. The
ablation of tapetal cells inside anthers results in failure of pollen development.
The barnase gene product (barnase) is an extracellular RNase found in Bacillus
amyloliquifaciens that protects the bacterium from microbial predators (Hartley
1989). The bacterium is protected by the cytotoxic effect of barnase by intracellular
barstar, a barnase-specific RNase inhibitor. A chimeric gene comprising tapetum-
specific gene promoter TA29 and barnase gene coding sequences was used to
develop transgenic male sterile tobacco and oilseed rape plants. These plants
showed ablation of tapetal cells and impaired pollen development. However,
these transgenic plants did not show any adverse effect on tapetal cells. When
male sterile plants containing TA29/barnase genes were crossed with male fertile
plants containing TA29/barstar gene, the resultant progeny was male fertile
(Mariani et al. 1992). The barnase/barstar system has been used extensively to
obtain male sterile lines in oilseed rape, tomato, cotton, corn and wheat (De Block
et al. 1997). Following the pioneering efforts of Mariani et al. (1990), several
biotechnological strategies to generate genic male sterile plants have been tested
(Perez-Prat and van Lookeren Campagne 2002), which include:
1. Tissue-specific expression of a gene encoding a protein that can disrupt the cell
function and thereby development of tissues that are vital for pollen development
– barnase/barstar is one such system. Chemical induction of male sterility by
selective expression of genes encoding a protein that converts a pro-herbicide into
a herbicide only in male reproductive tissues (O’Keefe et al. 1994; Dotson et al.
1996; Kriete et al. 1996). Alternatively, a male sterility gene engineered such that
it is induced by the application of a chemical (Goff et al. 1990). Fertility restorer
gene or a repressor of male sterility under the control of a chemically inducible
promoter employed for fertility restoration (Ward et al. 1993).
2. Manipulation of metabolite levels essential for normal pollen development.
Genetic alteration of the levels of amino acids, sugars (Goetz et al. 2001),
flavonols (Derksen et al. 1999), jasmonic acids (McConn and Browse 1996;
Browse 1997; Sanders et al. 2000), biotin (Albertsen and Howard 1999) and
auxins (Spena et al. 1992) to develop sterile lines. In such systems, fertility is
inducible by applying the altered metabolite (McConn and Browse 1996;
Browse 1997; Albertsen and Howard 1999; Sanders et al. 2000).
3. Crossing two transgenic parental lines, each carrying a gene encoding for an
inactive part of a toxin in the male reproductive tissue. When brought together in
the progeny, these inactive parts combine together to form the active toxin,
which results in male sterility (Fabijanski and Arinson 1995; Gutterson and
Ralston 1998).
4. Use of transgenic maintainer lines for natural or induced GMS lines. These lines
allow the propagation of natural or induced mutations in such a way that male
sterile plants may be obtained or male fertile plants may be visually identified
and separated. Such maintainer lines are isogenic to the genic male sterile lines
except at the transgenic locus comprised two components: (a) A restorer gene
that renders the maintainer lines male fertile; and (b) genes or set of genes,
which upon crossing a maintainer line with an isogenic male sterile line, either
prevent the maintainer locus from passing into the progeny (such plants are
called pollen lethality maintainers) or reveal the maintainer locus by simulta-
neously expressing a gene that results in pigment accumulation (color main-
tainers). In the latter crosses, 50% plants are male fertile; however, these seeds
can be identified and separated according to pigment accumulation (Perez-Prat
and van Lookeren Campagne 2002).
GMS is limited in its use for hybrid seed production as it is inherited in
Mendelian fashion. The plants segregate for male fertility and sterility. This pre-
sents a practical problem of separating the male fertile and male sterile plants.
CMS, on the other hand, is inherited maternally. Breeders have widely exploited
CMS systems to develop male sterile lines. Engineering male sterility through
somatic hybridization has resulted in improved CMS systems as well as expansion
of the CMS systems to a larger group of crop plants (Pelletier and Budar 2007).
Somatic hybridization involves isolation, culture and fusion of protoplasts of two
distantly or closely related or even unrelated plants at interspecific, intergeneric,
intraspecific or interfamily levels. The heterokaryons resulting from fused proto-
plasts are selected based on a marker gene and then cultured to develop into
complete plants. In some of these heterokaryons, nucleo-cytoplasmic interactions
result in formation of cytoplasmic hybrids (cybrids), which carry the nuclear
genome of only one parent but the cytoplasmic genomes from both parents. The
nuclear genome of the mitochondrial donor should carry a restorer gene (Pelletier
and Budar 2007). Male sterile cybrids have been developed in Brassica species
(Budar et al. 2004), rice (Brar and Khush 2006) and tomato (Melchers et al. 1992).
Natural CMS system is not found in all crop plants and its use for breeding
purposes is limited if an appropriate restorer system is not present. Although
transgenic dominant genic male sterile plants can be developed, this approach is
restricted by the Mendelian segregation of the progeny. Stable, maternally inherited
male sterility can be induced in crop plants by organelle transformation
(Chap. 1-8). Male sterility induction has been reported by plastid transformation
using b-ketothiolase gene (phaA) in tobacco plants (Ruiz and Daniell 2005). The
phaA gene, under the control of light-inducible psbA promoter, alters the course of
fatty acid synthesis, which in turn leads to pollen grain collapse and male sterility.
Male sterility was reversed by growing these plants under continuous illumination.
Plastid transformation holds great promise for genetic transformation of crop
plants. It will be possible to take better advantage of this technology for developing
male sterile crop plants and their restorer systems as more plant species become
amenable to plastid transformation (Kuchuk et al. 2006).
Studies on GMS and CMS systems have not only revealed the fundamental
aspects of male developmental pathways, but also the molecular players in male
sterility and fertility restoration in plants. Also, genetic engineering was employed
to inhibit flower development and introduce reproductive sterility. Expression of a
ribosome-inactivating protein (DTA) gene under the control of PTD gene – homo-
logous to the MADS box genes DEFICIENS and APETALA3 – resulted in sterile
flowers and consequently impaired dissemination of the transgene and the confine-
ment of specific trait (Skinner et al. 2003; Wei et al. 2006). Biotechnological
advances have allowed the expansion of these male sterility systems in a wider
range of crop plants. Identification of mitochondrial genes controlling male sterility
and those that restore fertility and understanding their mode of function will allow a
better control over these systems. Advances in organellar transformation will
provide a better process for inducing male sterility/fertility restoration in plants.
Transformation of mitochondria remains the next hurdle to overcome and will
prove a breakthrough advance towards understanding the role of this organelle in
male development and sterility aspects.
5.4 Flowering Induction
Flower induction is under the control of several interconnected pathways involving

light irradiation, temperature, plant growth regulators and the physiological state of
the plant (Jaeger et al. 2006; Kobayashi and Weigel 2007; Wilkie et al. 2008). The
genetic basis of these pathways are the subject of intense research in Arabidopsis
(Bäurle and Dean 2006), as well as in maize, rice (Chap. 4.2) (Izawa 2007),
soybean (Wong et al. 2009) and other plants of economic interest. The control of
flower induction is a major agronomical event in crop productivity, e.g., cereal
(Cockram et al. 2007), fruit (Carmona et al. 2008), plant fibers (Abdurakhmonov
et al. 2007) or wood industry (Yuceer et al. 2003; Böhlenius et al. 2006). In potato,
more or less the same genetic components regulate flowering and tuberization
(Rodrı́guez-Falcón et al. 2006). In cereal crops, regulation of flower induction is
critical for seed set and development. The day length and temperature were found to
be the key elements for the growth attributes of wheat and maize when initially
grown in the Fertile Crescent and Central America, respectively. In recent times,
however, the area for growing cereals has widely expanded during centuries of
selection and improvement (Cockram et al. 2007).
In Arabidopsis, promotion of flowering involves long-day photoperiod, gibber-
ellins, an autonomous route, and vernalization (Jack 2004; Putterill et al. 2004). The
photoperiod pathway perceives light quantity and circadian clock – long day
promotes flowering in Arabidopsis. In order to induce flowering at an appropriate
time, the day length has to be in sync with an internal time keeper (Bäurle and
Table 5.3 Genes regulating flowering time in plants

Gene Function Phenotype Biotechnology Reference
intervention
CO TranscriptionAltered flowering time. Constitutive Martı́nez-
Arabidopsis factor Impaired overexpression Garcı́a
potato et al.
tuberization (2002)
FT Kinase inhibitor Early flowering Overexpression Kardailsky
Arabidopsis Forms CO/ et al.
FT (1999)
regulatory
module
pTFT1 Kinase inhibitor Early flowering. Timing of Overexpression Böhlenius
Populus FT ortholog flowering and seasonal et al.
growth cessation (2006)
in trees
Ehd 2 (rice) Zinc finger Promotes flowering Mutation Matsubara
protein et al.
ortholog of (2008)
maize
INDETER
MINATE1
OsCO3 (rice) Transcription Inhibition of flowering
factor
Leafy (LFY) Reduced juvenile phase Overexpression Peña et al.
Apetala1 (2001)
(AP1)
Dean 2006). Gibberellins promote flowering, especially under short-day photope-

riod. The autonomous pathway is independent of photoperiod control, and causes
flowering in Arabidopsis under short days. Vernalization refers to promotion of
flowering by extended cold exposure, as naturally happens in plants during winter.
Different pathways of flower-regulation genes converge at a few defined floral
pathway integrators that include Flowering Locus T (FT), Suppressor of Over-
expression of Constans 1 (SOC1) and Leafy (LFY), which activate floral meristem
identity genes and trigger the transition from vegetative to reproductive phase
(Table 5.3) Details of the transition from vegetative meristem to floral transition
are described in Chap. 4.2.
Two genes, Constans (CO) and Flowering Transition (FT), whose expression is
tuned to the change in day length and involved in flowering, were initially isolated
from Arabidopsis (Kobayashi and Weigel 2007). CO is a transcription activator that
belongs to the B Box Zinc finger protein family. FT is a 23-kDa protein from the
RAF kinase inhibitor family involved in regulatory cascade and signal transduction
(Turck et al. 2008). FT protein is synthesized in the companion cell of the leaf
phloem and mobilized to the shoot apical meristem to initiate the flowering
(Cockram et al. 2007; Turck et al. 2008). In Arabidopsis, FT protein accumulation
is directly under the CO control.
CO protein does not bind DNA by itself but interacts with transcriptional
complex (Kobayashi and Weigel 2007). The biochemical mode of action of CO
is still unclear. Orthologs of CO have been identified in rice (Izawa 2007), potato
(Martı́nez-Garcı́a et al. 2002) and aspen tree (Böhlenius et al. 2006). CO transcrip-
tion is under the control of circadian regulation through CDF1 (cycling DOF
Factor). Its transcripts accumulate from the afternoon to the middle of the night
either in long day (LD) or short day (SD) but with a slight shoulder at the end of the
afternoon during LD (Fig. 5.2; Imaizumi et al. 2005). CO transcripts are degraded
in LD plants at the end of the day and during the night but in SD this takes place
only during the night (Valverde et al. 2004). Proteosome-mediated degradation of
CO protein occurs in the dark as well as under red light. During LD conditions, CO
protein accumulates at the end of the light cycle and activates FT transcription but
under SD it is rapidly degraded and therefore unable to activate FT transcription
(Fig. 5.2). In rice, a CO ortholog Hd1, activates flowering only slightly under the
SD condition but inhibits the accumulation of FT under LD. Patterns of CO and
Hd1 transcript accumulation are the same (Izawa 2007). Transgenic potato expres-
sing Arabidopsis CO exhibited reduced growth and delayed tuberization, indicating
an inhibitory effect of AtCO on tuberization (Martı́nez-Garcı́a et al. 2002).
CO-FT regulatory module also controls timing of flowering and seasonal growth
cessation in aspen trees (Böhlenius et al. 2006). Overexpression of FT ortholog,
PtFT1 in aspen, reduced the flowering delay in transgenic tree from several years to
6 months. Unexpectedly, it also regulated the SD-induced growth cessation and bud
setting in the fall. This may explain growth cessation displayed by aspen trees
a c
CO CO CO CO
CO CO CO CO
CO COCO CO CO COCO CO
CO
b d
CO CO CO
CO CO
CO CO CO CO
CO CO COCO
CO
CO CO
CO
DegradedCO FLOWERING
CO
ActiveCO
Fig. 5.2 Diurnal regulation of CO transcripts (—) CO protein (▪ ▪ ▪) and FT protein (- - -)

accumulation of in plants under long day and short day growth conditions. (a) Accumulation of CO
transcripts is regulated by circadian clock but not by the light status, (b) whereas accumulation the
CO protein is light dependent (adapted from Baürle and Dean 2006). (c) Under the short day
conditions CO protein is rapidly degraded and inactivated by the proteasome during the dark
period, (d) but when plants are grown under long day conditions it accumulates at the end of the
afternoon before the beginning of dark period. Active CO proteins induce the flowering (adapted
form Valverde et al 2004)
sampled across a latitudinal gradient spanning northern Europe. LFY or APETALA1

(AP1) are other genes that promote flower initiation in Arabidopsis. When these
genes were overexpressed under CaMV 35S promoter in hybrid citrus (Citrus
sinensis L. Osbeck x Poncirus trifoliata L. Raf.), juvenile phase was reduced
from 7 years to about 15–20 months (Peña et al. 2001). Expression of AP1 was as
efficient as LFY in the initiation of flowers, and no severe developmental abnor-
mality was observed. Modifying transition phase from juvenile to mature plants
should greatly improve the possibilities for breeding of trees for agronomical and
industrial use.
5.4.1 Flower Senescence
Senescence results in significant losses of cut flowers and offers challenge for post-
harvest researchers trying to delay this process. The process of flower senescence
has been shown to be a genetically programmed event (Stead et al. 2006; Arora
2008). Flower petals are ideal tissues for cell death studies as they are relatively
homogeneous, short-lived tissue that can be manipulated by exogenous chemical
treatment without substantial wounding. Plant hormones, membrane stability, water
availability, cellular proteolysis and carbohydrate metabolism act in concert to
determine the differential rate of senescence for different floral organs. However,
the death of floral tissues is affected by several factors including the developmental
stage, pro-senescence signals (e.g., pollination-induced petal senescence), and
stress-related metabolism in response to temperature, wounding, and nutrient
starvation (Stead et al. 2006; Arora 2008).
Floral senescence is a developmental continuum in the flower that is preceded by
tissue differentiation, growth and maturation of the petal, growth and development
of seeds. Genome-wide searches have resulted in isolation and identification of a
number of genes associated with flower senescence from several species, including
Alstroemeria (Breeze et al. 2004), carnations (Verlinden et al. 2002), Chrysanthe-
mum (Narumi et al. 2005), daffodil (Hunter et al. 2002), daylily (Panavas et al.
1999), rose (Channeliere et al. 2002), iris (van Doorn et al. 2003), Sandersonia
aurantiaca (Eason et al. 2002), Petunia (Jones et al. 2005) and Gladiolus (Arora
and Singh 2004; Arora et al. 2006). Characterization of their function has provided
insights into the roles played by ethylene signaling, proteolysis, nucleic acid and
chlorophyll breakdown, and lipid and nitrogen remobilization in the progression of
flower senescence (Gan and Amasino 1997; Stead et al. 2006). The future genetic
analysis of floral senescence will likely identify molecular mechanisms that help
maintain a non-senescent “juvenile” state and provide novel interventions to extend
post-harvest life of not only cut flowers but also fruit and vegetable crops (Mattoo
and Handa 2008).
Ethylene is one of the plant hormones with profound effects on plant growth
and development including senescence and ripening processes (Fluhr and Mattoo
1996; Mattoo and Handa 2004). Key genes regulating biosynthesis of ethylene
include S-adenosylmethionine (SAM) synthase, 1-aminocyclopropane-1-carboxy-

late (ACC) synthase and ACC oxidase (Fluhr and Mattoo 1996). Different plant
organs show responses to ethylene to different degrees. Even cultivars within
flower species exhibit a significant variability in sensitivity to ethylene. Most
modern carnation cultivars exhibit much reduced sensitivity to ethylene indicating
possibilities of genetically engineering flower vase life by reducing ethylene bio-
synthesis or perception. Impairing ethylene biosynthesis by expressing an antisense
ACC oxidase gene resulted in dramatic reduction of ethylene production with a
concomitant increase in the longevity of carnation and Torenia fournieri flowers
(Savin et al. 1995; Aida et al. 1998). However, similar attempts in Begonia failed
and transgenic flowers did not show prolonged flower life (Einset and Kopperud
1995). Increasing longevity of carnation cut flowers was also achieved by reducing
expression of ACC synthase by co-suppression (Michael et al. 1993).
Another avenue to control floral and leaf senescence is to manipulate perception
of ethylene. Characterization of an ethylene receptor gene from Arabidopsis,
AtETR1 (Chang et al. 1993), and tomato (Zhou et al. 1996a,b) led to the identifica-
tion of a family of ethylene receptor genes in plants. Tomato genome contains six
members, LeERT1–LeETR6, with each of the receptor genes having a distinct
expression pattern during plant development and in response to external stimuli
(Klee and Tieman 2002). Ethylene-insensitive 3 (EIN3) encodes a transcription
factor that functions downstream from the ethylene receptors in the ethylene signal
transduction pathway (Wang et al. 2002). EIN3 possesses two mitogen-activated
protein kinase (MAPK) phosphorylation sites that have opposing effects on EIN3
stability. Homologs of the Arabidopsis EIN3 gene are present in tomato: LeEIL,
LeEIL1, LeEIL2 and LeEIL3. EIN3-BINDING F-BOX1 and 2 (EBF1/2) coordi-
nately control 26S proteasome degradation of the transcription factors, EIN3 and
EIL1. ETHYLENE-INSENSITIVE5 (EIN5), which encodes the exoribonuclease
XRN4, represses expression of EBF1/2. However, functional significance of each
member of the ethylene receptor family is not fully understood (Kevany et al. 2007,
2008).
A mutated ERS gene (ethylene response sensor, an Arabidopsis gene uncovered
by cross-hybridization with the Arabidopsis ETR1 gene) conferred dominant
ethylene insensitivity to wild-type Arabidopsis (Hua et al. 1995). Overexpression
of a mutated dominant Arabidopsis ethylene resistance gene etr1-1 under the
CaMV 35S promoter conferred ethylene insensitivity to Petunia (Wilkinson et al.
1997; Clark et al. 1999; Gubrium et al. 2000; Celvenger et al. 2004). Among the
effects produced by the introduction of ethylene-related transgenes in plants were:
enhanced ethylene production in pollinated flowers; slightly faster flowering; poor
root development of cuttings; lower seed weight; less-efficient seed germination
and rooting; delayed seedling growth; and reduced flower senescence, abscission
and fruit ripening. The delayed flower senescence was seen both in the presence and
absence of ethylene in pollinated and non-pollinated flowers. Most of these effects
can be explained by the roles played by ethylene in different developmental
processes. The enhanced ethylene production in flowers is probably due to the
uncoupling of the feedback regulation from the plant’s response to pollination.
However, the poor rooting ability of cuttings limits the use of this methodology.
Overexpression of a mutated ers homolog from Brassica oleracea (Boers) also
imparted ethylene insensitive to Petunia and induced prolonged flower longevity
(Shaw et al. 2002). An unintended effect of this transformation, however, was
increased susceptibility of the transgenic plant to fungal diseases, most likely
because of an interference with the ethylene-induced defense activated by ethylene.
The use of a flower-specific promoter from Petunia (fbp1) to express etr1-1 in
carnation resulted in stronger insensitivity to ethylene without the undesired side
effects as observed in earlier experiments (Bovy et al. 1999). Transformation of an
ornamental Nemesia strumosa with a mutated etr1 melon homolog under a consti-
tutive promoter also exhibited enhanced flower longevity by altering ethylene
perception (Cui et al. 2004).
Overexpression of isopentenyl phosphotransferase (ipt gene) from Agrobacter-
ium tumefaciens under a senescence-regulated promoter Psag12 increased cyto-
kinin levels with concomitant delay in ethylene production and enhanced ethylene
tolerance of the flowers of the transgenic plants (Chang et al. 2003). The flower
longevity was prolonged by 100% in non-pollinated flowers and approximately
450% in pollinated flowers. Interactions between ethylene, cytokinins, sugars and
various hydrolytic enzymes are known to differentially mediate the progression of
flower senescence. However, individual signals appear to be species-specific and
vary among the plant variety and floral organs. The challenge for post-harvest
scientists is to identify a hierarchy of regulators or specific patterns underlying
the progression of flower senescence. The use of tissue-specific promoters
(Chap. 1-5) is recommended since these may reduce undesired effects of the
introduced gene, particularly in modifying plant hormone levels (Clark et al.
1999; Shaw et al. 2002). Conventional breeding has made significant advances in
increasing the number of flowering buds, extending the longevity of inflorescence
and improving post-harvest performance, as demonstrated in Lilium (Van der
Meulen-Muisers et al. 1999). Coupling transgenic approach with molecular breeding
might pave a way to greatly improve flowers, fruits and vegetables with desirable
traits including shelf life.
5.5 Fruit
5.5.1 Fruit Shape, Size and Mass
Domestication of almost all fruit species has invariably led to the selection for
increased fruit size. In addition to size, fruit crops have been bred for shape, texture,
flavor, shelf life and nutrient content. Genetic evaluation of various species, espe-
cially tomato, has provided a wealth of information about loci that control fruit size,
weight and shape (Frary et al. 2000; Tanksley 2004; Cong et al. 2008). However,
their quantitative nature has impeded characterization of individual genes regulating
these fruit attributes (Grandillo et al. 1999). Table 5.4 lists some of the genetic
Table 5.4 Transgenic genetics of fruit developmental attributes

Enzyme/protein Transgene Phenotype Reference
Fruit shape, size and mass
fw 2.2 Negative Overexpression Reduced fruit size, Fray et al. (2000)
regulator of cell fruit weight
division; possibly
involved in cell-to-
cell communication
SUN, IQ67 domain– Overexpression More elongated fruit Xiao et al. (2008)
containing family shape
Ethylene regulated ripening
ACC oxidase Underexpression Prolonged fruit shelf life Xiong et al. (2005)
ACC synthase Antisense Impaired ethylene Oeller et al. (1991)
production and fruit
ripening. Extended
shelf life
Ethylene receptor LeETR1 Delayed abscission, Whitelaw et al. (2002)
antisense shorter internode
length & reduced
auxin transport. No
effect on
pigmentation and
fruit softening
SAM decarboxylase Overexpression Spermidine and spermine Mehta et al. (2002)
accumulation. High
lycopene, improved
juice quality and
longer vine life
Ripening mutations
Ripening-inhibitor (rin) Overexpression Complements mutation Vrebalov et al. (2002)
MADS-box Regulates fruit
transcription factor ripening
Colorless non-ripening VIGS SPB-box transcription Manning et al. (2006)
(Cnr) Factor
Inhibits fruit ripening
high-pigment- Overexpression Elevated carotenoid and Davuluri et al. (2005)
2 DETIOLATED1 flavonoid
accumulation
Fruit Texture
Polygalacturonase (PG) Overexpression Increased pectin Giovannoni et al. (1989)
solubilization
Antisense Slightly firmer fruit. Langley etal. (1994),
Increased juice Schuch et al. (1991),
viscosity. Reduced Kramer et al. (1992),
pectin solubilization Brummell and
Labavitch (1997)
PG-b-subunit Antisense Increased fruit softening, Watson et al. (1994),
decreased middle Chun and Huber
lamella cohesion. (1998)
Reduced tissue
integrity
(continued)

Enzyme/protein Transgene Phenotype Reference
Pectin methylesterase Antisense No effect on softening but Tieman et al. (1992),
reduced tissue Hall et al. (1993),
integrity in overripe Tieman and Handa
fruit. Increased (1994), Thakur et al.
degree of (1996a, b)
methylesterification.
Reduced pectin
degradation. Increased
soluble solids.
Increased juice and
paste viscosity.
Increased serum
viscosity
Pectate lyase Antisense Increased fruit firmness Jiménez-Bermúdez et al.
in strawberry. (2002)
b-galactosidase1 Antisense No effect on cell wall Carey et al. (2001)
galactose.
No effect on fruit
softening.
b-galactosidase3 Antisense Reduction in TBG1 and de Silva and Verhoeyen
TBG4 transcripts. (1998)
Higher GalA in cell wall.
Reduced fruit
deterioration.
b-galactosidase4 Antisense Reduced tomato fruit Smith et al. (2002)
softening.
Higher GalA in cell wall.
Expansin Antisense Increased firmness; Brummell et al. (1999b)
reduced polymer.
Expansin and PG Both antisense Increased fruit firmness Kalamaki et al. (2003)
Modified juice
rheology.
PG and PME Both antisense Modified juice rheology. Errington et al. (1998)
Cellulase Antisense No reduction in Harpster et al. (2002a)
xyloglucan.
depolymerization,
pepper.
endo-1,4-b –glucanase Overexpression No increase in Harpster et al. (2002b)
xyloglucan.
depolymerization,
pepper
loci and genes that play a role in determining fruit shape, size and weight. Although
so far these genes have not been used to develop cultivars with altered fruit physical
attributes, they provide a resource to genetically engineer fruit crops for different
phenotypic attributes.
Fruit size and shape seem closely related as more extreme shapes were more
confined to larger fruit phenotype than smaller fruits (van der Knaap and Tanksley
2003). Mutation in three genes (ovate, sun and fs8.1) affected fruit shape through
modulation of early stages of carpel development (Gonzalo and van der Knaap
2008). About 30 quantitative trait loci (QTLs) have been identified regulating
tomato fruit size and shape but less than 10 loci could account for majority of the
changes associated with tomato domestication (Grandillo et al. 1999; Tanksley
2004). The effect of these loci appears to be largely confined to fruit mass except
fw3.1 (van der Knaap et al. 2002; van der Knaap and Tanksley 2003). The fw2.2 has
the strongest effect on fruit size and is associated with a hyper mitotic index during
cell division stage just after anthesis. It encodes a negative repressor of cell division
particularly during fruit development and has a sequence similarity to the human
oncogene c-H-ras p21A (Frary et al. 2000; Cong et al. 2002).
Introduction of fw2.2 into a large-fruited cultivar causes expected reduction in
fruit size. fasciated locus on chromosome 11 and the locule number locus on
chromosome 2 are reported to increase fruit size by increasing the number of
carpels in the flower, which after fertilization develops into locules resulting in
larger and wide fruits (Lippman and Tanksley 2001; Barrero et al. 2006). All the
large-fruited, multilocular tomatoes carry mutations in either one or both of these
loci while plants carrying mutated alleles of fasciated can produce more than 15
locules that affect the shape of the fruit. Although the exact nature of the genes at
these loci is yet to be characterized, putative tomato homologs for fascinated locus
have been identified in Arabidopsis.
Thus far, a complete separation between the loci that control fruit size and those
that control fruit shape has not been achieved. The organ-determining genes
fasciated and locule number affect both the final size and shape of the fruit.
However, the fruit size loci, fw1.1, fw2.2, fw3.1 and fw4.1 exert their effects largely
on fruit growth resulting in changes in size with no or little change in shape. Three
major loci, ovate (chromosome 2), sun (chromosome 8), and fs8.1 (chromosome 8),
that modulate fruit shape have minimal effect on size. Ovate locus could account
for both pear and elongated shape of tomato and the sequence comparisons of
OVATE alleles indicated that all tested pear-shaped varieties of tomato share the
same nonsense mutation that causes truncation of the predicted protein and could
account for the loss-of-function (recessive) nature of these alleles (Ku et al. 1999;
Liu et al. 2002). A single major locus, fs8.1, is responsible for both blocky and
slightly elongated appearance of processing tomatoes (Grandillo et al. 1996).
Changes in fruit shape caused by fs8.1 are initiated very early in floral/carpel
development (Ku et al. 2000).
5.5.2 Ripening Mutant Genes
A large number of genes that regulate fruit development and ripening have been
cloned and functionally characterized (Alba et al. 2005; Giovannoni 2007;
Seymour et al. 2008). Genes responsible for attenuated ripening in ripening-
impaired tomato mutants have provided an attractive model to modify fruit ripening.
Table 5.4 lists some of the genes that have been used to modify fruit ripening
process. Several single gene tomato-ripening mutants that exhibit attenuated

ripening have been known for quite some time. Identification and molecular
characterization of these mutant genes have opened new avenues for modifying
ripening process in fruit crops by transgenic approaches. RIPENING-INHIBITOR
(Vrebalov et al. 2002) and COLOURLESS NON-RIPENING (CNR) mutants
encode a MADS-box and SPB-box (Manning et al. 2006) genes, respectively.
These genes are required for ripening and their study has begun to provide an
insight into mechanisms that control ripening upstream of the plant ripening
hormone, ethylene. Delving into the Never-ripe mutant led to the cloning of the
first ethylene receptor gene from tomato (Wilkinson et al. 1995). Studies on
Green-ripe identified a gene that encodes a fruit-specific, novel component that
likely affects ethylene receptor-copper homeostasis during ethylene signaling
(Barry and Giovannoni 2006).
Gene mutations responsible for the high-pigment 1 (Damaged DNA Binding
Protein 1 (DDB1)) and high-pigment 2 (Detiolated1 (DET1)) phenotypes should
lead to dissection of regulatory pathways that impact metabolic content and asso-
ciated fruit quality (Mustilli et al. 1999; Liu et al. 2004).
5.5.3 Ethylene-Regulated Fruit Ripening
Fruit ripening is a genetically regulated transitional period during which many

dynamic processes occur which are manifested into perceivable and altered changes
including firmness, pigmentation, and weight loss. Although these changes are
desirable for human consumption, overripeness leads to wastage and discarding
large portions of harvested fruit. Once initiated, ripening is a relatively irreversible
process. Treatment with inhibitors of ethylene biosynthesis (e.g., aminoethoxy-
vinylglycine) or ethylene perception [e.g., silver salts or 1-methylcyclopropene
(1-MCP)] results in delaying fruit ripening (Mattoo and Suttle 1991; Lurie and
Paliyath 2008). However, the recognition of involvement of ethylene-dependent as
well as ethylene-independent processes in ripening and fruit quality is impacted by
preventing the ethylene responses in most fruits. Nevertheless, since ethylene plays
a critical role in fruit ripening (Oeller et al. 1991), especially in climacteric fruits,
approaches altering ethylene biosynthesis or its perception have been largely used
to engineer ethylene insensitivity in several plant species.
Fruit ripening control has been achieved by downregulating the expression of
ACC synthase (Oeller et al. 1991) or ACC oxidase (Hamilton et al. 1990). Over-
expression of a bacterial ACC deaminase (Klee et al. 1991) or SAM hydrolase
(Good et al. 1994) also resulted in attenuated fruit ripening in tomato by reducing
the levels of ethylene biosynthesis substrates ACC and SAM, respectively.
Engineered suppression of a member of ethylene receptor family, LeETR4, in a
fruit-specific manner was found to result in early fruit ripening without large effects
on fruit size, flavor or yield (Kevany et al. 2008). These authors suggested that
ethylene receptors might work as biological clocks to regulate the onset of fruit
ripening. A progeny from a cross between two transgenic tomato lines impaired in
the expression of LeEtr1 or LeERT2 using antisense RNA technology had a
phenotype similar to that observed for LeERT1 antisense plants indicating its role
in ethylene signaling during tomato growth and development (Wang et al. 2006a, b).
The functional significance of members of ethylene-receptor family and down-
stream signaling is complex (Kendrick and Chang 2008). Degradation of ethylene
receptor was found correlated with enhanced sensitivity to ethylene (Kevany et al.
2007).
5.5.4 Transgenic Interventions to Alter Fruit Texture
The economic value of post-harvest fruit softening has generated considerable

interest in understanding the biochemical changes associated with fruit textural
modifications (Brummell 2006; Vicente et al. 2007; Negi and Handa 2008). Ripen-
ing-associated fruit textural changes usually involve structural modification of the
polysaccharide components of the primary cell wall and middle lamella. Structural
proteins, such as expansins, also play an important role in this process (Brummell
2006; Vicente et al. 2007). The overall interplay between primary cell wall and
middle lamella components is a very complex process. Loosening the structure of
and cross-links between the cell wall and middle lamella have been implicated in
the transition of a firm unripe fruit to a softer/crispy and juicy fruit (Brummell
2006). Genetic engineering approaches have led to the identification of the com-
plexity of the cell wall chemistry and the role various cell wall modifying enzymes
play in fruit softening and texture. Table 5.4 lists the phenotypes of transgenic
plants obtained by decreasing the expression of genes encoding some of these
enzymes.
Antisense inhibition of polygalacturonase (PG) had only a slight effect on fruit
softening but significantly impacted depolymerization of pectins, resulting in
increased juice viscosity due to higher molecular size of the constituent pectins
(Thakur et al. 1997). Similarly, transgenic plants expressing an antisense gene of
pectin methylesterase (PME) showed >95% reduction in fruit PME activity con-
comitant with markedly improved juice viscosity and increased total soluble solids
(Tieman et al. 1992; Gaffe et al. 1994; Thakur et al. 1996a,b). Although softening of
transgenic fruits with reduced PME activity occurred to similar levels as the non-
transgenic fruits, the transgenic fruits exhibited loss of tissue integrity after
extended storage (Tieman and Handa 1994). Homology-dependent silencing of
TBG1 resulted in about 90% reduction in its transcript accumulation but it did not
affect the total exogalactanase activity, cell wall galactose content or fruit soften-
ing, indicating little, if any, role of this gene in fruit texture (Carey et al. 2001).
Suppression of TBG3 by antisense RNA caused reduction in TBG1 and TBG4
transcript levels and ~75% loss in extractable exo-galactanase activity with reten-
tion of the cell wall galactose content but with little effect on fruit softening
(de Silva and Verhoeyen 1998). However, reduction in TGB4 transcripts by antisense
RNA caused about 90% reduction in extractable exogalactanase activity, reduced

galactose levels in mature green fruits, and produced firmer fruits than the control
(Smith et al. 2002).
Specific members of the ß-galactosidase, expansin and pectate lyase (PL) gene
families partially regulate softening in tomatoes and strawberry (Brummell et al.
1999b; Jiménez-Bermúdez et al. 2002; Smith et al. 2002). However, functional
analyses of specific PGs, PMEs, xyloglucan endotransglucosylase/hydrolases
(XTHs) and 1,4-endoglucanases (EGases) in tomatoes, strawberries and peppers
have failed to establish their roles in softening (Lashbrook et al. 1998; Brummell
et al. 1999a; Woolley et al. 2001; Harpster et al. 2002a,b). Fruit firmness was not
significantly altered when PG or expansin was suppressed using transgenic toma-
toes, but when genes encoding both proteins were simultaneously downregulated
fruit firmness was retained for longer duration (Powell et al. 2003).
5.6 Cuticle Modification
Cuticle is the outermost layer of plants (Fig. 5.3), which plays important roles in
preventing plant dehydration by limiting non-stomatal water loss, improving resis-
tance of plants to biotic and abiotic stresses (See Chaps. 2-1 and 2-2), controlling
plant morphology, and regulating organ fusion (Pollard et al. 2008; Samuels et al.
2008). As a barrier to pathogen attack, cuticle negates germination of pathogen
spores (Gniwotta et al. 2005). Further, cuticle plays a role in osmotic adjustment as
well as in protecting plants against the negative effects of light, temperature and
pollution (Shepherd and Griffiths 2006). By regulating gas exchange, this layer
likely plays a significant role in extending post-harvest shelf life of fruits and
vegetables (Saladie et al. 2007).
The cuticle layer is composed of two main lipophilic components: cutin
and cuticular wax. Cutin is lipid-derived polyester composed mainly of a- and
o-hydroxyl and epoxy C16 and C18 fatty acids and glycerol (Nawrath 2006). The
wax component of cuticle is made up of aliphatic C24–C34 compounds made
Fig. 5.3 Section of a leaf showing the cuticle with respect to other tissues. Adapted from Purves
et al. (1997)
entirely of carbon and hydrogen or including functional groups like alcohol and
ketone (Kunst and Samuels 2003). The pathways responsible for the formation of
this component including its transport to the outside of a plant organ as well as the
intercellular and extracellular assembly remain to be elucidated (Pollard et al.
2008). Microarray analysis of transcripts (Chaps. 2-10) from the top epidermis of
Arabidopsis led to the isolation of 85 upregulated genes playing a role in lipid
metabolism (Chung et al. 2005), whereas the same analysis in the wax deposition
zone in barley leaf led to the isolation of five upregulated wax-deposition genes
(Richardson et al. 2007). Although more experimental evidence is needed, these
data suggest that the number of genes involved in cuticle assembly is rather few
compared to the number of genes participating in the biosynthetic pathway of the
cuticle components. Double knockouts of glycerol-3-phosphate acyltransferase
(GPAT), gpat4/gpat8, caused significant reduction in cutin. The mutant plants
were less resistant to desiccation and infection by the fungus Alternaria brassici-
cola. Also, overexpression of GPAT4 or GPAT8 led to about 80% increase in the
levels of C16 and C18 cutin monomers in leaves and stems (Li et al. 2007b). In
order to modify cutin composition, these authors overexpressed the acyltransferase
GPAT5 and the cytochrome P450-dependent fatty acyl oxidase CYP86A1, two
enzymes associated with suberin biosynthesis. The simultaneous overexpression of
both enzymes caused accumulation of new C20 and C22 o-hydroxyacids and a,o-
diacids typical of suberin with altered fine structure and water-barrier function of
the cuticle (Li et al. 2007b).
Substantial experimental evidence has accumulated on the roles that cuticle
plays in various developmental and physiological processes. Modification of cuticle
has been shown to result in a number of attributes such as:
(a) Altered leaf and petal epidermal cell structure, trichome number and branching
as well as density of stomata (Aharoni et al. 2004)
(b) Reduced leaf size and plant growth, seed production and seed germination
(Schnurr et al. 2004)
(c) Abnormal bending of embryos, ectopic adhesion between cotyledons, a perme-
able epidermal structure and an altered distribution pattern of stomata in several
tissues (Tsuwamoto et al. 2008)
(d) Dwarf appearance due to amorphous and smaller cells and enhanced tendency
to dehydrate (Cominelli et al. 2008)
(e) Altered morphology of trichomes and pavement cells (Panikashvili et al. 2007)
(f) Post-genital organ fusions and stunted growth (Sieber et al. 2000; Bird et al.
2007; Luo et al. 2007; Ukitsu et al. 2007)
(g) Hypersensitivity to drought (Chen et al. 2004; Ukitsu et al. 2007)
(h) Disruption of microspore normal development along with reduced pollen
development (Jung et al. 2006)
(i) Irregular shape of pollen (Ukitsu et al. 2007).
In tomato fruits, the manipulation of cuticle by expressing the gene cwp1
(cuticular water permeability 1), which imparts the microfissure/dehydration
phenotype in the wild species Solanum habrochaites, caused a microfissured fruit
Table 5.5 Genes with potential to modify cuticles

Gene Function Physiological effect Biotechnology Reference
intervention
CaCAF1 Transcription and Growth enhancement, Overexpression Sarowar
DQ672569 mRNA decay thicker cell walls and pCaMV35S et al.
cuticle layers. Increased (2007)
resistance against
Phytophtora infestans
WXP1 Ethylene response Enhanced drought and Overexpression Zhang
TC107019 transcription freezing tolerance pCaMV35S et al.
factor (2007b)
WXP2 Ethylene response Enhanced drought Overexpression
TC94548 transcription tolerance. Increased pCaMV35S
factor low-temperature
sensitivity. Reduced
growth.
GPAT5 Glycerol acyl Increased drought tolerance Overexpression Li et al.
At3g11430 transferase pCaMV35S (2007b)
CUTE Cutinase protein Total resistance to Botrytis Overexpression Chassot
M29759 cinerea pCaMV35S et al.
(2007)
SHN1 AP2/EREBP Increased cuticular wax, Overexpresion Aharoni
At1g15360 transcription drought tolerance and pCaMV35S et al.
SHN2 factors recovery. (2004)
At5g11190 regulating lipid
SHN3 biosynthesis
At5g25390
cuticle leading to a dehydrated fruit (Hovav et al. 2007). Table 5.5 lists some of the
genes that have been manipulated to modify cuticle and their effects on plant
growth and development.
Transgenic tomato plants overexpressing CaCAF1, a CCR4-associated factor 1
protein belonging to the CCR4-NOT complex that plays an important role in the
control of transcription and mRNA decay in yeast and mammals, exhibited signifi-
cant growth enhancement, with thicker leaves with twofold enlarged cell size,
thicker cell walls and cuticle layers, and enhanced resistance against Phytophthora
infestans compared to the control plants (Sarowar et al. 2007). In the same study,
virus-induced silencing (Chap. 1-2) of CaCAF1 in pepper resulted in significant
growth retardation and enhanced susceptibility to bacterial pathogen Xanthomonas
axonopodis pv. vesicatoria.
Introduction of putative ethylene-responsive transcription factor (ERF) genes,
WXP1 and WXP2, from Medicago truncatula into Arabidopsis led to increased
leaf wax accumulation and improved drought tolerance, but differential response
to freezing tolerance (Zhang et al. 2007b). Both WXP1 and WXP2 transgenic
plants showed increase in n-alkanes, the major wax component in Arabidopsis, but
only the WXP1 transgenic plants exhibited increase in the amount of primary
alcohols. The WXP1 transgenic plants showed no change, but the WXP2 plants
exhibited increased chlorophyll bleaching. Both WXP1 and WXP2 transgenic
plants retained more water than the control and exhibited significantly enhanced
plant drought tolerance. On the basis of electrolyte leakage from detached leaves,
the WXP1 plants showed increased freezing tolerance while the WXP2 plants were
more sensitive to low-temperature stress than the control plants. WXP1 overex-
pressing plants showed no obvious effects on plant growth and development, but
the expression of WXP2 resulted in slower plant growth. It is becoming increasing
clear that the manipulation of cuticle will help designing plants with higher
resistance to pathogens and abiotic stresses. Stress due to fungi and water loss
are significant factors causing post-harvest losses of fruit and vegetable crops
(Troncoso-Rojas and Tiznado-Hernández 2007). The cuticle engineering has a
high potential to reduce these losses.
5.7 Abscission
Abscission is a process by which plant organs (leaves, flowers and fruits) detach
(are shed) from the parent plant. This process is under the cue of developmental
signals as well as environmental stimuli. Thinning or reduction of crop load, a
common practice in the production of fruit crops, involves manual or chemical-
induced detachment of excess fruits to optimize fruit size. Harvesting of fruit crops
is one of the most demanding aspects of fruit production both in terms of labor as
well as economic inputs. There is currently renewed interest in mechanical harvest-
ing to circumvent these issues. Efficiency of mechanical harvesting systems can be
greatly improved by facilitating abscission-related processes. However, untimely
detachment of immature fruit can lead to crop loss and low profitability, and
therefore needs to be avoided. In seed crops, pod shattering and seed loss can be
prevented through better understanding of dehiscence, a process that is somewhat
similar to abscission. Hence, knowledge of abscission and its manipulation can
greatly benefit crop production.
Abscission occurs at specialized regions, termed abscission zones (AZ),
which are located proximal to the organ that will subsequently detach
(Fig. 5.4). Cells within the AZs are often small and rounded, and usually undergo
extensive cell division prior to organ separation (Sexton and Roberts 1982;
Goren 1993). Cell separation in the AZ occurs within a group of cells called
the separation layer. Activity of cell wall hydrolytic enzymes, in response to the
abscission stimulus, leads to dissolution of the primary cell wall and the middle
lamella within cells of the separation layer (Taylor and Whitelaw 2001; Roberts
et al. 2002). Subsequent loss of adhesion or collapse of cells in the separation
layer leads to organ detachment due to the weight of the subtending organ. Either
during the process of separation or immediately following it, cells within
the layer proximal to the separation layer expand to form a protective layer
(Patterson 2001).
Abscission JOINTLESS; SHATTERPROOF

Zone BOP1; BOP2 SHATTERPROOF2 FRUITFULL
Differentiation (AZ) (DZ)
AZ (Separation Layer)
ETHYLENE
(ACC SYNTHASE; ETR1; EIN2)
Abscission AUXIN
Signaling (ARF2)
ETHYLENE INDEPENDENT
(DAB; IDA)
Cell Wall
hydrolases
Cell
Separation IDA
Fig. 5.4 Progression of abscission is represented here in three phases involving: abscission zone
(AZ) differentiation; abscission signaling; and cell separation. AZ differentiation is regulated by
genes such as the JOINTLESS gene in tomato and the BLADE ON PETIOLE (BOP1; BOP2) genes
in Arabidopsis. The dehiscence zone (DZ) differentiation is regulated by expression of the
SHATTERPROOF genes which are in turn inhibited by the FRUITFULL gene in Arabidopsis
siliques. Abscission signaling is antagonistically regulated by ethylene and auxin. Additionally,
ethylene-independent mechanisms involved in Arabidopsis flower abscission have been reported
in the delayed abscission (dab) and inflorescence deficient in abscission (ida) mutants. Cell
separation is facilitated by the degradation of the middle lamella and the cell wall, activities that
are largely facilitated by cell wall hydrolases. Additionally, the IDA gene may also be involved in
regulating final stages of cell separation
5.7.1 Development of the Abscission Zone
Development and differentiation of AZs are under tight genetic control and usually
occur at specific sites within the plant (Fig. 5.4). Several genes associated with the
development of AZ and those that have an impact on the abscission process are
Table 5.6 Genes with potential to modify abscission

Gene Function Role in Biotechnology Reference
abscission intervention
JOINTLESS MADS-box AZ Mutation; over Mao et al.
Development expression; (2000)
silencing
SHATTERPROOF, MADS-box Dehiscence zone Mutation Liljegren et al.
SHATTERPROOF2 development (2000)
FRUITFULL MADS-box Dehiscence zone Mutation; Ferrandiz et al.
development overexpression (2000),
Østergaard
et al.
(2006)
AGL15 MADS-box Progression of Mutation Fernandez
abscission et al.
(2000)
CEL1, CEL2 Cell wall Promote cell Silencing Lashbrook
modification separation et al.
(1998),
Brummel
et al.
(1999a)
Polygalacturonase Cell wall Promote cell Silencing Gonzalez-
modification separation Carranza
et al.
(2007),
Jiang et al.
(2008)
ACC synthase Ethylene Promotes floral Overexpression; Lanahan et al.
biosynthesis organ silencing (1994),
abscission Ecker and
Theologis
(1994)
ETR1; EIN2 Ethylene Ethylene- Mutation; Bleecker and
perception mediated silencing Patterson
and signaling abscission (1997),
signaling Whitelaw
et al.
(2002)
DAB; IDA Unknown Delayed floral Mutation Patterson and
organ Bleecker
abscission (2004),
Butenko
et al.
(2006)
ARF2 Auxin response Delayed floral Mutation Ellis et al.
factor organ (2005)
(transcription abscission
factor)
listed in Table 5.6. One of the key genes regulating AZ development was isolated
using a spontaneous mutation in tomato, jointless. The jointless mutants do not
possess the AZ that typically forms midway along the flower/fruit pedicel. The
JOINTLESS gene was isolated using map-based cloning and found to encode a
MADS-box transcription factor that is essential for the development of the pedicel
AZ (Mao et al. 2000). Overexpression of the JOINTLESS gene in the mutant
background restored AZ development albeit not in the same location as in the
wild type, while antisense suppression of this gene resulted in loss of the flower
pedicel AZ in tomato (Mao et al. 2000). Several other MADS-box genes have been
implicated in dehiscence. Two such genes are SHATTERPROOF (SHP1) and
SHATTERPROOF2 (SHP2) both of which redundantly control cell separation by
affecting dehiscence zone formation and its lignification in Arabidopsis siliques
(Liljegren et al. 2000; Lewis et al. 2006). In contrast, FRUITFULL, another MADS-
box gene, negatively regulates dehiscence zone development by inhibiting expres-
sion of the SHATTERPROOF genes (Ferrándiz et al. 2000). Another MADS-box
gene, AGL15, is also implicated in regulating abscission through delaying progres-
sion of the process (Fernandez et al. 2000). Recently, two redundant BLADE ON
PETIOLE genes, BOP1 and BOP2, have been identified as essential regulators of
abscission zone development in Arabidopsis. These genes belong to the non-
expressor of PR1 protein (NPR1) family and appear to be involved in the establish-
ment of the floral AZ as well as the vestigial cauline leaf AZ in Arabidopsis
(McKim et al. 2008). Such genes affecting AZ zone development have immense
potential for genetic manipulation of organ abscission in crops. In fact, the jointless
mutant background has been widely used for developing “stemless” processing
tomatoes that are not physically damaged during storage (Mao et al. 2000). Also,
ectopic expression of FRUITFULL has been used to prevent unwanted pod dehis-
cence in Brassica (Østergaard et al. 2006).
5.7.2 Hormonal Control of Abscission
Ethylene has long been known to be a primary natural regulator of abscission

(Jackson and Osborne 1970). Increase in ethylene evolution is often associated
with the abscission process (Goren 1993; Taylor and Whitelaw 2001). Experiments
involving genetic manipulation of ethylene biosynthesis, perception and signaling
support a role for ethylene in promoting abscission of various plant organs. Consti-
tutive expression of ACC synthase resulted in enhanced ethylene production and
earlier flower abscission in tomato (Lanahan et al. 1994), whereas antisense sup-
pression of ACC synthase delayed abscission in Arabidopsis (Ecker and Theologis
1994). The ethylene receptor mutant, etr1, and the ethylene insensitive mutant,
ein2, exhibit delayed abscission of floral organs in Arabidopsis and tomato
(Bleecker and Patterson 1997; Whitelaw et al. 2002; Patterson and Bleecker
2004). One of the main mechanisms through which ethylene aids cell separation
is by enhancing expression and activity of cell wall hydrolyzing enzymes in the AZ.
However, recent work with the delayed abscission (dab) and inflorescence deficient
in abscission (ida) mutants, which exhibit altered floral abscission responses in an
ethylene-independent manner, have led to the suggestion that ethylene may not be
absolutely required for abscission in Arabidopsis but may act as an accelerator of
the abscission process (Patterson 2001; Patterson and Bleecker 2004; Butenko et al.
2006). Nonetheless, alteration of ethylene biosynthesis and/or perception continues
to be a promising approach for genetically altering abscission in plants.
Auxin plays a dual role in regulating abscission. While auxin generally inhibits
progression of abscission during early stages of the process, application of auxin at
later stages accelerates abscission by increasing ethylene biosynthesis (Brown
1997). In fact, the ratio of ethylene and auxin levels at the AZ is considered a key
factor determining progression of abscission (Sexton and Roberts 1982; Brown
1997). A flux of auxin across the AZ is thought to be essential to prevent abscission
(Sexton and Roberts 1982). Auxin inhibits cell separation by decreasing the activity
of cell wall hydrolyzing enzymes (Taylor and Whitelaw 2001). While rapid prog-
ress has been made in understanding auxin biosynthesis, transport and signaling, its
role in regulating abscission has not been adequately addressed at the molecular and
genetic level.
Evidence is accumulating for the involvement of auxin signaling genes in
regulating abscission. Expression of several AUX/IAA genes is altered in an
auxin-dependent manner at the leaf and stem AZs in Mirabilis (Meir et al. 2006).
Auxin response factors (ARFs) are transcription factors that, in conjunction with
AUX/IAA genes, either activate or inhibit downstream auxin responsive genes. Loss
of ARF2 function in Arabidopsis delayed floral organ abscission supporting a role
for auxin signaling in the progression of abscission (Ellis et al. 2005). Further
analysis of the molecular machinery involved in auxin transport and signaling
during abscission should lead to the identification of potential candidates that can
be utilized to alter abscission in plants.
5.7.3 Cell Wall Degradation during Cell Separation

in the Abscission Zone
Enzymes facilitating dissolution of the middle lamella and the cell wall, and
degradation of the cell membrane are obvious candidates for manipulating abscis-
sion. These include cellulases (Cels), PGs, pectin methylesterases (PME), expan-
sins and phospholipases (Cho and Cosgrove 2000; Roberts et al. 2002; Malladi and
Burns 2008). AZ-specific Cels and PGs have been identified and isolated from
many plants (Tucker et al. 1988; Kalaitzis et al. 1995; del Campillo and Bennett
1996; Kazokas and Burns 1998). Antisense inhibition of the cellulase gene, Cel1,
reduced floral abscission in tomato (Lashbrook et al. 1998). Similarly, antisense
inhibition of the Cel2 gene increased the force required for detachment at the
pedicel AZ in tomato by almost 50% (Brummell et al. 1999a). Loss of a PG gene
in Arabidopsis resulted in delayed floral organ abscission in air or in the presence
of the abscission-promoting hormone, ethylene (Gonzalez-Carranza et al. 2007).

Virus-induced gene silencing (Chap. 1–2) of a tomato abscission-related PG gene,
TAPG1, delayed ethylene-regulated petiole abscission and increased petiole break
strength (Jiang et al. 2008). An analysis of the ida mutant in Arabidopsis revealed a
function for this gene in regulating the final steps of cell separation, possibly
through the secretion of arabinogalactan proteins. Constitutive expression of IDA
results in enhanced floral organ abscission as well as ectopic abscission at several
locations in Arabidopsis (Stenvik et al. 2006). These studies demonstrate practical
applicability of altering cell wall/membrane modifying enzymes to manipulate
organ abscission.
5.8 Programmed Cell Death
PCD is a highly coordinated and complex process (Fig. 5.5) that involves disman-
tling of the cellular apparatus. It serves an important function in the development
and defense of the organism. Plants use a well-defined and self-regulated program
of cell death during vascular bundle formation, defense management and senes-
cence of leaf, flower or fruit. Whether senescence in plants is a form of PCD or
precedes PCD is being debated in the literature; one school of thought considers
senescence as a complete overlap with PCD (van Doorn and Woltering 2005), while
others view the latter as culmination of senescence events (Mattoo and Handa 2004;
Reape et al. 2008). The molecular events during senescence and hypersensitive
response (HR)-induced cell death share some common features but vary in the time
frame of their completion. PCD is believed to be more spontaneous and has a shorter
span than the senescence. Both plants and animals employ PCD to complete their
developmental process and manage defense against pathogenic (biotic) or abiotic
stresses. Advances in our understanding of PCD have provided opportunities for
biotechnological improvements in the plants ranging from a markedly improved
tolerance against stress to delayed senescence for higher biomass (Table 5.7)
Cell death is fundamental to the life cycle of an organism. In nature, it forms a
link in the evolutionary process that facilitates every form of life to adapt to the
natural vagaries. In multicellular organisms, the cell death serves the purpose of
removing damaged or redundant cells. In animals, PCD occurs in two morphologi-
cally distinct ways – apoptosis and autophagy. Another mode of cell death is
necrosis whose initial events bear footprints of PCD. Autophagy is a highly con-
served mechanism involving degradation of cytoplasmic contents by the lysosomal
vacuoles. It helps in the mobilization of cell constituents before the cell death. An
autophagic type of PCD is often observed in plants during growth and development
of growing cells (tissues), e.g., in tracheary elements or root growth and expansion.
Animal cell apoptosis is characterized by cell shrinkage, nuclear fragmentation,
formation of apoptotic bodies and their engulfment by the lysosomic action of
another cell (Adrain and Martin 2001). In plants, the engulfment of apoptotic bodies
by another cell is unknown. Characteristics such as retraction of protoplast, DNA
Fig. 5.5 A model of programmed cell death in plants. JA jasmonic acid; MAPK mitogen-activated
protein kinase; NO nitric oxide; PAMPs pathogen-associated molecular patterns; PK protein
kinase; PM plasma membrane; PRI pathogen R-gene protein interaction; RLK receptor-like-
kinase(s); ROS reactive oxygen species; SOD superoxide dismutase
fragmentation and involvement of caspase-like proteins are shared by plant

apoptosis. However, owing to yet-to-be-found other animal apoptotic characteri-
stics, some investigators have preferred to identify the phenomenon in plants as
apoptotic-like PCD (AL-PCD) (van Doorn and Woltering 2005; Reape and
McCabe 2008).
In plants, HR-induced PCD is a key defense mechanism to restrict the spread of
pathogens during a compatible host–pathogen interaction. Also, induction of HR
takes place during abiotic stresses due to cold, heat, drought, salinity, high light
intensity, or ultraviolet radiation to mitigate the stress effect. The core components
5
Table 5.7 Genes related to programmed cell death and their potential to modify plant development or agronomic traits
Gene Product/Function Effect Biotechnology intervention Reference
Fungal or bacterial resistance
CaPO2 Extracellular peroxidase Resistance to P. syringae pv. Overexpression of pepper gene in Arabidopsis Choi et al.
tomato (2007)
OsAOS2 Allene oxide synthase Resistance to Magnaporthe Overexpression of the gene in rice Mei et al.
grisea (rice blast) (2006)
RCT1 TIR-NBS-LRR - R gene protein Resistance to anthracnose Expression of M. truncatula gene in alfalfa Yang et al.
disease (2008)
Pto Serine/threonine protein kinase – Resistance to P. syringae Overexpression of tomato gene Tang et al.
R-gene protein (1999)
Rpi-blb1 CC-NBS-LRR-R gene protein Resistance to Phytophthora Expression of Solanum bulbocastanum in potato van der Vossen
infestans (late blight) (Expression of wild species potato gene in et al. (2003)
cultivated potato)
Rxo1 NBS-LRR-R gene protein Resistance to Xanthomonas Expression of maize gene in rice Zhao et al.
oryzae (2005)
N1141-flaA Flagellin Resistance to M. grisea (rice Expression of bacterial gene in rice Takakura et al.
blast) (2008)
AtNPR1 NPR1 protein Resistance to Fusarium Expression of Arabidopsis gene in wheat Makandar et al.
head blight (2006)
Cf9 & Avr9 R-gene proteins Resistance to Leptosphaeria Expression of tomato genes in Brassica napus Hennin et al.
maculans (2001)
Viral protection
Bcl-xL Or Animal cell death suppressor protein Protection from virus- Expression of animal gene(s) in tomato Xu et al. (2004)
Ced-9 induced necrosis
Biotechnological Interventions to Improve Plant Developmental Traits
Insect resistance
Prosyste- A signal protein for JA synthesis Resistance to herbivores Overexpression in tomato Li et al. (2002)
min gene
Tolerance to abiotic stress
Antioxidant Removal of superoxide radical Enhanced salt, drought Overexpression See Ashraf
genes tolerance (2008)
(continued)
229
230
Gene Product/Function Effect Biotechnology intervention Reference

IPT Isopentenyl transferase – in cytokinin Enhanced drought tolerance Expression of IPT gene Rivero et al.
biosythesis (2007)
MAPKKK Phosphorylation of regulatory proteins Freezing tolerance Expression of tobacco gene in maize Shou et al.
(2004a)
NPK1 Protein kinase Enhanced drought tolerance Expression of tobacco gene in maize Shou et al.
(2004b)
DREB1 Transcription factor Improved tolerance to Overexpression of the gene Ito et al. (2006)
drought, cold, and
salinity
PARP Poly (ADP-ribose) polymerase Tolerance to high light, Suppression of the gene De Block et al.
heat, and drought (2005)
Bcl-xL or Animal cell death suppressor protein Salinity, cold, and wound Overexpression of animal gene(s) in tobacco Qiao et al.
Ced-9 tolerance (2002)
Delayed senescence
AtNAP Transcription factor Delayed leaf senescence Suppression of the gene Guo and Gan
(2006)
OsDOS Nuclear-localized CCCH-type zinc Delayed leaf senescence Overexpression in rice Kong et al.
protein – negative regulator of JA (2006)
Plastid A component of chloroplast NDH Delayed leaf senescence Suppression of the gene in tobacco Zapata et al.
NDH complex (2005)
gene
A.K. Handa et al.
of PCD include perception of stimulus (extrinsic or intrinsic), signaling cascade and

execution of cell death. The mechanism of perception of biotic stimulus is relatively
better understood than that of any abiotic stimulus. The pathogens are recognized
by receptors at the cell surface through pathogen-associated molecular patterns
(PAMPs), which are highly conserved structural components required to support
the lifestyle of a pathogen. The host receptors are termed pattern-recognition
receptors, which resemble in function with toll-like receptors in animals
(Nurnberger et al. 2004; Zipfel et al. 2004). A receptor–pathogen interaction triggers
HR that helps to contain the proliferation and spread of pathogens to neighboring
cells. An endeavor to survive – a basic principle of evolution – allows certain
pathogens to evade host resistance by blocking the interaction-driven HR through
secretion of “effectors.” Interestingly, recognition of these effectors by “R” (resis-
tance) gene products enables plants to become resistant (Chisholm et al. 2006).
Characterization of R genes has become a potential tool in marker-assisted breeding
and genetic engineering to develop disease-resistant plants. Once pathogen is recog-
nized, a defense response is initiated and decision on the fate of cell is made. The
available information suggests the involvement of a multicomponent mechanism,
which is conserved in animals and plants in its basic form (Hoeberichts and
Woltering 2003; Franklin-Tong and Gourlay 2008; Williams and Dickman 2008).
Some regions of the R-gene product show significant similarity with apoptotic
proteins from humans (APAF) and Caenorhabditis elegans (CED4). The apoptotic
proteins recruit caspases, which are specific type of cysteine proteases capable of
acting on large number of substrates to effect cell death. In plants, though true
caspases are yet to be identified, caspase-like proteases called metacaspases have
been detected (see Mattoo and Handa 2004). The animal cell apoptosis is regulated
by pro-apoptotic (e.g., CED4, APAF, Bax) and anti-apoptotic (e.g., CED9, Bcl-2,
Bcl-xL) proteins. Bioinformatics has allowed predicting the presence of anti-
apoptotic proteins in plants despite the lack of sequence similarity with the animal
counterparts. An ectopic expression of animal anti-apoptotic proteins suppressed
apoptosis and imparted tolerance against certain type of stresses in plants (Table 5.7).
In HR-induced PCD, reactive oxygen species (ROS) and nitric oxide (NO) have
emerged as early messengers in the signaling cascade leading to defense response.
The most notable species are superoxide radical (O2-1) and hydrogen peroxide
(H2O2). A superoxide radical is highly reactive and dismutates to H2O2 by super-
oxide dismutase (SOD). The perception of a stimulus due to a pathogen or an
abiotic stress is marked by an oxidative burst resulting in the synthesis and
accumulation of ROS in large amounts. A similar increase in NO has been observed
in plants (Zeidler et al. 2004). It is thought that a fine-tuned NO/ROS balance is
responsible for the induction of genes during HR (Grun et al. 2006). ROS act in
concert with MAPK to activate a host of biochemical responses, such as cell wall
cross-linking, lignification, synthesis of pathogenesis-related (PR) proteins and
phytoalexins. They are also known to regulate the synthesis of hormones, such as
jasmonic acid, which in turn may initiate a defense response. NO interacts with
salicylic acid and jasmonic acid, particularly during wounding, which indicates its
role in stress signaling (see Grun et al. 2006). Miller et al. (2008) have reviewed the
mechanisms of ROS signaling. Besides mediating a stress signal, ROS can delete-
riously affect pathogen or host cell through oxidative damage of its biochemical
constituents. Further, recent studies suggest that both ROS and NO act as signaling
molecules in the development of plants (Gapper and Dolan 2006; Grun et al. 2006).
Because ROS play a crucial role in the cell function, their intracellular level is
tightly controlled. Different mechanisms are known to operate in the production
and removal of ROS. Following pathogen recognition, the accumulation of ROS in
apoplast is attributed to the action of more than just one enzyme. Respiratory burst
oxidase homologs (Rboh) with similarity in function to mammalian neutrophil
oxidase are recognized to play a dominant role (see Apel and Hirt 2004). More
recently, extracellular peroxidases have also been shown to participate in the
synthesis process (Bindschedler et al. 2006; Choi et al. 2007). During PCD in
mammalian cells, mitochondria serve as a source of ROS when there is a transient
change in their permeability because of stress and cytochrome-c is released to the
cytoplasm. In plants, chloroplasts seem to significantly contribute to ROS genera-
tion. Accordingly, the degeneration of chloroplast is observed prior to leaf senes-
cence and necrosis. Several antioxidant enzymes – dismutases, catalases and
peroxidases – are active during HR-induced PCD, and their suppression in trans-
genic plants has been observed to severely compromise the ability of these plants to
withstand stress. On the other hand, the increased antioxidant activity through
overexpression of the genes results in enhanced tolerance against a variety of
environmental stress (Table 5.7). Even though progress has been made in our
understanding of ROS role in plants, their signaling pathways and metabolic cross-
talk with other components are yet to be fully elucidated. Similarly, as roles of
NO in plant development and stress responses are revealed (Parani et al. 2004;
Lindermayr et al. 2005; Abat et al. 2008), the manipulation of its intracellular levels
can serve yet another tool for biotechnological improvements in crop plants.
5.9 Future Perspectives
Molecular and genetic dissection of plant developmental processes has provided a

map of complex interactions that regulate desirable plant traits. This knowledge is
enabling scientists to search for regulatory genes that can be manipulated to enhance
desirable developmental traits in agronomical and horticultural crops (Rothstein
2007). Details on the interactions among hormones, environment and physiology
are being unraveled and have begun to reveal how differentially mediated progres-
sion of various processes regulate the life span of plants. The challenge is to identify
a hierarchy of regulators or a specific pattern of events that control desirable
attributes and then genetically modify critical and beneficial processes without
negatively impacting other beneficial attributes in crop plants. Thus far, studies
on transgenic plants and effectiveness of a gene construct have reinforced the
advantage of using tissue-specific and regulatable promoters to tightly control the
expression of introduced transgene and avoid affecting non-targeted developmental
processes. Thus, the need is for identifying and characterizing tissue-specific pro-
moters and promoter elements that control both tissue specificity and the level of
expression of the introduced transgene. We also need to develop transgenic plants
particularly suited to grow well in ecofriendly, sustainable agricultural systems that
have minimal reliance on chemical input and synergistically (positively) influence
plant metabolism (Neelam et al. 2008; Mattoo and Teasdale 2009).
References
Aarts MGM, Dirkse WG, Stlekema WJ, Perelra A (1993) Transposon tagging of a male sterility
gene in Arabidopsis. Nature 363:715–717
Abat JK, Mattoo AK, Deswal R (2008) S-nitrosylated proteins of a medicinal CAM plant
Kalanchoe pinnata – ribulose-1, 5-bisphosphate carboxylase/oxygenase activity targeted for
inhibition. FEBS J 275:2862–2872
Abdurakhmonov IY, Kushanov FN, Djaniqulov F, Buriev ZT, Pepper AE, Fayzieva N, Mavlonov
GT, Saha S, Jenkins JN, Abdukarimov A (2007) The role of induced mutation in conversion of
photoperiod dependence in cotton. J Hered 98:258–266
Adrain C, Martin SJ (2001) The mitochondrial apoptosome: a killer unleashed by the cytochrome
seas. Trends Biochem Sci 26:390–397
Aguilar-Martı́nez JA, Poza-Carrión C, Cubas P (2007) Arabidopsis BRANCHED1 acts as an
integrator of branching signals within axillary buds. Plant Cell 19:458–472
Aharoni A, Dixit S, Jetter R, Thoenes E, van Arkel G, Pereira A (2004) The SHINE clade of AP2
domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers
drought tolerance when overexpressed in Arabidopsis. Plant Cell 16:2463–2480
Aida R, Yoshida T, Ichimura K, Goto R, Shibata M (1998) Extension of flower longevity in
transgenic Torenia plants incorporating ACC oxidase transgene. Plant Sci 138:91–101
Alba R, Payton P, Fei Z, McQuinn R, Debbie P, Martin G, Tanksley S, Giovannoni J (2005)
Transcriptome and selected fruit metabolite analysis reveal multiple points of ethylene regu-
latory control during tomato fruit development. Plant Cell 17:2954–2965
Albertsen MC, Howard JA (1999) Induction of male sterility in plants by expression of high levels
of avidin. Patent Application No WO 99/04023
Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal trans-
duction. Annu Rev Plant Biol 55:373–399
Arite T, Iwata H, Ohshima K, Maekawa M, Nakajima M, Kojima M, Sakakibara H, Kyozuka J
(2007) DWARF10, an RMS1/MAX4/DAD1 ortholog, controls lateral bud outgrowth in rice.
Plant J 51:1019–1029
Arora A (2008) Biochemistry of flower senescence. In: Paliyath G, Murr DP, Handa AK, Lurie S
(eds) Postharvest biology and technology of fruits vegetables and flowers. Blackwell, Ames,
IA, pp 51–85
Arora A, Singh VP (2004) Cysteine protease gene expression and proteolytic activity during floral
development and senescence in ethylene-insensitive gladiolus. J Plant Biochem Biotech
13:123–126
Arora A, Watanabe S, Ma B, Takada K, Ezura H (2006) A novel ethylene receptor homolog gene
isolated from ethylene-insensitive flowers of gladiolus (Gladiolus grandiflora hort.). Biochem
Biophys Res Comm 351:739–744
Ashraf M (2008) Biotechnological approach of improving plant salt tolerance using antioxidants
as markers. Biotechnol Adv 26:503–510
Balk J, Leaver CL (2001) The PET1-CMS mitochondrial mutation in sunflower is associated with
premature programmed cell death and cytochrome c release. Plant Cell 13:1803–1818
Barrero LS, Cong B, Wu F, Tanksley SD (2006) Developmental characterization of the fasciated

locus and mapping of Arabidopsis candidate genes involved in the control of floral meristem
size and carpel number in tomato. Genome 49:991–1006
Barry CS, Giovannoni J (2006) Ripening inhibition in the tomato Green-ripe mutant results from
ectopic expression of a novel protein which disrupts ethylene signal transduction. Proc Natl
Acad Sci USA 103:7923–7928
Bäurle I, Dean C (2006) The timing of developmental transitions in plants. Cell 125:655–664
Bentolila S, Alfonso AA, Hanson MR (2002) A pentatricopeptide repeat-containing gene restores
fertility to cytoplasmic male-sterile plants. Pro Natl Acad Sci USA 99:10887–10892
Bessire M, Chassot C, Jacquat AC, Humphry M, Borel S, Petetot JMC, Metraux JP, Nawrath C
(2007) A permeable cuticle in Arabidopsis leads to a strong resistance to Botrytis cinerea.
EMBO J 26:2158–2168
Bindschedler LV, Dewdney J, Blee KA, Stone JM, Asai T, Plotnikov J, Denoux C, Hayes T,
Gerrish C, Davies DR, Ausubel FM, Bolwell GP (2006) Peroxidase-dependent apoplastic
oxidative burst in Arabidopsis required for pathogen resistance. Plant J 47:851–863
Bird D, Beisson F, Brigham A, Shin J, Greer S, Jetter R, Kunst L, Wu XW, Yephremov A, Samuels
L (2007) Characterization of Arabidopsis ABCG11/WBC11, an ATP binding cassette (ABC)
transporter that is required for cuticular lipid secretion. Plant J 52:485–498
Bleecker AB, Patterson SE (1997) Last exit: senescence, abscission and meristem arrest in
Arabidopsis. Plant Cell 9:1169–1179
Böhlenius H, Huang T, Charbonnel-Campaa L, Brunner AM, Jansson S, Strauss SH, Nilsson O
(2006) CO/FT regulatory module controls timing of flowering and seasonal growth cessation in
trees. Science 312:1040–1043
Bovy AG, Angenent GC, Dons HJM, van Altvorst A-C (1999) Heterologous expression
of the Arabidopsis etr1–1 allele inhibits the senescence of carnation flowers. Mol Breed 5:
301–308
Brar DS, Khush GS (2006) Cytogenetic manipulation and germplasm enhancement of rice (Oryza
sativa L). In: Singh RJ, Jauhar PP (eds) Genetic resources, chromosome engineering and crop
improvement, vol 2, Cereal crops. Taylor and Francis, Boca Raton, FL, pp 115–158
Breeze E, Wagstaff C, Harrison E, Bramke I, Rogers HJ, Stead AD, Thomas B, Buchanan–
Wollaston V (2004) Gene expression patterns to define stages of postharvest senescence in
Alstroemeria petals. Plant Biotechnol J 2:155–168
Brown KM (1997) Ethylene and abscission. Physiol Plant 100:567–576
Brown GG, Formanová N, Jin H, Wargachuk R, Dendy C, Patil P, Laforest M, Zhang J, Cheung
WY, Landry BS (2003) The radish Rfo restorer gene of Ogura cytoplasmic male sterility
encodes a protein with multiple pentatricopeptide repeats. Plant J 35:262–272
Browse J (1997) Conditionally male-fertile plants and methods and compositions for restoring the
fertility thereof. Patent Application No WO 97/10703
Brummell DA (2006) Cell wall disassembly in ripening fruit. Funct Plant Biol 33:103–119
Brummell DA, Labavitch JM (1997) Effect of antisense suppression of endopolygalacturonase
activity on polyuronide molecular weight in ripening tomato fruit and in fruit homogenates.
Brummell DA, Hall BD, Bennett AB (1999a) Antisense supression of tomato endo-1, 4-b-
glucanase Cel2 mRNA accumulation increases the force required to break fruit abscission
zones but does not affect fruit softening. Plant Mol Biol 40:615–622
Brummell DA, Harpster MH, Civello PM, Bennett AB, Dunsmuir P (1999b) Modification of
expansin protein abundance in tomato fruit alters softening and cell wall polymer metabolism
during ripening. Plant Cell 11:2203–2216
Budar F, Delourme R, Pelletier G (2004) Male sterility. In: Pua EC, Douglas CJ (eds) Biotechno-
logy in agriculture and forestry, vol 54. Springer, Berlin, pp 43–64
Butenko MA, Stenvik GE, Alm V, Saether B, Patterson SE, Aalen RB (2006) Ethylene-dependant
and -independent pathways controlling floral abscission are revealed to converge using pro-
moter::reporter gene constructs in the ida abscission mutant. J Exp Bot 57:3627–3637
Carey AT, Smith DL, Harrison E, Bird CR, Gross KC, Seymour GB, Tucker GA (2001) Down-
regulation of a ripening-related beta-galactosidase gene (TBG1) in transgenic tomato fruits.
J Exp Bot 52:663–668
Carmona MJ, Chaı̈b J, Martı́nez-Zapater JM, Thomas MR (2008) A molecular genetic perspective
of reproductive development in grapevine. J Exp Bot 59:2579–2596
Celvenger DJ, Barret JE, Klee JH, Clark DG (2004) Factors affecting seed production in trans-
genic ethylene-insensitive petunias. J Am Soc Hortic Sci 129:401–406
Chang C, Kwok SF, Bleeker AB, Meyerowitz EM (1993) Arabidopsis ethylene-response gene
ETR1: similarity of product to two component regulators. Science 262:539–544
Chang H, Jones ML, Banowetz GM, Clark DG (2003) Overproduction of cytokinins in petunia
flowers transformed with PSAG12-IPT delays corolla senescence and decreases sensitivity to
ethylene. Plant Physiol 132:2174–2183
Channeliere S, Riviere S, Scalliet G, Szecsi J, Jullien F, Dolle C, Vergne P, Dumas C, Bend-
ahmane M, Hugueney P, Cock JM (2002) Analysis of gene expression in rose petals using
expressed sequence tags. FEBS Lett 515:35–38
Chase CD (2006) Genetically engineered cytoplasmic male sterility. Trends Plant Sci 11:7–9
Chase CD (2007) Cytoplasmic male sterility: a window to the world of plant mitochondrial–
nuclear interactions. Trends Genet 23:81–90
Chassot C, Nawrath C, Metraux JP (2007) Cuticular defects lead to full immunity to a major plant
pathogen. Plant J 49:972–980
Chaudhury AM (1993) Nuclear genes controlling male fertility. Plant Cell 5:1277–1283
Chen GX, Sagi MS, Song WN, Krugman T, Fahima T, Korol AB, Nevo E (2004) Wild barley eibi1
mutation identifies a gene essential for leaf water conservation. Planta 219:684–693
Chisholm ST, Coaker G, Day B, Staskawicz BJ (2006) Host-microbe interactions: shaping the
evolution of the plant immune response. Cell 124:803–814
Cho HT, Cosgrove DJ (2000) Altered expression of expansin modulates leaf growth and pedicel
abscission in Arabidopsis thaliana. Proc Natl Acad Sci USA 97:9783–9788
Choi HW, Kim YJ, Hong LSC, JK HBK (2007) Hydrogen peroxide generation by the pepper
extracellular peroxidase CaPO2 activates local and systemic cell death and defense response to
bacterial pathogens. Plant Physiol 145:890–904
Chrispeels MJ, Sadaya DE, Martinj C (2002) Plants genes and crop biotechnology. Jones and
Bartlett, Boston, MA
Chun JP, Huber DJ (1998) Polygalacturonase-mediated solubilization and depolymerization of
pectic polymers in tomato fruit cell walls – regulation by pH and ionic conditions. Plant
Physiol 117:1293–1299
Chung Suh M, Lacey SA, Jetter R, Kunst L, Pollard M, Ohlrogge J, Beisson F (2005) Cuticular
lipid composition, surface structure, and gene expression in Arabidopsis stem epidermis. Plant
Physiol 139:1649–1665
Clark DG, Gubrium EK, Barrett JE, Nell TA, Klee HJ (1999) Root formation in ethylene-
insensitive plants. Plant Physiol 121:53–59
Cockram J, Jones H, Leigh FJ, O’Sullivan D, Powell W, Laurie DA, Greenland AJ (2007) Control
of flowering time in temperate cereals: genes, domestication, and sustainable productivity.
J Exp Bot 58:1231–1244
Cominelli E, Sala T, Calvi D, Gusmaroli G, Tonelli C (2008) Over-expression of the Arabidopsis
AtMYB41 gene alters cell expansion and leaf surface permeability. Plant J 53:53–64
Commenil P, Belingheri L, Bauw G, Dehorter B (1999) Molecular characterization of a lipase induced
in Botrytis cinerea by components of grape berry cuticle. Physiol Mol Plant Pathol 55:37–43
Cong B, Liu J, Tanksley SD (2002) Natural alleles at a tomato fruit size quantitative trait locus
differ by heterochronic regulatory mutations. Proc Natl Acad Sci USA 99:13606–13611
Cong B, Barrero LS, Tanksley SD (2008) Regulatory change in YABBY-like transcription factor
led to evolution of extreme fruit size during tomato domestication. Nat Genet 40:800–804
Conley CA, Hanson MR (1995) How do alterations in plant mitochondrial genomes disrupt pollen
development? J Bioenerg Biomembr 27:447–457
Cui ML, Takada K, Ma B, Ezura H (2004) Overexpression of a mutated melon ethylene receptor
gene Cm-ETR1/H69A confers reduced ethylene sensitivity in a heterologous plant, Nemesia
strumosa. Plant Sci 167:253–258
Davuluri GR, van Tuinen A, Fraser PD, Manfredonia A, Newman R, Burgess D, Brummell DA,
King SR, Palys J, Bramley UJ, PM PHMJ, Bowler C (2005) Fruitspecific RNAi-mediated
suppression of DET1 enhances carotenoid and flavonoid content in tomatoes. Nat Biotechnol
23:890–895
Dawson J, Wilson ZA, Aarts MGM, Braithwaite AF, Briarty LG, Mulligan BJ (1993) Microspore
and pollen development in six male sterile mutants of Arabidopsis thaliana. Can J Bot
71(629):638
De Block M, Debrouwer D, Moens T (1997) The development of a nuclear male sterility system in
wheat Expression of a barnase gene under the control of tapetum specific promoters. Theor
Appl Genet 95:125–131
De Block M, Verduyn C, De Brouwer D, Cornelissen M (2005) Poly(ADP-ribose) polymerase in
plants affects energy homeotasis, cell death and stress tolerance. Plant J 41:95–106
de Silva J, Verhoeyen ME (1998) Production and characterization of antisense-exogalactanase
tomatoes. In: Kuiper HA (ed) Report of the demonstration programme on food safety evalua-
tion of genetically modified foods as a basis for market introduction. The Netherlands Ministry
of Economic Affairs, The Hague, pp 99–106
del Campillo E, Bennett A (1996) Pedicel break strength and cellulase gene expression during
tomato flower abscission. Plant Physiol 111:813–820
Derksen J, van Wezel R, Knuiman B, Ylstra B, van Tunen AJ (1999) Pollen tubes of flavonol
deficient petunia show striking alterations in wall structure leading to tube disruption. Planta
207:575–581
Dewey RE, Timothy DH, Levings CS III (1991) Chimeric mitochondrial genes expressed in the C
male-sterile cytoplasm of maize. Curr Genet 20:475–482
Dickman MB, Ha YS, Yang Z, Adams B, Huang C (2003) A protein kinase from Colletotrichum
trifolii is induced by plant cutin and is required for appressorium formation. Mol Plant Microbe
Interact 16:411–421
Doebley J, Stec A, Hubbard L (1997) The evolution of apical dominance in maize. Nature
386:485–488
Dotson SB, Lanahan MB, Smith AG, Kishore GM (1996) A phosphonate monoester hydrolase
from Burkholderia caryophilli PG2982 is useful as a conditional lethal gene in plants. Plant J
10:383–392
Doust A (2007) Architectural evolution and its implications for domestication in grasses. Anal Bot
100:941–950
Eason JR, Ryan DJ, Pinkey TT, Donoghue EMO (2002) Programmed cell death during flower
senescence: isolation and characterization of cysteine proteinases from Sandersonia auran-
tiaca. Funct Plant Biol 29:1055–1064
Ecker JR, Theologis A (1994) Ethylene: a unique plant signaling molecule. In: Meyerowitz EM,
Somerville CR (eds) Arabidopsis. Cold Spring Harbor Press, Plainview, NY, pp 485–521
Einset JW, Kopperud C (1995) Antisense ethylene genes for Begonia flowers. Acta Hortic
405:190–195
Ellis CM, Nagpal P, Young JC, Hagen G, Guilfoyle TJ, Reed JW (2005) AUXIN RESPONSE
FACTOR1 and AUXIN RESPONSE FACTOR2 regulate senescence and floral organ abscission
in Arabidopsis thaliana. Development 132:4563–4574
Errington N, Tucker GA, Mitchell JR (1998) Effect of genetic down-regulation of PG and pectin
esterase activity on rheology and composition of tomato juice. J Sci Food Agric 76:515–519
Fabijanski SF, Arinson PG (1995) Binary cryptocytotoxic method of hybrid seed production. US
Patent 5,426,041
Fatima T, Rivera-Domı́nguez M, Troncoso-Rojas R, Tiznado-Hernández ME, Handa AK,
Mattoo AK (2008) Tomato. In: Kole C, Hall TC (eds) Transgenic vegetable crops, vol 6.
Wiley-Blackwell, Oxford, pp 1–46
Fernandez DE, Heck GE, Perry SE, Patterson SE, Bleecker AB, Fang S-C (2000) The embryo
MADS domain factor AGL15 acts postembryonically: inhibition of perianth senescence and
abscission via constitutive expression. Plant Cell 12:183–198
Ferrándiz C, Liljegren SJ, Yanofsky MF (2000) Negative regulation of the SHATTERPROOF
genes by FRUITFULL during Arabidopsis fruit development. Science 289:436–438
Fluhr R, Mattoo AK (1996) Ethylene – biosynthesis and perception. Crit Rev Plant Sci 15:479–523
Franklin-Tong VE, Gourlay CW (2008) A role for actin in regulating apoptosis/programmed cell
death: evidence spanning yeast, plants and animals. Biochem J 413:389–404
Frary A, Nesbitt TC, Frary A, Grandillo S, van der Knaap E, Cong B, Liu J, Meller J, Elber R,
Alpert KB, Tanksley SD (2000) Cloning and transgenic expression of fw2.2: a quantitative trait
locus key to the evolution of tomato fruit size. Science 289:85–88
Fuji S, Kazama T, Toriyama K (2008) Molecular studies on cytoplasmic male sterility-associated
gene and restorer genes in rice. In: Hirai A, Sano Y, Hirano H, Sasaki T (eds) Rice biology
in genomics era: biotechnology in agriculture and forestry, vol 62. Springer, New York,
pp 205–214
Gaffe J, Tieman DM, Handa AK (1994) Pectin methylesterase isoforms in tomato (Lycopersicon
esculentum) tissues: effects of expression of a pectin methylesterase antisense gene. Plant
Physiol 105:199–203
Gan S, Amasino RM (1997) Making sense of senescence. Plant Physiol 113:313–319
Gapper C, Dolan L (2006) Control of plant development by reactive oxygen species. Plant Physiol
141:341–345
Giovannoni JJ (2004) Genetic regulation of fruit development and ripening. Plant Cell 16:170–180
Giovannoni J (2007) Fruit ripening mutants yields insights into ripening control. Curr Opin Plant
Biol 10:283–289
Giovannoni JJ, DellaPenna D, Bennett AB, Fischer RL (1989) Expression of a chimeric poly-
galacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide
degradation but not fruit softening. Plant Cell 1:53–63
Gniwotta F, Vogg G, Gartmann V, Carver TLW, Riederer M, Jetter R (2005) What do microbes
encounter at the plant surface? Chemical composition of pea leaf cuticular waxes. Plant
Physiol 139:519–530
Goetz M, Godt DE, Guivarc’h A, Kahmann U, Chriqui D, Roitsch T (2001) Induction of male
sterility in plants by metabolic engineering of the carbohydrate supply. Proc Natl Acad Sci
USA 98:6522–6527
Goff SA, Crossland LD, Privalle LS (1990) Control of gene expression in plants by receptor
mediated transactivation in the presence of a chemical ligand. US Patent 5,880,333
Gonzalez-Carranza ZH, Elliott KA, Roberts JA (2007) Expression of polygalacturonases and
evidence to support their role during cell separation processes in Arabidopsis thaliana. J Exp
Bot 58:3719–3730
Gonzalo MJ, van der Knaap E (2008) A comparative analysis into the genetic bases of morphology
in tomato varieties exhibiting elongated fruit shape. Theor App Genet 116:647–656
Good X, Kellogg JA, Wagoner W, Langhoff D, Matsumura W, Bestwick RK (1994) Reduced
ethylene synthesis by transgenic tomatoes expressing S-adenosylmethionine hydrolase. Plant
Mol Biol 26:781–790
Goren R (1993) Anatomical, physiological and hormonal aspects of abscission in citrus. Hort Rev
15:145–182
Grandillo S, Ku H-M, Tanksley SD (1996) Characterization of fs8.1, a major QTL influencing fruit
shape in tomato. Mol Breed 2:251–260
Grandillo S, Ku H-M, Tanksley SD (1999) Identifying loci responsible for natural variation in fruit
size and shape in tomato. Theor Appl Genet 99:978–987
Grun S, Lindermayr C, Sell S, Durner J (2006) Nitric oxide and gene regulation in plants. J Exp
Bot 57:507–516
Gubrium EK, Clevenger DJ, Clark DG, Barret JE, Neil TA (2000) Reproduction and horticultural
performance of transgenic ethylene insensitive Petunias. J Am Soc Hort Sci 125:277–281
Guo Y, Gan S (2006) AtNAP, a NAC family transcription factor, has an important role in leaf
senescence. Plant J 46:601–612
Gutterson N, Ralston E (1998) Two component plant cell lethality methods and compositions.
Patent Application No WO 98/32325
Hall LN, Tucker GA, Smith CJS, Watson CF, Seymour GB, Bundick Y, Boniwell JM, Fletcher JD,
Ray JA, Schuch W, Bird C, Grierson D (1993) Antisense inhibition of pectin esterase gene
expression in transgenic tomatoes. Plant J 3:121–129
Hamilton A, Lycett G, Grierson D (1990) Antisense gene that inhibits synthesis of the hormone
ethylene in transgenic plants. Nature 346:284–287
Hanson MR, Bentolila S (2004) Interactions of mitochondrial and nuclear genes that affect male
gametophyte development. Plant Cell 16:S154–S169
Hanson MR, Conde MF (1985) Functioning and variation of cytoplamsic genomes:Lessons
from cytoplasmic-nuclear interactions affecting male fertility in plants. Int Rev Cytol 94:
213–267
Harpster MH, Brummell DA, Dunsmuir P (2002a) Suppression of a ripening-related endo-1, 4-b-
glucanase in transgenic pepper fruit does not prevent depolymerization of cell wall polysac-
charides during ripening. Plant Mol Biol 50:345–355
Harpster MH, Dawson DM, Nevins DJ, Dunsmuir P, Brummell DA (2002b) Constitutive over-
expression of a ripening-related pepper endo-1, 4-b-glucanase in transgenic tomato fruit does
not increase xyloglucan depolymerization or fruit softening. Plant Mol Biol 50:357–369
Hartley RW (1989) Barnase and barstar: two small proteins to fold and fit together. Trends
Biochem Sci 14:450–454
Hennin C, Hofte M, Diederichsen E (2001) Functional expression of Cf9 and Avr9 genes in
Brassica napus induces enhanced resistance to Leptosphaeria maculans. Mol Plant Microbe
Interact 14:1075–1085
Hoeberichts FA, Woltering EJ (2003) Multiple mediators of plant programmed cell death:
interplay of conserved cell death mechanisms and plant-specific regulators. BioEssays
25:47–57
Homme YL, Stahl RJ, Li X-L, Hameed A, Brown GG (1997) Brassica nap cytoplasmic male
sterility is associated with expression of a mtDNA region containing a chimeric gene similar to
the pol CMS-associated orf224 gene. Curr Genet 31:325–335
Horn R, Kusterer B, Lazarescu E, Prüfe M, Friedt W (2003) Molecular mapping of the Rf1 gene
restoring pollen fertility in PET1-based F1 hybrids in sunflower (Helianthus annuus L.). Theor
Appl Genet 106(4):599–606
Hovav R, Chehanovsky N, Moy M, Jetter R, Schaffer AA (2007) The identification of a gene
(Cwp1), silenced during Solanum evolution, which causes cuticle microfissuring and dehydra-
tion when expressed in tomato fruit. Plant J 52:627–639
Hua J, Chang C, Sun Q, Meyerowitz EM (1995) Ethylene insensitivity conferred by Arabidopsis
ERS gene. Science 269:1712–1714
Hunter DA, Steele BC, Reid MS (2002) Identification of genes associated with perianth senes-
cence in daffodil (Narcissus pseudonarcissus L.). Plant Sci 163:13–21
Ida R, Yoshida T, Ichimura K, Goto R, Shibata M (1998) Extension of flower longevity in
transgenic Torenia plants incorporating ACC oxidase transgene. Plant Sci 138:91–101
Imaizumi T, Schultz TF, Harmon FG, Ho LA, Kay SA (2005) FKF1 F-box protein mediates cyclic
degradation of a repressor of CONSTANS in Arabidopsis. Science 309:293–297
Ishikawa S, Maekawa M, Arite T, Onishi K, Takamure I, Kyozuka J (2005) Suppression of tiller
bud activity in tillering dwarf mutants of rice. Plant Cell Physiol 46:79–86
Ito Y, Katsura K, Maruyama K, Taji T, Kobayashi M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K
(2006) Functional analysis of rice DREB1/CBF-type transcription factors involved in
cold-responsive gene expression in transgenic rice. Plant Cell Physiol 47:141–153
Izawa T (2007) Daylength measurements by rice plants in photoperiodic short-day flowering. Int
Rev Cytol 256:191–222
Jack T (2004) Molecular and genetic mechanisms of floral control. Plant Cell 16:S1–S17
Jackson MB, Osborne D (1970) Ethylene, natural regulator of leaf abscission. Nature 225:
1019–1022
Jaeger KE, Graf A, Wigge PA (2006) The control of flowering in time and space. J Exp Bot
57:3415–3418
Jiang C-Z, Feng L, Imsabai W, Meir S, Reid MS (2008) Silencing polygalacturonase expression
inhibits tomato petiole abscission. J Exp Bot 59:973–979
Jiménez-Bermúdez S, Redondo-Nevado J, Muñoz-Blanco J, Caballero JL, López-Aranda JM,
Valpuesta V, Pliego-Alfaro F, Quesada MA, Mercado JA (2002) Manipulation of fruit soften-
ing by antisense expression of a pectate lyase gene. Plant Physiol 128:751–759
Jin J, Huang W, Gao JP, Yang J, Shi M, Zhu MZ, Luo D, Lin HX (2008) Genetic control of rice
plant architecture under domestication. Nat Genet 40:1365–1369
Jones ML, Chaffin GS, Eason JR, Clark DG (2005) Ethylene sensitivity regulates proteolytic
activity and cysteine protease gene expression in petunia corollas. J Exp Bot 56:2733–2744
Jung KH, Han MJ, Lee DY, Lee YS, Schreiber L, Franke R, Faust A, Yephremov A, Saedler H,
Kim YW, Hwang I, An G (2006) Wax-deficient anther1 is involved in cuticle and wax
production in rice anther walls and is required for pollen development. Plant Cell 18:
3015–3032
Kalaitzis P, Koehler S, Tucker M (1995) Cloning of a tomato polygalacturonase expressed in
abscission. Plant Mol Biol 28:647–656
Kalamaki MS, Harpster MH, Palys JM, Labavitch JM, Reid DS, Brummell DA (2003) Simulta-
neous transgenic suppression of LePG and LeExp1 influences rheological properties of juice
and concentrates from a processing tomato variety. J Agric Food Chem 51:7456–7464
Kardailsky I, Shukla VK, Ahn JH, Dagenais N, Christensen SK, Nguyen JT, Chory J, Harrison MJ,
Weigel D (1999) Activation tagging of the floral induceur FT. Science 286:1962–1965
Kaul MLH (1988) Male sterility in high plants. In: Frankel R, Grossman M, Maliga P, Riley R
(eds) Monographs of theoretical applied genetics 10. Springer, Heidelberg, pp 775–797
Kazama T, Toriyama K (2003) A pentatricopeptide repeatcontaining gene that promotes the
processing of aberrant atp6 RNA of cytoplasmic male-sterile rice. FEBS Lett 544:99–102
Kazokas WC, Burns JK (1998) Cellulase activity and gene expression in citrus fruit abscission
zones during and after ethylene treatment. J Am Soc Hortic Sci 123:781–786
Kendrick MD, Chang C (2008) Ethylene signaling: new levels of complexity and regulation. Curr
Opin Plant Biol 11:479–485
Kevany BM, Tieman DM, Taylor MG, Cin VD, Klee HJ (2007) Ethylene receptor degradation
controls the timing of ripening in tomato fruit. Plant J 51:458–467
Kevany BM, Taylor MG, Klee HJ (2008) Fruit-specific suppression of the ethylene receptor
LeETR4 results in early-ripening tomato fruit. Plant Biotechnol J 6:295–300
Kim H, Hwang YW, An G (2006) Wax-deficient anther1 is involved in cuticle and wax production
in rice anther walls and is required for pollen development. Plant Cell 18:3015–3032
Kim SK, Yun CH, Lee JH, Jang YH, Park HY, Kim JK (2008) OsCO3, a CONSTANS-LIKE gene,
controls flowering by negatively regulating the expression of FT-like genes under SD condi-
tions in rice. Planta 228:355–365
Klee H, Tieman D (2002) The tomato ethylene receptor gene family: form and function. Physiol
Plant 115:336–341
Klee HJ, Hayford MB, Kretzmer KA, Barry GF, Kishore GM (1991) Control of ethylene synthesis
by expression of a bacterial enzyme in transgenic tomato plants. Plant Cell 3:1187–1193
Klein RR, Klein PE, Mullet JE, Minx P, Rooney WL, Schertz KF (2005) Fertility restorer locus
Rf1 of sorghum (Sorghum bicolor L.) encodes a pentatricopeptide repeat protein not present in
the colinear region of rice chromosome 12. Theor Appl Genet 111:994–1012
Kobayashi Y, Weigel D (2007) Move on up, it’s time for change–mobile signals controlling
photoperiod-dependent flowering. Gene Dev 21:2371–2384
Koizuka N, Imai R, Fujimoto H, Hayakawa T, Kimura Y, Kohno-Murase J, Sakai T, Kawasaki S,
Imamura J (2003) Genetic characterization of a pentatricopeptide repeat protein gene orf687
that restores fertility in the cytoplasmic male-sterile Kosena radish. Plant J 34:407–415
Koltunow AM, Truettner J, Cox KH, Wallroth M, Goldberg RB (1990) Different temporal and
spatial gene expression patterns occur during anther development. Plant Cell 2:1201–1224
Kong Z, Li M, Yang W, Xu W, Xue Y (2006) A novel nuclear-localized CCCH-type zinc finger
protein, OsDOS, is involved in delaying leaf senescence in rice. Plant Physiol 141:1376–1388
Kramer M, Sanders R, Bolkan H, Waters C, Sheehy RE, Hiatt WR (1992) Postharvest evaluation
of transgenic tomatoes with reduced levels of polygalacturonase: processing, firmness and
disease resistance. Postharvest Biol Technol 1:241–255
Kriete G, Niehaus K, Perlick AM, Puhler A, Broer I (1996) Male sterility in transgenic tobacco
plants induced by tapetum-specific deacetylation of the externally applied non-toxic compound
N-acetyl-L-phosphinothricin. Plant J 9:809–818
Ku H-M, Doganlar S, Chen KY, Tanksley SD (1999) The genetic basis of pear shaped tomato fruit.
Theor Appl Genet 99:844–850
Ku H-M, Grandillo S, Tanksley SD (2000) fs8.1, a major QTL, sets the pattern of tomato carpel
shape well before anthesis. Theor Appl Genet 101:873–878
Ku SJ, Yoon HJ, Suh HS, Chung YY (2003) Male-sterility of thermosensitive genic male-sterile
rice is associated with premature programmed cell death of the tapetum. Planta 217:559–565
Kuchuk N, Sytnyk K, Vasylenko M, Shakhovsky A, Komarnytsky I, Kushnir S, Gleba Y (2006)
Genetic transformation of plastids of different Solanaceae species using tobacco cells as
organelle hosts. Theor Appl Genet 113:519–527
Kumar A, Bernier J, Verulkar S, Lafitte HR, Atlin GN (2008) Breeding for drought tolerance:
direct selection for yield, response to selection and use of drought-tolerant donors in upland
and lowland-adapted populations. Fields Crop Res 107:221–231
Kunst L, Samuels AL (2003) Biosynthesis and secretion of plant cuticular wax. Progr Lipid Res
42:51–80
Lanahan MB, Yen HC, Giovannoni JJ, Klee H (1994) The never ripe mutation blocks ethylene
perception in tomato. Plant Cell 6:521–530
Langley KR, Martin A, Stenning R, Murray AJ, Hobson GE, Schuch WW, Bird CR (1994)
Mechanical and optical assessment of the ripening of tomato fruit with reduced PG activity.
J Sci Food Agri 66:547–554
Lashbrook CC, Giovannoni JJ, Hall BD, Bennett AB (1998) Transgenic analysis of tomato endo-
ß-1, 4-glucanase gene function. Role of cel1 in floral abscission. Plant J 13:303–310
Lewis MW, Leslie ME, Liljegren SJ (2006) Plant separation: 50 ways to leave your mother. Curr
Lewis JM, Mackintosh CA, Shin S, Gilding E, Kravchenko S, Baldridge G, Zeyen R,
Muehlbauer GJ (2008) Overexpression of the maize Teosinte Branched1 gene in wheat
suppresses tiller development. Plant Cell Rep 27:1217–1225
Li X-Q, Jean M, Landry BS, Brown GG (1998) Restorer genes for different forms of Brassica
cytoplasmic male sterility map to a single nuclear locus that modifies transcripts of several
mitochondrial genes. Proc Natl Acad Sci USA 95:10032–10037
Li C, Williams MM, Loh YT, Lee GI, Howe GA (2002) Resistance of cultivated tomato to cell
content-feeding herbivores is regulated by the octadecanoid-signaling pathway. Plant Physiol
130:494–503
Li X, Qian Q, Fu Z, Wang Y, Xiong G, Zeng D, Wang X, Liu X, Teng S, Hiroshi F, Yuan M,
Luo D, Han B, Li J (2003) Control of tillering in rice. Nature 422:618–621
Li P, Wang Y, Qian Q, Fu Z, Wang M, Zeng D, Li B, Wang X, Li J (2007a) LAZY1 controls rice
shoot gravitropism through regulating polar auxin transport. Cell Res 17:402–410
Li YH, Beisson F, Koo AJK, Molina I, Pollard M, Ohlrogge J (2007b) Identification of acyl-
transferases required for cutin biosynthesis and production of cutin with suberin like mono-
mers. Proc Natl Acad Sci USA 104:18339–18344
Liljegren SJ, Ditta GS, Eshed Y, Savidge B, Bowman JL, Yanofsky MF (2000) SHATTERPROOF
MADS-box genes control seed dispersal in Arabidopsis. Nature 404:766–770
Lindermayr C, Saalbach G, Durner J (2005) Proteomic identification of S-nitrosylated proteins in
Arabidopsis. Plant Physiol 137:921–930
Linke B, Borner T (2005) Mitochondrial effects on flower and pollen development. Mitochondrion
5:389–402
Lippman Z, Tanksley SD (2001) Dissecting the genetic pathway to extreme fruit size in tomato
using a cross between the small-fruited wild species L. pimpinellifolium and L. esculentum, var.
Giant Heirloom. Genetics 158:413–422
Liu J, Van Eck J, Cong B, Tanksley SD (2002) A new class of regulatory genes underlying the
cause of pear-shaped tomato fruit. Proc Natl Acad Sci USA 99:13302–13306
Liu Y, Roof S, Ye Z, Barry C, van Tuinen A, Vrebalov J, Bowler C, Giovannoni J (2004)
Manipulation of light signal transduction as a means of modifying fruit nutritional quality in
tomato. Proc Natl Acad Sci USA 26:9897–9902
Luo B, Xue XY, Hu WL, Wang LJ, Chen XY (2007) An ABC transporter gene of Arabidopsis
thaliana, AtWBC11, is involved in cuticle development and prevention of organ fusion. Plant
Lurie S, Paliyath G (2008) Enhancing postharvest shelf life and quality in horticultural commo-
dities using 1-MCP technology. In: Paliyath G, Murr DP, Handa AK, Lurie S (eds) Postharvest
biology and technology of fruits, vegetables and flowers. Blackwell, Ames, IA, pp 139–161
Lurin C, Andres C, Aubourg S, Bellaoui M, Bitton F, Bruyere C, Caboche M, Debast C, Gualberto J,
Hoffmann B, Lecharny A, Le Ret M, Martin-Magniette M-L, Mireau H, Peeters N, Renou J-P,
Szurek B, Taconnat L, Small I (2004) Genome-wide analysis of Arabidopsis pentatricopeptide
repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16:2089–2103
Makandar R, Essig JS, Schapaugh MA, Trick HN, Shah J (2006) Genetically engineered resistance
to Fusarium head blight in wheat by expression of Arabidopsis NPR1. Mol Plant Microbe
Interact 19:123–129
Malladi A, Burns JK (2008) CsPLDa1 and CsPLDg1 are differentially induced during leaf and
fruit abscission and diurnally regulated in Citrus sinensis. J Exp Bot 59:3729–3739
Manning K, Maw GA (1975) Distribution of acid invertase in the tomato plant. Phytochemistry
14:1965–1969
Manning K, Tor M, Poole M, Hong Y, Thompson A, King G, Giovannoni J, Seymour G (2006)
A naturally occurring epigenetic mutation in a gene encoding an SPB-box transcription factor
inhibits tomato fruit ripening. Nat Genet 38:949–952
Mao L, Begum D, Chuang HW, Budiman MA, Szymkoqiak EJ, Irish EE, Wing RA (2000)
JOINTLESS is a MADS-box gene controlling tomato flower abscission zone development.
Nature 406:910–913
Mariani C, De BM, Truettner J, Leemans J, Goldberg RB (1990) Induction of male sterility in
plants by a chimaeric ribonuclease gene. Nature 347:737–741
Mariani C, Gossele V, De Beuckeleer M, De Block M, Goldberg RB, De Greef W, Leemans J
(1992) A chimaeric ribonuclease inhibitor gene restores fertility to male sterile plants. Nature
357:384–387
Martı́nez-Garcı́a JF, Virgós-Soler A, Prat S (2002) Control of photoperiod-regulated tuberization
in potato by the Arabidopsis flowering-time gene CONSTANS. Proc Natl Acad Sci USA
99:15211–15216
Matsubara K, Yamanouchi U, Wang ZX, Minobe Y, Izawa T, Yano M (2008) Ehd2, a rice
ortholog of the maize INDETERMINATE1 gene, promotes flowering by up-regulating
Ehd1. Plant Physiol 148:1425–1435
Mattoo AK, Handa AK (2004) Ethylene signaling in plant cell death. In: Nooden L (ed) Plant cell
death processes. Academic, New York, pp 125–142
Mattoo AK, Handa AK (2008) Higher polyamines restore and enhance metabolic memory in
ripening fruit. Plant Sci 174:386–393
Mattoo AK, Suttle JC (eds) (1991) The plant hormone ethylene. CRC press, Boca Raton, FL
Mattoo AK, Teasdale JR (2009) Ecological and genetic systems underlying sustainable horticulture.
Hort Rev (in press)
McConn M, Browse J (1996) The critical requirement for linoleic acid is pollen development not
photosynthesis in an Arabidopsis mutant. Plant Cell 8:403–416
McKim SM, Stenvik G-E, Butenko MA, Kristiansen W, Cho SK, Hepworth SR, Aslen RB,
Haughn GW (2008) The BLADE-ON-PETIOLE genes are essential for abscission zone
formation in Arabidopsis. Development 135:1537–1546
Mehta RA, Cassol T, Li N, Ali N, Handa AK, Mattoo AK (2002) Engineered polyamine accumu-
lation in tomato enhances phytonutrient content, juice quality and vine life. Nat Biotechnol
20:613–618
Mei C, Qi M, Sheng G, Yang Y (2006) Inducible overexpression of a rice allene oxide synthase
gene increases the endogenous jasmonic acid level, PR gene expression, and host resistance to
fungal infection. Mol Plant Microbe Interact 19:1127–1137
Meir S, Hunter DA, Chen JC, Halaly V, Ried MS (2006) Molecular changes occurring during
acquisition of abscission competence following auxin depletion in Mirabilis jalapa. Plant
Physiol 141:1604–1616
Melchers G, Mohri Y, Watanabe K, Wakabayashi S, Harada K (1992) One-step generation of
cytoplasmic male sterility by fusion of mitochondrial-inactivated tomato protoplasts with
nuclear-inactivated Solanum protoplasts. Proc Natl Acad Sci USA 89:6832–6836
Michael MZ, Savin KW, Baudinette SC, Graham MW, Chandler SF, Lu CY (1993) Cloning of
ethylene biosynthetic genes involved in petal senescence of carnation and petunia, and their
antisense expression in transgenic plants. In: Pech JC, Latché A, Balagué C (eds) Cellular and
molecular aspects of the plant hormone ethylene. Kluwer, Dordrecht, pp 298–303
Miller G, Shulaev V, Mittler R (2008) Reactive oxygen signaling and abiotic stress. Physiol Planta
133:481–489
Mustilli AC, Fenzi F, Ciliento R, Alfano F, Bowler C (1999) Phenotype of the tomato high
pigment-2 mutant is caused by a mutation in the tomato homolog of DEETIOLATED1. Plant
Cell 11:145–157
Napoli CA, Fahy D, Wang HY, Taylor LP (1999) White anther: a petunia mutant that abolishes
pollen flavonol accumulation induces male sterility and is complemented by a chalcone
synthase transgene. Plant Physiol 120:615–622
Narumi T, Kanno Y, Suzuki M, Kishimoto S, Ohmiya A, Satoh S (2005) Cloning of a cDNA
encoding an ethylene receptor (DG-ERS1) from chrysanthemum and comparison of its mRNA
level in ethylene sensitive and -insensitive cultivars. Postharvest Biol Tech 36:21–30
Nawrath C (2006) Unraveling the complex network of cuticular structure and function. Curr Opin
Neelam A, Cassol T, Mehta RA, Abdul-Baki AA, Sobolev AP, Goyal RK, Abbott J, Segre AL,
Handa AK, Mattoo AK (2008) A field-grown transgenic tomato line expressing higher levels of
polyamines reveals legume cover crop mulch-specific perturbations in fruit phenotype at the
levels of metabolite profiles, gene expression, and agronomic characteristics. J Exp Bot
59:2337–2346
Negi PS, Handa AK (2008) Structural deterioration of the produce – The breakdown of cell wall
components. In: Paliyath G, Murr DP, Handa AK, Lurie S (eds) Postharvest biology and
technology of fruits, vegetables and flowers. Wiley-Blackwell, Ames, IA, pp 162–194
Newton KJ, Gabay-Laughnan S, DePaepe R (2004) Mitochondrial mutations in plants. In: Day
DA, Millar HA, Whelan J (eds) Plant mitochondria: from genome to function advances in
photosynthesis and respiration, vol 17. Kluwer, London, pp 121–142
Nurnberger T, Brunner F, Kemmerling B, Piater L (2004) Innate immunity in plants and animals:
striking similarities and obvious differences. Immunol Rev 198:249–266
O’Keefe DP, Tepperman JM, Dean C, Leto KJ, Erbes DL, Odell JT (1994) Plant expression of a
bacterial cytochrome P450 that catalyzes activation of a sulfonylurea pro-herbicide. Plant
Physiol 105:473–482
Oeller PW, Wong LM, Taylor LP, Pike DA, Theologis A (1991) Reversible inhibition of tomato fruit
senescence by antisense 1-aminocyclopropane-1-carboxylate synthase. Science 254:427–439
Ongaro V, Leyser O (2008) Hormonal control of shoot branching. J Exp Bot 59:67–74
Østergaard L, Kempin SA, Bies D, Klee HJ, Yanofsky MF (2006) Pod shatter-resistant Brassica
fruit produced by ectopic expression of the FRUITFULL gene. Plant Biotechnol J 4:45–51
Panavas T, Pikula A, Reid PD, Rubinstein B, Walker EL (1999) Identification of senescence-

associated genes from daylily petals. Plant Mol Biol 40:237–248
Panikashvili D, Savaldi-Goldstein S, Mandel T, Yifhar T, Franke RB, Hofer R, Schreiber L, Chory
J, Aharoni A (2007) The Arabidopsis DESPERADO/AtWBC11 transporter is required for
cutin and wax secretion. Plant Physiol 145:1345–1360
Parani M, Sairam S, Myers R, Weirich H, Smith B, Leaman DW, Goldman SL (2004) Microarray
analysis of nitric oxide responsive transcripts in Arabidopsis. Plant Biotechnol J 2:359–366
Patterson SE (2001) Cutting loose: abscission and dehiscence in Arabidopsis. Plant Physiol
126:494–500
Patterson SE, Bleecker AB (2004) Ethylene-dependant and -independent processes associated
with floral organ abscission in Arabidopsis. Plant Physiol 134:194–203
Pelletier G, Budar F (2007) The molecular biology of cytoplasmically inherited male sterility and
prospects for its engineering. Curr Opin Biotechnol 18:121–125
Peña L, Martı́n-Trillo M, Juárez J, Pina JA, Navarro L, Martı́nez-Zapatero JM (2001) Constitutive
expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time.
Perez-Prat E, van Lookeren Campagne MM (2002) Hybrid seed production and the challenge of
propagating male sterile plants. Trends Plant Sci 7:199–203
Pollard M, Beisson F, Li YH, Ohlrogge JB (2008) Building lipid barriers: biosynthesis of cutin and
suberin. Trends Plant Sci 13:236–246
Powell ALT, Kalamaki MS, Kurien PA, Gurrieri S, Bennett AB (2003) Simultaneous transgenic
suppression of LePG and LeExp1 influences fruit texture and juice viscosity in a fresh market
tomato variety. J Agric Food Chem 51:7450–7455
Purves WK, Orians GH, Heller HC, Sadava D (eds) (1997) Life: the science of biology. W.H.
Freeman, San Francisco, CA
Putterill J, Laurie R, Macknight R (2004) It’s time to flower: the genetic control of flowering time.
Bioessays 26:363–373
Qiao J, Mitsuhara I, Yazaki Y, Sakano K, Gotoh Y, Miura M, Ohashi Y (2002) Enhanced
resistance to salt, cold and wound stresses by overproduction of animal cell death suppressors
bcl-xl and ced-9 in tobacco cells – their possible contribution through improved function of
organella. Plant Cell Physiol 43:992–1005
Raghavan V (1997) Pollen abortion and male sterility. In: Molecular embryology of flowering
plants. Cambridge University Press, Cambridge, pp 120-151
Reape TJ, McCabe PF (2008) Apoptotic-like programmed cell death in plants. New Phytol
180:13–26
Reape TJ, Molony EM, McCabe PF (2008) Programmed cell death in plants: distinguishing
between different modes. J Exp Bot 59:435–444
Richardson A, Boscari A, Schreiber L, Kerstiens G, Jarvis M, Herzyk P, Fricke W (2007) Cloning
and expression analysis of candidate genes involved in wax deposition along the growing
barley (Hordeum vulgare) leaf. Planta 226:1459–1473
Rick CM (1948) Genetics and development of nine male-sterile tomato mutants. Hilgardia
18:599–633
Rivero RM, Kojima M, Gepstein A, Sakakibara H, Mittler R, Gepstein S, Blumwald E (2007)
Delayed leaf senescence induces extreme drought tolerance in a flowering plant. Proc Natl
Acad Sci USA 104:19631–19636
Roberts JA, Elliott KA, Gonzalez-Carranza ZH (2002) Abscission, dehiscence, and other cell
separation processes. Annu Rev Plant Biol 53:131–158
Rodrı́guez-Falcón M, Bou J, Prat S (2006) Seasonal control of tuberization in potato: conserved
elements with the flowering response. Annu Rev Plant Biol 57:151–180
Rogers HJ (2006) Programmed cell death in floral organs: how and why do flowers die? Ann Bot
97:309–315
Rothstein SJ (2007) Returning to our roots: making plant biology research relevant to future
challenges in agriculture. Plant Cell 19:2695–2699
Ruiz ON, Daniell H (2005) Engineering cytoplasmic male sterility via the chloroplast genome.
Sabar M, De PR, Kouchkovsky Y (2000) Complex I impairment respiratory compensations and
photosynthetic decrease in nuclear and mitochondrial male sterile mutants of Nicotiana
sylvestris. Plant Physiol 124:1239–1250
Saladie M, Matas AJ, Isaacson T, Jenks MA, Goodwin SM, Niklas KJ, Xiaolin R, Labavitch JM,
Shackel KA, Fernie AR, Lytovchenko A, O’Neill MA, Watkins CB, Rose JKC (2007) A
reevaluation of the key factors that influence tomato fruit softening and integrity. Plant Physiol
144:1012–1028
Samuels L, Kunst L, Jetter R (2008) Sealing plant surfaces: cuticular wax formation by epidermal
cells. Annu Rev Plant Biol 59:683–707
Sanders PM, Sanders PM, Lee PY, Biesgen C, Boone JD, Beals TP, Weiler EW, Goldberg RB
(2000) The Arabidopsis DELAYED DEHISCENCE1 gene encodes an enzyme in the jasmonic
acid synthesis pathway. Plant Cell 12:1041–1061
Sarowar S, Oh HW, Cho HS, Baek KH, Seong ES, Joung YH, Choi GJ, Lee S, Choi D (2007)
Capsicum annuum CCR4-associated factor CaCAF1 is necessary for plant development and
defence response. Plant J 51:792–802
Savin KW, Baudinette SC, Graham MW, Michael MZ, Nugent GD, Lu CY, Chandler SF, Cornish
EC (1995) Antisense ACC oxidase RNA delays carnation petal senescence. HortScience
30:970–972
Schnable PS, Wise RP (1998) The molecular basis of cytoplasmic male sterility and fertility
restoration. Trends Plant Sci 3:175–180
Schnurr J, Shockey J, Browse J (2004) The acyl-CoA synthetase encoded by LACS2 is essential
for normal cuticle development in Arabidopsis. Plant Cell 16:629–642
Schuch W, Kanczler J, Robertson D, Hobson G, Tucker G, Grierson D, Bright S, Bird C (1991)
Fruit quality characteristics of transgenic tomato fruit with altered polygalacturonase activity.
HortScience 26:1517–1520
Sexton R, Roberts JA (1982) Cell biology of abscission. Annu Rev Plant Physiol 33:133–162
Seymour G, Poole M, Manning K, King GJ (2008) Genetics and epigenetics of fruit development
and ripening. Curr Opin Plant Biol 11:58–63
Shaw JF, Chen HH, Tsai MF, Kuo CI, Huang LC (2002) Extended flower longevity of Petunia
hybrida plants transformed with boers, a mutated ERS gene of Brassica oleracea. Mol Breed
9:211–216
Shepherd T, Griffiths DW (2006) The effects of stress on plant cuticular waxes. New Phytol
171:469–499
Shikanai T (2006) RNA editing in plant organelles: machinery physiological function and evolu-
tion. Cell Mol Life Sci 63:698–708
Shou H, Bordallo P, Fan JB, Yeakley JM, Bibikova M, Sheen J, Wang K (2004a) Expression of an
active tobacco mitogen-activated protein kinase kinase kinase enhances freezing tolerance in
transgenic maize. Proc Natl Acad Sci USA 101:3298–3303
Shou H, Bordallo P, Wang K (2004b) Expression of the Nicotiana protein kinase (NPK1)
enhanced drought tolerance in transgenic maize. J Exp Bot 55:1013–1019
Sieber P, Schorderet M, Ryser U, Buchala A, Kolattukudy P, Metraux JP, Nawrath C (2000)
Transgenic Arabidopsis plants expressing a fungal cutinase show alterations in the structure
and properties of the cuticle and postgenital organ fusions. Plant Cell 12:721–737
Singh M, Hamel N, Menassa R, Li X-Q, Young B, Jean M, Landry BS, Brown GG (1996) Nuclear
genes associated with a single Brassica CMS restorer locus influence transcripts of three
different mitochondrial gene regions. Genetics 143:505–516
Skinner JS, Meilan R, Ma C, Strauss SH (2003) The Populus PTD promoter imparts floral-
predominant expression and enables high levels of floral-organ ablation in Populus, Nicotiana
and Arabidopsis. Mol Breed 12:119–132
Smith DL, Abbott JA, Gross KC (2002) Down-regulation of tomato ß-galactosidase 4 results in
decreased fruit softening. Plant Physiol 129:1755–1762
Sofi PA, Rather AG, Shafiq AW (2007) Genetic and molecular basis of cytoplasmic male sterility
in maize. Commun Biometr Crop Sci 2:49–60
Spena A, Estruch JJ, Prinsen E, Nacken W, Onckelen H, Van Sommer H (1992) Anther-specific
expression of the rolB gene of Agrobacterium rhizogenes increases IAA content in anthers and
alters anther development and whole flower growth. Theor Appl Genet 84:520–527
Srivastava A, Handa AK (2005) Hormonal regulation of tomato fruit development: a molecular
perspective. J Plant Growth Regul 24:67–82
Stead AD, van Doorn WG, Jones ML, Wagstaff C (2006) Flower senescence: fundamental and
applied aspects. Annu Plant Rev 20:261–296 Ainsworth C (ed) Flowering and Its Manipula-
tion. Blackwell Publ
Stenvik GE, Butenko MA, Urbanowicz BR, Rose JKC, Aalen RB (2006) Overexpression of
INFLORESCENCE DEFICIENT IN ABSCISSION activates cell separation in vestigial
abscission zones in Arabidopsis. Plant Cell 18:1467–1476
Stern DB, Hanson MR, Barkan A (2004) Genetics and genomics of chloroplast biogenesis: maize
as a model system. Trends Plant Sci 9:293–301
Takakura Y, Che FS, Ishida Y, Tsutsumi F, Kurotani K, Usami S, Isogai A, Imaseki H (2008)
Expression of a bacterial flagellin gene triggers plant immune responses and confers disease
resistance in transgenic rice plants. Mol Plant Pathol 9:525–529
Takeda T, Suwa Y, Suzuki M, Kitano H, Ueguchi-Tanaka M, Ashikari M, Matsuoka M, Ueguchi
C (2003) The OsTB1 gene negatively regulates lateral branching in rice. Plant J 33:513–520
Tan L, Li X, Liu F, Sun X, Li C, Zhu Z, Fu Y, Cai H, Wang X, Xie D, Sun C (2008) Control of a
key transition from prostrate to erect growth in rice domestication. Nat Genet 40:1360–1364
Tang X, Xie M, Kim YJ, Klessig DF, Martin GB (1999) Overexpression of Pto activates defense
responses and confers broad resistance. Plant Cell 11:15–29
Tang D, Simonich MT, Innes RW (2007) Mutations in LACS2, a long-chain acyl-coenzyme A
synthetase, enhance susceptibility to avirulent Pseudomonas syringae but confer resistance to
Botrytis cinerea in Arabidopsis. Plant Physiol 144:1093–1103
Tanksley SD (2004) The genetic, developmental, and molecular bases of fruit size and shape
variation in tomato. Plant Cell 16:S181–S189
Taylor LP, Jorgensen R (1992) Conditional male fertility in chalcone synthase-deficient petunia.
J Hered 83:11–17
Taylor EJ, Whitelaw JA (2001) Signals in abscission. New Phytol 151:323–339
Thakur BR, Singh RK, Handa AK (1996a) Effects of an antisense pectin methylesterase gene on
the chemistry of pectins in tomato (Lycopersicon esculentum) fruit juice. J Agri Food Chem
44:628–630
Thakur BR, Singh RK, Tieman DM, Handa AK (1996b) Quality of processed tomato products
from transgenic tomato fruits with reduced levels of pectin methylesterase activity. J Food Sci
61:245–248
Thakur BR, Singh RK, Handa AK (1997) Chemistry and uses of pectin – A review. Crit Rev Food
Sci Nutr 37:47–73
Tieman DM, Handa AK (1994) Reduction in pectin methylesterase activity modifies tissue
integrity and cation levels in ripening tomato (Lycopersicon esculentum Mill.) fruits. Plant
Physiol 106:429–436
Tieman DM, Harriman RW, Ramamohan G, Handa AK (1992) An antisense pectin methylesterase
gene alters pectin chemistry and soluble solids in tomato fruit. Plant Cell 4:667–679
Troncoso-Rojas R, Tiznado-Hernández ME (2007) Natural compounds to control fungal diseases
in fruits and vegetables. In: Troncoso-Rojas R, Tiznado-Hernández ME, González-León
A (eds) Recent advances in alternative postharvest technologies to control fungal diseases
in fruits and vegetables. Editorial Transworld Research Network, Kerala, pp 127–156
ISBN:81-7895-244-0
Tsuwamoto R, Fukuoka H, Takahata Y (2008) GASSHO1 and GASSHO2 encoding a putative
leucine-rich repeat transmembrane-type receptor kinase are essential for the normal develop-
ment of the epidermal surface in Arabidopsis embryos. Plant J 54:30–42
Tucker ML, Sexton R, del Campillo E, Lewis LN (1988) Characterization of a cDNA clone and
regulation of gene expression by ethylene and auxin. Plant Physiol 88:1257–1262
Turck F, Fornara F, Coupland G (2008) Regulation and identity of florigen: Flowering Locus T
moves center stage. Annu Rev Plant Biol 59:573–594
Ukitsu H, Kuromori T, Toyooka K, Goto Y, Matsuoka K, Sakuradani E, Shimizu S, Kamiya A,
Imura Y, Yuguchi M, Wada T, Hirayama T, Shinozaki K (2007) Cytological and biochemical
analysis of COF1, an Arabidopsis mutant of an ABC transporter gene. Plant Cell Physiol
48:1524–1533
Valverde F, Mouradov A, Soppe W, Ravenscroft D, Samach A, Coupland G (2004) Photoreceptor
regulation of CONSTANS protein in photoperiodic flowering. Science 303:1003–1006
Van der Knaap E, Tanksley SD (2003) The making of a bell pepper-shaped tomato fruit:
identification of loci controlling fruit morphology in Yellow Stuffer tomato. Theor Appl
Genet 107:139–147
Van der Knaap E, Lippman ZB, Tanksley SD (2002) Extremely elongated tomato fruit controlled
by four quantitative trait loci with epistatic interactions. Theor Appl Genet 104:241–247
Van der Meulen-Muisers JJM, Van Oeveren JC, Jansen J, Van Tuyl JM (1999) Genetic analysis of
postharvest flower longevity in Asiatic hybrid lilies. Euphytica 107:149–157
van der Vossen E, Sikkema A, Hekkert BL, Gros J, Stevens P, Muskens M, Wouters D, Pereira A,
Stiekema W, Allefs S (2003) An ancient R gene from the wild potato species Solanum
bulbocastanum confers broad-spectrum resistance to Phytophthora infestans in cultivated
potato and tomato. Plant J 36:867–882
van Doorn WG, Woltering EJ (2005) Many ways to exit? Cell death categories in plants. Trends
van Doorn WG, Balk PA, van Houwelingen AM, Hoeberichts FA, Hall RD, Vorst O, van der
Schoot C, van Wordragen MF (2003) Gene expression during anthesis and senescence in Iris
flowers. Plant Mol Biol 53:845–863
Verlinden S, Boatright J, Woodson WR (2002) Changes in ethylene responsiveness of senescence-
related genes during carnation flower development. Physiol Plant 116:503–511
Vicente AR, Saladie M, Rose JKC, Labavitch JM (2007) The linkage between cell wall meta-
bolism and fruit softening: looking to the future. J Sci Food Agric 87:1435–1448
Vrebalov J, Ruezinsky D, Padmanabhan V, White R, Medrano D, Drake R, Schuch W, Giovannoni
J (2002) A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin)
locus. Science 296:343–346
Wang KLC, Li H, Ecker JR (2002) Ethylene biosynthesis and signaling networks. Plant Cell
16:S131–S151
Wang Z, Zou Y, Zhang X, Chen L, Wu H, Su D, Chen Y, Guo J, Luo D, Long Y, Zhong Y, Liu Y-
G (2006a) Cytoplasmic male sterility of rice with Boro II cytoplasm is caused by a cytotoxic
peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing.
Wang ZF, Ying T, Zhang Y, Bao B, Huang X (2006b) Characteristics of transgenic tomatoes
antisensed for the ethylene receptor genes LeERT1 and LeERT2. J Zhejiang Univ Sci B 7:591–595
Ward ER, Ryals JA, Miflin BJ (1993) Chemical regulation of transgene expression in plants. Plant
Mol Biol 22:361–366
Wei H, Meilan R, Brunner AM, Skinner JS, Ma C, Strauss SH (2006) Transgenic sterility in
Populus: expression properties of the poplar PTLF, Agrobacterium NOS and two minimal 35S
promoters in vegetative tissues. Tree Physiol 26:401–410
Whitelaw CA, Lyssenko NN, Chen L, Zhou D, Mattoo AK, Tucker ML (2002) Delayed abscission
and shorter internodes correlate with a reduction in the ethylene receptor LeETR1 transcript in
transgenic tomato. Plant Physiol 128:978–987
Wilkie JD, Sedgley M, Olesen T (2008) Regulation of floral initiation in horticultural trees. J Exp
Bot 59:3215–3228
Wilkinson JQ, Lanahan M, Yen H, Giovannoni J, Klee H (1995) An ethylene-inducible compo-
nent of signal transduction encoded by Never-ripe. Science 270:1807–1809
Wilkinson JQ, Lanahan MB, Clark DG, Bleecker AB, Chang C, Meyerowitz EM, Klee HJ (1997)
A dominant mutant receptor from Arabidopsis confers ethylene insensitivity in heterologous
plants. Nat Biotechnol 15:444–447
Williams B, Dickman M (2008) Plant programmed cell death: can’t live with it; can’t live without
it. Mol Plant Pathol 9:531–544
Wise RP, Gobelman-Werner K, Pei D, Dill CL, Schnable PS (1999) Mitochondrial transcript
processing and restoration of male fertility in T-cytoplasm maize. J Hered 90:380–385
Wong CE, Singh MB, Bhalla PL (2009) Molecular processes underlying floral transition in the
soybean shoot apical meristem. Plant J 57:832–845
Woolley LC, James DJ, Manning K (2001) Purification and properties of an endo-beta-1,
4-glucanase from strawberry and down-regulation of the corresponding gene. Planta 214:
11–21
Xiao H, Jiang N, Schaffner E, Stockinger EJ, van der Knaap E (2008) A retrotransposon-mediated
gene duplication underlies morphological variation of tomato fruit. Science 319:1527–1530
Xiong AS, Yao QH, Peng RH, Li X, Han PL, Fan HQ (2005) Different effects on ACC oxidase
gene silencing triggered by RNA interference in transgenic tomato. Plant Cell Rep 23:639–646
Xu P, Rogers SJ, Roossink MJ (2004) Expression of antiapoptotic genes bcl-xL and ced-9 in
tomato enhances tolerance to viral-induced necrosis and abiotic stress. Proc Natl Acad Sci
USA 101:15805–15810
Yang S, Gao M, Xu C, Gao J, Deshpande S, Lin S, Roe BA, Zhu H (2008) Alfalfa benefits from
Medicago truncatula: the RCT1 gene from M. truncatula confers broad-spectrum resistance to
anthracnose in alfalfa. Proc Natl Acad Sci USA 105:12164–12169
Yoshihara T, Iino M (2007) Identification of the gravitropism-related rice gene LAZY1 and
elucidation of LAZY1-dependent and -independent gravity signaling pathways. Plant Cell
Physiol 48:678–688
Yu B, Lin Z, Li H, Li X, Li J, Wang Y, Zhang X, Zhu Z, Zhai W, Wang X, Xie D, Sun C (2007)
TAC1, a major quantitative trait locus controlling tiller angle in rice. Plant J 52:891–898
Yuceer C, Kubiske ME, Harkess RL, Land SB Jr (2003) Effects of induction treatments on
flowering in Populus deltoides. Tree Physiol 23:489–495
Zapata JM, Guera A, Esteban-Carrasco A, Martin M, Sabate B (2005) Chloroplasts regulate leaf
senescence: delayed senescence in transgenic ndhF-defective tobacco. Cell Death Differ
12:1277–1284
Zeidler D, Zaähringer U, Gerber I, Dubery I, Hartung T, Bors W, Hutzler P, Durner J (2004) Innate
immunity in Arabidopsis thaliana: lipopolysaccharides activate nitric oxide synthase (NOS)
and induce defence genes. Proc Natl Acad Sci USA 101:15811–15816
Zhang H, Li S, Yi P, Wan C, Chen Z, Zhu Y (2007a) A Honglian CMS line of rice displays
aberrant F0 of F0F1-ATPase. Plant Cell Rep 26:1065–1071
Zhang JY, Broeckling CD, Sumner LW, Wang ZY (2007b) Heterologous expression of two
Medicago truncatula putative ERF transcription factor genes, WXP1 and WXP2, in Arabi-
dopsis led to increased leaf wax accumulation and improved drought tolerance, but differential
response in freezing tolerance. Plant Mol Biol 64:265–278
Zhao B, Lin X, Poland J, Trick H, Leach J, Hulbert S (2005) A maize resistance gene functions
against bacterial streak disease in rice. Proc Natl Acad Sci USA 102:15383–15388
Zheng Z, Xia Q, Dauk M, Shen W, Selvaraj G, Zou J (2003) Arabidopsis AtGPAT1 a member of
the membrane-bound glycerol-3-phosphate acyltransferase gene family is essential for tapetum
differentiation and male fertility. Plant Cell 15:1872–1887
Zhou D, Kalaitzis P, Mattoo AK, Tucker ML (1996a) The mRNA for an ETR1 homologue in
tomato is constitutively expressed in vegetative and reproductive tissues. Plant Mol Biol
30:1331–1338
Zhou D, Mattoo AK, Tucker ML (1996b) Molecular cloning of a tomato cDNA (Genbank
U47279) encoding an ethylene receptor. Plant Physiol 110:1435–1436
Zipfel C, Robatzek S, Navarro L, Oakeley EJ, Jones JD, Felix G, Boller T (2004) Bacterial disease
resistance in Arabidopsis through flagellin perception. Nature 428:764–767
Zou J, Zengxiang Chen Z, Zhang S, Zhang W, Jiang G, Zhao X, Zhai W, Pan X, Zhu L (2005)
Characterizations and fine mapping of a mutant gene for high tillering and dwarf in rice (Oryza
sativa L.). Planta 222:604–612
Zou J, Zhang S, Zhang W, Li G, Chen Z, Zhai W, Zhao X, Pan X, Xie Q, Zhu L (2006) The rice
HIGH-TILLERING DWARF1 encoding an ortholog of Arabidopsis MAX3 is required for
negative regulation of the outgrowth of axillary buds. Plant J 48:687–698
Chapter 6
Transgenics for Biofuel Crops
Anjanabha Bhattacharya, Pawan Kumar, and Rippy Singh
6.1 Introduction
Fossil fuels, like petroleum and coal, are fast-depleting, nonrenewable sources that
were produced over million years ago, when the higher- and lower-order plants
were buried under the surface of the earth by volcanic activities and sedimentation.
These were further acted upon by microorganisms leading to the formation of
nonrenewable fuels. There is an urgent need to find alternative sources of energy
in order to cope with the energy demands of the human race and the much-debated
global climate change in the coming years. So intense is the problem that the world
has been divided into energy-independent producers and energy-dependent con-
sumer economies, triggering trade war and political unrest, leading to monopoly
over production and pricing issues. In such a scenario, there is a greater urge for the
fossil-fuel-dependent economies to look for alternative sources of energy to
become more self-reliant when it comes to their own energy needs (Hill et al.
2006). Several alternative energy sources, which exist and are being exploited
today, include wind, solar, hydroelectric, nuclear energy and possibly methane
gas reserves entrapped in the underground seabed. These may provide a part of
the solution. There are practical problems associated with harnessing, storage and
transport of these new renewable resources compared with other popular non-
renewable sources.
Biofuels or agrofuels are basically carbon-derived fuels (solid, liquid or gaseous
state) where the source of carbon is either plants or animals, and are therefore
indirectly solar energy sources. They can be derived from animal fats, vegetable
oils (biodiesel), or from agro-residues (bioethanol, biomethanol, or biobutanol), or
can be derived from solid forms (like refuse fuels, pellets, wood, sewage, and
A. Bhattacharya (*)
National Environmental Sound Production Agriculture Laboratory, University of Georgia, Tifton,
GA 31794, USA
e-mail: anjan@uga.edu

250 A. Bhattacharya et al.
Quadrillion Btu
250
History Projections
200
Liquids
150
100 Coal
Natural Gas Renewables

50
Nuclear
0
1990 2000 2005 2010 2020 2030
Fig. 6.1 Present and projected global energy demand. Adapted from Energy Information Admin-
istration (EIA), Official Energy Statistics from US government site available at http://www.eia.
doe.gov/oiaf/ieo/world.html. Original source: International energy annual report 2005 and EIA,
World energy projection plus (2008).
briquettes) (Petrou and Pappis 2009), which can be used to produce energy. They
are generally less efficient than fossil fuels but their usage results in less emission of
greenhouse gasses (Vogt et al. 2009). Besides, biofuels are termed green fuels
because they emit less carbon dioxide compared to the fossil fuels, contain no sulfur
and thus decrease the global warming effect (Vogt et al. 2009). They are bio-
degradable and contribute to sustainability (Puppan 2002).
The concept of biofuel usage in automobiles is in fact not new, but dates back to
the early nineteenth century, when Rudolf Diesel used peanut oil to drive engines
used in agriculture machinery (ijalwan et al. 2006; Murugesan et al. 2009). How-
ever, the availability of low-cost fossil fuels in practical abundance resulted in the
neglect of biofuel research. Rapid industrialization and fast economic growth of
several developing nations, including China and India, in the twentieth century
resulted in greater consumption of once abundant resources, which led to investiga-
tions of the feasibility of alternative fuels, and biofuels in particular. The annual
global fossil fuel consumption and biodiesel production is estimated to be about
4.018 and 0.107 billion tons, respectively (Demirbas 2008), and is increasing every
year (Fig. 6.1). Initially, biofuel production came from food crops like sugarcane,
sunflower, soybean, sugarbeet, rapeseed, and corn, which have been blamed for
triggering a food crisis in recent years. This raises the following question: Which
should be given a priority when it comes to making a choice between energy and
food resources? The answer to this paradox lies in the recently identified potential
biofuel crops, for example, poplar, hemp, members of the grass family (switch grass,
burmuda grass, miscanthus, prairie and wheat) and oil-rich crops like Jatropha
and Pongamia sp.
6 Transgenics for Biofuel Crops 251
Biofuels
Solid Liquid Gaseous
Briquettes Bioethanol
Pellets Biomethanol Biodiesel Biogas
Wood Biobutanol
Municipal
Sugar and
Agriculture & Oil rich crop Decomposing
Starch rich
Forestry plants Biomass
crop plants
residue
Fig. 6.2 Classification of biofuels
A prerequisite to using crops in efficient biofuel production is to design the most

effective way to break down complex carbohydrates into simple sugars. One of the
ways to circumvent this issue is to biotechnologically engineer transgenic crops
with modified lignin, tannin and cellulosic biosynthetic pathway, which will make
their decomposition into biofuels quicker and more efficient when acted upon by
microbes. Plants provide us with food, feed and fiber. We can genetically engineer
parts of plants often thrashed away, like the straw in wheat and paddy, stalks of
cotton, corn and several other crop plants, to degrade more quickly than they do
naturally. This will not only enhance the rate of production of biofuels but also
improve efficiency of the whole production process. At the same time, we can also
genetically engineer the microbial strains used in this process to increase their rate
of action.
Some biofuel-related terminology has been briefly explained in this section and
illustrated in Fig. 6.2.
6.1.1 Bioalcohol
The simple and complex polysaccharide plant food reserves are broken down into
simple sugars, and fermented to produce bioalcohol. They are specifically
designated as Bioethanol and Biobutanol.
ðPhotosynthesisÞ
1:6CO2 þ 6H2 O þ Solar energy ! C6 H12 O6 þ 6O2
Glucose=simplesugar
ðFermentationÞ
2: C6 H12 O6 ! 2C2 H5 OH þ 2CO2 þ energy
Glucose ðBioÞethanol
6.1.2 Biogas and Biohydrogen
Biogas is produced from decomposed organic substances by pyrolysis in the

presence of anaerobic bacteria, releasing gaseous substances (generally a mixture
of hydrogen, methane and carbon dioxide). Recently, scientists have developed
hydrogen and nitrogen-fixing bacteria capable of producing biogas.
6.1.3 Biodiesel
Plant lipids are composed of long-chain fatty acids which are broken down into
short-chain fatty acids by esterification to produce biodiesels.
Typical source material for biofuels include:
(a) Starch and sugars. The first generation of biofuel crops included food crops
like sugarcane, corn, barley, wheat and cassava.
(b) Lignocellulasic material. Unlocking the cellulose from plant cell walls and
efficiently converting it to ethanol is the key to make ethanol a universal, inexpen-
sive form of fuel for the future. Aquatic plants like lotus, lily, etc. are naturally low
in lignin content, which is less than 10% (Gunnarsson and Petersen 2007), and they
grow at a rapid pace (Ripley et al. 2006).
(c) Plant lipids. Crop plants like soybean, corn, sunflower, oil palm, castor seed,
rapeseed, and mustard, along with primitive photosynthetic microrganisms like
algae (including microalgae, kelps and fucoids), cynobacteria and phytoplanktons,
are naturally rich in oil or starch, which can be converted into biofuel in mutistage
processing. Biodiesel is better than conventional diesel fuel with respect to sulfur
content, flash point, aromatic content, and biodegradability (Hanna et al. 2005).
6.2 Transgenics Approach to Biofuels
Conventional breeding has been largely responsible for the introduction of novel
traits and has played an important role in the development of new cultivars. This
includes domestication of wild relatives and selection of novel sports from popular
cultivated species. However, complex polygenic traits are difficult to manipulate
using this method and can take considerable time (Zuker et al., 1995; Mishra and
Srivastava 2004). Crop plants in the past were never selected on the basis of their
amenability for biofuel production, and traits such as low lignin content or high
cellulose were lost systematically from the gene pool. Thus, limited gene pool and
failure of distant crosses in conventional breeding have lead to the exploitation of
genetic transformation in generating plants of relevance for the rapidly growing
biofuel industry. Growth and crop productivity are polygenic traits making their
breeding difficult under natural conditions. Successful genetic manipulation
depends upon the integration of transgene(s) into the host genome, the regeneration
capacity of the transformed cells often via tissue culture, and subsequent expression
and integration over several generations with exceptions in vegetatively propagated
plants (Robinson 1998). However, a transgenic crop production approach of mod-
ifying plant architecture by making available the gene of interest (manipulating
biofuel productivity traits or pathways) in a cloned form and robust plant regenera-
tion and transformation system paves the way for integration of transgene(s) in a
highly directed manner. This will ensure the development of new transgenic
varieties previously difficult to obtain through the conventional breeding.
With the advent of high-throughput, low-cost sequencing technology, genomes
of many plant species have been sequenced and annotated. An example of the
possible pathway, which could be adopted to genetically modify crop plants to
yield biofuel is described in Fig. 6.3. Many genes of importance in relation to
polysaccharide biosynthesis pathways have been identified using database infor-
mation, and a number of expression studies have been conducted to verify such
results (Ragauskas et al. 2006). For example, overexpression of expansin genes,
which bring about the loosening of the cell walls of plants, may be considered for
modification in biofuel crops (Cosgrove 2005). Also, emphasis must be given to
identification of quantitative trait loci (QTLs) controlling lignification in plants,
and breeding programs for biofuel production must emphasize selection of culti-
vars with low lignin content. RNA interference (RNAi) and antisense approaches
can also be adopted to selectively silence the genes involved in complex carbo-
hydrate synthesis.
6.2.1 Manipulating Genes and Pathways Involved in Starch

and Sugar Metabolism
Starch is produced by all green plants for energy storage (Fig. 6.4a). It is a large
polysaccharide made up of glucose linked via a-1,4 and a-1,6 glycosidic linkages
(amylose and amylopectin). These are stored in plant in a form of dense semicrys-
talline structure. Gelatinization of starch is required for enzymatic digestion and
fermentation, which can be achieved by heating starch at 60 C. Hydrolysis by
glucoamylase enzyme converts the starch molecule into D-glucose. Sucrose, on the
other hand, is a disaccharide of glucose and fructose. Enzymatic hydrolysis of
sucrose is catalyzed by invertase, converting sucrose into glucose and fructose
(both C6H12O6).
Sequence data of model plants and

other species available online
(NCBI, TIGR, EBI and AtEnsembl)
Identify major genes and / QTL involved in complex polysaccharide biosynthesis

by bioinformatics and subsequently find analogs or orthologs in other plant
species.
Generate ectopic expressor /knockout (T-DNA insertion or by

TILLING)/antisense/ RNAi induced silenced lines to hasten tissue
degradation
Genetically engineer complex metabolic pathway like cellulose, lignin, tannin

biosynthesis. Harvest and subsequent storage of the biomass
Pretreatment with genetically modified micro- Genetically manipulate

organism and other enhancer chemicals microorganism / enzymes
Recovery of product and

residue mix Residue can be used
as sillage or
biofertilzer or solid /
Liquid Biofuel biohydrogen
biofuels
Fig. 6.3 Possible pathway to genetically engineer crops to yield biofuel
C12 H22 O11 ! C6 H12 O6 þ C6 H12 O6

ðSucroseÞ ðGlucoseÞ ðFructoseÞ
Enzymatic hydrolysis is followed by a fermentation process for the production

of bioethanol. Commercial yeast such as Saccharomyces cerevisiae converts glu-
cose molecule into ethanol under anaerobic conditions.
ðFermentationÞ
C6 H12 O6 ! 2 C2 H5 OH þ2CO2
ðGlucoseÞ ðEthanolÞ
a
Sucrose
Cytosol
Uridine diphosphate
Glucose
Glucose 1-
phosphate Adenosine glucose
pyrophosphorylase
Adenosine diphosphate
glucose
Debranching
Plastid
enzymes
Amylose, Amylopectin
(Starch Granule)
Fig. 6.4 (Continued)

Fig. 6.4 Strategies for manipulating (a) Starch (b) Cellulose (c) Lignin (d) Lipids
Therefore, cloning genes responsible for rapid conversion to ethanol and then
expression of such genes in plant system will enhance the production of ethanol.
Myers et al. (2000) reported that transgenic plants with suppressed debranching
enzyme produce soluble phytoglycogen, thus no gelatinization by pretreatment is
required in this condition. Aside from genetic engineering of plants for reducing the
production costs, specific genetic manipulation may be required depending on the
source categories of biofuel. For example, in sugarcane, sugar content was
increased by targeting sugar accumulated in the vacuoles and subsequently con-
verted to isomaltulose by the enzymatic rearrangement of the glycosidic linkage
from a (1–2)-fructoside in sucrose to a (1–6)-fructoside (Wu and Birch 2007). This
new arrangement resulted in doubling the total sugar concentration in these trans-
genic lines (Gressel 2008). The increase in sugars in transgenic lines directly
translates into higher yield of biofuel. Smith (2008) reported that manipulation of
ADPglucose pyrophosphorylase (which plays a role in the sugar signaling pathway)
to increase the yields of starch have been met with limited success. They also
advocated partitioning of photosynthate in storage organs and increasing the flux of
photoassimilate from source to sink. Earlier studies concentrated on manipulating
carbohydrate composition of major food crops by altering the enzymes which act on
photosynthesis.
6.2.2 Manipulating Genes and Pathways Involved in Cellulose

and Lignin Biosynthesis
With the recent advent of increasing prices of food products and their shortage, the
first generation of biofuel crops were discouraged. Instead, research was vastly
concentrated on non-food crops and waste (nonutilizable biomass) from food crops,
which could be converted into biofuels without compromising the food needs of
mankind. This strategy could lead to potential sources of employment, and at the
same time help in land reclamation with little or no subsequent maintenance. Here,
we needed more advanced technologies to extract biofuels.
Cellulose (long chains of glucose molecules bound by b-glycosidic bonds) is a
component of plant cell walls, making up between a quarter and half of them in
terms of mass. However, it is not easy to extract, as the cellulose in a plant is
embedded in a cross-linked matrix with other components such as hemicellulose
(branched polymers of xylose, arabinose, galactose, mannose, and glucose that bind
bundles of cellulose strands together), lignin (a complex polymer into which the
above-described bundles are matrixed and thus unavailable for conversion to
fermentable sugar), pectins, xylene, and proline-rich proteins. Further, lignin limits
the access of cellulase enzymes needed for fermentation of sugars.
The area of ethanol production showing the most promise, both in terms of
potential energy savings and in terms of national security interests, is ethanol
derived from cellulose. Current processes for producing cellulosic ethanol are
time-consuming, low-yielding, and, above all, expensive. The process of cellulose
biochemistry is still poorly understood. Cellulose degradation pathways require

multiple steps of varying temporal, environmental, and efficiency penalties. The
major hurdle to producing biofuel is the high cost of conversion of cellulose and
hemicellulose into ethanol. Therefore, the price of cellulosic ethanol is two- to
threefold higher than bioethanol produced from corn grains. These production costs
could be cut down by bioengineering crop plants in such a way that the necessary
cell wall degrading enzymes, such as cellulase and hemicellulase, are produced
within the plant system, thereby reducing the dependency on microbial bioreactors
for enzymes. The concept of self-destructive plants could be coined to such
transgenic varieties. Ziegler et al. (2000) advocated the restriction of the hydrolytic
enzymes produced by the transgenic plants in the apoplast region of the cell to
avoid reaction with other symplast elements.
Further, Schillberg et al. (2003) advocated the potential of selectively degrading
protein bodies in subcellular compartments of the endoplasmic reticulum (ER),
thereby preventing interference with other cellular activities. Afterwards, the same
enzyme will help in increasing the degradation process upon extraction from the
transgenic plants. It is very important to ensure that enzymes produced for cell wall
degradation should not interfere with other cellular activities. The other strategy
could be directing the expression of these enzymes in the chloroplast of the plant
cells. Chloroplast transformation with transgenes has been shown to be consistently
predictable and less prone to silencing. The other strategy could involve genetically
engineering plants in a tissue-specific manner, so that the transgene does not
interfere with parts used for human consumption (Dai et al. 2000). Further cost
reduction could also be achieved by genetically engineering the cellulose biosyn-
thetic pathway in plants so that there is a minimal need for expensive pretreatment.
Another strategy is to upregulate the cellulose biosynthesis pathway enzymes for
increased polysaccharide production, which in turn will increase the yield of
cellulosic biofuel. Good et al. (2005) showed that ectopic expression of glutamate
dehydrogenate from bacteria resulted in increase in biomass of tobacco.
Oxenboll Sorensen et al. (2000) reported that pectin arrangement in rice and
potato plants was drastically changed (confirmed by Fourier transform infrared
microspectroscopy, immune-gold labeling, and fermentable sugar accumulation
studies) by the ectopic expression of endo-galactanase gene from fungus under
the influence of a tissue-specific promoter without altering the plant architecture.
Skjot et al. (2002) also reported decrease in arabinose content in the cell wall of
potato plants.
Sticklen (2008) reviewed the production of Acidothermus cellulolyticus E1
endo-1,4-b-glucanase in different crop plants for efficient conversion to biofuels,
which is a stepping stone in the production of biofuel from cellulosic material.
Attempts have been made to increase cellulose-binding molecules in plants, which
results in increase in cellulose production without subsequent increase in lignin
production. This may provide an opportunity to increase cellulose content in plants
and hence increase proportion of fermentable sugars (Fig. 6.4b). Another strategy
could be manipulating C3 mechanism of photosynthesis in favor of C4 mechanism,
which is present in the grass family and can only be grown in tropical regions.
Van Camp (2005) overexpressed two enzymes from cyanobacteria in tobacco,

resulting in enhanced rate of photosynthesis that translated into increase in biomass.
This could ensure equal distribution and usage of biomass over the globe. Jing et al.
(2004) reported increase in tree height and hence biomass by ectopically expressing
glutamine synthase gene (GS1).Also, manipulating genes for enhanced solar energy
capture and conversion of photosynthate to sugars, and genes which provide
endurance to plants growing under saline conditions, or land unutilizable for
cultivation, should be emphasized (Antizar-Ladislao and Turrion-Gomez 2008).
In plants, lignin (composed of phenylpropanoid groups) acts as a polymer
around the hemicellulose microfibrils, binding the cellulose molecules together
and protecting them against chemical degradation. Lignin cannot be converted
into sugars. Thus, it is not practical in biofuel production. Their degradation is a
high-energy process. A simplified illustration of genetic manipulation of lignin has
been described in Fig. 6.4c. Vanden Wymelenberg et al. (2006) reported a large
number of genes involved in breakdown of lignin from the fungus Phanerochaete
chrysosporium genome. Such genes could be expressed in plants to induce cell wall
breakdown in the latter stages of plant maturity. Ralph et al. (2006) reported genetic
manipulation of monolignins in such a way that they continue to provide cross-
linkage to cellulose and hemicellulose microfibrils required for the firm framework
of the plant and at the same time increase biomass conversion efficiency to biofuels.
Studies with the model plant Arabidopsis have shown that secondary wall
thickening is lost when knockout lines (nst1, nst2) are produced, without affecting
vascular bundle architecture (Mitsuda et al. 2007). This provides documented
evidence that lignin biosynthesis can be selectively altered without rendering the
plants prone to lodging by vascular collapse or increasing their susceptibility to
insects, pests, and disease.
The major share of the cost in biofuel production is the expense incurred in the
production of cellulase enzymes in microbial bioreactors. Secondly, the cost of
pretreating lignocellulosic matter for its breakdown to the intermediate compo-
nents, and subsequent removal of the lignin, is very high. Three families of enzymes
responsible for digesting lignin, laccases, manganese dependent peroxidases and
lignin peroxidases (Kirk and Farrell 1987) could be used in transgenic plant
production to produce plants with low lignin content amenable for biofuel produc-
tion. Li et al. (2003) showed that by downregulating a single lignin biosynthetic
gene, 4-hydroxycinnamoyl CoA ligase (4CL), a prominent reduction in lignin
composition was observed along with an increase in biomass. Ralph et al. (2006)
found that decreasing expression of 4-coumarate 3-hydroxylase (C3H) in alfalfa
(Medicago sativa) drastically altered the lignin profile and structure. This could
enhance the fermentation process in the production of biofuels. Gressel (2008)
reported the isolation of brown midrib (bmr) mutations in sweet-stemmed sorghum
and maize with low lignin content that were highly digestible by industrial carbo-
hydrases. Besides the grain component, a large portion of biomass is left unutilized
as lignocellulosic material. Lignin is a polyphenolic polymer (composed of mono-
lignin units) and is highly resistant to degradation. In a typical study, downregula-
tion of a lignin biosynthesis gene, cinnamyl alcohol dehydrogenase (CAD) in
alfalfa resulted in altered lignin biosynthesis, thus increasing the processibility

(Baucher et al. 1999).
Chabannes et al. (2001) found that reduction in expression of cinnamoyl CoA
reductase (CCR) in transgenic tobacco resulted in reduced lignin content with a
corresponding increase in glucose in plant cells. Blaschke et al. (2004) reported that
downregulation of O-methyl transferase (OMT) enzyme in Nicotiana tabacum
enhanced biomass content, though proportion of lignin content was decreased.
Ragauskas et al. (2006) suggested that often downregulation of a lignin biosynthe-
sis gene results in altered lignin architecture in transgenic plants. Similarly, Chen
and Dixon (2007) reported that downregulation of six different lignin biosynthetic
pathway enzymes in alfalfa may eliminate the pretreatment process associated with
extraction of fermentable sugars. Hu et al. (1999) reported alteration in lignin
content and subsequent increase in cellulose content in quaking aspen (Populus
tremuloides) by decreasing the enzyme hydroxycinnamate- CoA/5-hydroxyferu-
loyl-Co-A ligase. Bate et al. (1994) similarly advocated that suppression of phenyl-
alanine ammonia lyase (PAL) may decrease lignin content in poplar plants. Ralph
et al. (2006) found that flexible polymerization is possible in plants, whereby
monoligins can be actively substituted by polyphenols. Similarly, Riboulet et al.
(2009) identified lignin biosynthesis genes from microarray analysis in maize,
which are differentially expressed during plant development. Such information
will be vital in developing new transgenic crop programs and plant breeding.
This in turn may not affect the development process of plants, but will facilitate
the extraction of saccharides needed for biofuel production from the plants.
Among several other renewable sources biomass (includes residual non-food
parts of cultivated crop plants, garden and kitchen waste) is probably the most
promising alternative to fossil fuel resources. It has several advantages, such as it
will be sustainably available in the future and it is considered to be environmentally
friendly, since it has low net release of carbon dioxide and sulfur content. This will
also help us to mitigate the problem of landfilling in industrial and urban generated
waste. Biomass is a complex material primarily made up of cellulose (30–50%),
hemicellulose (20–40%) and lignins (15–30%). Complex carbohydrates like cellu-
lose and hemicellulose are first converted into their component sugars through a
hydrolysis process and then the sugars are anerobically fermented into biofuel such
as bioethanol. At present, only the cellulose and hemicellulose components of plant
biomass can be converted into biofuels by the action of anaerobic microbes. The
major obstacle in increased biofuel production is to find an efficient way to break
down complex plant polymers into simpler derivatives.
6.2.3 Manipulating Genes and Pathways Involved in Lipid

Metabolism
Oils from crops like palm, castor, soybean and oilseed rape are presently being used
to make biodiesel. The biosynthesis of oils in plants starts de novo in the plastids
where photosynthesis provides the carbon source, which are assembled into short
chains to produce palmatic (C16), stearic (C18), oleic and linoleic acids (C20). In
general, a greater proportion of short-chain fatty acids is more desirable in biodiesel
production (Fig. 6.4c). The higher proportion of short-chain fatty acids like oleic
acid will render oxidative stability to the oils even at high temperature (Metzger and
Bornscheuer 2006). The fatty acids are then transported to the endoplasmic reticu-
lum (ER) by an energy-dependent pathway, and there long-chain fatty acids are
assembled from the short-chain ones. The final products (oil or fatty acids) are
transported out of the ER to be stored in vacuoles as oil bodies, which act as a
source of reserve energy for the plants. Therefore, the pathway to synthesize fatty
acids and triacylglycerols will need to be modified to facilitate the production of
biodiesel from these crops (Durrett et al. 2008). All the genes acting on the lipid
biosynthesis have been cloned by Zhang et al. (2005). They further suggested that
improving the composition of the fatty acid components of plant oils will enhance
the acceptability of biodiesel in temperate regions of the world. For example,
Lardizabal et al. (2008) achieved increase in biodiesel production from soybean
by ectopically expressing diacylglycerol acyltransferase 2A (DGAT2A) from
Umbelopsis ramanniana fungus during the seed development stage. Similar
results have been reported in rapeseed by overexpressing a laurate-specific acy-
lACP thioesterase gene from California bay tree and a laurate-specific LPAAT
gene from coconut (Knutzon et al. 1999; Wiberg et al. 2000). Wiberg et al. (2000)
also described that an increase in the content of caprylic acid (C8) and capric acid
(C10) in rapeseed had resulted in a 30% increase of short-chain fatty acid in the
seed.
Also, the production of short chain fatty acids or oils should be enhanced from
the non-oil-producing tissues of the plant. Recent studies have shown that plant
storage tissue can store high quantities of oil bodies without any drastic effect of
cell membrane polarity and hence on plant growth (Napier 2007). Increasing the
percentage of short-chain fatty acids by a transgenic approach will reduce the cost
associated with the production of biodiesel. Most vegetable oils are large, branched
molecules; therefore, could not be directly used in diesel engines. These large and
branched molecules of bio-oils are converted into smaller and straight-chain mole-
cules through transesterification, making them suitable for regular diesel combus-
tion engines. Production of high-quality short-chain fatty acids by using advanced
biotechnological techniques (TILLING, RNAi, antisense approach, T-DNA inser-
tion lines) may come to our rescue. Targeted induced local lesion in genomes
(TILLING) is a very popular technique used today to identify mutant lines for
any gene of interest. For example, a mutation in fatty acid gene (FAD1 in peanut)
will cause the plant to accumulate short-chain fatty acids, as its capability to convert
the short-chain fatty acids into long-chain fatty acids will decrease. Again, RNAi
and antisense expression of the fatty acid gene will reduce translation of the gene,
and thus the amount of long fatty acids will be decreased. Random T-DNA insertion
is a technique introduced to prevent the production of a gene of interest. Random
T-DNA may get integrated in the exonic region of the gene and thus translation may
come to a premature stop.
6.3 Conclusion
The era of cheap fossil fuel is rapidly coming to an end. Therefore, the need of the
hour is to look at the alternative forms of energy, a strategy that was hardly
emphasized in the past. There are also concerns about global climate change and
severe food shortage. Biomass is the least expensive and most globally available
resource. Therefore, priority should be shifted towards utilizing biomass, leaving
aside food for human consumption. Plant architecture have been naturally modified,
in fact, ever since the evolution of land plants commenced several million years ago
and through the selection of desirable phenotypes by crop improvement (around
10000 BC, when man started cultivating plants for food and shelter) to the more
sophisticated methods of using genetic manipulation and plant biotechnology to
produce modern-day transgenic biofuel crops. Modified carbohydrate and lignin
biosynthetic pathway has been a major target for chemical or genetic intervention in
recent years. The use of transgene genes could also underpin conventional breeding
to modify biofuel production, in view of the growing public concern over the
potential environment damage and health hazards by the use of fossil fuel, and
the limited availability of suitable genes to modify synthesis of sugars, lignin and
lipids in plants, by adopting classical breeding approaches. Tissue-specific anti-
sense expression of transgenes involved in biofuel production in vegetative parts
will prevent unwanted assimilate partitioning and direct photosynthate towards
storage organs (seeds or underground storage like bulbs, corms and rhizomes),
increasing productivity besides contributing to biofuel processing and production.
One of the aspects for biofuel production is synchronous maturity of the crops used
for biofuel production. Many chemicals that promote synchronous maturity like
NAA (napthalene acetic acid) could be useful. Also, application of gibberelic acid
(GA) may result in high yield. Anti-GA substances like pacrobutrazol can be used
to control plant height, or ectopic expression of GA2-oxidase will result in dwarf
plants (Dijkstra et al. 2008). This will allow easy harvest of the produce with low
cost on resources and saving time. Compact plants are also less prone to damage by
biotic and abiotic stress.
Genetic manipulation strategies should lay emphasis on downregulating lignin
biosynthesis genes without drastically affecting plant architecture and making
plants prone to infestation of disease, insects and pests. Therefore, production of
transgenic plants with the above mentioned traits would help in developing a more
robust plant phenotype that was not always possible through conventional or
molecular breeding. This was because traits amenable for biofuel production did
not mostly go hand in hand with increased food crop production. Thus, suitable
traits for biofuels were lost in rounds of selection of superior genotypes responsible
for increased crop production practiced over several thousand years. Now, crop
breeders have been combining tissue culture approaches with traditional breeding
in order to broaden the gene pool available for improvement of productivity and to
introduce new, desirable traits. With the development of DNA microarray chips, it
is now possible to identify novel genes and to generate plants with novel traits
(in this case, traits amenable for biofuel production), and not just the development of
varieties restricted to insect pests (Wang et al. 1996), disease (Marchant et al. 1998)
and herbicide tolerance (Slater et al. 2003). Manipulating monolignins, and perhaps
modifying three-dimensional lignin architecture, could also be an additional possi-
bility. Therefore, modification of traits related to biofuel production may require
manipulation of the multiple hormones, complex carbohydrate synthesis pathways,
which may involve the introduction of multiple genes into plants. Such studies may
also lead to a better understanding of the roles of the different hormone classes in
biofuel crop development. Future research must be directed towards identifying
tissue-specific or species-specific inducible promoters that could, for example,
drive gene expression in the vegetative tissues, such as stem, internodes, and leaf
petioles, thus ensuring that flowering and seed set, both qualitatively and quantita-
tively, are not affected. Furthermore, the possibility of using constructs carrying
genes associated with enhanced biofuel production, but lacking the nptII selectable
maker gene (Holn et al. 2001), should be explored in order to facilitate the
acceptance of transformation technology to regulate plant biomass. This can be
achieved by selecting plants directly, based on phenotype, such as with dwarf
growth. It is also possible to use T-DNA related sequences, which have originated
from plant genomes. These have been referred to as cisgenic plants, as their
transformation vectors, including promoters and terminators, are derived from the
same plant species, which is transformed with the gene of interest (Chandler and
Tanaka 2007). Also, vectors from plant-originated selectable marker, recombinase
recognition sequences (Cre-lox system) can also be beneficial (Zuo et al. 2001). The
use of co-transformation (Komari et al. 1996) or the use of chimeric plants
(De Vetten et al. 2003) may also be considered. Further, introduction of transgenes
in male-sterile plants may be another viable option, or the use of terminator gene
technology may be considered. The exact role of each lignin biosynthetic gene on
plant development must be accessed to pinpoint genes (in turn enzymes) bringing
about the greatest effect.
The use of a specific promoter may be pivotal in driving gene expression under
study. Low transformation efficiency can be correlated with a constitutive pro-
moter, such as CaMV 35S (Zhu et al. 2008), which may result in poor regeneration
and low recovery of transgenic plants (Petty et al. 2003; Zhu et al. 2004; Busov
et al. 2006). The use of a viral promoter, like 35S, may result in gene silencing in
plants as observed (Bhattacharya et al. unpub.). Furthermore, an ubiquitin-like
promoter (Christensen and Quail 1996) may result in high efficiency of transforma-
tion compared to 35S promoter, and future genetic manipulation studies should
include such ubiquitin-like promoters. Genetic modification paves the way for
the development of new cultivars by gradually incorporating one or more genes
(Miflin 2000). However, expression of genes across plant species is not always
entirely predictable, leading to a large number of trial and error methods to reach
a generalized conclusion. Another area of emphasis will be manipulating oil-
synthesizing genes in lower primitive plants like algae, cynobacteria and phyto-
plankton. Exploiting everyday-generated crop waste will also help to lay the
foundation of a carbon-balanced world.
Antagonism to this technology has also restricted the development of the

technology (Azevedo and Araujo 2003; Phillips 2004). There is no uniform system
for international regulation on genetically modified crop plants with significant
difference between countries (Halsberger 2006). Since transgenic biofuel crop
plants are not anyway intended for human consumption, they should be theoreti-
cally more acceptable to the public. The Cartagena protocol (http://bch.biodiv.org)
is the first attempt to bring uniformity in laws governing genetic modification, and
many countries are adding these laws to their existing law in order to come to a
unified attempt to regulate genetically regulated products.
In conclusion, biofuel may not replace the use of fossil fuel in the foreseeable
future; however, it can become a major component of alternative fuels, and may add
towards the carbon economy in the age of rapid industrialization.
References
Antizar-Ladislao B, Turrion-Gomez JL (2008) Second-generation biofuels and local bioenergy

systems. Biofuels Bioprod Biorefin 2:55–469. doi:10.1002/Bbb.97
Azevedo JL, Araujo WL (2003) Genetically modified crops: environmental and human health
concerns. Mut Res 544:223–233
Bate N, Orr J, Ni W, Meromi A, Nadler-Hassar T, Doerner P, Dixon R, Lamb C, Elkind Y (1994)
Quantitative relationship between phenylalanine ammonia-lyase levels and phenylpropanoid
accumulation in transgenic tobacco identifies a rate-determining step in natural product
synthesis. Proc Natl Acad Sci USA 91:7608–7612
Baucher M, Andrée Bernard-vailhé M, Chabbert B, Besle JM, Opsomer C, Montagu MV, Botter-
man J (1999) Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa
(Medicago sativa L) and the effect on lignin composition and digestibility. Plant Mol Biol
39:437–447
Bijalwan A, Sharma CM, Kediyal VK (2006) Bio-diesel revolution. Sci Rep (Jan) 13:14–17
Blaschke L, Legrand M, Mai C, Polle A (2004) Lignification and structural biomass production in
tobacco with suppressed caffeic/5-hydroxy ferulic acid-O-methyl transferase activity under
ambient and elevated CO2 concentrations. Physiol Plant 121:75–83
Busov V, Meilan R, Pearce DW, Rood SB, Ma C, Tschaplinski M, Strauss SH (2006) Transgenic
modification of gai or rgl1 causes dwarfing and altered gibberellins, root growth, and metabo-
lite profiles in Populus. Planta 224:288–299
Chabannes M, Barakate A, Capierre C, Marita JM, Ralph J, Peem M et al (2001) Strong decrease
in lignin content without significant alteration of plant development is induced by simultaneous
down-regulation of cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase
(CAD) in tobacco plants. Plant J 28:257–270
Chandler S, Tanaka Y (2007) Genetic modification in floriculture. Crit Rev Plant Sci 26:169–197
Chen F, Dixon RA (2007) Lignin modification improves fermentable sugar yields for biofuel
production. Nat Biotechnol 25:759–761
Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high level expression
of selectable and/or screenable marker gene in monocotyledonous plants. Transgen Res
5:213–218
Cosgrove DJ (2005) Growth of the plant cell wall. Nat Rev Mol Cell Biol 6:850–861
Dai Z, Hooker BS, Anderson DB, Thomas SR (2000) Improved plant-based production of
E1 endoglucanase using potato: expression optimization and tissue targeting. Mol Breed
6:277–285
De Vetten N, Wolters AM, Raemakers K, van der Meer I, Stege R, Heeres E, Heeres P, Visser R
(2003) A transformation method for obtaining marker-free plants of a cross-pollinating and
vegetatively propagated crop. Nat Biotechnol 21:439–442
Demirbas A (2008) New liquid biofuels from vegetable oils via catalytic pyrolysis. Energ Edu Sci
Technol 21:1–59
Dijkstra C, Adams E, Bhattacharya A, Page A, Anthony P, Kourmpetli S, Power J, Lowe K,
Thomas S, Hedden P (2008) Over-expression of a gibberellin 2-oxidase gene from Phaseolus
coccineus L. enhances gibberellin inactivation and induces dwarfism in Solanum species. Plant
Cell Rep 27:463–470
Durrett TP, Benning C, Ohlrogge J (2008) Plant triacylglycerols as feedstocks for the production
of biofuels. Plant J 54:593–607
Good AG, Shrawat AK, Meunch DG (2005) Can less yield more? Is reducing nutrient input into
the environment compatible with maintaining crop production? Trends Plant Sci 12:597–605
Gressel J (2008) Transgenics are imperative for biofuel crops. Plant Sci 174:246–263
Gunnarsson CC, Petersen CM (2007) Water hyacinths as a resource in agriculture and energy
production: a literature review. Waste Manag 27:117–129
Halsberger AG (2006) Need for an “Integrated Safety Assessment” of GMOs linking food safety
and environmental consideration. J Agric Food Chem 54:3173–3180
Hanna MA, Isom L, Campbell J (2005) Biodiesel: Current perspectives and future. J Sci Indust Res
64:854–857
Hill J, Nelson E, Tilman D, Polasky S, Tiffany D (2006) Environmental, economic, and energetic
costs and benefits of biodiesel and ethanol biofuels. Proc Natl Acad Sci USA 103:11206–11210
Holn B, Levy AA, Puchta H (2001) Elimination of selection markers from transgenic plants. Curr
Opin Biotechnol 12:139–143
Hu WJ, Harding SA, Lung J, Popke JL, Ralph J, Stokke DD (1999) Repression of lignin
biosynthesis promotes cellulose accumulation and growth in transgenic trees. Nat Biotechnol
17:808–812
Jing ZP, Gallardo F, Pascual MB, Sampalo R, Romero J, Torres de Navarra A, Cánovas FM (2004)
Improved growth in a field trial of transgenic hybrid poplar overexpressing glutamine synthe-
tase. New Phytol 164:137–145
Kirk TK, Farrell RL (1987) Enzymatic “combustion”: the microbial degradation of lignin. Annu
Rev Microbiol 41:465–505
Knutzon D, Hayes T, Wyrick A, Xiong H, Maelor Davies H, Voelker T (1999) Lysophosphatidic
acid acyltransferase from coconut endosperm mediates the insertion of laurate at the sn-2
position of triacylglycerols in lauric rapeseed oil and can increase total laurate levels. Plant
Physiol 120:739–746
Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T (1996) Vectors carrying two separate T-DNAs
for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segrega-
tion of transformants free from selection markers. Plant J 10:165–174
Lardizabal K, Effertz R, Levering C, Mai J, Pedroso M, Jury T, Aasen E, Gruys K, Bennett K
(2008) Expression of Umbelopsis ramanniana DGAT2A in seed increases oil in soybean. Plant
Physiol 148:89–96
Li L, Zhou Y, Cheng X, Sun J, Marita J, Ralph J, Chiang V (2003) Combinatorial modification of
multiple lignin traits in trees through multigene cotransformation. Proc Natl Acad Sci USA
100:4939–4944
Marchant R, Davey MR, Lucas JA, Lamb CJ, Dixon RA, Power JB (1998) Expression of a
chitinase transgene in rose (Rosa hybrida L.). Ann Bot 81:109–114
Metzger J, Bornscheuer U (2006) Lipids as renewable resources: current state of chemical and
biotechnological conversion and diversification. Appl Microbiol Biotechnol 71:13–22
Miflin BJ (2000) Crop biotechnology: where now? Plant Physiol 123:17–27
Mishra P, Srivastava AK (2004) Molecular investigations in floricultural plants. J Plant Biotechnol
6:131–143
Mitsuda N, Iwase A, Yamamoto H, Yoshida M, Seki M, Shinozaki K, Ohme-Takagi M (2007)

NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary
walls in woody tissues of Arabidopsis. Plant Cell 19:270
Murugesan A, Umarani C, Subramanian R, Nedunchezhian N (2009) Bio-diesel as an alternative
fuel for diesel engines-A review. Renew Sustain Energ Rev 13:653–662
Myers AM, Morell MK, James MG, Ball SG (2000) Recent progress toward understanding the
biosynthesis of the amylopectin crystal. Plant Physiol 122:989–998
Napier J (2007) The production of unusual fatty acids in transgenic plants. Annu Rev Plant Biol
58:295–319
Oxenboll Sorensen S, Pauly M, Bush M, Skjot M, McCann M, Borkhardt B, Ulvskov P (2000)
Pectin engineering: modification of potato pectin by in vivo expression of an endo-1, 4-b-D-
galactanase. Proc Natl Acad Sci USA 97:7639–7644
Petrou EC, Pappis CP (2009) Biofuels: A Survey on Pros and Cons. Energ Fuel 23:1055–1066
Petty LM, Harberd NP, Carre IA, Thomas B, Jackson SD (2003) Expression of the Arabidopsis gai
gene under its own promoter causes a reduction in plant height in chrysanthemum by attenua-
tion of the gibberellin response. Plant Sci 164:175–182
Phillips AL (2004) Genetic and transgenic approaches to improving crop performance. In: Davies
PJ (ed) Plant hormones: biosynthesis, signal transduction, action! Kluwer, Dordrecht, The
Netherlands, pp 582–609
Puppan D (2002) Environmental evaluation of biofuels. Periodica Polytechnica Soc Manag Sci
10:95–116
Ragauskas A, Williams C, Davison B, Britovsek G, Cairney J, Eckert C, Frederick W, Hallett J,
Leak D, Liotta C (2006) The path forward for biofuels and biomaterials. Science 311:484–489
Ralph J et al (2006) Effects of coumarate 3-hydroxylase down-regulation on lignin structure. J Biol
Chem 281:8843–8853
Riboulet C, Guillaumie S, Mechin V, Bosio M, Pichon M, Goffner D, Lapierre C, Pollet B,
Lefevre B, Martinant JP, Barriere Y (2009) Kinetics of Phenylpropanoid gene expression in
maize growing internodes: relationships with cell wall deposition. Crop Sci 49:211–223
Ripley BS, Muller E, Behenna M, Whittington-Jones GM, Hill MP (2006) Biomass and photosyn-
thetic productivity of water hyacinth (Eichhornia crassipes) as affected by nutrient supply and
mirid (Eccritotarus catarinensis) biocontrol. Biol Con 39:392–400
Robinson A (1998) Developing sustainable global agriculture for the 21st century. In: Rice
Reports, John Innes Centre, Norwich, UK, pp 1–8
Schillberg S, Fischer R, Emans N (2003) Molecular farming of recombinant antibodies in plants.
Cell Mol Life Sci 60:433–445
Skjot M, Pauly M, Bush M, Borkhardt B, McCann M, Ulvskov P (2002) Direct interference with
rhamnogalacturonan I biosynthesis in Golgi vesicles. Plant Physiol 129:95–102
Slater A, Scott N, Fowler M (2003) Plant biotechnology – the genetic manipulation of plants.
Oxford University Press, Oxford, UK, pp 7–18
Smith AM (2008) Prospects for increasing starch and sucrose yields for bioethanol production.
Plant J 54:546–558
Sticklen MB (2008) Plant genetic engineering for biofuel production: towards affordable cellu-
losic ethanol. Nat Rev Genet 9:433–443
Van Camp W (2005) Yield enhancement genes: seeds for growth. Curr Opin Biotechnol 16:147–153
Vanden Wymelenberg A, Sabat G, Mozuch M, Kersten PJ, Cullen D, Blanchette RA (2006)
Structure, organization, and transcriptional regulation of a family of copper radical oxidase
genes in the lignin-degrading basidiomycete Phanerochaete chrysosporium. Appl Environ
Vogt KA, Vogt DJ, Patel-Weynand T, Upadhye R, Edlund D, Edmonds RL, Gordon JC, Suntana
AS, Sigurdardottir R, Miller M, Roads PA, Andreu MG (2009) Bio-methanol: how energy
choices in the western United States can help mitigate global climate change. Renew Energ
34:233–241
Wang G, Castiglione S, Chen Y, Li L, Han Y, Tian Y, Gabriel DW, Mang K, Sala F (1996) Poplar
(Populus nigra L.) plants transformed with a Bacillus thuringiensis toxin gene: insecticidal
activity and genome analysis. Transgen Res 5:289–301
Wiberg E, Edwards P, Byrne J, Stymne S, Dehesh K (2000) The distribution of caprylate, caprate
and laurate in lipids from developing and mature seeds of transgenic Brassica napus L. Planta
212:33–40
Wu LG, Birch RG (2007) Doubled sugar content in sugarcane plants modified to produce a sucrose
isomer. Plant Biotechnol J 5:109–117
Zhang P, Jarnefeld J, Wen B (2005) New process for biodiesel production from waste cooking oils.
Abstr Pap Am Chem Soc 229:U55–U55
Zhu LH, Li XY, Ahlman A, Xue ZT, Welander M (2004) The use of mannose as a selection agent
in transformation of the apple rootstock M26 via Agrobacterium tumefaciens. Acta Hort
663:503–506
Zhu LH, Li XY, Walender M (2008) Overexpression of the Arabidopsis gai gene in apple
significantly reduces plant size. Plant Cell Rep 27:289–296
Ziegler MT, Thomas SR, Danna KJ (2000) Accumulation of a thermostable endo-1, 4-d-glucanase
in the apoplast of Arabidopsis thaliana leaves. Mol Breed 6:37–46
Zuker A, Chang P, Ahroni A, Cheah K, Woodson WR, Bressan RA, Watad AA, Hasegawa PM,
Vainstein A (1995) Transformation of carnation by microprojectile bombardment. Sci Hort
64:177–185
Zuo J, Niu QW, Moller SG, Chua NH (2001) Chemically-regulated site-specific DNA excision in
transgenic plant. Nat Biotechnol 19:157–161
Chapter 7
Plant-Produced Biopharmaceuticals
Jared Q. Gerlach, Michelle Kilcoyne, Peter McKeown,

Charles Spillane, and Lokesh Joshi
7.1 Introduction
The term “biopharmaceutical” is commonly used to denote a therapeutic protein

produced by recombinant (genetic) engineering. In this process, genes encoding
proteins or peptides of interest from humans or other organisms are identified,
cloned, inserted into an expression vector and the protein or enzyme produced
within a prokaryotic or eukaryotic expression host “production platform” organism.
Such production platforms may typically be bacteria, yeasts, insects, mammals or
plants. Batch cultures of cell suspensions are also commonly used for large-scale
production of recombinant therapeutic proteins, a method often referred to as
“fermentation culture.” Bacteria and yeast can be grown in closed-loop bioreactors,
as can insect, mammal and plant cell-lines, which are generated from whole
organisms. This ensures several generations of stable cell line propagation, allowing
production of the protein or enzyme of interest.
The use of animal cell lines and bacterial or yeast strains to produce therapeutic
proteins has resulted in several successful products of medical interest (Walsh
2006). Such pharmaceutical products include glycosylated human erythropoietin
(rhEPO), which acts as a red blood cell stimulating hormone, recombinant human
clotting factor VIII (rhCFVIII), used for treating hemophilia, and recombinant
insulin, used to treat diabetes mellitus. Sales of rhEPO were estimated at US
$10.7 billion for 2005, while those of rhCFVIII reached an estimated US$1 billion
by the same year (Walsh 2006). The total market for biopharmaceuticals is
expected to reach approximately US$70 billion by 2010 (Walsh 2006), with insulin
estimated to generate US$14 billion from 16,000 kg of protein (Moloney et al.
2003) and monoclonal antibodies contributing approximately US$20 billion (Hiatt
and Pauly 2006).
L. Joshi (*)
Glycoscience and Glycotechnology Group, Martin Ryan Institute and National Centre for
Biomedical Engineering Science, National University of Ireland, Galway, Ireland
e-mail: lokesh.joshi@nuigalway.ie

270 J.Q. Gerlach et al.
Non-human mammalian cell lines, such as Chinese hamster ovary (CHO) cells,
are currently considered to be the most desirable expression systems as they
produce proteins similar to those from human cells, particularly with regard to
glycosylation or other post translational modifications (Hamilton and Gerngross
2007). However, one of the major challenges associated with mammalian cell
culture technology of particular public concern is that contaminating pathogens,
which are capable of infecting other mammalian species (i.e., xenozoonosis), may
be transferred to humans during product recovery. These include viruses, endoge-
nous retroviruses and internalized bacteria and mycoplasmas, as well as prions.
This inherent biological limitation poses a particular challenge to the development
of pathogen-free Good Manufacturing Practice (GMP) procedures for mammalian
cell culture systems.
Furthermore, as complex growth media is required for mammalian cell bioreac-
tors, minute variations in media components and fermentation conditions can lead
to undesirable changes in the end product(s). Even under the most stringent condi-
tions, maintaining consistency from batch to batch is challenging as the interactions
of cells with each other and their environment may also create variations in end
products (Butler 2006). Also, endogenously produced immunogenic agents, such
as proteins or species-specific glycosylation, harvested as a co-extracted or
incorporated component of biopharmaceuticals produced in CHO cells, may create
unwanted effects in patients even at extremely low levels (Hamilton and Gerngross
2007).
In addition to the above, there are a number of other key drivers for the
development of improved production platforms for biopharmaceutical production.
The costs associated with growing and maintaining mammalian cells for pharma-
ceutical production remain relatively high compared to yeast and bacteria (Fischer
et al. 1999). Costs for establishing a pharmaceutical-grade mammalian cell culture
facility may exceed €250 million (Sardana et al. 2007), and each requires exten-
sive testing and certification with the result that only a relatively small number of
such facilities worldwide can produce biopharmaceuticals on a large (kilograms
per year) production scale. Hence, any approach that leads to a manufacturing
cost-saving will gain competitive advantages over time. Increasing numbers of
new protein therapeutics coming on stream are generating a production capacity
crisis within the available mammalian cell culture systems. This situation is likely
to worsen – as of 2006, there were 2,500 biotech drugs in discovery phase, 900 in
preclinical trials and 1,600 in clinical trials (Walsh 2006). Moreover, the advent
of high-volume protein therapeutics targeted at much larger numbers of people
(e.g., monoclonal antibody-based therapeutics such as Herceptin, Enbrel, Mylotarg,
Remicade, etc.) and the need to produce generics or biosimilars are placing strains
on current biopharmaceutical production platforms to deliver at the scale and cost
levels needed. To produce 300 kg of secretory IgA antibodies per year, plant-based
production systems have the lowest capital costs (maize or tobacco; €0.5–2.0 mil-
lion/300 kg per year) when compared to platforms based on transgenic goats or
mammalian cell culture (~€6–7 million/300 kg per year) (Richard McClosky,
7 Plant-Produced Biopharmaceuticals 271
Centocor). Finally, with escalating elderly populations in developed countries

increasing product requirement, and patients and governments demanding afford-
ability, there is a definite need to innovate and develop more cost-effective plat-
forms for large-scale production of biopharmaceuticals.
7.2 Transgenic Plants as Production Systems

for Biopharmaceuticals
Transgenic plants (or derived plant suspension cell lines) are considered very
attractive alternatives to mammalian cell systems for the production of recombinant
mammalian glycoproteins. Plants as multicellular eukaryotes have many advan-
tages as production platforms for recombinant proteins or enzymes, including
similar eukaryotic biosynthetic pathways, high protein yields, freedom from animal
pathogens and bacterial endotoxin contamination and low production cost per unit
protein overhead (Gomord et al. 2005). Indeed, cost savings associated with plant
technologies and end-product purification may lead to a 10- to 100-fold advantage
over currently used mammalian fermentation systems such as CHO cells (Vidi et al.
2007). Additionally, with the use of organ-specific promoters to drive protein
expression, the seeds, stems, roots, fruits, and leaves of whole plants suitable for
growth in fields or greenhouses can be targeted for transgenic protein production
(Fischer et al. 2004). To date, the highest yields have generally been achieved by
targeting recombinant proteins to chloroplasts, oil bodies and seed protein bodies
of whole plants (see below).
It has been estimated that 250 acres of greenhouse production of transgenic
plants producing hepatitis B vaccine could meet Southeast Asia’s demand. Indeed,
the scalability of plant platforms for biopharmaceutical production is one compel-
ling advantage of this approach, e.g., in response to rapid demand for therapeutics
for any future pandemics. The possibility of plant systems for facilitating freedom
from refrigeration would be a tremendous advantage for economically challenged
populations, where maintenance of a “cold chain” for handling and storage may
simply not be possible (Rademacher et al. 2008). The capacity of plant storage
organs (seeds or tubers) to act as protein storage systems is truly remarkable
(Fischer et al. 1999) – antibodies in plant seeds stored at room temperature have
been found to be stable and fully active after several years.
The market potential of plant-based recombinant technology was first demon-
strated in the production of pure, research-grade proteins. By 2005, three major
recombinant proteins had been produced in corn (Zea mays) – the biotin-receptor,
avidin, and the enzymes, trypsin and b-glucuronidase – and were commercially
available (Ma et al. 2005). Seed storage protein bodies (PBs) are of particular interest
as the recombinantly expressed proteins can be accumulated in a comparatively high
proportion into a relatively stable, compact mass, greatly contributing to relative

ease of harvest, storage and purification of products (Stoger et al. 2005).
Despite the clear value of seed storage targeting, for many purposes it is more
appropriate to make use of plant cells propagated as suspension cultures. Such
techniques also aid containment (see below). Plant suspension cells are derived
from calli, clusters of undifferentiated cells. Calli are first stimulated from mature
plant tissues through direct contact with hormone-enriched solid growth media.
After reaching a suitable size, the frangible calli are transferred to a liquid growth
medium, agitated to shake the cells apart and cells continue to divide within the
liquid growth medium (Hellwig et al. 2004). Plant suspension cells are grown using
media that is much simpler in formulation than that used for insect and mammalian
systems, typically containing just a few salts and minerals, sucrose and a plant
hormone (Kolewe et al. 2008).
A wide range of plant species are used commercially for molecular pharming
purposes, including well-known biological models (e.g., Arabidopsis thaliana,
Medicago truncatula), food crops, and non-food crop species specialized for
particular purposes (Twyman et al. 2003; Fischer et al. 2004; Stoger et al. 2005).
Amongst the seed crops, molecular pharming is underway in cereals (maize, wheat,
rice, barley), legumes (peas, soybean) and oilseeds (safflower, rapeseed). The root
and tuber crops, carrot and potato, are used for pharming, while lettuce, spinach,
alfalfa, banana and tomato are examples of fruit and green leaf crops being used for
pharming. Non-food crops in use for pharming include tobacco and falseflax,
while non-cultivated species include duckweed (Lemna spp.) and moss (Physcomi-
trella patens), and also unicellular photosynthetic organisms, such as microalgae
(Chlamydomonas reinhardtii).
Importantly, all these eukaryotes share the common characteristics of eukary-
otic protein machinery and being amenable to genetic transformation. Transfor-
mation of plants and plant cells is typically done using one of two species of
Agrobacteria which are able to transfer transgenes (encoding recombinant bio-
pharmaceuticals) into the host plant genome. Agrobacterium tumefaciens is com-
petent to transform foliar and floral tissues and cultured cells (Broothaerts et al.
2005), while A. rhizogenes is competent to transform root tissue or cultures (Shi
and Lindemann 2006). First, the Agrobacteria are transformed with DNA plas-
mids containing the gene of biopharmaceutical interest, under the control of a
suitable plant promoter. In response to the patent landscape (Dunwell 2005) and
the need to develop complementary approaches for transferring desirable genes
and associated traits to plants, a range of other techniques for plant transformation
have been developed (Broothaerts et al. 2005; Vain 2007). These include plant
transformation by particle bombardment, otherwise known as the gene gun
method (Brereton et al. 2007). Plasmid vectors adhered to minute particles of a
carrier agent, often gold particles, are accelerated by compressed air to penetrate
and transform the nuclear genome of the target cells. Vectors for transformation
can be rapidly assembled using high-throughput cloning techniques, such as
Invitrogen’s Gateway system, which allows combination of specific features
(Curtis and Grossniklaus 2003). Plasmid vectors may be constructed to allow
for selection of successfully transformed cells by transformation with genes

conferring antibiotic or herbicide resistance and subsequent growth in media
containing these agents or encoding reporter enzymes or fluorescent molecules
(Sala et al. 2003). Promoters may be chosen for constitutive high expression
throughout the plant, or tailored for expression in cell culture (i.e., inducible
systems) or a particular organ, such as the seed or tuber.
These transformation techniques can also be targeted for chloroplast expression,
a relatively new alternative (Hou et al. 2003). Directing genes for recombinant
proteins to chloroplasts in whole plants may allow yields of up to approximately
45% of total leaf protein (Arlen et al. 2008). The chloroplast is an endosymbiotic
organelle of prokaryotic origins with its own genome. Hence, chloroplast genes and
translational machinery differ from those of the nucleus, although there is regu-
latory interaction and protein trafficking between these two cellular compartments
(Pogson et al. 2008; Woodson and Chory 2008). An important attribute of trans-
genic chloroplast systems is that chloroplasts are maternally inherited and therefore
rarely transmitted via pollen (there are some species exceptions) and therefore
transgenes located in chloroplast genomes are typically biologically contained
(Ruf et al. 2007).
Lipoprotein particles found within chloroplasts (plastoglobules) may be specifi-
cally targeted as depositories for recombinant proteins. Vidi and colleagues fused
recombinant yellow fluorescent protein (YFP) to a naturally occurring plastoglo-
bulin protein to study the viability of the process (Vidi et al. 2007) and found that
plants expressing the YFP fusion protein did not have a significantly altered
phenotype. Moreover, such plants had similar growth characteristics to those
expressing recombinant proteins targeted to oil bodies. One disadvantage of chlo-
roplast expression systems is a lack of access to the post translational machinery of
the secretory pathway (see below). Hence, proteins produced by chloroplasts
cannot be endogenously glycosylated, deacylated or phosphorylated (Anisimov
et al. 2007). Some recombinant proteins may be improperly folded or even rendered
biologically inactive without these modifications (Gomord and Faye 2004; Byrne
et al. 2006; Eichler et al. 2006; Mitra et al. 2006).
High-level production of recombinant proteins within cytoplasmic compart-
ments of plant cells depends on promoters to drive the expression of the gene of
interest. Many promoters have been used in both monocot and dicot species to date,
and the sophistication of such promoters is increasing as understanding of plant
gene regulation grows (Venter 2007, see also Vol. 1, Chap. 5). Low-level expres-
sion using dicot promoters in cereals has highlighted the necessity of developing
monocot-specific promoters (Stoger et al. 2005). Additionally, the use of an engi-
neered plant virus-based transient expression systems (e.g., magnICON from Icon
Genetics; Gleba et al. 2005) can allow a significantly faster turnaround time and a
much higher protein yield than other stable plant transformation techniques
(Mallory et al. 2002; Gleba et al. 2005), although the higher mutation rate of
RNA-based viral expression systems is a consideration for the generation of
homogeneous protein product (Domingo et al. 2006).
7.3 Challenges
7.3.1 Regulatory Challenges
Despite the distinct advantages transgenic plants offer, some specific challenges
remain. As transgenic plants will be producing protein compounds with known
pharmaceutical effects, it is imperative for the success and acceptability of the
molecular pharming field that such production operates in a biologically contained
system and is also compliant with current GMP (cGMP) procedures for the produc-
tion of therapeutic grade proteins. Given the issues with controlling the abiotic and
biotic environment, open-field production of food crops or other cultivated species
producing high-potency pharmaceutical compounds may not be desirable for
molecular pharming industry development. Closed-loop production systems
which ensure that pharma-producing seeds or plant materials are not used for any
other purposes are essential. This would include ensuring that any biotic predators
or pests are not adversely impacted by the pharmaceutical-producing plants.
However, pharmaceutical-producing plants have been deliberately cultivated
since the first pharmacopeias were compiled for apothecaries in the Middle Ages.
Physic gardens, which trained the earliest physicians in the use of medicinal plants,
were the forerunners of today’s botanic gardens, many of which were originally
associated with the faculties and departments of pharmacy in universities. Today,
there are multiple examples of highly potent (non-transgenic) medicinal or toxic
plants (e.g., opium poppy, high erucic acid rapeseed), which are cultivated for the
production of pharmaceuticals without any adverse effects on humans or environ-
ment. Nonetheless, to further bolster public trust and to avoid any unforeseen
secondary consequences of plant-based recombinant technologies, it will be pru-
dent to apply efficient measures of biological containment so that pharma-producing
seeds or transgenes are not used outside of the closed-loop production systems for
which they were developed. Hence, methods for the containment of reproductive
material (e.g., male sterility, maternal inheritance systems such as chloroplasts
or autonomous apomixis) in conjunction with the use of genetically-linked, visible
phenotypic markers for any transgenic seeds/plants expressing pharma proteins
will be advisable. Other approaches worth considering are inducible expression
systems. In general, the most prudent containment will be to use the best green-
house-controlled environment conditions (for whole transgenic plant systems) or
photobioreactors (for cell culture suspensions) for commercial production of the
first generation of plant-derived biopharmaceuticals. It is certain that plant-made
pharmaceutical (PMP) production will be like any other pharmaceutical production
system in terms of its strict regulatory control.
The regulations for pharmaceutical production in plants are not currently
harmonized internationally with different governing bodies operating in the
USA and European Union (EU). In the USA, regulatory oversight for PMPs is
under the aegis of the following bodies: the US Department of Agriculture
(USDA: http://www.usda.gov/wps/portal/usdahome) regulates growth of plant and

defines the required safeguards for the production and transport of the product; the
Food and Drug Administration (FDA) Centre for Biologics Evaluation and
Research (CBER: http://www.fda.gov/%20BiologicsBloodVaccines/default.htm)
regulates biologic products, their manufacture and distribution; the FDA Centre
for Food Safety and Applied Nutrition (CFSAN: http://www.foodsafety.gov/list.
html) and Centre for Veterinary Medicine (CVM: http://www.fda.gov/cvm/default.
html) are involved in consultations on food and feed safety, and finally the PMP
developer is responsible for safety assessments, drug master files, chemistry
manufacturing controls and biologics license application for the client’s drug
registration (Streatfield 2005).
In the European Union, regulatory procedures for PMPs are in flux due to a
need to tailor the existing regulations to deal with PMPs. Any field-grown
plant pharming would require notification under EU Directive 2001/18/EC (http://
eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32001L0018:EN:HTML),
which covers both food and non-food transgenic crops. Containment-grown plant
pharming would fall under EU Directives 90/219/EEC (http://eur-lex.europa.eu/
LexUriServ/LexUriServ.do?uri=CELEX:31990L0219:EN:HTML) and 98/81/EC
(http://ec.europa.eu/environment/biotechnology/pdf/dir98_81.pdf). For any com-
mercial release, the European Food Standards Agency (EFSA) would be involved
in the review process under 1829/2003/EU Food and Feed Guidelines (http://
eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2003:268:0001:0023:EN:
PDF). All pharma products produced from plants within the EU must adhere to
the 2309/93/EU regulations (http://www.biosafety.be/EMEA/2309_93en.pdf). All
decisions at the clinical trial stage for the PMP would be dealt with by the relevant
national authorities in each EU Member State. At the commercial evaluation stage,
the PMP would be regulated by the European Agency for Evaluation of Medicinal
Products (EMEA). See also Vol. 2, Chap. 11 for discussion.
7.3.2 Biological Challenges
The potential allergenicity of any co-extracted endogenous protein is a further

consideration. For example, targeting expression in plant seeds, which naturally
produce high levels of storage proteins, is a means of generating high yields of
recombinant products (Reggi et al. 2005; Nykiforuk et al. 2006). However, species
such as certain legumes contain endogenous proteins which can induce allergic
reactions. Several potent protein allergens have been identified in peanut (Arachis
hypogeae) including storage proteins Arah1, Arah2 and Arah3 (Barre et al. 2005),
and soybean (Glycine max) such as the vicilin Bd 28k (Tsuji et al. 2001). In plant
species and organs where there are known allergens, isolation of recombinant
protein products from the endogenous components requires stringent and exhaus-
tive purification, generally requiring multiple steps, to minimize risk of allergic
reaction.
7.3.3 Glycosylation and the Production of Clinical Grade

Biopharmaceuticals
One of the major roadblocks in the use of plants as recombinant protein production
platforms is the absence of human-type glycosylation (Wilson 2002; Gomord and
Faye 2004; Paccalet et al. 2007; Bakker et al. 2008). Protein glycosylation is an
issue that has to be taken into account for all protein-production platforms for
biopharmaceuticals that are glycosylated for parenteral administration by injection
or diffusion, whether mammalian, yeast, plant, insect or E. coli (Fig. 7.1). Typi-
cally, the objective will be to replicate the glycosylation profile of the protein
generated when the protein is expressed in the cells of the species in which the
particular protein or gene variant is found. However, in some instances differences
in glycosylation profiles may have no functional consequences in terms of thera-
peutic efficacy (Ko et al. 2003).
Plants are capable of assembling oligosaccharides with linkages not found in
humans and these moieties are immunogenic in humans (Fötisch and Vieths 2001;
Sourrouille et al. 2008). These motifs include b(1!2)-linked xylose (Xyl),
a(1!3)-linked fucose (Fuc) and b(1!3)-linked galactose (Gal). The latter con-
trasts with the b(1!4)-Gal extension typically found at the distal ends of mamma-
lian N-linked oligosaccharides which allows the correct attachment of terminal
sialic acid structures (Sourrouille et al. 2008). There are a number of strategies
Bacteria Yeast Insect Plant Mammalian
xylose N-acetylneuraminic acid

N-acetylglucosamine
galactose
fucose
mannose polypeptide
Fig. 7.1 Typical N-linked oligosaccharides from bacterial to mammalian

being developed to “humanize” the plant glycosylation machinery (Lerouge et al.

2000; Bardor et al. 2006) to better refine the efficacy of their products, boost useable
production and ultimately to lower costs (Castilho et al. 2008). These strategies are
based on (a) inhibition of plant-specific glycosylation enzymes and pathways
(Strasser et al. 2004) and/or (b) introduction of human-specific glycosylation
enzymes and pathways (Bakker et al. 2001). On the other hand, it should be noted
that topical or orally administered recombinant therapeutics may often contain plant
glyco-epitopes with no negative consequences to test subjects. While plants do
have many advantages, there are also competing (and complementary) technology
platforms such as yeast where there are parallel efforts to develop humanized
glycosylation pathways (Chiba and Jigami 2007; Hamilton and Gerngross 2007).
Transformation of plants with a gene for human b(1!4) galactosyltransferase
(b1,4GalT) is one way to combat the formation of natural plant glyco-epitopes on
recombinant proteins (Sourrouille et al. 2008). Human b1,4GalT in its native state
and fused with the Golgi targeting domain of a natural plant GlcNAc transferase
were found to be effective at a1!3Fuc and b(1!2)-Xyl linkage-containing
structures on plant proteins when cloned into alfalfa. The same study also found
that siRNA-mediated silencing of sequences encoding alfalfa XylTs and FucTs
was effective at decreasing the occurrence of plant-specific glyco-motifs on recom-
binant protein products (Sourrouille et al. 2008).
The moss P. patens has been engineered to reduce endogenous synthesis of
a(1!3)-Fuc and b(1!2)-Xyl linkages within N-linked oligosaccharides (Weise
et al. 2007). P. patens plants were transformed with the gene encoding human
erythropoietin (hEPO) and produced a highly glycosylated protein which still
contained the essential N-linked oligosaccharides, but lacked the immunogenic
a(1!3)-Fuc and b(1!2)-Xyl motifs. In small scale bioreactors (maximum
10 L), the moss plants secreted a maximum accumulation of rhEPO (250 mg g–1
dry plant weight) after 6 days of growth.
In humans, a predominant terminal sugar structure is the nine-carbon sialic acid,
5-N-acetyl-D-neuraminic acid (Neu5Ac), which is most often found in either an
a(2!3) or a(2!6) linkage to Gal or N-acetylgalactosamine (GalNAc), although
polysialic acid termination with a(2!8) linkages also occurs. The efficacy of
recombinant proteins can be greatly enhanced by the inclusion of complete
human-type oligosaccharides which allow them to mimic naturally occurring
blood-borne proteins and evade rapid removal by the body, thereby increasing the
half-life (Goochee et al. 1991; Paccalet et al. 2007). hEPO is reported to have only a
2 min half-life without sialylation, but this is extended to 3 h when sialylated
(Ngantung et al. 2006). Clotting Factor VIII has an unsialylated half-life of just
5 min while that of the fully sialylated version is 4 h (Ngantung et al. 2006).
The presence of sialic acids has been reported in A. thaliana suspension cells,
buckwheat and other plant species (Mayer et al. 1964; Onodera et al. 1966;
Bourbouze et al. 1982; Shah et al. 2003). These findings have been subject to much
debate in the literature, partly based on the identification methods used and partly due
to the lack of plant homologs corresponding to mammalian or bacterial enzymes
in their complete sialic acid biosynthetic pathways (Fig. 7.2; Lerouge et al. 2000;
CYTOSOL
UDP-GlcNAc GOLGI
Sialylated
ATP glycoconjugate
GNE
ManNAc-6-P STr
PEP
NANS
glycoconjugate
Neu5Ac-9-P
ST
NANP
CTP
Neu5Ac CMP-Neu5Ac
CMAS
NUCLEUS
Fig. 7.2 Mammlian biosynthetic pathway of sialic acid after Castilho et al. (2008). Enzyme
abbreviations are GNE, UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase; NANS,
N-acetylneuraminic acid phosphate synthase; NANP, Neu5Ac-9-phosphate phosphatase; CMAS,
CMP-Neu5Ac synthetase; ST, CMP-Neu5Ac transporter; STr, sialyltransferase
Faye et al. 2005; Zeleny et al. 2006). Recently, it has been reported that plants may
be able to transport sialic acid (Bakker et al. 2008) and some species may have
transferases capable of attaching sialic acid structures (Takashima et al. 2006).
However, present or not, based on the quantities of sialic acid reported, plants would
not produce sufficient sialylation to be of use in recombinant technologies regardless
of the endogenous mechanism and, thus, molecular engineering to augment any
endogenous pathways would be required (Paccalet et al. 2007).
It would be useful to discover and use plant genes whose products perform the
same function as those from mammals (Lerouge et al. 2000; Bakker et al. 2001;
Bakker et al. 2008). However, this goal comes with the caveat that genes for
enzymes of similar function in plants and mammals may not display high sequence
homology. For example, the discovery of a typically mammalian Gal-b-(1!3)
GalNAc-a-O-Ser/Thr structure on proteins from rice seeds (Kishimoto et al. 1999)
implies the presence of a GalNAc transferase (GalNAcT) gene encoding the
enzyme responsible for mucin-type (short oligosaccharide chains with GalNAc-a-
Ser/Thr linkage to the protein backbone) O-glycosylation initiation. Despite the
functional homology, no gene with significant sequence homology could be found
in the rice genome via an in silico search (Kilcoyne et al., 2009). This could imply
that certain enzymes of homologous function can differ widely from plant to
mammals, or that the same function is fulfilled by other enzymes in planta.
While the basic mechanisms of asparagine-linked core glycosylation in plants are
now considered to be well understood (Lerouge et al. 2000; Wilson 2002; Faye
et al. 2005), many questions still remain concerning exactly how plants perform
fully extended N- and O-linked glycosylation of proteins.
7.4 Plant-Produced Biopharmaceuticals
Biopharmaceuticals have already had an enormous global impact from both health
and economic perspectives. As demand is expected to exceed current supply
capabilities, less expensive alternative production platforms are under intense
investigation with plants firmly at the forefront in terms of biological and cost-
efficiency (Giddings et al. 2000; Daniell et al. 2001; Goldstein and Thomas 2004;
Yano and Takekoshi 2004). To date, many experimental products produced using
plant recombinant technologies have been explored and validated. These will be
discussed as three different groups of biopharmaceuticals: vaccines, antibodies and
protein therapeutics (Table 7.1).
7.4.1 Vaccines
Vaccines comprise the largest class of recombinant biopharmaceuticals (Carter

2006). Vaccinations, either therapeutic or prophylactic, are a cost-effective method
of reducing the occurrence of infectious disease in animals and humans. In general,
vaccines are composed of an antigenic agent from the bacterium or virus containing
an epitope known to induce an immunogenic response in the host, or else are based
on an inactive or attenuated pathogen (e.g., Sabin polio vaccine). Effective vaccines
have been developed and produced for a wide range of diseases, such as hepatitis
B (Keating and Noble 2003; Sojikul et al. 2003; Greco et al. 2007) and rabies (see
below), and are in development for many other diseases, such as HIV (see below).
However, there are large populations vulnerable to major disease burdens, espe-
cially in developing nations, who remain unprotected due to the prohibitive costs
of accessing even traditional or existing vaccines.
Oral delivery of vaccines could address some of the issues that increase vaccine
cost. Elimination of needles and reduced levels of training, expertise and equipment
for delivery of oral vaccines would be beneficial for reducing the cost of vaccine
delivery. Oral delivery can target the antigen to the mucosal surface of the intestinal
lining, which can prime other mucosal surfaces, and this is especially relevant for
gastrointestinal or sexually transmitted diseases. However, this delivery method
also typically leads to degradation of the vaccination agent in the gastrointestinal
Table 7.1 Three groups of biopharmaceuticals: vaccines, antibodies and protein therapeutics with
examples and references
Product Expression platform References
Vaccines
NV-VLP Tobacco, potato Mason et al. (1996), Zhang et al. (2006)
HBsAg Tobacco, potato, tomatillo Mason et al. (1992), Richter et al. (2000),
Kong et al. (2001), Gao et al. (2003),
Huang et al. (2005), Youm et al.
(2007), Sojikul et al. (2003)
HIV-1/HBV fusion Arabidopsis, tobacco Greco et al. (2007), Guetard et al. (2008)
protein
FI-V fusion protein Tobacco, tomato Jones et al. (2003), Williamson et al.
(2005), Mett et al. (2007), Alvarez
et al. (2006), Santi et al. (2006),
Arlen et al. (2008)
LTB Tobacco, corn, potato, Wagner et al. (2004), Lamphear et al.
soybean, carrot (2002), Moravec et al. (2007),
Mason et al. (1998)
PyMSP4/5 Tobacco, tomato Wang et al. (2008), Chowdhury and
Bagasra (2007)
Antibodies
Anti-CD4/28 Wheat Brereton et al. (2007)
receptor scFv
Anti-HBsAg Mab Tobacco Yano and Takekoshi (2004)
Anti-ErbB-2 scFv Tobacco Galeffi et al. (2006)
2G12 Mab Corn Rademacher et al. (2008), Ramessar et al.
(2008)
hsv81sc IgA Micro algae Mayfield and Franklin (2005)
Anti-HBsAg scFv Tobacco Pujol et al. (2007)
Therapeutics
MuIL-12 Tobacco Liu et al. (2008)
hGM-CSF Tobacco, rice, sugarcane Wang et al. (2005)
rh-insulin Safflower, Arabidopsis Moloney et al (2003), Markley et al.
(2006), Nykiforuk et al. (2006)
IFN-a Rice Shirono et al. (2006)
Gcase Tobacco Reggi et al. (2005)
Novokinin Soybeans Yamada et al. (2008)
tract, and to overcome this, large quantities of antigen have to be administered to

ensure that enough will survive to induce immunogenicity. The amount of vaccine
required for delivery of oral vaccines will place strains on the ability of current
mammalian systems to cost-effectively deliver the quantities required.
Ideally, a plant-produced vaccine would require minimal downstream proces-
sing. In this case, an edible (or orally ingested) plant vaccine would deliver the
antigen to the intestinal mucosa in sufficient quantities and could protect the antigen
from degradation in the harsh stomach environment as it is encapsulated within
plant cells. A current drawback for making an edible vaccine a viable option is the
low levels of accumulation of the recombinant protein in plant tissues, but advances
in gene expression systems for plants will allow such issues to be overcome.
Biosafety concerns about transgenic foods (i.e., ensuring production and delivery
is within a strictly controlled closed-loop production and regulatory system) and
consistency (cGMP-related) concerns regarding batch-to-batch consistency are also
barriers to the implementation of edible vaccines. Biosafety issues for the produc-
tion of medicinal plants (e.g., opium poppies) or toxic organs of food plants (e.g.,
poisonous leaves of Solanaceous species, such as potatoes) have been dealt with for
decades and, based on these previous experiences, should be possible to address. In
terms of the consistency issues, low-cost downstream processing approaches
involving concentration or partial purification step(s) could make plant-produced
vaccines feasible for commercial development.
Plant-produced vaccines for livestock have made more progress in getting to
market than those for humans, as they face fewer regulatory issues. Dow Agro
Sciences LLC received the first regulatory approval in January 2006 for a plant-
made injectable poultry vaccine against Newcastle Disease virus (NDV) from the
USDA Center for Veterinary Biologics. The antigen is a haemagglutinin neuramin-
idase protein from NDV that was produced in a transgenic tobacco cell culture
system. Diseases targeted for animal vaccine trials include foot and mouth disease
virus, canine parvovirus, rabies virus, porcine epidemic diarrhea virus and porcine
transmissible gastroenteritis virus (Ma et al. 2005). Diseases and antigens currently
targeted for human vaccinations are discussed below.
Noroviruses (NoV) belong to the Caliciviridae family and consist of one species,
Norwalk virus (NV; Xi et al. 1990). The species is genetically diverse and geno-
types are distributed over five genogroups (GGI–GGV; Ramirez et al. 2008). NoVs
are a group of non-enveloped, single-stranded RNA viruses and are the leading
cause of viral gastroenteritis in humans worldwide (Xi et al. 1990; Vinje et al. 1997;
Fankhauser et al. 1998). They are transmitted directly or indirectly by the fecal-oral
route but may also become airborne and are highly contagious. There is currently no
commercially available vaccine for norovirus but the economic and public health
impact of gastroenteritis epidemics make NoV an ideal candidate for vaccine
development.
Previously, when the capsid protein was expressed in insect cells by recombinant
baculovirus, it self-assembled into empty 38-nm virus-like particles (VLPs), which
were similar to the native virus in morphology and antigenicity (Jiang et al. 1992;
Prasad et al. 1994). These VLPs were immunogenic in CD1 and BALB/c mice
when orally administered (Ball et al. 1998) and the intranasal route induced higher
serum IgG and fecal IgA responses (Guerrero et al. 2001). Additionally, recombi-
nant Norwalk (rNV) VLPs orally administered without an adjuvant to humans in a
phase I study were found to be safe and produce a dose-dependent serum IgG
response (Ball et al. 1999). The potential of rNV VLPs for use as an oral immuno-
gen for a mucosal vaccine has made it a popular target for production in plants.
Partially purified rNV VLPs from transgenic tobacco plants elicited humoral
and mucosal antibody responses specific for rNV in mice, as did feeding with
transformed potato tubers (Mason et al. 1996). In another study by this group, the
plant optimized gene for rNV capsid protein was expressed in tomato and potato
plants and the product successfully assembled VLPs. Lyophilized tomato fruit
induced dose-dependent NV-specific serum IgG and mucosal IgA production in
mice. However, larger quantities of freeze-dried potato tuber (1 g) were required to
elicit the same response. It was found that rehydrated potato tuber was less
immunogenic due to VLP instability caused by phenolic compound oxidation and
that air-dried tomato fruit was more immunogenic than lyophilized tomato fruit and
dried potato tubers (Zhang et al. 2006). Furthermore, when raw transgenic potato
expressing rNV capsid protein was fed to human volunteers, 19 out of 20 volunteers
developed an immune response, although the increase in serum antibody level was
of limited magnitude (Tacket et al. 2000). These VLPs have also been produced in
Nicotiana benthamiana leaves using an engineered plant virus-based transient
expression system (magnICON). Oral immunization with partially purified rNV
VLPs without adjuvant induced serum IgG and fecal and vaginal IgA response in
mice (Santi et al. 2008). Coupled with a low-cost concentration and partial purifica-
tion step, as demonstrated in this study (Santi et al. 2008), the goal of manufacturing
oral vaccines in plant tissue for humans is becoming more achievable.
Over two billion people worldwide are infected with hepatitis B virus (HBV),
which is associated with chronic liver disease and liver cancer (WHO 1998). Hence,
there is a need for an inexpensive vaccine for widespread immunization, especially
in the developing world. Engerix-B1 (GlaxoSmithKline) and Recombivax HB1
(Merck and Co.) are commercial vaccines that confer seroprotection and consist of
recombinant hepatitis B surface antigen (HBsAg) produced in the yeast Saccharo-
myces cerevisiae (Adkins and Wagstaff 1998; Keating and Noble 2003). The
surface envelope glycoprotein exists in three isoforms produced by alternative
splicing and initiation – large (L), middle (M) and small (S) – of which the
commercial vaccine is the recombinantly produced S form (Fig. 7.3). This envelope
protein is reported to have an important role in attachment to the host cell surface
and subsequent infection (Paran et al. 2003).
HBsAgs assemble VLPs and, when expressed in tobacco leaves, the subviral
particles formed were similar to those produced in yeast (Mason et al. 1992).
SHBsAg
MHBsAg
12 108 109 163 164 389

pre- pre- S
LHBsAg
Fig. 7.3 The three isoforms of hepatitis B virus surface envelope glycoprotein (HBsAg) produced
by alternative splicing and initiation – large (L), middle (M) and small (S)
The partially purified antigen from tobacco elicited B- and T-cell responses when
injected in a mouse model and the T-cell antigen epitope was found to be a partial
sequence of the S region of HBsAg (Thanavala et al. 1995). HBsAg VLPs were then
produced in potato tubers with some improvements to increase the yield, such as
targeting the gene product for retention in the plant cell endoplasmic reticulum
(ER; Richter et al. 2000). Mice fed HBsAg-transgenic potatoes with a cholera toxin
adjuvant generated HBsAg-specific serum antibodies. A long-lasting secondary
antibody response was obtained on parenteral boosting (Kong et al. 2001) and,
conversely, by parenteral prime followed by an oral boost (Richter et al. 2000).
HBsAg has also been produced in transgenic cherry tomatillo (Physalis ixocarpa)
that was orally immunogenic in mice (Gao et al. 2003). Additionally, the serum
anti-HBsAg titre increased in over half the group of immunized humans who were
fed uncooked transgenic potatoes without a mucosal adjuvant or buffering for
stomach pH (Thanavala et al. 2005). MHBsAg from transformed tobacco resulted
in an enhanced antibody titre in mice after intraperitoneal (i.p.) injection (Huang
et al. 2005). This same result was also observed when mice were fed potato-derived
MHBsAg (Youm et al. 2007).
In an attempt to overcome low accumulation of antigenic proteins in plant
tissues, a fusion protein of a soybean signal protein with HBsAg was introduced
into suspension-cultured tobacco cells which resulted in greater accumulation.
The fusion protein was more immunogenic in mice than the unmodified HBsAg
and this may have been due to greater antigen stability, improved presentation of
the antigenic determinant in the S domain and increased oligomerization (Sojikul
et al. 2003).
The highly immunogenic hepatitis B core antigen (HBcAg) has also been
recombinantly produced in transgenic tobacco and was found to assemble into
spherical particles 25 to 30 nm in diameter. In the hemagglutination-inhibition
test, partially purified VLPs demonstrated serologic properties comparable to those
produced in E. coli (Tsuda et al. 1998). High levels of production of HBcAg (up to
7.14% of total soluble protein (TSP)) were reported 7 days post-infection using the
magnICON system (Icon Genetics). The product also self-assembled into VLPs and
the partially purified product evoked strong serum antibody response in mice when
injected i.p. Moreover, mucosal immunization (oral and nasal) with no adjuvant
gave HBcAg-specific serum IgG and intestinal IgA response (Huang et al. 2006).
This rapid, high-level antigen production of strong immunogenicity makes a poten-
tial oral vaccine from plants more feasible.
Enterotoxin produced by pathogenic strains of E. coli is the major cause of death
in developing countries and claims 1.6 million lives per annum (Tacket 2007). The
heat-labile toxin is similar to cholera toxin and is composed of the toxic A 27 kDa
subunit and the non-toxic B subunit (LTB) which is a 55 kDa homopentamer of
11.6 kDa subunits. LTB is a strong immunogen that binds to the GM1 ganglioside
on enterocytes and is a popular choice for a vaccine candidate. It has been expressed
in potato (Mason et al. 1998), maize (Lamphear et al. 2002), tobacco (Wagner et al.
2004) and soybean (Moravec et al. 2007); volunteers who consumed transgenic
potato developed serum and/or mucosal immune response (Tacket et al. 1998).
Transgenic defatted corn meal was generally well tolerated and was immunogenic
in volunteers (Tacket et al. 2004). However, as raw potato and uncooked corn meal
can be unpalatable to many, LBT was expressed in transgenic carrot taproots
(Rosales-Mendoza et al. 2008), which can be eaten raw. The product was found
to be immunogenic and also protected against cholera toxin challenge in mice
(Rosales-Mendoza et al. 2008).
A bivalent vaccine of recombinant HIV-1/HBV fusion protein VLPs has been
produced in A. thaliana and tobacco (Greco et al. 2007). The HIV-1 portion
consisted of a polyepitope comprised of eight epitopes from five major HIV-1
proteins. These particular epitopes were expressed together as HIV-1 and HBV
have similar transmission pathways and co-infection with HBV occurs in more
than 30% of HIV-1 patients. Oral administration to mice elicited anti-HIV-1
specific CD8+ T-cell activation (Guetard et al. 2008).
Malaria is another major effective vaccine target. Its prevalence in areas of
extreme poverty that lack infrastructure also makes it an ideal candidate for an oral
or edible vaccine. Malaria is caused by the parasite protozoan genus Plasmodium
transmitted to humans through the bites of infected female Anopheles mosquitoes.
The species Plasmodium vivax and P. falciparum cause most infections in humans
resulting in death and morbidity and are thus the main targets of vaccine develop-
ment (Chowdhury and Bagasra 2007). However, the parasite’s complexity, its
ability to change through its life cycle in humans and mosquitoes, and its ability
to evade the immune system make this a challenge (Chowdhury and Bagasra 2007).
An edible vaccine using transgenic tomatoes of different sizes, shapes and colors to
deliver multiple antigens for the various stages of malarial infection has been
proposed (Chowdhury and Bagasra 2007).
In an attempt to immunize against one life stage, surface protein 4/5 (PyMSP4/5)
of the murine P. yoelii merozoite, the homolog of P. falciparum merozoite surface
proteins 4 and 5, has been selected. The merozoite life stage takes place in
hepatocytes and PyMSP4/5 has been shown to provide protection for mice against
lethal challenge (Kedzierski et al. 2001). PyMSP4/5 from tobacco parenterally
delivered to mice induced antigen-specific antibodies but antibody levels were
not high enough to provide protection against lethal challenge (Wang et al. 2008).
The virulent Gram-negative bacterium, Yersinia pestis, is the cause of plague.
The most common form, bubonic plague, is spread to humans by bites from fleas
that have previously fed on infected animals and is then distributed systematically
in the body. The disease results in swollen, tender lymph nodes (buboes), which can
hemorrhage and become necrotic. The pneumonic form of the disease is almost
always fatal and can also be transmitted by inhalation of infected aerosolized
droplets, making plague a potential bioterrorism agent. Recently, there have been
plague outbreaks in India, Algeria, Congo, Zambia and Malawi (WHO 2007) and
over 2,000 cases are reported annually. Vaccines that are currently available
include killed whole cells, which are not protective against pneumonic plague
and have many undesirable side effects, and a live attenuated vaccine, which also
suffers from unacceptable side effects and has not been approved for use in the
United States (Anisimov and Amoako 2006). However, vaccines based on the
antigenic subunits of F1 (fraction 1 anti-phagocytic capsular envelope glycopro-

tein), V (a secreted immunomodulatory protein) and an F1–V fusion protein have
been intensely investigated and were successful immunogens in animal and human
trials (Jones et al. 2003; Williamson et al. 2005; Mett et al. 2007).
The F1–V fusion protein was expressed in tomato in an attempt to make a
useable oral vaccine. The transgenic tomatoes were immunogenic in mice primed
subcutaneously (s.c.) with bacterially produced F1–V and orally boosted by feeding
with freeze-dried transgenic tomato fruit (Alvarez et al. 2006). In this case, no
pathogen challenge was performed. The first report of a protective vaccination of
animals using plant-produced antigenic material, which appeared in 2006, made use
of the magnICON system to produce high levels of recombinant F1, V (2 mg g–1
fresh leaf weight) and F1–V (1 mg g–1 fresh leaf weight) antigens in tobacco leaves
(Santi et al. 2006). Female guinea pigs were vaccinated s.c. with the partially
purified antigens and alum adjuvant. After exposure to aerosolized virulent
Y. pestis, V-vaccinated animals had the highest survival rate.
The F1–V fusion antigen has been expressed in tobacco leaf chloroplasts, giving
a maximum yield of 14.8% of product out of the total soluble protein. Mice were
s.c. primed with enriched crude F1–V fusion protein from plant with alum adjuvant
and boosted either s.c., with adjuvanted doses, or orally, with unadjuvanted doses.
Oral F1–V mice had higher prechallenge serum IgG1 titers than s.c. injected F1–V
mice. After exposure to an inhaled lethal dose of aerosolized Y. pestis, 33% of the
adjuvanated F1–V s.c.-boosted mice and 88% of the orally boosted mice with
unadjuvanted F1–V survived. Hence, it may be concluded that oral booster doses
induce protective immune responses in vivo (Arlen et al. 2008).
7.4.2 Antibodies
Antibodies are the second largest class of biopharmaceuticals (Carter 2006). Serum
from animals immune to particular infectious disease antigens have been used as
therapeutics for at least a century (Yano and Takekoshi 2004). Orthoclone, a
monoclonal antibody used to treat organ rejection after transplants, was approved
for human use in 1986 (reviewed in Hiatt and Pauly 2006). The year 1987 marked
the first studies on the feasibility of antibody production using plants as expression
hosts, and the first report of recovered recombinant protein from plants followed in
1989 (Pujol et al. 2007). Monoclonal antibodies (MAbs) produced in plants are
often referred to as “plantibodies,” though use of this designation remains arbitrary.
Plants and plant cells may be engineered to produce antibodies by either stable or
transient expression.
A transient expression approach that has been used in plant suspension cell
cultures has been reported to decrease the timescale for producing milligram
quantities of antibodies from approximately a year (i.e., for whole transgenic
plant systems) to under a month (Hiatt and Pauly 2006). One component of the
process, termed magnifection (magnICON, Icon Genetics), is the result of adapting
viral transcripts to the expression host machinery by including additional introns

which better match those of mRNAs encoding endogenous plant proteins, and by
shortening overall mRNA lengths (Gleba et al. 2005). When used with a single viral
vector, magnifection is sufficient for the enhanced expression of individual light
chains but not complex hetero-oligomeric antibodies. Complete MAbs (consisting
of both light and heavy chains) were produced using magnifection and co-infection
with two separate viral vectors (Giritch et al. 2006). In contrast to stable plant
expression of antibodies, it is claimed that this approach offers more rapid deve-
lopment and better possibilities for efficient biological containment.
Many examples of methods for producing plantibodies have appeared in the
literature. Wheat (Triticum aestivum cv. Westonia), a monocot species, was trans-
formed to produce single light chain antibody (scFv) fragments suitable for use in
ocular disease treatment. The need to ensure very low levels of bacterial endo-
toxins, which may be present in bacterial expression systems, was cited as a major
benefit of producing antibody fragments in plants. ScFv chains, which bound to
CD4 or CD28 receptors on the surface of thymocytes, were produced using particle
bombardment transformation of T. aestivum calli. The maximum expression was
180 mg g–1 in crude extracts made from mature seeds of T. aestivum (Brereton et al.
2007). The affinity of the plant-produced scFvs was approximately equivalent to
that of single-chain fragments produced in bacteria.
Hepatitis B infection may be treated by the administration of antibodies against
the surface proteins of the viral envelope. Tobacco suspension cultures have been
used to produce recombinant antibodies against HBV (Yano et al. 2004) and
required a two-step transformation process. Cells derived from a patient carrying
natural antibodies against HBV surface antigens were immortalized by transforma-
tion with Epstein–Barr virus (EBV). From these EBV cells, RNA coding for the
MAbs was extracted and used to make cDNA copies which were cloned into plant
suspension cells. Proteins harvested directly from EBV transformed cells cannot be
used as human therapeutics due to the risk of infecting patients with EBV (Yano and
Takekoshi 2004). Hence, plant suspension cultures were used to produce the
monoclonal IgGs destined for use as human therapeutics. IgGs were purified and
compared with their counterparts from EBV TAPC301-CL4 cells for efficacy. The
plant-produced antibody was shown to produce a similar complement-dependent
cytotoxicity (Yano et al. 2004). The Cuban Centre for Biotechnology and Genetic
Engineering (CIGB) has been producing a monoclonal antibody (CB-Hep1) against
HBV in transgenic tobacco plants since April 2006 when regulatory use approval
was granted for this plantibody. The plant production platform for CB-Hep1 is used
for large-scale purification of the active component of CIGB’s hepatitis B vaccine,
sold under the trade name Heberbiovac-HB.
Aggressive carcinomas have been experimentally treated with antibodies bind-
ing to ErbB-2, a surface receptor that is a member of the epidermal growth factor
class of signaling proteins (Galeffi et al. 2006). A multi-platform approach was
used to produce stable transgenic tobacco plants expressing anti-ErbB-2 scFv
chains. scFv clones made from murine hybridomas were produced with and
without peptide spacer arms and used to transform N. tabacum plants through
A. tumefaciens infection. The resulting purified scFvs demonstrated a similar

binding constant to the parental hybridoma-derived MAbs and were also shown
to be useful in histochemical staining of tumor tissue (Galeffi et al. 2006).
It may be possible to prevent human HIV infection in developing countries by
using plant-produced antibodies as topical viruscides (Rademacher et al. 2008;
Ramessar et al. 2008). The antibodies function to neutralize the virus particles,
stopping them from entering hosts. Secretable forms of heavy and light antibodies
(2G12) directed against the coat proteins of HIV-1 were made in transgenic corn
(Zea mays L.). Antibodies accumulated in the endosperm of corn seeds, allowing
them to be dried and handled without need for refrigeration. Furthermore, a
threefold increase in the neutralization activity of the corn-produced antibodies
was reported when compared to 2G12 antibodies derived from CHO cells (Ramessar
et al. 2008). This increase in efficacy was suggested to be a result of multivalent
presentation as a result of aggregation of the recombinant antibodies as they were
deposited in the endosperm protein bodies (Ramessar et al. 2008).
Microalgae (C. reinhardtii) have also been explored as a system for producing
single scFv fragments of human A-type immunoglobulins (Mayfield and Franklin
2005). Chlamydomonas spp. are able to grow by phototrophy or heterotrophy and
may take in carbon through acetate (Mayfield and Franklin 2005). Genomic and
chloroplast DNA was transformed by the particle bombardment method. While the
yield was conditional on a high degree of codon optimization specific both for the
species and organelle transformed (nuclear genome or chloroplast), the authors
reported a yield of up to 0.5% (w/w total soluble protein) for hsv81sc when
optimized and used with atpA or rbcL promoters and accompanying 5’ untranslated
regions (Mayfield and Franklin 2005).
Apart from direct therapeutic uses, antibodies produced in plants may also have
a role in the purification of co-expressed biopharmaceuticals. MAbs have been
made in plants specifically to capture plant-manufactured antigens used in hepatitis
vaccines (Pujol et al. 2007). Two transgene constructs, one for a scFv fragment and
one encoding a complete copy of a mouse anti-hepatitis B surface protein antibody,
have been produced in tobacco seeds and in suspension cultures. In the case of
the suspension cultures, antibodies were transiently expressed after transformation
with A. tumefaciens and samples collected five days after initial transformation.
Recovery of whole “plantibody” structures was estimated to be on the order of 0.3
to 0.5 mg mL–1 of suspension culture (Pujol et al. 2007).
7.4.3 Therapeutic Proteins
Many therapeutic proteins can be produced in transgenic plants. For instance,

cytokines are a class of bioactive therapeutic proteins with stimulatory and/or
immune suppression functions. Cytokines have many possible therapeutic uses,
including as pro-immune supplements co-administered during treatments for other
disorders.
Human interleukin 12 (IL-12) is a large, heterodimeric pro-inflammatory cyto-

kine composed of the heavily glycosylated p35 subunit covalently linked by a
disulphide bond to a lightly glycosylated p40 subunit. Mice are frequently used
as an in vivo model for human immune characteristics and murine IL-12 (MuIL-12)
is homologous in both amino acid sequence and function to that of humans. A
single-chain construct of mouse cytokine MuIL-12 was expressed in N. tabacum
whole plants and hairy roots (Liu et al. 2008). The p35/p40 fusion MuIL-12 consists
of an IL-12p40 sequence joined by a triplet repeat of Gly–Gly–Gly–Gly–Ser to a p35
sequence (Mattner et al. 1993). A single chain was used instead of co-expression to
ensure a 1:1 ratio of subunits and assembly of both IL-12 subunits. Mouse spleno-
cyte proliferation was induced by the purified plant-produced MuIL-12 comparable
to the effect of MuIL-12 produced in animal cells (Liu et al. 2008). Protein
expression was up to 40 mg g–1 from fresh leaf tissue, while the hairy root cultures
produced up to 33 mg g–1 of fresh tissue.
Neutropenia, a condition characterized by a lack of granulocytes in the blood-
stream, is often encountered as a side effect of treatments, which suppress immune
function (Sardana et al. 2007). One of the therapies used to treat neutropenia relies
on the replacement of granuloctyte-macrophage colony-stimulating factor (hGM-
CSF). hGM-CSF is a cytokine normally found in the human body which stimulates
neutrophil and monocyte production as part of innate (antimicrobial) immune
function. Production of recombinant hGM-CSF has been achieved in rice suspen-
sion culture cells, tobacco and rice seeds, and the vegetative tissue of sugarcane
(Wang et al. 2005; Joo et al. 2006; Sardana et al. 2007).
Seeds of Oryza sativa cv. Xiushui 11 accumulated 1.3% (w/w) of total soluble
protein when transformed with the gene for hGM-CSF driven by a glutelin pro-
moter, Gt1, which allowed protein body targeting of the recombinant protein
(Wang et al. 2005; Sardana et al. 2007). Biologically active hGM-CSF has also
been produced in sugarcane (Saccharum hybrid; Wang et al. 2005). The hGM-CSF
gene was introduced by particle bombardment of embryogenic callus tissue and
resulted in a maximum yield of 0.02% of total soluble protein (Wang et al. 2005).
An ER-retention tag was necessary to achieve detectable accumulation levels in the
transgenic plants and, over the course of 14 months, this accumulation rate
remained stable. As sugarcane plants do not reach flowering under cultivation
and are harvested prior to flower budding, no pollen-mediated gene transfer is
possible (Wang et al. 2005).
SemBioSys Genetics (Calgary, Alberta, Canada, http://www.sembiosys.ca), has
begun efforts to produce human insulin at a very large scale in plants to meet the
16,000 kg demand expected by 2010 (Moloney et al. 2003). The innovative
SemBioSys production system targets recombinant insulin to the oil bodies and
allows traditional oil/water phase separation and purification to harvest the recom-
binant insulin from the seeds (Moloney et al. 2003; Nykiforuk et al. 2006).
Oleosins, proteins which occupy the outer membrane of oil bodies, are used in
the targeting of the recombinant oleosin fusion proteins to the oil bodies (Markley
et al. 2006) by creating fusion constructs containing an oleosin-targeting peptide
fused to the protein product of interest. Because of the lipid association of the oil
body membranes and localized protein, liquid–liquid phase separation can be used
to efficiently recover the biopharmaceuticals. Oil bodies in seeds of A. thaliana
have also been targeted for the transgenic production of human insulin and shown
to produce a maximum yield of 0.13% (w/w total soluble seed protein) with
comparable activity to insulin produced in yeast expression systems (Nykiforuk
et al. 2006).
Shirono and co-workers have used transgenic rice suspension cells derived from
dwarf rice plants (O. sativa L. cv. Hosetsu-dwarf) to produce active interferon-a
(IFN-a). IFN-a is used to treat disorders resulting from the intrusion of some classes
of chemicals and foreign bodies, including micoorganisms and viruses (Shirono
et al. 2006). Transformation of the rice suspension cells with A. tumefaciens
containing a transgene for IFN-a under the control of the constitutive 35S promoter
resulted in stable production of active protein for at least ten generations. The
transgene construct included the first intron of the rice cytosolic superoxide dis-
mutase gene, a 10 kDa prolamin signal sequence, a GUS reporter sequence, and a
thrombin cleavage sequence followed by the human IFN-a gene (Shirono et al.
2006).
Current treatment for Gaucher disease requires replacement therapy utilizing
human b-glucosyl-N-acylsphingosineglycohydrolase (also known as b-glucosidase,
EC 3.2.1.45, abbreviated GCase), an enzyme that cleaves glucosylceramide into
glucose and ceramide (Reggi et al. 2005). Gaucher disease is a fatal autosomal
disorder manifesting itself in homozygous recessive infants in the general popula-
tion at approximately 1:200,000 but approximately 1:640–10,000 in some Jewish
subpopulations (Reggi et al. 2005; Weinstein 2007). While it is possible that
functional, native GCase can be purified from human placental tissue, low yield
and risk of contamination make the process less than desirable for therapeutic use.
Recombinant GCase from tobacco plants was purified and found to be enzymati-
cally active and readily taken up by human fibroblasts (Reggi et al. 2005). Further-
more, although the presence of an N-linked glycan at one site is required for the
protein to be catalytically active, it was free from plant-specific glyco-epitopes
containing Xyl and Fuc, thus greatly reducing the possibility of an immune response
in patients.
Novokinin is a hypotensive therapeutic hexapeptide with the sequence Arg–
Pro–Leu–Lys–Pro–Trp, originally derived from the naturally occurring protein
ovalbumin found in avian eggs (Yamada et al. 2008). A transgene vector coding
for a modified form of naturally occurring a’ subunit of b-conglycinin soy protein,
which incorporated four copies of the Arg–Pro–Leu–Lys–Pro–Trp peptide
sequence, was introduced into soy plants and produced a yield of 0.5% of total
soluble seed protein for the peptide of interest. Defatted flour from the transgenic
soybeans administered orally reduced systolic blood pressure in spontaneously
hypertensive rats (Yamada et al. 2008).
Extracts from plants of the Digitalis genus have been used for centuries as
cardiovascular therapeutics (Michael 2006). Lanatosides are secondary metabolites
harvested from Digitalis lanata EHRH for the production of cardiotonic drugs
(Shi and Lindemann 2006). After the leaves are processed to isolate the lanatosides,
the enzyme cardenolide 16’-O-glucohydrolase (CGH-1) is recovered and used to

remove glucosylation from the lanatosides. This enhances human resorption of the
secondary metabolites and allows the formation of the active medicinal chemicals,
digitoxin and digoxin. Purification of active CGH-1 from D. lanata leaves is a
relatively expensive process. Therefore, recombinant expression of the enzyme
has been explored to produce larger quantities of CGH-1 more economically.
Since bacterially produced CGH-1 did not have comparable activity to that
harvested from D. lanata leaves, a plant-based eukaryotic expression strategy
was investigated. CGH-1 was produced in induced Cucumis sativis hairy root
but it too was observed to have less activity than the foliar D. lanata enzyme (Shi
and Lindemann 2006).
7.5 Plants as Model Systems for Biopharmaceutical

Development for Humans and Other Mammals
Due to their limited foliar mass and small seeds, whole A. thaliana plants may not
be a suitable volume expression system for most commercial biopharmaceutical
production efforts. However, Cobento Biotech in Denmark is using A. thaliana for
cGMP production of human intrinsic factor (rhIF), which is used for treatment
of vitamin B12 deficiency. Nonetheless, A. thaliana has made a tremendous con-
tribution to the understanding of plants, protein expression and the intricacies of
molecular interaction within eukaryotes. To the biomedical and pharmaceutical
research community, plants at first may seem unlikely candidates to study the
effects of chemicals and pathogens on human health. However, from an evolution-
ary and comparative biochemistry perspective, plants can be used instead of
animals to better understand many aspects of eukaryote gene regulation and
biochemistry. Indeed, a recent study has highlighted that many significant discov-
eries with direct relevance to biomedical science and medicine have been achieved
using the model plant A. thaliana, while many biological processes of relevance to
human health are easier to study in this model plant than in mammalian or model
animal (e.g., Drosophila, C. elegans) systems (Jones et al. 2008). Relative ease of
care, rapid generation maturity in many model and crop plants and complete or
near-complete genome sequence availability contribute to these uses (van Baarlen
et al. 2007).
The fully sequenced and extremely well-annotated genome of this model plant
(in conjunction with its powerful genetics) allows the use of A. thaliana for studies
directly linked to human drug metabolism, which may prove invaluable for the
development of new drugs. The pharmacogenetic effects of small molecules on
A. thaliana have been characterized to model individual organism sensitivity to
drugs. Attributes that make this non-animal platform desirable for such studies
include availability of a growing range of genetic and molecular tools including
recombinant inbred lines (RILs), near-isogenic lines (NILs), a high degree of
phenotype variation among accessions, and high polymorphism rates on a per-

nucleotide basis. Current efforts to re-sequence the genomes of 1,001 A. thaliana
accessions (Ossowski et al. 2008) as well as the generation of extremely dense SNP
maps of this model plant (Kim et al. 2007) will make it an even more powerful
genetic model. These ongoing advances are making A. thaliana increasingly suited
for “rapid molecular genetic characterization of alleles” (Zhao et al. 2007). For
example, Zhao and co-workers (Zhao et al. 2007) elucidated the effects of gluco-
sylation of the chemical hypostatin in subpopulations of A. thaliana plants, which
would normally suffer growth retardation from this treatment. It was found that
individuals which expressed large amounts of the enzyme HYR-1 produced gluco-
sylated hypostatin which stunted growth of the plants. This led to a lower inhibitory
concentration (IC50) value in connection with the stunted phenotype (Zhao et al.
2007). These kinds of data mirror the roles of various factors in drug efficacy and
toxicity found in humans.
7.6 Conclusions
Clearly, transgenic PMPs have major promise for the efficient and cost-effective
production of protein-based biopharmaceuticals. Many reports now demonstrate
that transgenic plant systems can produce vaccines, antibodies and other protein-
based therapeutics cost-effectively and potential human pathogen-free. In the case
of some vaccines, it may be possible using plant-based systems to develop
approaches for these to be administered orally, which confers the potential to
reduce vaccine delivery costs to poorer patients (e.g., in developing countries).
One of the major barriers to the commercial realization of a PMP industry is the
path-dependency and capital inertia in the current mammalian cell culture produc-
tion paradigm. The concept of path-dependency is frequently used to analyze trends
in innovation where path-dependency is associated with the idea of “lock-in” (Patel
and Pavitt 1997). While a technology may be quite flexible when first developed,
over time more fixed pathways become established which act as barriers to entry for
new innovations. Examples of technology lock-in include the QWERTY keyboard
or the VHS video format. The extremely high capital costs of producing therapeutic
proteins in mammalian cell culture systems are inherently linked to an expensive
and detailed regulatory approval system that has been developed specifically for
mammalian, yeast and bacterial cell culture systems for production of protein-based
biopharmaceuticals.
In some jurisdictions (e.g., EU), the regulatory pathways for approval of recom-
binant protein production from disruptive innovations such as transgenic plant-
produced pharmaceuticals are still under development and until such regulatory
pathways are developed, a disincentive will remain for commercial development
of plants as production platforms for biopharmaceuticals.
While the regulations are catching up with the science, there is a need for
continued research to improve further the efficiency, safety and cost-effectiveness
of such systems. For instance, the glycoprotein biochemistry of plants differs from
that of humans, requiring remodeling of biosynthetic pathways to avoid allergic or
immunological response. Each plant system also has potential for further optimiza-
tion of the expression system and yields, and both fundamental and applied research
on understanding gene regulation, protein expression and post-translational biology
in plants will drive further advances in this area.
There is a growing acceptance in the PMPs community that the first wave of
biopharmaceuticals to be commercially produced in plants will need to be produced
in closed-loop systems ideally under biological containment (i.e., in reverse pres-
sure controlled environment greenhouses or in photobioreactors). This will help
address issues such as batch-to-batch variation and also any possible biosafety risks
that could arise. As many of the biopharmaceutical products intended for produc-
tion in transgenic plants are already under commercial production as recombinant
(genetically engineered) products in mammalian cell culture, it is difficult to
envisage logical objections to the commercial production of such recombinant
proteins in transgenic plants under biological containment.
Overall, it is clear that as the technology, regulatory systems and business
models evolve for PMPs, a greater proportion of our therapeutics will be produced
in plants in the future – hopefully, at a more competitive cost for society and public
health than current therapeutic costs.
References
Adkins JC, Wagstaff AJ (1998) Recombinant hepatitis B vaccine: a review of its immunogenicity
and protective efficacy against hepatitis B. BioDrugs 10:137–158
Alvarez ML, Pinyerd HL, Crisantes JD, Rigano MM, Pinkhasov J, Walmsley AM, Mason HS,
Cardineau GA (2006) Plant-made subunit vaccine against pneumonic and bubonic plague is
orally immunogenic in mice. Vaccine 24:2477–2490
Anisimov AP, Amoako KK (2006) Treatment of plague: promising alternatives to antibiotics.
J Med Microbiol 55:1461–1475
Anisimov A, Koivu K, Kanerva A, Kaijalainen S, Juntunen K, Kuvshinov V (2007) Cloning of
new rubisco promoters from Brassica rapa and determination of their activity in stably
transformed Brassica napus and Nicotiana tabacum plants. Mol Breed 19:241–253
Arlen PA, Singleton M, Adamovicz JJ, Ding Y, Davoodi-Semiromi A, Daniell H (2008) Effective
plague vaccination via oral delivery of plant cells expressing F1-V antigens in chloroplasts.
Infect Immun 76:3640–3650
Bakker H, Bardor M, Molthoff JW, Gomord V, Elbers I, Stevens LH, Jordi W, Lommen A, Faye L,
Lerouge P, Bosch D (2001) Galactose-extended glycans of antibodies produced by transgenic
plants. Proc Natl Acad Sci USA 98:2899–2904
Bakker H, Routier F, Ashikov A, Neumann D, Bosch D, Gerardy-Schahn R (2008) A CMP-sialic
acid transporter cloned from Arabidopsis thaliana. Carbohydr Res 343:2148–2152
Ball JM, Hardy ME, Atmar RL, Conner ME, Estes MK (1998) Oral immunization with recombi-
nant Norwalk virus-like particles induces a systemic and mucosal immune response in mice.
J Virol 72:1345–1353
Ball JM, Graham DY, Opekun AR, Gilger MA, Guerrero RA, Estes MK (1999) Recombinant
Norwalk virus-like particles given orally to volunteers: Phase I study. Gastroenterology
117:40–48
Bardor M, Cabrera G, Rudd PM, Dwek RA, Cremata JA, Lerouge P (2006) Analytical strategies to
investigate plant N-glycan profiles in the context of plant-made pharmaceuticals. Curr Opin
Struct Biol 16:576–583
Barre A, Borges J-P, Rougé P (2005) Molecular modelling of the major peanut allergen Ara h 1
and other homotrimeric allergens of the cupin superfamily: a structural basis for their IgE-
binding cross-reactivity. Biochimie 87:499–506
Bourbouze R, Akiki C, Chardonloriaux I, Percheron F (1982) The evidence for neuraminic acid
derivatives in vegetable glycoproteins. Carbohydr Res 106:21–30
Brereton HM, Chamberlain D, Yang R, Tea M, McNeil S, Coster DJ, Williams KA (2007) Single
chain antibody fragments for ocular use produced at high levels in a commercial wheat variety.
J Biotechnol 129:539–546
Broothaerts W, Mitchell HJ, Weir B, Kaines S, Smith LMA, Yang W, Mayer JE, Roa-Rodriguez C,
Jefferson RA (2005) Gene transfer to plants by diverse species of bacteria. Nature
433:629–633
Butler M (2006) Optimisation of the cellular metabolism of glycosylation for recombinant proteins
produced by mammalian cell systems. Cytotechnology 50:57–76
Byrne SL, Leverence R, Klein JS, Giannetti AM, Smith VC, MacGillivray RT, Kaltashov IA,
Mason AB (2006) Effect of glycosylation on the function of a soluble, recombinant form of the
transferrin receptor. Biochemistry 45:6663–6673
Carter PJ (2006) Potent antibody therapeutics by design. Nat Rev Immunol 6:343–357
Castilho A, Pabst M, Leonard R, Veit C, Altmann F, Mach L, Glossl J, Strasser R, Steinkellner H
(2008) Construction of a functional CMP-sialic acid biosynthesis pathway in Arabidopsis.
Chiba Y, Jigami Y (2007) Production of humanized glycoproteins in bacteria and yeasts. Curr
Opin Chem Biol 11:670–676
Chowdhury K, Bagasra O (2007) An edible vaccine for malaria using transgenic tomatoes of
varying sizes, shapes and colors to carry different antigens. Med Hypotheses 68:22–30
Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional
analysis of genes in planta. Plant Physiol 133:462–469
Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular farming: production of antibodies,
biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 6:219–226
Domingo E, Martin V, Perales C, Grande-Perez A, Garcia-Arriaza J, Arias A (2006) Viruses as
quasispecies: biological implications. Curr Top Microbiol Immunol 299:51–82
Dunwell JM (2005) Review: intellectual property aspects of plant transformation. Plant Biotech-
nol J 3:371–384
Eichler R, Lenz O, Garten W, Strecker T (2006) The role of single N-glycans in proteolytic
processing and cell surface transport of the Lassa virus glycoprotein GP-C. Virol J 3:41
Fankhauser RL, Noel JS, Monroe SS, Ando T, Glass RI (1998) Molecular epidemiology of
“Norwalk-like viruses” in outbreaks of gastroenteritis in the United States. J Infect Dis
178:1571–1578
Faye L, Boulaflous A, Benchabane M, Gomord V, Michaud D (2005) Protein modifications in the
plant secretory pathway: current status and practical implications in molecular pharming.
Vaccine 23:1770–1778
Fischer R, Liao YC, Hoffmann K, Schillberg S, Emans N (1999) Molecular farming of recombi-
nant antibodies in plants. Biol Chem 380:825–839
Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM (2004) Plant-based production of
biopharmaceuticals. Curr Opin Plant Biol 7:152–158
Fötisch K, Vieths S (2001) N- and O-linked oligosaccharides of allergenic glycoproteins. Glyco-
conj J 18:373–390
Galeffi P, Lombardi A, Pietraforte I, Novelli F, Di Donato M, Sperandei M, Tornambe A, Fraioli
R, Martayan A, Natali P, Benevolo M, Mottolese M, Ylera F, Cantale C, Giacomini P (2006)
Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems.
J Transl Med 4:39
Gao Y, Ma Y, Li M, Cheng T, Li S-W, Zhang J, Xia N-S (2003) Oral immunization of animals
with transgenic cherry tomatillo expressing HBsAg. World J Gastroenterol 9:996–1002
Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharma-
ceuticals. Nat Biotechnol 18:1151–1155
Giritch A, Marillonnet S, Engler C, van Eldik G, Botterman J, Klimyuk V, Gleba Y (2006) Rapid
high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral
vectors. Proc Natl Acad Sci USA 103:14701–14706
Gleba Y, Klimyuk V, Marillonnet S (2005) Magnifection – a new platform for expressing
recombinant vaccines in plants. Vaccine 23:2042–2048
Goldstein DA, Thomas JA (2004) Biopharmaceuticals derived from genetically modified plants.
QJM 97:705–716
Gomord V, Faye L (2004) Posttranslational modification of therapeutic proteins in plants. Curr
Gomord V, Chamberlain P, Jefferis R, Faye L (2005) Biopharmaceutical production in plants:
problems, solutions and opportunities. Trends Biotechnol 23:559–565
Goochee CF, Gramer MJ, Andersen DC, Bahr JB, Rasmussen JR (1991) The oligosaccharides
of glycoproteins: bioprocess factors affecting oligosaccharide structure and their effect on
glycoprotein properties. Biotechnology (NY) 9:1327–1355
Greco R, Michel M, Guetard D, Cervantes-Gonzalez M, Pelucchi N, Wain-Hobson S, Sala F, Sala
M (2007) Production of recombinant HIV-1/HBV virus-like particles in Nicotiana tabacum
and Arabidopsis thaliana plants for a bivalent plant-based vaccine. Vaccine 25:8228–8240
Guerrero RA, Ball JM, Krater SS, Pacheco SE, Clements JD, Estes MK (2001) Recombinant
Norwalk Virus-like particles administered intranasally to mice induce systemic and mucosal
(fecal and vaginal) immune responses. J Virol 75:9713–9722
Guetard D, Greco R, Cervantes Gonzalez M, Celli S, Kostrzak A, Langlade-Demoyen P, Sala F,
Wain-Hobson S, Sala M (2008) Immunogenicity and tolerance following HIV-1/HBV plant-
based oral vaccine administration. Vaccine 26:4477–4485
Hamilton SR, Gerngross TU (2007) Glycosylation engineering in yeast: the advent of fully
humanized yeast. Curr Opin Biotechnol 18:387–392
Hellwig S, Drossard J, Twyman RM, Fischer R (2004) Plant cell cultures for the production of
recombinant proteins. Nat Biotechnol 22:1415–1422
Hiatt A, Pauly M (2006) Monoclonal antibodies from plants: a new speed record. Proc Natl Acad
Sci USA 103:14645–14646
Hou B-K, Zhou Y-H, Wan L-H, Zhang Z-L, Shen G-F, Chen Z-H, Hu Z-M (2003) Chloroplast
transformation in oilseed rape. Transgenic Res 12:111–114
Huang Z, Elkin G, Maloney BJ, Beuhner N, Arntzen CJ, Thanavala Y, Mason HS (2005) Virus-
like particle expression and assembly in plants: hepatitis B and Norwalk viruses. Vaccine
23:1851–1858
Huang Z, Santi L, LePore K, Kilbourne J, Arntzen CJ, Mason HS (2006) Rapid, high-level
production of hepatitis B core antigen in plant leaf and its immunogenicity in mice. Vaccine
24:2506–2513
Jiang X, Wang M, Graham DY, Estes MK (1992) Expression, self-assembly, and antigenicity of
the Norwalk virus capsid protein. J Virol 66:6527–6532
Jones SM, Griffin KF, Hodgson E, Williamson ED (2003) Protective efficacy of a fully recombi-
nant plague vaccine in the guinea pig. Vaccine 21:3912–3918
Jones AM, Chory J, Dangl JL, Estelle M, Jacobsen SE, Meyerowitz EM, Nordborg M, Weigel D
(2008) The impact of Arabidopsis on human health: diversifying our portfolio. Cell
133:939–943
Joo S-Y, Lee K-H, Lee Y-I, Kim D-I (2006) Enhanced production of hGM-CSF by
medium exchange in transgenic Oryza sativa L. suspension cultures. Enzyme Microb Technol
39:486–489
Keating GM, Noble S (2003) Recombinant hepatitis B vaccine (Engerix-B1): a review of its
immunogenicity and protective efficacy against hepatitis B. Drugs 63:1021–1051
Kedzierski L, Black CG, Stowers AW, Goschnick MW, Kaslow DC, Coppel RL (2001) Compari-
son of the protective efficacy of yeast-derived and Escherichia coli-derived recombinant
merozoite surface protein 4/5 against lethal challenge by Plasmodium yoelii. Vaccine
19:4661–4668
Kilcoyne M, Shah M, Gerlach JQ, Bhavanandan V, Nagaraj V, Smith AD, Fujiyama K, Sommer
U, Costello CE, Olszewski N, Joshi L (2009) O-glycosylation of protein subpopulations in
alcohol-extracted rice proteins. J Plant Physiol 166:219–232
Kim S, Plagnol V, Hu TT, Toomajian C, Clark RM, Ossowski S, Ecker JR, Weigel D, Nordborg M
(2007) Recombination and linkage disequilibrium in Arabidopsis thaliana. Nat Genet
39:1151–1155
Kishimoto T, Watanabe M, Mitsui T, Hori H (1999) Glutelin basic subunits have a mammalian
mucin-type O-linked disaccharide side chain. Arch Biochem Biophys 370:271–277
Ko K, Tekoah Y, Rudd PM, Harvey DJ, Dwek RA, Spitsin S, Hanlon CA, Rupprecht C,
Dietzschold B, Golovkin M, Koprowski H (2003) Function and glycosylation of plant-derived
antiviral monoclonal antibody. Proc Natl Acad Sci USA 100:8013–8018
Kolewe ME, Gaurav V, Roberts SC (2008) Pharmaceutically active natural product synthesis and
supply via plant cell culture technology. Mol Pharm 5:243–256
Kong Q, Richter L, Yang YF, Arntzen CJ, Mason HS, Thanavala Y (2001) Oral immunization
with hepatitis B surface antigen expressed in transgenic plants. Proc Natl Acad Sci USA
98:11539–11544
Lamphear BJ, Streatfield SJ, Jilka JM, Brooks CA, Barker DK, Turner DD, Delaney DE, Garcia
M, Wiggins B, Woodard SL, Hood EE, Tizard IR, Lawhorn B, Howard JA (2002) Delivery of
subunit vaccines in maize seed. J Control Release 85:169–180
Lerouge P, Bardor M, Pagny S, Gomord V, Faye L (2000) N-Glycosylation of recombinant
pharmaceutical glycoproteins produced in transgenic plants towards a humanisation of plant
N-glycans. Curr Pharm Biotechnol 1:347
Liu J, Dolan MC, Reidy M, Cramer CL (2008) Expression of bioactive single-chain murine IL-12
in transgenic plants. J Interferon Cytokine Res 28:381–392
Ma JKC, Chikwamba R, Sparrow P, Fischer R, Mahoney R, Twyman RM (2005) Plant-derived
pharmaceuticals – the road forward. Trends Plant Sci 10:580–585
Mallory AC, Parks G, Endres MW, Baulcombe D, Bowman LH, Pruss GJ, Vance VB (2002) The
amplicon-plus system for high-level expression of transgenes in plants. Nat Biotechnol
20:622–625
Markley NA, Nykiforuk CL, Boothe JG, Moloney M (2006) Producing proteins using transgenic
oilbody-oleosin technology. BioPharm Int 19:34–57
Mason HS, Lam DM, Arntzen CJ (1992) Expression of hepatitis B surface antigen in transgenic
plants. Proc Natl Acad Sci USA 89:11745–11749
Mason HS, Ball JM, Shi JJ, Jiang X, Estes MK, Arntzen CJ (1996) Expression of Norwalk virus
capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc Natl
Mason HS, Haq TA, Clements JD, Arntzen CJ (1998) Edible vaccine protects mice against
Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene.
Vaccine 16:1336–1343
Mattner F, Fischer S, Guckes S, Jin S, Kaulen H, Schmitt E, Rude E, Germann T (1993) The
interleukin-12 subunit p40 specifically inhibits effects of the interleukin-12 heterodimer. Eur
J Immunol 23:2202–2208
Mayer FC, Dam R, Pazur JH (1964) Occurence of sialic acid in plant seeds. Arch Biochem
Biophys 108:356–357
Mayfield SP, Franklin SE (2005) Expression of human antibodies in eukaryotic micro-algae.
Vaccine 23:1828–1832
Mett V, Lyons J, Musiychuk K, Chichester JA, Brasil T, Couch R, Sherwood R, Palmer GA,
Streatfield SJ, Yusibov V (2007) A plant-produced plague vaccine candidate confers protection
to monkeys. Vaccine 25:3014–3017
Michael WF (2006) Plants, medicines and man. J Sci Food Agric 86:1797–1804
Mitra N, Sinha S, Ramya TN, Surolia A (2006) N-linked oligosaccharides as outfitters for
glycoprotein folding, form and function. Trends Biochem Sci 31:156–163
Moloney M, Boothe J, Van Rooijen G (2003) Sembiosys: oil bodies and associated proteins as
affinity matrices. US Patent 6509453
Moravec T, Schmidt MA, Herman EM, Woodford-Thomas T (2007) Production of Escherichia
coli heat labile toxin (LT) B subunit in soybean seed and analysis of its immunogenicity as an
oral vaccine. Vaccine 25:1647–1657
Ngantung FA, Miller PD, Brushett FR, Tang GL, Wang DIC (2006) RNA interference of sialidase
improves glycoprotein sialic acid content consistency. Biotechnol Bioeng 95:106–119
Nykiforuk CL, Boothe JG, Murray EW, Keon RG, Goren HJ, Markley NA, Moloney MM (2006)
Transgenic expression and recovery of biologically active recombinant human insulin from
Arabidopsis thaliana seeds. Plant Biotechnol J 4:77–85
Onodera K, Hirano S, Hayashi H (1966) Sialic acid and related substances. 4. Sialic acid content of
some biological materials. Agric Biol Chem 30:1170–1172
Ossowski S, Schneeberger K, Clark RM, Lanz C, Warthmann N, Weigel D (2008) Sequencing of
natural strains of Arabidopsis thaliana with short reads. Genome Res 18:2024–2033
Paccalet T, Bardor M, Rihouey C, Delmas F, Chevalier C, D’Aoust MA, Faye L, Vezina L,
Gomord V, Lerouge P (2007) Engineering of a sialic acid synthesis pathway in transgenic
plants by expression of bacterial Neu5Ac-synthesizing enzymes. Plant Biotechnol J 5:16–25
Paran N, Cooper A, Shaul Y (2003) Interaction of hepatitis B virus with cells. Rev Med Virol
13:137–143
Patel P, Pavitt K (1997) The technological competencies of the world’s largest firms: Complex and
path-dependent, but not much variety. Res Policy 26:141–156
Pogson BJ, Woo NS, Forster B, Small ID (2008) Plastid signalling to the nucleus and beyond.
Trends Plant Sci 13:602–609
Prasad BVV, Rothnagel R, Jiang X, Estes MK (1994) Three-dimensional structure of Baculovirus-
expressed Norwalk Virus capsids. J Virol 68:5117–5125
Pujol M, Gavilondo J, Ayala M, Rodrı́guez M, González EM, Pérez L (2007) Fighting cancer with
plant-expressed pharmaceuticals. Trends Biotechnol 25:455–459
Rademacher T, Sack M, Arcalis E, Stadlmann J, Balzer S, Altmann F, Quendler H, Steigler G,
Kunert R, Fischer R, Stoger E (2008) Recombinant antibody 2G12 produced in maize
endosperm efficiently neutralizes HIV-1 and contains predominantly single-GlcNAc
N-glycans. Plant Biotechnol J 6:189–201
Ramessar K, Rademacher T, Sack M, Stadlmann J, Platis D, Stiegler G, Labrou N, Altmann F, Ma
J, Stoger E, Capell T, Christou P (2008) Cost-effective production of a vaginal protein
microbicide to prevent HIV transmission. Proc Natl Acad Sci USA 105:3727–3732
Ramirez S, Giammanco GM, De Grazia S, Colomba C, Martella V, Arista S (2008) Genotyping
of GII.4 and GIIb norovirus RT-PCR amplicons by RFLP analysis. J Virol Methods
147:250–256
Reggi S, Marchetti S, Patti T, Amicis FD, Cariati R, Bembi B, Fogher C (2005) Recombinant
human acid b-glucosidase stored in tobacco seed is stable, active and taken up by human
fibroblasts. Plant Mol Biol 57:101–113
Richter LJ, Thanavala Y, Arntzen CJ, Mason HS (2000) Production of hepatitis B surface antigen
in transgenic plants for oral immunization. Nat Biotechnol 18:1167–1171
Rosales-Mendoza S, Soria-Guerra R, López-Revilla R, Moreno-Fierros L, Alpuche-Solı́s Á (2008)
Ingestion of transgenic carrots expressing the Escherichia coli heat-labile enterotoxin B
subunit protects mice against cholera toxin challenge. Plant Cell Rep 27:79–84
Ruf S, Karcher D, Bock R (2007) Determining the transgene containment level provided by
chloroplast transformation. Proc Natl Acad Sci USA 104:6998–7002
Sala F, Manuela Rigano M, Barbante A, Basso B, Walmsley AM, Castiglione S (2003) Vaccine
antigen production in transgenic plants: strategies, gene constructs and perspectives. Vaccine
21:803–808
Santi L, Giritch A, Roy CJ, Marillonnet S, Klimyuk V, Gleba Y, Webb R, Arntzen CJ, Mason HS
(2006) Protection conferred by recombinant Yersinia pestis antigens produced by a rapid and
highly scalable plant expression system. Proc Natl Acad Sci USA 103:861–866
Santi L, Batchelor L, Huang Z, Hjelm B, Kilbourne J, Arntzen CJ, Chen Q, Mason HS (2008) An
efficient plant viral expression system generating orally immunogenic Norwalk virus-like
particles. Vaccine 26:1846–1854
Sardana R, Dudani A, Tackaberry E, Alli Z, Porter S, Rowlandson K, Ganz P, Altosaar I (2007)
Biologically active human GM-CSF produced in the seeds of transgenic rice plants. Transgenic
Res 16:713–721
Shah MM, Fujiyama K, Flynn CR, Joshi L (2003) Sialylated endogenous glycoconjugates in plant
cells. Nat Biotechnol 21:1470–1471
Shi H-P, Lindemann P (2006) Expression of recombinant Digitalis lanata EHRH. cardenolide
160 -O-glucohydrolase in Cucumis sativus L. hairy roots. Plant Cell Rep 25:1193–1198
Shirono H, Morita S, Miki Y, Kurita A, Morita S, Koga J, Tanaka K, Masumura T (2006) Highly
efficient production of human interferon-α by transgenic cultured rice cells. Plant
Sojikul P, Buehner N, Mason HS (2003) A plant signal peptide-hepatitis B surface antigen fusion
protein with enhanced stability and immunogenicity expressed in plant cells. Proc Natl Acad
Sci USA 100:2209–2214
Sourrouille C, Marquet-Blouin E, D’Aoust M-A, Kiefer-Meyer M-C, Seveno M, Pagny-
Salehabadi S, Bardor M, Durambur G, Lerouge P, Vezina L, Gomord V (2008) Down-
regulated expression of plant-specific glycoepitopes in alfalfa. Plant Biotechnol J 6:702–721
Stoger E, Ma JKC, Fischer R, Christou P (2005) Sowing the seeds of success: pharmaceutical
proteins from plants. Curr Opin Biotechnol 16:167–173
Strasser R, Altmann F, Mach L, Glossl J, Steinkellner H (2004) Generation of Arabidopsis
thaliana plants with complex N-glycans lacking beta1, 2-linked xylose and core alpha1,
3-linked fucose. FEBS Lett 561:132–136
Streatfield SJ (2005) Regulatory issues for plant-made pharmaceuticals and vaccines. Expert Rev
Vaccines 4:591–601
Tacket CO (2007) Plant-based vaccines against diarrheal diseases. Trans Am Clin Climatol Assoc
118:79–87
Tacket CO, Mason HS, Losonsky G, Clements JD, Levine MM, Arntzen CJ (1998) Immunoge-
nicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nat Med
4:607–609
Tacket CO, Mason HS, Losonsky G, Estes MK, Levine MM, Arntzen CJ (2000) Human immune
responses to a novel Norwalk Virus Vaccine delivered in transgenic potatoes. J Infect Dis
182:302–305
Tacket CO, Pasetti MF, Edelman R, Howard JA, Streatfield S (2004) Immunogenicity of recom-
binant LT-B delivered orally to humans in transgenic corn. Vaccine 22:4385–4389
Takashima S, Abe T, Yoshida S, Kawahigashi H, Saito T, Tsuji S, Tsujimoto M (2006) Analysis of
sialyltransferase-like proteins from Oryza sativa. J Biochem 139:279–287
Thanavala Y, Yang YF, Lyons P, Mason HS, Arntzen C (1995) Immunogenicity of transgenic
plant-derived hepatitis B surface antigen. Proc Natl Acad Sci USA 92:3358–3361
Thanavala Y, Mahoney M, Pal S, Scott A, Richter L, Natarajan N, Goodwin P, Arntzen CJ, Mason
HS (2005) Immunogenicity in humans of an edible vaccine for hepatitis B. Proc Natl Acad Sci
USA 102:3378–3382
Tsuda S, Yoshioka K, Tanaka T, Iwata A, Yoshikawa A, Watanabe Y, Okada Y (1998) Applica-
tion of the human hepatitis B virus core antigen from transgenic tobacco plants for serological
diagnosis. Vox Sang 74:148–155
Tsuji H, Hiemori M, Kimoto M, Yamashita H, Kobatake R, Adachi M, Fukuda T, Bando N, Okita
M, Utsumi S (2001) Cloning of cDNA encoding a soybean allergen, Gly m Bd 28K. Biochim
Biophys Acta 1518:178–182
Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R (2003) Molecular farming in plants:
host systems and expression technology. Trends Biotechnol 21:570–578
Vain P (2007) Thirty years of plant transformation technology development. Plant Biotechnol
J 5:221–229
van Baarlen P, van Belkum A, Thomma BPHJ (2007) Disease induction by human microbial
pathogens in plant-model systems: potential, problems and prospects. Drug Discov Today
12:167–173
Venter M (2007) Synthetic promoters: genetic control through cis engineering. Trends Plant Sci
12:118–124
Vidi P-A, Kessler F, Brehelin C (2007) Plastoglobules: a new address for targeting recombinant
proteins in the chloroplast. BMC Biotechnol 7:4
Vinje J, Altena SA, Koopmans MPG (1997) The incidence and variability of small round-
structured viruses in outbreaks of gastroenteritis in The Netherlands. J Infect Dis 176:
1374–1378
Wagner B, Hufnagl K, Radauer C, Wagner S, Baier K, Scheiner O, Wiedermann U, Breiteneder H
(2004) Expression of the B subunit of the heat-labile enterotoxin of Escherichia coli in tobacco
mosaic virus-infected Nicotiana benthamiana plants and its characterization as mucosal
immunogen and adjuvant. J Immunol Methods 287:203–215
Walsh G (2006) Biopharmaceutical benchmarks 2006. Nat Biotechnol 24:769–776
Wang M-L, Goldstein C, Su W, Moore PH, Albert HH (2005) Production of biologically active
GM-CSF in sugarcane: a secure biofactory. Transgenic Res 14:167–178
Wang L, Webster DE, Campbell AE, Dry IB, Wesselingh SL, Coppel RL (2008) Immunogenicity
of Plasmodium yoelii merozoite surface protein 4/5 produced in transgenic plants. Int J
Parasitol 38:103–110
Weinstein LB (2007) Selected genetic disorders affecting Ashkenazi Jewish families. Fam Com-
munity Health 30:50–62
Weise A, Altmann F, Rodriguez-Franco M, Sjoberg ER, Baumer W, Launhardt H, Kietzmann M,
Gorr G (2007) High-level expression of secreted complex glycosylated recombinant human
erythropoietin in the Physcomitrella D-fuc-t D-xyl-t mutant. Plant Biotechnol J 5:389–401
WHO (1998) The World Health Report 1998: life in the 21st century – a vision for all. World
Health Organisation, Geneva
WHO (2007) The World Health Report 2007 – a safer future: global public health security in the
21st century. World Health Organisation, Geneva
Williamson ED, Flick-Smith HC, LeButt C, Rowland CA, Jones SM, Waters EL, Gwyther RJ,
Miller J, Packer PJ, Irving M (2005) Human immune response to a plague vaccine comprising
recombinant F1 and V antigens. Infect Immun 73:3598–3608
Wilson IBH (2002) Glycosylation of proteins in plants and invertebrates. Curr Opin Struct Biol
12:569–577
Woodson JD, Chory J (2008) Coordination of gene expression between organellar and nuclear
genomes. Nat Rev Genet 9:383–395
Xi JN, Graham DY, Wang KN, Estes MK (1990) Norwalk virus genome cloning and characteri-
zation. Science 250:1580–1583
Yamada Y, Nishizawa K, Yokoo M, Zhao H, Onishi K, Teraishi M, Utsumi S, Ishimoto M,
Yoshikawa M (2008) Anti-hypertensive activity of genetically modified soybean seeds accu-
mulating novokinin. Peptides 29:331–337
Yano A, Takekoshi M (2004) Transgenic plant-derived pharmaceuticals – the practical approach?
Expert Opin Biol Ther 4:1565–1568
Yano A, Maeda F, Takekoshi M (2004) Transgenic tobacco cells producing the human monoclo-
nal antibody to hepatitis B virus surface antigen. J Med Virol 73:208–215
Youm J-W, Won Y-S, Jeon JH, Ryu CJ, Choi Y-K, Kim H-C, Kim B-D, Joung H, Kim HS (2007)
Oral immunogenicity of potato-derived HBsAg middle protein in BALB/c mice. Vaccine
25:577–584
Zeleny R, Kolarich D, Strasser R, Altmann F (2006) Sialic acid concentrations in plants are in the
range of inadvertent contamination. Planta 224:222–227
Zhang X, Buehner NA, Hutson AM, Estes MK, Mason HS (2006) Tomato is a highly effective
vehicle for expression and oral immunization with Norwalk virus capsid protein. Plant
Zhao Y, Chow TF, Puckrin RS, Alfred SE, Korir AK, Larive CK, Cutler SR (2007) Chemical
genetic interrogation of natural variation uncovers a molecule that is glycoactivated. Nat Chem
Biol 3:716–721
Chapter 8
Biotech Crops for Ecology and Environment
Saikat Kumar Basu, François Eudes, and Igor Kovalchuk
8.1 Introduction
Environmental pollution is a serious problem plaguing humanity, modern human

society and the quality of human life all across the globe. Chemical pollution is a
potent source of ecological and environmental degradation in recent times because
of the extensive use of chemicals in our modern life (Gray 2006; Datta Banik et al.
2007). Environmentally toxic chemicals range from a wide diversity of different
functional groups and species. They include: toxicants, irritants, mutagens, clasto-
gens, carcinogens, teratogens, plastics, xenobiotics, pesticides and fertilizers, heavy
metals, metalloids, pharmaceutical compounds, organic compounds, industrial
effluents, untreated domestic and industrial wastes, different radioactive wastes,
radionuclides, and abandoned military ammunition chemicals (Schnoor et al. 1995;
Schnoor 1997; Salt et al. 1998; Thompson et al. 1998; Lucero et al. 1999; Hooker
and Skeen 1999; Yoon et al. 2002).
Aggressive industrialization and urbanization, rapid depletion of forest areas to
extensive agriculture, mining, metal smelting, fuel production and energy genera-
tion, indiscriminate and unplanned dumping of industrial and domestic wastes
(such as sludge dumping), and the enormous increase in human population in
different parts of the world have all further aggravated issues and concerns about
environmental pollution (Cunningham et al. 1995; Salt et al. 1998; Zayed 2004;
Willey 2007). These factors have synergistically contributed towards an increase in
inefficient and improper chemical waste disposal, seriously reducing the quality of
land as well as human and animal life (Black 1995; Cunningham et al. 1995; Salt
et al. 1998; Zayed 2004; Ghosh and Singh 2005).
I. Kovalchuk (*)
Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge,
AB, Canada T1K 3M4
e-mail: igor.kovalchuk@uleth.ca

302 S.K. Basu et al.
Let us consider one of the most serious examples of chemical pollution in our
recent history, the problem of mercury (Hg) contamination. According to Pacyna
and Pacyna (2002), the global Hg emissions approximate to 1,900 tons, and ~75%
of that originates from the use of fossil fuels, when coal is burnt for thermal power
generation and related anthropogenic activities. The remaining 25% are supplied by
waste disposal sites, cement manufacturers and waste incineration centers. The bulk
of the pollution problem (about 50%) comes from countries in Asia while North
American and European Union (EU) member countries share the other half of Hg
emissions (Renneberg and Dudas 2001; Wagner-Dobler 2003). More than half of
the total Hg emissions include elemental Hg, while the remaining half contains
divalent and particulate forms (Rugh et al. 1996; Heaton et al. 1998; Pacyna and
Pacyna 2002). The most serious concern regarding Hg emissions is related to the
deposition of Hg in the form of snow and rainfall. In this case, it is converted
into more toxic forms, such as ionic and organic Hg, thus posing a considerable
health and environmental threat for arctic regions of Canada and the northeastern
United States (Rugh et al. 1996; Boyajian and Carrieira 1997; Heaton et al. 1998;
Renneberg and Dudas 2001; Pacyna and Pacyna 2002). It has been rightly pointed
out that the majority of serious pollutants introduced into the natural environment
and ecosystems are almost always anthropogenic in nature (Gratao et al. 2005;
Datta Banik et al. 2007).
Cleaning the polluted environment is a major task of our time. Plants having
unique physiological and metabolic processes have always served as an excellent,
handy, economical and eco-friendly source of natural remediation effort (Black
1995; Boyajian and Carrieira 1997; Salt et al. 1998). Nowadays, genetically
engineered higher plants, called transgenic plants, popularly called Biotech
Crops (BCs), play a very important role in the phytoremediation of toxic pollutants
(Suresh and Ravishankar 2004; Cherian and Oliveira 2005; Willey 2007). Also,
different microbes (Liu and Suflita 1993; Pollard et al. 1994; Banat 1995; Shann
1995; Cassidy et al. 1996; White et al. 1998; Juhasz and Naidu 2000; Lovely 2003;
Denton 2007; Mendez and Maier 2008), animals (Hammer 1996; Meier et al.
1997; Milanese et al. 2003; MacKenzie et al. 2004; Gifford et al. 2005, 2007;
Giangrande et al. 2006; Stabili et al. 2006), algae (Olguin 2003), fungi (Singh
2006), and even lichens (McLean et al. 1998) have been reported to be involved
in bioremediation of toxic pollutants. Using biotechnology innovations during the
past few decades, researchers have exploited transgenic plants to reduce impacts
of harmful pollutants in nature (Salt et al. 1998; Zayed 2004; Willey 2007;
Aken 2008).
In this review, we have provided a detailed historical overview of biotechnology
progress in utilizing transgenic plants for phytoremediation of pollutants; we also
discuss their applications, advantages and limitations. Possible outcomes and
projected advances in genetic engineering of plants to be used for phytoremediation
have also been provided. In addition, a very brief outline of other related applica-
tions of biotech crops in ecology and environment, such as biomonitoring and
production of biopolymers and bioplastics, has been included.
8 Biotech Crops for Ecology and Environment 303
8.2 Phytoremediation
8.2.1 Definition
The word phytoremediation is derived from the Greek prefix phyto-, meaning
“plant,” and the Latin word remedium, meaning “to cure, clean” (Gray 2006).
Professor Ilya Raskin at the Rutgers University in New Jersey, US, is credited
with coining this term (Black 1995). Other alternative terms for phytoremediation,
as reported in primary literature, are “green remediation” and “botanical remedia-
tion” suggested by Chaney et al. (1997) to indicate environmental cleanup by green
plants. Very recently, Hassinen et al. (2007) coined the term “green technology” for
environmental detoxification based on phytoremediation.
Phytoremediation is a natural, green-plant-mediated and solar-energy-driven
process that is economically feasible and environment-friendly. It cleans up harm-
ful and toxic pollutants from the environment by biodegrading, trapping and
accumulating them in their specific organs, tissues and cells. Phytoremediation
can also be defined as the use of plants to transform environmental contaminants
into less toxic/non-bioavailable forms and even to stimulate soil microbial com-
munities to either biodegrade or accumulate them, thereby restricting their move-
ment or migration to nonpolluted sites and groundwater resources and protecting
the environment (Salt et al. 1998; Reeves and Baker 2000; Suresh and Ravishankar
2004; Azevedo and Azavedo 2006; Gifford et al. 2007). As already mentioned, a
large number of technical terms are associated with phytoremediation research and
used by researchers. Several of them are used interchangeably or as alternative
terms for almost the same applications (Black 1995; Salt et al. 1995, 1998; Datta
and Sarkar 2004; Suresh and Ravishankar 2004; Zayed 2004; Azevedo and
Azavedo 2006). To avoid unnecessary confusion over these widely used terms,
we have provided a comprehensive table of terms and terminologies commonly
used in phytoremediation research and in literature sources dealing with phyto-
remediation (Table 8.1). For further simplification of different types and activities
related and/or associated with phytoremediation, a simple schematic representation
of currently available techniques has been illustrated in Fig. 8.1.
8.2.2 Uses and Applications of Phytoremediation
Conventional procedures of eradicating contaminants and pollutants include expen-

sive treatments such as excavation, dredging, electrolytic extraction processes,
chemical and acid leaching, soil washing, solidification or stabilization, vitrifica-
tion, chemical oxidation or reduction, electrokinetical treatment, use of incinera-
tors, pumping and treating of contaminated water, vapor stripping, thermal
desorption, and other expensive physical and chemical treatments (Raskin et al.
1994; Salt et al. 1995; Barcelo and Poschenrieder 2003; Gratao et al. 2005). Among
Table 8.1 Glossary of important terms and terminology associated with using plants for environment remediation
304
Terminologies Definitions References

Bioremediation Use of living organisms (predominantly microbes) for removal McGloughlin and Burke (2000), Reeves and Baker (2000),
and/or detoxification of pollutants within a given Mendez and Maier (2008)
environment
Phytoremediation Application of green plants for degradation, removal and Raskin et al. (1994), Cunningham et al. (1995), Raskin
detoxification of environmental pollutants. Also known as (1996), Chaney et al. (1997), Reeves and Baker (2000),
“Green Remediation” or “Botanical Remediation” or Sursala et al. (2002), Eapen and D’Souza (2005),
“Green Technology” Pilon-Smits (2005), Hassinen et al. (2007), Najmanova
et al. (2007), Aken (2008)
Phycoremediation Phytoremediation achieved by the use of different algal species Olguin (2003)
Mycoremediation Phytoremediation achieved by the use of different mushrooms Singh (2006)
and other fungal species
Phytodecontamination Removal of contamination from the site using Cunningham et al. (1995), Sadowsky (1999)
phytoremediation only
Phytodetoxification Complete detoxification of contaminated sites by using green Cunningham et al. (1995), Salt et al. (1995), Bizily et al.
plants (2000), Zayed (2004)
Phytoextraction Harvest, extraction and subsequent treatment of above-ground Raskin et al. (1994), Nanda Kumar et al. (1995), Salt et al.
plant biomass involved in phytoremediation, specifically by (1995), Trapp and Karlson (2001), Sursala et al.
using hyperaccumulating plant species (2002), Gratao et al. (2005), Pilon-Smits (2005),
Gifford et al. (2007)
Phytoaccumulation Plant roots taking up metal contaminants and accumulating Raskin (1996), Blaylock et al. (1997), Salt et al. (1998),
them in the stem and leaves. Also termed phytoextraction Suresh and Ravishankar (2004), Pilon-Smits (2005)
and often used interchangeably
Phytostabilization Application of plant roots for preventing further migration of Raskin et al. (1994), Cunningham et al. (1995), Chaney
toxic pollutants in the soil, thereby regulating their et al. (1997), Salt et al. (1995), Reeves and Baker
uncontrolled migration towards groundwater and converting (2000), Trapp and Karlson (2001), Zayed (2004),
them into less bioavailable forms Pilon-Smits (2005), Gifford et al. (2007)
Phytovolatilization Conversion of harmful and toxic pollutants to less toxic forms Cunningham et al. (1995), Salt et al. (1995), Raskin
by green plants (1996), Chaney et al. (1997), Vroblesky et al. (1999),
Trapp and Karlson (2001), Zayed (2004), Pilon-Smits
(2005)
S.K. Basu et al.
8
Phytotransformation/ Application of plants in biodegrading toxic organic compounds Salt et al. (1995, 1998), Dietz and Schnoor (2001), Suresh
Biotransformation into less toxic chemical forms. The toxicity of several and Ravishankar (2004), Gifford et al. (2007)
metals and metalloids can be reduced in plants by chemical
reduction of the element because of its incorporation into
available organic compounds (biotransformation)
Phytodegradation Enzymatic breakdown of organic pollutants both internally and Newman et al. (1997), Salt et al. (1998), Trapp and
externally by secreted enzymes. An alternate term for the Karlson (2001), Suresh and Ravishankar (2004), Pilon-
above Smits (2005), Gifford et al. (2007)
Plant hyperaccumulator/ Plants species reported to accumulate toxic heavy metals in the Chaney et al. (1997), Salt et al. (1998), Reeves and Baker
Phytohyperaccumulators/ ranges of >100 mg/kg for Cd, Cr, Co, Pb; or >1,000 mg/kg (2000), Sursala et al. (2002), Gifford et al. (2007)
Hyperaccumulators for Ni, Cu, Se, As, Al; or 10,000 mg/kg for Zn, Mn in their
above-ground dry weight biomass
Phytotolerance/ Ability of plants to survive and thrive in heavily contaminated de Crombrugghe (1964), Chaney et al. (1997), Cluis
Phytoresistance/ sites contaminated with heavy metals or metalloids (2004)
Hypertolerance
Phytostimulation Green plants promoting the subsequent breakdown of Cluis (2004), Suresh and Ravishankar (2004), Pilon-Smits
environmental pollutants by microbial communities (2005)

Phytorestoration Complete remediation of a contaminated/polluted site to fully Bradshaw (1997)
functional decontaminated soil
Phytomining Exploitation of sub-economic ore bodies using plants Brooks et al. (1998, 1999), Gratao et al. (2005)
Phytotoxicity When a potentially harmful substance has accumulated in the Beckett and Davis (1988), Naidu et al. (2003)
plant tissue to a level affecting plant growth and
development
Phytosiderophores Siderophores (metallophores, chelating agents) that are non- Raskin et al. (1994), Chaney et al. (1997), Salt et al. (1998)
synthetic and are exclusively of a plant origin. They help in
metal binding in the soil
Phytoindicators/ Plant assemblages associated with specific environmental Raskin et al. (1994), Simon et al. (1996), Mendez and
Bioindicators/Biological conditions and/or ecosystems that are referred to as Maier (2008)
indicators phytoindicators or plant indicators. When biological
organisms (plants, animals) are meant, the term
bioindicators are used. For animals. the term zooindicator is
used; in case of microbes the terms microbial/bacterial
indicators are used
305
(continued )
306
Terminologies Definitions References

Plant biomonitor/ A plant providing complete quantitative information on Kovalchuk and Kovalchuk (2001, 2003, 2008)
Phytomonitor environment quality
Plant-assisted bioremediation Remediation of soils contaminated with organic pollutants by Salt et al. (1995)
plant roots in association with rhizosphere-inhabiting
microbial communities
Rhizosecretion This is a subset of molecular farming designed to produce and Gleba et al. (1999)
secrete valuable natural products and recombinant proteins
from roots
Rhizofiltration/Phytofiltration Use of live plant roots for the removal of toxic heavy metals and Raskin et al. (1994), Dushekov et al. (1995), Raskin et al.
other pollutants from water or any liquid source. Also (1997), Salt et al. (1998), Trapp and Karlson (2001),
referred to as phytofiltration Zayed (2004), Pilon-Smits (2005)
Mycofiltration Fungal mycelial mats used as biological filters Stamets and Sumerlin (undated)
Rhizosphere degradation/ Plant roots and/or root exudates provide a local environment Schnoor et al. (1995), Salt et al. (1998), Ramaswami et al.
Rhizodegradation rich in nutrients and enzymes in the rhizosphere that (2003), Suresh and Ravishankar (2004), Pilon-Smits
promotes degradation of soil contaminating pollutants by (2005)
resident microbial communities. Microbial breakdown of
organic pollutants in the rhizosphere
Organic pump/Tree pump Trees with dense root systems accumulate greater volume of Salt et al. (1998), Trapp and Karlson (2001), Suresh and
water, thereby reducing possibilities of surface pollutants to Ravishankar (2004)
migrate downwards towards the groundwater table and
contaminate freshwater resources. Extensively used for
regulating run off from agricultural fields and leaching of
toxic pollutants from landfill sites
Land farming Used for land affected by oil pollution. Sludge is ploughed onto Trapp and Karlson (2001)
topsoil, fertilizers are applied, and grasses mostly rye
(Secale cereale) or alfalfa (Medicago sativa) are then
sown on it
Oil is degraded rapidly in the rooted, aerated and fertilized
topsoil zone
S.K. Basu et al.
CUMULATOR
HYPER AC
PHYTOREMEDIATION PHYTOVOLATILIZATION
PHYTODECONTAMINATION PHYTOTRANSFORMATION
PHYTODETOXIFICATION PHYTODEGRADATION
PHYTORESTORATION PHYTOCONVERSION
Less toxic pollutant

PHYTOEXTRACTION forms
PHYTOACCUMULATION
Highly toxic pollutant

forms
PHYTOSTABILIZATION
RHIZOFILTRATION
PHYTOMINING
PHYTOSTIMULATION
ORGANIC PUMP RHIZOSPHERE DEGRADATION
GROUND WATER
Fig. 8.1 Schematic representation of different types of phytoremediation and relationships

between them
biological soil treatments, the most common ones are landfarming (see Table 8.1)
and ex situ techniques such as biopiles, slurry reactors and composting (Cunningham
et al. 1995).
Phytoremediation has been considered to be an environmentally compatible,
sustainable, easily monitored, efficient and less expensive approach for the removal
and detoxification of harmful environmental pollutants, compared to other chemical
engineering alternatives (Baker and Brooks 1989; Baker et al. 1994; Nanda Kumar
et al. 1995; Raskin et al. 1997; Datta and Sarkar 2004; Gray 2006). Reliable cost
estimates associated with phytoremediation have been evaluated earlier by Cun-
ningham et al. (1995) and recently by Pilon-Smits (2005). An important message as
indicated by Gratao et al. (2005) is that phytoremediation processes are cost-
effective and safe alternatives to conventional physical and chemical treatments.
Phytoremediation is a process with a lower impact on the surrounding environment
and without any disruption of highly fragile and vulnerable ecosystems (Barcelo
and Poschenrieder 2003; Zayed 2004). Although a large number of plants (known
as hyperaccumulators) are capable of bioaccumulating high concentrations of toxic
metals, they generally do not generate sufficient biomass and are not efficient for
phytoremediation over a longer period of time. Hence, an alternative solution could
be the creation of transgenic plants with greater biomass, faster growth rate, and
better phytoremediation characters.
Phytoremediation is an advantageous process in the sense that it helps remove
toxic components in situ, and there are no direct risks of environmental contamina-
tion exposure during handling and transfer of pollutants from the contaminated
Soil stabilization, Restoration of Immobilization

reclamation, nutritional and and trapping of
amelioration & biological
revegetation pollutants before
qualities of
Cost-effective, reaching groundwater
contaminated soil
sustainable,
non-intrusive,
environment-friendly Reduction of
soil erosion
Reduction of:
Agricultural surface
run-offs, loss of Decontamination
top soil, sediment of contaminated sites
run-offs, soil moisture Phytoremediation
Benefits
Biodegradation and
Landscaping,
detoxification of
increased value
military munitions
of remediated
compounds
landfills
Improving qualities of
dump sites, landfills,
agricultural lands, forests,
Wildlife Improvement of Aesthetic and wetlands, abandoned
habitat the local micro recreational industrial sites, mining
restoration climate values areas, marginal lands
Fig. 8.2 Benefits of phytoremediation
sites. Moreover, the process itself does not generate secondary waste products
(Baker and Brooks 1989; Baker et al. 1994; Shann 1995; Chaney et al. 1997;
Zayed 2004; Willey 2007). Benefits of phytoremediation have been compared to
conventional physical and chemical remediation treatments (Fig. 8.2).
8.2.3 Historical Background
The basic and empirical studies of phytoremediation focused mainly on and around
natural plant species capable of detoxifying harmful substances and on their natural
properties of hyperaccumulation of toxic metals and environmentally detrimental
toxic chemical compounds (Baker and Brooks 1989; Nanda Kumar et al. 1995; Salt
et al. 1995; Shann 1995; Chaney et al. 1997; Raskin et al. 1997; Datta and Sarkar
2004). Over the past few decades, extensive investigations have been conducted on
different phytoremediation strategies and techniques used by plants (Banuelos et al.
1993; Baker et al. 1994; Salt et al. 1998).
The role of a number of phytoremediating species involved in complete and
partial degradation of chemical explosives (as reviewed in Hannink et al. 2002),
heavy metals and metalloids (Terry et al. 2003), and their biochemical pathways for
uptakes has been extensively studied. Basic research associated with phytoreme-
diation was primarily focused on identifying and screening plants capable of
withstanding heavy metals and radionuclide pollution, such as natural hyperaccu-

mulators. Plants capable of decontamination of polluted sites by rapid biosorption,
bioaccumulation, biodegradation, and their conversion (biotransformation) to less
toxic, non-bioavailable forms have also been extensively studied and reviewed in
primary literature sources (Freeman et al. 2004; Suresh and Ravishankar 2004;
Cherian and Oliveira 2005; Pilon-Smits 2005; Denton 2007).
A large number of chemicals such as heavy metals, xenobiotics, nitramines and
nitraromatics, herbicides, pesticides, fertilizers, complex organic and inorganic
salts and compounds, pharmaceutically important compounds, and radioactive
chemicals have been reported to be successfully phytoremediated by different
plant species and model plants used in the laboratories around the globe (Ellis
et al. 2004; Freeman et al. 2004; Suresh and Ravishankar 2004; Cherian and
Oliveira 2005; Mezzari et al. 2005; Willey 2007). The promising potential roles
played by different metallothionein (MT) and phytochelatin (PC) genes have been
reviewed by Eapen and D’Souza (2005).
Recently, Hassinen et al. (2007) reported that ~400 plant species have been
identified as hyperaccumulators, and the majority of these plants are Brassicaceae
(Arabidopsis family) members hyperaccumulating nickel (Ni). A large number of
papers are also available on physiology, biochemistry, metabolism, transport
mechanisms, sequestration patterns of toxic heavy metals and other pollutant
compounds (Baker and Brooks 1989; Baker et al. 1994; Nanda Kumar et al.
1995; Shann 1995; Chaney et al. 1997; Pence et al. 2000; Ellis et al. 2004; Freeman
et al. 2004; Denton 2007).
8.2.4 Transgenics Research on Herbs and Shrubs
Compared to natural and conventional phytoremediators, genetically engineered

plants or transgenic plants (transgenics) have proved to be a rather handy tool in
generating actively phytoremediating plants because of their enhanced ability to
effectively reduce, degrade, transform and accumulate toxic pollutants from the
environment; their growth rates are rapid, and they have better bioaccumulation
characteristics (Cherian and Oliveira 2005; Willey 2007).
Conventional plant breeding can only utilize available and rather limited genetic
resources within species and genera of plants for phytoremediation. On the con-
trary, transgenic plants have specific pollutant-detoxifying or binding genes form
widely divergent sources and are better equipped to address challenges associated
with effective phytoremediation of contaminated sites and water bodies (Chaney
et al. 1997). Another important reason for interest in producing transgenics in
nondomesticated species is explained by the fact that growth rates of normal
phytoremediating plants are slow and are often seasonally variable, and the amount
of sequestration and degradation reported for such plants are often low (Terry et al.
2003). Hence, decontamination or detoxification does not always reach the required
values accepted and set by regulatory agencies (Suresh and Ravishankar 2004).
Even worse, at some contaminated sites, the level of pollutants could be at toxic
concentration to plants or may be recalcitrant to degradation or bioaccumulation,
rendering plant services completely ineffective (Terry et al. 2003). Hence, one of
the most direct approaches for making phytoremediation by target plant species
more successful is overexpression of genes involved in metabolism, uptake, trans-
port, sequestration, or detoxification of harmful chemical pollutants (Suresh and
Ravishankar 2004; Cherian and Oliveira 2005; Willey 2007).
Since the genetic diversity among natural phytoremediators is not very high,
their ability to clean the environment is comparatively low (Fladung and Ewald
2006). Hence, the idea of transferring genes from bacterial, yeast, and animal
members and even from other plants to produce target laboratory plants that are
better equipped to be strong phytoremediator has recently gained enormous promi-
nence (Oksman-Caldentey and Barz 2002; Fladung and Ewald 2006; Willey 2007).
Terry et al. (2003) have discussed in details the significance of works on over-
expression of enzymes catalyzing rate-limiting steps in sulfate assimilation and PC
biosysnthetic pathways in transgenic plants exhibiting an increased resistance to
selenium (Se) and cadmium (Cd).
In addition, conventional contaminated site cleanup technologies have an exten-
sive overhead cost that could only be addressed by promoting transgenic plants as
phytoremediators, because they have been recently estimated to be more cost-
effective than traditional in situ or ex situ processes. They are easier to be used
for cleaning up sites located in distant areas, difficult mountainous terrain, or other
less inaccessible localities. Moreover, higher adaptability of transgenic phytoreme-
diators to toxic compounds and their better survival rates make them a better choice
for cleaning the environment (Suresh and Ravishankar 2004; Cherian and Oliveira
2005; Fladung and Ewald 2006; Willey 2007).
Till date, a large number of plants from 45 different plant families have been
identified for phytoremediation abilities (Raskin 1996; Salt et al. 1998); the maxi-
mum number of phytoremediation species has been reported from the Brassicaceae
family (Reeves and Baker 2000; Gratao et al. 2005). The first transgenic plants
developed for bioremediation purposes were reported by Misra and Gedamu
(1989). These plants expressed the human MT gene to develop tolerance to Cd
toxicity. The next major breakthrough in phytoremediation research was reported
by Rugh et al. (1996). To increase the tolerance of Arabidopsis thaliana to mercury,
they generated transgenic plants overexpressing the mercuric reductase gene.
The most important factors for considering plants for phytoremediation as
suggested by Newman et al. (1997) and Tong et al. (2004) are: rapid plant growth,
large biomass, easy multiple harvesting (3–4 times per year), better than average
uptake. Although several plant breeding approaches have been targeted to develop
plant varieties demonstrating better phytoremedaiation performance, the success
has not been phenomenal. Hence, nowadays the emphasis is being made on genetic
engineering and development of transgenic lines for producing better phytoreme-
diating lines in target species (Salt et al. 1998; Clements et al. 2002; Willey 2007).
However, it is important to note that transgenes procured from different sources
need to be carefully tracked, and their expression needs to be targeted to appropriate
cell compartments for maximizing the benefits of transgene incorporation (Salt

et al. 1998; Clements et al. 2002; Bizily et al. 2003; Pilon et al. 2003). A com-
prehensive summary of diverse transgenes inserted into different target plant
species used in phytoremediation and their corresponding responses after genetic
engineering are presented in Table 8.2.
8.2.5 Transgenic Trees in Phytoremediation
Nowadays, trees are extensively used in phytoremediation (Sykes et al. 1999)

because of their advantages over other plant species. These advantages are: greater
biomass, larger size, strong extensive and proliferating root systems capable of
spreading to a considerable depth for efficient phytoremediation, better ability to
accumulate pollutants and prevent rapid soil erosion, better ability to withstand low
water-stress conditions, and perennial growth habits. Other advantages include
easier maintenance, no need for constant monitoring, easy harvesting, effective
removal of pollutants from contaminated sites, and better ability to survive in
diverse biogeographical and widely fluctuating climatic conditions.
Both Clements et al. (2002) and Sykes et al. (1999) suggested introduction of
“hyperaccumulation genes” into target tree species for making them more amena-
ble to better phytoremediation in challenging sites and localities, where it was
difficult to achieve success using other plant species. Recently, for the first time
Doty et al. (2003) reported that the tropical leguminous tree Leucaena leucocephala
is an excellent phytoremediating species that can take up and metabolize both toxic
organic pollutants 2.4,6-trichloroethylene (TCE) and ethylene dibromide (EDB).
The authors reported that this plant’s ability to debrominate makes it an interesting
candidate to explore in terms of genetic engineering, if it is not recalcitrant to
genetic modifications.
8.3 Phytoremediation of Inorganic Pollutants

by Transgenic Plants
8.3.1 Mercury
Hg pollution is a serious threat to a wide array of living organisms ranging from

humans, animals, plants, and agriculturally important crops to beneficial microbes
residing in the soil (Rugh 2001). Hg is commonly released into the environment in
its ionic form (Hg+2), and then it is converted into methylmercury mostly by the
process of methylation aided by methanogenic anaerobic microbes from aquatic
sediment deposits (Meagher 2000). Methylmercury is approximately 200 times
more toxic than the ionic form of Hg and is even considered to be more detrimental
Table 8.2 Summary of transgenes inserted into different target plant species used in phytoremediation and their corresponding responses after genetic
312
engineering
Target plant Family Transgene Phytoremediation response(s) reported References
Arabidopsis halleri Brassicaceae NAS Better Ni hyperaccumulation Becher et al. (2004)
Arabidopsis thaliana Brassicaceae g-GCS, arsC Increased fresh weight and shoot accumulation of Dhanker et al. (2002)
arsenates
A. thaliana Brassicaceae merA, merB Better resistance to Hg toxicity Bizily et al. (2000)
A. thaliana Brassicaceae merB Better Hg volatilization Bizily et al. (2003)
A. thaliana Brassicaceae SAT Better tolerance to Ni Freeman et al. (2004)
A. thaliana Brassicaceae AtPCS1 Increase in PC concentration Lee et al. (2003)
A. thaliana Brassicaceae YCF1 Bigger biomass and higher Cd uptake in leaves Gong et al. (2003)
A. thaliana Brassicaceae merApe9 Better resistance to Hg contamination Rugh et al. (1996)
A. thaliana Brassicaceae SL Slightly increased Se accumulation and slightly
lowered Se incorporation in proteins
A. thaliana Brassicaceae SMT Increase in Se tolerance and accumulation Ellis et al. (2004)
A. thaliana Brassicaceae ZAT1 Higher tolerance to Zn Van der Zaal et al.
(1999)
A. thaliana Brassicaceae AtNramp3 Increase intake of Fe and better tolerance to Cd Thomine et al. (2000)
A. thaliana Brassicaceae PsMTA Higher Cu accumulation Evans et al. (1992)
A. thaliana Brassicaceae OASTL Better tolerance to Cd and accumulation of Cd
A. thaliana Brassicaceae Lip, MnP PCB degradation Sonoki et al. (2007)
A. thaliana (Ac/Ds transposon Brassicaceae Ds transposon + GUS PCB degradation Sonoki et al. (2007)
tagging transformant)
A. thaliana Brassicaceae LAC1 Detoxification of phenolic compounds like TCP Wang and Chen (2007)
A. thaliana (cadl-3 mutant Brassicaceae TaPCS1 Low CD accumulation; higher transport rate in Gong et al. (2003)
line) leaves
Atropa belladonna (hairy root Solanaceae P450 2E1 Rapid TCE metabolism Banerjee et al. (2002)
culture)
Brassica juncea Brassicaceae gshII Better Cd accumulation Liang et al. (1999)
B. juncea Brassicaceae g-GCS Increase in Zn and Cd uptake Bennett et al. (2003)
B. juncea Brassicaceae GC Increase in Zn and Cd uptake Bennett et al. (2003)
B. juncea Brassicaceae gshI Increase in Cd tolerance and accumulation Zhu et al. (1999)
B. juncea Brassicaceae SMT Increase in Se tolerance and accumulation LeDuc et al. (2004)
S.K. Basu et al.
8
B. juncea Brassicaceae APS1 High accumulation of Se in a plant body Pilon-Smits et al.

(1999)
B. juncea Brassicaceae GR Increased tolerance to Cd Pilon-Smits et al.
(2000)
Brassica napa Brassicaceae ACC Increased accumulation and tolerance to arsenate Nie et al. (2002)
Brassica napus Brassicaceae ACC Higher heavy metal tolerance (Ni, Zn, Cu, Pb) Stearns et al. (2007)
B. napus Brassicaceae MT II Increased resistance to Cd toxicity Misra and Gedamu
(1989)
Liriodendron tulipifera Magnoliaceae merApe9 Better phytovolatilization of Hg contaminants Rugh et al. (1998)
L. tulipifera Magnoliaceae gsh1 Increased accumulation of Cd, Cu, Zn Arisi et al. (2000)
Nicotiana benthamiana Solanaceae MT II Increased resistance to Cd toxicity Misra and Gedamu
(1989)
N. benthamiana Solanaceae todC1, todC2 Increased phytoremediation of toluene Novakova et al. (2007)
Nicotiana glauca Solanaceae TaPCS1 Higher Pb and Cd accumulation Gisbert et al. (2003)
Nicotiana glutinosa Solanaceae MT Increased tolerance to Cd Liu et al. (2002)
Nicotiana tabacum Solanaceae merA Better resistance to Hg toxicity Heaton et al. (1998)
N. tabacum Solanaceae merA Better resistance to Hg toxicity Meagher et al. (2000)

N. tabacum Solanaceae nfs1 Biotransformation of TNTs Hannik et al. (2001)
N. tabacum Solanaceae onr Better detoxification of nitroglycerin compounds French et al. (1999)
N. tabacum Solanaceae merA About a fivefold increase in Hg volatilization in He et al. (2001)
roots compared to leaves and shoots
N. tabacum Solanaceae CUP1, GUS, HisCUP, Increased accumulation of Cd and Ni Pavlikova et al. (2004)
HisGUS
N. tabacum Solanaceae merA, merB Better Hg phytovolatilization and Bizily et al. (2003)
phytoaccumulation
N. tabacum Solanaceae NtCBP4 Better tolerance to Ni and improved Arazi et al. (1999),
hyperaccumulation of Pb Sunkar et al. (2000)
N. tabacum Solanaceae AtMHX1 Reduce tolerance to Mg and Zn Shaul et al. (1999)
N. tabacum Solanaceae MT1 Higher tolerance to Cd Pan et al. (1994)
N. tabacum Solanaceae CUP1 Better tolerance to Cu Thomas et al. (2003)
N. tabacum Solanaceae bphC Phytodegradation of PCBs Chrastilova et al.
(2007)
Solanaceae merA, merB Ruiz et al. (2003)
313
(continued )
314
Target plant Family Transgene Phytoremediation response(s) reported References

N. tabacum (Chloroplast
genome engineering)
Oryza sativa Poaceae merA Higher Hg phytoremediation in wetland areas Heaton et al. (2003)
O. sativa Poaceae cbnA Biodegradation of chlorinated aromatic Shimizu et al. (2002)
compounds
O. sativa Poaceae CYP1A1, CYP2B6, CYP2C9, Enhanced resistance to herbicides Ohkawa and Ohkawa
CYP2C18, CYP2C19 (2002)
Populus deltoides Salicaceae merA9, merA18 Better resistance to Hg toxicity and greater Che et al. (2003)
biomass generation
Populus tremula Populus Salicaceae CYP2E1 Increased phytoremediation of different Doty et al. (2007)
alba (Hybrid clone) hydrocarbons
Solanum tuberosum Solanaceae P450CYP1A1 Herbicide detoxification Yamada et al. (2002)
S. tuberosum Solanaceae CYP1A, CYP2B6, CYP2C Herbicide detoxification Ohkawa and Ohkawa
(2002)
Spartina alteriniflora Cyperaceae merA, merB Enhanced ability to withstand Hg toxicity and Czako et al. (2006)
better phytovolatilization
Thlaspi caerulescens Brassicaceae ZNT1 Increase in Zn uptake and tolerance Pence et al. (2000)
Thlaspi goesingense Brassicaceae TgMTP1 Efficient Ni hyperaccumulation Persans et al. (2001)
S.K. Basu et al.
because of its enormous biomagnification property up in the ecological food chain

(Ruiz et al. 2003). Hg, compared to other toxic heavy metals detected in the
environment, is extremely detrimental even at considerably low concentrations
(Meagher 2000). Methylmercury is exceptionally toxic because of its unique
property of hydrophobicity that enables it to migrate inside the cell and then
deactivate several key metabolically important enzyme systems (Castoldi et al.
2001; Rugh 2001).
A number of bacterial species have been reported that can demethylate methyl-
mercury and transform it back to its less toxic native forms (Rugh et al. 1996; Bizily
et al. 2003). Elemental Hg0 is extremely volatile and is readily transformed from the
liquid phase to the vapor phase. Bacterial species capable of converting methyl-
mercury ! Hg+2 ! Hg0 are characterized by the presence of a Hg-responsive
operon consisting of: (1) Hg-responsive regulatory proteins; (2) transport proteins
that bind and carry Hg into the cell; (3) a specific enzyme known as an organo-
mercuric lyase (merB gene) that catalyzes the removal of CH3 group from methyl-
mercury and transforms it to ionic mercury (Hg+2); and (4) mercuric ion reductase
(merA gene) transforming ionic mercury to elemental mercury Hg0 (Rugh et al.
1996, 1998). Gene products are expressed only when species is exposed to Hg
(Rugh et al. 1996, 1998). In the past, researchers exploited these Hg-responsive
operons in bacteria for effective phytoremediation of toxic Hg-contaminated sites
(Meagher 2000; Rugh 2001; Suresh and Ravishankar 2004).
Earlier attempts to express merA in plant systems were not successful, because
the gene was reported to be G + C rich (about 67%), and hence it expressed itself
only in bacterial systems (Rugh et al. 1996). In addition, it also had a higher number
of CpG motifs (sites for methylation and gene silencing). Rugh et al. (1996) for the
first time exploited the merA gene by replacing codons 287–336 and thereby devel-
oping a modified gene merApe9 that was efficiently expressed in the A. thailiana
system. Transgenic lines with the merApe9 gene produced viable seeds, and the
corresponding seedlings survived on agar plates containing 25–100 mM HgCl2 com-
pared to their corresponding non-transformed controls. Hg vapor analysis also con-
firmed that transgenic lines successfully phytovolatilized ionic mercury to elemental
forms with approximately 50 ng Hg0/mg fresh tissue weight. The authors detected
that plants expressing merApe9 were also resistant to gold.
In an attempt to further improve key technology and extend its application to
other plant species in addition to model laboratory plants, Rugh et al. (1998) used
bigger biomass generating plants. The researchers further modified the previously
developed transgene merApe9 to include an additional 9% of the coding sequence
DNA fragment for better codon optimization and effective expression in a particu-
lar species of yellow poplar (Liriodendron tulipifera). The gene was delivered into
plant embryonic masses using a particle bombardment approach. The authors
reported transgenic seedlings growing and surviving on agar plates incorporated
with 25 and 50 mM HgCl2 compared to their non-transformed controls and phyto-
volatilization rates detected in the species were also appreciable.
Bizily et al. (1999) reported A. thaliana lines containing the bacterial merB gene
and successfully surviving on higher concentrations of HgCl2 and phenyl mercuric
acetate compared to their controls. The original bacterial merB gene was modified
using PCR techniques to contain flanking sites incorporated with consensus plant
sequences and restriction sites. Using Western blot analysis, the authors provided
substantial evidence that a sufficient amount of the gene product (organomercurial
lyase) was synthesized by A. thaliana transgenic lines. Later, Bizily et al. (2000)
reported production of A. thaliana lines with enhanced abilities of Hg phytoreme-
diation. In this case, separate transgenic A. thaliana lines carrying merA and merB
genes, respectively, were crossed and hybrid F2 plants and screened for expression
of both gene products. Plants with double gene expression (merA/merB) survived
better in methylmercury-incorporated agar plates containing the concentration of
methylmercury higher than 10 mM; compared to plants that expressed either merA
or merB separately, which could survive at concentrations up to 5 mM of methyl-
mercury. Western blot analysis confirmed the simultaneous expression of gene
products. However, Bizily et al. (2000) suggest that transgenic lines were less
hardy compared to control plants in most experimental trials. The authors attributed
this to the transgene capable of reducing a wide diversity of ions (particularly
merA), many of which being vital for physiology and metabolism of plants. In
another study, He et al. (2001) reported detection of an approximately fivefold
increase in Hg volatilization in transgenic tobacco (Nicotiana tabacum) roots
compared to the above-ground biomass, thus suggesting the organ-specific and
species-specific plant response to Hg phytoremediation.
An important limiting factor for Hg phytoremediation is the diffusion rate of
methylmercury to the cytoplasmically expressed MErB proten (Bizily et al. 2000).
It was addressed later by Bizily et al. (2003) in developing a specific merB cassette
that targets MerB proteins on the cell wall to avoid the slower diffusion rate of
methylmercury in the cytoplasm. The authors showed that transgenic lines having
this specific merB construct along with the merA cassette were 10–70 times better
than any other competing lines developed.
Ruiz et al. (2003) reported for the first time integration of a native operon with
merA and merB bacterial genes into the chloroplast genome of tobacco by a single
transformation event. The authors detected high levels of tolerance to phenylmer-
curic acetate (100, 200 and 400 mM) in the stable transgenic lines compared to the
controls. These plants were highly resistant to toxic levels of organomercurials and
had higher chlorophyll per unit dry leaf weight. The major advantages of this
innovative approach have been better transgene expression without the necessity
of expensive and laborious codon optimization that is usually necessary for expres-
sion in higher plant systems and lower levels of unwanted gene silencing (Ruiz
et al. 2003).
In addition to extensive work on engineering model plants for Hg phytoremedia-
tion, in a fairly recent study, Heaton et al. (2003) reported development of trans-
genic rice (Oryza sativa) with merA construct delivered through the biolistic gene
delivery process. This is the first report on developing a transgenic wetland phyto-
remediation plant capable of detoxifying toxic mercury form aquatic sediments.
Che et al. (2003) reported introduction of the merA gene into another wetland
species, eastern yellow poplar (Populus deltoids), and it is good news as they
confirm that genetic engineering technology is not just restricted to laboratory

model plants and actively pursue developing actively remediating plant lines
or transgenic plants suitable for specific ecosystems. Future studies need to investi-
gate divergent species representing different ecosystems and habitats, especially
those with higher Hg phytoaccumulation and inefficient phytovolatilization of the
accumulated Hg for a safer, cleaner and healthier environment.
Recently, Czako et al. (2006) reported developing green leaf tissue-specific
merA expression cassettes using the wheat rbcS promoter in mature green leaves
of A. thaliana and tobacco transgenic lines for enhanced merA gene expression,
increased tolerance to Hg toxicity and better phytovolatilization. The authors also
reported transgenic lines of wetland species Spartina alteriniflora (salt-water cord-
grass/smooth cordgrass) with merA and merB genes under the control of two
constitutive promoters 35S and Ubi. These ransgenic plants had higher resistance
to both ionic and organic Hg toxicity. Researchers are reported to have been
working on a number of other wetland species such as Arundo spp. (giant reed),
Phragmites spp. (common reed), Typha spp. (cattail) and Schoenoplectus spp.
(common threesquare). If this group is successful in developing a diversity of
wetland-specific transgenic plants, it would mean a big step in the decontamination
of Hg toxicity in different wetland ecosystems.
In a very recent work, Hussein et al. (2007) reported developing transgenic
tobacco plants with merA and merB genes engineered through the chloroplast
genome. Transgenic lines exhibited a steady growth in the concentration of about
200 mg/g of phenylmercuric acetate or HgCl2 compared to non-transformed lines;
and they also showed the ability to phytoaccumulate both organic and inorganic
forms of Hg, with better absorption rates of organic Hg over inorganic forms. The
authors for the first time reported a 100-fold increase in Hg phytoaccumulation and
very rapid rates of phytovolatilization, varying between 2 and 7 days depending
upon the chemical form of Hg used under experimental conditions compared to
control lines. This is an interesting study that shows high success rate with respect
to both Hg phytoaccumulation and phytovolatilization and holds great promise for
the development of future transgenics for Hg decontamination.
8.3.2 Cadmium, Lead, Nickel and Zinc
Heavy metals like Cd, Pb, Ni, Fe (iron), zinc (Zn), copper (Cu), and Mg (mag-
nesium) are important sources of environmental pollution because of their
toxic effects on human and animal health. They have been under constant inves-
tigations by different research groups for their effective phytoremediation (Pilon-
Smits 2005). In the late 1990s, Misra and Gedamu (1989) transferred human
metallothionein-II (MT II) into tobacco (N. tabacum) and Brassica napus. Metal-
lothioneins (MTs) represent a broad family of low molecular weight cysteine-rich
chelator proteins that bind a number of heavy metals via the thiol group of
its cysteine residues and transport them for sequestration into the plant vacuole
(Pilon-Smits 2005). The transgene expressed the MT protein constitutively, and

seeds form self-fertilized transgenic plants were able to survive in agar plates
containing up to 100 mM CdCl2. Later overexpressed the metallothionein (MT)
gene under the control of the constitutive cauliflower mosaic virus (CaMV) 35S
promoter in tobacco (N. glutinosa) delivered via AMGT. The authors reported that
transgenic lines expressing MT grew successfully in the presence of 200 mM CdSO4
compared to non-transgenic lines that failed to withstand concentrations higher than
50 mM CdSO4. PCR analysis of T2 transgenic and non-transgenic seedlings demon-
strated a strong correlation between phytotolerance to Cd and the presence of the
transgene. This is an interesting strategy of increasing phytoremediation abilities in
plants by overexpressing MT genes in the genome of higher plants.
Phytochelatins (PCs) represent a big family of heavy metal-inducible peptides
that play an important role in intracellular heavy metal detoxification by chelation in
different organisms such as microbes, yeasts (Schizosaccharomyces pombe) and
higher plants (Arabidopsis); but it has not been reported in animals yet (Ha et al.
1999; Gong et al. 2003). In 2003, Lee et al. reported overexpression of A. thaliana
phytochelatin synthase (AtPCS1) back into A. thaliana with an increase (1.3- and
2.1-fold) in PC concentration. However, the authors reported hypersensitivity of
transgenic lines to Cd stress measured as related to the proportion of root growth of
transgenic plants compared to their wild types. The authors postulated that this might
be due to higher concentration of glutathione in transformed plants. On the contrary,
Gong et al. (2003) targeted wheat TaPCS1 (involved in PC synthesis) cDNA
expression in Arabidopsis under the Arabidopsis alcohol dehydrogenase promoter
and also developed special PC-deficient Arabidopsis lines (cadl-3) under the influ-
ence of the CaMV 35S promoter. The researchers detected lower Cd accumulation in
transgenic plants compared to the original cadl-3 mutant lines and observed a higher
rate of Cd transport into leaves. In the same year, Song et al. (2003) reported using a
yeast vacuolar glutathione Cd transporter (YCF1) in A. thaliana that resulted in
greater biomass and an approximately twofold increase in Pb and Cd tolerance and
uptake compared to non-transformed controls. This technology has the potential to
be used in developing advanced phytoremediators that can pump heavy metals into
safe compartments, requiring only a very low expression of transporters compared to
greater quantity of chelating peptides (Tong et al. 2004).
Similarly, Bennett et al. (2003) generated transgenic B. juncea lines using micro-
bial g-GCS and glutathione synthetase (GC) (both associated with PC synthesis)
separately and reported approximately a 1.5-fold increase in Zn and Cd uptake
compared to their corresponding controls. Researchers detected that overexpression
of both genes enhanced soil-borne Cd by 25%, and they expected to increase the
phytoremediation potential of transgenic lines from 1.5- to 3-folds, compared to their
non-transformed control plants. Working on shrub tobacco (N. glauca) in Eastern
Spain, Gisbert et al. (2003) genetically modified the species through AMGT using a
wheat gene encoding phytochelatin synthase (TaPCS1). The authors reported a
significant phytotolerance to Pb and Cd and an increase in root length by approxi-
mately 160%. Another significant achievement was reported by Zhu et al. (1999)
when they transferred the Escherichia coli gene gshI (involved in PC synthesis) and
overexpressed it in B. juncea with a fivefold increase in ECS and GC activity and

better Cd accumulation (approximately 40–90%). Liang et al. (1999) in their study
on Cd phytoremediation overexpressed the gshII gene encoding GC into the cytosol of
B. juncea and exhibited higher Cd accumulation compared to wild-type plants.
Vacchina et al. (2003) first reported a possible role of nicotinamine (NA) in
the process of detoxification of a microelement Ni by Thlaspi caerulescens as
a phytoremediator. Another group of researchers (Becher et al. 2004) besides
Vacchina et al. (2003) found consistent results by overexpressing the nicotinamine
synthase (NAS) gene in Brassicaceae family members such as A. halleri and
T. caerulescens. The Cation Diffusion Facilitator (CDF) family homologs such as
ZTP1 in T. caerulescens and AhMTP1 in A. halleri have been reported to be
constitutively expressed at higher concentrations (Assuncao et al. 2001; Becher
et al. 2004). Pence et al. (2000) cloned ZNT1 cDNA (a heavy metal transporter)
from T. caerulescens via functional complementation in the yeast strain ZHY3 and
found higher and lower affinities for Zn+2 and Cd+2, respectively. The authors also
reported that ZNT1 (a heavy metal transporter that forms a complex with heavy
metals and carries them inside plant cells for sequestration into the plant vacuole) is
heavily expressed in roots and shoots of target plants. A comparative analysis was
made between ZNT1 and high-affinity Zn+2 accumulation in roots of T. caerulescens
(a hyperaccumulator) and T. arvense (a non-accumulator), and it established
an excellent correlation between alterations in ZNT1 expression and species-
specific Zn accumulation status resulting in overexpression of the target gene in
T. caerulescens. This study highlights the pattern of regulation and molecular
control mechanisms of heavy metal uptake systems in plants.
In another interesting study, Pavlikova et al. (2004) tested four transgenic
tobacco lines carrying transgenes: CUP1 (encoding MTs), the GUS reporter gene,
HisCUP (CUP combined with a polyhistidine tail) and HisGUS (GUS with a
polyhistidine tail) under the constitutive CaMV 35S promoter for Cd, Zn and Ni
phytoaccumulation. The GUS line accumulated all the three metals. The HisCUP
line showed the best Cd accumulation with the shoot content of Cd enhanced by
90% and the subsequent reduction in root accumulation by 40%. The HisGUS line
confirmed the best performance in Ni accumulation, while no significant accumu-
lation was detected for Zn in any of the lines tested. Other lines exhibited higher
phytoaccumulation of only one specific metal, as discussed above. Recently,
Stearns et al. (2007) reported efficient tolerance to Ni, Zn, Cu, and Pb in canola
(B. napus) by transferring to the genome the bacterial 1-aminocyclopropane-
1-caboxylate (ACC) gene associated with ethylene biosynthesis for inducing better
environmental stress response to metal toxicity indirectly.
8.3.3 Arsenic
The first clear evidence of arsenic phytoremediation by transgenic canola

(B. napus) was reported by Nie et al. (2002). The researchers expressed the
Enterobacter cloacae UW4 1-aminocyclopropane-1-caboxylate (ACC) deaminase

in canola. The reason for expressing this particular enzyme is that it is associated
with lower levels of stress-induced ethylene. Hence, these transgenic lines are
expected to tolerate a wide variety of environmental stresses including heavy
metal stress and toxicity. Transgenic plants survived quite well while grown in
the presence of 2 mM arsenate; they exhibited higher seed germination percentage,
enhanced fresh and dry weight of both roots and shoots, and higher concentrations
of protein and chlorophyll in plant cells and tissues. The most important improve-
ment reported in transgenic plants compared to their corresponding controls was a
fourfold increase in their ability to accumulate arsenate. The authors also explored
the role of the plant growth-promoting bacterium E. cloacae CAL2 in both trans-
genic and control lines with no significant observation with respect to arsenate
uptake. They proposed the combined use of transgenic lines along with the growth-
promoting bacteria as a better strategy to deal with arsenic contaminated sites.
In the same year, Dhanker et al. (2002) were successful in increasing As
tolerance in the model plant A. thaliana using two different genes, a bacterial
g-glutamyl cysteine synthetase (g-GCS) of the glutathione biosynthetic pathway
(involved in PC synthesis) and the arsenate reductase (arsC) from E. coli (for
transforming highly toxic arsenates to less toxic arsenites). Constitutive overex-
pression of the g-GCS gene along with leaf-specific expression of arsC allowed to
achieve increased phytoremediation of arsenate by transgenic plants with respect to
fresh weight (~fivefold) and shoot accumulation (~threefold), as compared to non-
transformed controls. Such advances may play a significant role especially in the
parts of world that are adversely affected by severe arsenic pollution like the Indo-
Gangetic plain and the Ganges delta (Ruiz and Romero 2002).
8.3.4 Selenium
One of the most comprehensive and detailed study on Se toxicity in plants was
conducted by Banuelos et al. (1997). The authors investigated selenium-induced
growth reduction in two different Brassica species (B. juncea and B. carinata).
Several other studies have been conducted on Se phytoremediation with particular
emphasis on plant physiology and biochemistry, Se toxicity and Se hyperaccumu-
lation (see Suresh and Ravishankar 2004; Cherian and Oliveira 2005). Pilon et al.
(2003) studied overexpression of a mouse selenocysteine lyase (SL) that breaks
down selenocysteine into elemental selenium and alanine in A. thaliana. The
overexpression resulted in a minor increase in the rate of Se accumulation, slightly
lowering the amount of Se incorporation in plant proteins. It is very important to
note that the researchers reported that chloroplastic SL reduced tolerance to Se,
while vacuolar SL enhanced tolerance to Se, indicating the importance of precise
localizations of transgenic proteins at the subcellular level (Pilon et al. 2003).
Overexpression of metallothionein expressing selenocysteine methyltransferase
(SMT) from A. bisulcatus to A. thaliana (Ellis et al. 2004) and B. juncea (LeDuc
et al. 2004) resulted in an approximately two- to threefold increase in Se tolerance
and accumulation in transgenic plants compared to their untransformed controls.
This specific enzyme (SMT) is capable of detoxification of selenocysteine to a non-
protein amino acid methylselenocysteine via methylation, thereby reducing
chances of toxic incorporation of Se in plant proteins (LeDuc et al. 2004). Recently,
Banuelos et al. (2007) reported a twofold increase in Se accumulation and 1.8-fold
increase in Se leaf accumulation by transgenic Indian mustard (Brassica juncea (L.)
Czern.) overexpressing SL.
8.4 Phytoremediation of Organic Pollutants by Transgenic

Plants
8.4.1 Organic Hydrocarbons
Organic pollutants are another significant group of environmental toxicants used for
rapid phytoremediation (Gratao et al. 2005; Pilon-Smits 2005). In an interesting
recent study, Doty et al. (2007) reported the development of a transgenic poplar
line from an original base population of the hybrid poplar clone INRA 717-1B4
(P. tremula P. alba) via overexpression of rabbit CYP2E1 under the control of
CaMV 35S. Stable transgenic lines showed excellent phytoremediation of different
hydrocarbons (trichloroethylene, vinyl chloride, carbon tetrachloride, benzene and
chloroform) from hydroponic solutions, compared to their controls. The plants also
exhibited better phytovolatilization for trichloroethylene, chloroform, and benzene.
Recently, Novakova et al. (2007) successfully transferred the bacterial todC1
and todC2 genes into N. benthamiana. The todC1 C2 genes were cloned into the
plant genome to synthesize ISPTOL (a bacterial component of toluene dioxygenese),
causing rapid oxidation of toluene and other organic pollutants. The overall per-
formances of transgenic lines are still in progress to record their phytoremediation
ability to degrade toluene and other organic compounds.
8.4.2 Trichloroethylene (TCE)
Doty et al. (2000) for the first time developed a transgenic tobacco line capable of
phytoremediating TCE. The authors introduced the mammalian cytochrome P450
E1 gene (CYP2E1) into tobacco leaf disks, and transgenic plants were regenerated.
Oxidoreductases of plant and mammalian origins being substantially similar, the
mammalian P450 could successfully interact with its tobacco counterpart. Intro-
duction of this specific gene resulted in a significant increase in both TCE and
ethylene dibromide metabolism. The authors reported a 600-fold increase in TCE

metabolism in transgenic lines, compared to their non-transformed controls. This
specific gene encodes enzymes that can successfully oxidize a wide diversity of
organic pollutants such as TCE, ethylene dibromide (EDB), carbon tetrachloride,
chloroform and vinyl chloride, and many others (Aken 2008). Later on, the
same group of researchers reported another intriguing finding: they successfully
expressed the P450 E1 gene (CYP2E1) in hairy root cultures of the medicinal plant
Atropa belladonna (Banerjee et al. 2002).
8.4.3 Polychlorinated Biphenyls (PCB)
PCBs are considered to be a formidable source of environmental pollution, and they

are extremely toxic in nature (Sonoki et al. 2007). Chrastilova et al. (2007)
transferred the 35S-driven bphC gene derived from the bacterial PCB degradation
pathway into tobacco. Significant PCBs degradation was observed in the transgenic
lines.
Sonoki et al. (2007) also reported PCB degrading transgenic lines of A. thaliana.
One of the lines developed contains fungal lignin-degrading enzymes (Lip, MnP),
while the other line is an enhancer-trap Ac/Ds transposon tagging transformant of
A. thalina containing a nonautonomous mobile Ds transposon linked to the reporter
gene GUS. According to the researchers, the minute promoter-driven GUS can only
drive gene expression when the Ds transposon GUS is shifted towards the enhancer
region of the gene in the Arabidopsis genome. Hence, efficient monitoring of the
genes(s) involved in Polyhydroxy butyrate (PHB) degradation is indirectly
achieved by monitoring the expression of the reporter gene. Both lines have been
found to efficiently degrade PCBs under laboratory conditions.
8.4.4 Phenolic Compounds
Recently, Wang and Chen (2007) reported transferring the laccase enzyme (LAC1)
from cotton plants (Gossypium arboreum) into A. thaliana. Transgenic plants were
efficient in degrading 2,4,6-tricholorophenol (TCP) by simple oxidation. In another
study, Floco and Giulietti (2007) reported developing hairy root cultures of Armor-
acia lapathifolia using A. rhizogenes for phytoremediation of aromatic compounds
like phenol from Argentina. This particular plant species have high concentrations
of the enzyme peroxidase (E.C. 1.11.1.7) that is capable of detoxifying phenolic
compounds. The authors exposed 30-day-old hairy root cultures to aqueous solu-
tions of phenols of different concentrations (25, 50 and 100 mg/mL). They reported
70% phenol removal in the cultures after 3 h of incubation at all concentrations in
the presence of hydrogen peroxide, and approximately 30–55% in the absence of an
oxidant and co-substrate, suggesting a significant role of a plant peroxidase in

phytodetoxification of phenols.
8.4.5 Herbicides, Pesticides and Organic Solvents
Herbicides, pesticides and organic solvents are other significant sources of environ-
mental toxicants that have a detrimental impact on our ecosystems (Gratao et al.
2005). Shimizu et al. (2002) reported transferring the bacterial cbn4 gene from
Ralstonia eutropha NH9 into rice (O. sativa) under the constitutive CaMV 35S
promoter. Transgenic rice calli were successful in converting 3-chlorocatechol
to 2-chloromucote. Such techniques may be suitable for phytoremediation of
chlorinated aromatic compounds represented by herbicides, pesticides and several
organic solvents.
In another related study, Ohkawa and Ohkawa (2002) reported producing trans-
genic rice and potato (Solanum tuberosum) lines with mammalian cytochrome p450
monooxigenase genes. The researchers introduced five P450 genes in rice, namely,
CYP1A1, CYP2B6, CYP2C9, CYP2C18 and CYP2C19. All stable transgenic lines
(with the exception of one line) exhibited enhanced tolerance to herbicides meta-
chlor, alochlor and acetochlor (inhibiting protein biosynthesis) and trifluralin (inhi-
biting cell division). T1 seeds of CYP2C9 showed resistance to such herbicides like
chlortoluron, mefenacet, phenylurea herbicide, pyridazinone herbicide etc; while
CYP1A1 exhibited resistance to phenylurea herbicide, mefenacet and quizalofop-
ethyl. Rice lines carrying the CYP2C9 gene were resistant to chlorosulfuron and
imazosulfuron; however, the line with the CYP2C18 gene did not show any specific
resistance to any herbicide.
A similar approach was used for generating transgenic lines of potato. Four lines
(S1965, S1972, S1974 and T1977), each with three transgenes (CYP1A, CYP2B6
and CYP2C), were selected for testing. The T1977 line showed tolerance to
atrazine, chlortoluron, methabenzthiazuron, acetochlor and metolachlor; while the
S1972 line had tolerance to chlortoluron and methabenzthiazuron and was suscep-
tible to all these herbicides except acetochlor and metolachlor; the S1974 line was
partially tolerant to atrazine and tolerant to acetochlor and metolachlor.
8.4.6 Explosive Chemicals
Bioremediation of explosives is not an innovation; several researchers success-

fully used different fungal and bacterial members to degrade explosive and ammu-
nition chemicals like TNT (trinitrotoluene), RDX (hexahydro-1,3,5-trinitro-1,3,5
triazine), HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) and TETRYL
(N-methyl-N, 2, 4, 6-tetranitroaniline) (Hooker and Skeen 1999; Jhonston 2002).
In the last few decades, phytoremediation of explosive chemicals has received great
attention in a large number of laboratory studies by different research groups
(French et al. 1998, 1999). An excellent review by Hannink et al. (2002) covered
in details energetic, metabolic, biochemical and transformation mechanisms asso-
ciated with phytoremediation of explosive chemicals.
TNT is one of the most dangerous explosive chemicals that require considerable
time for complete biodegradation (Jhonston 2002; Cluis 2004). However, a soil
bacteria E. cloacae PB2 has been reported to be able to use TNTs as its primary
nitrogen source for growth and metabolism because of the presence of two unique
enzymes, pentaerythritol tetranitrate (PETN) reductase and nitroreductase (French
et al. 1998). Both these enzymes utilize NADPH as an electron donor source, and
thereby can easily reduce TNTs into less toxic compounds. French et al. (1999)
introduced the PCR-modified gene encoding PETN reductase (onr) into the tobacco
genome with a plant consensus start sequence for better expression in the plant
system. The authors reported that seeds from transgenic lines germinated and grew
successfully in the presence of 1 mM glycerol trinitrate (GTN) or 0.05 mM TNT,
compared to their non-transformed wild types. The resultant transgenic seedlings
grown in liquid medium with 1 mM GTN exhibited faster and complete degradation
(denitration) of GTN than non-transformed lines.
In another related study, expressed the PCR-modified bacterial (E. cloacae
NCIMB101011) gene encoding for nitroreductase (nfs1) with a consensus start
sequence to facilitate translation in tobacco plants. The nitroreductase enzyme
catalyzed the reduction of TNT to hydroxyaminodinitrotoluene, following the
subsequent reduction to aminodinitrotoluene derivatives. Transgenic plants expres-
sing nitroreductase exhibited a significant increase in TNT uptake, tolerance, and
subsequent detoxification compared to wild-type plants.
The ability of plants to metabolize xenobiotic nitrate ester and glycerol trinitrate
(nitroglycerin) in sugar beet (Beta vulgaris) cells and cell extracts has been con-
vincingly demonstrated by Goel et al. (1997). Here, it is important to note that the
authors suggested that GTNs could not be completely denitrated, they could only be
transformed to mono- or dinitrated glycerols. Hence, there are opportunities for
future researchers to explore these data and develop new transgenic lines for
complete denitration of nitroglycerin compounds.
In Fig. 8.3, we have illustrated some of the common pathways of phytoremedia-
tion within a plant cell.
8.5 Immunological Approach for Phytoremediation
Immunological approaches for phytoremediation represent entirely new concepts in

the field of phytoremediation research. This technology can be called plant
immuno-remediation or phytoimmuno-remediation. Drake et al. (2002) for the
first time demonstrated the importance of hydroponicaly grown transgenic tobacco
plants used for phytoimmuno-remediation. These plants express a murine IgG1
HMT HM
PC/MT HM
C 2
1 L
M
C Tonoplast
GC Cell Wall
M
PC/MT HM Cell Membrane

ER
HM
N
Vacuole
L I GSH
O GSH Cell Sap
O 3 C
4
3 O GSH
3 Cytoplasm C I GSH
O M
C L 4
C 4
M
I
I
Fig. 8.3 Schematic representation of a plant cell and several phytoremediation pathways of target
toxic pollutants. C Chloroplast; ER Endoplasmic reticulum; GC Golgi complex; HM Heavy metal;
HMT Heavy metal transporters; I Inorganic pollutant; L Lysosome; M Mitochondrion; MT
Metallothioneins; N Nucleus and nucleolus; O Organic pollutant; PC Phytochelatins. Pathway
1: PCs and heavy metals form complexes are translocated across the tonoplast and finally
sequestered in the vacuole (Gong et al. 2003). Pathway 2: HMTs detoxifies toxic heavy metals
by transporting across the vacuole to less toxic forms (Song et al. 2003; Pilon-Smits 2005).
Pathway 3: Organic contaminants are phytoremediated by either getting adsorbed on the cell
wall during entry or moving into the cytoplasm depending on the nature of pollutants. Within the
cell cytoplasm, they are either attacked by series of enzymes and get transformed and degraded or
form conjugated complexes with glucose and GSH and get sequestered in the plant cell wall or the
vacuole (Pilon-Smits 2005). Pathway 4: Inorganic pollutants may form complexes with nicotina-
mine and organic acids and get adsorbed on the cell wall; if they can enter the cell cytoplasm, they
often form conjugates with PCs and GSH and are finally sequestered in the plant vacuole (Pilon-
Smits 2005)
monoclonal antibody either to neutralize toxic bioactive molecules in the rhizo-

sphere, or to accumulate and concentrate molecules in the above-ground biomass.
In their experiment, two different types of transgenic tobacco plants were used.
First, a functional antibody was subjected to rhizosecretion and directed to
bind with antigen in the surrounding media to generate an immune complex.
In the second case, a monoclonal antibody was retained in plant leaves via a
transmembrane sequence. It is interesting to report that the antigen incorporated in

the medium was actively transported to axial plant leaves within a day; there, it
underwent sequestration from binding to the antibody on the cellular membrane.
The immune complex density remained the same even 3 days following antigen
removal form the media. Such innovative technology could empower researchers to
develop transgenic species that could phytoremediate any pollutant for which it is
possible to develop a monoclonal antibody. It could also contribute to phytodecon-
tamination and phytorestoration of contaminated sites using transgenic plants.
8.6 Limitations of Phytoremediation Research
In spite of its relevance and importance, phytoremediation still has several pro-
blems such as reduced growth rate and poor biomass of phytoremediating plants,
limited remediation, and high plant mortality rates (Barcelo and Poschenrieder
2003; Cluis 2004; Gray 2006). In addition, there is always a permanent risk of
bioaccumulation of toxic elements and compounds by plants and their transmission
initially to immediate secondary consumers and subsequently into higher orders of
food chains and food webs (Raskin et al. 1994; Barcelo and Poschenrieder 2003;
Cluis 2004; Gratao et al. 2005). There are a number of concerns and issues
associated with future effectiveness and potential of transgenic phytoremediators
from food and feed crops (Dietz and Schnoor 2001; Cluis 2004; Ghosh and Singh
2005; Gratao et al. 2005).
Although many plant species have been reported to show uptake, biodegradation
and sequestration of several explosive chemicals, such as TNT and RDX residuals,
these activities were low. Some chemicals, such as RDX, were only partially
degraded or transformed, leaving space for engineered plants to take over in this
area in the not-so-distant future (Goel et al. 1997; Dietz and Schnoor 2001; Ghosh
and Singh 2005).
Among other technical factors associated with phytoremediation, a subtle one is
that of a gap between the scientist and the lay person, and misconceptions about
benefits of the process and its scientific management (Trapp and Karlson 2001). A
list of concerns and challenges haunting the successful development of transgenic
lines has been presented in Fig. 8.4. Developing efficient phytoremediators for
contaminated sites lab has always been extremely challenging for researchers
(Black 1995; Cunningham et al. 1995; Jhonston 2002; McIntyre 2003).
Among these challenges are genotype environment interactions of responding
plant species and variability of performance across the years (Cunningham et al.
1995; McIntyre 2003; Zayed 2004; Ghosh and Singh 2005; Willey 2007). More-
over, phytoaccumulation of toxic pollutants is also dependent on root growth of
plant species involved. Restricted root growth in natural contaminated sites may or
may not allow plants to effectively accumulate toxicants in the above-ground
biomass or to immobilize pollutants preventing them from leaching into the
groundwater table (Black 1995; Pulford and Watson 2003; Gratao et al. 2005).
1. Phytotoxic concentrationof pollutants

often detrimental to plants
2. Phytoremediation affected by locality,

seasonal and climatic variations
3. Phytoremediation success depends on

plant species and varieties and specific
plant communities
4. Often difficult to establish a successful

plant colony for effective and economical
remediation
5. Ecological risk associated with

introduction of new species
6. Phytoremediation limited by root

length and root growth rate in plants
CHALLENGING QUESTIONS 7. Uncertainty regarding production of

secondary pollutants and effective
CONFRONTING FUTURE AND
disposal of phytoremediating plants
PROGRESS OF TRANSGENIC LINE
DEVELOPMENT FOR 8. Economic and ecological cost of
PHYTOREMEDIATION generating transgenic plants
Fig. 8.4 Future of transgenic phytoremediating plants
According to Salt et al. (1998) metal-accumulating plants could be either

disposed off or used for metal recovery depending on economics of the process.
However, it is important to note that reliable data and information regarding such
disposal and success are not easily available (Jhoanston 2002). Although compost-
ing and compaction have been suggested by Cunningham et al. (1995) as pretreat-
ment for volume reduction before actual disposal, it is important to make sure that
such practices do not promote accidental leaching from compaction (Ghosh and
Singh 2005). Incineration of harvested plants from contaminated sites after reme-
diation has been strongly advocated by Ghosh and Singh (2005) to avoid the
possibility of secondary waste generation through the application of phytoremedia-
tion. However, the impact of such technologies on the environment and ecosystems
has not been well documented yet.
Lastly, another important question to answer is whether transgenic plants can
eventually become a source of secondary pollutants in the process of phytoreme-
diation, and whether their effective disposal becomes another challenge and threat
to ecology and environment (Black 1995; McIntyre 2003; Gratao et al. 2005;
Willey 2007). It is very important to look for easy recycling of plant biomass to
retrieve toxic pollutants and for the opportunity to reuse them after recycling to
reduce our ecological footprints on the nature. In addition, it is also very important
to look for enhancing the quality of phytovolatilization or phytotransformation
of toxic pollutants within phytoremediating plants to reduce the possibility of

generating secondary pollutants in the long run. The interaction between the plant
genome and hyperaccumulation of toxic heavy metals needs to be investigated in
further details to understand molecular genetics and physiological aspects of heavy
metal uptake in phytoremediating plants (Mejare and Bulow 2001).
8.7 Transgenics in Phytomonitoring and in Biopolymer

and Bioplastic Productions
In addition to phytoremediation, transgenics have made a significant impact on

some other areas too. Although a detailed discussion would be too voluminous and
is beyond the scope of this review, however, we would like to point out some
features very briefly in the following paragraphs. One of them deals with the
development of transgenic plants for rapid detection of environmental pollution
and contamination using efficient molecular screening techniques (plant biomoni-
toring or phytomonitoring), and the second thriving field deals with the synthesis of
bioplastics and biopolymers in transgenic plants.
8.7.1 Transgenic Plants in Biomonitoring
In recent years, substantial progress has been achieved in the realm of transgenic
biomonitoring plants (phytomonitors) (Lebel et al. 1993; Kovalchuk et al. 1998,
1999a,b, 2000a,b,c, 2001a,b; Ries et al. 2000; Besplug et al. 2004; Boyko et al.
2006; Li et al. 2006; van der Auwera et al. 2008). One of the most important aspects
in the development of transgenic biosensors is the option to customize the assay
according to specific biomonitoring requirements (Kovalchuk and Kovalchuk
2008). Two most important assays that are reportedly used in biomonitoring in
recent times are the Recombination Reporter Assay and the Point Mutation
Reporter Assay (Kovalchuk et al. 2000b, c). The biggest success attributed to the
transgenic recombination assay has been its application in detecting radioactive
pollution in soil and water (Kovalchuk et al. 2001a, b). Transgenic Arabidopsis and
tobacco biomonitoring lines have been reported to be excellent tools for detecting
genotoxicity of radioactively contaminated sites (Kovalchuk et al. 1998, 1999a, b).
In case of the Point Mutation Reporter Assay, Kovalchuk et al. (2000b, c) has
developed a system in which they introduced a stop codon at the 50 -end of the
GUS (uidA) gene by means of a single nucleotide substitution that completely
inactivated the transgene. Transgenic plants responded to mutagens (either physical
or chemical agents) by increasing levels of point mutations. They led to the
restoration of the uidA gene activity. Cells where such restorations occurred were
visualized as blue sectors on white plants after histochemical staining. Further
details of these works are beyond the scope of the current article and are available
in a series of papers (Hohn et al. 1999; Kovalchuk et al. 1999a,b, 2000a,b,c;

Kovalchuk and Kovalchuk 2001, 2003, 2008; Filkowski et al. 2003).
8.7.2 Biopolymer Production in Transgenic Plants
Transgenic plants are catching up rapidly in the areas of biopolymer production.

Industrially important biopolymers include proteins, enzymes, recombinant anti-
bodies, vaccines and other biopharmaceutical products (Torres et al. 1999; Doran
2000; Fischer and Emans 2000; Fischer et al. 2000; Giddings et al. 2000; Langridge
2000; Walmsley and Arntzen 2000). Several industrially important proteins have
been produced in transgenic plants: human milk proteins in potato (Chong and
Langridge 2000; Chong et al. 1997); the oleosin–hirudin fusion protein in canola
(Parmenter et al. 1999); phytase in canola (Ponstein et al. 2002); collagens in
tobacco (Ruggiero et al. 2000); spider silk protein (spidroins) in tobacco and potato
(Gosline et al. 1999); bio-elastic proteins in tobacco and avidin in maize (Giddings
et al. 2000); biopolymers in peas and rice and wheat (Frigerio et al. 2000; Perrin
et al. 2000; Stoger et al. 2000).
Transgenic plants offer the following advantages over microbial and animal
expression systems: lower rates of contamination, low production cost, easier and
economic protein extraction and purification steps. Lastly, rapid advances in the
areas of plant proteomics and protein targeting are also promoting the development
of transgenic lines producing different biopolymers (Doran 2000; Giddings et al.
2000; Fischer et al. 2000; Nawrath and Bonetta 2001).
8.7.3 Bioplastics from Transgenic Plants
Transgenic plants producing bioplastics (polyhydroxy butyrate/PHB) are still not a

cheaper alternative compared to the microbial PHB production (Scheller and
Conrad 2005). According to Scheller and Conrad (2005), PHB production in
transgenic plants still needs to reach a comfortable yield target to be economically
feasible. Nawrath et al. (1994) first reported PHB production in the model plant
A. thaliana. The majority of works on PHB transgenics are restricted to Arabidopsis.
However, there are reports of the PHB accumulation in other plants: 5.7% of dry
weight has been reported in maize (Zea mays) by Moire et al. (2003); 7.7% in
oilseed rape (B. napus) by Houmiel et al. (1999), and 5% in sugar beet (B. vulgaris)
by Menzel et al. (2003).
Bohmert et al. (2000) reported 4% PHB production in Arabidopsis; however,
researchers detected a negative correlation between the PHB accumulation and
plant growth. Analysis of T2 plants indicated the loss of PHB synthesis to a
biologically significant amount over generations. Among other plants, tobacco
plants producing PHB are reported to have stunted growth (Nakashita et al. 2001;
Lossl et al. 2003). As to fiber-yielding crops, in cotton the amounts of PHB are
small – 0.34% fiber weight (John and Keller 1996), in flax – 0.5% fiber weight
(Wrobel et al. 2004). Most studies highlighted rapid depletion of other essential
plant metabolites because of the increase in PHB production. Recent progress in
bioplastic production in transgenic plants is aimed at developing lines with higher
PHB production without impacting on growth qualities of targeted plant species
(Scheller and Conrad 2005).
8.8 Conclusions and Future Directions of Phytoremediation

Research
Overall, it is important to note that in the past few decades, phytoremediation

research has moved from its initial emphasis on physiological, biochemical and
genetic investigations and screening of phytoremediating species towards advances
and applications in plant biotechnology and genetic engineering. The future success
of phytoremediation as an emerging agro-industry will depend on how successful
we are in acquiring knowledge regarding the plant genome of model plants and
applying this knowledge to “real-life situations” and “real-life nondomesticated
plant species” to tackle the challenges of complex environmental pollution induced
by chemicals. Tree species will certainly have priority over herbs and shrubs
because of their better phytoremediation abilities.
It has been reported that 64% of contaminated sites contain both organic and
inorganic pollutants; hence, it is important to attract our attention towards generating
transgenics with multitasking abilities (MTTs). Some authors have been successful
in introducing 13 different transgenes in rice using biolistics. Although these
transgenes functioned separately, multiple gene insertion with synergistic functions
was successful in other plants (Hooker and Skeen 1999). Such approach would be
necessary to introduce multiple genes for phytoremediation into a target plant
to make it suitable for phytoremediation under diverse conditions and at sites
contaminated with mixed toxicants. This approach named as a “molecular tool
box” approach by Raskin (1996) could be efficient in dealing with future phytor-
emediation. Plant omics will help regulate future phytoremediation technologies
and strategies to guide us towards a platform where it could be established as a
formidable future industry (Fig. 8.5).
Phytoremediation research and applications have been previously restricted to
North American and European continents only (Raskin 1996; Salt et al. 1998;
Pilon-Smits 2005). Now they have been adopted and extensively pursued in
emerging giant economies such as India, China, Brazil, Russia, and several smaller
EU countries, as well as in Australia and New Zealand (Gratao et al. 2005; Willey
2007). Since the focus on agriculture has shifted back (Raskin 1996), newer
initiatives like phytoremediation can open up opportunities as full-scale agro-
industries in the long run. The future directions guiding phytoremediation research
has been envisioned and highlighted into three separate sections below.
Fig. 8.5 Phytoremediation and its relationships of with different disciplines
8.8.1 Transgenic Trees: A Better Solution for Phytoremediation
Trees with promising phytoremediation genes from other bacterial, yeast, human
and animal sources, and even from other plants could possibly be an essential tool
for future phytoremediation of contaminated sites and soils (Barcelo and Poschen-
rieder 2003; Zayed 2004). It may be a challenging but provocative idea to transfer
multiple phytoremediation genes into candidate tree species for an efficient multi-
phytoremediation approach. This plant would be more desirable than phytoreme-
diating species carrying a single transgene. Such transgenic species can work at two
or more different polluted sites contaminated with totally different chemical pollu-
tants, making the process more efficient and cost-effective.
8.8.2 Multidisciplinary Research Approach
Huge progress has been made in the characterization and modification of the
chemical nature of soil to facilitate phytoremediation of contaminated sites (Datta
and Sarkar 2004) and also in understanding the basic mechanics of pollutant uptake,
translocation, detoxification, and storage mechanisms in plants (as reviewed in
Suresh and Ravishankar 2004; Pilon-Smits 2005). However, there is much still to
be investigated to completely identify all the factors that interact in the process of
phytoremediation, including soil and site, nature and types of a pollutant affecting
contamination, and plants involved in phytoremediation. This realm of research
involves a serious multidisciplinary approach, and it needs collaboration among
scientists working in different fields (Suresh and Ravishankar 2004; Willey 2007).
Although phytoremediating species are slowly turning into crop plants in real
life situations, a lab-to-land transition will involve a lot of support research in
related disciplines to facilitate and speed up the process. For example, in addition to
developing transgenic plant lines, it will also be necessary to engineer plant growth-
promoting rhizobacteria and arbuscular mycorrhizal fungi residing in the same
contaminated soil to further facilitate the efficiency in the natural cleanup of
contaminated sites (Salt et al. 1998). In future, more comprehensive efforts will
be necessary to deal with sites contaminated with complex pollutants, pollutants
representing different chemical species and products generated by their interac-
tions. A dynamic and integrative approach should be used to address future
challenges of phytoremediation.
8.8.3 Applications of Plant Omics for Advancing

Phytoremediation Techniques
Research progress in this field with respect to a proteomic and metabolomic

approach, and metallomic investigations of metal bindings and metalloproteins
are evidently lacking (Azevedo and Azavedo 2006; Garcia et al. 2006). According
to Cobbett and Meagher (2002), approximately 80–90% of all phytoremediation
genes and gene families have been detected in the genome of A. thaliana. This
offers great potential for manipulating and genetic engineering of phytoremediating
plant species. It has been estimated that about 5% of the Arabidopsis genome
constitute membrane transport proteins involved in the transportation of different
ions and metals across the plasma membrane and other organellar plant cell
membranes. Several putative metal transporters have not been identified yet, and
even a handful of those identified are not fully characterized (Maser et al. 2001;
Zayed 2004). According to Heinekamp and Willey (2007), the whole-genome
sequence of A. thaliana will give researchers a grand opportunity to identify and
analyze all genes associated with phytoremediation.
Vacuolar compartmentalization has been suggested to be a second-generation
approach to genetic engineering of phytoremediating species (Heinekamp and
Willey 2007). According to the authors, targeting the accumulation of toxic meta-
bolites inside plant vacuoles makes it possible to enhance the ability of plants to
withstand high toxic concentrations of different target pollutants, and at the same
time it enables plants to increase uptake and accumulation within a restricted
growth period, thereby maximizing the scale of phytoremediation.
References
Aken BV (2008) Transgenic plants for phytoremediation: helping nature to clean up environmen-
tal pollution. Trends Biotechnol 26(5):225–227
Arazi T, Sunker R, Kaplan B, Fromm H (1999) A tobacco plasma membrane calmodulin-binding
transporter confers Ni+2 tolerance and Pb+2 hypersenitivity in transgenic plants. Plant J 20
(2):171–182
Arisi ACM, Mocquot B, Lagriffoul A, Mench M, Foyer CH, Jouanin L (2000) Responses to
cadmium in leaves of transformed poplars overexpressing g-glutamylcysteine synthetase.
Physiol Plant 109:143–149
Assuncao A, Martins P, De Folter S, Vooijs R, Schatt H, Aarts MGM (2001) Elevated expression
of metal transporter genes in three accessions of the metal hyperaccumulator Thlaspi caer-
ulescens. Plant Cell Environ 24:217–226
Azevedo JA, Azavedo RA (2006) Heavy metals and oxidative stress where do we go from here?
Commun Biomet Crop Sci 1(2):135–138
Baker AJM, Brooks RR (1989) Terrestrial higher plants with hyper-accumulate metallic
elements – a review of their distribution, ecology and phytochemistry. Biorecover 1:81–126
Baker AJM, McGrath SP, Sidoli CMD, Reeves RD (1994) The possibility of in situ heavy metal
decontamination of polluted soils using crops of metal-accumulating plants. Resour Conservat
Recycl 11:41–49
Banat IM (1995) Biosurfactants production and possible uses in microbial enhanced oil recovery
and oil pollution remediation: a review. Bioresour Technol 51(1):1–12
Banerjee S, Shang TQ, Wilson AM, Moore AL, Strand SE, Gordon MP, Doty SL (2002)
Expression of functional mammalian P450 2E1 in hairy root cultures. Biotechnol Bioeng
77:462–466
Banuelos GS, Cardon G, Mackey B, Ben-Asher J, Wu L, Beuselinck P, Akohoue S,
Zambruzuski S (1993) Boron and selenium removal in boron-laden soils by four sprinkler
irrigated plant species. J Environ Qual 22:786–792
Banuelos GS, Ajwa HA, Wu L, Guo X, Akohoue S, Zambruzuski S (1997) Selenium-induced
growth reduction in Brassica land races considered for phytoremediation. Ecotoxicol Environ
Saf 36:282–287
Banuelos G, Leduc DL, Pilon-Smits EAH, Terry N (2007) Transgenic indian mustard overexpres-
sing selenocysteine lyase or selenocysteine methyltransferase exhibit enhanced potential for
selenium phytoremediation under field conditions. Environ Sci Technol 41(2):599–605
Barcelo J, Poschenrieder C (2003) Phytoremediation: principles and perspectives. Contribut Sci
2(3):333–344
Becher M, Talke I, Krall L, Kramer U (2004) Cross-species microarray transcript profiling reveals
high constitutive expression of metal homeostasis genes in shoots of the zinc hyperaccumulator
Arabidopsis halleri. Plant J 37:251–268
Beckett PHT, Davis RD (1988) Upper critical levels of toxic elements in plants. New Phytol
79:95–106
Bennett LE, Burkhead JL, Hale KL, Terry N, Pilona M, Pilon-Smits EAH (2003) Analysis of
transgenic Indian mustard plants for phytoremediation of metal-contaminated mine tailings.
J Environ Qual 32(2):432–440
Besplug J, Filkowski J, Burke P, Kovalchuk I, Kovalchuk O (2004) Atrazine induces homologous
recombination but not point mutation in the transgenic plant-based biomonotoring assay. Arch
Environ Contam Toxicol 46(3):296–300
Bizily S, Rugh C, Meagher R (2000) Phytodetoxification of hazardous organomercurials by
genetically engineered plants. Nat Biotechnol 18:213–217
Bizily SP, Kim T, Kandasamy MK, Meagher RB (2003) Subcellular targeting of methylmercury
lyase enhances its specific activity for organic mercury detoxification in plants. Plant Physiol
131:463–471
Black H (1995) Absorbing possibilities: phytoremediation. Environ Health Perspect 103

(12):1106–1108
Blaylock MJ, Salt DE, Dushenkov V, Zakharova O, Gussman C, Kapulnik Y, Raskin I (1997)
Enhanced accumulation of lead in Indian mustard by soil applied chelating agents. Environ Sci
Technol 31:860–865
Bohmert K, Balbo I, Kopka J, Mittendorf V, Nawrath C, Poirier Y, Tischendorf G, Trethewey RN,
Willmitzer L (2000) Transgenic Arabidopsis plants can accumulate polyhydroxybutyrate to up
to 4% of their fresh weight. Planta 211:841–845
Boyajian GE, Carrieira LH (1997) Phytoremediation: a clean transition from laboratory to
marketplace? Nat Biotechnol 15:127–128
Boyko A, Greer M, Kovalchuk I (2006) Acute exposure to UVB has more pronounced effect on
plant genome stability than chronic exposure. Mutat Res 602:100–109
Bradshaw A (1997) Restoration of mined lands-using natural processes. Ecol Eng 8:255–269
Brooks RR, Chambers MF, Nicks LJ, Robinson BH (1998) Phytomining. Trends Plant Sci 3
(9):359–362
Brooks RR, Anderson C, Stewart RB, Robinson BH (1999) Phytomining: growing a crop of a
metal. Biologist 46(5):201–205
Cassidy MB, Lee H, Trevors JT (1996) Environmental applications of immobilized microbial
cells: a review. J Ind Microbiol Biotechnol 16(2):79–101
Castoldi AF, Coccini T, Ceccatelli S, Manzo L (2001) Neurotoxicity and molecular effects of
methylmercury. Brain Res Bull 55(2):197–203
Chaney RL, Malik M, Li YM, Brown SL, Angel JS, Baker AJM (1997) Phytoremediation of soil
metals. Curr Opin Biotechnol 8:279–284
Che DS, Meagher RB, Heaton AC, Lima A, Merkle SA (2003) Expression of mercuric ion
reductase in eastern cottonwood confers mercuric ion reduction and resistance. Plant Biotech-
nol 1(4):311–319
Cherian S, Oliveira M (2005) Transgenic plants in phytoremediation: recent advances and new
possibilities. Environ Sci Qual 39(24):9377–9390
Chong DKX, Langridge WHR (2000) Expression of full-length bioactive antimicrobial human
lactoferrin in potato plants. Transgenic Res 9:71–78
Chong DKX, Roberts W, Arakawa T, Illes K, Bagi G, Slattery CW, Langridge WHR (1997)
Expression of human milk protein b-casein in transgenic potato plants. Transgenic Res 6:289–296
Chrastilova Z, Mackova M, Novakova M, Maeck T, Szekeres M (2007) Transgenic plants for
effective phytoremediation of persistent toxic organic pollutants present in the environment.
J Biotechnol, Abstr No 5: S38. doi:10.1016/j.biotec.2007.07.64
Clements S, Plamgren MG, Kramer U (2002) A long way ahead: understanding and engineering
plant metal accumulation. Trends Plant Sci 7:309–315
Cluis C (2004) Junk-greedy greens: phytoremediation as a new option for soil decontamination.
Cobbett CS, Meagher RB (2002) Arabidopsis and the genetic potential for the phytoremediation of
toxic elemental and organic pollutants. In: Arabidopsis book. Am Soc Plant Biol, pp 1–22.
http://www.aspb.org/publications/arabidopsis/toc.cfm. Accessed 21 Jul 2008
Cunningham SD, Berti WR, Huang JW (1995) Phytoremediation of contaminated soils. Trends
Czako M, Feng X, He Y, Liang D, Pollock R, Marton L (2006) Phytoremediation with transgenic
plants. Acta Hortic 725:753–770
Datta Banik S, Basu SK, De AK (2007) Environment concerns and perspectives. APH Publ,
New Delhi, India, pp 113–138
Datta R, Sarkar D (2004) Effective integration of soil chemistry and plant molecular biology in
phytoremediation of metals: an overview. Environ Geosci 11(2):53–63
de Crombrugghe SA (1964) Vergleich einiger amerikanischer Wildschadenverhütungsmittel mit
deutschen Präparaten. Z Jagdwiss 10(2):62–68
Denton B (2007) Advances in phytoremediation of heavy metals using plant growth promoting
bacteria and fungi. Microbiol Mol Genet 3:1–5
Dhanker OP, Li Y, Rosen BP, Shi J, Salt D, Senecoff JF, Sashti NA, Meagher RB (2002)
Engineering tolearance and hyperaccumulation of arsenic in plants by combining arsenate
reductase and gamma-glutamylcysteine synthetase expression. Nat Biotechnol 20:1140–1145
Dietz AC, Schnoor JL (2001) Advances in phytoremediation. Environ Health Perspect 109
(1):163–168
Doran PM (2000) Foreign protein production in plant tissue cultures. Curr Opin Biotechnol
11:199–204
Doty SL, Shang TQ, Wilson AM, Tangen J, Westergreen AD, Newman LA, Strand SE,
Gordon MP (2000) Enhanced metabolism of halogenated hydrocarbons in transgenic plants
containing mammalian cytochrome P450 E1. Proc Natl Acad Sci USA 97:6287–6292
Doty SL, Shang TQ, Wilson AM, Moore AL, Newman LA, Strand SE, Gordon MP (2003)
Metaboism of the soil and groundwater contaminants, ethylene dibromide and trichoethylene,
by the tropical leguminous tree, Leucaena leucocephala. Water Res 37(2):441–449
Doty SL, James CA, Moore AL, Vajzovic A, Singleton GL, Ma C, Khan Z, Xin G, Kang JW,
Park JY, Meilan R, Strauss SH, Wilkerson J, Farin F, Strand SE (2007) Enhanced phytoreme-
diation of volatile environmental pollution with transgenic trees. Proc Natl Acad Sci USA
104(43):16816–16821
Drake PMW, Chargelegue D, Vine ND, Van Dolleweerd GJ, Obregon P, Ma JK-C (2002)
Transgenic plants expressing antibodies: a model for phytoremediation. FASEB J 16:
1855–1860
Dushekov V, Nanda Kumar PBA, Motto H, Raskin I (1995) Rhizofiltration: the use of plants to
remove heavy metals from aquatic streams. Environ Sci Technol 29:1239–1245
Eapen S, D’Souza SF (2005) Prospects of genetic engineering of plants for Phytoremediation of
toxic metals. Biotechnol Adv 23(2):97–144
Ellis DR, Sors TG, Brunk DG, Albrecht C, Orser C, Lahner B, Wood KV, Harris HH, Pickering IJ,
Salt DE (2004) Production of Se-methylselenocysteine in transgenic plants expressing seleno-
cysteine methyltransferase. BMC Plant Biol 4:1. doi:10.1186/1471-2229-4-1
Evans KM, Gatehouse JA, Lindsay WP, Shi J, Tommey AM, Robinson NJ (1992) Expression of
pea metallothionein-like gene PsMTA function. Plant Mol Biol 20:1019–1028
Filkowski J, Besplug J, Burke P, Kovalchuk I, Kovalchuk O (2003) Recombination- and point
mutation-monitoring transgenic plants reveal the genotoxicity of commonly used herbicides 2,
4-D and dicamba. Mutat Res 542(1–2):23–32
Fischer R, Emans N (2000) Molecular farming of pharmaceutical proteins. Transgenic Res 9:
279–299
Fischer R, Hoffmann K, Schillberg S, Emans N (2000) Antibody production by molecular farming
in plants. J Biol Regul Homeost Agents 14:83–92
Fladung M, Ewald D (2006) Tree transgenesis – recent developments. Springer, Berlin,
pp 137–148
Floco CG, Giulietti AM (2007) In vitro hairy root cultures as a tool for phytoremediation research.
In: Willey N (ed) Phytoremediation: methods and reviews. Humana Press, Totowa, NJ,
pp 161–173
Freeman JL, Persans MW, Nieman K, Albrecht C, Peer WA, Pickering IJ, Salt DE (2004)
Increased glutathione biosynthesis plays a role in nickel tolerance in Thlaspi nickel hyper-
accumulators. Plant Cell 16:2176–2191
French CE, Nicklin S, Bruce NC (1998) Aerobic degradation of 2, 4, 6-trinitrotoluene by
Enterobacter cloacae PB2 and by pentaerythritol tetranitrate reductase. Appl Environ Micro-
biol 64(8):2864–2868
French CE, Rosser SJ, Davies GJ, Nicklin S, Bruce NC (1999) Biodegradation of explosives by
transgenic plants expressing pentaerythritol tetranitrate reductase. Nat Biotechnol 17:491–494
Frigerio L, Vine ND, Pedrazzini E, Hein MB, Wang F, Ma JKC, Vitale A (2000) Assembly,
secretion, and vacuolar delivery of a hybrid immunoglobulin in plants. Plant Physiol
123:1483–1493
Garcia JS, Magalhaes CS, Arruda MAZ (2006) Trends in metal-binding and metalloprotein
analysis. Talanta 69:1–15
Ghosh M, Singh SP (2005) A review on phytoremediation of heavy metals and utilization of its
byproducts. Appl Ecol Environ Res 3(1):1–18
Giangrande A, Cavallo A, Licciano M, Mola E, Pierri C, Trianni L (2006) Utilization of the filter-
feeder polychaete Sabella spallanzanii Gmelin (Sabellidae) as bioremediator in aquaculture.
Aquacult Int 13:129–136
Giddings G, Allison G, Brooks D, Carter C (2000) Transgenic plants as factories for biopharma-
Gifford S, Dunstan H, O’Connor W, Macfarlane GR (2005) Quantification of in situ nutrient and
heavy metal remediation by a small pearl oyster (Pinctada imbricata) farm at Port Stephens,
Australia. Mar Pollut Bull 50(4):417–422
Gifford S, Dunstan RH, O’Conor W, Koller CE, MacFarlane GR (2007) Aquatic zooremediation:
deploying animals to remediate contaminated aquatic environments. Trends Biotechnol 25
(2):60–65
Gisbert C, Ros R, Haro AD, Walker DJ, Bernal MP, Serrano R, Avino JN (2003) A plant
genetically modified that accumulates Pb is especially promising for phytoremediation.
Bichem Biophys Res Commun 303:440–445
Gleba D, Borisjuk NV, Borisjuk LG, Kneer R, Poulev A, Skarzhinskaya M, Dushenkov S,
Logendra S, Gleba YY, Raskin I (1999) Use of plant roots for phytoremediation and molecular
farming. Proc Natl Acad Sci USA 96(11):5973–5977
Goel A, Kumar G, Payne GF, Dube SK (1997) Plant cell biodegradation of a xenobiotic nitrate
ester, nitroglycerin. Nat Biotechnol 15:174–177
Gong JM, Lee D, Schroeder JI (2003) Long-distance root-to-shoot transport to phytochelatins and
cadmium in Arabidopsis. Proc Natl Acad Sci USA 100:10118–10123
Gosline JM, Guerette PA, Ortlepp CS, Savage KN (1999) The mechanical design of spider silks:
from fibroin sequence to mechanical function. J Exp Biol 202:3295–3303
Gratao PL, Prasad MNV, Cardoso PF, Lea PJ, Azevedo RA (2005) Phytoremediation: green
technology for the clean up of toxic metals in the environment. Braz J Plant Physiol 17(1):
53–64
Gray KA (2006) General phytoremediation. Section 3. www.civil.northwestern.edu/EHE/
HTML_KAG/Kimweb/MEOP/Section3.htm. Accessed 14 Jul 2008
Ha S-B, Smith AP, Howden R, Dietrich WM, Bugg S, O’Connell MJ, Goldsbrough PB, Cobbett
CS (1999) Phytochelatin synthase genes from Arabidopsis and the yeast Schizosaccharomyces
pombe. Plant Cell 11:1153–1164
Hammer J (1996) Improving water quality in eutrophied fjord system with mussel farming. Ambio
25:356–362
Hannik N, Rosser SJ, French CE, Basran A, Murray JAH, Nicklin S, Bruce NC (2001) Phytor-
emediation of TNT by transgenic plants expressing a bacterial nitroreductase. Nat Biotechnol
19:11168–11172
Hannink NK, Rosser SJ, Bruce NC (2002) Phytoremediation of explosives. Crit Rev Plant Sci
21(5):511–538
Hassinen VH, Tervahauta AI, Karenlampi S (2007) Searching for genes involved in metal
tolearance, uptake and transport. In: Willey N (ed) Phytoremediation: methods and reviews.
Humana Press, New Jersey, USA, pp 265–289
He YK, Jg S, Feng XZ, Czako M, Marton L (2001) Differential mercury volatilization by tobacco
organs expressing a modified bacterial merA gene. Cell Res 11:231–236
Heaton A, Rugh C, Wang N, Meagher R (1998) Phytoremediation of mercury and methylmercury
polluted soils using genetically engineered plants. J Soil Contam 7:497–509
Heaton AC, Rugh CL, Kim T, Wang NJ, Meagher RB (2003) Towards detoxifying mercury-
polluted aquatic sediments with rice genetically engineered for mercury resistance. Environ
Toxicol Chem 12:2940–2947
Heinekamp Y-J, Willey N (2007) Using real-time PCR polymerase chain reaction to quantify gene
expression in plants exposed to radioactivity. In: Willey N (ed) Phytoremediation: methods and
reviews. Humana Press, New Jersey, USA, pp 59–70
Hohn B, Kovalchuk I, Kovalchuk O (1999) Transgenic plants sense radioactive contamination.
BioWorld 6:13–15
Hooker BS, Skeen RS (1999) Transgenic phytoremediation blasts onto the scene. Nat Biotechnol
17:428
Houmiel KL, Slater S, Broyles D, Casagrande LS, Colburn S, Gonzalez K, Mitsky TA, Reiser SE,
Shah D, Taylor NB, Tran M, Valenti HE, Gruys KJ (1999) Poly(b-hydroxybutyrate) produc-
tion in oilseed leucoplasts of Brassica napus. Planta 209:547–550
Hussein HS, Ruiz ON, Terry N, Daniell H (2007) Phytoremediation of mercury and organomer-
curials in chloroplast transgenic plants: enhanced root uptake, translocation to shoots, and
volatilization. Environ Sci Technol 41:8439–8446
John ME, Keller G (1996) Metabolic pathway engineering in cotton: biosynthesis of polyhydrox-
ybutyrate in fiber cells. Proc Natl Acad Sci USA 93:12768–12773
Juhasz AL, Naidu R (2000) Bioremediation of high molecular weight polycyclic aromatic hydro-
carbons: a review of the microbial degradation of benzo[a]pyrene. Int Biodeterior Biodegra-
dation 45(1–2):57–88
Kovalchuk I, Kovalchuk O (2001) Bioindication with transgenic plants. ISB News Rep, Sep 2001,
pp 1–3
Kovalchuk I, Kovalchuk O (2008) Transgenic plants as sensors of environmental pollution
genotoxicity. Sensors 8:1539–1558
Kovalchuk I, Kovalchuk O, Arkhipov A, Hohn B (1998) Transgenic plants are sensitive bioindi-
cators of nuclear pollution caused by the Chernobyl accident. Nat Biotechnol 16:1054–1059
Kovalchuk I, Kovalchuk O, Hohn B (1999a) Transgenic plants as bioindicators of environmental
pollution, vol 1. AgBiotechNet, Oct 1999, ABN 030
Kovalchuk O, Kovalchuk I, Titov V, Arkhipov A, Hohn B (1999b) Radiation hazard caused by the
Chernobyl accident in inhabited areas of Ukraine can be monitored by transgenic plants. Mutat
Res 446:49–55
Kovalchuk I, Kovalchuk O, Fritsch O, Hohn B (2000a) Des bio-indicators de pollution radioactive.
Biofutur 205:48–51
Kovalchuk I, Kovalchuk O, Hohn B (2000b) Genome-wide variation of the somatic mutation
frequency in transgenic plants. EMBO J 19:4431–4438
Kovalchuk O, Arkhipov A, Dubrova Yu, Hohn B, Kovalchuk I (2000c) Wheat mutation rate after
Chernobyl. Nature 407:583–584
Kovalchuk I, Kovalchuk O, Hohn B (2001a) Biomonitoring of genotoxicity of environmental
factors with transgenic plants. Trends Plant Sci 6:306–310
Kovalchuk O, Titov V, Hohn B, Kovalchuk I (2001b) A sensitive transgenic plant system to detect
toxic inorganic compounds in the environment. Nat Biotechnol 19:568–572
Langridge WHR (2000) Edible vaccines. Sci Am 283:48–53
Lebel EG, Masson J, Bogucki A, Paszkowski J (1993) Stress-induced intrachromosomal recombi-
nation in plant somatic cells. Proc Natl Acad Sci USA 90:422–426
LeDuc DL, Tarun AS, Montes-Bayson M, Meija J, Malit MF, Wu CP, Abdel Samie M,
Chiang CY, Tagmount A, deSouza M, Neuhier B, Bock A, Caruso J, Terry N (2004) Over-
expression of selenocysteine methyltransferase in Arabidopsis and Indian mustard increases
selenium tolerance and accumulation. Plant Physiol 135:377–383
Lee S, Moon J, Ko TS, Petros D, Goldsbrough PB, Korban SS (2003) Overexpression of
Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress.
Li L, Jean M, Belzile F (2006) The impact of sequence divergence and DNA mismatch repair on
homeologous recombination in Arabidopsis. Plant J 45:908–916
Liang ZY, Pilon-Smits EA, Jouanin L, Terry N (1999) Overexpression of glutathione synthetase
in Indian mustard enhances cadmium accumulation and tolerance. Plant Physiol 119(1):
73–80
Liu S, Suflita JM (1993) Ecology and evolution of microbial populations for bioremediations.
Trends Biotechnol 11(8):344–352
Lossl A, Eibl C, Harloff J, Jung C, Koop UH (2003) Polyester synthesis in transplastomic tobacco
(Nicotiana tabacum L.): significant contents of polyhydroxybutyrate are associated with
growth reduction. Plant Cell Rep 21:891–899
Lovely DR (2003) Cleaning up with genomics: applying molecular biology to bioremediation. Nat
Rev Microbiol 1:35–44
Lucero ME, Mueller W, Hubstenberger J, Phillips GC, O’Conell MA (1999) Tolerance to
nitrogenous explosives and metabolism of TNT by cell suspensions of Datura innoxia. In
Vitro Cell Dev Biol Plant 35:480–486
MacKenzie BR, Almesjö L, Hansson S (2004) Fish, fishing, and pollutant reduction in the Baltic
Sea. Environ Sci Technol 38(7):1970–1976
Maser P, Thomine S, Schroeder J, Wrad J, Hirschi K, Sze H, Talke I, Amtmann A, Maathuis F,
Sanders D, Harper J, Tchieu J, Gribskov M, Persans M, Salt D, Kim S, Guerinot M (2001)
Phylogenetic relationships within cation transporter families of Arabidopsis. Plant Physiol
126:1646–1667
McGloughlin MN, Burke JI (2000) Biotechnology – present position and future developments.
Teagasc, Ireland, UK, pp 118–127
McIntyre T (2003) Phytoremediation of heavy metals from soils. Adv Biochem Eng Biotechnol
78:97–123
McLean J, Purvis OW, Williamson BJ, Bailey EH (1998) Role for lichen melanins in uranium
remediation. Nature 391:649–650
Meagher RB (2000) Phytoremediation of toxic elemental and organic pollutants. Curr Opin Plant
Biol 3:153–162
Meagher RB, Rugh CL, Kandasamy MK, Gragson G, Wang NJ (2000) Engineered phytoremedia-
tion of mercury pollution in soil and water using bacterial genes. In: Terry N, Banuelos G (eds)
Phytoremediation of contaminated soil and water. Lewis, Boca Raton, FL, pp 201–221
Meier JR, Chang LW, Jacobs S, Torsella J, Meckes MC, Smith MK (1997) Use of plant and
earthworm bioassays to evaluate remediation of soil from a site contaminated with polychlori-
nated biphenyls. Environ Toxicol Chem 16(5):928–938
Mejare M, Bulow L (2001) Metal-binding proteins and peptides in bioremediation and phytor-
emediation of heavy metals. Trends Biotechnol 19(2):67–73
Mendez MO, Maier RM (2008) Phytostabilization of mine tailings in arid and semiarid environ-
ments-an emerging remediation technology. Environ Health Perspect 116(3):278–283
Menzel G, Harloff HJ, Jung C (2003) Expression of bacterial poly(3-hydroxybutyrate) synthesis
genes in the hairy roots of sugar beet (Beta vulgaris L.). Appl Microbiol Biotechnol 60:
571–576
Mezzari MP, Walters K, Jelı́nkova M, Shih M-C, Just CL, Schnoor JL (2005) Gene expression and
microscopic analysis of Arabidopsis exposed to chloroacetanilide herbicides and explosive
compounds. A phytoremediation approach. Plant Physiol 138(2):858–869
Milanese M, Chelossi E, Manconi R, Sarà A, Sidri M, Pronzato R (2003) The marine sponge
Chondrilla nucula Schmidt, 1862 as an elective candidate for bioremediation in integrated
aquaculture. Biomol Eng 20(4–6):363–368
Misra S, Gedamu L (1989) Heavy-metal tolerant transgenic Brassica napus L. and Nicotaina
tabacum L. plants. Theor Appl Genet 78:161–1168
Moire L, Rezzonico E, Poirier Y (2003) Synthesis of novel biomaterials in plants. J Plant Physiol
160:831–839
Naidu R, Oliver D, McConnell S (2003) Heavy metal phytotoxicity in soils. In: Langley A, Gilbey
M, Kennedy B (eds) Proceedings of the 5th national workshop on the assessment of soil
contamination. National Environment Protection Council, Adelaide, Australia, pp 235–241
Nakashita H, Arai Y, Shikanai T, Doi Y, Yamaguchi I (2001) Introduction of bacterial metabolism
into higher plants by polycistronic transgene expression. Biosci Biotechnol Biochem 65:
1688–1691
Nanda Kumar PBA, Dushenkov V, Motto H, Raskin I (1995) Phytoextraction: the use of plants to
remove heavy metals from soils. Environ Sci Technol 29:1232–1238
Nawrath C, Poirier Y, Somerville C (1994) Targeting of the polyhydroxybutyrate biosynthetic
pathway to plastids of the Arabidopsis thaliana results in high levels of polymer accumulation.
Newman LA, Strand SE, Choe N, Duffy J, Ekuan G (1997) Uptake and biotransformation of
tricholoroethylene by hybrid poplars. Environ Sci Technol 31:1062–1067
Nie L, Shah S, Rashid A, Burd GI, Dixon DG, Glick BR (2002) Phytoremediation of arsenate
contaminated soil by transgenic canola and the plant growth-promoting bacterium Enterobac-
ter cloacae CAL2. Plant Physiol Biochem 40:355–361
Novakova M, Mackova M, Sylvestre M, Macek T (2007) Preparation of genetically modified
plants bacterial dioxygenase-toll for preferable phytoremediation. J Biotechnol 131S, Abstr No
1: S36. doi:10.1016/j.biotec.2007.07.60
Ohkawa Y, Ohkawa H (2002) Transgenic rice and potato plants expressing human cytochrome
P450S show cross-tolerance to herbicides by detoxifying them. http://www.agnet.org/library/
tb/159/. Accessed 14 Jul 2008
Oksman-Caldentey K-M, Barz WH (2002) Plant biotechnology and transgenic plants. Marcel
Dekker, New York, USA, pp 665–669
Olguin EJ (2003) Phycoremediation: key issues for cost-effective nutrient removal processes.
Biotechnol Adv 22:81–91
Pacyna E, Pacyna J (2002) Global emissions of mercury from anthropogenic sources in 1995.
Water Air Soil Pollut 137:149–165
Pan A, Yang M, Tie F, Li L, Chen Z, Ru B (1994) Expression of mouse metallothionein-I gene
confers cadmium resistance in transgenic tobacco plants. Plant Mol Biol 24:341–351
Parmenter DL, Boothe JG, van Rooijen GJ, Yeung EC, Moloney MM (1999) Production of
biologically active hirudin in plant seeds using oleosin partitioning. Plant Mol Biol 29:
1167–1180
Pavlikova D, Macek T, Mackova M, Sura M, Szakova J, Tlustos P (2004) The evaluation of
cadmium, zinc and nickel accumulation ability of transgenic tobacco bearing different trans-
genes. Plant Soil Environ 50:513–517
Pence NS, Lasen PB, Ebbs SD, Letham DLD, Last MM, Garvin DF (2000) The molecular
physiology of heavy metal transport in Zn/Cd hyperaccumulator Thlaspi caerulescens. Proc
Natl Acad Sci USA 97(9):4956–4960
Perrin Y, Vaquero C, Gerrard I, Sack M, Drossard J, Stoger E, Christou P, Fischer R (2000)
Transgenic pea seeds as bioreactors for the production of a single-chain Fv fragment (scFV)
antibody used in cancer diagnosis and therapy. Mol Breed 6:345–352
Persans MW, Nieman K, Salat DE (2001) Functional activity and role cation-efflux family
members in Ni hyper accumulation in Thlaspi goesingense. Proc Natl Acad Sci USA
98:9995–10000
Pilon M, Owen JD, Garifullina GF, Kurihara T, Mihara H, Esaki N, Pilon-Smits EAH (2003)
Enhanced selenium tolerance and accumulation in transgenic Arabidopsis expressing a mouse
selenocysteine lyase. Plant Physiol 131:1250–1257
Pilon-Smits E (2005) Phytoremediation. Annu Rev Plant Biol 56:15–39
Pilon-Smits EA, Hwang S, Mel Lytle C, Zhu Y, Tai JC, Bravo RC, Chen Y, Leustek T, Terry N
(1999) Overexpression of ATP surfurylasev in Indian mustard leads to increased selenate
uptake, reduction and tolerance. Plant Physiol 119(1):123–132
Pilon-Smits EAH, Zhu YL, Sears T, Terry N (2000) Overexpression of glutathione reductase in
Brassica juncea: effects on cadmium accumulation and tolerance. Physiol Plant 110:455–460
Pollard SJT, Hrudey SE, Fedorak PM (1994) Bioremediation of petroleum- and creosote-con-
taminated soils: a review of constraints. Waste Manag Res 12(2):173–194
Ponstein AS, Bade JB, Verwoerd TC, Molendijk L, Storms J, Beudeker RF, Pen J (2002) Stable
expression of Phytase (phyA) in canola (Brassica napus) seeds: towards a commercial product.
Mol Breed 10:31–44
Pulford I, Watson C (2003) Phytoremediation of metals using transgenic plants. Crit Rev Plant Sci
21(5):439–456
Ramaswami A, Rubin E, Bonola S (2003) Non-significance of rhizosphere degradation during
phytoremediation of MTBE. Int J Phytoremediation 5(4):315–331
Raskin I (1996) Plant genetic engineering may help with environmental cleanup. Proc Natl Acad
Sci USA 93:3164–3166
Raskin I, Nanda Kumar PBA, Dushenkov S, Salt DE (1994) Bioconcentration of heavy metals by
plants. Curr Opin Biotechnol 5:285–290
Raskin I, Smith RD, Salt DE (1997) Phytoremediation of metals: using plants to remove pollutants
from the environment. Curr Opin Biotechnol 8:221–226
Reeves RD, Baker AJM (2000) Metal-accumulating plants. In: Raskin I, Ensley BD (eds)
Phytoremediation of toxic metals: using plants to clean up the environment. Wiley,
New York, USA, pp 193–229
Renneberg A, Dudas J (2001) Transformation of elemental mercury to inorganic and organic
mercury and hydrocarbon co-contaminated soils. Chemosphere 45(6–7):1103–1109
Ries G, Heller W, Puchta H, Sandermann H, Seidlitz HK, Hohn B (2000) Elevated UV-B radiation
reduces genome stability in plants. Nature 406:98–101
Ruggiero F, Exposito JY, Bournat P, Gruber V, Perret S, Comte J, Olagnier B, Garrone R,
Theisen M (2000) Triple helix assembly and processing of human collagen produced in
transgenic tobacco plants. FEBS Lett 469:132–136
Rugh CL (2001) Mercury detoxification with transgenic plants and other biotechnological break-
throughs for phytoremediation. In Vitro Cell Mol Dev Plant 37(3):321–325
Rugh CL, Wilde D, Stack NM, Thompson DM, Summers AO, Meagher RB (1996) Mercuric ion
reduction and resistance in transgenic Arabidopsis thaliana merA gene. Proc Natl Acad Sci
USA 93:3182–3187
Rugh CL, Senecoff JF, Meagher RB, Merkle SA (1998) Development of transgenic yellow poplar
for mercury phytoremediation. Nat Biotechnol 16:925–928
Ruiz JN, Romero L (2002) First evidence of arsenic phytoremediation. Trends Plant Sci 7(9):384
Ruiz ON, Hissein HS, Terry N, Daniell H (2003) Phytoremediation of organomercurial
compounds via chloroplast genetic engineering. Plant Physiol 132:1344–1352
Sadowsky MJ (1999) Phytoremediation: past promises and future. In: Bell CR, Brylinsky M,
Jhonson-Green P (eds) Microbial biosystems: new frontiers, section-plant microbe interac-
tions. Proc 8th Int Symp of Microbiol. Atlantic Canada Soc Microbial Ecol, Halifax, Canada,
pp 1–7. http://socrates.acadiau.ca/isme/Symposium26/sadowsky.PDF. Accessed 16 Jul 2008
Salt DE, Blaylock M, Kumar NPBA, Dushnekov V, Ensley BD, Chet I, Raskin I (1995) Phytor-
emediation: a novel strategy for the removal of toxic metals from the environment using plants.
Salt DE, Smith RD, Raskin I (1998) Phytoremediation. Annu Rev Plant Physiol Plant Mol Biol
49:643–668
Scheller J, Conrad U (2005) Plant based material, protein and biodegradable plastic. Curr Opin
Schnoor J (1997) Phytoremediation: ground water remediation technologies analysis center
evaluation report, TE-98-01, p 37
Schnoor JL, Licht LA, Mc Cutcheon SC, Wofe NL, Carreira LH (1995) Phytoremediation of
organic and nutrient contaminants. Environ Sci Technol 29:318–323
Shann JR (1995) The role of plant/microbial systems in the reduction of exposure. Environ Health
Perspect 103(5):13–15
Shaul O, Hilgemann DW, de Almeida-Engler J, van Montagu M, Inze D, Galili G (1999) Cloning
and characterization of a novel Mg+2/H+ exchanger. EMBO J 18:3973–3980
Shimizu M, Kimura T, Koyama T, Suzuki K, Ogawa N, Miyashita K, Sakka K, Ohmiya K (2002)
Molecular breeding of transgenic rice plants expressing bacterial chlorocatechol dioxygenase
gene. Appl Environ Microbiol 68(8):4061–4066
Simon L, Martin HW, Adriano DC (1996) Chicory (Cichorium intybus L.) and dandelion
(Taraxacum officinale Web.) as phytoindicators of cadmium contamination. Water Air Soil
Pollut 91(3–4):351–362
Singh H (2006) Mycoremediation: fungal bioremediation. Wiley, NY, USA, pp 1–15
Song WY, Sohn EJ, Martinoia E, Lee YJ, Yang YY, Jasinski M, Forestier C, Hwang I, Lee Y
(2003) Engineering tolerance and accumulation of lead and cadmium in transgenic plants. Nat
Sonoki S, Fujihiro S, Hisamatsu S (2007) Genetic engineering of plants for phytoremediation of
polychlorinated biphenyls. In: Willey N (ed) Phytoremediation: methods and reviews. Humana
Press, New Jersey, USA, pp 3–13
Stabili L, Licciano M, Giangrande A, Fanelli G, Cavallo RA (2006) Sabella spallanzanii filter-
feeding on bacterial community: ecological implications and applications. Mar Environ Res
61(1):74–92
Stearns JC, Shah S, Glick BR (2007) Increasing plant tolerance to metals in the environment. In:
Willey N (ed) Phytoremediation: methods and reviews. Humana Press, New Jersey, USA,
pp 15–26
Stoger E, Vaquero C, Torres E, Sack M, Nicholson L, Drossard J, Williams S, Keen D, Perrin Y,
Christou P, Fischer R (2000) Cereal crops as viable production and storage systems for
pharmaceutical scFv antibodies. Plant Mol Biol 42:583–590
Sunkar R, Kaplan B, Bouche N, Arazi T, Dolev D, Talke IN, Frans JM, Maathuis FJM, Sanders D,
Bouchez D, Fromm H (2000) Expression of truncated tobacco NtCBP4 channel in transgenic
plants and disruption of the homologous Arbaidopsis CNGC1 gene confers Pb+2 tolerance.
Plant J 24(4):533–542
Suresh B, Ravishankar GA (2004) Phytoremediation- a novel and promising approach for envi-
ronmental clean up. Crit Rev Biotechnol 24(2–3):97–124
Sursala S, Medina VF, McCutcheon SC (2002) Phytoremediation: an ecological solution to
organic chemical contamination. Ecol Eng 18(5):647–658
Sykes M, Yang V, Blankenburg J, AbuBakr S (1999) Biotechnology: working with nature to
improve forest resources and products. In: Proc TAPPI Int Pulping Conf, vol 2, April 18–22,
1999, Nashville, TN. TAPPI Press, Atlanta, GA, pp 631–637
Terry N, Sambukumar SV, LeDuc DL (2003) Biotechnological approaches for enhancing phytor-
emediation of heavy metals and metalloids. Acta Biotechnol 23(2–3):282–288
Thomas JC, Davies EC, Malick FK, Endreszl C, Williams CR, Abbas M, Petrella S, Swisher K,
Perron M, Edwards R, Ostenkowski P, Urbanczyk N, Wiesend WN, Murray KS (2003) Yeast
metallothionein in transgenic tobacco promotes copper uptake from contaminated soils.
Biotechnol Prog 19(2):273–280
Thomine S, Wang R, Ward JM, Crawford NM, Schroeder JI (2000) Cadmium and ion transport by
members of a plant metal transporter family in Arabidopsis with homology to Nramp genes.
Thompson P, Ramer L, Guffey AP, Schnoor JL (1998) Decreased transpiration in poplar trees
exposed to 2, 4, 6,-trinitrotoluene. Environ Toxicol Chem 17:902–906
Tong Y-P, Kneer R, Zhu Y-G (2004) Vascular compartmentalization: a second-generation
approach to engineering plants for phytoremediation. Trends Plant Sci 9:7–9
Torres E, Vaquero C, Nicholson L, Sack M, Stoger E, Drossard J, Christou P, Fischer R, Perrin Y
(1999) Rice cell culture as an alternative production system for functional diagnostic and
therapeutic antibodies. Transgenic Res 8:441–449
Trapp S, Karlson U (2001) Aspects of phytoremediation of organic pollutants. J Soils Sediments
1:1–7
Vacchina V, Mari S, Czernic P, Marques L, Pianeli K, Schaumloffe D, Lebru M, Lobinski R
(2003) Speciation of nickel in a hyperaccumulating plant by high-performance liquid chroma-
tography-inductively couple plasma mass spectrometry and electrospray MS/MS assisted by
cloning using yeast complementation. Anal Chem 75:2740–2745
Van der Auwera G, Baute J, Bauwens M, Peck I, Piette D, Pycke M, Asselman P, Depicker A
(2008) Development and application of novel constructs to score C:G-t0T: a transitions and
homologous recombination in Arabidopsis. Plant J 45:908–916
Van der Zaal BJ, Neuteboom LW, Pinas JE, Chadonnens AN, Schat H, Verkleij JAC, Hooykaas
PJJ (1999) Overexpression of a novel Arabidopsis gene related to putative zinc-transporter
genes from animal can lead to enhanced zinc resistance and accumulation. Plant Physiol
119:1047–1055
Vroblesky DA, Nietch CT, Morris JT (1999) Chlorinated ethanes from groundwater in tree trunks.
Environ Sci Technol 33:277–281
Wagner-Dobler I (2003) Pilot plant for bioremediation of mercury-containing industrial waste-
water. Appl Microbiol Biotechnol 62:124–133
Walmsley AM, Arntzen CJ (2000) Plants for delivery of edible vaccines. Curr Opin Biotechnol
11:126–129
Wang G-D, Chen X-Y (2007) Detoxification of soil phenolic pollutants by plant secretory enzyme.
In: Willey N (ed) Phytoremediation: methods and reviews. Humana Press, New Jersey, USA,
pp 49–57
White C, Shaman AK, Gadd GM (1998) An integrated microbial process for the bioremediation of
soil contaminated with toxic metals. Nat Biotechnol 16:572–575
Willey N (2007) Phytoremediation: methods and reviews. Humana Press, New Jersey, USA,
pp 351–468
Wrobel M, Zebrowski J, Szopa J (2004) Polyhydroxybutyrate synthesis in transgenic flax.
J Biotechnol 107:41–54
Yamada T, Ishige T, Shiota N, Inui H, Ohkawa H, Ohkawa Y (2002) Enhancement of metaboliz-
ing herbicides in young tubers of transgenic potato plants with the rat CYP1A1 gene. Theor
Appl Genet 105:515–520
Yoon JM, Oh B-T, Just CL, Schnoor JL (2002) Uptake and leaching of octahydro-1, 3, 5,
7-tetranitro-1, 3, 5, 7-tetrazocine by hybrid poplar trees. Environ Sci Technol 36:4649–4655
Zayed A (2004) Phytoremediation: advances toward a new clean up technology. In: Goodman RE
(ed) Encyclopaedia of plant crop science. Taylor and Francis, Oxford, UK, pp 924–927:
doi:10.1081/E-EPCS 120005584
Zhu YL, Pilon-Smits EA, Tarun AS, Weber SU, Jouanin L, Terry N (1999) Cadmium tolerance
and accumulation in Indian mustard is enhanced by overexpressing gamma-glutamylcysteine
synthetase. Plant Physiol 121:1169–1178
Chapter 9
Algal Biotechnology: An Emerging Resource
with Diverse Application and Potential
Stephen Cunningham and Lokesh Joshi
9.1 Introduction to Algae
Algae include a wide variety of species that range from diatoms, which are micro-
scopic unicellular organisms, to seaweeds extending over 30 m (Fig. 9.1). They
constitute a group of approximately 40,000 species, a heterogeneous group that
describes a life-form, not a systematic unit; hence, a broad spectrum of phenotypes
exists in this grouping. Algae are grouped into six main classes, mainly on the basis
of their color (Fogg 1953). Algae are found in fresh or salt water, with a few being
terrestrial (e.g., Chrysophyta and Cyanophyta). The eukaryotic algae are placed in
the kingdom Protista, classified as euglenoids (phylum Euglenophyta), dinoflage-
llates (phylum Pyrrophyta) and diatoms (phylum Bacillariophyta). All have chloro-
plasts and carry out photosynthesis similar to that of plants. Prokaryotic blue-green
algae belong to the phlyum Cyanobacteria.
Unlike land plants, algae do not have true roots, stems or leaves. Algae of
different size and shape not only occupy aquatic ecosystems, but also occur in a
number of different habitats, some of which are extreme environments (Hallmann
2007). The environmental conditions have led to the development of adaptive
tolerances, such as temperature, salt and pressure selection; such adaptive selection
is widely observed in bacteria.
Large forms of algae are often referred to as seaweeds or microalgae, which are
widely distributed in the ocean, occurring from the tide level to considerable
depths, free-floating or anchored, holding an important role in providing marine
primary productivity. Eukaryotic green (phylum Chlorophyta), red (phylum Rho-
dophyta) and brown algae (phylum Phaeophyta) are all grouped as seaweeds. They
are often found to produce considerable biomass, evident from natural population
L. Joshi (*)
Glycoscience and Glycotechnology Group and the Martin Ryan Institute National Centre for
Biomedical Engineering Science, National University of Ireland, Galway, Ireland
e-mail: lokesk.joshi@nuigalway.ie

344 S. Cunningham and L. Joshi
Fig. 9.1 Demonstration of 1 μm Osteococcus tauri

size diversity among algae Cyanidioschyzon merolae
species
Thalassiosira pseudonana
10 μm Chlamydomanas reinhardtii
Phaeodactylum tricornutum
100 μm Asterionella formosa
1 mm Volvox carteri
1 cm Neomeris annulata
10 cm
Ulva lactuca
1m Fucus vesiculosus
10 m
Macrocystis pyrifera
100 m
of brown algae Macrocystis in Northern America and the cultivated populations of

artificially cultivated brown algae Laminaria and Undaria in East Asia. Geographi-
cally, algae have been used as a food source in the Asian countries, while in Europe
algae has been traditionally used for the production of phycocolloids such as agar
and carrageenan both from red algae and alginate from brown algae (McHugh
2003; Hallmann 2007). In most cases, algae are used in human and animal foods for
their mineral contents or for the functional properties of their polysaccharides.
As in the case of terrestrial plants, development of key techniques of genetic
manipulation specific for algae was necessary to enable creation of good cultivars
and to transform seaweed into multiple, functional marine bioreactors. This has led
to the application of algae being maximized in:
1) Eliminating heavy metal pollutants,
2) Reducing factors of eutrophication (e.g., nitrogen and phosphorus),
9 Algal Biotechnology: An Emerging Resource with Diverse Application and Potential 345
3) Improving the feed quality for maricultured animals using transgenic algae
delivering/introducing genes encoding immunologically active peptides (mole-
cular pharming), and
4) The production of high-value materials such as oral vaccines and drugs for
humans
Great strives have been made in the genetic engineering of plants and micro-
organisms over the last two decades. It is established that an effective transformation
model consists of elements permitting an effective transformation methodology, that
applicable vectors carry recognizable promoters, and that a screening mechanism to
select transformants is in place to isolate the transformants from the endogenous
mass. These are sufficient for single-celled organisms; however, multicellular
organisms require further factors for successful recombination.
This chapter describes the application, development and status of algae as a
source of natural and recombinant molecules for industrial and human health
applications.
9.2 The Application of Non-Transgenic Algae
9.2.1 Nutritional and Health-Related Properties
Algae have been utilized both historically and currently as food sources for
human, animal and mariculture to date. In relation to animal feed, as with
human feed, algae have been used to enhance the nutritional content of conven-
tional feed preparations (Spolaore et al. 2006). Recognition as a source of fatty
acids such as polyunsaturated fatty acid family (o3) and sterols, has increased
their consumption in health-orientated diets (Cardozo et al. 2007). The protein
and amino acid nutritional attributes of algae have been reviewed elsewhere by
Fleurence (1999) and MacArtain et al. (2007).
9.2.1.1 Polyunsaturated Fatty Acids
Human beings and higher plants lack the required enzymes to synthesize long o3
polyunsaturated fatty acids; therefore, they need to obtain them from external
dietary sources. Several o3 polyunsaturated fatty acids including eicosapentaenoic
acid (EPA), an important dietary supplement (Yokoyama et al. 2007; Doughman
et al. 2007), have been identified in a number of algae species. Although a number
of algae species are cultivated as natural sources of these fatty acids, only a few
species have demonstrated the potential to be capable of industrial scale production
because of low growth rates and low cell number in culture (Wen and Chen 2003).
The application of transgenic algae may act as an alternative, like transgenic oilseed
crops, providing an alternative sustainable source of these essential oils for human
consumption (Abbadi et al. 2001; Doughman et al. 2007).
9.2.1.2 Sterols
Sterols are one of the most important chemical constituents of algae and a major
nutritional component in the diet of aquacultured organisms. Microalgae are an
important component in the diet of many hydrobionts, such as bivalves (Ponomarenko
et al. 2004). The ability of bivalves to synthesize or bioconvert sterols de novo
varies among different species, but is generally low and sometimes completely
absent. This implies that a dietary supply of sterol is necessary for bivalve growth
(Soudant et al. 1998). The type of algae used as a food source determines the quality
and sterol composition; seasonal shifts also occur. Therefore, this is used as a
criterion for species selection for the culture of bivalves (Park et al. 2002). Plant
and algae sterols have been shown to reduce cholesterol by blocking absorption,
resulting in reduced quantities of cholesterol reaching the liver (Plat and Mensink
2005; Charest et al. 2004). Despite the ability of these sterols to block cholesterol
absorption, the human intestine poorly absorbs them (Cater and Grundy 1998).
9.2.1.3 Carotenoids
The natural pigments, carotenoids, are produced in bacteria, algae and plants
(Polı́vka and Sundström 2004). These carotenoids have important biological func-
tional roles including optimal photosynthesis and indeed protection from potential
damage arising from UV light exposure. To date, over 600 different carotenoids
exercising important biological functions in bacteria, algae, plants and animals
have been identified (Polı́vka and Sundström 2004). Animals lack the ability to
synthesize carotenoids endogenously and thus obtain these compounds by nutri-
tional intake. For human nutritional purposes, a number of carotenoids offer
provitamin A activity (Mayne 1996). Vitamin A deficiency is a major health risk
that has surfaced in the developing countries as the leading cause of preventable
blindness in children and also leads to the increased risk of disease and death from
severe infections (WHO, Micronutrient deficiencies: http://www.who.int/nutrition/
topics/vad/en/: accessed July 20 2009). They are also biological antioxidants,
protecting cells and tissues from free radicals and singlet oxygen. Carotenoids are
utilized in pharmaceuticals, dietary supplements, cosmetics, and as food additives.
Dunaliella salina and Spirulina maxima have been utilized for cartenoid astax-
anthin, a red-orange pigment used widely in the food industry (Meyers and Latscha
1997). The green algae Haematococcus pluvialis has been the focus of biotechnology
companies for the commercial development of this carotenoid (Hussein et al. 2006).
The potent antioxidant property of astaxanthin has been implicated in its various
biological activities demonstrated in both experimental animals and clinical studies,
with potential beneficial roles in human health (Hussein et al. 2006).A second group, the
phycobiliproteins, consists of proteins with covalently bound phycobilins. Phycobili-
proteins, primarily composed of a- and b-polypeptides, are a brilliantly colored group
of disc-shaped proteins (Samsonoff and MacColl 2001; Liu et al. 2005). These have
been implemented within laboratories as labels for biomolecules (Spolaore et al. 2006).
9.2.1.4 Polysaccharides
Phycocolloids are unique polysaccharides produced by several seaweed species.

Carrageenan and agar are sulfated polysaccharides extracted from some Rhodophy-
ceae. The principal feature distinguishing the highly sulfated carrageenans from
the less-sulfated agars is the presence of anhydro-d-galactose in the former and
l-galactose or anhydro-l-galactose in the latter (Craigie 1990). Agar and carra-
geenan are extracted from red algae, while alginates are extracted primarily from
brown algae. Phycocolloids are used in a wide range of products including foods,
pharmaceutics and medical devices (Walker et al. 2005).
Carrageenan, extracted from red algae is subdivided into kappa carrageenan, iota
carrageenan and lambda carrageenan, each of which has different characteristics.
They are used as gelling agents, stabilizers, texturants, thickeners, and viscosifiers
for a wide range of food products (Cardozo et al. 2007). Alginates, the salts of
alginic acid and their derivatives, are extracted from the cell walls of brown
macroalgae. These carboxylated polysaccharides are used for a wide variety of
applications within the food industry. Alginates are required for production of dyes.
The water absorbing properties of alginates are utilized in slimming aids and in the
production of textiles and paper. Calcium alginate has been used in the production
of medical products, including burn dressings (Cardozo et al. 2007). Owing to its
biocompatibility and simple gelation with divalent cations, it is also used for cell
immobilization and encapsulation. Additionally, alginates are widely used in pros-
thetics and for dental molds.
Use within the food sector and as a source of phycocolloids has lead to a need
for cultivation for sufficient global demand, with current estimates of global
production greater than 6.5 million tons in terms of fresh weight (McHugh 2003).
Such large-scale production places this in a similar economic and global impor-
tance as land crops (McHugh 2003), with abilities to fix high volumes of carbon
dioxide, absorb and consume eutrophied nitrogen and phosphorus transforming
them into large amounts of seaweed biomass. In doing so, they serve to protect the
marine environment.
9.2.1.5 Lectins
Lectins or agglutinins, defined as proteins that bind to carbohydrates without

initiating any further modifications (Weis and Drickamer 1996), have been found
widely in all organisms. They are involved in numerous biological processes
including host–pathogen interactions, cell–cell communication, cancer, metastasis,
differentiation and immune regulation. The presence of lectins in marine algae was
first reported by Boyd and colleagues in 1966 (Boyd et al. 1966). Marine algal
lectins differ from higher plant lectins (agglutinins) typically in exhibiting (1) lower
molecular mass ranging from 4,000 to 25,000 Da, (2) occurrence mainly in monomer
form, and (3) independence from divalent cations to maintain their structure
(Rogers and Hori 1993). The applications for algal lectins are at an early stage of
development, though many have emerged as potential therapeutic agents deserving

further elucidation.
To date, a number of lectins have been discovered in algae, which demonstrate
antiviral activity, among them are cyanovirin-N (CV-N), scytovirn (SVN) and
Microcystis viridis lectin (MVL) (Ziółkowska and Wlodawer 2006). Antiviral
properties of lectins is due to their interaction with glycans, which in turn disturbs
the interactions between proteins of the viral envelope and the cells of the host
(Botos and Wlodawer 2005; Balzarini 2006). As a high proportion of current
antiviral therapeutics act through inhibition of the viral life cycle, lectins can
prevent viral penetration of host cells. Algae lectins with anti-HIV activity have
been reviewed previously (Ziółkowska and Wlodawer 2006).
Seaweeds are rarely promoted for the nutritional value of their proteins. Their
protein contents differ according to the species and seasonal conditions. To date,
little information is available on the nutritional value of algae proteins and on the
compounds that affect their digestibility.
9.2.1.6 Antibacterial and Antiviral Properties
A high number of marine algae produce antibiotic and antiviral substances capable
of inhibiting bacteria, viruses, and fungi. The antimicrobial activity of aquatic
microalgae was first reported for Chorella vulgaris in the 1940’s (Pratt and Fong
1940, 1944). This concept was strengthened with growing literature and reports of
the antiviral properties of polysaccharides from marine algae towards mumps virus
and influenza B virus in the 1960s. The antibiotic/antiviral properties are dependent
on factors including the species of algae, the agent in question, the season, and the
growth conditions (Pesando and Caram 1984; Centeno and Ballantine 1999).
The antibacterial activity of marine algae has generally been assayed using
extracts in various organic solvents (Liao et al. 2003). A number of these chemicals
are toxic to microorganisms and therefore may be responsible for the antibiotic
activity reported (Ohta 1979). However, this does not reflect the antibacterial
activity of marine algae under natural conditions. Earlier investigations have
demonstrated the effects of the release of phenolics as antifouling substances, the
release of organic substances, which hold both an inhibitory effect on growth of
adjacent diatoms or a stimulatory effect depending on source (Liao et al. 2003).
Polysaccharide fractions from red algae were found to inhibit a number of viruses
including herpes simplex virus (HSV). At that time, these findings did not generate
much interest because the antiviral action of the compounds was considered to be
largely nonspecific (Witvrouw and De Clercq 1997). Isolation from algae of poly-
saccharides and sulphated polysaccharides and other compounds with antiviral
activity against enveloped viruses increased the interest in algae as a source of
antiviral compounds (Schaeffer and Krylov 2000). Enveloped viruses include HIV,
HSV type 1 and HSV type 2, influenza A virus, RSV, simian immunodeficiency
virus (SIV), pseudorabies virus, bovine herpes virus, and human cytomegalovirus
(HCMV) (Schaeffer and Krylov 2000; Ziółkowska and Wlodawer 2006).
Anticancer Agents
Brown algae grown in the Black Sea, among other global regions such as Japanese
coastline, have been demonstrated to be potential sources of antitumor agents
(Apryshko et al. 2005). Carotenoid fucoxantine (Fx) extracted from these algae
possess antitumor properties, which have been tested using prostate cancer per-
formed in Russia. During these studies, patients typically received dried brown
algae Laminaria daily. Dosage of Fx was estimated to be 10–15 mg daily. During
treatment, there was gradual improvement of general state and blood indices.
Disease course became stabilized and survival rate increased. Similarly, the rate
of breast cancer is greatly reduced in populations consuming brown algae
(Apryshko et al. 2005).
Anti-HIV Activity
Most of the research on the anti-HIV activity of marine algae has focused upon red
and brown macroalgae (Schaeffer and Krylov 2000). The initial studies using these
algae isolated sulfated polysaccharides with antiviral activity and later investigators
continued interest in this class of compounds. However, other classes of compounds
with anti-HIV activity have been identified including polysaccharides, fucoidan and
carrageenans (Schaeffer and Krylov 2000).
A number of natural polysulfates isolated from algae and synthetic polysulfates
exhibit differential inhibitory activity against different HIV strains, which suggests
differences in the target molecules with which these compounds interact (Witvrouw
and De Clercq 1997; Table 9.1). They inhibit the cytopathic effect of HIV and also
prevent HIV-induced syncytium formation (Ziółkowska et al. 2006). Antiviral
activity increases with increasing molecular weight and degree of sulfation (Witv-
rouw and De Clercq 1997).
Anti-HIV polysaccharides and polyphenols have been isolated from brown algae,
Fucus vesiculosus, inhibition of both HIV-induced syncytium and HIV reverse
transcriptase (RT) activity at nontoxic levels (Béress et al. 1993). The mechanism
of this effect remains to be further elucidated. A sulphated polysaccharide isolated
Table 9.1 Anti-HIV activity Lectin EC50 (nM)

of algal lectins Jacalin >227
Myrianthus holstii lectin 150
Urtica diocia agglutinin 105
Concanavalin A 98
N. pseudonarcissus lectin 96
P. tetragonolobus lectin 52
MVL* 30 (IC50 used)
SVN 0.3
CV-N 0.1
GRFT 0.04
Data from Ziółkowska et al. (2006)
*IC50 rather than EC50 reported
from S. horneri demonstrated potent antiviral activity against HSV-I, cytomegalo-

virus and HIV. Other anti-HIV polysaccharides have been found in Agardhiella
tenera, Cochlodinium polykrikoides, and Nothogenia fastigiata. Carrageenans and
their cyclized derivatives isolated from G. skottsbergii are potent inhibitors of herpes
viruses, and inhibit HIV to a lesser extent.
A number of cyanobacteria (blue-green algae) species have been found to
produce highly anti-HIV active sulfoglycolipids. In addition to their activity, the
sulfoglycolipids constitute part of the chloroplast membrane and are therefore
abundant. Their abundance and accessibility should be highlighted for potential
future development. Extracts of cultured marine cyanobacteria, Lyngbya lagerheimii
and Phormidium tenue, have been screened for anti-HIV compounds leading to
the discovery of sulfonic acid-containing glycolipids as a novel class of HIV-1-
inhibitory compounds (Gustafson et al. 1989). A number of cyanobacteria extracts
have been screened for antiviral compounds including Phormidium cebennse,
Oscillatoria raciborskii, Scytonema burmanicum, Calothrix elenkinii, and Ana-
baena variabilis. Compounds in all were found to be anti-HIV and extracts found
to contain sulfolipids. Cyanobacteria antiviral polysaccharides have also been
demonstrated.
9.3 Application of Transgenic Algae
The ease of growth, biomass content and low cost of production of algae make them
immensely attractive for both pharmaceutical and therapeutic compound discovery
and for recombinant engineering (Fig. 9.2). In the absence of cell differentiation,
algae would provide a much simpler system for genetic manipulations compared
with higher plants. Manipulation of algae by metabolic and genetic methods would
both permit (1) selection of beneficial pathways redirecting cellular function toward
the synthesis of preferred products and (2) introduction of non-algae genes for the
generation of algal recombinant protein. The selection of favorable pathways may
include increased resistance to environmental or stress changes on the culturing/life
cycle of the algae. The potential of this system remains to be optimized as an
alternative protein expression system.
9.3.1 Transformation and Engineering
Reports of plant-produced recombinant proteins began to be published in the late

1980s when antibodies were produced in tobacco (Hiatt et al. 1989) and human
serum albumin was expressed in tobacco and potato (Sijmons et al. 1990). Genetic
transformation of unicellular algae began in the 1970s (Shestakov and Khuyen
1970) with cyanobacteria, followed by in the 1980s with the work in marine algae
(Stevens and Parter 1980). Transformation of algae is still an area of research in its
Potential Resources Oxygen Environmental and

Produced Culture Conditions
Recombinant proteins
Environmental Conditions
Hydrogen
Light, Water, CO2
BioDiesel
Lipids
Photosynthesis
Nutraceuticals
Calvin
Cycle Transgenic Engineering
Ethanol Carbohydrates
Nutrition,
Carotenoids, Biomass
Aquaculture
Nucleus
Organic compounds
Gasoline Nutrients
Fig. 9.2 Downstream potential for algae production, applications and uses
infancy. With continuous expansion of genome information, the application of algal

models for the production of proteins will become an attractive alternative to the
existing systems of recombinant protein expression.
Recombinant engineering technology has evolved globally into an industry
sector encompassing food, agriculture, pharmaceutics, biofuel (see sect. 6.1.3),
and environmental areas. Recombinant production of proteins now produces a
myriad of protein-based industrial and biopharmaceutical products in crop plants,
aquatic plants and algae (Giddings et al. 2000). Stability of transformation and
efficiency have, to date, been the key determinants upon experimental success.
These are relative to the size and complexity of the algae used in each case
(Hallmann 2007). Transformation has been focused on transient expression of
reported genes, typically antibiotic resistance genes as these confer traits regardless
of genotype (Qin et al. 2005; Hallmann 2007).
9.3.1.1 Status of Alga Genome Information
For the development and application of engineered algae for scale-up of endoge-
nous molecules and for their application as a recombinant tool for protein produc-
tion, highly annotated genome data are required. Complete genome annotation and
sequenced expressed sequence tag (EST) mapping is currently available for a
number of species. Like other genome projects, data increase almost exponentially.
Sequencing and annotation of the 16.5 Mb Cyanidioschyzon merolae genome
(Matsuzaki et al. 2004), the 34 Mb Thalassiosira pseudonana genome (Armbrust

et al. 2004) and the ~120 Mb genome of Chlamydomonas reinhardtii has been
completed (Merchant et al. 2003). Genomes of over 50 species are all currently
undergoing annotation or nearing sequencing completion, 30 of which are genomes
of cyanobacteria (NCBI, Entrez Genome Project: www.ncbi.nlm.nih.gov/sites/
entrez: accessed 20 July 2009). Owing to their small-sized genomes, completed
genome information is being produced at a faster rate for blue-green algae.
9.3.1.2 Current Status of Algae Engineering
The genetic engineering and modification of algae is an area, which has received a
lot of attention over the last few years (León-Bañares et al. 2004; Walker et al.
2005). Reports detailing the introduction of DNA into the diatom Phaeodactylum,
the green algae Chlamydomonas, and the blue-green algae Synechococcus and
Synechocystis have been circulated (Raja et al. 2008). Genetic engineering of the
expression of mosquito larvicidal properties in blue-green algae has also been
reported (Boussiba et al. 2000). However, at the time of publication there has not
been a report on the commercial use of any transgenic algae for the application of a
functional transformation system. Literature supports the development of such
methodologies demonstrated with the manipulation of diatoms to enhance lipid
production (Dunahay 1996), the expression of a functional glucose transporter in
the obligate phototrophic Phaeodactylum enabling this diatom to grow on glucose
in the dark (Zaslavskaia et al. 2001) and the advancements using genetically
modified strains of Chlamydomonas for hydrogen production as an alternative
biofuel (Melis et al. 2000). Chlamydomonas is particularly relevant as a model
algae system for genetic manipulation and is detailed further below. The potential
of algae to be genetically modified, permitting the synthesis of recombinant pro-
teins, opens up alternatives to the current recombinant systems, and presents a
simpler model with respect to minimal or removed system contaminants for the use
in expression of human antibody and therapeutic proteins.
9.3.1.3 Maintenance in Bioreactors
The ability to culture single-celled plants and aquatic plants in bioreactors offers
two advantages over the use of terrestrial plants: (1) the growth conditions can be
controlled precisely, insuring optimal growth conditions and batch-to-batch product
reproducibility (yield, activity); and (2) growth in bioreactors is contained in-house,
thus removing environmental biosafety issues associated with “release” of trans-
genic terrestrial plants.
With respect to their high protein levels and their amino acid composition the red
seaweed appear to be an interesting potential source of food proteins. With large
scale production in bioreactors possible, this is a developing area of research and
industry. Algae may represent functional foods, which remain to be utilized. Use
of algae, as a food source for mariculture permits the delivery of target genes,
influencing both the organism feeding and also downstream consumers. Such an
approach to molecular pharming would permit edible therapeutics and health
management. The amino acid content is of nutritional value, however, their protein
digestibility in vivo remains to be completely elucidated.
9.3.2 Models of Alga Engineering
9.3.2.1 Laminaria japonica
The brown algae, Laminaria japonica, commonly referred to as Kelp is a cultivated

seaweed, which has been utilized as both a foodstuff and as a raw material for
iodine, mannitol and alginate production (Tseng and Qin 1999). The production of
L. japonica is a low-cost, high-yield crop in China. Extensive research has been
carried out for the breeding and strain development for the establishment of
improved traits. Transformation of L. japonica has been hindered by a lack of
knowledge in relation to kelp viruses or symbiotic bacteria, which could be used for
direct gene transfer. Therefore, until this point particle bombardment and ultra-
sound have been the transformation methodologies applied. Research on a virus
(ESV-1) infecting Ectocarpus, a filamentous brown algae found as epiphytic to kelp
has been performed, including viral genome annotation (Delaroque et al. 2001).
This virus infects unicellular spores or gamates, transmitting its genome by mitosis
to all cells of the growing host plant (Müller et al. 1998). The transmission of its
genome presents this virus as a potential vector for gene delivery. As no usable
promoter from kelp has yet been identified, a number of promoter regions from
terrestrial plants, unicellular algae and algal viruses have been introduced without
much success. Stable expression has been shown for transforants using two pro-
moters, fcp (diatom fucoxanthin-chorophyll a/c binding protein gene) and the SV40
(simian virus) (Qin et al. 2005). The potential of L. japonica in the production of
recombinant protein was further demonstrated by the introduction of the vaccine
gene encoding human hepatitis B surface antigen gene (HBsAg) (Qin et al. 2005).
Expression levels in transgenic kelp were compared to that of transgenic tobacco.
Comparable levels of protein were expressed from both systems (Qin et al. 2005),
demonstrating the potential for edible/direct delivery of proteins.
9.3.2.2 Chlamydomonas Reinhardtii
The green algae, Chlamydomonas reinhardtii, has been utilized as a model organ-
ism in the study of photosynthesis and light-regulated gene expression. Recently, it
has been explored as a potential host for recombinant protein synthesis. Production
of several forms of human IgA antibody directed against glycoprotein D and HSV
have been reported to date in C. reinhardtii (Mayfield et al. 2003; Franklin and
Mayfield 2005). The production of these monoclonal antibodies in algae demon-

strated that the production is both possible and is comparable to terrestrial plant
production. Algae offers several advantages over terrestrial plants including: (1)
transgenic forms can be generated quickly (only weeks between generation of
initial transformants and their scale-up for production); (2) both the chloroplast
and nuclear genome of algae can be genetically transformed by standard methodol-
ogies, i.e. microprojectile particle bombardment or electroporation, providing
scope for the production of several different proteins simultaneously; and (3) the
ability to culture volumes ranging from a few milliliters to 500,000 liters makes
such an expression system advantageous at an industrial level (Franklin and
Mayfield 2004).
9.4 Summary
The demand for improved systems of production of nutraceuticals and cost-effective

protein expression systems (both industrial and pharmaceutical applications) lend
themselves to the potential useful capacities of algae. Currently, recombinant
expression of proteins is performed in both mammalian and nonmammalian systems
routinely. As a system, algae provide a simpler model than plants. Limitations of
manufacturing are a bottleneck limited on size, cost, time and levels of purification,
especially with reference to mammalian recombinant proteins. The progress being
made in relation to sequencing of algal genomes will permit the cloning and
manipulation of genes and allow “omics” technologies to be applied. This advance-
ment will aid in the identification of key regulators of metabolism and enable the
eventual manipulation of cellular pathways.
Such advances in transformation techniques will permit in future more sophisti-
cated forms of recombinant engineering to be performed. Ultimately, algal biotech-
nology offers the potential to have impact on the advancement of recombinant
technologies. This is only the beginning of this field of research and much remains
to be achieved to optimize the full potential of algae.
References
Abbadi A, Domergue F, Meyer A, Riedel R, Sperling P, Zank TK, Heinz E (2001) Transgenic
oilseeds as sustainable source of nutritionally relevant C20 and C22 polyunsaturated fatty
acids? Eur J Lipid Sci Technol 103:106–113
Apryshko GN, Ivanov VN, Milchakova NA, Nekhoroshev MV (2005) Mediterranean and Black
Sea organisms and algae from mariculture as sources of antitumor drugs. Exp Oncol 27:94–95
Armbrust EV, Berges JA, Bowler C, Green BR, Martinez D, Putnam NH, Zhou S, Allen AE,
Apt KE, Bechner M, Brzezinski MA, Chaal BK, Chiovitti A, Davis AK, Demarest MS,
Detter JC, Glavina T, Goodstein D, Hadi MZ, Hellsten U, Hildebrand M, Jenkins BD,
Jurka J, Kapitonov VV, Kröger N, Lau WW, Lane TW, Larimer FW, Lippmeier JC,
Lucas S, Medina M, Montsant A, Obornik M, Parker MS, Palenik B, Pazour GJ,

Richardson PM, Rynearson TA, Saito MA, Schwartz DC, Thamatrakoln K, Valentin K,
Vardi A, Wilkerson FP, Rokhsar DS (2004) The genome of the diatom Thalassiosira pseudo-
nana: ecology, evolution, and metabolism. Science 306:79–86
Balzarini J (2006) Inhibition of HIV entry by carbohydrate-binding proteins. Antiviral Res
71:237–247
Béress A, Wassermann O, Tahhan S, Bruhn T, Béress L, Kraiselburd EN, Gonzalez LV, de Motta
GE, Chavez PI (1993) A new procedure for the isolation of anti-HIV compounds (polysacchar-
ides and polyphenols) from the marine algae Fucus vesiculosus. J Nat Prod 56:478–488
Botos I, Wlodawer A (2005) Proteins that bind high-mannose sugars of the HIV envelope. Progr
Biophys Mol Biol 88:233–282
Boyd WC, Almodovar IR, Boyd IG (1966) Agglutinins in marine algae for human erythrocytes.
Transfusion 6:82–83
Cardozo KH, Guaratini T, Barros MP, Falcão VR, Tonon AP, Lopes NP, Campos S, Torres MA,
Souza AO, Colepicolo P, Pinto E (2007) Metabolites from algae with economical impact.
Comp Biochem Physiol C Toxicol Pharmacol 146:60–78
Cater NB, Grundy SM (1998) Lowering serum cholesterol with plant sterols and stanols: historical
perspectives. J Postgrad Med, 6-14
Centeno POR, Ballantine DL (1999) Effects of culture conditions on production of antibiotically
active metabolites by the marine algae Spyridia filamentosa. I. Light. J Appl Phycol 11:217–
224
Charest A, Desroches S, Vanstone CA, Jones PJH, Lamarche B (2004) Unesterified plant sterols
and stanols do not affect LDL electrophoretic characteristics in hypercholesterolemic subjects.
J Nutr 134:592–595
Craigie JS (1990) Cell Walls. In: Cole KM, Sheath RG (eds) Biology of the Red Algae. Cambridge
Univ Press, Cambridge, UK, pp 226–236
Delaroque N, Müller DG, Bothe G, Pohl T, Knippers R, Boland W (2001) The complete DNA
sequence of the Ectocarpus siliculosus Virus EsV-1 genome. Virology 287:112–132
Doughman SD, Krupanidhi S, Sanjeevi CB (2007) Omega-3 fatty acids for nutrition and medi-
cine: considering microalgae oil as a vegetarian source of EPA and DHA. Curr Diabet Rev
3(3):198–203
Dunahay TG (1996) Manipulation of microalgal lipid production using genetic engineering. Appl
Biochem Biotechnol 57:223–231
Fleurence J (1999) Seaweed proteins: biochemical, nutritional aspects and potential uses. Trends
Food Sci Technol 10:25–28
Fogg GE (1953) The Metabolism of Algae. Methuen, London, UK
Franklin SE, Mayfield SP (2004) Prospects for molecular farming in the green algae Chlamydo-
monas. Curr Opin Plant Biol 7:159–165
Franklin SE, Mayfield SP (2005) Recent developments in the production of human therapeutic
proteins in eukaryotic algae. Expert Opin Biol Ther 5:225–235
Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharma-
Gustafson KR, Cardellina JH, Fuller RW, Weislow OS, Kiser RF, Snader KM, Patterson GM,
Boyd MR (1989). AIDS: antiviral sulfolipids from cyanobacteria. J. Natl. Cancer Inst.
81:1254–1258
Hallmann A (2007) Algal transgenics and biotechnology. Transgen Plant J 1:81–98
Hiatt A, Cafferkey R, Bowdish K (1989) Production of antibodies in transgenic plants. Nature
342:76–78
Hussein G, Sankawa U, Goto H, Matsumoto K, Watanabe H (2006) Astaxanthin, a carotenoid with
potential in human health and nutrition. J Nat Prod 69:443–449
León-Bañares R, González-Ballester D, Galván A, Fernández E (2004) Transgenic microalgae as
green cell-factories. Trends Biotechnol 22:45–52
Liao WR, Lin JY, Shieh WY, Jeng WL, Huang R (2003) Antibiotic activity of lectins from marine
algae against marine vibrios. J Indust Microbiol Biotechnol 30:433–439
Liu LN, Chen XL, Zhang YZ, Zhou BC (2005) Characterization, structure and function of linker
polypeptides in phycobilisomes of cyanobacteria and red algae: an overview. Biochim Biophys
Acta 1708:133–142
MacArtain P, Gill CI, Brooks M, Campbell R, Rowland IR (2007) Nutritional value of edible
seaweeds. Nutr Rev 65:535–543
Matsuzaki M, Misumi O, Shin IT, Maruyama S, Takahara M, Miyagishima SY, Mori T, Nishida K,
Yagisawa F, Nishida K, Yoshida Y, Nishimura Y, Nakao S, Kobayashi T, Momoyama Y,
Higashiyama T, Minoda A, Sano M, Nomoto H, Oishi K, Hayashi H, Ohta F, Nishizaka S,
Haga S, Miura S, Morishita T, Kabeya Y, Terasawa K, Suzuki Y, Ishii Y, Asakawa S, Takano H,
Ohta N, Kuroiwa H, Tanaka K, Shimizu N, Sugano S, Sato N, Nozaki H, Ogasawara N, Kohara Y,
Kuroiwa T (2004) Genome sequence of the ultra small unicellular red algae Cyanidioschyzon
merolae 10D. Nature 428:653–657
Mayfield SP, Franklin SE, Lerner RA (2003) Expression and assembly of a fully active antibody in
algae. Proc Natl Acad Sci USA 100:438–442
Mayne ST (1996) Beta-carotene, carotenoids, and disease prevention in humans. FASEB J
10:690–701
McHugh DJ (2003) A guide to the seaweed industry. FAO Fish Tech Pap 441:1–105
Melis A, Zhang L, Forestier M, Ghirardi ML, Seibert M (2000) Sustained photobiological
hydrogen gas production upon revers ible inactivation of oxygen evolution in the green algae
Chlamydomonas reinhardtii. Plant Physiol 122:127–136
Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A,
Fritz-Laylin LK, Maréchal-Drouard L, Marshall WF, Qu LH, Nelson DR, Sanderfoot AA,
Spalding MH, Kapitonov VV, Ren Q, Ferris P, Lindquist E, Shapiro H, Lucas SM,
Grimwood J, Schmutz J, Cardol P, Cerutti H, Chanfreau G, Chen CL, Cognat V, Croft MT,
Dent R, Dutcher S, Fernández E, Fukuzawa H, González-Ballester D, González-Halphen D,
Hallmann A, Hanikenne M, Hippler M, Inwood W, Jabbari K, Kalanon M, Kuras R,
Lefebvre PA, Lemaire SD, Lobanov AV, Lohr M, Manuell A, Meier I, Mets L, Mittag M,
Mittelmeier T, Moroney JV, Moseley J, Napoli C, Nedelcu AM, Niyogi K, Novoselov SV,
Paulsen IT, Pazour G, Purton S, Ral JP, Riaño-Pachón DM, Riekhof W, Rymarquis L,
Schroda M, Stern D, Umen J, Willows R, Wilson N, Zimmer SL, Allmer J, Balk J,
Bisova K, Chen CJ, Elias M, Gendler K, Hauser C, Lamb MR, Ledford H, Long JC,
Minagawa J, Page MD, Pan J, Pootakham W, Roje S, Rose A, Stahlberg E, Terauchi AM,
Yang P, Ball S, Bowler C, Dieckmann CL, Gladyshev VN, Green P, Jorgensen R, Mayfield S,
Mueller-Roeber B, Rajamani S, Sayre RT, Brokstein P, Dubchak I, Goodstein D, Hornick L,
Huang YW, Jhaveri J, Luo Y, Martı́nez D, Ngau WC, Otillar B, Poliakov A, Porter A,
Szajkowski L, Werner G, Zhou K, Grigoriev IV, Rokhsar DS, Grossman AR (2003) The
Chlamydomonas genome reveals the evolution of key animal and plant functions. Science
318:245–250
Meyers SP, Latscha T (1997) Carotenoids. In: D’Abramo LR, Conklin DE, Akiyama DM (Eds.),
Crustacean Nutrition, Advances in World Aquaculture, Vol. 6. World Aquaculture Society,
Baton Rouge, LA, pp. 164–193
Müller DG, Kapp M, Knippers R (1998) Viruses in marine brown algae. Adv Virus Res 50:49–67
Ohta K (1979) Chemical studies on biologically active substances in seaweeds. Proc Int Seaweed
Symp 9:401–411
Park DW, Jo Q, Lim HJ, Veron B (2002) Sterol composition of darkgrown Isochrysis galbana and
its implication in the seed production of pacific oyster, Crassostrea gigas. J Appl Phycol
14:351–355
Pesando D, Caram B (1984) Screening of marine algae from the French Mediterranean coast for
antibacterial and antifungal activity. Bot Mar 27:381–386
Plat J, Mensink RP (2005) Plant stanol and sterol esters in the control of blood cholesterol levels:
mechanism and safety aspects. Am J Cardiol 96:15D–22D
Polı́vka T, Sundström V (2004) Ultrafast dynamics of carotenoid excited States-from solution to

natural and artificial systems. Chem Rev 104:2021–2071
Ponomarenko LP, Stonik IV, Aizdaicher NA, Orlova TY, Popvskaya GI, Pomazkina GV, Stonik
VA (2004) Sterols of marine microalgae Pyramimonas cf. cordata (prasinophyta), Atteya
ussurensis sp. nov. (Bacollariophyta) and a spring diatom bloom form Lake Baikal. Comp
Biochem Physiol (B) 138:65–70
Pratt R, Fong J (1940) Studies on Chorella vulgaris. Am J Bot 27:431–436
Pratt R, Fong J (1944) Chlorellin, an antibacterial substance from Chorella. Science 99:351–352
Qin S, Jiang P, Tseng C (2005) Transforming kelp into a marine bioreactor. Trends Biotechnol
23:264–268
Raja R, Hemaiswarya S, Kumar N Ashok, Sridhar S, Rengasamy R (2008) A Perspective on the
Biotechnological Potential of Microalgae. Critical Reviews in Microbiology 34:77–88
Rogers DJ, Hori K (1993) Marine algal lectins: new developments. Hydrobiologia 260:589–593
Samsonoff WA, MacColl R (2001) Biliproteins and phycobilisomes from cyanobacteria and red
algae at the extremes of habitat. Arch Microbiol 176:400–405
Schaeffer DJ, Krylov VS (2000) Anti-HIV activity of extracts and compounds from algae and
cyanobacteria. Ecotoxicol Environ Saf 45:208–227
Shestakov SV, Khuyen NT (1970) Evidence for genetic transformation in blue-green algae
Anacystis nidulans. Mol Gen Genet 107:372–375
Sijmons PC, Dekker BM, Schrammeijer B, Verwoerd TC, van den Elzen PJ, Hoekema A (1990)
Production of correctly processed human serum albumin in transgenic plants. Biotechnology
8:217–221
Soudant P, Coz JR, Marty Y, Moal J, Robert R, Samain JF (1998) Incorporation of microalgae
sterols by scallop Pecten maximus (L.) larvae. Comp Biochem Physiol (A) 119:451–457
Spolaore P, Joannis-Cassan C, Duran E, Isambert A (2006) Commercial applications of micro-
algae. J Biosci Bioeng 101:87–96
Stevens SE, Parter RD (1980) Transformation in Agmenellum quadruplicatum. Proc Natl Acad Sci
USA 77:6052–6056
Tseng CK, Qin S (1999) Mariculture and genetic transformation of Laminaria japonica in China.
In: Xu HS, Colwell RR (eds) Proc Int Symp on Progress and Prospect of Marine Biotechnol.
China, Ocean Press, pp 11–16
Walker TL, Purton S, Becker DK, Collet C (2005) Microalgae as bioreactors. Plant Cell Rep
24:629–641
Weis WI, Drickamer K (1996) Structural basis of lectin-carbohydrate recognition. Annu Rev
Biochem 65:441–473
Wen ZY, Chen F (2003) Heterotrophic production of eicosapentaenoic acid by microalgae.
Biotechnol Adv 21:273–294
Witvrouw M, De Clercq E (1997) Sulfated polysaccharides extracted from sea algae as potential
antiviral drugs. Gen Pharmacol 29:497–511
Yokoyama M, Origasa H, Matsuzaki M, Matsuzawa Y, Saito Y, Ishikawa Y, Oikawa S, Sasaki J,
Hishida H, Itakura H, Kita T, Kitabatake A, Nakaya N, Sakata T, Shimada K, Shirato K, Japan
EPA lipid intervention study (JELIS) Investigators (2007) Effects of eicosapentaenoic acid on
major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label,
blinded end point analysis. Lancet 369:1090–1098
Zaslavskaia LA, Lippmeier JC, Shih C, Erhardt D, Grossman AR, Apt K (2001) Trophic conver-
sion of an obligate photoautotrophic organism through metabolic engineering. Science
292:2073–2075
Ziółkowska NE, Wlodawer A (2006) Structural studies of algal lectins with anti-HIV activity.
Acta Biochim Pol 53:617–626
Ziółkowska NE, O’Keefe BR, Mori T, Zhu C, Giomarelli B, Vojdani F, Palmer KE, McMahon JB,
Wlodawer A (2006) Domain-swapped structure of the potent antiviral protein griffiths in and
its mode of carbohydrate binding. Structure 7:1127–1135
Chapter 10
Biotech Crops and Functional Genomics
Narayana M. Upadhyaya, Andy Pereira, and John M. Watson
10.1 Introduction
The increase in human population, poor performance of crop cultivars under

increasingly adverse environmental conditions and a decline in the available land
for sustainable crop production are contributing to a shortage of global food supply
and increase in its demand. Conventional breeding efforts in crops such as rice over
the last three decades have resulted in a doubling of agricultural productivity
(Khush 1997). However, for sustained increase in the agricultural productivity,
crops which can resist pests, pathogens and tolerate salinity, drought and tempera-
ture extremes need to be developed and deployed.
The deployment of a handful of gene classes developed as transgenes for insect
and/or herbicide resistance in crops such as maize, soybean, canola, cotton, squash,
papaya, alfalfa, and sugar beet reduces yield losses and increases agricultural
profitability. However, commercial application of transgenes conferring more
complex traits such as abiotic stress tolerance, yield, vigor and nutritional quality
are yet to be achieved because of the lack of adequate knowledge of the critical
genes controlling such traits and the complexity of their behavior under different
environmental conditions. Long development times from discovery to commercial
release, intellectual property constraints, high development costs, and regulatory
constraints and costs are also slowing down the process of commercial deployment
(Birch 2000). With continual improvements in transgene delivery, integration and
expression modulation, several other transgenes of agronomic importance are being
deployed and tested for efficacy in various crop plants. Major crops such as rice,
wheat, barley and sugarcane are bound to become biotech crops as more and more
novel genes, with high potential to be deployed as transgenes, are discovered, and
N.M. Upadhyaya (*)

CSIRO Plant Industry, GPO Box 1600, Canberra ACT 260, Australia
e-mail: Narayana.upadhyaya@csiro.au

360 N.M. Upadhyaya et al.
the concerns over the biosafety of their large-scale deployment are addressed
scientifically and politically.
Mapping and DNA sequencing of plant genomes and analysis of the information
content present in genomic sequences commonly termed as “genomics” have the
potential to provide valuable insight into genes controlling some of the complex
traits mentioned above. Various genome-wide high-throughput “functional geno-
mics” tools and resources are being developed worldwide. The ultimate aim of
these approaches is to define the structures of all genes, and the functions of all gene
products, as well as all the processes that occur during plant growth and develop-
ment. Such knowledge could be used effectively, not only in molecular marker-
assisted breeding, but also in transgenic breeding. Arabidopsis, a model dicot, and
rice, a model cereal, have emerged as front-runners with near-complete sequencing
of the Arabidopsis ecotype Columbia (TAGI 2000) and two rice genotypes, namely,
japonica cv. Nipponbare (IRGSP 2005) and indica cv. 93-11 (Yu et al. 2002, 2005).
Genome sequencing efforts are now underway for several other food grain and
tuber crops (barley, cassava, maize, mungbean, potato, sorghum and wheat),
vegetable crops (tomato, cabbage and field mustard), fruit crops (grape, papaya,
orange and apple), oil crops (rapeseed, Indian mustard, black mustard, soybean and
castor), forage crops (barrel medic), biofuel crops (jatropha, miscanthus, switch-
grass, pine, madhuca, arundo and pongamia) and other commercial crops such as
tobacco and cotton (Tables 10.1 and 10.2; http://www.arabidopsis.org/portals/gen
Annotation/other_genomes/#sequence).
In this chapter, we give a brief overview of transgenic crops, plant genomics and
plant functional genomics and then discuss various transgenic strategies being used
to establish gene–phenotype relationships and elaborate on how they are facilitating
the functional characterization of genes and gene control sequences.
10.2 Transgenic Plants
Transgenic crops have great potential for alleviating some of the production con-
straints such as insect pests, pathogens, salinity and drought. Since the first demon-
stration of the introduction and expression of foreign genes in tobacco in 1984,
more than 150 plant species in at least 50 plant families have been experimentally
transformed and transgenic events reported. Today, 13 transgenic crops are grown
commercially in 25 different countries, including 15 developing countries (James
2008). Regulatory approvals for 24 transgenic crops, for the importation for food
and feed use and for release into the environment, have been granted in another 30
countries (James 2008).
Worldwide acreages of transgenic crops are increasing at the rate of ~12%
each year (James 2008). However, the current successes in transgenic crops still
have a narrow base with respect to traits (>95% are insect resistance and/or her-
bicide tolerance traits), crops (>95% are soybean, corn, cotton and canola) and
countries (>95% are grown in US, Argentina, Brazil, Canada, India, and China).
Table 10.1 Current and future biotech plants for which full-scale genomics studies have been initiated or completed
10
Common Plant Type Biotech Maps ESTs Sequencing Chromo- Haploid Sequencing group/consortium NCBI
name status status somes (n) genome size project
(Mb) ID
Apple Malus Fruit Future na* 256,249 In progress 17 750 IASMA, Institoto Agrario S. 12882
domestica Michele all’Adige
(http://www.ismaa.it/)
Arabidopsis Arabidopsis Dicot model Model Yes 1,526,124 Completed 5 120 The Arabidopsis Information 9506
(thale thaliana resource
cress) (http://www.arabidopsis.org/)
Arabidopsis Thellungiella Dicot model Model na 38,022 In progress 7 260 DOE Joint Genome Institute 18765
relative halophila (http://www.jgi.doe.gov/
sequencing/why/50029.html)
Banana Musa Fruit Future Yes 5,524 In progress 11 600 The Global Musa Genomics 15719
acuminata Consortium
(http://www.musagenomics.org/)
Barley Hordeum Food Future Yes 501,366 In progress 7 5,000 International Barley Genome 9511
vulgare Sequencing Consortium

(http://www.public.iastate.edu/
~imagefpc/IBSC%20Webpage/
IBSC%20Template-home.html)
Barrel medic Medicago Forage legume Future Yes 260,238 Completed 8 500 Medicago truncatula Sequencing 9508
truncatula model Resources (http://www.
medicago.org/genome/)
Brachypodium Brachypodium Model cereal Model na 20,449 In progress 5 300 DOE Joint Genome Institute 18703
distachyon (http://www.brachypodium.org/)
Broccoli Brassica Vegetable Future na na In progress 9 600 TIGR Brassica oleracea Genome 12577
oleracea Project (http://www.tigr.org/
tdb/e2k1/bog1/)
Cassava Manihot Food Future Yes 79,444 In progress 18 760 DOE Joint Genome Institute 13628
esculenta (http://www.jgi.doe.gov/
Castor bean Ricinus Oil, industrial Future na 62,592 Draft assembly 10 400 J. Craig Venter Institute 16957
communis (http://www.jcvi.org/cms/
research/projects/castor-
bean-database/overview/)
361
(continued)
362
(Mb) ID
Eucalyptus Eucalyptus Oil, tree Future na 10,003 In progress 11 600 The International Eucalyptus 12504
(blue globulus Genome Network
gum) (http://www.fabinet.up.ac.za/
eucagen)
Field mustard Brassica rapa Vegetable Future na 33,398 In progress 10 500 The Multinational Brassica 12578
Genome Project (MBGP)
(http://www.brassica.info/
resource/sequencing.php)
Giant cane Arundo donax Biofuel, grass Future na na In progress 12 2,744 Nandan Biometrix Ltd 32663
(http://www.nandan.biz/)
Japanese bitter Poncirus Model for citrus Future na 62,344 In progress 9 380 International Citrus Genome 12948
orange trifoliata Consortium
(http://www.citrusgenome.
ucr.edu/)
Jatropha Jatropha Biofuel Future na 1,012 In progress 11 416 Nandan Biometrix Ltd 32385
curcas (http://www.nandan.biz/)
Jatropha Jatropha Biofuel Future na na In progress 11 416 Nandan Biometrix Ltd 32643
tanjorensis (http://www.nandan.biz/)
Karanj Pogamia Biofuel, tree Future na na In progress 11 1,700 Nandan Biometrix Ltd
pinnata (http://www.nandan.biz/)
Leaf mustard Brassica Oil, vegetable Future na 193 In progress 18 1,200 The Brassica Genome Gateway 18137
juncea (http://www.brassica.bbsrc.
ac.uk/)
Loblolly pine Pinus taeda Biofuel Future na 328,628 In progress 12 30,000 The Pine Genome initiative 30775
(http://pinegenomeinitiative.
org/)
Lotus Lotus japonicus Model legume Model yes 157,951 Draft assembly 6 470 Kazusa DNA Research Institute 10747
(http://www.kazusa.or.jp/
lotus/clonelist.html)
Lyreleaf Arabidopsis Dicot model for Model na 561 In progress 8 230 DOE Joint Genome Institute 15628
rockress lyrata comparative (http://www.jgi.doe.gov/
genomics sequencing/why/3066.html)
N.M. Upadhyaya et al.
10
Mahwa Madhuca Biofuel tree Future na na In progress 13 na Nandan Biometrix Ltd 32407
longifolia (http://www.nandan.biz/)
var.
latifolia
Maiden grass Miscanthus Biofuel Future na na In progress 19 na Nandan Biometrix Ltd 32661
sinensis (http://www.nandan.biz/)
Maize Zea mays Food and fodder Current Yes 2,018,337 In progress 10 2,400 The Maize Genome Sequencing 9514
Project (http://www.
maizesequence.org/
overview.html)
Monkey Mimulus Model Future na 14,587 In progress 14 430 DOE Joint Genome Institute 15658
flower guttatus (http://www.jgi.doe.gov/
Moss Physcomitrella Model for Model Yes 20,453 Draft assembly 27 510 DOE Joint Genome Institute 12940
patens comparative (http://www.jgi.doe.gov/
genomics sequencing/why/3141.html)
Moss Selaginella Model for Model na 93,806 Completed 8 100 DOE Joint Genome Institute 13079
moellen evolutionary (http://www.jgi.doe.gov/

dorffii study sequencing/why/3153.html)
Oilseed rape Brassica napus Oil Current na 596,249 In progress 19 (2)** 1,100 The Brassica Genome Gateway 12478
(http://brassica.bbsrc.ac.uk/)
Papaya Carica papaya Fruit Current na 77,158 Draft assembly 9 370 The Hawaii Papaya Genome 16103
Project (http://asgpb.
mhpcc.hawaii.edu/papaya/)
Pink Capsella Dicot Model na na In progress 8 686 DOE Joint Genome Institute (http:// 15659
Shep- rubella model, www.jgi.doe.gov/sequencing/
herd’s Arabidopsis why/3066.html)
purse relative
Poplar Populus Tree model Current na 89,943 Draft assembly 19 480 The International Populus 10770
trichocarpa Genome Consortium
(http://www.ornl.gov/
sci/ipgc/)
Potato Solanum Food crop Future Yes 231,704 In progress 12 840 The potato Genome Sequencing 12984
tuberosum Consortium (http://www.
potatogenome.net/)
(continued)
363
364
(Mb) ID
Rapeseed Brassica nigra Oil, vegetable Future na 1 In progress 8 700 The Brassica Genome Gateway 18141
(http://www.brassica.
bbsrc.ac.uk/)
Rice Oriza sativa Food Future Yes na Completed 12 375 BGI Rice Information System 9512
indica (http://rice.genomics.org.
cn/rice/index2.jsp)
Rice Oryza sativa Food Future Yes na Completed 12 390 International Rice Genome 9512
japonica Sequencing Project
(http://rgp.dna.affrc.go.jp/IRGSP/)
Safed musli Chlorophytum Medicinal crop Future na na In progress 14 (2) 540 Nandan Biometrix Ltd (http://www. 32621
borivi- nandan.biz/)
lianum
Sorghum Sorghum Food crop Future Yes 209,814 Completed 10 760 DOE Joint Genome Institute 10785
bicolor (http://www.phytozome.
net/sorghum)
Soybean Glycine max Oil, vegetable Current Yes 1,386,618 In progress 20 1,200 DOE Joint Genome Institute 9507
(http://www.phytozome.
net/soybean)
Squash Cucurbita pepo Vegetable Current na 27 In progress 20 539 Cucurbit Genomics Database na
(http://www.icugi.org)
Switchgrass Panicum Biofuel Future na 436,535 In progress 18–36 1,911–2,303 DOE Joint Genome Institute 17453
virgatum (2–4) (http://www.jgi.doe.gov/
Tobacco Nicotiana Commercial crop Future Yes 240,440 In progress 24 (2) 4,500 Tobacco genome Initiative 13234
tabacum (http://www.tobaccogenome.
org/)
Tomato Solanum Fruit Current Yes 258,830 In progress 12 950 International tomato sequencing 9509
lycoper- Project (http://www.sgn.
sicum cornell.edu/about/tomato_
sequencing.pl)
10
Upland cotton Gossypium Fibre, oil Current Yes 268,779 In progress 26 (2) 2,100 International cotton genome 12542
hirsutum initiative (http://icgi.tamu.edu/
developing.html)
Valencia Citrus sinensis Fruit Future na 207,500 In progress 9 380 International Citrus Genome 9597
orange Consortium (http://www.
citrusgenome.ucr.edu/)
Western Aquilegia Flower model Model na na Pending 7 350 DOE Joint Genome Institute 18647
columbine formosa (http://www.jgi.doe.gov/
Wheat Triticum Food crop Future Yes 1,051,736 Pilot scale 21 (3) 16,000 International Wheat Genome 9513
aestivum Sequencing consortium
(http://www.wheatgenome.org/)
Wine grape Vitis vinifera Fruit and wine Future Yes 353,688 Draft assembly 19 500 International Grape Genome 12992
Program (http://www.
vitaceae.org/index.php/
International_Grape_Genome_
Program)
Yellow owl’s Triphysaria Parasitic weed Model na 49,006 Pending 11 1,200 DOE Joint Genome Institute 15684
clover versicolor (http://www.jgi.doe.gov/
*na ¼ not available or not determined or not assigned
** ¼ x denotes ploidy level
Data source: NCBI, relevant web pages and Plant DNA C-values database (Bennett and Leitch 2005, http://data.kew.org/cvalues/)
365
Table 10.2 Current and future biotech plants for which full-scale genomics studies are yet to be initiated
366
Common name Plant Biotech status ESTs Maps Type Haploid genome Chromosome NCBI Project ID
size (Mb) number (n)
Alfalfa Medicago sativa Current 11,090 Yes Forage 900 8 13214
Almond Prunus dulcis Future 3,864 na Nut 300 8 12944
Apricot Prunus armeniaca Future 15,105 Yes Fruit 300 8 20685
Asparagus Asparagus officinalis Future 8,422 Yes Vegetable 1,323 10 16855
Cardamom Amomum sp, Elettaria sp Near future na na Condiment na 12 na
Cabbage Brassica oleracea var capitata Near future 26,692 na Vegetable 600 9 12577
California poppy Eschscholzia californica Near future 9,083 na Medicinal 1,103 6 na
Capsicum Capsicum annuum Current 33,311 Yes Vegetable 3,000 12 12486
Carnation Dianthus caryophyllus Current 387 na Flower 613 15 na
Cauliflower Brassica oleracea var botrytis Near future 202 na Vegetable 760 9 na
Chicory Cichorium intybus Near future 53,973 na Beverage na 9 na
Chinese broccoli Brassica oleracea var. alboglabra Near future 30,759 na Vegetable 760 9 na
Coffee Coffea arabica Future 1,577 na Beverage 1,176 11 10702
Common bean Phaseolus vulgaris Future 83,448 na Vegetable 630 11 12933
Cotton Gossypium arboreum Current 41,768 na Fibre, oil 2,132 13 12946
Cowpea Vigna unguiculata Near future 183,751 Yes Food 588 11 31169
Creeping bentgrass Agrostis stolonifera Near future 9,020 na Lawn grass 3,430 28 (2)** na
Cucumber Cucumis sativus Near future 6,662 na Vegetable 370 7 36671
Cuphea hybrid Cuphea Near future na na Ornamental, oil na 8 na
Egg plant Solanum melongena Near future 3 Yes Vegetable 1,100 12 15625
Ethiopian mustard Brassica carinata Near future 2,482 na Oil 1,544 17 na
Flax Linum usitatissimum Near future 7,929 na Fruit 686 15 na
Lettuce Lactuca sativa Near future 80,781 na Vegetable 2,597 9 12869
Lilly Nuphar advena Near future 20,589 na Flower 2,400 17 na
Mung bean Vigna radiata Near future 829 na Food 515 11 17571
Musk mellon Cucumis melo Near future 5,943 na Fruit 931 12 na
Norway spruce Picea abies Near future 10,217 na Timber tree 18,228 12 na
Opium poppy Papaver somniferum Near future 20,340 na Medicinal 3,724 11 na
Peach Prunus persica Future 79,023 Yes Fruit 290 8 12949
Petunia Petunia axillaris subsp axillaris Current 1,696 na Flower 1,372 7 na
Pineapple Ananas comosus Near future 5,649 na Fruit 569 25 na
Poplar Populus tremula Near future 37,313 na Tree na 19 13278
Poplar Populus tremuloides Near future 12,813 na Tree na 19 13258
10
Rose Rosa chinensis Near future 1,794 na Flower 564 7 na

Rose Rosa hybrid cultivar Near future 5,563 na Flower 578 7 na
Rose Rosa luciae Near future 1,932 na Flower na 7 na
Runner bean Phaseolus coccineus Near future 391,138 na Flower, food 662 11 12925
Safflower Carthamus tinctorius Near future 41,011 na Oil na 9 na
Sugar beet Beta vulgaris Current 26,870 Yes Sugar and herb 760 9 12562
Sugarcane Saccharum officinarum Near future 246,379 na Food 3,969 40 (4) 12961
Sunflower Helianthus annuus Near future 133,682 na Oil 3,000 17 12865
Tall fescue Festuca arundinacea Near future 44,377 na Forage 5,629 21 (3) na
Tea Camellia sinensis Near future 6,416 na Beverage 3,824 15 31167
Tobacco relative Nicotiana benthamiana Future 42,658 na Narcotics 3,136 19 (2) 12911
Torenia Torenia fournieri Near future na na Flower na 17 na
Watermellon Citrullus lanatus Near future 7,891 na Fruit 424 11 na
White clover Trifolium repens Near future 46 na Forage 956 16 (2) na
White spruce Picea glauca Near future 284,329 na Wood, ornamental 19,796 12 32251
*na ¼ not available or not determined or not assigned
** ¼ x denotes ploidy level

Data source: NCBI, relevant web pages and Plant DNA C-values database (Bennett and Leitch 2005, http://data.kew.org/cvalues/)
367
Long development times from discovery to commercial release, intellectual prop-

erty and regulatory constraints, and high development and regulatory costs are
partly responsible for this (Birch 2000). So far, only transgenic crops and traits with
high commercial return potential have been commercialized by large multinational
companies in countries which allow the deregulation of genetically modified (GM)
crops or have regulatory frameworks in place for commercial releases. In such
countries and with such crops, companies, farmers and the consumers (to some
extent) have reaped the financial benefits. It is worth noting that 20 countries have
banned the importation and/or commercial cultivation of GM crops with another
five countries having a moratorium on the same (http://www.centerforfoodsafety.
org/geneticall5.cfm). As such, the issue of GM food crops for human consumption
is still contentious because of the perceived health and environmental risks asso-
ciated with the widespread usage of certain transgenes.
Commercial deployments of transgenes conferring more complex traits such as
abiotic stress tolerance, yield, vigor and nutritional quality are yet to be achieved.
This is largely due to the lack of adequate knowledge of the genes controlling such
traits and to the complexity of their behavior under different environmental condi-
tions and in different genetic backgrounds. Quite often “green house champion”
transgenic lines fail miserably under field conditions. Although very high transfor-
mation efficiencies are now achievable in many crop species, they are still highly
genotype dependent. This is mainly due to the genotype dependency of tissue
culturability and transformability (mainly via Agrobacterium). It is important to
note that the proportion of transformation events resulting in an acceptable com-
mercial phenotype is very low. Even with an optimized gene construct (gene
promoter-coding region-gene terminator), this frequency could be one in 400
events (Birch 2000). Somaclonal variations introduced during tissue culture, pleio-
tropic effects of transgene expression, insertional inactivation of endogenous genes,
integration position effects and transgene silencing are likely to reduce the fre-
quency of “useful transformation events” (Birch 2000). Recent progress and appli-
cations in genomics and functional genomics are not only increasing the number of
potential transgenes and gene control sequences but are also improving our under-
standing of the complexity of gene expression under different environmental con-
ditions and in different genetic backgrounds.
Progress with transgenics has been more rapid with rice than with any other
cereals due to the very efficient rice tissue culture and transformation systems
developed over the past 20 years (Upadhyaya et al. 2000). The use of appropriate
embryogenic target tissues, gene delivery methods, gene promoters, selectable
marker genes, selection regimes and reporter genes have all contributed to this
success. Protoplasts derived from embryogenic callus have been used for gene
delivery by either electroporation or polyethylene glycol treatment. Embryo or
embryogenic calli have been the target tissues for biolistics and Agrobacterium-
mediated gene delivery. Although the initial successes were restricted to reporter
and selectable marker transgenes, genes of agronomic value such as herbicide,
insect, bacterial, fungal and viral resistances have also been introduced into rice and
transgenic lines have been studied under field conditions. Transgenic rice is now an
10 Biotech Crops and Functional Genomics 369
ideal tool for elucidating various aspects of gene expression and regulation, espe-
cially of genes from other monocots, which are not as amenable as rice to genetic
manipulation. Some of the challenges in producing sustainable transgenic rice
currently being addressed include the removal of selectable markers, targeted
gene delivery (gene replacement), stability of transgene expression over many
generations and the spatial, temporal and developmental control of transgene
expression.
10.3 Plant Genomics
As proposed by Hieter and Boguski (1997), genomics can be broadly classified into
two disciplines: “structural genomics” and “functional genomics.” Structural geno-
mics corresponds to the initial phase of genome analysis resulting ultimately in the
definition of the complete DNA sequence of an organism, while functional geno-
mics makes use of the genome sequence to assess, on a large-scale, the functions of
genes as well as their expression and interaction. With the near completion of the
sequencing of their genomes (Table 10.3), Arabidopsis and rice are generally
accepted as model dicot and monocot species, respectively for genetic and genomic
studies. This is because of their small genome sizes (~135 Mb and 430 Mb,
respectively), the ease with which they can be grown, transformed and used in
genetic experiments, and the similarity of their respective gene orders and gene
Table 10.3 Arabidopsis and rice genome sequence assembly and annotation – current status
Arabidopsis Rice Rice
TAIRa TIGRb BGIc
Genotype/ecotype Columbia Japonica indica
Cultivar – Nipponbare 93-11
Genome version TAIR8 Release 5 Release 2
Chromosomes 5 12 12
Sequenced genome size (bp) 119,186,497 372,077,801 360,157,649d
(374,545,499)e
Estimated complete genome size (bp) 134,634,692 388,820,000 NA
Unassigned sequences (bp) – – 104,840,190
Predicted genes including transposable 33,282 56,278 (66,710)f 59,660
element (TE) genes and noncoding RNAs (38,963)
Predicted non-TE genes 27,235 41,046 (51,286)f 49,088
Mapped Full-length cDNA 13,066 32,775 25,645
ORF cDNA 24,235 – –
a
The Arabidopsis Information Resource (http://www.arabidopsis.org/)
b
TIGR = The Institute for Genomic Research. TIGR is now merged with The J. Craig Venter
Institute (http://www.jcvi.org/) and TIGR’s Rice Annotation Project is moved to Michigan State
University (http://rice.plantbiology.msu.edu/)
c
BGI (http://rice.genomics.org.cn/index2.jsp)
d
Genome sizes based on the sum total of assigned contigs
e
Figures in the parenthesis are the genome sizes as the sum total of genome assigned scaffolds
f
Figures in the parenthesis are the total genes including splice variants
sequences with other dicots (e.g., crucifers) or monocot cereals, (e.g., barley, wheat
and maize). Thanks to the recent rapid developments in high-throughput nucleic
acid sequencing technologies, genome sequencing and/or expressed sequence tag
(EST) sequencing efforts are underway for the majority of crop plants (Table 10.1).
10.4 Plant Functional Genomics
Functional genomics is a rapidly expanding field of biological science. Various

high-throughput technologies are being employed in the large-scale profiling of
genes, mRNAs, proteins and metabolites that participate in various cellular pro-
cesses during plant growth and development. These include: (1) data mining tools
for structural similarities; (2) RNA level expression profiling with ESTs, oligonu-
cleotide or cDNA chips; (3) protein level expression profiling (proteomics);
(4) metabolite level expression profiling (metabolomics); (5) gene knock-outs or
loss-of-function studies with naturally occurring alleles, induced deletion and
insertional mutants; (6) gene expression knock-down (gene silencing) studies;
and (7) gain-of-function studies with overexpression or misexpression transgenes.
10.4.1 Gene Predictions Using DNA Sequence Comparison
The most straightforward way of predicting the function of an unknown gene from
one organism is by comparison of its DNA sequence with known gene sequences
from other organisms, as functionally similar genes normally have sequence simi-
larities at both the DNA and the protein sequence levels. The precision with which
sequences can be compared has increased tremendously with recent vast improve-
ments in computing power, gene prediction programs and various other bioinfor-
matics capabilities. Several laboratories have embarked on sequence annotation
using this approach (Antonio et al. 2007; Itoh 2007). Computational gene predic-
tions in rice suggest that there could be more than 50,000 rice genes with ~60%
having some evidence of expression. For example, according to the International
Rice Genome Sequencing Project (IRGSP)’s rice annotation project database
(RAP-DB; http://rapdb.dna.affrc.go.jp/), among the 53,461 predicted rice genes
31,439 show evidence of expression and 25,012 are protein-coding loci with full-
length cDNA support. The remainder is based on computer predictions without any
evidence of transcriptional activity. The validation of gene functions predicted by
sequence comparison needs to be done by other methods to avoid the progressive
build up of inaccurate gene function assignments in the genome sequence data-
bases. With the available japonica and indica genome sequences, attempts are
being made to unravel allelic variations between these two subspecies using various
functional genomics approaches. Transgenic approaches could be used to unravel
the function of an unknown gene by overexpression, knock-out or knock-down of
that gene as detailed later in this chapter.
10.4.2 Gene Expression Studies at the RNA Level
Although there could be more than 50,000 genes in a plant genome, not all of these
are transcribed into RNA at any given time, in any given tissue or under any given
environmental condition. Even some of the transcribed RNAs are suppressed,
broken down or rendered non-translatable. The characterization of all the transcribed
genes, referred to as the “transcriptome,” is normally attempted by collecting large
numbers of ESTs from diverse cDNA libraries. Currently, there are more than 17
million plant ESTs in the public database (http://www.ncbi.nlm.nih.gov/dbEST/)
with ~5 million coming from current transgenic crops and 7 million from future
transgenic crops. To date, there are 1,220,876 rice ESTs in the public database.
Recent advances in the technology for construction of full-length cDNA libraries
have made it possible to produce more than 30,000 rice full-length cDNAs (Kikuchi
et al. 2003; Satoh et al. 2007). This has helped in improving the rice genome
annotation, gene organization and genome-wide expression profiling. One other
significant EST and full-length cDNA collection from indica rice comes from
the Beijing Genomics Institute (BGI), which can be viewed through BGI-RIS
(http://rice.genomics.org.cn/index2.jsp).
Genome-wide expression profiling (including differential expression) of genes
in various crop species is being facilitated by high-throughput techniques, such as
microarrays, serial analyses of gene expression (SAGE), massively parallel signa-
ture sequencing (MPSS) and more recently by ultra-deep sequencing (e.g., 454,
Solexa and SOliD technologies). These procedures are typically used to compare
two mRNA populations derived from tissues of different developmental stages or
those subjected to different environmental stimuli, to yield information on the
comparative changes in gene expression in each tissue. The conceptual basis of
this method is that genes contributing to the same biological process are likely to
exhibit similar expression patterns and thus allow the putative assignment of gene
function. With all these new developments in deep sequencing technologies, we are
seeing an explosive increase in the RNA expression tag and small RNA datasets
from diverse plants under different environmental conditions and experimental
treatments. This will help in unraveling the complexities of the transcriptome,
including that of non-coding RNAs, in diverse biological systems. Thus, it is now
possible to study spatial and temporal RNA expression patterns which could
provide insights into their cellular and developmental functions. The regulatory
and developmental functions of a transgene could also be studied using these
techniques.
10.4.3 Gene Expression Studies at the Protein Level
With recent advances in high-resolution two-dimensional polyacrylamide gel elec-

trophoresis (2D-PAGE), staining, detection, peptide micro-sequencing and asso-
ciated computer software, “proteomics” is also emerging as a powerful functional
genomics tool. Here, instead of looking at gene expression, an assessment is made

on the gene product, i.e., the protein. In the 2D-PAGE-based approach, intact
proteins are separated by 2D-PAGE and protein abundance is determined by the
relative stain intensities of protein spots on the gel. The differential proteome is
confirmed by image analysis. The identity of a specific protein is generally deter-
mined by mass-spectrometric (MS) analysis of peptides after proteolysis of the
protein spot or by protein sequencing after blotting the gel to a membrane. Com-
parison of the amino acid sequences of fragments of proteins with those predicted
from DNA sequences greatly facilitates not only the validation of gene predictions
but also provides insight into the cellular and developmental regulation of gene
expression. Several public databases of 2D-PAGE-derived plant proteins are already
available, such as WORLD-2DPAGE (http://expasy.org/ch2d/2d-index.html), Rice
Membrane Protein Library (http://wardlab.cbs.umn.edu/rice/) and the Rice Prote-
ome Database (http://gene64.dna.affrc.go.jp/RPD/), that provide extensive infor-
mation on the progress of rice proteome research. In addition, progress is being
made in detecting post-translational modifications such as glycosylation, lipid
attachment, phosphorylation, methylation, disulfide bond formation and proteolytic
cleavage. Differential proteome analyzes of a particular transgenic plant and an
appropriate non-transgenic plant (i.e., a segregating null and/or wild type plant)
could highlight any unintended or flow-through effect of the transgene on the
expression of other genes.
10.4.4 Metabolomics
Metabolomics is the comprehensive analysis of low-molecular-weight compounds

in biological samples and is emerging as a biochemical phenotyping tool along with
transcriptomics and proteomics in functional genomics (Tarpley and Roessner
2007). Technologies used in metabolomics are normally based on the chro-
matographic separation of complex compound mixtures, using either liquid or gas
chromatography and mass-spectrometric detection. Nuclear magnetic resonance
(NMR) spectroscopy is also playing a major role in metabolomic approaches.
Fourier-transform ion cyclotron mass spectrometry (FT-ICR-MS) can mass-resolve
metabolites with a mass accuracy of <1 ppm (Dunn et al. 2005), and thus provides a
high-throughput method for metabolite fingerprinting. This technique has been used
in conjunction with proteomics for comparing leaves, panicles and calli of wild type
rice (cv. Nipponbare) and a transgenic line over expressing the YK1 gene, which is a
homolog of the maize HC-toxin reductase gene (Takahashi et al. 2005). The YK1
gene is known to confer increased tolerance to rice blast and multiple environmental
stresses. Although the global composition of organ-specific metabolites did not
differ significantly between the two lines, alterations in less than 10% of the
metabolites were observed. The transgenic line expressed several previously
reported stress-responsive proteins suggesting that ectopic overexpression of a
single gene (YK1) can affect the expression of unrelated proteins and metabolites.
The current limitations of routine metabolomic analyzes, as a part of plant

functional genomics programs, include access to expertise in the techniques of
plant metabolomics, the necessary instrumentation, the establishment of adequate
sample preparation procedures, the need forcoordinated comprehensive cataloging
and/or control of environmental variables, the availability of databases providing
storage and access to metabolome-specific data (Tarpley and Roessner 2007).
10.5 Transgenic Plants in Functional Genomics
Transgenic plants are being used in several functional genomics strategies. These
strategies can be broadly classified into two categories, namely forward genetics
and reverse genetics. The forward genetics strategies include gene disruptions
(knock-outs) with T-DNA and/or transposon insertions, gene/enhancer trap (with
promoter-less reporter genes) insertions, gene activations with promoter/enhancer
insertions and gene deletions with site-specific recombinases. The reverse genetics
strategies include site-selected insertional mutagenesis and gene knock-downs with
RNA-silencing transgenes and gene activity disruption with modified genes pro-
ducing mutant proteins.
10.5.1 Forward Genetics Strategies
10.5.1.1 Insertional Gene Disruptions (Knock-Outs)
One of the most direct approaches to determine gene function is the production of
insertion mutations and the study of their effects on the plant phenotype. Alterations
in a plant phenotype, as a consequence of the mutation, may then provide insight
into the gene’s function. As the inactivated gene in such plants contains a known
DNA insertion sequence, it is a relatively simple task to isolate the gene as it has
been effectively “tagged” by the inserted sequence. Such tagging can be achieved
by employing both non-transgenic and transgenic strategies. Endogenous transpo-
sons or “jumping genes” (both autonomous elements and their non-autonomous
counterpart elements) such as Activator (Ac)/Dissociation (Ds), Enhancer (En)/
Inhibitor (I) (also known as Suppressor-Mutator or Spm/dSpm) and Mutator
(MuDR/Mu) in maize, or retrotransposons such as Tos17 in rice have been used
to generate insertional mutants by non-transgenic means. Transgenic strategies
include Agrobacterium-mediated T-DNA insertions and heterologous transposons
delivered through T-DNA. In both cases, plants can be initially screened for
changes in phenotype (Fig. 10.1). One can then clone the mutated gene using the
inserted DNA tag as a reference point (commonly referred to as flanking sequence
tags or FSTs) and compare its sequence to sequences in the genome databases, thus
linking the mutant phenotype with a known gene sequence.
Introduce DNA tags ( )

into rice (insertion mutagenesis)
Rice lines each with a tag

disrupting a different gene
Any change due to

Screen lines for changes gene disruption associates
in plant form or function function with gene sequence
FUNCTIONAL GENE
TAG DISRUPTED GENE

TAG
Compare to
sequence
Clone database
GENE MACHINE
Poor leaf No flower Stunted Library of tagged lines and
development formation roots gene sequence information
Fig. 10.1 A schematic diagram describing gene tagging and identification in rice
For crop plants, such as rice, having efficient tissue culture, generation and
transformation systems, T-DNA has emerged as the preferred insertion mutagen
for generating large random libraries of insertional mutant lines. Research groups in
Korea, China, France and Taiwan are generating T-DNA insertion libraries, char-
acterizing T-DNA flanking sequences at insertion points and gathering phenotypic
information in web-accessible databases (Hirochika et al. 2004; Guiderdoni et al.
2007; Krishnan et al. 2009). To date, more than 460,000 T-DNA lines and ~118,000
FSTs have been produced. Although these resources will be useful for reverse
genetics, there are limitations in obtaining T-DNA insertions in smaller genes such
as single-exon genes, which may account for up to 40% of the genes in rice.
Furthermore, a large proportion of mutant phenotypes could be due to tissue
culture-induced, non-tagged “somaclonal” mutations. Non-tissue culture transfor-
mation techniques such as seed transformation (Feldmann 1991), vacuum infiltra-
tion (Bechtold and Pelletier 1998) or floral dipping (Clough and Bent 1998) have
partly overcome this problem in Arabidopsis. However, these techniques are yet to
be made workable in common transgenic crops.
The T-DNA system can also be used to deliver jumping genes or transposons,
which will cause many random insertions once activated. Although there are quite a
few groups of transposons (most of them originating from maize) being used in the
several plant systems, we will confine our discussion to the use of the maize
transposon Ac and its derivative Ds. The Ac element can excise and integrate
randomly throughout the plant genome (although it tends to transpose close to its
original position in the chromosome). A protein called transposase produced by Ac
mediates these transpositions. A deletion derivative of Ac called Dissociation (Ds)
has the capacity to jump but only in the presence of Ac, as it lacks the capacity to
produce its own transposase. By removing certain sequences from Ac it can be
made immobile (iAc), however it can still produce transposase. Since the first
report of the activity of the autonomous Ac element in transgenic rice 18 years
ago, sophisticated transposon tagging systems have been developed to improve
both tagging and screening efficiencies in rice (Zhu et al. 2007) and other crop
plants, and are primarily based on the two-component iAc/Ds (Chin et al. 1999) or
En/I (Greco et al. 2004) systems. Since the successful cloning of a gene (BFL1/
FZP), which mediates the transition from spikelet to floret meristem (Komatsu
et al. 2003; Zhu et al. 2003) several genes have been cloned by transposon tagging
in rice.
The process of producing stable Ds insertion lines in the two-component Ac/Ds
system is illustrated in Fig. 10.2. Essentially, this involves the production of
transgenic Ac lines and Ds lines by Agrobacterium-mediated transformation,
followed by the production of Ac/Ds mutagenic lines either by crossing or by co-
transformation or super-transformation. In this mutagenic population, under the
influence of a transposase produced by iAc, the normally-stable Ds element starts to
jump or undergo transposition. This transposition will continue while the iAc is
present. In the subsequent generations, the iAc can be segregated from Ds elements
that have integrated into new regions of the genome. Plant lines containing these
stable Ds insertions in new genomic locations are then analyzed. Regions flanking
the Ds element are then cloned and sequenced to create a database of flanking
sequences, or molecular flags, that represent disrupted genes. Public sequence
databases are then searched for similar sequences.
A wide variety of Ac/Ds gene constructs have been produced by several research
groups (Zhu et al. 2007). The additional features of these Ac and Ds constructs are
that they have improved tagging and screening efficiencies. These features include
a negative selection gene or a visual marker gene for Ac, a herbicide-resistance gene
for Ds selection or an antibiotic gene to detect Ds transposition, inducible or
developmental-specific promoters to control transposase activity to maximize ger-
minal transposition, and removal of Ac after Ds transposition by site-specific
recombinases. These features greatly assist in the elimination of plants without
Ds insertions and positively select for plants with stable Ds insertions in different
genomic locations.
A novel method of producing stable Ds insertion lines, using a transiently-
expressed transposase (TET) system, has been developed (Upadhyaya et al.
2006). Constructs suited for high-efficiency insertional mutagenesis in general,
and the TET system in particular, have also been developed. By super-infecting
callus tissue from single-copy Ds/T-DNA lines, having both Ds excision and
(I) N P Ac S S Ds R
(II)
N P Ac S S Ds R
(No mutant phenotype)

P Gene T
(III) N P Ac S S R
P Gene Ds T
(mutant phenotype)
(IV) a b
– GA3 + GA3
Fig. 10.2 The two-component Ac/Ds based gene (DsG) and enhancer (DeE) trapping system for
rice. (I) Ac and Ds constructs are first delivered to rice by Agrobacterium-mediated transformation
with hph as the selectable marker (S). The Ac construct also contains the negative selector such as
tms2 (N) while the Ds construct contains sgfpS65T (R) as the launching pad reporter gene.
(II) Selected homozygous progeny (containing single-copy Ac and Ds) are crossed. Alternatively,
this can be achieved by co- or super-transformation. (III) In the F1 progeny of the crosses or T0
plants of double transformants, Ds under the influence of the Ac transposase is excised from the
original location (launching pad) and reinserts into a new genomic location. The insertion in a
coding region can lead to gene expression knockout thus producing an insertional mutant. Progeny
seedlings with stable Ds (unlinked to Ac), either linked to the original launching pad (GFP +ve) or
unlinked to the Ds launching pad (GFP +ve) are identified using negative selection via the use of
naphaleneacetamide (NAM) selection. (IV) An acute dwarf mutant obtained in the screening
population was later identified as having an insertion in the ent-kaurene synthase gene, the second
gene in the GA biosynthetic pathway (Margis-Pinheiro et al. 2005). Panel A contains homozygous
dwarf plant and the normal looking heterozygous plants. Panel B contains mutant plants with or
without GA3 treatment showing the GA3 responsiveness
re-insertion markers, with Agrobacterium harboring iAc constructs containing a

visual marker, sgfpS65T, stable Ds insertion lines have been regenerated at a
frequency of ~5%, in addition to iAc/Ds double transformants (Upadhyaya et al.
2006). Mapped single-copy Ds/T-DNA launch pads produced using these
constructs are highly suitable for efficient chromosomal region-directed insertion

mutagenesis (Upadhyaya et al. 2006).
Insertional mutagenesis by transposons has distinct advantages over that by
T-DNA insertion mutagenesis. Large-scale, transposon-mutagenized populations
can be produced using a relatively-small number of starter lines, because many
independent insertions can be generated among the progeny of a single parental
line. The tagged gene can be confirmed by revertants resulting from excision of the
transposon due to secondary transposition. Transposons can also be remobilized to
produce new insertion lines in order to target closely-linked genes in a specific
chromosomal region, i.e., corresponding to a mapped quantitative trait locus
(QTL). The iAc/Ds-based systems in rice yield 5–10% unique stable insertion
mutant lines in the progeny of mutagenic lines (Zhu et al. 2007). The frequency
of Ds re-insertions linked to the original Ds/T-DNA launch pads vary from 36 to
67% with the majority being within one cM of the Ds launch pad (Zhu et al. 2007).
One potential drawback is that quite often a secondary transposition of Ds could
leave a DNA footprint (scar) at the initial transposition site which could lead to an
untraceable mutation.
To validate phenotype and gene sequence relationships, complementation
experiments can be carried out by introducing the corresponding wild type
sequence into the mutant line as a transgene. The availability of multiple mutant
alleles will also facilitate the validation. Alternatively, an RNAi system can be
employed to determine whether the mutant phenotype can be mimicked by a
targeted gene expression knock-out (discussed later in this chapter).
10.5.1.2 Insertional Enhancer/Gene Traps
Under normal growth conditions at a given growth stage, less than 3% of the
insertion lines have obvious phenotypes. Some of the subtle mutants require
specialized screening to visualize the phenotype and other conditional phenotypes
can be revealed only when challenged with appropriate environmental cues such
as biotic and abiotic stresses. The other major cause of the “phenotype gap” is
functional redundancy wherein two or more genes have the same function. The
expression patterns of disrupted genes can however be visualized by placing a
“reporter gene” in the tagging element (T-DNA or transposon) as illustrated in
Fig. 10.3. When such a promoter trapping element is inserted downstream of the
promoter of a given gene, it results in reporter gene expression with a pattern
mimicking that of the disrupted gene. Similarly, it is possible to detect enhancer
elements in the vicinity of a gene by using a tagging element with a reporter gene
also containing a minimal promoter. Enhancers can activate genes from a distance
in an orientation-independent manner and thus the frequency of enhancer detec-
tion is normally high. Quite often, expression patterns reveal more about the
functional category of the disrupted gene. This would facilitate further characteri-
zation of the gene with subsequent specialized screening. However, it should be
noted that the identity and location of the target gene is sometimes difficult to
a b
Enhancer Trap Ds (DsE) Gene Trap Ds (DsG)
Trap reporter (gus)

Plasmid rescue (bla, ori)
Trap tracer (nptII or bar)
Ds Ds Ds Ds
Transcriptional Activator (TA) Splice acceptor

Splice donor
P/E Ds Ds G P G Ds Ds
GUS RNA gene::gus RNA

Complete or partial intron splicing
gene disruption
GUS protein
GUS expression GENE::GUS protein
c1 c2 c3
Fig. 10.3 Ds enhancer and gene trapping systems. The Ds enhancer (DsE) trap (a) or the Ds gene
(DsG) trap (b). Both the constructs used contain uidA (gus) as a trap reporter, the ampicillin
resistance gene bla, E. coli origin of replication (ori, for one step cloning of flanking sequences by
plasmid rescue) and nptII or bar as a tracer. The DsE contains minimal transcriptional activator
(TA) sequences in front of gus. When DsE is inserted downstream of the promoter/enhancer
elements (P/E) of a particular gene, these elements along with the TA activate gus transcription
resulting in GUS expression faithful to the trapped promoter or enhancer. DsG contains an intron
with splice acceptors (in all three reading frames) in front of gus. DsG trapped insertion in an
intronic region of a particular gene (G) may result in correct splicing of the RNA (between the
splice donors of the intron disrupted and the splice acceptors in DsG), producing a GENE::GUS
fusion. As seen with DsE, the GUS expression pattern mirrors the activity of the gene disrupted
depicted in root tip (c1), leaf vascular (c2) and leaf non-vascular (c3) specific GUS expression in
different lines. Such insertions in some cases result in loss of the gene product or produce
disfunctional gene product may lead to mutant phenotypes. Flanking sequences are then cloned
and sequenced for further characterization
determine, especially with poorly or incompletely sequenced and annotated

genomes.
Another commonly-used type of tagging is “gene trapping.” Here the reporter
gene is carried within the tagging element along with an intron with splice acceptor
sites in all three potential reading fames. Insertion of such a “gene trapping”
element within an intronic region of an endogenous gene may result in the splicing
of this hybrid intron (i.e., the region between the splice donor of the disrupted gene
and the slice acceptor in front of the reporter gene) resulting in the production of
a hybrid endogenous gene/reporter gene transcript. Under these circumstances,
the reporter gene expression mirrors the expression of the disrupted gene. Most of
the gene-trapping constructs contain b-glucuronidase (gus) as a reporter gene, the
expression of which is visualized by the addition of a chromogenic substrate, which
can be broken down by the reporter gene protein into an insoluble colored product
(indigo).
Most of the T-DNA and transposon (Ds or I) constructs used as insertional
mutagens have been modified to act as gene traps or enhancer traps. Gene trapping
efficiencies of ~6% have been reported for these constructs in rice (Hirochika et al.
2004). The efficiency of T-DNA gene trapping depends on the frequency of “clean”
T-DNA insertions, i.e., insertions devoid of direct or inverted T-DNA repeats, or of
vector backbone (VB) sequences derived from outside the T-DNA borders (Sallaud
et al. 2004; Upadhyaya et al. 2006). A “clean” Ds-containing T-DNA is also
essential for the satisfactory mobilization of Ds.
10.5.1.3 Insertional Gene Activations
Any plant genome, including that of rice, contains a large number of dormant gene
sequences, pseudogenes or genes with suboptimal cellular, spatial and develop-
mental expression patterns. Activation tagging systems allow the trapping of these
cryptic genes. Activation tagging involves introducing foreign DNA containing
specific gene promoters and control sequences throughout the genome using ran-
dom T-DNA or transposon insertions. Some of the different activation tagging
systems being used are represented schematically in Fig. 10.4. In the classical
activation tagging approach, random insertions of a cauliflower mosaic virus
(CaMV) 35S enhancer element into the genome can result in the overexpression
of native genes (or even dormant genes) in all cell types of the plant. Such increased
gene expression can create mutants of essential and redundant genes that are either
not present, or have no phenotype in knock-out collections. This gain-of-function
approach reveals dominant mutations affecting the transcriptional control of genes,
without altering the functional gene product. A sizable number of T-DNA activa-
tion tagged lines have been produced by research groups in France, Korea, China
and Taiwan (Guiderdoni et al. 2007).
Further refinement of activation tagging comes from the development of extensive
GAL4 enhancer trapping resources in rice, which enable transgene expression to be
targeted to specific cell types (Johnson et al. 2007). In the first step of a two-step
process known as “transactivation,” a large number of GAL4 enhancer trapping
“driver” lines are generated and the patterns of reporter gene expression are char-
acterized. “Responder” lines are then produced in which genes of interest are placed
downstream of the upstream activator sequence (UAS) element to which GAL4 binds.
In progeny plants of crosses between the driver and the responder lines, the
target genes are transactivated by GAL4 revealing the specific expression profile
a
LB RB
b EEEE
LB RB
35S
c
LB RB
Reporter UAS GAL4

d
LB RB
GOI UAS
e
UAS UAS
Fig. 10.4 Schematic representations of the different activation tagging systems in plants; T-DNA
constructs appear in light gray, bordered by left and right borders (LB and RB), and plant genomic
elements in dark gray. (a) Classical activation tagging with a tetramer of the CaMV 35S enhancer
(E) cloned next to the left border of a T-DNA construct. An adjacent endogenous transcriptional
unit consisting of promoter (small dashed cylinder) and coding sequence (large dashed cylinder)
shows upregulated expression (indicated by arrow) due to interaction with the enhancer element.
(b) Activation tagging with the complete CaMV 35S promoter (35S) cloned next to the LB of
a T-DNA construct. Integration of the T-DNA directly 50 of an endogenous coding sequence
replaces the native promoter with the 35S promoter, resulting in constitutive overexpression of the
gene. (c) Enhancer trapping with the minimal promoter-equipped Gal4 gene (GAL4) cloned next
to the right border of a T-DNA construct. An endogenous enhancer element (hatched arrow)
drives transcription of the Gal4 gene, leading to the GAL4 transcriptional activator protein binding
to the UAS element (five 17 bp UAS repeats cloned in tandem, followed by a minimal promoter
TATA) and activating expression of a downstream reporter gene. The resulting pattern of GAL4/
reporter gene expression can be highly specific, depending on the genomic enhancer, and is the
defining characteristic of the driver line. (d) Activation of a responder construct in specific cell
types using GAL4 transactivation. A gene of interest (GOI) is placed immediately downstream of
the UAS element and the resulting construct is introduced, through sexual crosses or retransfor-
mation, into a driver line. The responder construct subsequently comes under transcriptional
control of the driver, forcing transcription of the GOI in the same pattern as GAL4/reporter gene
expression. (e) Cell type-specific activation tagging using GAL4 transactivation. UAS elements
are cloned next to the LB and RB of a T-DNA construct, creating a double-sided gene transacti-
vator that is capable of up-regulating endogenous gene expression from either border (reproduced
from Johnson et al. 2007)
of each individual driver. In addition, random deployment of the UAS element

into the rice genome, followed by crosses to specific driver lines, should enable
activation tagging to be carried out in specific cell types of the plant. Cell type-
specific activation tagging has the potential to uncover novel mutations that are
missed or “averaged out” by the classical activation tagging technique (Johnson
et al. 2007).
10.5.1.4 Gene Targeting and Site-Specific Deletions
Gene targeting refers to the in vivo modification of a specific endogenous gene

sequence to alter its function. Homologous recombination-dependent gene target-
ing is being used routinely in bacteria, yeast and lately in mice (Evans et al. 2001) to
create precise deletions, insertions or mutations of DNA sequences within their
native chromosomal contexts. The routine use of gene targeting in plants, however,
has not been achieved yet because of the very low frequency of homologous
recombination in plant cells (0.01–0.1% of homology-independent random illegiti-
mate recombination). Despite numerous efforts made over the years, there are only
three reports of the production of fertile transgenic plants with reproducible endog-
enous gene targeting, including two genes in Arabidopsis and one gene in rice
(Hanin et al. 2001; Terada et al. 2002; Shaked et al. 2005).
A few of the approaches tried, in order to overcome the barrier imposed by
illegitimate recombination, are: (1) positive (for integration events including true
gene targeting) and negative (for T-DNA border-associated random integrations)
selections (Terada et al. 2002), (2) enhancing homologous recombination relative to
illegitimate recombination by manipulating the plant’s recombination machinery.
Examples of the latter are the overexpressions of Escherichia coli recA or ruvC
genes in plants and the disruption of plant RAD50 homologs (Shalev et al. 1999;
Reiss et al. 2000; Gherbi et al. 2001). Chromosomal breaks at the target site have
been shown to stimulate the cell’s DNA repair system and, if a homologous
template is present, this repair is achieved through homologous recombination.
For example, enzymatic cleavage or Ac/Ds excision have been shown to increase
recombination frequencies by 100–1,000 fold in plants (Reiss 2003).
Recently, chimeric zinc-finger nucleases (ZFNs) have been used to create site-
specific chromosome breaks in the absence of pre-engineered target sites (Bibikova
et al. 2003). Zinc-finger nucleases have a DNA recognition domain composed of an
array of Cys2–His2 zinc fingers. The zinc fingers recognize and bind to specific
nucleotide triplets. Zinc fingers are available that recognize all GNN and ANN and
some CNN and TNN triplets, and multiple zinc fingers can be joined together to
generate DNA-binding arrays that recognize extended sequence patterns with great
specificity and high affinity. Fused to the zinc-finger array is a nuclease, typically a
nonspecific cleavage domain from a type IIS restriction endonuclease such as Fok I
(Kim et al. 1996). Fok I functions only as a dimer, and this has been capitalized
upon to enhance the target specificity of the ZFN. Each Fok I monomer is fused to a
zinc-finger array that recognizes a different DNA sequence; only when the two
recognition sites are in close proximity do the inactive monomers come together to
create a functional enzyme. By requiring DNA binding to activate the nuclease, a
highly site-specific restriction enzyme is created. Using a tobacco test system
Wright et al. (2005) have shown that chromosome breaks created by zinc-finger
nucleases greatly enhance the frequency of localized recombination. Homologous
recombination was measured by restoring function to a defective gus:nptII reporter
gene (also containing a ZFN recognition site) integrated at various chromosomal
sites in different transgenic tobacco lines. Protoplasts from each transgenic line
were electroporated with DNA encoding the nuclease and donor DNA to effect
repair of the reporter gene. Homologous recombination occurred in more than 10%
of the transformed protoplasts regardless of the reporter gene’s chromosomal
position. Approximately 20% of the gus:nptII reporter genes were repaired solely
by homologous recombination, whereas the remainder had associated DNA inser-
tions or deletions consistent with repair by both homologous recombination and
non-homologous end-joining. Using this strategy, it is possible to engineer the
DNA-binding domain encoded by zinc-finger nucleases to recognize a variety of
chromosomal target sequences in order to achieve high frequency gene targeting.
Thus, such transgenic strategies are currently being aggressively employed in gene
targeting studies in plants. If successful, gene targeting will have tremend-
ous application in plant functional genomics as well as in controlled transgene
“docking” or even transgene “upgrades” for the sustainable deployment of transgenes
of agronomic importance.
Specific genomic deletions are also useful in functional genomics. Heterologous
site-specific recombination systems like the bacteriophage P1 Cre-lox and the yeast
FLP–FRT systems have been shown to work in plants to generate specific deletions
(van Haaren and Ow 1993). Here, recombination occurs between two lox or FRT
sites, mediated respectively by the Cre- or FLP-recombinases. These systems can
also be delivered through transposons. For example, constructs carrying lox recom-
binase sites, both within the transposon (Ds-lox) and in the adjacent T-DNA have
been used to produce small deletions in lines with Ds-lox transposed to closely-
linked positions (Osborne et al. 1995). A Cre-lox site-specific recombination
system has also been used to remove Ac from Ds transposants by triggering cre
recombinase expression with Ds excision (Shaohong et al. 2004). The elimination
of the Ac transposase gene helps to stabilize the transposed Ds elements in the rice
genome.
10.5.2 Reverse Genetics Strategies
10.5.2.1 Site-Selected Insertions
In plant populations saturated with multiple endogenous transposon copies, it is

possible to identify knock-out mutations in a specific gene. For example, in maize,
snapdragon and petunia (Coen et al. 1989; Gerats et al. 1990; Walbot 1992) such
populations have provided genome saturation and knock-out mutations in specific
genes (Das and Martienssen 1995; Koes et al. 1995). Similarly, deletion mutant
populations generated by chemical and physical mutagens can be used to identify
mutants in specific genes by employing high-throughput screening methods includ-
ing PCR screening and targeting induced local lesions in genomes (TILLING).
Using the transgenic approach with T-DNA or Ac/Ds, it is theoretically possible
to achieve this saturation with substantial numbers of tagged lines, from which
DNA pools can be prepared. It is then possible to screen for mutations in a
particular gene by polymerase chain reaction (PCR) analysis, using pooled DNA as
a template and a gene-specific primer in combination with an insertion sequence-
specific primer. Recovered mutants can then be subjected to custom screening for
visible/obvious phenotypes. This type of reverse genetics approach can be very
powerful in identifying the functions encoded by unknown sequences which are
predicted to be important by other methods.
However, the chance of recovering an insert in a target gene is dependent on the
population size of the insertion lines, the size of the genome and the size of the gene
target. The two types of populations that are currently being created in different
laboratories worldwide include: (1) those with single- or low-copy stable insertion
elements such as T-DNA, Ds and I, and (2) those with multiple actively-transposing
elements such as Ac or En. In a population of ~110,000 Arabidopsis random
insertion mutants one would expect an insert every kb, with a ~99% chance of
mutating a gene of 5 kb (Krysan et al. 1999). The number of insertion mutants
needed to tag every gene in rice is estimated to be between 180,000 and 460,000
(Hirochika et al. 2004; Krishnan et al. 2009). Larger genomes such as barley, wheat
and maize would require much larger numbers of insertion lines.
10.5.2.2 Gene Knock-Downs with Silencing Transgenes
RNA silencing or gene silencing is a broad term used to describe mechanisms that
interfere with gene expression in most eukaryotic organisms. This interference
occurs either by suppression of gene transcription (transcriptional silencing) or
the initiation of sequence-specific mRNA degradation or inhibition of RNA trans-
lation (post-transcriptional gene silencing or PTGS). RNA silencing has evolved to
a high level of sophistication in the plant kingdom and is intimately involved in
viral defense, suppression of transposon activity, control of chromatin modification
and regulation of expression of genes involved in plant development (Waterhouse
et al. 2001). RNA silencing mechanisms may have several parallels with the
immune system of animals and there is evidence to suggest that it is likely to
have been a major factor in the evolution of eukaryotes from prokaryotes (Margis
et al. 2006).
Gene silencing has now become a powerful transgenic technology to selectively
suppress gene activity. Through the PTGS process, it is possible to block the
expression of endogenous genes by introducing synthetic gene constructs that
cause RNA interference (RNAi). These transgenes produce double-stranded RNA
transcripts having sequence identities with their target genes that specifically trigger
the degradation of endogenous gene transcripts. Theoretically, a specific gene or a
set of genes, of unknown function, can be selectively silenced and the consequence
of such “expression knock-outs” in the form of a phenotype can be studied.
However, quite often structurally-similar genes, having functions, which are spa-
tially and developmentally controlled in different ways, may be silenced simulta-
neously. This makes the functional characterization of individual genes potentially
more difficult. One way of circumventing this problem is to ensure that the
a LB P 35S RdRP M1 M3 Target Term SM RB

M2
Hairpin of target sequence

b LB 35S P Term SM RB
c LB 35S P Target Term SM RB
d Ub P
azzR
gus linker att R Term
att R1 CM ccd B att R2
e 35S P Target Target Target Target
f FMV P PG transgene
nos T spacer nos T
g A IV
5' II
IV I 3' 5' 3'
5'
III aniRNA
ANRina
A B
A B A B
III II
B
h T7 RdRP M1 M3 Target CP
M2
i LB 35 S RdRP M1 M3 Term RB
M2
j LB 35 S RdRP M Term RB
+
k LB 35 S CP Target Term RB
Fig. 10.5 Types of transgene constructs for RNA silencing in plants. (a) A plasmid containing
infectious Potato virus X (PVX) cDNA can be transcribed in vitro and inoculated onto the plant.
A component of the PVX cassette contains an inserted region of sequence from the targeted gene
(Helliwell and Waterhouse 2003). (b) A typical T-DNA plasmid that can express hairpin RNA in
plants. This construct can be introduced into the plant by DNA bombardment or stably transformed
by Agrobacterium-mediated transformation. The latter method requires a selectable marker. (c) A
T-DNA plasmid similar to the one above can express RNA with the target gene sequence located
upstream of the hairpin structure. This vector can potentially be used for high-throughput screen-
ing with a cDNA library. (d) The general structure of the pANDA construct with the Gateway
vector conversion system cloned in an anti-sense and sense direction and separated by gus linker.
PCR products corresponding to the targeted gene are cloned into the pENTRO/D-TOPO vector
followed by a LR clonase reaction to produce the final construct for transformation into rice
sequence selected for targeting is specific to an individual gene. On the other hand,
sequence-specific knock-outs can be used to block the expression of a whole class
of genes involved in a particular process by targeting a common conserved
sequence present in such gene families.
Perhaps the immediate use of RNAi technology is in elucidating the functions of
genes, which otherwise would show lethal phenotypes with insertion mutants. This
is because PTGS causes a reduced level of gene expression rather than a complete
gene inactivation. The molecular basis of PTGS remains to be fully elucidated
despite recent significant advances made in understanding the different silencing
pathways namely, (1) microRNA and trans-acting siRNA, (2) repeat-associated
siRNA and RNA-directed DNA methylation and the various key proteins involved
in these pathways (Curtin et al. 2007).
A number of gene-silencing platforms have been developed for delivering
gene silencing through transgenes in plants including sense and antisense
transgenes, amplicon transgenes, hairpin RNA transgenes, direct-repeat and
30 -inverted repeat transgenes, and artificial miRNA transgenes. These are described
in Fig. 10.5.
Several plant single-stranded RNA (ssRNA) viruses have also been effectively
used as silencing vectors. Virus-induced gene silencing (VIGS) was first demon-
strated in tobacco with an infectious tobacco mosaic virus (TMV) clone (Kumagai
et al. 1995). In principle, VIGS is achieved by producing a recombinant infectious
virus containing a 300–800 nucleotide plant target gene sequence. Viral infections
can be established with purified viral RNA in the absence of viral coat proteins.
Viral RNA transcripts, synthesized in vitro from a plasmid containing a cDNA
encoding the recombinant virus genome, have been widely used to initiate virus
infections. Alternatively, the recombinant viral cDNA can be cloned into T-DNA
vectors (with appropriate promoters) and delivered to the plant via Agrobacterium
infection. Inside the plant cell, virally encoded RNA-dependent RNA polymerase
generates both sense and antisense recombinant viral RNA (containing the target
gene sequences) that has the potential to hybridize to form dsRNA and thereby
trigger PTGS mechanisms to induce target gene silencing. Several plant viruses
such as potato virus X (PVX), tobacco rattle virus (TRV), cabbage leaf curl virus
(cbLCV) and tobacco mosaic virus (TMV) can be used to induce PTGS in various
plant species.
There are several advantages to using the VIGS system over other methods such
as insertional mutagenesis or transgene-derived hpRNA. VIGS is a rapid method
<
Fig. 10.5 (continued) (Miki and Shimamoto 2004). (e) Multiple direct repeats of chloramphenicol
acetyltransferase (CAT) and gus gene sequences were shown to trigger efficient PTGS called
direct repeat-induced PTGS (driPTGS). (f) Schematic representation of the SHUTR construct
containing an inverted repeat of the 30 -untranslated region from the Agrobacterium nos gene. (g)
Artificial miRNAs are constructed using overlapping PCR on an endogenous miRNA precursor.
Primers are designed to replace the existing miRNA and miRNA* sequences with artificial
sequences (gray). The artificial miRNA is generated by combing all three PCR products A-IV,
II-III and I-B in a single reaction with primers A and B (Schwab et al. 2006). (h) VIGS vectors
(reproduced from Curtin et al. 2007)
for generating multiple mutants (in either related or unrelated genes) and can be
applied to monocotyledonous plants such as barley and polyploids such as wheat
(Scofield et al. 2005). VIGS can be applied to both mature and juvenile plants to
induce the silencing of embryogenesis-related genes and genes required for germi-
nation, which may be intractable by other methods. VIGS can also be applied to
plants, which are difficult to transform. Some of the limitations of VIGS are the
non-availability of viral infectious clones for all crop plants (host range), masking
of the phenotype by viral symptoms, quarantine issues and target sequence size
restrictions (Watson et al. 2005).
10.5.2.3 Gene Activity Disruption with Mutant Transgenes
Transgenes encoding mutant proteins, especially regulatory proteins, have the

potential to disrupt the activity of the endogenous wild type protein thus producing
a dominant negative mutant phenotype (Herskowitz 1987). Such a transgenic
approach to introduce dominant mutations was demonstrated for the Arabidopsis
MADS box gene AG, with the overexpression of specific protein domains
(Mizukami et al. 1995), thus providing an insight into protein regulatory mechan-
isms in flower development.
10.6 Conclusion and Future Prospects
The commercial successes with the first wave of genetic engineering involving
herbicide and/or insect resistance have provided great impetus for transgenic
research worldwide in a wide variety of traits and crops. The scientific community
by and large is optimistic about the potential of transgenic crop plants, not only in
alleviating major production constraints such as insect pests, pathogens, salinity
and extremes of temperature and drought, but also in producing designer crops with
high product value. Besides Intellectual property and regulatory constraints, and
consumer perception issues, there are still some unresolved technical limitations
associated with plant transgenic technology. For many plant species, efficient
transformation is still highly genotype dependent and the “useful transformation”
frequency is still very low. This could be due to the use of inadequate promoter-
gene combinations, integration position effects, insertional inactivation of endoge-
nous genes, somaclonal variation, transgene silencing or pleiotropic effects arising
from expression of the introduced transgene.
The expectation is that functional genomics will reveal novel genes and gene
control sequences conferring more complex traits such as abiotic stress tolerance,
yield, vigor and nutritional quality. As a foundation for functional genomics,
genome sequencing has been completed for two model plants, Arabidopsis and
rice. Sequencing (DNA and cDNA) efforts are now underway for several other
so-called transgenic crop plants.
Functional genomics has also expanded the scope of biological investigation

from a one-gene approach to a more system-based holistic approach encompassing
genome, transcriptome, proteome and metabolome. Such an approach will
undoubtedly provide considerable, and possibly dramatic, insights into the epistatic
interactions between seemingly unrelated genes, which account for optimal plant
growth, performance and productivity. Because of the objective nature of these
powerful new bioinformatics technologies, they will also reveal a number of novel,
and potentially-valuable strategies for the design of future food crop plants
designed to keep pace with the uncertainties of predicted climate change. Trans-
genics has become an integral part of the various functional genomics strategies
being employed. For example, transgenically produced insertion lines and the
flanking sequences of model plants (rice and Arabidopsis) will enable the scientific
community to find one or more insertions in any given gene. Following the
association of a phenotype with a specific gene, the level or pattern of expression
of that gene can be altered to achieve the desired effect. This can be done using
transgenic methods to either reduce gene expression by RNAi technology, or to
overexpress the gene of interest using suitable promoters. These specifically altered
lines can then be incorporated into breeding programs. These transgenic solutions
may be used if public antipathy to GM food crops can be overcome. Until then,
novel gene sequences can at least be used as useful molecular markers in classical
breeding efforts.
Acknowledgments The authors wish to thank Dr. Jake Jacobsen for critical reading of the
manuscript.
References
Antonio BA, Buell CR, Yamazaki Y, Yap I, Perin C et al (2007) Informatics resources for rice
functional genomics. In: Upadhyaya NM (ed) Rice functional genomics - challenges, progress
and prospects. Springer, New York, USA, pp 355–394
Bechtold N, Pelletier G (1998) In planta Agrobacterium-mediated transformation of adult Arabi-
dopsis thaliana plants by vacuum infiltration. Methods Mol Biol 82:259–266
Bennett MD, Leitch IJ (2005) Plant DNA C-values database (release 4.0, Oct. 2005): http://www.
kew.org/cvalues/
Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designed
zinc finger nucleases. Science 300:764
Birch RG (2000) Application of gene transfer to crop improvement. In: O’Brien L, Henry RJ (eds)
Transgenic cereals. American Association of Cereal Chemists, Minnesota, USA, pp 267–276
Chin HG, Choe MS, Lee SH, Park SH, Koo JC et al (1999) Molecular analysis of rice plants
harboring an Ac/Ds transposable element-mediated gene trapping system. Plant J 19:615–623
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transfor-
mation of Arabidopsis thaliana. Plant J 16:735–743
Coen ES, Robbins TP, Almeida J, Hudson A, Carpenter R (1989) Consequences and mechanisms
of transposition in Antirrhinum majus. In: Berg DE, Howe MM (eds) Mobile DNA. American
Society for Microbiology, Washington DC, pp 413–436
Curtin SJ, Wang M-B, Watson JM, Roffey P, Blanchard CL et al (2007) RNA silencing and
its application in functional genomics. In: Upadhyaya N (ed) Rice functional genomics -
challenges, progress and prospects. Springer, New York, USA
Das L, Martienssen R (1995) Site-selected transposon mutagenesis at the hcf106 locus in maize.
Dunn WB, Bailey NJ, Johnson HE (2005) Measuring the metabolome: current analytical techno-
logies. Analyst 130:606–625
Evans MJ, Smithies O, Capecchi MR (2001) Mouse gene targeting. Nat Med 7:1081–1090
Feldmann KA (1991) T-DNA insertion mutagenesis in Arabidopsis: mutational spectrum. Plant J
1:71–82
Gerats AG, Huits H, Vrijlandt E, Marana C, Souer E et al (1990) Molecular characterization of a
nonautonomous transposable element (dTph1) of petunia. Plant Cell 2:1121–1128
Gherbi H, Gallego ME, Jalut N, Lucht JM, Hohn B et al (2001) Homologous recombination in
planta is stimulated in the absence of RAD50. EMBO Rep 2:287–291
Greco R, Ouwerkerk PB, Taal AJ, Sallaud C, Guiderdoni E et al (2004) Transcription and
somatic transposition of the maize En/Spm transposon system in rice. Mol Genet Genom
270:514–523
Guiderdoni E, An G, Yu S-M, Hsing Y-I, Wu C (2007) T-DNA insertion mutants as a resource for
functional genomics. In: Upadhyaya NM (ed) Rice functional genomics - challenges, progress
and prospects. Springer, New York, USA, pp 182–321
Hanin M, Volrath S, Bogucki A, Briker M, Ward E et al (2001) Gene targeting in Arabidopsis.
Plant J 28:671–677
Helliwell C, Waterhouse P (2003) Constructs and methods for high-throughput gene silencing in
plants. Methods 30:289–295
Herskowitz I (1987) Functional inactivation of genes by dominant negative mutations. Nature
329:219–222
Hieter P, Boguski M (1997) Functional genomics: it’s all how you read it. Science 278:601–602
Hirochika H, Guiderdoni E, An G, Hsing YI, Eun MY et al (2004) Rice mutant resources for gene
discovery. Plant Mol Biol 54:325–334
IRGSP (2005) The map-based sequence of the rice genome. Nature 436:793–800
Itoh T (2007) Rice genome annotation: beginning of functional genomics. In: Upadhyaya NM
(ed) Rice functional genomics - challenges, progress and prospects. Springer, New York, USA,
pp 21–30
James C (2008) Global status of commercialized biotech/GM crops: 2008. ISAAA Brief No 39.
ISAAA, Ithaca, NY
Johnson AAT, Yu S-M, Tester M (2007) Activation tagging systems in rice. In: Upadhyaya NM
(ed) Rice functional genomics - challenges, progress and prospects. Springer, New York, USA,
pp 333–353
Khush GS (1997) Origin, dispersal, cultivation and variation of rice. Plant Mol Biol 35:25–34
Kikuchi S, Satoh K, Nagata T, Kawagashira N, Doi K et al (2003) Collection, mapping, and
annotation of over 28,000 cDNA clones from japonica rice. Science 301:376–379
Kim YG, Cha J, Chandrasegaran S (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I
cleavage domain. Proc Natl Acad Sci USA 93:1156–1160
Koes R, Souer E, van Houwelingen A, Mur L, Spelt C et al (1995) Targeted gene inactivation in
petunia by PCR-based selection of transposon insertion mutants. Proc Natl Acad Sci USA
92:8149–8153
Komatsu M, Chujo A, Nagato Y, Shimamoto K, Kyozuka J (2003) FRIZZY PANICLE is required
to prevent the formation of axillary meristems and to establish floral meristem identity in rice
spikelets. Development 130:3841–3850
Krishnan A, Guiderdoni E, An G, Hsing YI, Han CD et al (2009) Mutant resources in rice for
functional genomics of the grasses. Plant Physiol 149:165–170
Krysan PJ, Young JC, Sussman MR (1999) T-DNA as an insertional mutagen in Arabidopsis.
Plant Cell 11:2283–2290
Kumagai MH, Donson J, Della-Cioppa G, Harvey D, Hanley K et al (1995) Cytoplasmic

inhibition of carotenoid biosynthesis with virus-derived RNA. Proc Natl Acad Sci USA
92:1679–1683
Margis R, Fusaro AF, Smith NA, Curtin SJ, Watson JM et al (2006) The evolution and diversifi-
cation of Dicers in plants. FEBS Lett 580:2442–2450
Margis-Pinheiro M, Zhou XR, Zhu QH, Dennis ES, Upadhyaya NM (2005) Isolation and
characterization of a Ds-tagged rice (Oryza sativa L.) GA-responsive dwarf mutant defective
in an early step of the gibberellin biosynthesis pathway. Plant Cell Rep 23:819–833
Miki D, Shimamoto K (2004) Simple RNAi vectors for stable and transient suppression of gene
function in rice. Plant Cell Physiol 45:490–495
Mizukami Y, Huang H, Tudor M, Hu Y, Ma H (1995) Functional domains of the floral regulator
AGAMOUS: characterization of the DNA binding domain and analysis of dominant negative
mutations. Plant Cell 8:831–845
Osborne BI, Wirtz U, Baker B (1995) A system for insertional mutagenesis and chromosomal
rearrangement using the Ds transposon and Cre-lox. Plant J 7:687–701
Reiss B (2003) Homologous recombination and gene targeting in plant cells. Int Rev Cytol
228:85–139
Reiss B, Schubert I, Kopchen K, Wendeler E, Schell J et al (2000) RecA stimulates sister
chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in
plants transformed by Agrobacterium. Proc Natl Acad Sci USA 97:3358–3363
Sallaud C, Gay C, Larmande P, Bes M, Piffanelli P et al (2004) High throughput T-DNA insertion
mutagenesis in rice: a first step towards in silico reverse genetics. Plant J 39:450–464
Satoh K, Doi K, Nagata T, Kishimoto N, Suzuki K et al (2007) Gene organization in rice revealed by
full-length cDNA mapping and gene expression analysis through microarray. PLoS ONE 2:e1235
Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing
by artificial microRNAs in Arabidopsis. Plant Cell 18:1121–1133
Scofield SR, Huang L, Brandt AS, Gill BS (2005) Development of a virus-induced gene-silencing
system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust
resistance pathway. Plant Physiol 138:2165–2173
Shaked H, Melamed-Bessudo C, Levy AA (2005) High-frequency gene targeting in Arabidopsis
plants expressing the yeast RAD54 gene. Proc Natl Acad Sci USA 102:12265–12269
Shalev G, Sitrit Y, Avivi-Ragolski N, Lichtenstein C, Levy AA (1999) Stimulation of homologous
recombination in plants by expression of the bacterial resolvase ruvC. Proc Natl Acad Sci USA
96:7398–7402
Shaohong Q, Billizzi M, Jeon J-S, Leach J, Ronald P et al (2004) Transposon tagging in rice using
a novel Ds element, cre-lox site-specific recombination and an inducible Ac transposase. In:
Plant and animal genomes XII conference, San Diego, CA
TAGI (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature
408:796–815
Takahashi H, Hotta Y, Hayashi M, Kawai-Yamada M, Komastu S et al (2005) High throughput
metabolome and proteome analysis of transgenic rice plants (Oryza sativa L.). Plant Biotechnol
22:47–60
Tarpley L, Roessner U (2007) Metabolomics: enabling systems-level phenotyping in rice func-
tional genomics. In: Upadhyaya NM (ed) Rice functional genomics - challenges, progress and
prospects. Springer, New York, USA, pp 91–107
Terada R, Urawa H, Inagaki Y, Tsugane K, Iida S (2002) Efficient gene targeting by homologous
recombination in rice. Nat Biotechnol 20:1030–1034
Upadhyaya NM, Zhou X-R, Zhu Q-H, Eamens A, Wang M-B et al (2000) Transgenic rice. In:
Henry RJ, O’Brien L (eds) Transgenic cereals. American Association of Cereal Chemists,
Minnesota, USA, pp 28–87
Upadhyaya NM, Zhu QH, Zhou XR, Eamens AL, Hoque MS et al (2006) Dissociation (Ds)
constructs, mapped Ds launch pads and a transiently-expressed transposase system suitable for
localized insertional mutagenesis in rice. Theor Appl Genet 112:1326–1341
van Haaren MJ, Ow DW (1993) Prospects of applying a combination of DNA transposition and
site-specific recombination in plants: a strategy for gene identification and cloning. Plant Mol
Biol 23:525–533
Walbot V (1992) Strategies for mutagenesis and gene cloning using transposon tagging and
T-DNA insertional mutagenesis. Annu Rev Plant Physiol Plant Mol Biol 43:49–82
Waterhouse PM, Wang MB, Lough T (2001) Gene silencing as an adaptive defence against
viruses. Nature 411:834–842
Watson JM, Fusaro AF, Wang M, Waterhouse PM (2005) RNA silencing platforms in plants.
FEBS Lett 579:5982–5987
Wright DA, Townsend JA, Winfrey RJ, Irwin PA, Rajagopal J et al (2005) High-frequency
homologous recombination in plants mediated by zinc-finger nucleases. Plant J 44:693–705
Yu J, Hu S, Wang J, Wong G, Li S et al (2002) A draft sequence of the rice genome (Oryza sativa
L. ssp. indica). Science 296:79–92
Yu J, Wang J, Lin W, Li S, Li H et al (2005) The genomes of Oryza sativa: a history of
duplications. PLoS Biol 3:e38
Zhu QH, Hoque MS, Dennis ES, Upadhyaya NM (2003) Ds tagging of BRANCHED FLORET-
LESS 1 (BFL1) that mediates the transition from spikelet to floret meristem in rice (Oryza
sativa L). BMC Plant Biol 3:6
Zhu Q-H, Eun MY, Han C-D, Kumar CS, Pereira A et al (2007) Transposon insertional mutants: a
resource for rice functional genomics. In: Upadhyaya NM (ed) Rice functional genomics -
challenges, progress and prospects. Springer, New York, USA, pp 223–271
Chapter 11
Deployment: Regulations and Steps
for Commercialization
Kelly D. Chenault Chamberlin
11.1 Introduction
Genetically modified (GM) crops produced by genetic engineering continue to

increase in production worldwide. In 2007, 23 countries produced 282.3 million
acres of GM crops, up 30.3 million acres since 2006 [International Service of
AgriBiotech Applications (ISAAA)]. The US ranks first in GM crop production,
followed by Argentina, Brazil and Canada, in that order. The potential for economic
and social gain from the production of GM crops is generally greater in developing
countries/economies, since there is usually a higher incidence of disease/pests and
larger potential for yield increase (Abdalla et al. 2003; Qaim 2005). According to
Nossal et al. (2008), GM crop production in 2007 increased by 20% in countries
with emerging economies versus 6% in developed countries.
There is no denying that in today’s economy, there is a great demand to decrease
production costs and increase yield, advantages that are offered by biotechnology.
However, before any GM crop can enter the public domain, it must first be
approved or “de-regulated” for commercial production and public consumption.
The emergence of GM crops that offer clear economic and/or health advantages to
producers and consumers has forced different countries to develop regulations
governing the release of transgenic plants for experimental or commercial pur-
poses. This chapter discusses the regulatory frameworks and policy principles
governing GM crop research and production in those countries playing major
roles in worldwide GM crop production.
K.D. Chenault Chamberlin

Wheat, Peanut, and Other Field Crops Unit, USDA-ARS, 1301 N. Western, Stillwater, OK 74075,
USA
e-mail: kelly.chamberlin@ars.usda.gov

392 K.D. Chenault Chamberlin
11.2 GM Crop Regulation in the United States
11.2.1 History
Oversight of genetically modified organisms (GMOs), including transgenic crops,

began in the US in the early 1970s with the advent of recombinant DNA (rDNA)
technology. The scientific community was first to recognize the huge potential and
possible risks associated with rDNA technology and outlined its own internal
operation guidelines in a meeting among concerned scientists held in Asilomar,
California, in 1975 (Berg et al. 1981). Shortly thereafter the US federal government
followed with their own set of rules regulating rDNA research in projects funded by
the government agencies such as the National Institutes of Health (NIH), United
States Department of Agriculture (USDA), Food and Drug Administration (FDA)
and the Environmental Protection Agency (EPA) (NIH 1976, 1978). As it became
apparent that crops would be improved using rDNA technology, groups began to
seriously examine the impact of the release of transgenic crops and the need for
regulatory action. In the early 1980s, several reports were issued on the risks of
transgenic plants by various organizations including the Organization for Economic
Co-operation and Development (OECD) and the National Institute of Health (NIH)
(OECD 1982; NIH 1983). These concerns coincided with the first official report of
transgenic plants, which described transgenic tobacco, which was resistant to
methotrexate and kanamycin (Herrera-Estrella et al. 1983; Schell et al. 1983).
The rapid advances being made by rDNA research prompted the US federal
government to form a committee at the Office of Science and Technology Policy
(OSTP) to create regulatory policy regarding rDNA research and products. The
result was a publication by the OSTP in 1986 (http://www.ostp.gov) outlining the
approach to be taken in regulating rDNA products, the basic concept of which is
still being used to regulate transgenic crops in the US today. The OSTP urged
regulators to focus less on the rDNA process and more on the risks of the products
of such research and thus assigned regulatory control to the relevant federal
agencies of the USDA, FDA and EPA. The roles of each agency are outlined in
Table 11.1. All three of these agencies have adopted this “product-based” approach
Table 11.1 US regulatory authorities for agricultural biotechnology products

Agency Jurisdiction Laws
USDA Plant pests, plant veterinary biologics Federal Plant Pest Act (FPPA)
FDA Food, feed, food additives, veterinary Federal Food, Drug, and Cosmetic Act
drugs, human drugs, medical devices (FFDCA)
EPA Microbial and plant pesticides, new uses Federal Insecticide, Fungicide, and
of existing pesticides, novel Rodenticide Act (FIFRA); FFDCA, Toxic
microorganisms Substances Control Act (TSCA)
Source: http://www.agbios.com
11 Deployment: Regulations and Steps for Commercialization 393
of GM crop regulation. The US regulatory framework for agricultural biotechnol-

ogy has been the subject of several reviews (Belson 2000; McHughen and Smyth
2008). For an overview of the US coordinated regulatory framework for biotech-
nology, visit the website: http://usbiotechreg.nbii.gov/index.asp.
11.2.2 US Regulatory Framework
11.2.2.1 The USDA
The USDA is composed of 17 government agencies, all dealing with various

aspects of food, agriculture, natural resources, rural development and related issues
based on sound public management. The USDA agency involved in GM crop
regulation is the Animal and Plant Health Inspection Service (APHIS: http://
www.aphis.usda.gov). APHIS is responsible for protecting and promoting US
agricultural health, administering the Animal Welfare Act, and carrying out wild-
life damage management activities. Within APHIS is the office of Biotechnology
Regulatory Services (BRS; http://www.aphis.usda.gov/biotechnology/brs_main.
shtml), which is responsible for regulating GM plants at all levels, including
research and development, import, interstate movement, field trials and commercial
release and cultivation. BRS derives its authority to write regulations from provi-
sions of the Plant Protection Act, which is a part of the larger Agriculture Risk
Protection Act of 2000 (APHIS 2000). Congress authorizes various parts of USDA
to regulate specified areas of US agriculture under these federal statutes.
APHIS uses the term biotechnology to mean the use of rDNA technology, or
genetic engineering (GE) to modify living organisms. APHIS regulates certain
GMOs that may pose a risk to plant or animal health. In addition, APHIS partici-
pates in programs that use biotechnology to identify and control plant and animal
pests. APHIS considers a plant to be a “regulated article” if the plant and/or its
progeny arose from a specific (single) transformation event, and such plants would
continue to be regulated under APHIS until such time that a petition for non-
regulated status was approved. Once the GM plant achieves non-regulated status,
it may be released commercially with no further USDA action required. APHIS
exercises its regulatory authority through a system that includes both permits and
notifications. The regulatory process generally begins as the regulated article
approaches the field trial stage.
Notifications
Most field trials are approved under the notification procedure. Notification is an
administratively streamlined alternative to the permit. The goal of the notification
procedure is the same as the permit system: preventing the unintended release of
the regulated article. In order to use the notification procedure, the genetically
engineered (GE) plant must first meet all of the six eligibility criteria listed below:
1. The recipient organism is not listed as a noxious weed or considered to be a weed
in the area of release into the environment
2. Stable integration of genetic material has been achieved
3. The function of the introduced genetic material is known and does not cause
plant disease
4. The characteristics of the introduced gene or gene product do not include the
production of an infectious entity, encode substances that are known or likely to
be toxic or hazardous to non-target organisms, or encode products intended for
pharmaceutical or industrial use
5. The introduced genetic material does not pose significant risk of creating new
plant viruses
6. The introduced genetic material does not contain nucleic acid or coding
sequences from human or animal pathogens
Next, the introduction (the importation, interstate movement, or environmental
release) must meet all performance standards. The performance standards are a set
of six conditions that must be met in order to ensure that the regulated article is
introduced in such a way that it is not inadvertently released beyond the proposed
introduction, allowing it to persist in the environment. Generally, performance
standards are characteristics associated with the act of introduction. The six
required performance standards are listed below:
1. Shipping and maintenance at destination must be done to ensure that plant
material is unlikely to be disseminated into the environment
2. Inadvertent mixing of materials in environmental releases is prevented
3. Identity of material is known and maintained while in use and plant parts are
devitalized after use
4. Viable vector agents are not associated with the regulated article
5. No persistence of the regulated article or its progeny in the environment is
permitted
6. No viable material of the regulated article is allowed to volunteer in subsequent
sessions
Protocols must be designed by the applicant to meet all performance standards
for regulated articles and must be implemented during field trials. Once an applicant
has determined that their regulated article meets all required eligibility criteria and
performance standards, a letter of notification may be submitted to the APHIS BRS
for review. Introductions cannot proceed without an acknowledgement letter from
APHIS. Once approved, field trials are subject to inspection by federal and/or state
inspectors and a field test report must be submitted to APHIS within 6 months of
termination of the release. Online application of notification is now possible on the
APHIS website. More details on the requirements of the notification procedure can
be found in the USDA-APHIS BRS User’s Guide (http://www.aphis.usda.gov/brs/
pdf/Notification_Guidance.pdf).
Permits
For those GM plants not eligible for notification approval, the APHIS BRS requires
a permit to be issued. The permit procedure is much more involved than that of the
notification, requiring more data and information from the applicant. In addition to
the information required for notification, permit applicants must provide a detailed
description of the problem or “need” being addressed by release of the regulated
article, along with a detailed description of the article itself. The article description
must include details on the donor and recipient of the gene being introduced,
method of introduction, and post-transformation analysis of resulting regulated
articles. Detailed information must also be provided regarding the field testing
protocol and containment issues, including assessment of the article’s capability
to escape containment and possible consequences of such actions. More informa-
tion of the permit application process can be found at http://www.aphis.usda.gov/
biotechnology/permits.shtml.
Deregulation and Deployment of GM Crops by the USDA
USDA-APHIS regulations provide a petition process for the determination of non-

regulated status. If a petition is granted, that organism will no longer be considered
a regulated article and will no longer be subject to oversight by USDA-APHIS.
APHIS grants non-regulated status if the GE organism poses no more of a plant pest
risk than an equivalent non-GE organism (substantial equivalence). If a regulated
article is very similar to a GE organism that has already been granted non-regulated
status, APHIS may extend non-regulated status to that organism. The petitioner
must supply information such as the biology of the recipient plant, experimental
data and publications, genotypic and phenotypic descriptions of the genetically
engineered organism, and field test reports. The agency evaluates a variety of issues
including the potential for plant pest risk; disease and pest susceptibilities; the
expression of gene products, new enzymes, or changes to plant metabolism;
weediness and impact on sexually compatible plants; agricultural or cultivation
practices; effects on non-target organisms; and the potential for gene transfer to
other types of organisms. A notice is filed in the Federal Register and public
comments are considered on the environmental assessment and determination
written for the decision on granting the petition.
The National Environmental Policy Act (NEPA) of 1970 requires federal agen-
cies to investigate environmental impacts prior to making decisions or taking
actions that could pose environmental risks. In compliance with the NEPA,
APHIS will respond to a petitioner by generating an Environmental Assessment
(EA) of the regulated article which reviews the environmental consequences of
releasing the article. If the EA is in favor of article release, a Finding of No
Significant Impact (FONSI) statement will then be issued which justifies the EA
and the agency’s decision for release.
If the EA is inconclusive, a Notice of Intent (NOI) may be issued, requiring a

more detailed analysis of the impact of article release and demand the preparation
of an Environmental Impact Statement (EIS) before a FONSI can be issued. For a
complete listing of the steps involved in deregulation of GM plants, visit the NEPA
website (www.nepa.gov). Copies of the USDA-APHIS documents are available to
the public.
Current Status of US Deployment of GM Crops
After more than a decade since the introduction of GM crops, only four countries
plant 99% of the world’s GM crops. The US represents 55% of the area grown,
while Argentina, Canada and Brazil account for the balance. The first GM crop to
achieve deregulated status in the US was the Flavr SavrTM tomato from Calgene in
1992, in which an antisense polygalacturonase transgene from tomato resulted in
delayed fruit ripening. Since that initial event, 74 additional petitions for deregula-
tion of GM crops have been granted, 10 are pending decision, and over 35 GM
crops are in development for market (www.trufoodnow.org/crop/pipeline.html).
Among the GM crops approved for deregulated commercial production in the US
are soybean, corn, rice, cotton, sugar beet, rapeseed, tobacco, potato, flax, beet,
plum, papaya and squash. Estimated percentage of crops that are GM produced in
the US as of 2008 are at 91% for soybean, 73% of corn, 87% of cotton, 75% of
canola and more than 50% of Hawaiian papaya. For a complete and updated listing
of GM crops achieving nonregulated status in the US, visit http://www.aphis.usda.
gov/brs/not_reg.html.
11.2.2.2 The US Food and Drug Administration
The Food and Drug Administration (http://www.fda.gov) is part of the US Depart-

ment of Health and Human Services and consists of nine internal centers, of which
the Center for Food Safety and Nutrition and the Center for Veterinary Medicine
are responsible for the evaluation of GE foods and feeds. The FDA is responsible
for protecting the public health by assuring the safety, efficacy, and security of
human and veterinary drugs, biological products, medical devices, our nation’s
food supply, cosmetics, and products that emit radiation. The FDA is also respon-
sible for advancing the public health by helping to speed innovations that make
medicines and foods more effective, safer, and more affordable; and helping the
public get the accurate, science-based information they need to use medicines and
foods to improve their health. The FDA receives its regulatory authority from the
Federal Food, Drug and Cosmetic Act. In 1992, the FDA published a policy
statement and testing guidelines for foods developed using all methods of plant
breeding, including the use of genetic engineering. These guidelines explain the
types of food safety questions that developers should address in evaluating the
safety of all plant-derived foods.
In general, the FDA evaluates GM crops to determine if they are substantially

equivalent (SE) to their non-modified counterparts, with focus on changes in
nutritional and anti-nutritional components such as allergens and toxins. GM
crops that do not differ significantly from a non-GM counterpart do not require
FDA review and approval, while those that have significant differences are consi-
dered “adulterated” and are subjected to FDA regulations.
Review of GM foods by the FDA is not legally mandated, but all GM crops
produced in the US have undergone FDA inspection. Most developers of GM crops
perform extensive analysis of their GM product, comparing its composition to that
of a non-modified version, before considering application for release. The deve-
loper then submits a synopsis of these data to the FDA for review. Such data will
include an evaluation of the crop’s genetic stability, compositional and nutritional
analysis of the product and changes in allergenicity and/or toxicity as compared to a
non-GM counterpart. To date, the FDA has not identified any examples of GM
foods or crops as “adulterated.”
The FDA has no information that the use of biotechnology creates a class of food
that is different in quality, safety or any other attribute from food developed using
conventional breeding techniques and therefore does not require the disclosure of
the inclusion of GM foods on food labels. Any significant differences between the
bioengineered food and its conventional counterpart do have to be disclosed in
labeling. These would include differences in nutritional properties, the presence of
an allergen that consumers would not expect in the food, or any property that would
require different handling, storage, cooking or preservation. For example, when a
manufacturer produced a line of soybeans whose oil had higher levels of oleic acid
than found in conventional soybean oil, the FDA agreed to name the product “high-
oleic soybean oil” to distinguish it from traditional soybean oil. The high-oleic oil
can be used in frying without the need for the chemical process of hydrogenation,
which produces trans-fat. Food processors may voluntarily label either the presence
or absence of a genetically engineered food in their products as long as the
information is truthful and not misleading to consumers. The FDA has produced
guidance to the industry for this type of labeling.
11.2.2.3 The US Environmental Protection Agency
The mission of the Environmental Protection Agency (EPA: http://www.epa.gov) is

to protect human health and the environment. The EPA is largely concerned with
pesticides and their effect on the environment and human health and thus regulates
not only pesticides but also their properties and usage. The EPA was given authority
to regulate GM crops under the Federal Insecticide, Fungicide and Rodenticide Act
and focuses mainly on the potential pesticidal properties of GM plants. Examples of
such regulated plants include those that produce plant incorporated protectants
(PiPs) like Bt or other insecticides and plants that are resistant to pesticides such
as Roundup ReadyTM crops.
The data required by the EPA are similar to that of the USDA and FDA and
include a summary of the genetic manipulation performed, an analysis of any
known pesticidal properties and their origin, analysis of genetic stability, details
on allergenicity and/or toxicity and effect on non-target organisms. The EPA also
requires a detailed analysis of the pesticidal protein itself, including its sequence
(amino acid and entire nucleic acid sequence of the construct), source of origin,
allergenicity profile and overall expression pattern. The sustainability of the PiP
must also be determined with respect to the environment (i.e., decay rate, soil
response, etc.). The EPA also requires that appropriate resistant management
practices are in place so as to decrease or avoid the generation of resistant popula-
tions of insects and crop species.
11.3 GM Crop Regulation in Canada
11.3.1 History
Unlike the US where the market place determines the success or failure of a
traditionally developed crop variety, the Canadian government regulates the release
of conventionally bred crop varieties. The regulatory system governing GM crops is
an extension of the framework used for non-GM crop releases. The agency respon-
sible for such regulation is the Canadian Food Inspection Agency (CFIA) (http://
www.inspection.gc.ca). In 1985, the Canadian government enacted the Seeds Act
(Department of Justice Canada 1985b), which mandates the performance standards
for new germplasm. The Seeds Act focuses on germplasm uniformity, stability and
uniqueness but also established thresholds for environmental safety risks such as
gene flow, weediness, invasiveness and the effect on non-target organisms. Two
other Acts involved in the regulatory framework are the Food and Drugs act
(Department of Justice Canada 1985a), which deals with rules set for human
consumption, and the Feeds Act (Department of Justice Canada 1983), which sets
maximum tolerances for nutrients in livestock feed. It is the intent of the Canadian
government that the integration of all three Acts in the regulatory framework for
new plant varieties will identify all potential risks and ensure that new varieties will
pose no more of a threat to human and animal consumption than those already
existing in the environment. For a new crop variety to be approved for release in
Canada, it must be at least of equal quality (in set parameters) of existing commer-
cial varieties.
In Canada, a plant breeder is responsible for risk management of the research
and development of a new variety until the potential cultivar is ready to be
examined for registration, at which time the government system becomes heavily
involved in the process. Even for conventionally bred varieties, field tests must be
designed not only to examine the agronomic traits of the new cultivar, but also its
environmental risks. Field trial data are submitted to a recommending committee
organized by the CFIA, which will make a decision on the potential variety’s merit.
If registration is recommended, an application is then submitted to the Variety
Registration Office (VRO), also part of the CFIA, which retains the final authority
to grant variety approval. Once approved, the Canadian Seed Trade Association
manages the breeder’s seed increase and the Canadian Grain Commission is
responsible for setting and monitoring the standards for seed trade.
Much like the GM crop regulatory system of the US, Canada bases its regulatory
policies on the end-product that is established, not the process by which it was
created. However, within the Canadian system, a new and unique classification of
regulated plants has been established. Plants with novel traits (Table 11.2; PNTs)
are defined as any plant which has a new trait, not present or previously character-
ized among existing crop systems. By definition, a plant does not have to be
produced by genetic engineering to be a PNT. Many varieties developed by
conventional methods have been considered PNTs due to their novel nature (see
“novel foods” and “major change,” Table 11.2). Furthermore, some, but not all GM
plants are classified as PNTs. If a plant was developed using genetic engineering but
is not expressing a novel trait, that plant is exempt from PNT regulation and must
only be governed by conventional regulations.
11.3.2 PNT Regulatory Framework
No regulatory framework for the development of GM crops was in place at the time
of the first GM crop field trials in Canada in the late 1980s, but soon after, the
federal government began to require permits for such experiments. Currently in
Canada, biotechnology products are overseen by three agencies: The Canadian
Food Inspection Agency, Environment Canada and Health Canada. For a compre-
hensive review on the topic of regulating GM crops in Canada, see Smyth and
McHughen (2008). The roles of each agency are outlined in Table 11.3.
The CFIA is responsible for PNTs, novel fertilizers, novel livestock feed and
veterinary biologics. Within the CFIA is the Office of Food Biotechnology (OFB),
which coordinates the safety evaluation of novel foods produced from PNTs. It is
not possible in Canada to obtain a split permit where the crop would be approved
for animal feed but not human consumption. Thus all PNTs are also subject to the
regulatory approval of the other two agencies involved. Environment Canada
oversees the regulation of all animal products of biotechnology not covered under
other federal legislation and derives its authority from the Canadian Environmental
Protection Act (Department of Justice Canada 1999). Health Canada oversees the
safety assessment of goods, drugs, cosmetics, medical devices and pest control
products, much like the Food and Drug Administration of the US.
Table 11.2 Definitions

Plants with A plant variety/genotype possessing Includes plants produced using rDNA
novel characteristics that demonstrate techniques, chemical mutagenesis,
traits neither familiarity nor substantial cell fusion and conventional cross
(PNTs) equivalence to those present in a breeding
distinct, stable population of a
cultivated seed in Canada and that
have been intentionally selected,
created or introduced into a
population of that species through
a specific genetic change
Novel foods A substance, including a microorganism, Includes food products from genetically
that does not have a history of safe engineered plants, but also any food
use as a food product without a history of safe use
A food that has been manufactured, (e.g., novel fibers, single-cell
prepared, preserved or packaged by a protein), or an existing food product
process that has not been previously manufactured or packaged in a
applied to that food and causes the manner that results in a major
food to undergo a major change change
A food that is derived from a plant,
animal, or microorganism that has
been genetically modified such that
the plant, animal or microorganism
exhibits characteristics that were not
previously observed in that plant,
animal or microorganism – one or
more characteristics of the plant,
animal or microorganism no longer
fall within the anticipated range for
that plant, animal or microorganism
Major change A change in the food that, based on the
manufacturer’s experience or
generally accepted nutritional or
food science theory, places the
modified food outside the accepted
limits of natural variations for that
food with regard to:
– The composition, structure or
nutritional quality of the food or its
generally recognized physiological
effects
– The manner in which the food is
metabolized in the body
– The microbiological safety, the
chemical safety or the safe use of the
food
11.3.2.1 The Canadian Food Inspection Agency
In Canada, most GM crops have been considered to be “novel” and have been
subjected to rigorous regulation by the CFIA. All plants are evaluated on a
Table 11.3 Canadian federal authorities responsible for agricultural biotechnology products
Department/ Products regulated Relevant legislation Regulations
agency
Canadian Food Plants and seeds, including Consumer Feeds regulations
Inspection those with novel traits; Packaging and
Agency animals; animal Labeling Act
(CFIA) vaccines and biologics; Feeds Act Fertilizers regulations
fertilizers; livestock Fertilizers Act Health of animals
feeds regulations
Food and Drugs Act Food and drug regulations
Health of Animals
Act
Seeds Act
Plant Protection Act
Environment Biotechnology products Canadian New substances notification
Canada under CEPA such as Environmental regulations (these
microorganisms used in Protection Act regulations apply to
bioremediation; waste (CEPA) products not regulated
disposal, mineral under other federal
leaching or enhanced legislation)
oil discovery
Health Canada Foods; drugs; cosmetics; Foods and Drugs Act Cosmetics regulations
medical devices; pest CEPA Food and drug regulations
control products Pest Control Novel foods regulations
Products Act Medical devices regulations
New substances notification
regulations
Pest control products
regulations
case-by-case basis so the information required for approval is not always the same.
Environmental safety is of utmost concern to the CFIA. The PNT must be exten-
sively described and characterized molecularly. Information generally required
includes molecular characterization and comparison with its conventional counter-
part. The Regulatory Directive Dir94-08 (AAFC 1994) details the information
required for the environmental impact analysis which includes:
1. Taxonomy and pedigree analysis
2. Method of modification
3. Description of the novel trait(s), including gene product activity, decay, by-
product activity and any adverse effects on non-target organisms, including
humans
4. Biology of the PNT, including reproductive cycle and survival biology
5. Agricultural practices or behavior, including release sites, habitat, cultivation
practices and management
6. Potential gene flow of the PNT to related species and subsequent consequences
Unlike the US where guidelines are suggested but not mandatory for the research
and development stage of GM crops, the CFIA begins regulating PNT development
at a very early stage, requiring confined use even before field trials. PNTs with
commercial release potential are then selected for evaluation in field trials. Con-
fined field trials may be conducted if approved by the CFIA, and must be directed to
obtain the environmental safety assessment data, which analyze the PNT with
regards to the five risk categories listed above. The risk assessment of GM crops
in Canada has recently been critically reviewed (Barrett and Abergel 2000).
Following a CFIA review of the risk assessment data, a decision is rendered as to
whether the crop variety is eligible to apply to the CFIA for unconfined commercial
production. This application process can be delayed if additional scientific data are
requested. The CFIA has been criticized for requesting additional data too late in
the process and delaying commercialization of GM crops, which has resulted in the
loss of millions of dollars to producers.
11.3.2.2 Environment Canada and Health Canada
Environment Canada is responsible for the regulation of new substances that may
pose a threat to the environment and receives its authority from the Canadian
Environmental Protection Act (CEPA) (Department of Justice Canada 1999). A
“substance” is defined as animate matter by the CEPA and a “new substance” is one
that is not listed on the Domestic Substances List (DSL). All PNTs would logically
have the potential to produce a new substance and are therefore regulated by this
process. The assessment of the PNT or its new substance includes a determination
that the substance is not toxic. If the substance is suspected of being toxic,
Environment Canada is responsible for controlling and/or prohibiting its import
or manufacture pending the submission and assessment of additional data.
New substances are also subjected to regulation by Health Canada, which
reviews data related to human exposure and potential human toxicity risks. PNTs
may produce novel foods, defined as foods resulting from a process not previously
used, to produce food, genetically engineered foods, or from products without a
history as safe to be used as food. Any GM or novel food proposed for sale in
Canada is regulated by Health Canada. Novel foods are further defined as a food
having a major deviation from the accepted limits of (1) composition, structure or
nutritional quality, (2) metabolic properties or (3) microbiological, chemical or
food safety. Health Canada requires that the developers of novel foods must
examine and describe:
1. How the novel food was developed, including molecular characterization
2. Composition of the novel food compared to the conventional counterpart
3. Nutritional profile of the novel food compared to the conventional counterpart
4. Toxicity potential/profile
5. Allergenic potential/profile
Unlike the CFIA and Environment Canada, Health Canada will accept a history
of safe production and consumption elsewhere (outside Canada) as evidence for
regulatory approval. Once the GM crop/food has been approved for release/human
consumption by the Canadian regulatory framework, no post-market monitoring or
long-term surveillance is performed, due to the assumption that the biotechnology
product has been judged no more risky to consumers than the conventional coun-
terpart.
Since the Canadian regulatory system approved GM canola as the first GM crop
released for production in Canada in 1995, several other GM crops have also passed
through the system and acquired approval, including GM alfalfa, cotton, corn, flax,
tomato, potato, rice, soybean, squash, sugar beet, sunflower and wheat. In 2007,
there were 7.0 million hectares of GM crops grown in Canada.
11.4 Regulation in the European Union (EU)
11.4.1 History
As of January 2007, the EU had 27 member states and was comprised of over 450
million consumers making it the largest developed market in the world. In the
1980s, member states realized the necessity for establishing a common food policy
that would harmonize their standards and ensure easy trade. Resulting from this
action was a number of fragmented laws that created a set of directives often
described as opaque and incoherent. Included in these directives were regulations
involving food additives, pesticide residues in foods and contaminants in foods.
Differing social values and risk concerns between the US and EU consumers
have probably resulted in the differential regulatory approaches taken regarding
GM crops. Many opinion papers have been written comparing the two contrasting
approaches to regulating GM products (Haniotis 2000; Kalaitzandonakes 2000;
Dale 2002; Morris 2006; Ramjouè 2007). In response to public fears about geneti-
cally modified organisms (GMOs) in food, the European Union (EU) adopted a
regulation in 2001 governing GM plants and two more regulations establishing an
EU-wide system to trace and label GMOs and to regulate the commercialization
and labeling of food derived from GMOs in July 2003 (http://www.gmo-compass.
org). Finally, in May of 2004, the European Commission put an end to the “de
facto” moratorium on approving new GM products for the European market, which
had been in place since 1998.
The EU and the member states are now of the opinion that using genetic
engineering in agriculture and food production is permissible. Unlike the US and
Canada where GM crops are judged using an end product base, the EU regulatory
system is representative of the “process-based” approach, meaning that all crops
developed by genetic engineering must undergo rigorous regulatory oversight
before commercial release.
Individual GMOs must receive approval before they can be sold as seed or used
in food and feed. Approval is granted only after satisfying conditions of safety,
freedom of choice, labeling and traceability. The product must be safe and cannot
pose threats to human or animal health or to the environment. Consumers, farmers,
and businesses must be given the freedom to either use or to reject products made
from GMOs. It must remain possible in the long term to produce foods without the
use of genetic engineering. The term used for this is coexistence. Genetically
modified plants must be grown and handled in such a way that prevents uncon-
trolled mixing with conventional products. Whenever GMOs are intentionally used
in a food product, it must be clearly stated on the label. Every consumer is thereby
entitled to make an “informed decision.” Labeling is required even if GM content
cannot be detected in the final product. This is why all producers, suppliers, and
retailers must inform their buyers if GMOs were used in their products. To do
this, stakeholders must set up systems for keeping and sharing information and
documentation.
11.4.2 EU Regulations/Directives for GM Crops
Current EU legislation on GMOs is regarded as the strictest in the world. It deals

with a plethora of issues, including rules relating to the release of GMOs into the
environment, the traceability and labeling of GMOs and GMOs in food and animal
feed. The co-existence of GM and conventional crops is currently under discussion
at EU level.
There are two different set of rules governing the authorization of genetically
modified products in the EU: one is for the use of GM plants, while the other is for
food and feed made from them. The EU Governing bodies within the member states
must abide by these regulations regarding the commercial production/release of
GM crops.
11.4.2.1 Directive on the Deliberate Release into the Environment

of Genetically Modified Organisms (2001/18)
In effect since April 17, 2001, Directive 2001/18/EC (Official Journal of the
European Communities 2001) repeals former Directive 90/220/EEC and regulates
the deliberate release and placing on the market of GMOs and the environmental
release of GM crops. A guide to help in navigating Directive 2001/18/EC was
published in 2002 by the Department of Environment, Food and Rural Affairs
(DEFRA; http://www.defra.gov.uk). This directive defines the risk assessment and
decision making process on the release of GMOs into the environment as well as the
information required to be given to the public regarding GMO release, labeling and
traceability at all stages. Authorizations granted under this directive will be good
for 10 years and subject to post-market monitoring.
GM crops can only be allowed on the market once they have received authori-
zation. The authorization process is carried out by the EU, and the resulting
decision applies to all EU Member States. Under the terms of the amended release
directive (2001/18/EC), a post-release monitoring plan must accompany applica-
tions for cultivating GM plants. Authorization is contingent upon a parallel, general
monitoring plan, which in some cases can include special stipulations addressing
crop-specific areas of concern. The purpose of monitoring is to identify hidden
effects of large-scale GMO production on the environment. It can also be useful for
determining if potential negative effects noticed during safety assessments actually
cause problems.
To obtain authorization to release a GM crop for commercial production, an
application must be submitted to federal authorities of the pertaining EU member
state. Authorizations under EU Regulation 2001/18 are for a 10-year period. An
initial assessment (scientific opinion) by national agencies is made and documents
are then forwarded to the national authorities of the Member States and to the
European Commission. Safety assessments are then made by the European Food
Safety Authority (http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_
home.htm).
11.4.2.2 European Food Safety Authority (EFSA)
The EFSA was established in 2002 as the central authority for the scientific
evaluation of food and feed safety in the EU. As of 2005, EFSA is permanently
based in Parma, Italy.
EFSA was established based on the legal mandate of regulation 178/2002/EC of
the European Parliament and European Council addressing basic principles of food
law. New food law legislation and hence the EFSA were created in response to a
number of food scandals that shook consumer confidence. EFSA addresses two
main areas: (1) Scientific risk evaluation for all questions related to food and feed
safety and (2) informing the public of potential risks.
To assist with safety evaluations, EFSA is supported by eight scientific panels
composed of independent researchers from various EU Member States. The GMO
Panel is responsible for GMOs (including GM crops) and genetically modified food
and feed. EFSA offers a solid scientific basis for making informed political deci-
sions. The decisions themselves, however, are the responsibility of political bodies
such as the European Parliament, the European Commission, and the Council of
Ministers. EFSA cooperates closely with national authorities of all Member States.
The safety assessments for GM crops (Fig. 11.1.), food or feed required by the
EFSA are similar in many ways to those in other countries. A detailed understand-
ing of the modification process, the genes or constructs introduced and their
products and the resulting alteration of the plant and how it differs from the
conventional counterpart are all necessary for proper assessment. Comparative
analysis of the agronomic and nutritional properties of the GM crop with those of
GM Crop Non-GM Crop
molecular genotype composition phenotype

characteristics
Identification of Differences
inserted genes expressed proteins metabolites
Decision on Further Testing
gene transfer allergenicity/toxicity toxicity
Estimation of Actual Consumers Exposure to Hazards
Risk Assessment of GM Food Crops
Fig. 11.1 EU risk assessment strategy (comparative analysis) for GM food crops
Source: European Commission Report; genetically modified crops in the EU: food safety assess-
ment, regulation and public concerns; 2000
the traditional counterpart is required. For those GM crops with no traditional

counterpart, a product-specific safety assessment is carried out.
Regulation on Genetically Modified Food and Feed (1829/2003)
Enacted on April 19, 2004, the EU Regulation 1829/2003 (Official Journal of the
European Communities 2003) regulates food and/or feed that is made from or
contains GM plants. This regulation is an extension of EU Regulation on novel
foods (258/97), and basically requires that GM food or feed produces no harmful
effects on human or animal health or on the environment and that consumers are not
mislead in the labeling process of GM materials. Authorizations for release into
the public market require a safety assessment, which involves the determination
that the GM food or feed is as safe as the conventional counterpart. Regulation
1929/2003 also governs labeling of, detections methods for and post-market moni-
toring of GM products. Applications for authorization of GM product release are
submitted directly to the EFSA, where a scientific evaluation from expert com-
mittee takes place and results in a recommendation to the European Commission.
As for GM crop release, the European Commission considers the EFSA recom-
mendation and sends a draft for vote to the Standing Committee for the Food Chain
and Food Safety. The European Commission’s draft may be accepted or rejected
with a qualified majority. If no qualified majority can be reached, the European
Commission submits its draft to the Council of Ministers. Finally, a vote in the
Council of Ministers results in approval or rejection by qualified majority – without
qualified majority the Commission’s draft takes effect. Authorizations under EU
Regulation 1929/2003 are for a 10-year period.
Currently, valid authorizations for production in the EU have been approved for
GM cotton, flowers, maize, rapeseed, soybean and sugar beet. For a comprehensive
list of the status of the application process for GM crops in the EU and more on the
EU regulatory system, go to http://www.gmo-compass.org/eng/gmo/db/.
11.5 Regulation in Argentina and Brazil
11.5.1 GM Crop Regulation in Argentina
Argentina is second only to the US in GM crop production, producing 19.1 million

hectares of GM crops in 2007 (www.isaaa.org) and like the US, regulates GMOs on
a product, not process basis. In Argentina, the regulatory framework for Genetically
Modified Plant Organisms (GMPOs) consists of Regulations and Resolutions
issued by the Secretariat of Agriculture, Livestock, Fisheries and Food (SAGPyA;
http://www.sagpya.gov.ar). The permit to commercially release a GMPO is granted
by the Secretary based on three independent reviews, each issued by advisory
committees within the scope of the SAGPyA. The committees involved are (1)
The National Advisory Commission on Agricultural Biotechnology (CONABIA;
http://www.sagpya.mecon.gov.ar/new/0-0/programas/biotecnologia/index_en.php),
(2) The Technical Advisory Committee on the Use of GMOs which belongs to the
National Agri-food Health and Quality Service (SENASA; http://www.senasa.gov.
ar) and (3) The Directorate of Agricultural Markets (DNMA).
Regulatory requirements for GM crops have been incorporated into Argentina’s
farming sector general regulatory system. CONABIA was established to provide
advice and support on the supervision of activities related to agricultural biotech-
nology and bio-safety, especially those concerning authorizations for the environ-
mental release and commercialization of genetically modified plants and animals
(to be used in agricultural/livestock and aquaculture activities). It is also devoted to
define policies, and to design specific regulations, and to aid in the public diffusion
of the SAGPyA activities related to biotechnology issues.
CONABIA assesses all Applications to release GMPOs into the environment

and advises the Secretary of Agriculture, Livestock, Fisheries and Food on the
convenience of approving or not such releases. The assessment has two phases:
1. Field trial (low scale) release assessment, which aims at determining that the
probability of having any environmental impact is non-significant
2. Large-scale release assessment, which aims at determining that the environmen-
tal impact of the released GMPO will not significantly differ from that of its non-
modified counterpart
Permits are required to conduct greenhouse or field trials and the application for
permits requires extremely detailed information on the GMO itself, the greenhouse/
field location and conditions, and post-harvest treatment and monitoring.
While CONABIA is responsible for the environmental safety assessment of GM
products, SENASA is responsible for their food and feed safety assessment and the
DNMA determines the effect of the GM product on local and international trade
markets. The SAGPyA considers reports from all three agencies before making
a final decision on whether or not to release the GM product for commercial
production/consumption. GM crops currently approved for commercialization in
Argentina include soybean, maize and cotton.
11.5.2 GM Crop Regulation in Brazil
Brazil ranked third in the world for GM crop production in 2007, producing 15
million hectares of GM soybean and cotton. The first bio-safety law in Brazil took
effect in 1995 (law no. 8974/95), but was replaced in 2005 with bio-safety law
number 11.105/05. Several federal departments have active roles in bio-safety
regulation with the national authority being the National Technical Commission
on Bio-safety (CTNBio; http://www.ctnbio.gov.br/index.php/content/view/4060.
html). The CTNBio is composed of members from the public and private sectors,
including scientists, ministerial representatives and specialists. The CTNBio has
developed bio-safety policies and a Code of Ethics for genetic engineering and has
determined the risk assessments to be performed on GMOs before experimental or
commercial release.
Before any GM plant is field tested, the environmental risks must be assessed,
including potential harm to human health, other organisms and the environment. An
application for approval, which includes a summary of this information, must be
submitted to the CTNBio. If the GM plant is not considered a potential environ-
mental risk, field release is granted, but otherwise the GM plant must undergo an
environmental impact study before field testing can take place. If the GM plant or
its products will enter the human food chain, the food and safety regulations of the
National Agency for Health and Surveillance of the Ministry of Health (ANVISA;
http://www.anvisa.gov.br) must be followed. Other important governing agencies
include the National Surveillance System, which regulates research laboratories,
field experiments and commercialization and the Inspection Agencies from the
Ministries of Agriculture, Environment and Health which regulate inspection,
registration, operating license, import license, and temporary field testing license.
If the GM crop produces a pesticidal substance, it must also be regulated under the
Pesticide law, which governs experimental research, production, packing and
labeling, transportation, storage and commercialization.
Commercial approval for GM crops in Brazil must also be granted by CTNBio.
A commercial release dossier must be submitted, which details the GM crop’s
molecular characterization, protein expression, nutritional composition and agro-
nomic/environmental risk assessment. Currently, a 2/3 favorable majority vote by
the CTNBio is required for GM crop release. Once approved for release by
CTNBio, the application is reviewed by the Council of Ministries which also
considers the effect of GM crop release on the economical and political interests.
Thus far the Brazilian bio-safety regulatory system has approved the commercial
release of four varieties of GM corn, two varieties of GM cotton and one variety of
GM soybean (www.ctnbio.gov/br).
11.6 Summary
Most GM crop regulatory policies can be categorized as either (1) “process-based,”

where any and all biotechnology research and its products are strictly regulated, or
(2) “product-based,” where each product of biotechnology is evaluated on a case by
case basis to determine if regulation is necessary. Thus far, the largest production of
GM crops comes from those countries adopting a “product-based” approach to
biotechnology regulation. Social acceptance of GM crops plays a large role in
directing the type of regulatory policies a country will adopt. Public view on the
need to regulate GM crops varies. Some believe that biotechnology is just an
extension of conventional breeding practices, while others see the need for exten-
sive detailed analysis of all possible consequences that GM crop release may have
on human, animal and environmental systems. Regardless of contrasting view-
points, there is an international agreement on the need for GM crop regulation. It
is certain that regulations governing GM crop research and commercial release will
continue to evolve as economic and social pressures change.
References
AAFC, Agriculture and Agri-Food Canada (1994) Assessment criteria for determining environ-
mental safety of plants with novel traits, regulatory directive 94-08. AAFC, Ottawa
Abdalla A, Berry P, Connell P, Tran Q, Buetre B (2003) Agricultural biotechnology: potential for
use in developing countries. ABARE eReport, vol 03.17. Canberra, Australia
APHIS (2000) Agriculture Risk Protection Act of 2000. http://www.aphis.usda.gov/brs/pdf/
AgRiskProtAct2000.pdf
Barrett K, Abergel E (2000) Genetically engineered crops. Breeding familiarity: environmental

risk assessment for genetically engineered crops in Canada. Sci Public Policy 27:2–12
Belson N (2000) US regulation of agricultural biotechnology: an overview. AgBioForum 3:268–280
Berg P, Baltimore D, Brenner S, Roblin RO, Singer MF (1981) Summary statement of the
Asilomar Conference on recombinant DNA molecules. Proc Natl Acad Sci USA 72:
1981–1984
Dale PJ (2002) The environmental impact of genetically modified (GM) crops: a review. J Agr Sci
138:245–248
Department of Justice Canada (1983) Feeds Act. http://laws.justice.gc.ca/en/ShowFullDoc/cs/
F-9///en
Department of Justice Canada (1985a) Food and Drugs Act. http://laws.justice.gc.ca/en/ShowFul-
lDoc/cs/F-27///en
Department of Justice Canada (1985b) Seeds Act. http://laws.justice.gc.ca/en/ShowFullDoc/cs/
S-8///en
Department of Justice Canada (1999) Canadian Environmental Protection Act. http://laws.justice.
gc.ca/en/C-15.31/text.html
Haniotis T (2000) Regulating agri-food production in the US and the EU. AgBioForum 3:84–86
Herrera-Estrella L, Depicker A, van Montagu M, Shell J (1983) Expression of chimaeric genes
transferred into plant cells using a Ti-plasmid-derived vector. Nature 303:209–213
ISAAA (International Service for the Acquisition of Agri-Biotech Applications). http://www.
isaaa.org
Kalaitzandonakes N (2000) Why does biotech regulation differ so much between the EU and the
US? AgBioForum 3:75–76
McHughen A, Smyth S (2008) US regulatory system for genetically modified [genetically
modified organism (GMO), rDNA or transgenic] crop cultivars. Plant Biotechnol J 6:2–12
Morris S (2006) EU biotech crop regulations and environmental risk: a case of the emperor’s new
clothes? Trends Biotechnol 25(1):2–6, Jan 2007
NIH (US National Institute of Health) (1983) Risk assessment in the federal government. Manag-
ing the process. National Academies Press, Washington, DC
NIH (National Institutes of Health) (1976) Recombinant DNA research. Guidelines. Federal
Register 41, 27902, 27911–27943
NIH (1978) Guidelines for research involving recombinant DNA molecules. Fed Regist 43
(60):108
Nossal K, Abdalla A, Curtotti R, Tran QT, Brown A (2008) GM crops in emerging economies.
Research Report 08.3. www.abare.gov.au
OECD (Organization for Economic Cooperation and Development) (1982) Biotechnology, inter-
national trends and perspectives. OECD, Paris, France
Official Journal of the European Communities (2001) Directive 2001/18/ec of the European
Parliament and of the Council. http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:
L:2001:106:0001:0038:EN:PDF
Official Journal of the European Communities (2003) Directive 1829/2003 of the European
Parliament and of the Council. http://www.gmo-compass.org/pdf/law/1829-2003.pdf
Qaim M (2005) Agricultural biotechnology adoption in developing countries. Am J Agr Econ
87:1317–1324
Ramjouè C (2007) The transatlantic rift in genetically modified food policy. J Agr Environ Ethics
20:419–436
Schell J, van Montagu M, Holsters M, Zambryski P, Joos H, Inze D, Herrera-Estrella L, Depicker A,
de Block M, Caplan A, Dhaese P, Van Haute E, Hernalsteens JP, de Greve H, Leemans J,
Deblaere R, Willmitzer L, Schroder J, Otten L (1983) Ti plasmids as experimental gene vectors
for plants. Advances in gene technology: molecular genetics of plants and animals. Miami
Winter Symp 20:191–209
Smyth S, McHughen A (2008) Regulating innovative crop technologies in Canada: the case of
regulating genetically modified crops. Plant Biotechnol J 6:213–225
Chapter 12
Patent and Intellectual Property Rights Issues
Jim M. Dunwell
12.1 Introduction
The present status and future prospects of genetically modified (GM or transgenic)
crops have been the subject of several recent reviews (Dunwell 2000, 2002, 2004,
2008). Although these reviews include some information extracted from patent
databases in order to provide a commercial perspective, this analysis has been
necessarily limited in extent. The present review will supplement the information
published previously on the patent and intellectual property rights (IPR) (Johns
2006) aspects of transgenic methodology (Dunwell 2005, 2006), horticultural crops
(Dixon and Ogier 2007; Clark and Jondle 2008; Dunwell 2009b) and haploid plants
(Dunwell 2009a) and will extend to a discussion of IPR relevant to the research
scientist (Shear and Kelley 2003) and of those interested in international develop-
ment (Koo et al. 2004), globalization (Parayil 2003; Aerni 2007; Beatty 2008),
and sociological (Cabanilla 2007) and ethical aspects of the public- and private-
sector relationships (Graff et al. 2003; Donnenwirth et al. 2004; Karapinar and
Temmerman 2008).
12.2 Historical Retrospective
The history of patents and plants extends back over a century. In July 1899, an
international conference on the subject of hybridization was organized by the Royal
Horticultural Society (RHS) and held in London. One of the many speeches given at
the conference banquet was that by the leading British judge, Lord Justice Lindley
(Anon 1900). In this presentation, he made the following prediction: “I have heard
J.M. Dunwell
University of Reading, Whiteknights, Reading RG6 6AS, UK
e-mail: j.dunwell@reading.ac.uk

412 J.M. Dunwell
something about hybridisation of which I know little. I have heard something which
leads me to suppose that the development of that art may react with the profession
to which I have the honor to belong. Without being a prophet, I seem to see before
me a vista of patent hybrids! What a treat for the patent lawyers! And what an
accession of work for her Majesty’s Judges!” He could clearly see the potential for
litigation and legal conflict even at that early stage.
In a prelude to later discussions, Assistant Secretary of Agriculture, Willet
M. Hays (Troyer and Stoehr 2003), at a meeting of the American Breeders’ Associa-
tion (Hays 1905; Kimmelman 1983; Allen 2000) in 1905, remarked: “Possibly laws
or business can be devised which will give private individuals, animal breeders,
seed firms and nursery firms practically a patent or a royalty on new blood lines.”
By 1906, the emphasis on patents was demonstrated in a review of the relations
between science and industry, particularly the chemical sector where it was
reported that “the German company Baeyer (sic) had achieved a monopoly position
in novel chemicals, with 1,000 patents at home and 1,200 overseas” (Anon 1906).
However, the first detailed discussion of patents in relation to plant breeding is
probably that from the subsequent Third International Conference on Genetics,
organized by the RHS in 1906 and most famous for the first public coining of the
term “genetics” by William Bateson (Dunwell 2007). During this meeting, there
was a session entitled “Copyright” for Raisers of Novelties (Anon 1907). In the
report of the session, it is stated that Mr. George Paul, whilst commenting on the
absence of several well-known plant breeders, remarked: “The fact is, these gentle-
men do not like to tell us, or to show, what they have done in their experiments,
because once their knowledge becomes public, they have not the slightest chance of
receiving any pecuniary reward for their labors. If they were properly protected
from being deprived of the due reward of their labors, they would no doubt be much
more willing to come forward and help us and place their experience at our
disposal.” During the subsequent discussion in this session, Professor Hanson
replied: “I believe, in law, a seedling is regarded as the gift of God, and it would
be hard to patent that; but could we not hope to have some law fashioned that would
give a bonus to the man who does such skilled and valuable work as that which has
come before us over and over again during the sessions of this conference?” The
chairman, although sympathizing with the Mr. Paul, concluded that it would be
unwise to pass a resolution on the subject since the discussions had demonstrated
“[w]hat very great difficulty there would be in enforcing such a law, because we
have gentlemen from all parts of the world maintaining that a thing is new, and
others, equally capable, maintaining that it is old.”
In the following years, there were to be several cases of speculation about the
possible consequences of patent coverage for plants. For example, David Fairchild
produced an interspecific hybrid in Actinidia and stated: “If I could patent it – this
hybrid – then, should some other breeder have been at work on the same problem
and made the same pollination a few days later, I would have the prior claim to a
patent. Unlike the invention, the hybrid is not a crude unfinished thing but a perfect
working machine from the moment it is made, and claims entered would not come
into interference as they do in inventions because when the date of the hybridization
12 Patent and Intellectual Property Rights Issues 413
was once established the date of the first perfected machine would be automatically
determined” (Fairchild 1927). Following these early prescient comments and
debates, it took several years, after much encouragement and lobbying by Luther
Burbank (Burbank 1914), before the first legal protection for plants, The Plant
Patent Act 1930, was enacted in the USA, and then only for clonal material (e.g.,
rose, apple and pear, though not potato) (Fowler 2000). The first US plant patent
(PP00001) was issued for a climbing rose in the following year, 1931 (Cook 1931a).
This was soon followed by further examples (Cook 1931b, 1933a), although it
should be noted that even in those days the topic was the subject of controversy
from scientific, legal, and financial experts (Anon 1931; Allyn 1933a,b; Cook
1933b, 1936; Barrons 1936; Rossman 1931; Fay 1937). Much of the controversy
today, more than 100 years after the first discussions, follows the same themes.
12.3 What Are Patents?
The history of patent law dates back several centuries, with the first example
sometimes considered to be a Venetian statute of 1474. A summary definition
states: “A patent gives an inventor a period of exclusive exploitation (up to
20 years in the UK) in return for a disclosure of the invention” (Huskisson 1996).
According to the UNCTAD site (http://www.iprsonline.org/guide/index.htm), a
patent application must satisfy the patent examiners that the invention is:
– Useful (i.e., has industrial application): ideas, theories, and scientific formulas
are not sufficiently useful or industrially applicable to be patentable
– Novel: the invention should be recent and original, but perhaps most importantly
it should not already be known (in the public domain). In most countries (except
the USA) the patent is awarded to the first person to apply, regardless of whether
this person was the first to invent
– Non-obvious or must involve an inventive step: not obvious to a person skilled in
the technology and more inventive than mere discovery of what already exists in
nature (such as a gene with no known function). The invention must be disclosed
to the patent examiners in a detailed way that would enable a skilled technician
to make and use it. In the case of an invented process, the patent can cover a non-
obvious way of making something already known (i.e., previously invented or
discovered). In the case of an invented product, the non-obvious/inventive step
requirement does not require it to be made by a novel method.
This disclosure of an invention (Fromer 2007) takes the form of a publication
from the relevant patent office. In the case of most authorities, the patent application
is published 18 months after the date of filing and is then available for inspection.
A former exception to this rule is that the United States Patent and Trademark
Office (USPTO) (http://www.uspto.gov/), until 15 March 2001, maintained secrecy
until the time the patent was granted, a period that can range from an average of
2–3 years upwards to more than 20 years. An extreme example of the length of time
414 J.M. Dunwell
sometimes involved is the approximate 20 years taken for the resolution of a dispute
concerning key patents which cover elements of Agrobacterium-mediated transfor-
mation. It was announced on 4 February 2005 that Monsanto, Bayer CropScience,
Max Planck Society and Garching Innovation had agreed to cross-license their
respective technologies worldwide. Bayer CropScience, Max Planck’s exclusive
licensee, and Monsanto agreed to provide each other, in selected areas of the world,
nonexclusive licenses related to the development, use and sale of transgenic crops.
Monsanto also agreed to provide Max Planck Society with a license in the United
States for research purposes.
An important difference between the US and the patent systems of other
international jurisdictions is that the 17-year duration of a US patent filed prior to
2001 only starts from the time at which it was granted, whereas in Europe (and now
in the US) the 20-year period of exclusivity starts from the time of filing the
application. Some of the consequences of this change are discussed in more detail
later in this review.
12.4 Sources of Patent and Other Related Information
The association between an active IPR system and a strong national or regional
economy has been demonstrated in many global studies (Griliches 1990). For
example, the Organization for Economic Cooperation and Development (OECD)
maintains various patent databases that can be used to assess global and regional
trends in all disciplines including biotechnology (Van Beuzekom and Arundel
2006; Anon 2008; Maraut et al. 2008).
One of the issues raised in the early discussion of the US Plant Patent system was
that access to the patent documents was extremely difficult. In the words of Cook
(1933b): “The patent authorities have taken the view that these patents are too
valuable to be disposed of casually, and an applicant for a copy must ‘show cause’
why he should be sold one. . . . Until copies of plant patents are available to
interested parties this situation can hardly be considered satisfactory from the
plant breeder’s point of view.” The situation is now much simpler (Krattiger
et al. 2007; Nottenburg 2007). During the preparation of this review extensive
use has been made of the freely available patent databases in the US (http://www.
uspto.gov/patft/index.html), Europe (http://ep.espacenet.com/), World Interna-
tional Patent Organization (http://pctgazette.wipo.int/) and other international
sites (e.g., http://www.surfip.gov.sg/; http://www.google.com/patents; http://www.
freepatentsonline.com/; http://www.pat2pdf.org/). The most comprehensive and
integrated international site is probably the Patent Lens section of BiOS, Biological
Innovation for Open Society, an initiative of CAMBIA (Center for the Application
of Molecular Biology to International Agriculture) (http://www.bios.net/daisy/bios/
patentlens.html). A very useful site relating to US ag-biotech patents granted during
the period from 1976 to 2000 is provided by the Economic Research Service (ERS)
of the US Department of Agriculture (USDA) (http://www.ers.usda.gov/Data/

AgBiotechIP/). It should be noted that the most detailed and sophisticated types
of patent analysis require commercial subscription. Companies providing this
service include Thompson Reuters (http://www.thomsonreuters.com/products_
services/scientific1/Delphion), Micro Patent (http://www.micropat.com/static/index.
htm), and patentmaps.com (http://patentmaps.com/shop/v2/shophome.htm).
12.5 Patents and Plant Biotechnology
Apart from the natural genetic protection provided by F1 hybrids (Duvick 1999;
Smith et al. 2008), there are a range of legalistic methods that can be used to protect
novel types of plants produced by one organization or commercial company from
being exploited by competitors, with these methods varying from one country to
another (Cahoon 2000; Llewelyn and Adcock 2006; Locke 2007). An introduction
to the various approaches, namely plant breeders rights (Kesan and Janis 2002;
Helfer 2004; Chen 2006; Ghijsen 2007) and patents, is available from several
authors (Brown 2003; Lenssen 2006; Janis and Smith 2007; Kock et al. 2007;
Rimmer 2003; Smith 2008), and from the Biological Innovation for Open Society
(BiOS) organization (http://www.bios.net/daisy/bios/patentlens/tutorials.html).
Information relating to individual countries is available at their respective patent
offices. For example, the latest note on patenting of plants in the UK “Examination
Guidelines for Patent Applications relating to Biotechnological Inventions in the
UK Intellectual Property Office” was published by the Intellectual Property Office
in September 2007 (http://www.ipo.gov.uk/biotech.pdf). Similar information is
available concerning the patentability of plants in the US (Merrill et al. 2004),
Europe (Fleck and Baldock 2003; Schrell et al. 2007), New Zealand (Ministry of
Economic Development 2002) and China. In a comparative analysis, the results of a
detailed survey of the actual practice of patent examiners in the three key patent
offices, US, Europe and Japan, have been published (Howlett and Christie 2003).
A complementary study restricted to the present and future position in the US
(Merrill et al. 2004) concluded that the continuing high rates of innovation suggest
that the patent system there is working well and does not require fundamental
changes, although the authors note that both legal and economic changes are putting
new strains on the system.
There have been several, extensive reviews of the consequences, and implica-
tions of applying patent (and other IPR) protection to plants (Farnley et al. 2004;
Temmerman 2006; Adcock 2007; Kock 2007) and the reader is referred to these
publications, most of which are freely available on the web. In one of the most
comprehensive of these reviews (Binenbaum et al. 2003), the important conclusion
is reached that as patenting becomes ever more prevalent in biotechnology (Wright
2006; Wright and Pardey 2006a; Chapotin and Wolt 2007) and elsewhere (Straus
2007), the diversity of innovations utilized in the development of modern cultivars
416 J.M. Dunwell
means that there is an associated increase in the number of separate rights needed to
produce each new product (Tokgoz 2003). Where ownership of the relevant IPR
rights is particularly dispersed, the problem of multilateral negotiation can become
difficult or even impossible to resolve. For example, those who develop new
technology by building on existing technologies often know neither the extent to
which the latter have been claimed as IP nor the strength of any claims. As a
consequence, both the conduct of research and development and any subsequent
commercialization entail navigating through a potential minefield of patent appli-
cations that have been filed but remain invisible pending publication by the patent
office. Fortunately, the uncertainty arising from such so-called “submarine” patents
is becoming less important as the US has harmonized with the rest of the world, first
by awarding a patent term of 20 years from the date of filing (previously 17 years
from the date the patent was awarded), and secondly by publishing (from November
2000) patent applications within 18 months of filing.
Despite the increasing complexity of biotechnological IPR (Eisenberg 2006;
Kukier 2006), and the difficulties of making accurate investment predictions over
extended time scales (Yerokhin and Moschini 2007) it should be noted that a
similar position exists in the electronics industry where sophisticated products are
assembled from numerous components, sourced internationally, and covered by a
multiplicity of patents.
12.6 Patents and Plant Transformation
During the period since the production of the first transgenic plants in the 1980s, a
wide diversity of patents have been sought, and granted, on all aspects of the
process, ranging from the underlying methods for tissue culture through to the
means of introducing the heterologous DNA, and to the composition of the DNA
construct so introduced (Dunwell 2005; Pray and Naseem 2007). It would be
impossible to summarize all this information in the space available here; the
amount of patent information available in the area of plant transformation alone
can be judged by the fact that a search of the US application database alone for
“transgenic plant” and “method” returned 5,995 records on 1 August 2008. Sum-
maries of relevant recent granted patents and patent applications in the US are given
in Tables 12.1 and 12.2.
For detailed analyzes of several of the key areas of this subject, the reader is
referred to comprehensive summaries published elsewhere, for example in the
series of extensive CAMBIA White Papers (Mayer et al. 2004; Roa-Rodrigues
2007; Roa-Rodrigues and Nottenburg 2007a,b); some aspects of these will be
considered below. Frequently, the main point of interest in such discussions is the
breadth of coverage of the patent(s) in question. There are some well known
examples of patents with very broad coverage and this is often a topic of debate
and the reason for concerted opposition. For example, European Patent 301749,
Table 12.1 Selection of US patents on transgenic plants published immediately prior to 8 July
2008
Number Date Named inventors Applicant Subject
7396979 8 July 2008 Alexandrov N et al. Ceres Increased biomass
7396978 8 July 2008 Miki B et al. Min Agri-Food Seed coat gene
Canada
7396977 8 July 2008 Usami S et al. Japan Tobacco PPDK gene
7393999 1 July 2008 Acevedo PAN et al. Pioneer Antimicrobials
7393998 1 July 2008 Streatfield S et al. ProdiGene Insulin
7393997 1 July 2008 Datla R et al. – Floral shape
7393948 1 July 2008 Sekar V et al. MS technologies Polyubiquitin promoter
7393947 1 July 2008 Diehn S et al. Pioneer Inducible promoter
7393946 1 July 2008 Memelink J et al. Rijksuniversiteit Transcription factor
Leiden
7393928 1 July 2008 Chang RC et al. FibriGen Gelatin
7393922 1 July 2008 Dean DH Ohio State Univ Cry4B Bt gene
7390937 24 June 2008 Good AG et al. Univ Alberta Nitrogen metab
7390936 24 June 2008 Van Rooijen G et al. SemBioSys Chymosin
7390655 24 June 2008 Hinchey B Monsanto Promoters
7390643 24 June 2008 Croteau RB, Burke – Geranyl diphosphate
CC synthase
7388126 17 June 2008 Duncan DR, Ubach C Monsanto NO modulation
7388125 17 June 2008 Ristic Z et al. Pioneer Elongation factor
7388091 17 June 2008 Erickson L, Zhang J Univ Guelph Inducible gene
7385123 10 June 2008 Sauer M et al. SunGene Ketolase
7385107 10 June 2008 Donovan WP et al. Monsanto CryET33 gene
7385106 10 June 2008 Stein H et al. Tel Aviv Univ Proline metab
7385105 10 June 2008 Medrano L et al. Ceres Root promoters
7385104 10 June 2008 Landschutze V Bayer Starch metab
7385048 10 June 2008 Fujii T et al. Kirin Beer Promoters
7385046 10 June 2008 Alexandrov N et al. Ceres Ethylene response
granted to Agracetus (then a subsidiary of WR Grace & Co) on 2 March 1994, is an

exceptionally broad “species patent,” which grants this company rights to all forms
of transgenic soybean varieties and seeds – irrespective of the genes used or the
transformation technique employed. Agracetus was purchased by Monsanto in
April 1996, after which it withdrew its previous opposition to this patent. However,
opposition continued from other companies and organizations, and a hearing was
finally agreed by the European Patent Office in May 2003, at which the patent was
upheld, with the exception of Claim 25 covering plants other than soybean (http://
www.epo.org/about-us/press/releases/archive/2003/06052003.html; http://www.
european-patent-office.org/news/pressrel/pdf/bginfo_soya_e.pdf). After a further
series of oppositions the patent was finally revoked on 3 May 2007 (http://www.
epo.org/about-us/press/releases/archive/2007/070503.html).
Interestingly, there are also IPR aspects of marker-assisted breeding (Henson-
Apollonio 2007), a method sometimes considered as an alternative to transgenic
technologies.
418 J.M. Dunwell
Table 12.2 Selection of US patent applications on transgenic plants published immediately prior
to 8 July 2008
Number Named inventors Applicant Subject
20080168587 Yao K et al. – High oleic acid
20080168586 Laga B et al. Bayer Mutant fatty acid desaturase
20080168585 da Costa e Silva O et al. BASF Stress-related GTP binding protein
20080168584 Zheng Z et al. NRCC Brassica seed metabolism
20080168578 da Costa e Silva O et al. BASF Stress-related protein kinase
20080166811 Maliga P et al. – Site-specific recombination of plastids
20080166754 Cahoon E et al. – Caffeic acid 3OMT homologs
20080163402 Wilkinson JQ et al. Pioneer Non-plant 30 termination sequences
20080163399 Carozzi N et al. Athenix Delta-endotoxin
20080163398 Kakefuda G et al. BASF AHAS small subunit protein
20080163397 Ratcliffe OJ et al. Mendel Biotech CCAAT-binding transcription factor
20080163396 Allen SM et al. – Gene expression
20080163395 Song H-S et al. BASF Gene expression
20080163394 Frankard V et al. CropDesign Cyclin and yield
20080163393 Spangenberg G et al. Agric Victoria CAD genes
Services
20080163392 Zink O et al. Bayer Herbicide resist
20080163391 Sussman SM et al. Penn State Stomatal closure
20080163390 Kachroo P et al. Univ Kentucky Fatty acids and disease
20080161191 Falco SC et al. Du Pont Methionine sulfoxide reductase
20080160162 Davies JP et al. Agrigenetics Improved oil
20080155717 Kaeppler SM et al. Wisconsin Polycomb genes
Alumni
20080155715 Komatsu S et al. NIAS Japan Stress response
20080155714 Gontier E et al. Total France Fatty acid synthase
20080155713 Yephremov A et al. Bayer Fatty acids
20080155712 Savidan Y et al. IRD Apomixis
12.6.1 Transformation Methods
There is a great diversity of techniques for the introduction of recombinant DNA

vectors containing heterologous genes of interest into plant cells, and the
subsequent regeneration of plants from such cells (Moeller and Kan Wang 2008).
The two principal methods exploited in commerce are indirect methods based on
the use of Agrobacterium tumefaciens (Chung et al. 2006; Lacroix et al. 2008) or
the direct introduction of DNA on microparticles of metal, usually gold, a technique
known as biolistics (http://www.bioportfolio.com/indepth/Biolistics.html). The
most extensive publication in this area is the CAMBIA White Paper (Roa-Rodrigues
and Nottenburg 2007a) on Agrobacterium-mediated transformation. This document
focuses on the patents directed to methods and materials used for transformation,
mainly of plants, and also of other organisms such as fungi. It should be noted that
although most of the early development of this technique was performed in
universities, most of the patents were consolidated in the hands of a few companies.
12.6.2 DNA Sequences
Almost all the functional components of the various constructs used in plant
transformation have been the subject of patent coverage. These include the “effect
gene” as well as its associated regulatory sequences (e.g., promoters), the selectable
or screenable marker, and additional sequences such as those that might be utilized
for the subsequent excision of the transgene (for examples see Dunwell and Ford
2005) or even allow IP protection in their own right (Heider and Barnekow 2007).
The present review does not cover details of the gene of interest and the reader is
referred to other recent reviews that include summaries of the range of present and
future transgenic crops (Dunwell 2002, 2004) (see also Tables 12.1 and 12.2).
Much of the debate in this area concerns the ability to apply for patents on DNA
sequences of unproven function. There have been several attempts to do so, and the
decisions on such applications have not been finalized (Howlett and Christie 2003;
Rimmer 2007). However, the important fact remains that patent databases contain a
great deal of useful sequence information that is frequently ignored by research
scientists in the academic sector. Specifically, it is estimated that some 30–40% of
all DNA sequences are only available in patent databases, since there is of course no
obligation for commercial (or other) applicants to submit their sequences to public
databases. Free access to some patent sequence data is now available via the latest
version of the Blast search system at NCBI, and via Patent Lens (http://search.
patentlens.net/sequence/blast/blast.html). Access to this information is also avail-
able via the GENESEQ system, a commercial service from Thompson Reuters
(http://scientific.thomsonreuters.com/bondplus/geneseq/).
Regulatory elements are crucial to gene expression in all organisms. The patent
landscape of transcriptional regulators that are constitutively active, spatially active
(e.g., tissue-specific), or temporally active (e.g., induced or active in response to a
certain chemical or physical stimulus) has been well summarized (Roa-Rodrigues
2007). In this review, an assessment is presented of the possibilities for, and
limitations on, further development of regulation of gene expression. Although
the inventions protected by individual patents cannot be exactly the same, in certain
cases there are patents that due to the breadth of their scope may encompass other
protected inventions or there may be patents which share common features. Is this
case, this review points out the juxtaposition of the different inventions and the
possible room left to maneuvre around the different entities in the field. The science
behind some of these issues has also been reviewed recently (Century et al. 2008).
It also needs to be considered that there are patents that while not totally directed to
promoters may have an effect on the control of gene expression. This is the case for
the restrictive reproductive technologies, for example, those termed as “Terminator”
technologies (Van Acker et al. 2007; Van Dooren 2007), which may have a
great impact on the use and development of methods to regulate the expression of
genes related to plant reproduction and seed generation (Dunwell and Ford 2005;
Hills et al. 2007).
420 J.M. Dunwell
12.6.3 Selection and Identification of Transformants
The production of transgenic organisms, including plants, involves the delivery of a

gene of interest and the use of a selectable marker that enables the selection and
recovery of transformed cells. This is necessary because only a minor fraction of the
treated cells become transgenic while the majority remain untransformed. It has been
estimated recently (Miki and McHugh 2004) that approximately 50 marker genes
used for transgenic and transplastomic plant research or crop development have been
assessed for efficiency, biosafety, scientific applications and commercialization.
These selectable marker genes can be subdivided into several categories depend-
ing on whether they confer positive or negative selection and whether selection is
conditional or nonconditional on the presence of external substrates. The most
common strategy currently used for selection is negative selection, the elimination
of nontransformed cells in conditions where the transformed cells are allowed to
thrive. Elimination is often effected by the treatment of cells with compounds, (e.g.,
antibiotics or herbicides) in conjunction with a transgene that confers resistance or
tolerance to the chemical through detoxification or modification of the chemical.
A summary of the most important scientific aspects of such resistance genes has
been published recently, together with an analysis of selected patents that relate to
the most widely used ARMs (Roa-Rodrigues and Nottenburg 2007b). Many of
these marker genes are covered by patents or patent applications with the most
thorough IP analysis available probably being that published on antibiotic markers
and Basta resistance by CAMBIA (Mayer et al. 2004).
12.7 Novel Products and the Freedom to Operate
One of the issues of overriding importance to all companies is whether or not they
are free to commercialize any particular product (Lence et al. 2002). Such “freedom
to operate” is determined by the status of any IPR that might cover the product in
question and analysis of such IPR requires continuous (and therefore expensive)
surveillance. A well known example that can be used to demonstrate the complexity
of this issue is “golden rice,” a transgenic line enhanced for beta-carotene (provita-
min A) (Ye et al. 2000; Mayer 2007) and provides hope for alleviating the severe
vitamin A deficiency that causes blindness in half a million children every year. It
has been suggested that extensive patenting has hampered delivery of this rice to
those in need since some 40 organizations hold 72 patents on the technology
underlying its production (Kryder et al. 2000). The range of patents covering
various components of the pBin 19 hpc plasmid used in the production of this
rice include ones on the phytoene trait genes, the promoter sequences, the selectable
marker and the transit peptide. This issue has now been overcome by a coordinated
international program designed to streamline the production and distribution of
this material (http://www.goldenrice.org/) (Potrykus 2007). However, perceived
problems with access to golden rice (Enserink 2008) and essential medicines have
stimulated debate within the US on the obligations of American universities to
facilitate the provision of goods for the public benefit (Kowalski and Kryder 2002;
Phillips et al. 2004), an issue also considered below.
12.8 Economic Benefits of Transgenic Crops
The exploitation of IPR associated with transgenic crops is central to the generation
of economic benefit (Moschini and Yerokhin 2007) for the companies producing
such crop and the farmers growing them. The global economic benefits of the first
decade of these crops have been summarized recently by Barfoot and Brookes
(2008) whereas the specific benefits of individual crops have also been described
(Anderson and Valenzuela 2008). One consequence of the IPR system is that
companies place considerable emphasis on legal protection of their investment
and this includes the prosecution of those deemed to be infringing any proprietary
IPR (Kesan and Janis 2002, 2003; Ziff 2005; Munzer 2006; Pila 2008).
12.9 Patents and Commercial Consolidation
Several summaries of this subject has been provided in the last few years (Brennan
et al. 2005; Bulut and Moschini 2005, 2006; Chan 2006; Schimmelpfennig and
King 2006; Karapinar and Temmerman 2008; Marco and Rausser 2008). The latter
authors conducted an analysis of agbiotech patents issued between 1976 and 2000,
classified by their original patent holders and their 2002 owners. These data showed
how 95% of patents originally held by seed or small agbiotech firms had been
acquired by large chemical or multinational corporations. Furthermore, none of the
smaller firms acquired patents from the larger ones, and none of the patents changed
hands among the different types of large firms. Specifically, chemical companies
retained all 651 patents that they originally owned, but they also acquired 219
patents from agbiotech firms and 451 patents from seed companies. A similar,
related study of the role of patents in the pharmaceutical industry has also been
published (Brusoni et al. 2005), together with a summary of the consequences of
having a large portfolio of patents (Chan 2008), and a legal discussion of using IPR
as collateral in trade (Dequiedt et al. 2007; Dunn and Seiler 2007).
12.10 Public and Private Sector Issues
The most detailed review of this aspect of agbiotech patents is probably that
conducted by Graff et al. (2003) who summarized both the ownership of critical
patents and compared the relative significance of the private and public sectors in
422 J.M. Dunwell
each area of research relevant to the commercialization of transgenic plants. The

main findings of this review and others (Heisey et al. 2005; Schimmelpfennig and
King 2006; Eaton 2007) are as follows. Six companies hold 75% of all agricultural
patents and it has been suggested that such concentration exacerbates the challenge
of delivering agricultural inventions to the neediest segments of the world’s popu-
lation. One solution could be the compulsory licensing of patented inventions that
have failed to reach the neediest markets (see below for further detail). An alterna-
tive would be based on the fact that while the public sector holds less than 3% of all
patents, it does have 24% of agricultural biotechnology patents, many covering
genes of great potential interest. By exploiting these resources, universities and
other public organizations, therefore, do have opportunities to deliver affordable
biotechnological innovations (Morris et al. 2006).
Concern has also been expressed about the potential dangers (financial or
otherwise) associated with the use of patented technologies by academic establish-
ments (Kesselheim and Avorn 2005). This is a complicated issue involving “exper-
imental use exception” (Faye 2005a,b), the policy that allows others to examine and
test a patented discovery, but not to use it routinely.
12.11 Patents, Ethics and International Development
The moral and ethical aspects of transgenic plants have recently been considered
(Myskja 2006; Cooley 2007; DeBeer 2007; Wilson 2007), and in a broader context,
some authors consider that the commercialization of biotechnology, especially
research and development by transnational pharmaceutical and ag-biotech compa-
nies, is already excessive and is increasingly dangerous to distributive justice,
human rights, and access of marginal populations to basic goods required for
human prosperity (Shrader-Frechette 2005). The various trends associated with
these socio-economic aspects of ag-biotech development have also been reviewed
(Parayil 2003; Lipton 2007; Lea 2008). Amongst the agencies involved, the various
Consultative Group on International Agricultural Research (CGIAR) centers add
value through selective breeding, and the superior varieties they generate are
widely distributed without charge, thereby benefiting both developing and deve-
loped countries (Anon 2001).
During the Gene Revolution, the situation changed, and much has been written
over the last few years on the potentially deleterious effects of plant IPR on the
freedom and commercial opportunities of farmers in developing countries (Bastuck
2006; Chiarolla 2006; Fukuda-Parr 2006; Garrison 2006; Hamilton 2006; World
Bank 2006; Wright and Pardey 2006b). One of the major reasons that IPR have
become an important factor in plant breeding (Lence et al. 2002; Louwaars et al.
2006; Kingston 2007) is through the greater use of utility patents (Summers 2003;
Kevles 2007). Such patents have stimulated greater investment in crop improve-
ment research in industrialized countries, but they are also creating major problems
and potentially significant additional expense for the already financially constrained
public-sector breeding programs that produce seeds for poor farmers (Spielman
2007; Spielman et al. 2007). For example, it has been calculated (Phillips et al.
2004) that developed countries spend about $5 in research and development for
every $100 in agricultural output whereas developing countries spend only 66 cents.
Patents on biotechnology methods and materials, and even on plant varieties
(Tripp et al. 2007), are thus potentially complicating and undermining the collabo-
rative relationships between international institutions. There is some concern that
public-sector research institutions in industrialized countries no longer fully share
new information and technology. Instead, they are inclined to patent and license
and have special administrative offices charged with maximizing their financial
return from licensing (Brazell 2000). Commercial production of any GM crop
variety requires dozens of patents and licenses (see above), and it is only the
large companies that can afford to assemble the IPR portfolios necessary to give
them the freedom to operate. Additionally, now, under the TRIPS agreement
of the World Trade Organization (http://www.iprsonline.org/unctadictsd/docs/
RB_Part2_2.5_nov02_fullpatents-updated.pdf), most developing countries are
required to put in place their own IPR systems, including IPR for plants (Giannakas
2001; Gaisford et al. 2007; Watal and Kampf 2007).
Several proposals have been made on how the international community should
deal with these present IPR realities affecting agriculture and horticulture (Delmer
et al. 2003; Delmer 2003; Ramanna 2005; Brewster et al. 2007; Lence et al. 2003).
With little competitive loss, seed companies could agree to use the Plant Variety
Protection (PVP) system (including provisions allowing seed saving and sharing by
farmers) in developing countries in cooperation with public plant-breeding agen-
cies, rather than using patents to protect their varieties (Singh 2004). To speed the
development of biotechnology capacity in developing countries (Louwaars et al.
2005; Salazar et al. 2006; Léger 2007), companies that have IPR claims over certain
key techniques or materials might agree to license these for use in developing
countries at no cost (Nottenburg et al. 2002). These authors also propose an
agreement to share the financial rewards from IPR claims on crop varieties or
crop traits of distinct national origin (Wu and Lu 2005; Mgbeoji 2006; Kartal 2007;
Mahop 2007; McManis 2007), such as Thailand’s Jasmine rice or South Asian
Basmati rice.
There have been several reviews of this topic each with a particular regional
emphasis. For example, biotechnology and GM crops has been discussed specifi-
cally in relation to development in Africa (Eicher et al. 2006; Juma and Seregeldin
2007; Virgin et al. 2007; Asante 2008), India (Demangue 2005; Sreedharan 2007),
China (Stone 2008), the Philippines (Cabanilla 2007) and South America (Orozco
et al. 2007; Scoones 2008). There is also a series of published case studies in which
the IPR aspects of specific transgenic crops have been explained. Such examples
include those for corn (Gracen 2007), papaya (Goldman 2007; Gonsalves et al.
2007), herbicide tolerant rice (Espinoza-Esquivel and Arrieta-Espinoza 2007) and
canola (Smyth 2006), insect resistant eggplant (Medakker and Vijayaraghavan
2007) and cotton (Rao and Antharvedi 2007), and biopharming (Durell 2006;
Krattiger and Mahoney 2007).
424 J.M. Dunwell
Many of the concerns about the scope and application of IPR in the area of
agriculture (Tencalla 2006; Laxmi et al. 2007) also extend to the more general issue
of food from the perspective of safety (Klaasen 2007; Key et al. 2008) as well as
long term security (Malik and Zafar 2005; Tansey 2006; Hossain 2007). There are
also discussions about the need to regulate the whole process (Rimmer 2006;
Rhodes 2007; Roff 2008).
For all the reasons discussed above, new organizations such as Public Intellec-
tual Property Resource for Agriculture (http://www.pipra.org/) and the African
Agricultural Technology Foundation (http://www.aftechfound.org/) have been
established as a means of rationalizing the huge proliferation of patents, especially
in plant biotechnology. It is the intention of these organizations to develop a
freedom-to-operate information database, and to help public sector agricultural
research institutions achieve their public missions (Cantley 2004) by ensuring
access to intellectual property required to develop and distribute improved staple
crops and specialty crops (Anon 2006; Dodds et al. 2007).
12.12 Conclusion
Although the 1906 “Genetics” conference (Anon 1907) did receive commercial
support and there were 20 pages of advertisements in the proceedings, this funding
was restricted to horticulture and associated gardening items. Bateson, in his after
dinner speech to foreign guests, concluded: “I expect a century must elapse before
the . . . complete union of Science and Practice will be achieved.” Some 25 years
after Bateson, the following comment was being made: “It will be extremely
interesting to follow the new developments in plant breeding in order to determine
the influence of the new patent protection on agriculture. In years to come, much of
the food consumed, many of the clothes worn and even the houses occupied by man
may be radically changed by the mass attack of plant breeders so that the future
generations may speak of a horticultural revolution rivaling, if not surpassing the
great industrial revolution” (Rossman 1931). A century has now elapsed since the
first of these comments and indeed the value of genetics in agriculture and horticul-
ture has been proven. Moreover, the role played by patent protection in this period
has been critical to the commercial exploitation of genetics during this period.
References
Adcock M (2007) Intellectual property, genetically modified crops and bioethics. Biotechnol J
2:1088–1092
Aerni P (2007) Agricultural biotechnology and its contribution to the global knowledge economy.
In: Fiechter A, Sautter C (eds) Green gene technology. Research in an area of social conflict,
vol 107. Springer, Berlin, pp 69–96 Adv Biochem Eng Biotechnol
Allen GE (2000) The reception of Mendelism in the United States, 1900–1930. CR Acad Sci Paris
Life Sci 323:1081–1088
Allyn RS (1933a) Plant patent queries. A patent attorney’s views on the law. J Hered 24:55–58
Allyn RS (1933b) Plant patent queries. J Pat Off Soc 15:180–186
Anderson K, Valenzuela E (2008) Recent and prospective adoption of genetically modified cotton:
a global computable general equilibrium analysis of economic impacts. Econ Dev Cult Change
56:265–296
Anon (1900) Report of the after dinner toasts. J Roy Hortic Soc 24:47 In: Wilks W (ed) Hybrid
Conference Report 1900
Anon (1906) The relations between science and industry. Nature 74:386–387
Anon (1907) “Copyright” for raisers of novelties. In: Wilks W (ed) Report of the Third Interna-
tional Conference 1906 on Genetics; hybridisation (the cross-breeding of genera or species),
the cross-breeding of varieties and general plant breeding. Roy Hort Soc, London, UK, pp
474–475
Anon (1931) Patenting of plants promises big profits – and big problems. Business Week, Aug 26, p 26
Anon (2001) Booklet of CGIAR centre policy instruments, guidelines and statements on genetic
resources, biotechnology and intellectual property rights. System-wide Genetic Resources
Programme (SGRP), Rome, Italy
Anon (2006) Access, benefit-sharing and intellectual property rights. Int Centre for Trade and
Sustainable Development (ICTSD), p 4, http://www.trade-environment.org/output/infoxch/
COP8_ICTSD_ABS.pdf
Anon (2008) Compendium of patent statistics 2008. OECD, p 38, http://www.oecd.org/dataoecd/
5/19/37569377.pdf
Asante DKA (2008) Genetically modified food – The dilemma of Africa. Afr J Biotechnol 7:
1204–1211
Barfoot P, Brookes G (2008) Global impact of biotech crops: socio-economic and environmental
effects, 1996-2006. AgBioForum 11:21–38 http://www.agbioforum.org
Barrons KC (1936) A defense of basic plant patents. From the plant breeder’s point of view.
J Hered 27:475–478
Bastuck C (2006) ‘Biopiracy’ and patents – developing countries’ fears are exaggerated. Master
Thesis for LLM by coursework and dissertation in International Law. Univ of Capetown, South
Africa, p 68
Beatty A (2008) The globalization of agriculture and social resistance. Master of Arts in Sociol-
ogy. Thesis. Humboldt State Univ, p 100
Binenbaum E, Nottenburg C, Pardey PG, Wright BD, Zambrano P (2003) South-north trade,
intellectual property jurisdictions, and freedom to operate in agricultural research on staple
crops. Econ Dev Cult Change 51:309–335
Brazell L (2000) Strategies for minimizing IP risks. Patent World 120:19–25
Brennan M, Pray C, Naseem A, Oehmke JF (2005) An innovation market approach to analyzing
impacts of mergers and acquisitions in the plant biotechnology industry. AgBioForum 8:89–99
Brewster AL, Hansen SA, and Chapman AR (2007) Facilitating humanitarian access to
pharmaceutical and agricultural innovation. In: Krattiger A, Mahoney RT (eds) Intellectual
Property Management in Health and Agricultural Innovation: A Handbook of Best Practices.
MIHR (Oxford, UK), PIPRA (Davis, USA), Oswaldo Cruz Foundation (Fiocruz, Rio de
Janeiro, Brazil) and bioDevelopments-International Institute (Ithaca, USA), pp 47–61, www.
ipHandbook.org
Brown WM (2003) Intellectual property law: a primer for scientists. Mol Biotechnol 23:213–224
Brusoni S, Criscuolo P, Geuna A (2005) The knowledge bases of the world’s largest pharmaceuti-
cal groups: what do patent citations to non-patent literature reveal? Econ Innov New Technol
14:395–415
Bulut H, Moschini G (2005) Parallel research, multiple intellectual property right protection
instruments, and the correlation among R&D projects. Center for Agricultural and Rural
Development, Iowa State Univ, Ames, Iowa, USA. Working Paper 05-WP 404, p 25
426 J.M. Dunwell
Bulut H, Moschini G (2006) Patents, trade secrets and the correlation among R&D projects. Econ
Lett 91:131–137
Burbank L (1914) How plants are trained to work for man, vol 1. PF Collier, New York, p 302
Cabanilla LS (2007) Socio-economic and political concerns for GM foods and biotechnology
adoption in the Philippines. AgBioForum 10:178–183
Cahoon RS (2000) Property rights and agricultural biotechnology. In: Hrazdina G (ed) Use of
agriculturally important genes in biotechnology. IOS Press, Amsterdam, pp 203–215
Cantley M (2004) How should public policy respond to the challenges of modern biotechnology?
Curr Opin Biotechnol 15:258–263
Century K, Reuber TL, Ratcliffe OJ (2008) Regulating the regulators: the future prospects for
transcription-factor-based agricultural biotechnology products. Plant Physiol 147:20–29
Chan HP (2006) International patent behavior of nine major agricultural biotechnology firms.
AgBioForum 9:59–68
Chan HP (2008) Does sequential innovation decline in the presence of large patent portfolios?
Examining future variety creation in the U.S. agricultural biotechnology industry, p 27, http://
ssrn.com/abstract¼1008970
Chapotin SM, Wolt JD (2007) Genetically modified crops for the bioeconomy: meeting public and
regulatory expectations. Transgenic Res 16:675–688
Chen J (2006) The parable of the seeds: interpreting the Plant Variety Protection Act in furtherance
of innovation policy. Notre Dame Law Rev 81, p 51, http://ssrn.com/abstract¼784189
Chiarolla C (2006) Commodifying agricultural biodiversity and development-related issues.
J World Intellect Prop 9:25–60
Chung SM, Vaidya M, Tzfira T (2006) Agrobacterium is not alone: gene transfer to plants by
viruses and other bacteria. Trends Plant Sci 11:1–4
Clark JR, Jondle RJ (2008) Intellectual property rights for fruit crops. In: Hancock JF (ed)
Temperate fruit crop breeding. Germplasm to genomics. Springer, Berlin, pp 439–455
Cook RC (1931a) The first plant patent. J Hered 22:313–319
Cook RC (1931b) Three more plant patents. J Hered 22:369–372
Cook RC (1933a) Other plant patents. J Hered 24:49–54
Cook RC (1933b) The administration of the plant patent law from the breeder’s point of view. J Pat
Trademark Off Soc 15:275–282
Cook RC (1936) Plant patent 110 declared invalid. J Hered 27:394–400
Cooley D (2007) Transgenic organisms, the European Union, and the World Trade Organization.
In: Kolb RW (ed) The ethics of genetic commerce. Blackwell, Oxford, pp 87–108
DeBeer JF (2007) The rights and responsibilities of biotech patent owners. UBC Law Review,
pp 343–373
Delmer D (2003) The congested intellectual property landscape and its effect on the use of
biotechnology for the development of horticultural and subsistence crops. Acta Hortic
625:439–443
Delmer DP, Nottenburg C, Graff GD, Bennett AB (2003) Intellectual property resources for
international development in agriculture. Plant Physiol 133:1666–1670
Demangue S (2005) Intellectual property protection for crop genetic resources. A suitable system
for India. Dissertation, Herbert Utz Verlag, Munich, Germany, p 467
Dequiedt V, Meniere Y, Trommettier M (2007) Collective management of intellectual property
rights. Working Paper GAEL; 2007-04, Laboratoire d’Economie Appliquée de Grenoble, p 25
Dixon GR, Ogier J (2007) Understanding and protecting horticulture’s intellectual property – a
review of opportunities. Acta Hortic 760:61–68
Dodds J, Krattiger A, Kowalski SP (2007) Plants, germplasm, genebanks, and intellectual prop-
erty: principles, options, and management. In: Krattiger A, Mahoney RT (eds) Intellectual
Janeiro, Brazil) and bioDevelopments-International Institute (Ithaca, USA), pp 389–400,
www.ipHandbook.org
Donnenwirth J, Grace J, Smith S (2004) Intellectual property rights, patents, plant variety
protection and contracts: a perspective from the private sector. IP Strategy Today 9:19–34
Dunn JD, Seiler PF (2007) Trade secrets and non-traditional categories of intellectual property
as collateral. Presented to: UNCITRAL 2nd Int Colloq on Secured Transactions: Security
Interests in Intellectual Property Rights, p 13
Dunwell JM (2000) Transgenic approaches to crop improvement. J Exp Bot 51:487–496
Dunwell JM (2002) Future prospects for transgenic crops. Phytochem Rev 1:1–12
Dunwell JM (2004) Transgenic crops: the current and next generations. In: Peña L (ed) Transgenic
plants: methods and protocols. Humana Press, Totowa, NJ, pp 377–396 Meth Mol Biol, vol
286
Dunwell JM (2005) Review: intellectual property aspects of plant transformation. Plant Biotechnol
J 3:371–384
Dunwell JM, Ford CS (2005) Technologies for biological containment of GM and non-GM
Crops: http://www.defra.gov.uk/science/project_data/DocumentLibrary/CB02036/CB02036_
3629_FRP.doc
Dunwell JM (2006) Patents and transgenic plants. Acta Hortic 725:719–732
Dunwell JM (2007) 100 years on: a century of genetics. Nat Rev Genet 8:231–235
Dunwell JM (2008) Transgenic wheat, barley and oats: future prospects. In: Jones HD, Shewry PR
(eds) Transgenic wheat, barley and oats: production and characterisation. Humana Press,
Totowa, NJ, pp 333–345 Meth Mol Biol, vol 478
Dunwell JM (2009a) Patents and haploid plants. In: Touraev A, Forster BP, Jain SM (eds)
Advances in haploid production in higher plants. Springer, Berlin, pp 97–113
Dunwell JM (2009b) Patents for plants: context and current status. Acta Hortic 72:241–247
Durell KL (2006) Intellectual property protection for plant derived vaccine technology: here they
come are we ready or not? Lex Electronica 10:29 http://www.lex-electronica.org/articles/
v10-3/durell.htm
Duvick DN (1999) Commercial strategies for exploitation of heterosis. In: Coors JG, Pandey S
(eds) Genetics and Exploitation of Heterosis in Crops. Am Soc Agron, Madison, Wisconsin,
USA, pp 295–304
Eaton D (2007) Intellectual property rights in plant breeding and biotechnology: a comparative
institutional analysis. Presented at: 11th Ann Conf Int Soc New Instit Econ (ISNIE), Reykja-
vik, p 25
Eicher CK, Marieda K, Sithole-Niang I (2006) Crop biotechnology and the African farmer. Food
Pol 31:504–527
Eisenberg RS (2006) Biotech patents: looking backward while moving forward. Nat Biotechnol
24:317–319
Enserink M (2008) Tough lessons from golden rice. Science 320:468–471
Espinoza-Esquivel AM, Arrieta-Espinoza G (2007) A multidisciplinary approach directed towards
the commercial release of transgenic herbicide-tolerant rice in Costa Rica. Transgenic Res
16:541–555
Fairchild D (1927) The fascination of making a plant hybrid. Being a detailed account of the
hybridization of Actinidia arguta and Actinidia chinensis. J Hered 18:49–62
Farnley S, Morey-Nase P, Sternfeld D (2004) Biotechnology – a challenge to the patent system.
Curr Opin Biotechnol 15:254–257
Fay AE (1937) Are plant patents “inventions”? J Hered 28:261–262
Faye DJ (2005a) Exploitation of research tools in plant biotechnology: Access through application
of the experimental use exception (Part 1). Bio-Science Law Report. 8th Feb 2005
Faye DJ (2005b) Exploitation of research tools in plant biotechnology: Access through application
of the experimental use exception (Part 2). Bio-Science Law Report. 12th April 2005
Fleck B, Baldock C (2003) Intellectual property protection for plant-related inventions in Europe.
Nat Rev Genet 4:834–838
Fowler C (2000) The plant patent act of 1930: a sociological history of its creation. J Pat
Trademark Off Soc 82:621–644
428 J.M. Dunwell
Fromer JC (2007) Invigorating the disclosure function of the patent system. NYU Law School,
Public Law Research Paper Series, 2 March 2007: http://ssrn.com/abstract=967560
Fukuda-Parr S (2006) The gene revolution: GM crops and unequal development. Earthscan,
London, p 280
Gaisford JD, Hobbs JE, Kerr WA (2007) Will the TRIPS agreement foster appropriate biotechno-
logies for developing countries? J Agric Econ 58:199–217
Garrison C (2006) Exceptions to patent rights in developing countries. International centre for
trade and sustainable development (ICTSD). Issue Paper No 17, p 104
Ghijsen H (2007) Plant breeder’s rights: a fair and balanced intellectual property right for plant
varieties. Tailoring Biotechnol 3:79–98
Giannakas K (2001) The economics of intellectual property rights under imperfect enforcement:
developing countries, biotechnology, and the Trips Agreement. EPTD Discussion paper No 80.
Int Food Policy Research Institute, Washington, USA, 50 p
Goldman M (2007) The IP Management of the PRSV-Resistant papayas developed by Cornell
University and the University of Hawaii and commercialized in Hawaii. In: Krattiger A,
Mahoney RT (eds) Intellectual Property Management in Health and Agricultural Innovation:
A Handbook of Best Practices. MIHR (Oxford, UK), PIPRA (Davis, USA), Oswaldo Cruz
Foundation (Fiocruz, Rio de Janeiro, Brazil) and bioDevelopments-International Institute
(Ithaca, USA), pp 1837–1844, www.ipHandbook.org
Gonsalves C, Lee DR, Gonsalves D (2007) The adoption of genetically modified papaya in Hawaii
and its implications for developing countries. J Dev Stud 43:177–191
Gracen V (2007) How intellectual property and plant breeding come together: corn as a case study
for breeders and research managers. In: Krattiger A, Mahoney RT (eds) Intellectual Property
Management in Health and Agricultural Innovation: A Handbook of Best Practices. MIHR
(Oxford, UK), PIPRA (Davis, USA), Oswaldo Cruz Foundation (Fiocruz, Rio de Janeiro,
Brazil) and bioDevelopments-International Institute (Ithaca, USA), pp 1819–1827, www.
ipHandbook.org
Graff GD, Cullen SE, Bradford KJ, Zilberman D, Bennett AB (2003) The public-private structure
of intellectual property ownership in agricultural biotechnology. Nat Biotechnol 21:989–995
Griliches Z (1990) Patent statistics as economic indicators: a survey. J Econ Lit 28:1661–1707
Hamilton C (2006) Biodiversity, biopiracy and benefits: what allegations of biopiracy tell us about
intellectual property. Dev World Bioeth 6:158–173
Hays WM (1905) Distributing valuable new varieties and breeds. In: Proc 1st Meet of the Am
Breeders’ Assoc, St Louis, Mo, USA, pp 58–65
Heider D, Barnekow A (2007) DNA-based watermarks using the DNA-Crypt algorithm. BMC
Bioinformat 8:176
Heisey PW, King JL, Rubenstein KD (2005) Patterns of public-sector and private-sector patenting
in agricultural biotechnology. AgBioForum 8:73–82
Helfer LR (2004) Intellectual property rights in plant varieties. International legal regimes and
policy options for national governments. FAO Legislative Study 85, p 113
Henson-Apollonio, V (2007) Impacts of intellectual property rights on marker-assisted selection
research and application for agriculture in developing countries. In: Guimarães EP, Ruane J,
Scherf BD, Sonnino A, Dargie JD (eds) Marker-assisted Selection. Current status and Future
Perspectives in Crops, Livestock, Forestry and Fish, FAO, Rome, Italy, pp 405–425, ftp://ftp.
fao.org/docrep/fao/010/a1120e/a1120e.pdf
Hills MJ, Hall L, Arnison PG, Good AG (2007) Genetic use restriction technologies (GURTs):
strategies to impede transgene movement. Trends Plant Sci 12:177–183
Hossain M (2007) Technological progress for sustaining food-population balance: achievement
and challenges. Agric Econ 37:161–172
Howlett MJ, Christie AJ (2003) An analysis of the approach of the European, Japanese and United
States Patent Offices to partial DNA sequences (ESTs). Int Rev Indust Prop Copyright Law
34:581–710
Huskisson FM (1996) Patents and biotechnology. In: Owen MRL, Pen J (eds) Transgenic plants: a
production system for industrial and pharmaceutical proteins. Wiley, New York, pp 313–328
Janis MD, Smith S (2007) Technological change and the design of plant variety protection
regimes. Chicago-Kent Law Rev 82:1556–1615
Johns A (2006) Intellectual property and the nature of science. Cult Stud 20:145–164
Juma C, Seregeldin I (2007) Freedom to innovate: biotechnology in Africa’s development. A
report of the High Level African Panel on Modern Biotechnology. African Union and new
Partnership for Africa´s Development (NEPAD), Addis Ababa and Pretoria, p 163, http://
www.nepadst.org/doclibrary/pdfs/biotech_africarep_2007.pdf
Karapinar B, Temmerman M (2008) Benefiting from biotechnology: pro-poor intellectual property
rights and public–private partnerships. Biotechnol Law Rep 27:189–202
Kartal M (2007) Intellectual property protection in the natural product drug discovery, traditional
herbal medicine and herbal medicinal products. Phytother Res 21:113–119
Kesan J, Janis MD (2002) U.S. Plant Variety Protection: Sound and Fury...? Univ Illinois Working
Paper No. LE03-002 Available at SSRN: http://ssrn.com/abstract¼384140 or DOI: 10.2139/
ssrn.384140
Kesan JP, Janis MD (2003) Intellectual property protection for plant innovation: unresolved issues
after J.E.M. v. Pioneer. Illinois Public Law Research Paper No 03-01: SSRN: http://ssrn.com/
abstract¼378820 or DOI: 10.2139/ssrn.378820
Kesselheim AS, Avorn J (2005) University-based science and biotechnology products: defining
the boundaries of intellectual property. J Am Med Assoc 293:850–854
Kevles DJ (2007) Patents, protections, and privileges – The establishment of intellectual property
in animals and plants. Isis 98:323–331
Key S, Ma JK, Drake PMW (2008) Genetically modified plants and human health. J R Soc Med
101:290–298
Kingston W (2007) Repairing incentives to invest in plant breeding. Int Prop Q 10:294–311
Kimmelman BA (1983) The American Breeders’ association: genetics and eugenics in an agricul-
tural context, 1903–13. Soc Stud Sci 13:163–204
Klaasen JA (2007) Commercialization of the agrarian ideal and arguments against the new Green
Revolution: feeding the world with “Frankenfoods”? In: Kolb RW (ed) The ethics of genetic
commerce. Blackwell, Oxford, pp 109–126
Kock MA (2007) Essentially biological processes: the interpretation of the exception under Article
53(b) of the European Patent Convention. J Intellect Prop Law Prac 2:286–297
Kock MA, Porzig S, Willnegger E (2007) The legal protection of plant-biotechnological inven-
tions and plant varieties in light of the EC Biopatent Directive. IIC. Int Rev Intellect Prop
Comp Law 37:135–156
Koo B, Nottenburg C, Pardey PG (2004) Intellectual property. Plants and intellectual property: an
international appraisal. Science 306:1295–1297
Kowalski SP, Kryder RD (2002) Golden rice: a case study in intellectual property management
and international capacity building. RISK Health Saf Environ 13:47–67
Krattiger A, Mahoney RT (2007) Specific IP Issues with molecular pharming: case study of plant-
derived vaccines. In: Krattiger A, Mahoney RT (eds) Intellectual Property Management in
Health and Agricultural Innovation: A Handbook of Best Practices. MIHR (Oxford, UK),
PIPRA (Davis, USA), Oswaldo Cruz Foundation (Fiocruz, Rio de Janeiro, Brazil) and bioDe-
velopments-International Institute (Ithaca, USA), pp 1809–1817, www.ipHandbook.org
Krattiger A, Mahoney RT, Nelsen L, Thomson JA, Bennett AB, Satyanarayana K, Graff GD,
Fernandez C, Kowalski SP (2007) Executive guide to intellectual property management in
health and agricultural innovation: a handbook of best practices. MIHR (Oxford, UK), PIPRA
(Davis, USA), Oswaldo Cruz Foundation (Fiocruz, Rio de Janeiro, Brazil) and bioDevelop-
ments-International Institute (Ithaca, USA), p 284, www.ipHandbook.org
Kryder RD, Kowalski SP, Krattiger AF (2000) The intellectual and technical property components
of pro-vitamin A rice (GoldenRiceTM): a preliminary freedom-to-operate review. ISAAA Brief
430 J.M. Dunwell
No 20. International Service for the Acquisition of Agri-biotech Resources. Ithaca, New York,
USA
Kukier KN (2006) Navigating the future(s) of biotech intellectual property. Nat Biotechnol
24:249–251
Lacroix B, Kozlovsky SV, Citovsky V (2008) Recent patents on Agrobacterium-mediated gene
and protein transfer, for research and biotechnology. Recent Pat DNA Gene Seq 2:69–81
Laxmi T, Krishna PSJ, Reddy GP (2007) Changing paradigms in agricultural research. Signifi-
cance of end-user involvement. Outlook Agric 36:119–125
Lea D (2008) The expansion and restructuring of intellectual property and its implications for the
developing world. Ethic Theor Moral Prac 11:37–60
Léger A (2007) The role(s) of intellectual property rights for innovation: a review of the empirical
evidence and implications for developing countries. Discussion papers 707. DIW Berlin.
German Inst Econ Res, p 51
Lence SH, Hayes DJ, McCunn A, Smith S, Niebur B (2003) Welfare Impacts of Property Rights in
the Seed Industry, p 32: http://www.econ.iastate.edu/department/seminar/ispw/Sergio-Der-
mot-seed-paper.pdf
Lenssen M (2006) The overlap between patent and plant variety protection for transgenic plants:
problems and a solution: SSRN: http://ssrn.com/abstract¼924343
Lipton M (2007) Plant breeding and poverty: can transgenic seeds replicate the “Green Revolu-
tion” as a source of gains for the poor? J Dev Stud 43:31–62
Llewelyn M, Adcock M (2006) European plant intellectual property. Hart Publishing, Oxford
Locke SD (2007) Intellectual property for the botanist and the plant breeder: an overview of
protection afforded by plant patents and plant variety protection certificates. Chicago-Kent J
Intellect Prop 6:198–212
Louwaars NP, Tripp R, Eaton D, Henson-Apollonio V, Hu R, Mendoza M, Muhhuku F, Pal S,
Wekundah J (2005) Impacts of strengthened intellectual property rights regimes on the plant
breeding industry in developing countries: a synthesis of five case studies. Wageningen, the
Netherlands, p 176
Louwaars NP, Tripp R, Eaton D (2006) Public research in plant breeding and intellectual property
rights: a call for new institutional policies. Washington, The World Bank, Agricultural & Rural
Development Notes issue 13, p. 4 p, http://go.worldbank.org/YPG9S2D5V2
Mahop MT (2007) Patents, scientific and commercial use of plant genetic resources and traditional
knowledge: how does public interest fit in this arena? Icfai J Intellect Prop Rights 6:8–24
Malik KA, Zafar Y (2005) Intellectual property rights in plant biotechnology: a contribution to
crop biosecurity. Asian Biotechnol Dev Rev 8:7–42
Maraut S, Dernis H, Webb C, Spiezia V, Guellec D (2008) The OECD REGPAT database: a
presentation, STI Working Paper 2008/2, Statistical Analysis of Science, Technology and
Industry. OECD DSTI/DOC(2008)2, p. 36, http://www.oecd.org/dataoecd/22/19/40794372.
pdf
Marco AC, Rausser GC (2008) The role of patent rights in mergers: consolidation in plant
biotechnology. Am J Agric Econ 90:133–151
Mayer JE (2007) Golden rice, golden crops, golden prospects. Rev Colomb Biotecnol 9:22–34
Mayer J, Sharples J, Nottenburg C (2004) Resistance to phosphinothricin. CAMBIA Intellectual
Property, Canberra, p 43
McManis CR (2007) Biodiversity and the law: intellectual property, biotechnology and traditional
knowledge. Earthscan, London, p 484
Medakker A, Vijayaraghavan V (2007) Successful commercialization of insect-resistant eggplant
by a public-private partnership: reaching and benefiting resource-poor farmers. In: Krattiger A,
Mahoney RT (eds) Intellectual property management in health and agricultural innovation: a
handbook of best practices. MIHR (Oxford, UK), PIPRA (Davis, USA), Oswaldo Cruz
Foundation (Fiocruz, Rio de Janeiro, Brazil) and bioDevelopments-International Institute
(Ithaca, USA), pp 1829–1831, www.ipHandbook.org
Merrill SA, Levin RC, Myers MB (2004) A patent system for the 21st century. The National
Academies Press, Washington DC
Mgbeoji I (2006) Global biopiracy: patents, plants, and indigenous knowledge. Cornell University
Press, Ithaca, NY, p 289
Miki B, McHugh S (2004) Selectable marker genes in transgenic plants: applications, alternatives
and biosafety. J Biotechnol 107:193–232
Ministry of Economic Development (2002) New issues in patentability 1: Biotechnological
patents. In: Review of the Patents Act 1953: Boundaries to Patentability. Wellington, New
Zealand, pp 29–40
Moeller L, Wang K (2008) Engineering with precision; tools for the new generation of transgenic
crops. Bioscience 58:391–401
Morris M, Edmeades G, Pehu E (2006) The global need for plant breeding capacity: what roles for
the public and private sectors? HortScience 41:30–39
Moschini G, Yerokhin O (2007) The economic incentive to innovate in plants: patents and plant
breeders’ rights. In: Kesan J (ed) Intellectual property protection for agricultural biotechno-
logy. CABI, Wallingford, pp 190–203
Munzer SR (2006) Plants, torts, and intellectual property. In: Endicott T, Getzler J, Peel E (eds)
Properties of Law: Essays in Honour of Jim Harris, Oxford Univ Press: SSRN: http://ssrn.com/
abstract¼886838
Myskja BK (2006) The moral difference between intragenic and transgenic modification of plants.
J Agric Environ Ethics 19:225–238
Nottenburg C (2007) How to read a biotech patent. In: Krattiger A, Mahoney RT (eds) Intellectual
Janeiro, Brazil) and bioDevelopments-International Institute (Ithaca, USA), pp 351–360,
www.ipHandbook.org
Nottenburg C, Pardey PG, Wright BD (2002) Accessing other people’s technology for non-profit
research. Aust J Agric Resour Econ 48:389–416
Orozco LA, Chavarro-Bohórquez DA, Olaya DL, Villaveces JL (2007) Methodology for measur-
ing the socio-economic impacts of biotechnology: a case study of potatoes in Colombia. Res
Eval 16:107–122
Parayil G (2003) Mapping technological trajectories of the green revolution and the gene revolu-
tion from modernization to globalisation. Res Pol 32:971–990
Phillips RL, Chen J, Okediji R, Burk D (2004) Intellectual property rights and the public good.
Scientist 18:8
Pila J (2008) Article 53(b) EPC: A challenge to the Novartis theory of European patent
history. Oxford Legal Studies Research Paper No. 21/2008, p 18, SSRN: http://ssrn.com/
abstract¼1160191
Potrykus I (2007) GMO-technology and malnutrition- public sector responsibility and failure.
In: Busch RJ (ed) Biotechnologie in Gesellschaftlicher Deutung. Herbert Utz Verlag, Munich,
pp 157–167
Pray CE, Naseem A (2007) Supplying crop biotechnology to the poor: opportunities and con-
straints. J Dev Stud 43:192–217
Ramanna A (2005) Intellectual property rights, agriculture, and rural communities. J Agric Saf
Health 11:423–430
Rao VCK, Antharvedi U (2007) IPR regime in plant biotechnology with reference to Bacillus
thuringiensis cotton, 25 p, SSRN: http://ssrn.com/abstract¼960207
Rhodes C (2007) Four key roles for regulation of biotechnology: are they being fulfilled at the
international level? J Int Biotechnol Law 4:1–11
Rimmer M (2003) Franklin barley: patent law and plant breeders’ rights. Murdoch Univ Electr J
Law 10, 42 p, SSRN: http://ssrn.com/abstract¼603228
Rimmer M (2006) After the gold rush: patent law and biological inventions. ANU College of Law
Research Paper No. 07–11 Law in Context 24
432 J.M. Dunwell
Rimmer M (2007) The new conquistadors: patent law and expressed sequence tags. J Law Inform
Sci 16:10–50
Roa-Rodrigues C (2007) Promoters used to regulate gene expression. CAMBIA Intellectual
Property, Canberra, p 191
Roa-Rodrigues C, Nottenburg C (2007a) Agrobacterium-mediated transformation of plants.
CAMBIA Intellectual Property, Canberra, p 229
Roa-Rodrigues C, Nottenburg C (2007b) Antibiotic resistance genes and their uses in genetic
transformation, especially in plants. CAMBIA Intellectual Property, Canberra, p 38
Roff RJ (2008) Preempting to nothing: neoliberalism and the fight to de/re-regulate agricultural
biotechnology. Geoforum 39:1423–1438
Rossman J (1931) Plant patents. J Pat Off Soc 13:7–21
Salazar R, Louwaars NP, Visser B (2006) On protecting farmers’ new varieties: new approaches to
rights on collective innovations in plant genetic resources. CAPRI Working Paper No 45,
IFPRI, p 40
Schimmelpfennig D, King J (2006) Mergers, acquisitions and flows of agbiotech intellectual
property. In: Evenson RE, Santaniello V (eds) International trade and policies for genetically
modified products. CABI Publishing, Wallingford, pp 97–109
Schrell A, Bauser H, Brunner H (2007) Biotechnology patenting policy in the European Union-as
exemplified by the development in Germany. Adv Biochem Eng Biotechnol 107:13–39
Scoones I (2008) Mobilizing against GM Crops in India, South Africa and Brazil. J Agrar Change
8:315–344
Shear RH, Kelley TE (2003) A researcher’s guide to patents. Plant Physiol 132:1127–1130
Shrader-Frechette K (2005) Property rights and genetic engineering: developing nations at risk.
Sci Eng Ethics 11:137–149
Singh H (2004) Intellectual property rights (IPR) issues in horticulture: an Indian scenario with
particular reference to medicinal plants. Acta Hortic 662:465–470
Smith S (2008) Intellectual property protection for plant varieties in the 21st century. Crop Sci
48:1277–1290
Smith JSC, Hussain T, Jones ES, Graham G, Podlich D, Wall S, Williams M (2008) Use of
doubled haploids in maize breeding: Implications for intellectual property protection and
genetic diversity in hybrid crops. Mol Breed 22:51–59
Smyth S (2006) Implication and potential impacts from the expiry of patents on herbicide tolerant
canola varieties. Saskatchewan Canola Development Commission, p 48, www.saskcanola.
com/pdfs/scdc-patent-report.pdf
Spielman DJ (2007) Pro-poor agricultural biotechnology: can the international research system
deliver the goods? Food Pol 32:189–204
Spielman DJ, Cohen JI, Zambrano P (2007) Are developing-country policies and investments
promoting research and research partnerships in agricultural biotechnology? Int J Biotechnol
9:509–529
Sreedharan SK (2007) Coming of age – the Indian product patent regime. Intellectual Asset
Management Magazine. Special report on: From Innovation to Commercialisation 2007:
www.iam-magazine.com/issues/article.ashx?g¼e6c6910d-4788-4006-aed5-b9ad1aa69937
Stone R (2008) Intellectual property: chinese province crafts pioneering law to thwart biopiracy.
Science 320:732–733
Straus J (2007) The impact of the new world order on economic development: the role of the
intellectual property rights system. Eur Rev 15:47–63
Summers TM (2003) The scope of utility in the twenty-first century: new guidance for gene-
related patents. Georgetown Law J 91:475–509
Tansey G (2006) Global rules, patent power and our food future: controlling the food system in the
21st century. IIIS Discussion Paper No. 130: SSRN: http://ssrn.com/abstract¼923771
Temmerman M (2006) The patentability of plant genetic inventions. NCCR Trade Regulation
Working Paper No 2007/04: SSRN: http://ssrn.com/abstract¼1069948
Tencalla F (2006) Science, politics, and the GM debate in Europe. Regul Toxicol Pharmacol
44:43–48
Tokgoz S (2003) R&D spillovers in agriculture: results from a trade model. Center for Agri Rural
Dev, Iowa State Univ, Ames, Iowa, USA, Working Paper 03-WP 344, p 27
Tripp R, Louwaars N, Eaton D (2007) Plant variety protection in developing countries. A report
from the field. Food Policy 32:354–371
Troyer AF, Stoehr H (2003) Willet M. Hays, great benefactor to plant breeding and the founder of
our association. J Hered 94:435–441
Van Acker RC, Szumgalski AR, Friesen LF (2007) The potential benefits, risks and costs of
genetic use restriction technologies. Can J Plant Sci 87:753–762
Van Beuzekom B, Arundel A (2006) OECD Biotechnology Statistics – 2006. Organisation for
Economic Co-Operation and Development. p. 156, http://www.oecd.org/dataoecd/51/59/
36760212.pdf
Van Dooren T (2007) Terminated seed: death, proprietary kinship and the production of (bio)
wealth. Sci Cult 16:71–94
Virgin I, Bhagavan M, Komen J, Kullaya A, Louwaars N, Morris EJ, Okori P, Persley G (2007)
Agricultural biotechnology and small-scale farmers in eastern and southern Africa. Stockholm
Environment Institute, Stockholm, p 62
Watal J, Kampf G (2007) The TRIPS agreement and intellectual property in health and agriculture.
In: Krattiger A, Mahoney RT (eds) Intellectual Property Management in Health and Agricul-
tural Innovation: A Handbook of Best Practices. MIHR (Oxford, UK), PIPRA (Davis, USA),
Oswaldo Cruz Foundation (Fiocruz, Rio de Janeiro, Brazil) and bioDevelopments-Interna-
tional Institute (Ithaca, USA), pp 253–263, www.ipHandbook.org
Wilson J (2007) GM crops: patently wrong? J Agric Environ Ethics 20:261–283
World Bank (2006) Intellectual Property Rights. Designing regimes to support plant breeding in
developing countries. Washington DC, World Bank Agriculture and Rural Development.
Report # 35517, p. 77, http://siteresources.worldbank.org/INTARD/Resources/IPR_ESW.pdf
Wright BD (2006) Plant genetic engineering and intellectual property protection. Agri Biotechnol
in California Series. Publication 8186, p 6
Wright BD, Pardey PG (2006a) The evolving rights to intellectual property protection in the
agricultural biosciences. Int J Technol Globalis 2:12–29
Wright BD, Pardey PG (2006b) Changing intellectual property regimes: implications for develop-
ing country agriculture. Int J Technol Globalis 2:93–114
Wu H, Lu B (2005) Prior consents: preventing offensive genetic engineering patents against
indigenous people’s rights. Global Jurist Frontiers 5(1) article 3
Ye X, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P, Potrykus I (2000) Engineering the
provitamin A (b-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Sci-
ence 287:303–305
Yerokhin O, Moschini G (2007) Intellectual property rights and crop-improving R&D under
adaptive destruction. Center for Agri Rural Devel, Iowa State Univ, Ames, Iowa, USA,
Working Paper 07-WP 449, p 32
Ziff B (2005) Travels with my plant: Monsanto v. Schmeiser revisited. Univ Ottawa Law and
Technol J 2:493–509: SSRN: http://ssrn.com/abstract¼894084
Chapter 13
Transgenic Crop Plants: Contributions,
Concerns, and Compulsions
Brian R. Shmaefsky
13.1 Introduction
No one discovery, event, person, or product alone defines or typifies plant biotech-
nology. Biotechnology plants, known scientifically as transgenic plants or geneti-
cally modified plants (GMPs), are derived from a blend of ancient agricultural
practices and modern genetics-based technologies. Traditionally, plants served
societies primarily for basic needs, such as food and shelter from the environment.
Some early cultures made use of whole plants and plant compounds for medical and
religious purposes. Plants took esthetic roles as civilizations grew. Many plants in
early civilizations were selected for their beauty and fragrance to grow in gardens
and in homes. Biotechnology significantly improved the traditional use of plants by
improving the way plants are grown and the quality of the plant products. It has also
greatly expanded the roles of plants within the past 20 years. Plants are now used for
biomanufacturing a variety of commercial and industrial products. They are also
put to work for a host of bioremediation purposes (Shmaefsky 2007).
The principal people of biotechnology plant development are from a variety of
scientific disciplines. Many of the contributors to plant biotechnology are biolo-
gists. However, the field also uses the research efforts of chemists, computer
information scientists, engineers, medical doctors, mathematicians, and physicists
to provide the knowledge base and application potentials for biotechnology plant
development. Plant biotechnology innovations and research are not restricted to the
wealthiest developed nations. Many new developments are coming out of China,
India, Korea, and Mexico. Plant biotechnology is now a global effort, with each
country using the technology to meet local and universal needs (Ernst and Young
2007). Venture capital and technology transfer agreements are providing many
B.R. Shmaefsky
Lone Star College – Kingwood, HSB 202V, 20,000 Kingwood Drive, Kingwood, TX77339-3801,
USA
e-mail: Brian.R.Shmaefsky@lonestar.edu

436 B.R. Shmaefsky
incentives for unprecedented growth of the field in both the basic and applied
scientific aspects. Unlike many sciences, marketplace demand is fueling new
directions in plant biotechnology.
Plant biotechnology developments have made many valuable contributions to
agriculture, environmental remediation, horticulture, and medicine. Many of these
advances go unnoticed. Few people are likely to be aware of the scope of biotech-
nology during a typical purchase of foods and products at a department store or a
supermarket (Fritz et al. 2003). In spite of the benefits, many advances in plant
biotechnology are not applauded by governmental officials, the public, and scien-
tists. In contrast, these developments are criticized for a wide variety of reasons.
Certain trepidations are founded on legitimate issues related to environmental
quality, food safety, public health, and sustainable economic development. Other
criticisms of plant biotechnology are provoked by compulsions rooted in culture,
morality and public perception (Morris 2007). Both categories of criticism have
hindered the progress of plant biotechnology. However, they have also contributed
to refinements in rationale for developing biotechnology plants. In addition, they
have fostered new ways of improving the safety of this upcoming technology.
13.2 Contributions of Plant Biotechnology Sciences
13.2.1 Contributions to Biotechnology
Plant biotechnology made use of ancient and modern discoveries and technologies
to produce new types of plants and to find new uses for plants. Researchers not only
improved the plants with innovative ideas, but they also advanced the field by
applying animal, bacterial, and fungal research to plant models. In some situations,
plants even became more viable substitutes for the original research model asso-
ciated with the discovery or new technology (Shmaefsky 2007). For example, the
production of human therapeutics produced in genetically modified (GM) animal
cell lines were proven to be safer if biomanufactured in plant cell cultures. Plants do
not carry the hidden cytoplasmic and genomic parasites and pathogens transmitted
through animal cell lines (Shmaefsky 2005).
For many centuries, people have been choosing and modifying the character-
istics of native and imported plants for agricultural and medical purposes. The
intentional cultivation of plants began in the Middle East about approximately
15,000 years ago as societies moved away from hunting-and-gathering lifestyles
(Mannion 1999). Small farms were set up to grow native plants used for human and
animal feed. Unique characteristics of the plants were preserved by collecting and
sowing only the seeds from plants with sought-after traits (Kaniewski et al. 2007).
This cultivation of plants gave communities a consistent supply of food from one
season to another. However, cultivation eventually led to the domestication of
plants having a combination of desirable qualities not available by standard
13 Transgenic Crop Plants: Contributions, Concerns, and Compulsions 437
Table 13.1 Major events leading to plant biotechnology

Year Event People/place
Pre-12000 BCE Origins of crop cultivation Middle and Near East
Circa 8000 BCE Selective domestication of crops Middle and Near East
Circa 8000 BCE Domestication of ornamental plants Asia, Europe, and Middle East
Circa 7000 BCE Corn domesticated South America
Circa 4000 BCE Most modern-day crops domesticated Africa, Northern Europe and
Far East
Circa 2000 BCE Globalization of domesticated crops Worldwide
700 BCE Crops irrigation and pest control Europe and Near East
300 BCE First books on plant domestication Theophrastus
100 BCE Sowing and harvesting technology China
1663 First description of cells R. Hooke
1860s Principles of trait inheritance G. Mendel
1890s Inheritance applied to crop production H. DeVries
1890s Natural selection applied to agriculture C. Darwin
1900s Gene locus mapping T.H. Morgan
1939 First successful plant tissue culture P. Nobécourt
1944 First transformation experiment O. Avery
1952 Transposable elements discovered B. McClintock
1953 Role and structure of DNA confirmed E. Chargaff , F. Crick, L.
Pauling, J. Watson
1972 DNA splicing with restriction enzymes P. Berg
1973 First genetically modified organism H. Boyer, S. Cohen
1977 DNA sequencing F. Sanger
1978 Discovery of RFLP and gene markers D. Botstein
1983 Polymerase chain reaction discovered K. Mullis
1983 First GMO plant – tobacco D.T. Gibson
1987 Bioinformatics developed E. Lander
1994 First commercial GMO Tomato Calgene Inc, Davis, CA
cultivation (Betz 1998). This desire to cultivate crops contributed to the cultivation
of food animals and thereby improved the likelihood of gathering a balanced diet
(Diamond 1999). Cattle and deer were attracted to cultivated fields and were then
corralled for easy gathering when preparing to harvest the animals (Mannion 1999).
The major events leading to plant biotechnology are furnished in Table 13.1.
Early attempts at selective breeding were used 10,000 years ago for domestica-
tion to produce a monoculture of plants that can be grown for consistent features
from one generation to the next. The early domesticated plants, known as Neolithic
founder crops, included purebred and hybridized cereals, fiber plants, and pulses
(Zohary and Hopf 2000). The production of custom-designed plants was generally
limited to food and medical plants (Woods and Woods 2000). However, ornamental
plants were developed in various countries including Asia and parts of Europe and
the Middle East (Harlan 1980). Greek, Persian, and Roman agriculture from
700 BC experimented with irrigation, pesticides, and weed control to improve
crop yield (Isager and Skydsgaard 1992). The domestication of plants gave people
the principles to selectively breed animals with characteristics favorable for
438 B.R. Shmaefsky
agricultural purposes (Mannion 1999). Iron Age people in northern Europe drank
the milk from domesticated cows over 3,000 years ago (Shmaefsky 2005, 2007).
After 1866, selective breeding of commercially important plants led to a finer
degree of precision upon the publication of G.J. Mendel’s lectures in the Proceed-
ings of the Natural History Society (Mendel 1866). In the 1900, H. de Vries,
C. Correns, and E. von Tschermack rediscovered Mendel’s cross ratios in plants
(Goldschmidt 1950). With this information they were able to breed field crops
with predictable characteristics. At the same time, this information was melded
with C. Darwin’s ideas of species selection in his work On the Origin of Species by
Means of Natural Selection, or The Preservation of Favoured Races in the Struggle
for Life, published in 1899 (Bowler 1989). Plants were being produced for what was
identified as superior qualities usually related to ease of cultivation and consistency
of quality (Mannion 1999). Later, the need for resistance to pathology and predation
was recognized as essential elements of plant worthiness. The evolutionary princi-
ples of plant selective breeding were then applied to agricultural animals and
microorganisms involved in the fermentation of beverages and foods (Shmaefsky
2005).
Trait variation and distribution in crop selective breeding were better understood
after T.H. Morgan’s work on mutations and gene locus mapping in the early 1900s
(Morgan et al. 1915). The knowledge of gene loci distances facilitated the predict-
ability of producing purebred crops with several desirable characteristics in a
particular variety of crop. In 1977, F. Sanger opened the door for analyzing crop
genes with the development of the chain termination method for DNA sequencing
(Sanger 2001). The identification and isolation of potentially valuable crop genes
was facilitated by D. Botstein’s 1978 work on restriction fragment length poly-
morphisms (RFLP) and gene markers (Tanksley et al. 1989; Heesacker et al. 2008).
Contributions of modern plant gene loci engineering are serving as models for other
eukaryotic systems (Roden et al. 2005). Work on plant genome sequences are
providing generalized data for molecular genetic studies at the gene locus and
chromosome levels. Many of the plant studies can directly translate to animal and
yeast genomic research.
Unfortunately, in the middle twentieth century Soviet Union, T.D. Lysenko
abused the concept of crop superiority by breeding for arbitrary crop characteristics
related to the political ideology of Russian botanist I. Vladimirovich Michurin
(Graham 1998). This work was an extension of the eugenics doctrine favored by
F. Galton who in 1883 misinterpreted C. Darwin’s and A.R. Wallace’s views of
selective fitness (Bulmer 2003). It forced the scientific community to establish a
realistic interpretation of crop fitness that is reflected today in contemporary
agronomic principles (Williams et al. 1998). Most conventional crop researchers
did not follow the premises and protocols of Lysenko contributing to the integrity of
evolutionary biology and plant genetics applications.
The technology of plant cultivation remained unchanged from classical Greek,
Persian, and Roman times until the advent of plant tissue culture. Initial advances in
animal cell culture in the 1920s conducted by Alexis Carrel paved the way for plant
tissue culture. In 1934, R.J. Gautheret performed preliminary studies on plant tissue
with limited success. He based his research on the animal cell culture work of
A. Carrel. Plant tissue culture became more feasible in 1939 following the work of
P. Nobécourt. Further refinements in growing conditions and media made it possi-
ble to grow a wide variety, based on the findings of L. Knudson, F. Skoog,
K.V. Thimann, F. Went, and P. White. They developed precision growing condi-
tions by identifying the roles of hormones on plant cell differentiation, growth, and
organogenesis (Gautheret 1983). Advances in plant hormone and growth factor
research provided the foundations for understanding similar cell-to-cell channel-
mediated communication systems in other organisms (Fleming 2005). These chan-
nels, which resemble plasmodesmata, convey critical developmental signals in
mammalian embryos (Hertzberg and Skibbens 1984).
Plant tissue culture permitted the rapid clonal propagation of plants in a disease-
free environment. Cloning eliminated the need for continually bred hybrid plants
each generation and it also facilitated the propagation of seedless varieties. Cultur-
ing plant tissues free from bacteria and viruses aided in the removal of untreatable
diseases during clonal propagation (Bhojwani and Soh 2003). Plant propagation
entered a new era with the first successful genetic engineering conducted by
P. Berg in 1972 and the first genetically modified organism produced by H. Boyer
and S. Cohen in 1973. Genetic engineering from bacteria and yeast models was
applied to plants in the middle 1980s with the development of genetically engi-
neered crops tobacco and tomato produced for field use (Christipeels and Sadava
2003). In turn, it became simple to grow experimental plants in vitro and as field
models for other transgenic eukaryotic systems (Piruzian et al. 2006; Bassett 2007).
The “biotechnology era” was fully established in agriculture in the 1980s with
the development of polymerase chain reaction (PCR) by K. Mullis’s team at Cetus
Corporation (Mullis et al. 1994). It made possible the amplification desirable genes
from minute samples collected from archived and fresh specimens. PCR also made
it possible to store isolated genes as well as whole genomes in germplasm banks
(Committee on Managing Global Genetic Resources 1993). Advances in transfec-
tion vectors with reporter genes for eukaryotic systems in the late 1980s increased
the feasibility of producing genetically modified crops (Marillonnet et al. 2005).
Another boon was the creation of reverse transcription PCR (RT-PCR) in the late
1980s (Stoflet et al. 1988). It permitted the amplification of genes from rare
transcripts extracted from a cell or synthesized from protein sequences. PCR studies
in plant pathology contributed to disease investigation in other agriculture and food
production fields (Kageyama et al. 2003). Soil PCR studies, in particular, have led
to advances in understanding the spread of animal and human pathogens on various
environmental surfaces.
Biotechnology plants have been developed for a variety of marketable applica-
tions since the first GMP was commercialized in 1994 (Piruzian et al. 2006). The
Flavr Savr tomato, produced by Calgene Incorporated of Davis, California, showed
that GM plants were safe for general human consumption, which is one of most
strictly regulated applications of novel plants (Martineau 2001). GM plants could
be developed rapidly with precise characteristics almost unattainable by breeding
crosses. New traits could be introduced without sacrificing other characteristics
440 B.R. Shmaefsky
unintentionally lost in the production of monoculture plants. However, this initial

success did not show that GM plants were economically viable alternatives to plants
produced by traditional selective breeding (Piruzian et al. 2006). The growing field
of extrachromosomal genetic engineering was established in plants during the
1990s. The genetic modification of chloroplasts contributed to metabolic engineer-
ing trial in algae and the mitochondria in animal cells (Shmaefsky 2007). Cultiva-
tion of GM plants in different countries in 2007 is shown in Fig. 13.1. The trend in
increase in cultivation of transgenic crop plants is depicted in Fig. 13.2.
The late 1990s proved different from early periods of plant development with the
creation of drought, herbicide, and insect-resistant crop plants (Table 13.2). Plants
were now being developed for dealing with environmental stress as well as market
quality and consistency. They were practicable to grow for field production and
inspired the other commercially feasible plants. Plant biotechnology in twenty-first
century moved out of the field and into the industrial manufacturing setting. GM
plants were diversified for uses in biopharming and chemical production (Berger
2007). Forestry also benefited with GM trees capable of doubling the output of
wood fiber per tree (Shmaefsky 2000). A new generation of GM field plants was
being developed and exploited during that period. Field trials of GM plants con-
tribute greatly to the field of bioremediation. Plants capable of bioremediation in the
field were developed to degrade and uptake a variety of environmental pollutants
(Mulligan 2002). This led to a new technique called phytoremediation that aug-
mented the roles of bacteria and fungi in bioremediation (Willey 2007).
Philippines 0.3
Uruguay 0.5
South Africa 1.8
Paraguay 2.6
China 3.8
India 6.2
Canada 7
Brazil 15
Argentina 19.1
United States 57.7
0 10 20 30 40 50 60
millions of hectares
Fig. 13.1 Cultivation of biotechnology plants in 2007 (From: EarthTrends, June 2008 Monthly
Update: Genetically Modified Crops and the Future of World Agriculture (http://earthtrends.wri.
org/updates/node/313))
100,000
90,000
World
80,000
70,000
Thousand hectares
60,000 Developed
Countries
50,000
40,000
Developing
Countries
30,000
20,000
10,000
0
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005
Fig. 13.2 Biotechnology plant cultivation from 1996 to 2005 (From: EarthTrends, June 2008
Monthly Update: Genetically Modified Crops and the Future of World Agriculture (http://earth-
trends.wri.org/updates/node/313))
Table 13.2 Global area of genetically engineered crops, 1996 to 2006: by trait (million hectares)
Trait HT IR (Bt) IR/HT VR/others Total
1996 0.6 1.1 – <0.1 1.7
1997 6.9 0.4 <0.1 <0.1 11
1998 19.8 7.7 0.3 <0.1 27.8
1999 28.1 8.9 2.9 <0.1 39.9
2000 32.7 8.3 3.2 <0.1 44.2
2001 40.6 7.8 4.2 <0.1 52.6
2002 44.2 10.1 4.4 <0.1 58.7
2003 49.7 12.2 5.8 <0.1 67.7
2004 58.6 15.6 6.8 <0.1 81
2005 63.7 16.2 10 <0.1 90
2006 69.9 19 13.1 <0.1 102
HT Herbicide tolerance; IR Insect resistance (mostly Bt); VR Resistance to virus diseases
Source: ISAAA, Clive James, 2006
(From: Global Status of Commercialized Biotech/GM Crops: 2006 (http://www.isaaa.org/
RESOURCES/PUBLICATIONS/BRIEFS/35/EXECUTIVESUMMARY/default.html)) Permission
to publish table granted by the International Service for the Acquisition of Agri-Biotech Applications
Bioinformatics heralded in another period of plant biotechnology advances. It

provided the computational power needed to interpret complex genomic expression
information (Lander et al. 1987). This branch of computational science integrates
information on plant gene expression, genotype, phenotype, and taxonomic
442 B.R. Shmaefsky
relationship with analytical tools for interpreting multivariate data. It is currently

providing unprecedented information for building models that give a better under-
standing about how biotechnology plants interact with abiotic and biotic environ-
mental factors. Bioinformatics is also paving the way for new types of plant derived
products from novel biotechnology plants (Shmaefsky 2007). Advances in plant
bioinformatics are contributing to other organismic models particularly related to
long-term environmental factors that regulate gene expression (Edwards 2007).
The complexity of data interpretation provided by bioinformatics has led to the
compartmentalization of plant biotechnology research into genomics, proteomics,
cellomics, metabolomics, physiomics, and enviromics (Edwards 2007; Shmaefsky
2007). Genomics is the oldest of the endeavors and is rooted in traditional genetics
that is continually updated by contemporary molecular biology investigations.
Genomics investigates the DNA level of plant function and is tentatively catego-
rized into subspecialties such as chromatinomics, chromonomics, and epigenomics.
Chromatinomics is defined as “the total differentiation capacity, gene expression
as well as other stem cell functional characteristics that vary throughout a cell cycle
transit” (Cerny and Quesenberry 2004). Although chromatinomics started out as an
animal cell field, recent advances in botany showed that plants have stem cell with
characteristics similar to animals. This adds to the validity that plants can serve as
accurate genomic models for animals for understanding organismic development
(Dinneny and Benfey 2008). Chromonomics, coined by H. Willard of Case Western
Reserve University, is similar in meaning to chromatinomics and deals with how
genetic sequences relate to the overall function of an organism.
Epigenomics is a holistic approach to understanding an organism’s genome. It is
described as an approach that views imprinting, metabolic networks, genetic
hierarchies in embryonic development, and epigenetic mechanisms of gene activa-
tion and other complex phenotypes from the genomic level down, rather than from
the genetic level up (Beck et al. 1999). Crop plant research in epigenomics is
contributing to a vast database of knowledge applicable to most multicellular
organisms. Plant epigenomics is also contributing in plant usage to a human
genomics field called ethnogenomics. Ethnogenomics was defined by E. K. Khus-
nutdinova as “The main task of ethnogenomics is to study the characteristics of
genomic polymorphism and genomic diversity of various groups of population:
separate communities, ethnoses, and ethnoterritorial communities” (Khusnutdinova
2003). Genomic properties of early cultivated and domesticated plants correlate
with population-specific single nucleotide polymorphisms that affect diet-related
health (Box 1981; Limborska 2004).
Proteomics investigates the protein makeup of an organism in contrast to the
DNA information. According to Stephen M. Beverley of the Mitsubishi Kagaku
Institute of Life Sciences (MITILS) of Japan, proteomics is study of the structure
and function of proteins, including the way they function and interact with each
other inside cells. It is related to the term proteogenomics and transcriptomics,
which also investigate the roles of transcribed RNA (Anon 1999). Plants serve as
excellent proteomic model organism and GM plants are being exploited for unique
and practicable transgenic proteins having a variety of agricultural, alternative
energy, commercial, and medical uses (Gibson 2008). It is hoped that GM plants
may serve as a sustainable and “green” source of biomanufacturing many produces
made from fossil fuels (Shmaefsky 2007).
Cellomics, metabolomics, and physiomics all investigate different levels of
biochemical functions within an organism. The term cellomics was used to describe
cell function particularly related to drug impacts at the cellular level (Russo 2000).
Transgenic plants that modify cellomic processes are under development for
precision disease treatment (Shmaefsky 2005). Metabolic and physiomics research
is conducted on the organismic levels. These fields have expanded beyond the study
of human pathology to an understanding of processes in any type of multicellular
organism and are now covered under the category of functional genomics (Fiehn
2001). Biotechnology plant metabolic engineering trials are serving as research
models for other colonial and multicellular eukaryotic organisms.
Enviromics is the most global study of genomic function. It was coined by J. C.
Anthony of Michigan State University School of Medicine to mean “The envirome
is the total complement of environmental characteristics, conditions, and processes
required for life form viability and successful adaptation. The genome dwells
within the environment, and genomic expression shapes and is shaped by environ-
ment.” Enviromics was originally used in the human context for studying drug
interactions. It is now being generalized for any organism. New developments in
GM medicinal plants are contributing to two fields of human enviromics called
pharmacomics and plant nutriomics (Yan et al. 2006). It is hoped to develop
medical plant products tailored to pharmacogenetic differences in animals and
humans.
Transgenic plants are valuable contributors to modern biotechnology as well
being benefactors of genetic research on other organisms. Many of the investiga-
tions on transgenic plants are leading to innovative technologies with far-reaching
economic potential. The biotechnology platform currently includes several spe-
cialty areas and research competencies centered on biotechnology plants. Included
in the transgenic plant platforms are efforts in biochemical and genetic diversity,
large scale biochemical and organic molecule production, metabolic pathway engi-
neering, phenotype development and improvement, and protein design (Hertzberg
and Skibbens 1984; Piruzian et al. 2006; Shmaefsky 2007).
13.2.2 Plant Platform Contributions
The development and uses of transgenic plants are still in the infancy stage.
However, transgenic plants are forming the foundation of biotechnology research
and development (R&D) platforms. The European Community developed an inter-
nationally acknowledged classification of biotechnology platforms according to a
particular industrial strategy unique to that type of biotechnology. These platforms
are the basis of R&D efforts that have agricultural, commercial, or medical applica-
tions (Hertzberg and Skibbens 1984; Piruzian et al. 2006; Shmaefsky 2007).
444 B.R. Shmaefsky
The Plant Industry Platform is primarily involved in investigating and producing

genetically exceptional plants used in agriculture, forestry, and horticulture. It also
provides a source of genes used in genetic engineering of microorganisms and
plants for commercial applications. This platform is investigating and developing
applications for plants that produce commercial proteins, dietary supplements,
herbal therapeutics hormones, medical diagnostics compounds, pharmaceutical
compounds, research chemicals, and vaccines. Another aspect of this platform is
phytoremediation or the use of plants to clean up contamination of air, soil, and
water with pollutants (Hertzberg and Skibbens 1984; Piruzian et al. 2006; Shimoda
1998; Shmaefsky 2007). Lower plants are also serving as a valuable source of genes
and are currently being used in environmental quality biomonitoring systems
(Martin 1998).
Transgenic plants are making significant contributions to the Plant Structural
Biology Industrial Platform. This platform focuses on the functional chemistry of
organisms. It includes investigations into the structural analysis of biological
molecules at every level of organization. This platform is studied using all
methods that lead to an understanding of biological function in terms of molecular
and supermolecular structure. Supermolecular structure refers to the forces that
cause molecules to interact with other molecules and carry out various tasks. The
Structural Biology Industrial Platform looks at the technology transfer potential
of carbohydrates, lipid, nucleic acids, and proteins applications (Hertzberg and
Skibbens 1984; Piruzian et al. 2006; Shmaefsky 2007). Current products of this
platform include commercial cements, industrial enzymes, medical adhesives,
nanotechnology devices, preservatives, and synthetic plastics all of which can be
derived from transgenic plants. For example, lignin pathways are modifiable to
produce a wide array of organic polymers ranging from synthetic fuels to plastics
(Hans-Joachim and Weiting 1998).
The Biotechnology for Biodiversity Platform is a basic research platform that
uses information about biodiversity for technology transfer into industrial applica-
tions. Biodiversity is generally defined within this platform as the number and
variety of living organisms. It takes into account the genetic diversity, species
diversity, and ecological diversity of all organisms on the Earth and even on
other planets. The biodiversity platform primarily investigates the potential com-
mercial applications of particular genes identified through biodiversity investiga-
tions. The botanical aspect of this platform identifies genes from wild plants
ancestors of crops. These genes can be used to help crops resist diseases, drought,
insects, herbicides, and poor soil quality (Eizenga et al. 2006). A significant
proportion of the research in this platform involves the establishment of gene
banks (Hertzberg and Skibbens 1984; Piruzian et al. 2006; Shmaefsky 2007). The
DNA information collected within this platform’s gene bank is stored as a bio-
informatics catalog of the DNA sequence and the various traits imparted by a
particular sequence of DNA.
One of the newest areas is the Environmental Biotechnology Industrial Platform.
It is engaged in the pursuit of novel environmental biotechnologies. Environmental
biotechnology is a broad field that includes a wide variety of agricultural and
industrial applications. The Environmental Biotechnology Industrial Platform uses

biological systems to remediate anthropogenic and natural chemical degradation of
the atmosphere, land, and water. Some current applications include soil and sedi-
ment remediation, water purification, the removal of organic and inorganic pollu-
tants, the breakdown or biodegradation of organic pollutants, introduction of
natural or genetically modified organisms to treat solid wastes, water treatment,
marine clean-up, and the conversion of wastes into other materials and energy
(Hertzberg and Skibbens 1984; Piruzian et al. 2006; Shmaefsky 2007). This plat-
form overlaps with the Plant Industry Platform. The Biotechnology for Biodiversity
Platform also contributes to this platform by identifying genes relevant to environ-
mental protection and remediation.
Transgenic plants are also making significant contributions to the In Vitro
Testing Industrial Platform. This platform was formed from economic, ethical,
political, moral, and scientific arguments in favor of reducing or replacing the
need for animal tests commonly used in medicine and research. The platform
investigates technologies that comply with the same governmental regulations
that set the guidelines for animal testing. It involves the development of in vitro
tests that model the chemistry and functions of animals, microorganisms, and
plants. The technologies used in this platform currently involve the use of animal
cell cultures to replace the role of whole live animals for testing the effectiveness
and safety of many consumer products (Hertzberg and Skibbens 1984; Piruzian
et al. 2006; Shmaefsky 2007). Model plant systems using hairy root cell cultures are
being used as a GM plant in vitro model within this platform (Hu and Du 2006).
However, plant models are also being investigated as adjuncts to animal and
microbial models. For example, it is known that cytoskeleton components of plants
can be generalized to other eukaryotic organisms (Grabski et al. 1998). In vitro
models are used for safety testing on chemicals such as cleaning agents, cosmetics,
dietary supplements, dyes, food ingredients, fragrances, inks, preservatives, and
soaps (Shmaefsky 2007). The plant models in this platform must be based on sound
scientific principles and have ample evidence to show that they provide equivalent
data to animal studies. Current European Community Platforms directly and indi-
rectly related to plant biotechnology are listed in Table 13.3.
13.3 Concerns
13.3.1 Assessing the Risks of Transgenic Plants
As with almost any scientific application or technology, biotechnology poses

potential risks associated with the benefits it provides. Most technologies are used
in spite of the risks they create. For example, the Internet has benefited many people
throughout the world with the conveniences of electronic commerce. However, the
risk of identity theft and credit fraud comes along with the advantages of shopping
446 B.R. Shmaefsky
Table 13.3 Current European community biotechnology platforms

ACTIPa Animal Cell Technology Industrial Platform: The Animal Cell Technology Industrial
Platform includes animal cell technologies involved in a variety of industrial and
medical applications.
BACIP Bacillus Subtilis Genome Industrial Platform: The main goal of this platform is bring
together information and technological applications related to the genetics the
Bacillus bacterial. Bacillus bacteria carry out a variety of metabolic activities
that have important commercial value.
BBPb Biotechnology for Biodiversity Platform: This is a basic research platform that uses
information about biodiversity for technology transfer into industrial applications.
EBIPb Environmental Biotechnology Industrial Platform: This is one of the newer platforms
and is engaged in the field of environmental biotechnology.
ENIPa European Neuroscience Industrial Platform: This platform focuses on medical and
pharmaceutical applications related to information about the nervous system.
FAIPa Farm Animal Industrial Platform: This platform is composed of small and large
agricultural operations involved in farm animal reproduction and selection.
FIPa Fungal Industry Platform: The Fungal Industry Platform represents research and
technology transfer efforts interested in biotechnology applications of filamentous
fungi.
HAE Healthy Aging Europe Industrial Platform: This platform combines research on human
aging with biotechnology innovations that may reduce ailments and diseases
attributed to age.
IPMb Industry Platform for Microbiology: This is a basic science platform that provides
information on microbial physiology, microbial ecology, microbial taxonomy, and
microbial biodiversity.
IVTIPb In Vitro Testing Industrial Platform: This platform was formed from economic, ethical,
political, moral, and scientific arguments in favor of reducing or replacing the need
for animal tests commonly used in medicine and research.
LABIP Lactic Acid Bacteria Industrial Platform: The main goal of this platform is coordinate
information and technological applications related to the genetics the lactic acid
producing bacteria.
PIPb Plant Industry Platform: The Plant Industry Platform is primarily involved in genetically
unique plants used in agriculture, forestry, and horticulture.
SBIPb Structural Biology Industrial Platform: This platform focuses more on the chemistry of
organisms. It includes any investigations into the structural analysis of biological
molecules at every level of organization.
TSE IP TSE Industrial Platform: This platform deals with research related to transmissible
spongiform encephalopathies.
YIPa Yeast Industry Platform: This platform is founded on any applications of yeast related
biotechnology.
a
Indirectly supplemented by plant biotechnology
b
Directly related to plant biotechnology
online. Over time, societies provide protective measures to reduce risk, and secure
sites that encrypt Internet data in order to reduce information theft. A risk is
traditionally defined as the probability of harmful consequences associated with
an entity or an action. However, it is important that the risks associated with
scientific applications and technologies are evaluated in a detectable or quantitative
context (Hansson 2004).
Risks of transgenic plants, as is done for other scientific and technological
developments, are determined using a risk assessment (Apostolakis 2004). A risk
assessment is the determination of qualitative and quantitative values of risk related

to an entity or situation and recognized as a measurable threat (Wilson and
Shlyakhter 1997). Transgenic plants are subject to different types of risk assessment
models based on the risks related to their applications, development, or platform
categories (Wilson and Shlyakhter 1997; Apostolakis 2004; Shmaefsky 2007).
A risk is first determined by identifying any hazards related to the entity of action.
The hazard identification determines the nature of the potential adverse conse-
quences of the entity or activity and the strength of the evidence supporting that it
can have that type of hazardous effect. Evidence of a hazard must be determined by
empirical studies that should have a cause and effect relationship or mechanism for
the type of harm being induced. It is also necessary to identify the recipient of the
hazard such as agricultural animals, an ecosystem, or humans (Wilson and Shlyakhter
1997; Apostolakis 2004).
It is generally accepted by governmental regulatory agencies that it is impracti-
cable and likely impossible to adopt a zero-risk policy (Wilson and Shlyakhter
1997). So, once a risk identification study is completed, an acceptable risk determi-
nation is calculated. There is no universally acceptable model for computing how
much harm is tolerable for a particular risk. However, the calculation must be
represented as a numerical value of what is determined to be a negligible increase in
risk or harm from the technology. The negative control for determining negligible is
a comparison to not having the technology (Apostolakis 2004). It is in many cases
unclear how the consensus on a particular figure is established. This apparent
arbitrariness in quantifying acceptable risk leaves the determination open to con-
troversy and debate amongst the public, regulators, and scientists (Flyvbjerg 2006).
The risk assessment must also take into account acute versus long-term or lifelong
risks (Wilson and Shlyakhter 1997; Apostolakis 2004).
A benefits assessment is essential to weight the importance of accepting a risk by
using a technological development (Wilson and Shlyakhter 1997; Flyvbjerg 2006).
The benefits are characterized in a variety of ways based on the intended application
of the technology. For almost all applications of transgenic plants, the benefits need
to be accurate, quantitative, and realistic. In addition, the benefits must be based on
empirical evidence supported by the mode of action of the technology (Flyvbjerg
2006; Shmaefsky 2007). Upon evaluating the benefits, risk–benefit determination is
then calculated for the technology (Fig. 13.3; Virine and Trumper 2008). The
assessment that it assumes exposure to personal risk is a normal aspect of everyday
life and that a risk should not be used to completely negate the value of the benefits.
Several types of risk–benefits analyses can be performed on transgenic plant
applications. A statistical risk–benefits analysis is determined using currently
available data on the related past benefits and risks. Novel products and technolo-
gies require at projected risk–benefits analysis. This analysis uses theoretical
models structured from historical on similar, but not identical, benefits and risks.
Some types of innovations are subject to perceived risk–benefit analyses. This
assessment is based on intuition and weighs emotional factors that can cause people
to overestimate the risks to the degree of diminishing the value of the benefits
(Virine and Trumper 2008). Many types of transgenic plants applications
448 B.R. Shmaefsky
Risk Assessment
Experimentation Risk
Hazard Entry of
Action
No Yes
Risk
Determination
No Action
Exposure
Estimation
Consequence
Benefits
Assessment
Risk
Yes Estimation No
Deterimnation
Risk No Action
Management
Program
Fig. 13.3 Risk assessment flowchart

unfortunately stir fears that compel regulators to carry out a perceived risk–benefit
study (Shmaefsky 2005; Morris 2007). Positive outcomes of perceived risk are
critically important in ensuring the marketability of biotechnology plants (Spehar
2000).
Transgenic plant risk assessment and regulations are carried out in the US by
three governmental agencies: the Department of Agriculture (USDA), the Environ-
mental Protection Agency (EPA), and the Food and Drug Administration (FDA).
Each agency is individually responsible for oversight of genetically engineered
plants and products developed in the US and imported from other countries (Farm
Foundation 2005). Their responsibilities are determined by the nature of the
biotechnology plant application.
For example, the USDA oversees applications related to animal feed and human
food. A division of the USDA called the Animal and Plant Health Inspection
Service (APHIS) has authority over the field cultivation of transgenic plants.
It also oversees veterinary biological substances, including animal vaccines that
are products of biotechnology under the Virus, Serum, Toxin Act. The EPA has
jurisdiction over the testing of transgenic plant products containing pesticidal
compounds. This covered primarily under the Toxic Substance Control Act Bio-
technology Program of the Office of Prevention and Toxic Substances. They also
develop regulations on plants grown in the field and used for phytoremediation. The
FDA is responsible for overseeing biotechnology plant products used for
manufacturing nutritional supplements and pharmaceutical agents (Farm Founda-
tion 2005; Shmaefsky 2007). The FDA usually regulates biotechnology plants
under the Federal Food, Drug, and Cosmetic Act. In many situations, the supervi-
sory authorities of these agencies have common characteristics that overlap, some-
times causing jurisdictional confusion. Current United States regulations over
biotechnology plants can be found on the USDA (USDA Animal and Plant Health
Inspection Service 2007), EPA (EPA Biotechnology 2008; EPA Pesticides 2008),
and FDA websites (FDA Biotechnology 2008).
As in the United States, transgenic plants are regulated to differing degrees in
other nations. Transgenic plants in Canada are regulated by the Canadian Food
Inspection Agency (Canadian Food Inspection Agency 2008). They are charged
with safeguarding Canada’s food supply and the plants and animals to ensure
consistent standards for safe and high quality foods and agriculture-related pro-
ducts. They share this responsibility with another government agency called Health
Canada (Health Canada/Sante 2008). Health Canada evaluates the human health
safety of products developed from transgenic plants including cosmetics, foods,
pharmaceuticals, medical devices, pesticides. Environmental health concerns of
biotechnology plants are also assessed by Health Canada. Canada also has the
Canadian Biotechnology Advisory Committee, which advises on biotechnology
plant risk policy (Canadian Biotechnology Advisory Committee 2008).
European Union (EU) nations regulate biotechnology risks under the European
Commission on Biotechnology (European Commission on Biotechnology 2008).
This role is carried out by the Secretariat-General who supervises Directorate-
General that oversee specialized areas of technology. The Directorate-General
450 B.R. Shmaefsky
over Life Sciences and Biotechnology works with transgenic plants. It develops
new policies and evaluates how the policies fit into other EU institutions. Risk
issues are covered under guidelines related to the Confidence in Science-Based
Regulatory Oversight recommendations (Commission of the European Commu-
nities 2002). The European Parliament deals with legal aspects of biotechnology
plant risks (European Parliament 2006). They have jurisdiction over biotechnology
risks categorized as industrial biotechnology and bioremediation, nonfood agricul-
tural and silvicultural biotechnology, and pharmaceuticals.
Many Asian nations are collectively investigating harmonization agreements
that are in agreement with America, Canadian and European risk assessments. The
Food and Agriculture Organization of the United Nations is serving as a neutral
forum for assessing biotechnology plant risks in Asia (Food and Agriculture
Organization of the United Nations 2008). The cooperating nations include
Bangladesh, China, India, Indonesia, Malaysia, Pakistan, Philippines, Sri Lanka,
Thailand, and Viet Nam. The Organization for Economic Co-operation and Devel-
opment is another harmonization agency with a more global charge of making
biotechnology plant risk evaluation studies for countries worldwide. They state
their mission as, “The main focus of the work is on international harmonization of
regulatory oversight in modern biotechnology, which will ensure that environmen-
tal health and safety aspects are properly evaluated, while avoiding nontariff trade
barriers to products of the technology” (Organization for Economic Co-operation
and Development 2008).
Biotechnology applications are likely the most highly scrutinized and regulated
contemporary technologies. Much of this is due to the newness of the applications
meaning that there is a large lack of past data about probable risks. It is also possible
that the strict regularly environment of biotechnology plants is to ensure that almost
no harm is produced to appease public misunderstanding and unwarranted pubic
fear of biotechnology (Fritz et al. 2003; Shmaefsky 2005, 2007). Tight regulation
has not stifled the advancement of transgenic plant development. However, it has
slowed the pace of getting products to market and produced a technology transfer
environment requiring high costs to accommodate for comprehensive risk assess-
ments (Caruso 2006).
13.3.2 Case Studies in Biotechnology Plant Risks
The regulations placed on biotechnology plant applications are meant to reduce the
chance of significant concerns of risks to the end user and the environment.
However, this does not always allay concerns about transgenic plants and their
products. Scientifically valid problems have arisen from transgenic plants causing
regulators to reconsider risk assessments. Four representative risk concerns came
about with the earliest successful transgenic plants released on the market: hori-
zontal gene transfer, genetic pollution and super weeds, biopesticide safety, long-
term toxicological effects, and biodiversity loss (Shmaefsky 2005).
13.3.2.1 Horizontal Gene Transfer
Horizontal gene transfer, which is also known as lateral gene transfer, is described
as the natural genetic transformation of bacteria believed to be the essential
mechanism for genetic plasticity within and between species (Davison 1999).
This inherent tendency of bacteria to exchange DNA in the environment raised
safety concerns about the production of transgenic bacteria. It was believed that
novel genes introduced into bacteria could spread to wild type bacteria thereby
producing potentially harmful wild bacteria. This genuine concern compelled the
biotechnology community to hold the Asilomar Conference on Recombinant DNA
in 1974 (Berg and Singer 1995). As a result of the conference, guidelines were
developed to reduce the risk of horizontal gene transfer from genetically modified
microorganisms.
The widespread field cultivation of transgenic plants in the late 1990s raised
concerns if horizontal gene transfer was possible in plants. Michael Syvanen in
1994 reported that studies on plant phylogeny showed horizontal gene transfer as a
natural phenomenon involved in evolutionary change (Syvanen 1994). Plants
contained genes from the genomes of other plants and of soil microorganisms.
His other supporting work consisted of various genetic studies dating back to the
early 1980s, including recent genomic investigations on Archaea. Other researchers
have reported evidence that functional chunks of DNA persisted in the environ-
ment. Plus they found that significant quantities of gene vector-like plasmid DNA
survived passage through a mouse digestive system and even stayed intact after
macrophage ingestion. At the time of his initial research, Syvanen had no confirmed
studies of this type of gene transmission occurring in nature. However, the compel-
ling laboratory studies were evidence enough to warrant Syvanen’s concern of
horizontal gene transfer in the environment by plants.
Much of the scientific community was not persuaded by Syvanen’s extrapola-
tions about horizontal gene transfer in plants. They felt the risk was unfounded
because of the lack of direct experimental evidence in the field (Ho 2002). This
skepticism was undermined in 2001 when a plausible mechanism for horizontal
gene transfer in plants was proposed. A study reported the transmission of complete
gene cassettes by rhizosphere Pseudomonas (Sengelov et al. 2001). A host of
phylogenetic, laboratory, and greenhouse studies further supported the risk of
horizontal gene transfer by a variety of rhizosphere and pathogenic bacteria includ-
ing Agrobacteria (Broothaerts et al. 2005). Concerns were now being raised about
wild plants picking up novel genes and soil bacteria obtaining kanamycin resistance
reporter genes from transgenic plants.
Unfortunately, there is not enough research about the frequency of horizontal
gene transfer in the field. So, it is difficult to make any policies or regulations that
reduce the incidence of horizontal gene transfer to other plants or to soil micro-
organisms. Estimates of horizontal gene transfer from transgenic plants to micro-
organisms in the environment is likely at a frequency one trillion times lower than
the current risk assessment from laboratory data (Heinemann and Traavik 2004).
Sensitive methods for finding reporter genes or other components of transgenic
452 B.R. Shmaefsky
gene vectors will have to be developed to come up with acceptable limits of

transfer.
Metabolic engineering of transgenic plants creates other concerns of horizontal
gene transfer. There is now experimental evidence that the plant organelle genes
can by transferred in the field (Richardson and Palmer 2007). The studies showed
that parasitic plants were donated and received mitochondrial genes when attached
to host plants. Data indicated that most of the transfer involved a single gene.
However, horizontal gene transfer of large sets of mitochondrial genes was also
demonstrated. The studies showed the transfer in bryophytes and eudicots. It is
shown that the same mechanism can transfer chloroplast genes. Parasitic plants are
common invaders of agricultural lands adjacent to natural areas. They are particu-
larly invasive in the ecotomes formed by plant cultivation (Pennisi 2006). Fre-
quency studies capable of doing a risk assessment are not available.
The apparent risk of horizontal gene transfer with transgenic plants is a valid
scientific claim. Laboratory evidence supports that intact functional genes can be
transmitted from plants somewhat readily to other plants and biosphere micro-
organisms. The precedence for transferring deleterious traits from one organism to
another is well supported in the microbiological literature. Phylogenetic studies on
plants confirm genomic and epigenomic sharing through the rhizosphere micro-
organisms and parasitic plants. In spite of these findings, there is no compelling
evidence that the amount of horizontal gene transfer from transgenic plants poses
harm to people or the environment. As follows, it is nearly impossible to come up
with rational risk assessments. In addition, there are no definitive strategies for
reducing horizontal gene transfer from transgenic plants.
13.3.2.2 Genetic Pollution and Super weeds
Natural plant breeding mechanisms raised another set of legitimate risk concerns
for transgenic plants grown in the field. A growing number of studies confirmed the
gene flow between closely related plants during normal pollination mechanisms
(Arias and Rieseberg 1994). The consequence of gene flow is the transmission of
traits between crop plants and wild type ancestors living within pollination distance.
Earlier papers used the term genetic pollution to describe this contamination of the
environment with domesticated plant genes (Dubois and M’orere 1980). Many
proponents of transgenic plants see this as a mechanism for producing uncontroll-
able wild plants called super weeds (Ellstrand and Schierenbeck 2000). The term
super weed was coined in 1949 by E. Anderson to explain newly invasive plant
lines that resulted from hybridization between crops and related wild plants. N. C.
Ellstrand reintroduced the term to mean wild plants that picked transgenes from
transgenic plant relatives. He mainly was concerned about weeds picking up
characteristics that make them more invasive and difficult to control.
Genetic pollution at first was held with the same skepticism as horizontal gene
transfer by the scientific community. Initially, there was not a body of evidence
supporting the creation of super weeds through gene flow. However, in 2000,
N.C. Ellstrand used DNA analyses to document 28 examples of hybridizations that

increased the aggressiveness of plants (Ellstrand and Schierenbeck 2000). This
increase in vigor promoted invasive traits in the recipients of the gene flow. He
showed pollen transfer and compatibility as the mechanism of gene flow. Compel-
ling data was presented for the following gene flow events: Male sterile canola
(Brassica napus) picked up fertility genes from wild radish (Raphanus raphanis-
trum) and birdsrape mustard (B. rapa L.), oilseed rape (canola) passed transgenes
for herbicide resistance to turnip rape (B. rapa syn. B. campestris), and sorghum
(Sorghum bicolor) exchanged flowering time genes with Johnsongrass (S. halepense).
Transgenes also make their way from genetically modified alfalfa, berries, cotton,
cucumbers, potatoes, rice, strawberries, and sunflowers. The probability of the
transgenes appearing randomly through mutation in the wild population was highly
unlikely.
The possible deleterious effects of herbicide resistance genes were particularly
being criticized by opponents to transgenic plants. A concern about gene flow
patterns that produce herbicide-resistant weeds was fueled by the abundance of
studies on the transmission of other types of transgenes (Cavan et al. 2008). Gene
flow of herbicide resistance genes would in effect render weed plants resistant to
the popularly used broad spectrum agricultural herbicides. This valid concern
compelled government investigators to seek proactive strategies for controlling
herbicide-resistant super weeds.
The early research did not evaluate transgene transfer frequency and did not
establish an accurate determination of the environmental proximity needed for
successful transgene transmission in the field. However, the studies did establish
the fact that traits that are most likely to be retained in hybrids are those, which
improve fitness by encouraging invasiveness, drought tolerance, disease resistance,
herbicide resistance, and winter hardiness. The research also recognized that the
repeated introduction of transgenes produces an effect called swamping, which
ensures establishment of the new genes in the population (Rissler and Mellon
1996). Evidence of super weeds being created in the field was confirmed in a
2004 study conducted by the Department for Environment, Food and Rural Affairs
in the UK. The study showed gene flow of herbicide resistance genes from GM
oilseed rape to wild rape (Daniels et al. 2004).
Initially, transgenic plants grown in the field were designed with excision genes
and genes that cause male infertility (Daniell 2002). Excision genes remove the
transgene when not expressed. Infertility genes prevented pollen formation. These
technologies were used to prevent the spread of transgenic plants into the wild.
Concerns were raised that these strategies were not effective at preventing gene
flow and even horizontal gene transfer (Jackson et al. 2002). For example, the
excision genes could be separated from the desirable genes on another expression
vector.
In 1999, J. Gressel of the Weizman Institute of Sciences proposed another
approach to reducing the risk of gene flow between GM crops and wild plants.
He suggested using tandem construct vectors to reduce the chance of successful
gene transfer through pollination (Gressel 1999). Tandem repeat technology
454 B.R. Shmaefsky
piggybacks two genes that restrict gene flow. For example, genes affecting germi-
nation by altering seed dormancy, ripening, and dissemination closely linked to
genes for the desired trait in the construct. Other strategies include adding traits that
cause dwarfing, inhibit flower production, and prevent maturation. The basis of the
constructs were to use tightly linked genes that do not segregate separately, use
traits that are harmless to crops but deleterious to typical weeds, and use genes that
are disadvantageous to the successful reproduction of weeds within a population
lacking the construct trait.
The risk analysis of gene flow in transgenic plants requires an understanding of
several factors that affect the successful expression of transgenes in nontarget
plants. First, the probability of gene flow must be determined. This involves looking
at the potential gene flow candidate plants growing within pollination distance to
the transgenic plants. Factors designed to limit gene flow are taken into account
when determining the probability. Another factor that must be assessed is the effect
of gene flow on improving the fitness of the wild plant receiving the transgenes.
Another consideration in a risk analysis is the ability for gene flow that disables
factors such as suicide genes in the transgenic crop plants.
Gene flow is a valid scientific risk that is already being addressed in the design of
transgenic plants. It is more likely a greater risk than horizontal gene transfer. There
is ample evidence that the gene flow has occurred. Improved strategies for reducing
the back and forth gene exchange between transgenic plants and wild relatives will
be needed for the development of each novel genetically modified crop grown in
greenhouses and in the field. Continuous monitoring of super weeds and transgenic
plants disabled by gene flow is required to develop future strategies to reduce gene
flow. The scientific community needs to compromise on accepting worst-case
scenario analysis models until more information is collected to fully evaluate any
risks of deleterious gene flow (Thompson et al. 2003).
13.3.2.3 Biopesticide Safety
Some of the earlier biotechnology plant applications were intended at reducing

pesticide use in agriculture while seeking less crop damage by herbivorous pests.
This was driven by a growing number of environmental and public health regula-
tions mandating reductions in agricultural chemical use (Shmaefsky 2005; Piruzian
et al. 2006). In the late 1980s in the United States, transgenic plant development
was influenced by a revitalization of Delaney Amendment-type food safety con-
cerns. In 1958, Vermont Senator Charles L. Delaney added a provision to the US
Food, Drug, and Cosmetic Act, which stated that no food additive shall be deemed
safe after it is found to induce cancer when ingested by human beings or animals, at
any dose level. Such an additive therefore must not be used (Benders 1995).
This zero tolerance thinking became the acceptable risk held by the public. It
was spurred by media reports about the possible harmful effects of agricultural
chemicals, food additives, food irradiation, food-borne microorganisms, and drugs
used in agricultural animals (Jones 1992). As a result, crop growers were looking
for ways of reducing pesticide use. Pesticides in particular were targeted for the
harmful effects as possible carcinogens and endocrine disrupters (Colborn et al.
1996). Harmless natural pesticides such as pyrethrums were not efficacious for
large scale field crop production (Shmaefsky 2005). So, other options to safely
control crop pests had to be evaluated resulting in the development of biopesticides.
The EPA defines biopesticides as “Certain types of pesticides derived from such
natural materials as animals, plants, bacteria, and certain minerals. For example,
canola oil and baking soda have pesticidal applications and are considered biopes-
ticides” (Environmental Protection Agency 2008). There are approximately 200
registered biopesticides found in a variety of products. Developers of transgenic
plants took advantage of microbial pesticide genes with a cytotoxic specificity
towards insect digestive systems. The most effective of these was Bacillus thur-
ingiensis toxins, or Bt toxins, derived from a Gram-positive soil bacterium that is
also found in the guts of insects. Bt toxins are delta-endotoxin that lyse cells lining
the digestive system of insects and nematodes susceptible to a specific type of
Bt toxin. The toxin binds cadherin-like proteins and forms ion channels that cause
an efflux of potassium ions from the cells. Bt toxins are active within hours and
ultimately result in starvation within a few days (Harper 1974).
There are many Bt toxin genes. Common ones used in biotechnology plant insect
control applications are the Cry1Ab, Cry1Ac, Cry1F, Cry3Bb, Cry34Ab1, and
Cry35Ab1. The genes are modified in various ways to increase transcriptional
functionality in plant cells and are inserted into cauliflower mosaic virus (CaMV)
35S promoter expression vector (Lewin et al. 1998). Bt gene products proved safe
for agricultural use by the EPA and USDA. This led to the release of Bt corn, cotton,
and potatoes as field crops in the 1990s (Piruzian et al. 2006). The field cultivation
of these crops was uneventful until concerns arose about the potentially harmful
effects of Bt on nontarget insects, pest insect resistance to Bt, and vector–virus
recombination.
Bt corn carrying the Cry1ab gene used against corn borer became the focus of
criticism about its effects on nontarget insects. Two factors precipitated this con-
cern. Corn is wind-pollinated and the corn borer is a lepidopteran. The wind
pollination factor raised legitimate concerns about the spread of the Bt toxin into
nontargeted areas. The Bt toxin was meant to be effective as the corn borer larvae
were feeding on plant tissues expressing the gene. Several studies cautioned that it
was possible that Bt corn pollen could be toxic to beneficial Lepidoptera in areas
downwind from the fields (Sears et al. 2001).
The study focused on monarch butterflies and correlated with an apparent
decline in monarch population migrating between Eastern North American Bt fields
and Mexico. It also contained a risk assessment model that supported the potential
hazardous effects of Bt toxin corn pollen on wild insect populations outside of the
field. Studies by the EPA confirmed that harmful levels of Bt toxin protein were
present in the corn pollen and that monarch larvae feeding on the pollen could be
harmed (US Environmental Protection Agency 1995). However, there was no study
confirming a direct link between dwindling monarch populations related to the
distribution of Bt corn fields.
456 B.R. Shmaefsky
Researchers at the University of Illinois conducted a study in 2001 to look into

the corn pollen density of the monarch feeding areas adjacent to corn fields. They
discovered that within a corn field the pollen densities are below 600 grains cm–2 of
leaf for 95% of the plants. The highest pollen density was 1,400 grains cm–2 under
rainless conditions just after anthesis (Pleasants et al. 2001). The USDA discovered
in 2002 that “eating leaves with pollen coating densities below 1,000 grains cm–2
had no effect on caterpillars’ weight or survival rate. Above 1,000 grains cm–2,
caterpillars were smaller than those from the control treatments, but their survival
rate was no different from that of controls” (Kaplan 2002). So, the USDA report
invalided the claims that Bt corn was responsible for monarch population loss. It is
now believed that the monarch population incongruity way have been due to errors
in reporting the population or due to a variety of density-independent and density-
independent environmental factors (Borland et al. 2004).
Pesticide resistance is a realistic and scientifically valid risk of using any type of
pesticide. Biopesticides are equally likely candidates for being counteracted by
evolutionary mechanisms of target organisms. There are three major mechanisms
for pesticide resistance at the biochemical level against biopesticidal compounds:
alterations of pesticide toxicity target proteins, the development of enzyme-
mediated resistance, and thermal stress response (Patil et al. 1996). Biopesticides
that bind to surface proteins, such as Bt toxin, malathion, and pyrethroids, require a
specific ligand binding conformation site on the particular target protein. Mutations
that affect the amino acid composition of the binding site can render the biopesti-
cide less effective by reducing binding tenacity (Williamson et al. 1996). For
Bt toxin, this lack of binding specificity to the target receptor means reduced
capability to lyse intestinal cells of the insect pest.
Biopesticides are treated as xenobiotics by target organisms and are thereby
subject to degradation by detoxification metabolic pathways. Typical detoxification
enzymes found in many organisms include esterases, glutathione S-transferases
(GST), and oxidases (Cygler et al. 1993). Most types of Bt toxin crystal proteins
were initially thought to be resistant to enzymatic decay. Studies confirm that
multiple types of toxins derived from B. T. israelensis are subject to resistance by
enzymatic degradation in certain lepidopterans (Keller et al. 1996). Heat or thermal
shock proteins likely play a role in Bt toxin resistance (Patil et al. 1996). They have
a homeosis in which exposure to high levels of a toxin inhibits the defenses while
low or subtoxic exposure stimulates protective mechanisms against the toxin
(Cohen 2006). As with other biopesticides, Bt toxin is likely to induce thermal
shock because they induce lysis by apoptosis. Apoptosis induction is one factor
having a homeosis effect (Samali and Cotter 1996). Methods have been developed
in which the Cry2A class of Bt toxin genes can be modified to reduce thermal shock
induced resistance (Mandal et al. 2007).
Transgenic plants using the CaMV expression vector, and related vector types,
have been criticized by M.-W. Ho of the Open University in the UK as being a
“promiscuous” sequence of genetic material. She used this term to describe the
vector’s ability to recombine readily in unpredictable ways with genomic DNA,
extrachromosomal DNA, and intracellular organisms such as viruses (Ho et al.
2000). The ability for Bt CaMV expression vectors to combine with plant viruses
during field infections is supported in the literature (Kohli et al. 1999). There is
concern that this ability to recombine with wild type viruses can produce new types
of viruses with harmful characteristics produced by vector–virus recombination
(De Vries and Wackernagel 1998). Ho provides compelling theoretical evidence
that the CaMV vector can transfer Bt cassettes to viruses that then transfer the
functional gene to wild plants growing in the vicinity of transgenic plants. She
believes that this hazard can spread far from the fields of transgenic plants (Ho and
Steinbrecher 1998).
Laboratory studies and knowledge of the behavior of transgenic plant expression
vectors support the idea of possible hazards associated with biopesticides expres-
sing plants. Evidence is available to assess nontarget insect toxicity, biopesticide
resistance, and vector–virus recombination as measurable risks associated with
transgenic plants that express biopesticides. However, as with other concerns
about transgenic plants, there is little field evidence about the degree of risk
posed by the technology. So, it is difficult to determine the severity of the risk.
Consequently, it is not possible to calculate if the risk outweighs the benefits of
the biotechnology plants. Older regulations were apparently not adequate for
preventing these risks based on the current findings (Rechcigl 1998).
13.3.2.4 Long-Term Toxicological Effects
A growing number of biotechnology plants are being genetically modified for the
food supplement and nutraceutical (or nutriceutical) markets (Hugenholtz and Smid
2002). This usually involved enhancing plant metabolite expression or introducing
transgenes for a nutritional supplement such as an amino acid or a vitamin. The
most common types of nutraceutical biotechnology plants are engineered to pro-
duce antioxidants, phytoestrogens, prebiotic compounds, and vitamins (Ajjawi and
Shintani 2004). These plants were developed partly based on global consumer
demand for nutritional supplements and to a certain extent to provide higher
nutrition plants for developing nations (Piruzian 2005; Shmaefsky 2005). Many
of the ingredients of transgenic plants go into functional foods. Functional foods are
foods formulated to provide a certain wellness benefit and may contain plants
materials designed to overexpress omega-3 fatty acids that are believed to reduce
heart disease (International Life Sciences Institute 2002).
For the good they were intended to provide, certain biotechnology-based food
supplement and nutraceutical plants were received with much criticism from the
scientific community (Shmaefsky 2005). Two products of particular concern were
golden rice and high-isoflavone soybeans. There were fears that improper control
over the consumption of these products can lead to toxicological dosing (Millstone
et al. 1999; Domingo 2000).
Golden rice was developed by P. Beyer and I. Potrykus in 1999 to investigate the
possibility of b-carotene production in grains (Ye et al. 2000). It used the phytoene
synthase (psy) gene from daffodil and the carotene desaturase (crtI) gene from the
458 B.R. Shmaefsky
bacterium Erwinia uredovora to produce b-carotene, which in turn is converted to

vitamin A in the human digestive system. The ultimate goal of overexpressing
vitamin A was to reduce malnutrition-induced blindness primarily in the develop-
ing nations. Rice is normally low in vitamin A. Yet, rice makes up a bulk of the
caloric intake of people in the developing nations. This first generation of golden
rice did not produce sufficient enough quantities of b-carotene to meet the 300 mg
per day vitamin A requirement for young children. So, a new generation of
transgenic rice called golden rice 2 was produced by modifying the expression
vector. It synthesized 20-times the amount of b-carotene as the original golden rice
(Paine et al. 2005).
Nutritional concerns about golden rice ranged from furthering malnutrition to
inducing vitamin A toxicity. Research shows that improper or prolonged storage of
golden rice significantly reduces the b-carotene content (Zimmermann and Qaim
2004). People not educated about storing the rice may end up not being benefited by
the golden rice. It is feared that they may even avoid other forms of vitamin A
supplementation provided by public aid organizations. Vitamin A toxicity is par-
ticularly a concern with golden rice 2 because of its high b-carotene content
(Anderson et al. 2004). Pregnant women were a special precautionary group
because of the known teratogenic effects of vitamin A in large doses. Overdosing
concerns were a valid argument based on experience with foods fortified with iron
(Hurrell 1997; Van den Berg 1999). This concern haunts the use of transgenic rice
developed by the International Rice Research Institute to sequester iron equivalent
to food fortification levels (Florentino 2001). These concerns are being addressed
and remedied by risk–benefit studies related to food distribution and public
education programs (Al-Babili and Beyer 2005).
Tomatoes and other crops developed for high flavonoid content created scientifi-
cally valid concerns about phytoestrogens in the diet. These crops were primarily
grown for the purported health benefits of the antioxidant properties of flavonoids
(Reddy et al. 2007). Ample in vitro research shows that dietary antioxidants can
reduce the incidence of cancer and cardiac diseases (Goodman et al. 2004). Other
studies show that antioxidants reduce inflammation induced disease by suppressing
serum C-reactive protein (Chun et al. 2008). In addition, some plants were devel-
oped for the phytoestrogen effect of flavonoids as a hormone supplement therapy
(Ji and Peterson 2004). The plants are developed by inserting or enhancing flavonoid
biosynthetic pathways (Jung et al. 2000).
Studies on increased phytoestrogens in diet showed risks that in some people
may outweigh the benefits. A high to moderate intake of dietary phytoestrogens can
promote breast cancer in women with breast cancer susceptibility genes (Bernstein
2002). Other studies show that increased levels of dietary phytoestrogens can
impair estrogen function in any individual (Kuiper et al. 1998). Phytoestrogens
may inhibit testosterone function during human male embryogenesis in mothers
who consume large levels of estrogen-like compounds (Hoei-Hansen et al. 2004).
The US FDA currently places precautions on antioxidant and phytoestrogen nutra-
ceuticals, under Title 21 – Part 101, because of the lack of adequate long-term
human health risk–benefit studies (Code of Federal Regulations 2008). Other
precautions and guideline about functional food safety and utility are provided by
the Institute of Food Technologists (Institute of Food Technologists 2005).
13.3.2.5 Biodiversity Loss
The danger of GMOs to biodiversity has been a concern to many scientists since the
1986 field trials of ice-minus Pseudomonas syringae that were genetically engi-
neered to protect plants from frost damage (Amarger 2003). It was released with
great protest from various environmental protection groups by Advanced Genetic
Sciences of Oakland, California. Greenpeace International was one of the earlier
groups to mount an anti-GMO campaign (Greenpeace International 2008). They
support their opposition to field GMO crops by stating, “GMOs should not be
released into the environment as there is not adequate scientific understanding of
their impact on the environment and human health.” Greenpeace believes that there
is a long-term risk of biodiversity loss created by field grown biotechnology plants.
They rightfully defend their view with the fact that there are no adequate studies
showing that biodiversity would not be affected. However, there is little direct
scientific evidence that biodiversity would be significantly and irreversibly be
harmed. Their outlook on biodiversity impairment by biotechnology is supported
by other conservation organizations such as World Wildlife Fund (World Wildlife
Fund 2008).
A science based nonprofit organization called the Union of Concerned Scientists
(UCS) uses extrapolative field studies and laboratory research to support their
opposition to GMOs. In particular, they are opposed to field grown transgenic
plants (Union of Concerned Scientists 2008). They state, “UCS does not support
or oppose genetic engineering per se. With respect to some applications, such as the
production of pharmaceuticals by genetically engineered bacteria, the benefits are
clear and compelling. In the food system, however, we find the risk–benefit calculus
more difficult.” As consistent with Greenpeace, the UCS feels the risks of GMO
field crops have not been fully explored and that these crops have the potential to
disrupt biodiversity.
A variety of research studies support the biodiversity hazards of field grown
transgenic plants. Some studies show that GMO crops could affect fauna and flora
through intensification of agricultural activities into wilderness fueled by easier
cultivation in previously non-arable areas and by greater profits from transgenic
plants (Manteo 1998; Johnson 2000). Nontarget wild insect deaths have also been
confirmed with filed use of biopesticides (Birch et al. 1997; Jensen 2000). The
impacts of horizontal gene transfer and gene flow on plant biodiversity near
agricultural lands has also been studied and assessed for potential biodiversity
impairment risks (Ammann et al. 1999; Hodgson 2000; Crawley et al. 2001).
Aquatic biodiversity is also been studied because of the possible deleterious effects
caused by phytoremediation and biopesticides biotechnology plants. A variety of
studies also investigated the impacts of biotechnology field crops on different
460 B.R. Shmaefsky
aspects of soil biodiversity (Donegan et al. 1997; Crecchio and Stotzky 1998; Rosi-
Marshall et al. 2007).
Accurate and research based risk assessments are currently being determined for
the impacts of transgenic plants on local and global biodiversity. The risks asso-
ciated with agricultural development have to be assessed separately from the direct
impacts of the plants on the environment. Earlier risk assessments are being
combined with contemporary findings to develop rational risk–benefit models for
field grown transgenic plants (Regal 1989; Williamson 1993; Ammann et al. 1996;
Clark and Lehman 2004; Garcia and Altieri 2005; Raybould 2006; Raybould 2007).
So far, there is currently no measurable biodiversity loss directly associated with
the unique attributes of transgenic plants compared to traditional field grown plants.
13.4 Compulsions
There are many public health fears of transgenic plants in spite of the best regula-
tions that assure low risks of harm to humans and the environment (Fig. 13.4).
Many of these fears in the early days of biotechnology were not founded on
scientific evidence (Lewis 1998). These fears are usually fueled by pseudoscientific
claims from anti-biotechnology groups or are founded on anecdotal evidence.
Unfortunately, these compulsions are reinforced by negative or inaccurate media
coverage and are promulgated by certain opponents of biotechnology (Baker 2005).
Today, many of the compulsions are due to a lack of public understanding of the
science behind transgenic plants and the regulations that reduce risks to environ-
mental and public health (Gaskell and Bauer 2001).
The emotions behind compulsions are not something to be ignored or taken
nonchalantly. They become myths that are difficult to erase from public memory.
Many of the myths remain on anti-biotechnology websites that are regularly
cited as factual information by consumer and public interest groups. Public
Fig. 13.4 Perceived risk

misunderstanding of transgenic plants in the past has led to anti-biotechnology

legislative policies, decreasing capital investments in biotechnology companies,
governmental restrictions on biotechnology funding, product prohibitions, protests
by public interest groups, and terrorist attacks against biotechnology facilities
(Querling 2000). This negative publicity is compelling some companies such as
Unilever in Europe to avoid using plant materials made with transgenic plants
(Informa Economics, Inc. 1999).
Typical new coverage of transgenic plant protests and moratoriums include:
1. 60 Arrested In Sacramento Bio-Tech Protests Demonstrators Dress As Corn,
Butterflies And Tomatoes. Oakland Tribune, June 24, 2003: More than 1,500
protesters rallied at the state Capital then marched through downtown as the
Ministerial Conference and Expo on Agricultural Science and Technology
began and discussed for three days on genetically engineered crops (BNET
Business Network 2008)
2. Eight arrests at GM crop protest. BBC News, July 14, 2001: Approximately 40
protesters broke into the field and began ripping up the crop of GM fodder maize
(BBC News On-line 2008)
3. French Farmers Protest Against GMO Maize Destruction. Reuters News Ser-
vice, August 2, 2006: Around 300 maize growers protested on Tuesday in
southwest France in defense of a local farmer whose field of genetically mod-
ified (GMO) maize was destroyed at the weekend by activists linked to Jose
Bove (Planet Ark 2008)
4. GM crops banned in Switzerland until 2012. Animal Feed & Animal Nutrition
News, May 29, 2008: The Swiss Federal Council (government) has voted to
extend the country’s moratorium on genetically modified (GM) plants for a
further 3 years beyond the current expiry date of November 2010, Dow Jones
reports (Reed Business 2008)
5. Mass Protests against GM Crops in India. ISIS Press Release, March 30, 2008.
As India edges closer to what is probably the last year of field trials for Bt Brinjal
(eggplant, aubergine) before commercial approval may be granted, large scale
resistance has been building up all over the country. Bt Brinjal, if allowed in
India, would be the first food crop in the world with the Bt gene inserted into it
that is to be directly consumed by human beings. Indians feel that they are about
to be made guinea pigs by USAID, and by Monsanto and Cornell University that
have developed this crop (Institute of Science in Society 2008)
6. Kenya’s Biosafety Bill faces opposition. African Science News Service, August
21, 2007: The debate on agricultural biotechnology in Kenya boiled over again
last week when peasant farmers and GMO critics staged a demo against GMO
proponents who see it as a panacea to low yields against . . . (African Science
News Service 2008)
7. Landless peasants occupy Syngenta plants (Justiça mantêm multa de 1 milhão
para Syngenta). Reuters News Service, December 15, 2007: Hundreds of acti-
vists broke into a Swiss-owned Syngenta agrochemical plant in the state of Sao
Paulo, expelling 50 employees and shutting down production, a company
462 B.R. Shmaefsky
spokeswoman told Reuters. Members of the Landless Rural Workers’ Move-

ment, or MST, and the allied group Via Campesina also destroyed genetically
modified corn and soy seedlings at a Syngenta farm in the northeastern state of
Ceara, the groups said. The groups demand Syngenta leave Brazil, accusing the
company of attacking landless workers and violating environmental laws (Terra
de Direitos 2008)
8. GM Crops in Australia – will the moratoria end? GMO Compass, August 31,
2007. The cultivation of GM crops is banned in all Australian states except
Queensland. However, moratoria will expire in New South Wales and Victoria
next year. A report written on behalf of the Australian government now supports
the commercial use of GM plants to promote competitive agricultural produc-
tion. This has raised the debate on the future role of GM plants in Australia
(GMO Compass 2007)
It is difficult to predict the full extent of human behavior when compulsions are
based on misunderstanding and fear of a technology. Some people react by avoid-
ing the technology and others react by violently opposing it. Given normal percep-
tions of a technology, risk is assessed in the mind as the threat impact multiplied by
the probability of impact. Fear and misunderstanding can elevate either or both the
threat impact and probability values. People generally prioritize the dangers of a
technology by multiplying the risk by their ability to control that risk (Gardner
2008). Misunderstanding and fear exaggerate a person’s inability to control thereby
greatly decreasing their acceptance of the technology. Major compulsions about
biotechnology fall into the categories of “Frankenfoods,” food allergies and vaccine
resistance, and erosion of the global economy.
13.4.1 Frankenfoods
The pejorative term “Frankenfood” was coined in 1992 by Boston University

English Professor Paul Lewis in a letter about biotechnology foods to the New
York Times (Katz 2005). This term became a symbol of the perceived dangers of
GMO foods. It is commonly used in publications and dialogs by opponents of
transgenic plants for animal and human consumption. The Frankenfood term has
effectively associated transgenic plants with the unethical and unnatural methodo-
logies used to produce the fictitious monster in Mary Shelley’s book Frankenstein
or The Modern Prometheus. Unfortunately, the use of the term has been very
successful in producing public compulsions about transgenic plants (Rowan 1994).
A news story entitled Frankenfood concerns are valid, published in 2000 in the
Albuquerque (New Mexico) Journal is a moderate view of the public sentiment
about Frankenfoods. It stated:
The United States moved away from its near-isolationist opposition to the labeling of
genetically modified foods last weekend when it accepted a new international agreement
that will speed the labeling of such foods on the world market.
But it has yet to shed fully its unconditional support of the biotech industry, as
evidenced by the compromise it exacted in the UN Biosafety Protocol: For 2 years after
the protocol takes effect, labels need merely say a product has been engineered, without
specifics. During those two years, negotiators will work out more specific labels.
The United States had only a handful of allies in opposing labeling; 125 other nations
supported the move, including members of the European Union. EU resistance to so-called
“Frankenfoods” is strong in light of member nations’ experiences with food scares, from
Mad Cow fatalities in the ’90s to historical episodes of famine.
While there is no evidence genetically modified foods are unsafe to eat, consumers do
have legitimate concerns that make detailed labeling imperative. (Albuquerque Journal
Online 2000)
More extreme news coverage sternly denigrates any benefits of transgenic plants
while stressing unsubstantiated risks to human health.
Greenpeace International promoted the following news story in Europe entitled
Monsanto maize approved for human consumption potentially toxic warns new
study. It was released by Media-Newswire in 2007:
The study, carried out by French scientific research institute CRIIGEN on the results of rat
feeding trials using a GE maize made by biotech firm Monsanto, highlights 60 significant
differences between the rats that were fed the GE maize and those fed normal maize (all for
90 days). The first group showed differences in their kidney, brain, heart and liver
measurements, as well as significant weight differences. These could be warning signs of
toxicity, but have not been further investigated. (Media Newswire 2007)
This story had a high impact on public sentiment in Europe even though the
study finding were not substantiated by follow-up research that indicated any
causality or mechanisms of possible toxicity from the transgenic plants (Miller
and Conko 2004).
The health fears of Frankenfoods fall into four commonly publicized categories:
there is little scientific study about their health risks, safety testing technology is
inadequate to assess potential harm, they can carry unpredictable toxins, and they
may increase the risk of allergenic reactions (Ewen and Pusztai 1999; Pusztai
2000). Unfortunately, it is nearly impossible and unrealistic to do a complete risk
assessment about the potential hazards of any foods (Kuiper et al. 1999). In spite of
this lack of information, most food causes few unpredictable health problems if
consumed in normal quantities. In addition, foods derived from transgenic plants
should not be any more at risk for toxicological dangers than traditionally cultivated
plants (Momma et al. 1999). Their metabolic processes are known just as thor-
oughly as other plants. This does not quell the fears that the risks of genetically
modified foods are not fully known. The epidemiological evidence of no ill harm to
human populations consuming genetically modified foods is not compelling enough
to quell concerns. Therefore, the compulsion over Frankenfoods will not likely be
completely eliminated from public sentiment (Domingo 2000).
Evidently, the bad press perpetuating the unsubstantiated health risks of trans-
genic plants is effective and contributing to consumer buying trends and legislative
decisions (Fox 1999). An informal 2008 poll of attitudes about biotechnology
foods conducted by CBS (Columbia Broadcasting System) found that “87% of
(American) consumers would like GMO ingredients to be labeled, just as they are in
464 B.R. Shmaefsky
Europe, Japan and Australia.” In the same poll they discovered that “53 percent of
Americans say they won’t buy food that has been genetically modified (CBS
Evening News 2008).” A 2008 commentary in “The Economist entitled Son of
Frankenfood?” reported that biotechnology companies, food manufactures, and
food retailers will have a difficult time convincing consumers to purchase biotech-
nology food products in spite of regulatory approvals that ensure safety (The
Economist 2008). Books dispelling the Frankenfood myths have done little to
improve consumer confidence in transgenic plants (Schacter 1999; Fedoroff and
Brown 2004).
13.4.2 Food Allergies and Vaccine Resistance
Food allergies and other immunological problems are probably one of the most
common compulsions associated with transgenic plants used for consumer chemi-
cals, foods, and pharmaceuticals (CNN.com 2000; Shmaefsky 2007) There is a
general belief that some unknown factor in transgenic plants can induce allergies.
Others biotechnology opponents argue that certain known components, such as
specific fragments of bacterial and viral nucleic acids, can induce immune hyper-
sensitivies and autoimmune disease. Another criticism of edible vaccine transgenic
plants claims that they contribute to the growing number of disease organisms
resistant to vaccines. These compulsions are seemingly supported by compelling
research studies and epidemiological data (US. EPA. FIFRA Scientific Advisory
Panel 2001; Genetically Engineered Organisms 2008)
A typical example of public hysteria caused by a fear of transgenic plants
involved the Taco Bell brand packaged taco shell scare in 2000. Kraft Corporation
of the US voluntarily recalled approximately three million boxes of taco shells
unintentionally contaminated with StarLink corn intended for animal feed (CNN.
com 2000). StarLink was genetically modified to express Bt toxin using the Cry1A
for the cry9C protein and was not approved for human use (Halford 2005). Most of
the news coverage included statements such as the Taco Bell shells “contained at
toxin” or “the new protein could be an allergen to humans” (Genetically Engineered
Organisms 2008).
These news stories led to 210 people complaining about allergic reactions after
purportedly eating the contaminated taco shells (US, EPA, FIFRA Scientific Advi-
sory Panel 2001). Seventy-four of the people went to a physician with various
medical complaints and another 20 admitted themselves to an emergency room
seeking treatment for self-reported allergic reaction symptoms. This is almost five
times the number of people that normally report about illnesses from eating related
corn products. Ultimately, the Centers for Disease Control (CDC) formed a study to
investigate the allergy claims to the relationship of the illnesses to a GMP (Centers
for Disease Control 2001).
The CDC established that 28 of the people who were confirmed to have eaten
corn products containing the Cry9C protein had experienced a true allergic reaction
unrelated to any other medical condition. However, none of the subjects possessed
immunoglobin E (Ig E) antibodies that responded to Cry9C protein. Ig E is the
definitive indicator of an allergic response in humans. So, the allergic response was
determined by the CDC to be due to another factor. Plus, purposed illnesses in the
subjects not investigated by the CDC were unrelated to food allergies. Critics
debate the validity of the CDC study and argue that allergic reactions to the Bt
protein or other transgenic plant proteins have not been ruled out (Organic Con-
sumers Association 2008).
Another compulsion about transgenic plants is illness associated with the con-
sumption of bacterial and viral DNA and RNA used in the transgene vectors (Ho
and Ching 2004). M.-W. Ho and A. Pusztai are the primary supporters of this view
and foresee the possibility of autoimmune diseases developing from the consump-
tion of these nucleic acids (Reisner 2001). Their arguments are very convincing and
they cite laboratory animal studies that allude to the possibility of antibody produc-
tion in response to high levels of transgene vectors in the diet (Violand et al. 1994;
Schubbert et al. 1997). Much of this is based on the claims that the CaMV virus
found in normal foods is not highly infectious and cannot be absorbed by mammals.
It is believed that instability of the CaMV vector can possibly recombine in ways
that initiate autoimmunity in consumers (Green and Allison 1994). However, no
distinct mechanism explaining how the vector causes autoimmunity has been
confirmed (Kuiper et al. 2001).
Edible vaccines have created a different set of compulsions that are hampering
their development for human use (Bonetta 2002). The primary concern, which is
scientifically valid if proper quality control is not followed, is their effectiveness at
inducing adequate immunity (Toonen 1996). However, this may be true with any
vaccination program. The main compulsion is the fear that edible vaccines are
highly likely to induce resistance in the organisms targeted by the vaccination
(Sharma et al. 1999). This in turn will make the edible vaccine plants and the
tradition vaccines ineffective. However, there is no evidence that edible vaccines
are more likely to do this than traditional vaccines (Daniell et al. 2001). Overall, a
consensus of the scientific community is that edible vaccines are effective and
likely safer than traditionally vaccine administration strategies (Washam 1997).
13.4.3 Erosion of the Global Economy
There are many types of criticisms about the professed economic benefits provided
by transgenic plants. Much of this negative sentiment is targeted at agricultural field
applications (Piruzian et al. 2006). A global disapproval of agricultural biotechnol-
ogy plants is their alleged interference with sustainable development programs in
developing nations (World Summit on Sustainable Development 2002). The senti-
ment of the 2002 World Summit on Sustainable Development was reflected in the
following statement: “It was impossible to determine whether GMPs could contri-
bute to sustainable agriculture in the absence of a high level of scientific knowledge
466 B.R. Shmaefsky
and technology to carry out credible risk assessments.” It was debated that the costs
of remediating any risks may outweigh long-term economic benefits of GM crops.
In addition, the conference sentiment believed the R&D and production costs of
GM crops was contrary to economic sustainability in developing nations and in
small farms in developed nations.
Apparently, the general model of sustainable development rejects the current
contributions of transgenic plants (Arrow et al. 2003). Sustainability is best defined
as, “a pattern of resource use that aims to meet human needs while preserving the
environment so that these needs can be met not only in the present, but in the
indefinite future” (United Nations 1987). The United Nations has several economic
criteria of sustainability that are not currently met by transgenic plant development.
Major economic determinants of sustainability include the use of technologies that
encourage the ratio of share in national income of highest to lowest quintile,
increase the gross domestic product (GDP) per capita, raise the investment share
in GDP, and develop material intensity of the economy (The United Nations
Division for Sustainable Development 2008). The World Health Organization
believes that transgenic plants currently used in developed and developing nations
do not overall contribute to the equal distribution of wealth and resources. In
developing nations the plants are perceived as not contributing much to the long-
term sharing of a nation’s wealth. They also do not improve the country’s share in
GDP particularly if the plants are owned or managed by foreign entities (U.S.
Department of Agriculture 1999a, U.S. Department of Agriculture 1999b).
Another purported limitation of GM crops is that the improvements they provide
do not necessarily provide economic benefits to the availability and affordability of
human foods. Due to safety regulations, most of the food-related plant products are
used as animal feed (U.S. Department of Agriculture 1999b). A criticism presented
to the US Department of State by Jennifer Kuzma, Director of the Center for
Science, Technology, and Public Policy at the University of Minnesota, reflects a
common view of transgenic crop plants. She stated, “But these and other applica-
tions carry risks that need to be addressed through regulatory and safety regimes.
Governments and other organizations also need to step in and invest in biotechno-
logy research and development tailored toward products that can help developing
countries and assist these nations in building the capacity to benefit from bioinno-
vation” (Kuzma 2005). Little has been done to remediate the economic investment
situation since her comments were made in 2005. Apparently, transgenic plants still
require serious economic commitments and a research infrastructure available
primarily in wealthier nations. The efforts of wealthier nations do not necessarily
contribute to sustainable economic growth of the transgenic plant sector for deve-
loping nations (Suter and Oegerli 2002).
There is data disputing the economic value of transgenic crop plants. A 2006
assessment evaluated the economic gains of using transgenic crop plants by the
developing and developed nations. A conclusion was made that, “In terms of the
division of the economic benefits obtained by farmers in developing countries
relative to farmers in developed countries, data shows that in 2006, just over half
of the farm income benefits (53%) have been earned by developing country
farmers. The vast majority of these income gains for developing country farmers
have been from GM IR cotton and GM HT soybeans. Over the 11 years, 1996–
2006, the cumulative farm income gain derived by developing country farmers was
$16.4 billion (48.5% of the total)” (Brookes and Barfoot 2007). The authors’
conclusions show that substantial gains in net economic benefits were made at the
farm level amounting to $6.94 billion in 2006 and $33.8 billion for the period 1996–
2006. Some critics debate the sustainability and equitable distribution of this
increased wealth. They believe that most of the profits go consistently to the
developers of the plants and not necessarily the growers (International Service for
the Acquisition of Agri-Biotech Applications 2004).
Many Asian countries are finding economically sustainable to invest in local
niche market and global specialty market transgenic plants. They are already seeing
a demand for plant technologies that would provide equitable economic develop-
ment for their nations (International Food Policy Research Institute 2007).
Countries, such as India, see a great need for developing food and pharmaceutical
GM plants that meet the needs of Asian consumers. In addition, they are hoping that
they produce a large export market of functional food GM plants. African nations
are convinced that investing in the potential for economic benefits is not worth the
risk. They have a greater infrastructure to build for achieving a sustainable trans-
genic plant market (Eicher et al. 2005). Overall, the information about the benefits
of transgenic plants to the global economy is filled with emotionally fueled con-
cerns. Many of these concerns are based on the perceived economic gain compared
to the overall picture of the risks.
13.5 Conclusion
Transgenic plants have their obvious merits to science and society. Plus, the science
behind transgenic plants have contributed to the overall growth of biotechnology
and supplemented developments in botanical knowledge. However, there are also
real and perceived society benefits and risks that must be addressed for the full
utility of these plants to be applied globally (Doyle 1995; EPA Biotechnology
Program 2008; Mae-Won 2000). There are measurable attributes to the benefits and
real risks have data that can be used to make rather reliable prognostications about
the economic value and safety of transgenic plants. Unfortunately, the compulsions
or perceived risks are not fully measurable. However, they have significant impacts
on the future of transgenic plants. Efforts are being made to help reconcile per-
ceived risks of transgenic plants. A report by the Riso National Laboratory in
Roskilde, Denmark recognized that, “Rapid developments in, and the controversial
nature of, biotechnology call for communication, networks, partnerships, and
collaboration in research, not just among researchers but also between researchers
and research ‘users’ in industry, government, and elsewhere. Technological
468 B.R. Shmaefsky
foresight appears to offer a coordinating method for developing and strengthening

those linkages” (Borch and Rasmussen 2003).
The study in the Riso National Laboratory report concluded:
However, the current debate characteristically involves sharply opposed fronts. In it,
stakeholders and experts on both side of the conflict advocate widely differing opinions.
Without a proper, generally intelligible dialog, the broader public audience finds it hard to
comprehend this type of debate. The study pursues the notion that public dialog can act as a
driver of future applications in the technological domain, specifically GM crops. The study
concluded with a stakeholder workshop that revealed three key issues that might provide
helpful starting points for a more free-flowing and open-minded debate about the future of
GM crops. The issues were those arising from the following statements: a broad perspective
on risk is crucial; international regulation must make allowance for developing countries; a
better configuration of the risk debate is needed. These issues are discussed in more detail in
the report, along with the foresight method we used to reveal these issues.
Currently, there are few systemic global initiatives that follow the recommenda-
tions of this report.
References
African Science News Service (2008) http://africasciencenews.org/asns/index.php?option=com_

content&task=view&id=32&Itemid=1. Accessed 25 Aug 2008
Ajjawi I, Shintani D (2004) Engineered plants with elevated vitamin E: a nutraceutical success
story. Trends Biotechnol 22(3):104–107
Al-Babili S, Beyer P (2005) Golden rice – five years on the road – five years to go? Trends Plant
Sci 10(12):565–573
Albuquerque Journal Online (2000) http://www.abqjournal.com/. Accessed 24 Aug 2008
Amarger N (2003) Genetically modified bacteria in agriculture. Biochimie 84(11):1061–1072
Ammann K, Jacot Y, Al R, Mazyad P (1996) Field release of transgenic crops in Switzerland: an
ecological assessment of vertical gene flow. In: Schulte E, Käppeli O (eds) Schwerpunktpro-
gramm Biotechnologie. BATS, Basel, Switzerland, pp 101–157
Ammann K, Jacot Y, Kjellsson G, Simonsen V (1999) Methods of risk assessment of transgenic
plants, III. Ecological risks and prospects of transgenic plants, where do we go from here? In:
A dialogue between biotech industry and science. Birkhäuser, Basel, Switzerland
Anderson K, Jackson LA, Pohl Nielsen C (2004) Genetically modified rice adoption: implications
for welfare and poverty alleviation. Discussion Paper No 0413. Centre for International
Economic Studies
Anonymous (1999) Proteomics, transcriptomics: what’s in a name? Nature 402(6763):715
Apostolakis GE (2004) How useful is quantitative risk assessment? Risk Anal 24(3):515–520
Arias DM, Rieseberg HL (1994) Gene flow between cultivated and wild sunflower. Theor Appl
Genet 89:655–660
Arrow K, Dasgupta P, Mäler K-G (2003) Evaluating projects and assessing sustainable develop-
ment in imperfect economies. Environ Res Econ 26(4):647–685
Baker V (2005) Greenpeace v. Shell: media exploitation and the Social Amplification of Risk
Framework (SARF). J Risk Res 8(7-8):679–691
Bassett CL (ed) (2007) Regulation of gene expression in plants: the role of transcript structure and
processing. Springer, New York
BBC News On-line (2008) http://news.bbc.co.uk/2/hi/uk_news/wales/1438969.stm. Accessed 26

Aug 2008
Beck S, Olek A, Walter J (1999) From genomics to epigenomics. Nat Biotechnol 17(12):1144
Benders AE (1995) Dictionary of nutrition and food technology, 7th edn. CRC, Boca Raton, FL
Berg P, Singer MF (1995) The recombinant DNA controversy: twenty years later. Proc Natl Acad
Sci USA 92(20):9011–9013
Berger RB (2007) Flavours and fragrances: chemistry, bioprocessing and sustainability. Springer,
Berlin
Bernstein L (2002) Epidemiology of endocrine-related risk factors for breast cancer. J Mammary
Gland Biol Neoplasia 7:3–15
Betz VM (1998) Early plant domestication in Mesoamerica. Athena Rev 2(1):24–31
Bhojwani SS, Soh W-Y (eds) (2003) Agrobiotechnology and plant tissue culture. Science Publish-
ers, Enfield, NH
Birch ANE, Geoghegan IE, Majerus MEN, Hackette C, Allen J (1997) Interactions between plant
resistance genes, pest aphid populations and beneficial aphid predators. In: Scottish Crop
Research Institute Annual Report, pp 68–72
BNET Business Network (2008) http://findarticles.com/p/articles/mi_qn4176/is_20030624/ai_
n14552002?tag=artBody;col1. Accessed 25 Aug 2008
Bonetta L (2002) Edible vaccines: not quite ready for prime time. Nat Med 8:94
Borch K, Rasmussen B (2003) Debating the future of genetically modified plants – bridging
knowledge dimensions. A technology foresight study. In: Report Risø-R-1421(EN) http://
www.risoe.dk/rispubl/SYS/syspdf/ris-r-1421.pdf. Accessed on January 29, 2009
Borland J, Johnson CC, Crumpton TW III, Thomas M, Saltizer SM, Oberhauser KS (2004)
Characteristics of fall migratory monarch butterflies, Danaus plexippus, in Minnesota and
Texas. In: Oberhauser KS, Solensky MJ (eds) The monarch butterfly: biology and conserva-
tion. Cornell University Press, Ithaca, NY, pp 97–104
Bowler PJ (1989) The Mendelian revolution: the emergence of hereditarian concepts in modern
science and society. Johns Hopkins University Press, Baltimore, MD
Box L (1981) Cultivation and adaptation: an essay in the sociology of agriculture. Rural Sociol
21(2):160
Brookes G, Barfoot P (2007) Global impact of biotech crops: socio-economic and environmental
effects, 1996–2006. AgBioForum 11(1):21–38
Broothaerts W, Mitchell HJ, Weir B, Kaines S, Smith LMA, Yang W, Mayer JE, Roa-Rodriguez
C, Jefferson RA (2005) Gene transfer to plants by diverse species of bacteria. Nature 433
(7026):629–633
Bulmer M (2003) Francis Galton: pioneer of heredity and biometry. Johns Hopkins University
Press, Baltimore, MD
Canadian Biotechnology Advisory Committee (2008) http://cbac-cccb.ca/epic/site/cbac-cccb.nsf/
en/ah00186e.html. Accessed 6 Jun 2006
Canadian Food Inspection Agency/Agence Canadienne d’inspection des aliments (2008) http://
www.inspection.gc.ca/english/sci/biotech/bioteche.shtml. Accessed 10 Jan 2008
Caruso D (2006) Intervention: confronting the real risks of genetic engineering and life on a
biotech planet. Hybrid Vigor Press, San Francisco, CA
Cavan G, Biss P, Moss SR (2008) Herbicide resistance and gene flow in wild-oats (Avena fatua
and Avena sterilis ssp. ludoviciana). Ann Appl Biol 133(2):207–217
CBS Evening News (2008) http://www.cbsnews.com/stories/2008/05/11/eveningnews/
main4086518.shtml. Accessed 22 Aug 2008
Centers for Disease Control (2001) Investigation of human health effects associated with potential
exposure to genetically modified corn. A report to the US Food and Drug Administration from
the Centers for Disease Control and Prevention. Availablet at: http://www.cdc.gov/nceh/ehhe/
Cry9CReport/executivesummary.htm Accessed on January 29, 2009
Cerny J, Quesenberry PJ (2004) Chromatin remodeling and stem cell theory of relativity. J Cell
Physiol 201(1):1–16
470 B.R. Shmaefsky
Christipeels MJ, Sadava DE (2003) Plants, genes, and crop biotechnology. Jones and Bartlett,
Sudbury, MA
Chun OK, Chung S-J, Claycombe KJ, Song WO (2008) Serum C-reactive protein concentrations
are inversely associated with dietary flavonoid intake in U.S. adults. J Nutr 138:753–760
Clark EA, Lehman H (2004) Assessment of GM crops in commercial agriculture. J Agric Environ
Ethics 14(1):3–28
CNN.com: Safeway recalls its taco shells (2000) News story posted Oct12, 2000. Available at:
http://archives.cnn.com/2000/FOOD/news/10/12/safeway.taco.shells/. Accessed on January
29, 2009
Code of Federal Regulations (CFR) (2008) Main Page. Available at: http://www.gpoaccess.gov/
cfr/index.html. Accessed 5 Aug 2008
Cohen E (2006) Pesticide-mediated homeostatic modulation in arthropods. Pestic Biochem
Physiol 85(1):21–27
Colborn T, Dumanoski D, Myers JP (1996) Our stolen future. Dutton, New York, USA
Commission of the European Communities (2002) Communication from the commission to the
council, the European Parliament, the economic and social committee and the committee of the
regions: life sciences and biotechnology – a strategy for Europe. Available at: http://ec.europa.
eu/biotechnology/pdf/policypaper_en.pdf. Accessed on January 30, 2009
Committee on Managing Global Genetic Resources (1993) Agricultural crop issues and policies.
Natl Acad Press, Washington, DC
Crawley MJ, Brown SL, Hails RSD, Kohn DD, Rees M (2001) Transgenic crops in natural
habitats. Nature 409:682–683
Crecchio C, Stotzky G (1998) Insecticidal activity and biodegradation of the toxin from
Bacillus thuringiensis subsp. kurstaki bound to humic acids from soil. Soil Biol Biochem
30:463–470
Cygler M, Schrag JD, Sussman JL, Harel M, Silman I, Gentry MK, Doctor BP (1993) Relationship
between sequence conservation and three-dimensional structure in a large family of esterases,
lipases and related proteins. Protein Sci 2:366–382
Daniell H (2002) Molecular strategies for gene containment in transgenic crops. Nat Biotechnol
20(8):581–586
Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular farming: production of antibodies,
biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 6:219–226
Daniels R, Boffey C, Mogg R, Bond J, Clarke R (2004) Final report to DEFRA: the potential for
dispersal of herbicide tolerance from genetically-modified, herbicide-tolerant oilseed rape
crops to wild relatives. Available at: http://www.defra.gov.uk/environment/gm/research/pdf/
epg_1-5-151.pdf. Accessed on January 30, 2009
Davison J (1999) Genetic exchange between bacteria in the environment. Plasmid 42:73–91
De Vries J, Wackernagel W (1998) Detection of nptII (kanamycin resistance) genes in genomes of
transgenic plants by marker-rescue transformation. Mol Gen Genet 257:606–613
Diamond J (1999) Guns, germs, and steel. WW Norton, New York, USA
Dinneny JR, Benfey PN (2008) Plant stem cell niches: standing the test of time. Cell 132(4):
553–557
Domingo JL (2000) Health risks of genetically modified foods: many opinions but few data.
Science 288:1748–1749
Donegan KK, Seidler RJ, Fieland VJ, Schaller DL, Palm CJ, Ganio LM, Cardwell DM,
Steinberger Y (1997) Decomposition of genetically engineered tobacco under field conditions:
persistence of proteinase inhibitor I product and effects on soil microbial respiration and
protozoa, nematode and microarthropod populations. J Appl Ecol 34:767–777
Doyle JD, Stotzky G, McClung G, Hendricks CW (1995) Effects of genetically engineered
microorganisms on microbial populations and processes in natural habitats. Adv Appl Micro-
biol 40:337
Dubois A, M’orere JJ (1980) Pollution genetique et pollution culturelle. CR Soc Biogeogr
488:5–22
Edwards D (2007) Plant bioinformatics – methods and protocols. Springer, New York
Eicher CK, Maredia K, Sithole-Niang I (2005) Biotechnology and the African Farmer. Staff Paper
2005–08. Michigan State University Press, East Lansing, MI, USA
Eizenga GC, Agrama HA, Lee FN, Yan W, Jia Y (2006) Identifying novel resistance genes in
newly introduced blast resistant rice germplasm. Crop Sci 46(5):1870–1878
Ellstrand NC, Schierenbeck K (2000) Hybridization as a stimulus for the evolution of invasiveness
in plants? Proc Natl Acad Sci 97:7043–7050
Environmental Protection Agency (2008) What are biopesticides? Available at: http://www.epa.
gov/opp00001/biopesticides/whatarebiopesticides.htm. Accessed 21 Jul 2008
EPA Biotechnology Program Under Toxic Substances Control Act (TSCA) (2008) http://www.
epa.gov/oppt/biotech/. Accessed 16 Jul 2008
EPA Pesticides (2008) Regulating pesticides. Available at: http://www.epa.gov/pesticides/biopes-
ticides/. Accessed 30 Apr 2008
Ernst & Young Global Ltd (2007) Beyond borders: global biotechnology report 2007. Ernst &
Young Global Ltd, UK
European Commission on Biotechnology (2008) http://ec.europa.eu/biotechnology/index_en.htm.
Accessed 24 Jul 2008
European Parliament (2006) http://www.europarl.europa.eu. Accessed 12 Dec 2006
Ewen SWB, Pusztai A (1999) Effects of diets containing genetically modified potatoes expressing
Galanthus nivalis lectin on rat small intestine. Lancet 354:1353–1354
FDA Biotechnology (2008) http://www.cfsan.fda.gov/~lrd/biotechm.html. Accessed 25 Apr 2008
Fedoroff N, Brown NM (2004) Mendel in the kitchen: a scientist’s view of genetically modified
foods. Joseph Henry Press, Washington, DC
Fiehn O (2001) Combining genomics, metabolome analysis and biochemical modelling to under-
stand metabolic networks. Comp Funct Genomics 2(2):155–168
Fleming AJ (ed) (2005) Intercellular communication in plants. Annual Plant Reviews, vol 16.
Blackwell, Oxford and CRC, Boca Raton
Florentino R (2001) Experiences on rice fortification in the Philippines. In: Conference on
Proceedings of Forging Effective Strategies to Combat Iron Deficiency, 7–9 May 2001,
Atlanta, GA, USA
Flyvbjerg B (2006) From Nobel prize to project management: getting risks right. Proj Manag J
37(3):5–15
Food and Agriculture Organization of the United Nations (2008) http://www.fao.org/. Accessed 15
Aug 2008
Foundation F (2005) Economics of regulation of agricultural biotechnologies: issue report. Farm
Foundation, Oak Brook, IL
Fox M (1999) “Frankenfood” headlines scare public, study shows. Reuters World Report. Avail-
able at: http://www.global-reality.com/biotech/articles/news081.htm. Accessed on January 30,
2009
Fritz S, Husmann D, Wingenbach G, Rutherford T, Egger V, Wadhwa P (2003) Awareness and
acceptance of biotechnology issues among youth, undergraduates, and adults. J Agrobiotech-
nol Manag Econ 6(4):178–184
Garcia MA, Altieri MA (2005) Transgenic crops: implications for biodiversity and sustainable
agriculture. Bull Sci Technol Soc 25(4):335–353
Gardner D (2008) The science of fear: why we fear the things we shouldn’t and put ourselves in
greater danger. Penguin Group, New York
Gaskell G, Bauer MW (eds) (2001) Biotechnology 1996–2000: the years of controversy. Science
Museum, London
Gautheret RJ (1983) Plant tissue culture: a history. J Plant Res 9(4):393–410
Genetically Engineered Organisms (2008) Public Education Issues Project: StarLink: GE corn in
taco shells. Available at: http//www.geo-pie.cornell.edu/issues/starlink.html. Accessed 24 Aug
2008
Gibson G (2008) The environmental contribution to gene expression profiles. Nat Rev Genet
9(8):575–581
472 B.R. Shmaefsky
GMO Compass (2007) News: GM crops in Australia – will the moratoria end? Available at: http://
www.gmo-compass.org/eng/news/stories/285.gm_crops_australia_will_moratoria_end.html.
Accessed 31 Aug 2007
Goldschmidt RB (1950) The impact of genetics upon science. In: Dunn LC (ed) Genetics in the
twentieth century. Essays on the progress of genetics in its first fifty years. Macmillan, New
York, pp 1–23
Goodman GE, Thornquist MD, Balmes J, Cullen MR, Meyskens FL Jr, Omenn GS, Valanis B,
Williams JH Jr (2004) The beta-carotene and retinol efficacy trial: incidence of lung cancer and
cardiovascular disease mortality during 6-year follow-up after stopping beta-carotene and
retinol supplements. J Natl Cancer Inst 96:1743–1750
Grabski S, Arnoys E, Busch B, Schindler M (1998) Regulation of actin tension in plant cells by
kinases and phosphatases. Plant Physiol 116(1):279–290
Graham L (1998) What have we learned about science and technology from the Russian experi-
ence? Stanford University Press, Palo Alto, CA
Green AE, Allison RF (1994) Recombination between viral RNA and transgenic plant transcripts.
Viruses and transgenic crops. Science 263:1423–1424
Greenpeace International (2008) Say no to genetic engineering. Available at: http://www.green-
peace.org/international/campaigns/genetic-engineering. Accessed 24 Aug 2008
Gressel J (1999) Tandem constructs: preventing the rise of superweeds. Trends Biotechnol
17(9):361–366
Halford NG (2005) Prospects for genetically modified crops. Ann Appl Biol 145(1):17–24
Hans-Joachim GJ, Weiting N (1998) Lignification of plant cell walls: impact of genetic manipu-
lation. Proc Natl Acad Sci 95(22):12742–12743
Hansson SO (2004) Fallacies of risk. J Risk Res 7(3):353–360
Harlan JR (1980) Studies on the origin and evolution of plants since NI Vavilov. In: Well-being of
Mankind and Genetics. Proceedings of XIV International Congress of Genetics, vol 1, Book 1.
MIR, Moscow, USSR, pp 35–38
Harper JD (1974) Forest insect control with Bacillus thuringiensis. Survey of current knowledge.
Agricultural Experimental Station, Auburn University, Auburn, AL
Health Canada/Sante Canada (2008) http://www.hc-sc.gc.ca/index-eng.php. Accessed 18 Aug
2008
Heesacker A, Kishore VK, Gao W, Tang S, Kolkman JM, Gingle A, Matvienko M, Kozik A,
Michelmore RM, Lai Z, Rieseberg LH, Knapp SJ (2008) SSRs and INDELs mined from the
sunflower EST database: abundance, polymorphisms, and cross-taxa utility. Theor Appl Genet
117(7):1021–1029
Heinemann JA, Traavik T (2004) Problems in monitoring horizontal gene transfer in field trials of
transgenic plants. Nat Biotechnol 22(9):1105–1109
Hertzberg EL, Skibbens RV (1984) A protein homologous to the 27,000 dalton liver gap junction
protein is present in a wide variety of species and tissues. Cell 39(1):61–69
Ho M-W (2002) Astonishing denial of transgenic contamination. Sci Soc 15:13–14
Ho M-W, Ching LL (2004) GMO free: exposing the hazards of biotechnology to ensure the
integrity of our food supply. Vital Health Publ, Ridgefield, CT
Ho M-W, Ryan A, Cummins J (2000) Health risks: hazards of transgenic plants containing the
cauliflower mosaic viral promoter. Availablet at: http://www.biotech-info.net/hazards.html.
Accessed 6 Sep 2000
Ho M-W, Steinbrecher R (1998) fatal flaws in food safety assessment: critique of the joint FAO/
WHO biotechnology and food safety report. Environ Nutri Interact 2:51–84
Hodgson J (2000) GMO roundup. Nat Biotechnol 18:1023
Hoei-Hansen CE, Nielsen JE, Almstrup K, Brask S, Graem N, Skakkebaek NE, Leffers H,
Rajpert-De Meyts E (2004) Transcription factor AP-2 is a developmentally regulated marker
of testicular carcinoma in situ and germ cell tumors. Clin Cancer Res 10:8521–8530
Hu Z-B, Du M (2006) Hairy root and its application in plant genetic engineering. J Integr Plant
Biol 48(2):121–127
Hugenholtz J, Smid EJ (2002) Nutraceutical production with food-grade microorganisms. Curr

Opin Biotechnol 13(5):497–507
Hurrell RF (1997) Preventing iron deficiency through food fortification. Nutr Rev 55:210–222
Informa Economics, Inc (1999) Unilever, Nestle to remove GMO ingredients from British product
lines. Food & Drink Weekly. Available at: http://findarticles.com/p/articles/mi_m0EUY/
is_17_5?tag=content;col1. Accessed 3 May 1999
Institute of Food Technologists (2005) Functional foods: opportunities and challenges. Available at:
http://members.ift.org/IFT/Research/IFTExpertReports/functionalfoods_report.htm. Accessed
on February 1, 2009
Institute of Science in Society (2008) http://www.i-sis.org.uk/gmProtestsIndia.php. Accessed 25
Aug 2008
International Food Policy Research Institute (2007) Economic considerations of biosafety and
biotechnology regulations in India. In: Proceedings of Conference, 24–25 Aug 2006, New
Delhi, India, IFPRI Publ, Washington, DC, USA
International Life Sciences Institute (2002) Functional foods-scientific and global perspectives.
In: ILSI Eur Sr, Summ Symp, Oct 2001, International Life Science Institute Press, Washington,
DC, USA
International Service for the Acquisition of Agri-Biotech Applications (2004) ISAAA Briefs No
32. Executive summary. Global status of commercialized biotech/GM crops: 2004. Available
at: http://www.isaaa.org/kc/bin/ESummary/index.htm. Accessed on February 1, 2009
Isager S, Skydsgaard JE (1992) Ancient Greek agriculture: an introduction. Routledge, New York
Jackson JF, Linskens HF, Inman RB (2002) Testing for genetic manipulation in plants. Springer, Berlin
Jensen MN (2000) Silk moth deaths show perils of biocontrol. Science 290(5500):2230–2231
Ji LL, Peterson DM (2004) Aging, exercise, and phytochemicals promises and pitfalls. Ann NY
Acad Sci 1019:453–461
Johnson B (2000) Genetically modified crops and other organisms: implications for agricultural
sustainability and biodiversity. In: Persley GJ, Lantin MM (eds) Agricultural biotechnology
and the poor. CGIAR, Washington, DC, pp 131–138
Jones J (1992) Food safety. Eagan Press, St. Paul, MN
Jung W, Yu O, Lau S-MC, O’Keefe DP, Odell J, Fader G, McGonigle B (2000) Identification and
expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes.
Nat Biotechnol 18(2):208–212
Kageyama K, Komatsu T, Suga H (2003) Refined PCR protocol for detection of plant pathogens in
soil. J Gen Plant Pathol 69(3):153–160
Kaniewski D, Paulissen E, De Laet V, Dossche K, Waelkens M (2007) A high-resolution late
Holocene landscape ecological history inferred from an intramontane basin in the Western
Taurus Mountains, Turkey. Quat Sci Rev 26(17/18):2201–2218
Kaplan JK (2002) Bt corn not a threat to monarchs. Agric Res Mag 50(2):16–18
Katz SB (2005) Biotechnology and global miscommunication with the public: rhetorical assump-
tions, stylistic acts, ethical implications. In: Proceedings of International Professional Com-
munication Conference 2005, 10–13 July 2005, pp 274–280
K-C Ma J, Drake PMW, Christou P (2003) Genetic modification: the production of recombinant
pharmaceutical proteins in plants. Nat Rev Genet 4:794–805
Keller M, Sneh B, Strizhov N, Prudovsky E, Regev A, Koncz C, Schell J, Zilberstein A (1996)
Digestion of delta-endotoxin by gut proteases may explain reduced sensitivity of advanced
instar larvae of Spodoptera littoralis to CryIC. Insect Biochem Mol Biol 26:365–373
Khusnutdinova EK (2003) The ethnogenomics and genetic history of eastern European peoples.
Herald Russ Acad Sci 73(4):365–372
Kohli A, Griffiths S, Palacios N, Twyman RM, Vain P, Laurie DA, Christou P (1999) Molecular
characterization of transforming plasmid rearrangements in transgenic rice reveals a recombi-
nation hotspot in the CaMV 35S promoter and confirms the predominance of microhomology
mediated recombination. Plant J 17:591–601
Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van der Burg B,
Gustafsson JA (1998) Interaction of estrogenic chemicals and phytoestrogens with estrogen
receptor beta. Endocrinology 139:4252–4263
474 B.R. Shmaefsky
Kuiper HA, Kleter GA, Noteburn HPJM, Kok EJ (2001) Assessment of the food safety issues
related to genetically modified foods. Plant J 27:503–528
Kuiper HA, Noteborn HPJM, Peijnenburg AACM (1999) Adequacy of methods for testing the
safety of genetically modified foods. Lancet 354:1315–1316
Kuzma J (2005) eJournal USA: economic perspectives, Oct 2005. Available at: http://usinfo.state.
gov/journals/ites/1005/ijee/regulation.htm. Accessed on January 30, 2009
Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln ES, Newburg L (1987)
MAPMAKER: an interactive computer package for constructing primary genetic linkage
maps of experimental and natural populations. Genomics 1:174–181
Lewin A, Jacob D, Freytag B, Appel B (1998) Gene expression in bacteria directed by plant-
specific regulatory sequences. Transgen Res 7(6):403–411
Lewis R (1998) Public expectations, fears reflect biotech’s diversity. Scientist 12(7):1
Limborska SA (2004) Human molecular genetics: research in medical genomics and ethnoge-
nomics. Mol Biol 38(1):100–109
Mae-Wan Ho, M-W., Ryan, A. & Cummins, J (2000) Health risks: hazards of transgenic plants
containing the cauliflower mosaic viral promoter. Last updated on September 6, 2000. Web
site: http://www.biotech-info.net/hazards.html. Accessed on January 29, 2009
Mandal CC, Gayen S, Basu A, Ghosh KS, Dasgupta S, Maiti MK, Sen SK (2007) Prediction based
protein engineering of domain I of Cry2A entomocidal toxin of Bacillus thuringiensis resulted
enhancement of toxicity against lepidopteran insects. Protein Eng Des Sel 20:599–606
Mannion AM (1999) Domestication and the origins of agriculture: an appraisal. Prog Phys Geogr
23(1):37–56
Manteo N (1998) Wild biodiversity: the last frontier? In: Yves CL, Bedford BM (eds) Agricultural
biotechnology in international development. CABI Publ, Oxfordshire, UK
Marillonnet S, Thoeringer C, Kandzia R, Klimyuk V, Gleba Y (2005) Systemic Agrobacterium
tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants.
Nat Biotechnol 23(6):718–723
Martin MH (1998) The use of plants for biomonitoring industrial pollution. Acta Hortic 457:
211–224
Martineau B (2001) First fruit: the creation of the flavr savr tomato and the birth of genetically
engineered foods. McGraw-Hill, New York
Media Newswire (2007) Monsanto maize approved for human consumption potentially toxic,
warns new study. Available at: http://media-newswire.com/release_1052430.html. Accessed
15 June 2007
Mendel G (1866) Meteorologische Beobachtungen aus Mähren und Schlesien für das Jahr 1865.
Verhandlungen des naturforschenden Vereines, Abhandlungen, Brünn 4:318–330
Miller HI, Conko G (2004) The Frankenfood myth how protest and politics threaten the biotech
revolution. Greenwood, Westport, CT
Millstone E, Brunner E, Mayer S (1999) Beyond substantial equivalence. Nature 401:525–526
Momma K, Hashimoto W, Ozawa S, Kawai S, Katsube T, Takaiwa F, Kito M, Utsumi S, Murata K
(1999) Quality and safety evaluation of genetically engineered rice with soybean glycinin:
analyses of the grain composition and digestibility of glycinin in transgenic rice. Biosci
Biotechnol Biochem 63:314–318
Morgan TH et al (1915) The mechanism of Mendelian heredity. Holt Rinehart & Winston, New
York Reprinted, Johnson Reprint Co with an Introduction by Garland E Allen, 1978
Morris J (2007) The ethics of biotechnology. Chelsea House Publ, New York
Mulligan CN (2002) Environmental biotreatment. ABS Consulting/Government Institutes,
Rockville, MD
Mullis KB, Ferré F, Gibbs RA (1994) The polymerase chain reaction. Birkhäuser, Boston, MA
Organic Consumers Association (2008) Scientific critique of FDA whitewash of Starlink Corn
AllergyScandal. Available at: http://www.organicconsumers.org/gefood/fdaallergyscandal.
cfm. Accessed 24 Aug 2008
Organization for Economic Co-operation and Development (2008) http://www.oecd.org/.

Paine JA, Shipton CA, Chaggar S, Howells RM, Kennedy MJ, Vernon G, Wright SY, Hinchliffe E,
Adams JL, Silverstone AL, Drake R (2005) A new version of golden rice with increased pro-
vitamin A content. Nat Biotechnol 23:482–487
Patil NS, Lole KS, Deobagkar DN (1996) Adaptive larval thermotolerance and induced cross-
tolerance to propoxur insecticide in mosquitoes Anopheles stephensi and Aedes aegypti. Med
Vet Entomol 10:277–282
Pennisi E (2006) Parasitic weed uses chemical cues to find host plant. Science 313(5795):1867
Piruzian ES (2005) From the mechanisms of genetic transposition to the functional genomics
Genetika. 41(4):440–454
Piruzian ES, Goldenkova-Pavlova IV, Abdeev RM, Komakhin RA, Brouskin AS, Abdeeva IA
(2006) Transgenic plants expressing bacterial genes as a model system for plant functional
genomics. Curr Genomics 7(1):33–42
Planet Ark (2008) http://www.planetark.com/dailynewsstory.cfm/newsid/37468/story.htm.
Pleasants JM, Hellmich RL, Dively GP, Sears M, Stanley-Horn DE, Mattila HR, Foster JE, Clark
P, Jones GD (2001) Corn pollen deposition on milkweeds in and near cornfields. Proc Natl
Acad Sci USA 98(21):11919–11924
Pusztai A (2000) The need for rigorous risk assessment. Chem Ind 8:280
Querling J (2000) Resistance takes root – protests against genetically modified crops – brief article.
Ecologist 30(9):57
Raybould A (2006) Problem formulation and hypothesis testing for environmental risk assess-
ments of genetically modified crops. Environ Biosafety Res 5:119–125
Raybould A (2007) Ecological versus ecotoxicological methods for assessing the environmental
risks of transgenic crops. Plant Sci 173:589–602
Rechcigl JE (1998) Biological and biotechnological control of insect pests. CRC Press, Boca
Raton, FL
Reddy AM, Reddy VS, Scheffler BE, Wienand U, Reddy AR (2007) Novel transgenic rice
overexpressing anthocyanidin synthase accumulates a mixture of flavonoids leading to an
increased antioxidant potential. Metab Eng 9(1):95–111
Reed Business (2008) All about Feed. Available at: http://www.allaboutfeed.net/news/id102-
50901/gm_crops_banned_in_switzerland_until_2012.html. Accessed 25 Aug 2008
Regal PJ (1989) The planned introduction of genetically engineered organisms: ecological con-
siderations and recommendations. Ecology 70(2):298–315
Reisner AE (2001) Social movement organizations’ reactions to genetic engineering in agricul-
ture. Am Behav Sci 44(8):1389–1404
Richardson AO, Palmer JD (2007) Horizontal gene transfer in plants. J Exp Bot 58(1):1–9
Rissler J, Mellon M (1996) The ecological risks of engineered organisms. MIT, Cambridge, MA
Roden LC, Göttgens B, Mutasa-Göttgens ES (2005) Protocol: precision engineering of plant gene
loci by homologous recombination cloning in Escherichia coli. Plant Methods 1:6
Rosi-Marshall EJ, Tank JL, Royer TV, Whiles MR, Evans-White M, Chambers C, Griffiths NA,
Pokelsek J, Stephen ML (2007) Toxins in transgenic crop byproducts may affect headwater
stream ecosystems. Proc Natl Acad Sci USA 104:16204–16208
Rowan KE (1994) The technical and democratic approaches to risk situations: their appeal,
limitations, and rhetorical alternatives. Argumentation 11:391–409
Russo E (2000) Merging IT and biology. Scientist 14(23):8
Samali A, Cotter TG (1996) Heat shock proteins increase resistance to apoptosis. Exp Cell Res
223(1):163–170
Sanger F (2001) The early days of DNA sequences. Nat Med 7(3):267–268
Schacter B (1999) Issues and dilemmas of biotechnology: a reference guide. Greenwood, West-
port, CT
476 B.R. Shmaefsky
Schubbert R, Renz D, Schmitz B, Doerfler W (1997) Foreign (m13) DNA ingested by mice
reaches peripheral leukocytes spleen and liver via intestinal wall mucosa and can be covalently
linked to mouse DNA. Proc Natl Acad Sci USA 94:961–966
Sears MK, Hellmich RL, Stanley-Horn DE, Oberhauser KS, Pleasants JM, Mattila HR, Siegfried
BD, Dively GP (2001) Temporal and spatial overlap between monarch larvae and corn pollen.
Proc Natl Acad Sci USA 98(21):11937–11942
Sengelov G, Kristensen KJ, Sorensen AH, Kroer N, Sorensen SJ (2001) Effect of genomic location
on horizontal transfer of a recombinant gene cassette between Pseudomonas strains in the
rhizosphere and spermosphere of barley seedlings. Curr Microbiol 42(3):160–167
Sharma AK, Mohanty A, Singh Y, Tyagi AK (1999) Transgenic plants for the production of edible
vaccines and antibodies for immunotherapy. Curr Sci 77(4):524–529
Shimoda SM (1998) Agricultural biotechnology: master of the universe? AgBioForum 1(2):62–68
Shmaefsky BR (2000) Building better trees through hormones. ISB News Report, Oct 2000
Shmaefsky BR (2005) Biotechnology on the farm and in the factory: agricultural and industrial
applications. Chelsea House Publ, New York
Shmaefsky BR (2007) Biotechnology 101. Greenwood, Westport, CT
Spehar T (2000) Biotechnology risk: assessment, communication, and regulation. Biotechnology
Law Report 19(5):560–575
Stoflet ES, Koeberl DD, Sarkar G, Sommer SS (1988) Genomic amplification with transcript
sequencing. Science 239(4839):491–494
Suter C, Oegerli T (2002) Participatory technology assessment in biotechnology: concept and
practice. In: Mehta MD (ed) Sociology of biotechnology. University of Toronto Press, Toronto,
Canada
Syvanen M (1994) Horizontal gene transfer: evidence and possible consequences. Annu Rev
Genet 28:237–261
Tanksley SD, Young ND, Paterson AH, Bonierbale MW (1989) RFLP mapping in plant breeding:
new tools for an old science. Nat Biotechnol 7(3):257–264
Terra de Direitos (2008) http://www.terradedireitos.org.br/2007/12/05/justica-mantem-multa-de-
1-milhao-para-syngenta/. Accessed 24 Aug 2008
The Economist (2008) The food industry: son of Frankenfoods? Available at: http://www.econo-
mist.com/business/displaystory.cfm?story_id=10534084. Accessed 22 Aug 2008
The United Nations Division for Sustainable Development (2008) CSD indicators of sustainable
development, 3rd edn. Available at: http://www.un.org/esa/sustdev/natlinfo/indicators/factsheet.
pdf. Accessed on January 30, 2009
Thompson CJ, Thompson BJP, Ades PK, Cousens SR, Garnier-Gere P, Landman K, Newbigin E,
Burgman MA (2003) Model-based analysis of the likelihood of gene introgression from
genetically modified crops into wild relatives. Ecol Modell 162(3):199–209
Toonen J (1996) Are edible vaccines a solution? Biotechnol Dev Mon 27:12–14
U.S. Department of Agriculture (1999a) Feed grains yearbook. USDA, Bethesda, MD
U.S. Department of Agriculture (1999b) Oil seeds yearbook. USDA, Bethesda, MD
U.S. Environmental Protection Agency (1995) Publ No EPA731-F-95-004. US Government
Printing Office, Washington, DC, USA
Union of Concerned Scientists (2008) UCS’s position on biotechnology. Available at: http://www.
ucsusa.org/food_and_environment/genetic_engineering/ucss-position-on-biotechnology.html.
Accessed 23 Jun 2008
United Nations (1987) Report of the world commission on environment and development. General
Assembly Resolution 42/187, 11 Dec1987. Available at: http://www.un.org/documents/ga/res/
42/ares42-187.htm. Accessed 16 Dec 1999
US EPA’S FIFRA Scientific Advisory Panel (2001) A set of scientific issues being considered by
the Environmental Protection Agency regarding: assessment of additional scientific informa-
tion concerning StarLink Corn. SAP Report No 2001-09. U.S. Environmental Protection
Agency, Arlington, VA, USA
USDA Animal and Plant Health Inspection Service (2007) http://www.aphis.usda.gov/biotech-

nology/index.shtml. Accessed 26 Nov 2007
Van den Berg H (1999) Responding to consumer needs: risk benefit analysis of fortification. Scand
J Nutr 43:112S–116S
Violand BN, Schlittler MR, Lawson CQ, Kane JF, Siegel NR, Smith CE, Kolodziej EW, Duffin
KL (1994) Isolation of Escherichia coli synthesized recombinant eukaryotic proteins that
contain epsilon-N-acetyllysine’. Protein Sci 3:1089–1097
Virine L, Trumper M (2008) Project decisions: the art and science. Management Concepts,
Vienna, VA
Washam C (1997) Biotechnology creating edible vaccines. Ann Intern Med 127(6):499
Willey N (2007) Phytoremediation: methods and reviews (methods in biotechnology). Humana
Press, Totowa, NJ
Williams MM II, Mortensen DA, Doran JW (1998) Assessment of weed and crop fitness in cover
crop residues for integrated weed management. Weed Sci 46:595–603
Williamson M (1993) Invaders, weeds, and the risk from genetically manipulated organisms.
Experientia 49:219–224
Williamson MS, Martinez-Torrez D, Hick CA, Devonshire AL (1996) Identification of mutations
in the housefly para-type sodium channel gene associated with knockdown resistance (kdr) to
pyrethroid insecticides. Mol Gen Genet 252:51–60
Wilson R, Shlyakhter A (1997) Uncertainty and variability in risk analysis. Fundamentals of risk
analysis and risk management. CRC, Boca Raton, FL
Woods M, Woods MB (2000) Ancient agriculture: from foraging to farming. Runestone,
Minneapolis, MN
World Summit on Sustainable Development (2002) Johannesburg, South Africa, 26 Aug–4 Sep
2002. Available at: http://www.un.org/events/wssd/. Accessed 16 Aug 2008
World Wildlife Fund (2008) Biodiversity loss puts people at risk: world wildlife fund. Science
Daily. Available at: http://www.sciencedaily.com/releases/2008/05/080516112715.htm.
Yan X, Wu P, Ling H, Xu G, Xu F, Zhang Q (2006) Plant nutriomics in China: an overview. Ann
Bot 98(3):473–482
Ye X, Al Babili S, Kloeti A, Zhang J, Lucca P, Beyer P, Potrykus I (2000) Engineering the
provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm.
Science 287:303–305
Zimmermann R, Qaim M (2004) Potential health benefits of golden rice: a Philippine case study.
Food Policy 29:147–168
Zohary D, Hopf M (2000) Domestication of plants in the old world, 3rd edn. Oxford University
Press, New York, pp 7–11
Index
A Antisense, 96, 218

Abiotic stress, 68 suppression, 225
Abscission, 212, 222 Antiviral, 348
Absicisic acid, 73 ANVISA, 408
Acclimation, 100 Aphid, 4
Acidothermus cellulolyticus, 258 APHIS, 393, 449
Acquired resistance, 38 Apoplast, 43
Activation tagging, 379 Aquaporin, 86
Activator, 373 Arabidopsis, 14, 44, 73, 170, 209
Adaptation, 116 A. thaliana, 277, 290
Agbiotech firm, 421 Arabinose, 258
Agglutinins, 347 Armyworm, 15
Agrobacterium, 26, 272, 418 Arsenic, 319
A. tumefaciens, 272, 287 Arthropod, 2
Algae, 343 Astaxanthin, 346
Allergenic, 142, 402 Atropa belladonna, 322
Allergenicity, 142, 188 Autoimmune disease, 465
Allergic Autoimmunity, 465
reaction, 465 Auxin, 200, 226
response, 465 Avicennia marina, 93
Allergies, 464
Amylase, 190
Annotation, 351 B
Anti-apoptotic, 231 Bacillus thuringiensis (Bt), 6
Antibacterial, 348 Bt corn, 455
Antibodies, 285 Bt cotton, 9
Anticancer, 349 Bt gene, 455
Antigen, 283 Bt maize, 7
Anti-HIV, 349 Bt pollen, 20
Antimicrobial, 41 Bt potato, 9
Antioxidant, 39, 70, 89, 232 Bt resistance, 10
Antirrhinum, 175 Bt toxin, 7, 134, 455
479
480 Index
Bamboo, 177 C
barnase gene, 206 Calmodulin, 81
barstar gene, 206 Canola, 26
Basmati rice, 423 Carcinogen, 455
b-carotene, 457 Carotenoid, 346
Beta vulgaris, 329 Carrageenan, 347
Billion dollar bug, 4 cDNA, 370
Bioaccumulation, 309 Cell line, 269
Bioalcohol, 251 Cellomics, 443
Biobutanol, 251 Cellulose, 257
Biodegradability, 252 Cellulosic, 257
Biodegradation, 309, 324 Centers for Disease Control, 464
Biodiesel, 186, 252 CEPA, 402
Biodiversity, 5, 444 CFIA, 399
loss, 459 CGIAR, 422
Biodiversity Platform, 444 Chaperone, 81
Bioengineering, 258 Chaperoning, 81, 102
Bioethanol, 251 Chenopodium album, 22
Biofuel, 249 Chickweed, 22
Biogas, 252 Chilling, 98
Bioinformatics, 442 resistance, 101
Biomagnification, 315 resistant, 101
Biomass, 91, 258 sensitive, 101
Biomolecule, 346 stress, 98, 101
Biomonitoring, 302 Chill injury, 98
Biopesticide, 11, 450 Chimeric gene, 205
safety, 454 Chitinase, 41
Biopharmaceutical, 269 Chlamydomonas, 352
Bioplastic, 328 C. reinhardtii, 272, 287, 352, 353
Biopolymer, 328 Chloroplast, 258
Bioreactor, 277, 352 Chromatinomics, 442
BiOS, 415 Circadian clock, 208
Biosafety, 281 Citrullus lanatus, 92
Biosimilar, 270 Climate change, 67
Biosorption, 309 Cloning, 257, 439
Biosynthesis, 259 Cold
Biotechnology, 436 acclimation, 98, 99
Biotic stress, 1 chain, 271
Biotransformation, 309 responsive, 103
Biotype, 25 stress, 97
Bradyrhizobium, 138 tolerance, 97, 99
Brassica, 83 Coleoptera, 7
B. carinata, 320 Colorado potato beetle, 4
B. juncea, 318 Compatible solute, 84
B. napus, 94 CONABIA, 407
B. rapa, 150, 453 Contamination, 302
Bromoxynil, 134 Copyright, 412
Brown planthopper, 12 Corn earworm, 7
Index 481
Cotton, 26 Ectopic, 231

Cry EFSA, 405
protein, 7 Electrolyte leakage, 222
toxin, 10 Electron transport chain, 100
cry gene, 7 Eleucine, 139
CTNBio, 408 Elicitor, 44
Cuticle, 219 Embryogenesis, 458
Cyanidioschyzon, 351 Endosymbiotic, 273
Cyanobacteria, 259, 350 Enhancer, 373
Cybrid, 207 Enterobacter cloacae, 320
Cytokinin, 177, 287 Enterotoxin, 283
Cytoplasmic male sterility, 203 Enviromics, 443
Cytotoxicity, 88 Environment, 307
Environment Canada, 402
D Environmental biotechnology, 444
Daktulosphaira vitifoliae, 3 Environmental Impact Statement, 396
Decontamination, 309 Enzyme inhibitor, 13
Defensin, 42 Epigenomics, 442
DEFRA, 404 Erwinia uredovora, 458
Dehiscence, 222 Escherichia, 318
Dehydration, 220 E. Coli, 381
Dehydrin, 102 Estrogen, 458
Deletion, 381 Ethanol, 257
Dendroclamus strictus, 177 Ethnogenomics, 442
Desaturation, 101 Ethylene, 38, 211
Desiccation, 90, 102, 220 receptor, 217
Detoxification, 5, 81, 93, 307 European corn borer, 7
Diacylglycerol, 80 European Union, 403, 449
Dietary supplement, 346 Eutrophication, 344
Digitalis, 289 Explosive, 323
Diptera, 7 Expressed sequence tag (EST), 351, 370
Disease resistance, 40, 43 Expression knock-outs, 383
Dissociation, 373
DNA
footprint, 377
F
sequence, 419
Fat hen, 22
sequencing, 438
Fermentation, 253
DNMA, 407
Fertility restorer (Fr/Rf ), 203
Domestication, 213, 436
Flagellin, 44
Domestic Substances List, 402
Flanking sequence tag, 373
Drought
Flavonoid, 203, 458
avoidance, 72
Floral transition, 168
resistance, 70
Florigen, 169
stress, 69
Flowering time, 169
tolerance, 69
FONSI, 395
E Food and Agriculture Organization, 450
Economic Research Service, 414 Food and Drug Administration (FDA),
Ecosystems, 302 396, 449
482 Index
Forward genetics, 373 Glutathione reductase, 93

Fossil fuel, 249 Glycine betaine, 84
Frankenfood, 462 Glycophyte, 88
Fucus vesiculosus, 349 Glycosylation, 270, 276, 277
Functional food, 457 Glyphosate
Functional genomics, 87 resistance, 25
Fusarium, 10 Glyphosate resistant (GR), 25, 133
Fusion protein, 15 alfalfa, 149
canola, 134
G cotton, 134
Gain-of-function, 379 crop, 136, 137
Gelatinization, 253 gene, 150
Gene maize, 134
discovery, 68 soybean, 134
disruption, 373 transgene, 150
expression, 44, 371 varieties, 134
flow, 32, 148, 452 GMPO, 407
knock-down, 383 Golden rice, 457
prediction, 370 Good manufacturing practice, 270
silencing, 316, 383 Gossypium hirsutum, 9
stacking, 15 Green leafhopper, 12, 13
targeting, 381 Greenpeace International, 459
transfer, 451 Green peach aphid, 18
trapping, 378 Gross domestic product, 466
Generics, 270 GUS expression, 45
Genetic
alternation, 206
diversity, 310 H
pollution, 452 Halophytic, 93
resource, 50 Health Canada, 401, 449
Genetically modified (GM), 6, 368, 391 Heavy metal, 108
GM cotton, 9 Helicoverpa zea, 7
GM crop, 396 Helminthosporium oryzae, 37
GM food, 406 Hemicellulose, 258
Genetically modified organisms, 392 Hepatitis B, 286
Genetically modified plants, 435 vaccine, 271
Genetic engineering, 393 Herbal therapeutic, 444
Genic male sterility, 203 Herbicide (HT), 24
Genome resistance, 25
genome-wide expression, 371 resistant crop (HRC), 133
organization, 204 tolerant, 27
replication, 204 canola, 28
Genomics, 442 cotton, 29–30
Gibberellic acid, 180 maize, 29
Gibberellin, 170 soybean, 30
Ginseng, 181 Heterosis, 202
Glufosinate, 134 Hexapeptide, 289
Glutathione, 94 Homeostasis, 186, 217
Index 483
Homoptera, 7 L
Honeybee, 30 Lacanobia oleracea, 16
Hordeum, 83 Laminaria, 344, 349
Human interleukin, 288 L. japonica, 353
Hybrid, 207 Late embryogenesis abundant, 81
Hybridoma, 286 LD50, 146
Hydrogen peroxide, 80 Lectin, 12, 347
Hydrolysis, 253 Lepidoptera, 7
Hydrophilicity, 83 Leucaena leucocephala, 311
Hymenoptera, 7 Leucine-rich repeats, 41, 44
Hyperaccumulation, 311 Leymus, 83
Hypersensitive response, 38 Lignin, 258
Hypersensitivity, 220 Lipoprotein, 273
Hypertensive, 289 Lipoxygenase, 189
Liriodendron tulipifera, 315
Locust, 4
I Lolium, 139
Imidazolinone, 150
Immunity, 465
Immunization, 282 M
Immunogenicity, 280 Maintainer line, 207
Immunoglobin, 465 Maize, 26
Inbred line, 187 Male sterility, 203
Insecticide, 4 Mammalian cell culture, 270
Insertion, 381 Mannitol, 84, 90
library, 374 Marker
line, 261 -assisted breeding, 417
Insertional mutagenesis, 377 -assisted selection, 87
Insulin, 269 gene, 420
Integrated pest management, 6 Medicago, 221
Intellectual Property Rights, 411 M. sative, 80
Interleukin, 288 Mercury, 311
Invasiveness, 33 Metabolic engineering, 452
Iron, 108 Metabolism, 260
Isogenic line, 143 Metabolite, 18, 372
Isopentyl transferase, 72 Metabolomics, 372, 443
Metallothionein, 112
gene (MT), 318
J Methylation, 46
Jasmonic acid, 16, 38 Methylmercury, 311
Jatropha, 250 Microalgae, 272, 348
Jumping gene, 373 Microarray, 16, 95, 220
Microcystis, 348
Microfissure, 220
K MicroRNA, 46, 47
Kanamycin resistance, 451 Mirabilis, 226
Klebsiella, 138 MITILS, 442
K. pneumoniae, 27 Mitochondrial DNA (mtDNA), 204
Knock-out, 373 Model plant, 445
484 Index
Molecular breeding, 213 O. sativa, 289, 316

Molecular pharming, 272 Osmoprotectants, 88
Monarch butterfly, 20 Osmoprotection, 84
Monoclonal antibodies (MAbs), Osmosensors, 79
269, 285 Osmotic, 219
Mouse, 288 Ostrinia nubilalis, 7
mRNA, 187 Overexpression, 45, 209
Mutagenesis, 375 Oxidative burst, 231
Mutagenic, 375
Mutation, 139, 174, 215
spontaneous mutation, 225 P
Mutator, 373 Panax gensing, 181
Papaya ringspot virus, 47
Patent, 413
N Patent discovery, 422
NCBI, 419 pat gene, 29
Near-isogenic lines (NILs), 290 Pathogen
Necrosis, 38 mimicry, 46
Nematode related (PR), 39
cyst nematodes, 13 Pathogenesis-related (PR), 231
foliar nematodes, 13 Pathogen-related (PR)
rootknot nematodes, 13 PR gene, 46
NEPA, 395 PR transcript, 46
Neutropenia, 288 Pearl millet, 177
Nicotiana Peroxidaton, 85
N. glutinosa, 318 Peroxiredoxin, 93
N. tabacum, 260, 286 Pesticide, 6
Nitrogen, 105 Petunia, 213
assimilation, 105 Phaeodactylum, 352
Notice of Intent (NOI), 396 Phanerochaete chrysosporium, 259
Novel trait, 401 Pharmaceutical, 444
Nucleotide binding site (NBS), 44 Pharmacomics, 443
Nutraceutical, 457 Phosphatidic acid, 79
Nutriomics, 443 Phospholipid, 100
Phospholipid-cleaving enzymes
(PLEs), 79
O Phosphorus, 107
OECD, 414 Photoperiod, 170, 208
Oleosin, 288 Photosynthate, 259
Oomycete, 36 Phragmites, 317
Organelle Phycocolloids, 347
biogenesis, 205 Phylloxera plaque, 3
transformation, 207 Physiomics, 443
Organic Phytoaccumulation, 317
agriculture, 6 Phytoalexin, 43
pollutant, 321 Phytochelatins (PCs), 318
Orobanche, 31 Phytoestrogen, 457
Oryza, 288 Phytoextraction, 112
Index 485
Phytohormone, 181 R
Phytoimmuno-remediation, 324 Ralstonia, 155
Phytomonitor, 328 R. eutropha, 323
Phytophthora, 221 Raphanus, 453
P. infestans, 36, 43 Reactive oxygen species (ROS), 79
Phytoremediation, 110, 302, 303, 444 Recombinant
Phytoremediator, 310 DNA (rDNA), 393, 451
Phytorestoration, 326 engineering, 351
Phytosensing, 45 protein technology, 271
Phytotoxin, 136 Recombinant inbred lines (RILs), 290
Phytovolatilization, 110, 315 Reporter gene, 377
Pink bollworm, 9 Resistance (R)
Pisum sativum, 181 durable resistance, 44
Plant Industry Platform, 444 major-gene resistance, 44
Plants with novel trait (PNT), 399 polygenic resistance, 44
regulation, 399 R-gene, 38
Plasmid, 272 single-gene resistance, 44
Plasmodium, 284 Restorer gene, 204
Plastid transformation, 207 Retrotransposon, 373
Pleiotropic, 72 Reverse genetics, 382
Pollutant, 302 Rhizobacteria, 38
Pollution, 302 Rhizosecretion, 325
Polyamine, 89 Ripening, 216
Polymerase chain reaction (PCR), 383 Risk assessment, 446
Pongamia, 250 RNA
Poplar, 316 datasets, 371
Populus, 111, 209, 316 expression, 371
P. tremuloides, 260 interference (RNAi), 261
Powdery mildew, 42 polymerase, 385
Programmed cell death (PCD), 227 silencing, 47, 373, 383
Prolamin, 289 RNA-interference (RNAi), 19
Proline, 84 Roses, 181
Protease inhibitor, 11 Roundup ready, 29
Protein Royal Horticultural Society (RHS), 411
bodies, 271 Ryegrass, 25
denaturation, 81
kinase, 80 S
trafficking, 273 Saccharomyces, 82, 254
Proteinase inhibitor, 16 SAGPyA, 407
Proteomics, 371 Salicylic acid, 38
Protoplast, 381 Salinity
Provitamin A, 346 stress, 88
Pseudomonas, 459 tolerance, 87
Pyrus communis, 89 Salt
sensitivity, 92
Q tolerance, 87
Quantitative trait loci (QTL), 44, 87, Schizosaccharomyces, 92
174, 377 Sclerotinia, 42
486 Index
Selenium, 320 Terminator, 419

SENASA, 407 Thalassiosira pseudonana, 352
Senescence, 211 Therapeutic proteins, 287
Sequencing, 351 Therapeutics, 277
Sequestration, 5, 114, 309 Thlaspi
Shoot apical meristem (SAM), 168 T. caerlescens, 114
Short interfering RNA (siRNA), 46 T. caerulescens, 319
Signal Tillering, 200
perception, 78 Tobacco budworm, 9
sensing, 78 Tomato moth, 16
transduction, 78, 96 Tonoplast, 85
Signaling, 169, 226, 231 Trademark, 413
cascade, 18, 231 Transactivation, 379
Signal transduction, 45, 72 Transcription, 95
Single-stranded RNA (ssRNA), 385 factor (TF), 46, 76, 95
Snowdrop lectin, 15 Transcriptome, 371
Solanum, 104 Transcript tryncation, 205
S. commersonii, 104 Transgene, 21, 148, 372
S. lycopersicum, 38 docking, 382
S. tuberosum, 9, 104 mitigation, 32
Somaclonal mutation, 374 upgrades, 382
Sorghum Transgenic goats, 270
S. bicolor, 453 Translation, 206
S. halepense, 453 Transporter, 91
Soybean, 26, 181 Transposase, 382
Spartina, 317 Transposon, 373
Specialty crops, 424 tagging, 322, 375
Spider mite, 16 Trehalose, 84, 90
Spodoptera frugiperda, 7 Trichederma, 82
Stellaria media, 22 Trichloroethylene (TCE), 321
Sterols, 346 Triticum aestivum, 286
Striga, 22 Trypsin inhibitor, 9
Structural genomics, 369 Typha, 317
Structural protein, 218
Substantially equivalent (SE), 397 U
Sulfonylurea, 27 Ubiquitin, 86
Superoxide, 42 Umbelopsis ramanniana, 261
Superweed, 32, 453 Undaria, 344
Suppressor, 373 Union of Concerned Scientists
Synechocystis, 352 (UCS), 459
USDA, 393, 415, 449
T USPTO, 413
Tamarix, 84
T-DNA, 261, 374
Technology transfer, 444 V
Teosinte, 200 Vaccine, 279
Teratogenic Variety Registration Office (VRO), 399
effects, 458 Vector, 272
Index 487
Vernalization, 170, 208 X

Vitamin A, 458 Xanthomonas, 41, 221
Vitis Xanthotoxin, 19
V. kabrusca, 3 Xenopus, 96
V. vinifera, 3
W Y
Water use efficiency (WUE), 73 Yeast, 289
Western corn rootworm, 4
Wild-type (WT), 73
Wilting, 94 Z
Witchweed, 22 Zea, 171
Wounding, 231 Zea mays, 171

Transgenic Crop Plants (PDFDrive)

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Transgenic Crop Plants (PDFDrive)

Uploaded by

Copyright:

Available Formats

Transgenic Crop Plants

Chittaranjan Kole Charles H. Michler

Albert G. Abbott Timothy C. Hall

Transgenic Crop Plants

Prof. Charles H. Michler Prof. Timothy C. Hall

ISBN: 978-3-642-04811-1 e-ISBN: 978-3-642-04812-8

# Springer-Verlag Berlin Heidelberg 2010

Cover design: WMXDesign GmbH, Heidelberg, Germany

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

Transgenic Plants – known also as Biotech Plants, Genetically Engineered Plants,

Chittaranjan Kole, Clemson, SC

1 Transgenic Crop Plants for Resistance to Biotic Stress . . . . . . . . . . . . . . . 1

2 Transgenic Plants for Abiotic Stress Resistance . . . . . . . . . . . . . . . . . . . . . . 67

3 Transgenic Crops for Herbicide Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . 133

4 Understanding and Manipulation of the Flowering

5 Biotechnological Interventions to Improve Plant

6 Transgenics for Biofuel Crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

7 Plant Produced Biopharmaceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

8 Biotech Crops for Ecology and Environment . . . . . . . . . . . . . . . . . . . . . . . . 301

9 Algal Biotechnology: An Emerging Resource with Diverse

10 Transgenic Crops and Functional Genomics . . . . . . . . . . . . . . . . . . . . . . . . . 359

11 Deployment: Regulations and Steps for Commercialization . . . . . . . . 391

12 Patent and Intellectual Property Rights Issues . . . . . . . . . . . . . . . . . . . . . . . 411

13 Transgenic Crop Plants: Contributions, Concerns,

Diaa Al-Abed Edenspace Systems Corporation, Manhattan, KS 66502, USA,

Ajay Arora Division of Plant Physiology, Indian Agricultural Research

Saikat Kumar Basu Department of Biological Sciences, University of

Anjanabha Bhattacharya National Environmental Sound Production Agricul-

Antonio L. Cerdeira Brazilian Department of Agriculture, Agricultural

Kelly D. Chenault Chamberlin USDA-ARS, Wheat, Peanut, and Other Field

Zhiping Deng Department of Plant Biology, Carnegie Institution of Washington,

Stephen O. Duke Agricultural Research Service, United States Department of

Jim M Dunwell University of Reading, Whiteknights, Reading RG6 6AS, UK,

François Eudes Bioproducts and Bioprocesses, Agriculture and Agri-Food

N. Ferry School of Biology, Institute for Research on Environment and Sustain-

Joel Gaffe Laboratoire Adaptation et Pathogénie des Microorganismes, LAPM,

A.M.R. Gatehouse School of Biology, Institute for Research on Environment

Jared Q. Gerlach Glycoscience and Glycotechnology Group and the Martin

Stephen L. Goldman Department of Environmental Sciences, The University of

Ravinder K. Goyal Department of Biochemistry and Microbiology, University

Avtar K. Handa Department of Horticulture and Landscape Architecture, Pur-

Michelle Kilcoyne Glycoscience and Glycotechnology Group and the Martin

Igor Kovalchuk Department of Biological Sciences, University of Lethbridge,

Pawan Kumar National Environmental Sound Production Agriculture Labora-

Anish Malladi Department of Horticulture, University of Georgia, Athens, GA

Autar K. Mattoo Sustainable Agricultural Systems Laboratory, The Henry A.

Peter McKeown Genetics and Biotechnology Laboratory, Department of

Michael G. Muszynski Department of Genetics, Development and Cell Biology,

Pradeep S. Negi Department of Horticulture and Landscape Architecture,

Andy Pereira Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA

Shobha D. Potlakayala Penn State Milton S. Hershey College of Medicine,

Sairam Rudrabhatla Environmental Engineering, College of Science,

Brian R. Shmaefsky Lone Star College – Kingwood, HSB 202V, 20,000

Rippy Singh National Environmental Sound Production Agriculture Laboratory,

Charles Spillane Genetics and Biotechnology Laboratory, Department of

Alka Srivastava Department of Horticulture and Landscape Architecture, Pur-

Cunningham Stephen Glycoscience and Glycotechnology Group and the

Martı́n-Ernesto Tiznado-Hernández Fisiologı́a y Biologı́a Molecular de

Narayana M. Upadhyaya CSIRO Plant Industry, GPO Box 1600, Canberra,

1-FFT Fructan:fructan 1-fructosyltransferase

APHIS Animal and Plant Health Inspection Service

chs chalcone synthase gene