Breeding For Ornamentals: Classical and Molecular Approaches

Breeding For Ornamentals:

Classical and Molecular Approaches
Edited by

Alexander Vainstein
The Hebrew University of Jerusalem,
Rehovot, Israel

SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

A C.I.P. Catalogue record for this book is available from the Library of Congress.

ISBN 978-90-481-5975-8 ISBN 978-94-017-0956-9 (eBook)

DOI 10.1007/978-94-017-0956-9

Printed on acid-free paper

All Rights Reserved

© 2002 Springer Science+Business Media Dordrecht
Originally published by Kluwer Academic Publishers in 2002
No part of the material protected by this copyright notice may be reproduced or
utilized in any form or by any means, electronic or mechanical,
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TABLE OF CONTENTS

Preface vii

SECTION ONE

IN1RODUCTION TO CLASSICAL AND MOLECULAR GENETICS

Evolution of Sexual Reproduction and Floral Diversity

R.J. Griesbach 1
Transmission Genetics
A. Ashri 7
Molecular Genetics: Gene Isolation, Characterization and Manipulation
B.R. Glick, D.M. Penrose 25

SECTION TWO

CLASSICAL BREEDING

Breeding Methods and Breeding Research

W.Hom 47
Interspecific Hybridization and Introgression
J.M. VanTuyl, K.B. Lim, M.S. Ramanna 85
Mutation Breeding of Vegetatively Propagated Ornamentals
A.M. Van Harten 105
Introduction of New Cut Flowers: Domestication of New Species and Introduction of
New Traits Not Found in Commercial Varieties
D. Weiss 129
Tissue Culture for Ornamental Breeding
A. C. Cassells 139

SECTION THREE

GENETIC MANIPULATION AT THE DNA LEVEL

Gene Transfer to Plants

S.C. Deroles, M.R. Boase, C.E. Lee, T.A. Peters 155
Vl

Molecular Approaches for Increasing Plant Resistance to Biotic and Abiotic Stresses
M. Lorito, G. Del Sorbo, F. Scala 197
Molecular Control of Light Sensing in Plant Development
A. Samach, M. Pineiro 219
Molecular Control of Flower Development
M. Vishnevetsky, E.M. Meyerowitz 239
Molecular Control of Floral Pigmentation: Anthocyanins
H. Ben-Meir, A. Zuker, D. Weiss, A. Vainstein 253
Molecular Control of Floral Pigmentation: Carotenoids
F.X. Cunningham, Jr., E. Gantt 273
Molecular Control of Floral Fragrance
N. Dudareva 295
Molecular Genetics of Flower Senescence
J. E. Thompson, T.-W. Wang 311
Molecular Markers as a Tool for Analyses of Genetic Relatedness and Selection in
Ornamentals
T. Debener 329
Plant-Specific Intellectual Property Rights
S. Berman 347

Index 381
PREFACE

The relationship between mankind and ornamentals has a very long and romantic
history. In more recent times, thanks to amateur and professional breeders, ornamentals
have become a highly important economic commodity. Today, they are sold worldwide,
to the tune of tens of billions of dollars. Every year, breeders driven by their search for
novelty create new and attractive varieties. Traits such as new colors, altered forms,
enhanced fragrance and increased longevity are in high demand by the consumer, who
is continually seeking novel products. From the grower's point of view, ornamentals
with improved agronomic performance are no less important.
Classical breeding, which is based on the search for exotic plants, mutants and/or
crosses within and between related species and selection of the most promising
offspring, has a proven track record. It has been responsible for the introduction of
many traits and the production of a large number of varieties in many ornamentals.
However, although classical breeding is still a powerful tool in the breeder's hands, the
available gene pool for new traits is limited. Furthermore, the selection of a desired trait
in the siblings is performed on the genetic background of their parents, which together
with the genetic variability of the offspring, complicates and limits controlled breeding.
The development of new tools for the introduction of foreign genes into plants,
combined with the growing knowledge and technology related to gene identification
and isolation, has revolutionized all aspects of the biological sciences, particularly
agriculture. Genetic engineering approaches have created almost unlimited possibilities
for the molecular breeding of novel/improved crops. Via this route, for example, it has
become possible to purposely alter single traits, e.g. disease resistance, in otherwise
successful cultivars; or alternatively, to generate a completely novel phenotype, e.g.
plant/flower architecture. However, despite the great progress and interest in gene
transfer to ornamental crops, the genetic engineering of ornamentals is currently lagging
far behind that of main food crops and is considered routine in only a very few
laboratories. The main reason is the lack of efficient transformation systems for
ornamental species; even when such procedures are available, they are generally not
suited to elite varieties. Moreover, each ornamental crop represents only a small
segment of a market that consists of numerous different species, each comprising
hundreds of varieties. Hence, the limited economic value of any single ornamental crop
has precluded the investment of the massive funds, such as those spent, for example, on
food crops, needed to advance ornamentals into the molecular breeding era. The high
cost of registering transgenic crops also constitutes a significant constraint in
ornamentals. Nevertheless, the transformation of numerous ornamentals has been
reported in the last few years and the first genetically engineered ornamentals have
already been put on the market.
In this book we bring together the most up-to-date information on developments,
both basic and applied, that already have or are expected to impact the field of
ornamental breeding. These include classical and molecular techniques, traditional and
high-throughput approaches and future trends. Since not only professional scientists, but

Vll
viii

also thousands of future scientists/students as well as amateur breeders around the world
contribute heavily to the field of ornamental breeding, an introductory section dealing
with the basics of molecular and classical genetics and the evolution of floral diversity
is included. Hopefully, this should enable the reader to bridge the gap between
traditional breeding and molecular genetics. Classical approaches to the
creation/selection of genetic variability, including mutation and tissue culture-aided
breeding, are presented in section two. The current knowledge of the processes affecting
ornamental and agronomic traits at the molecular level is presented in chapters of
section three, which also includes an in-depth analysis of developments in the protection
of intellectual property rights.
It is quite apparent from the leap made in biological sciences in the last few years-
deciphering plant genomes and developing high-throughput technologies that allow the
simultaneous detailing of the activities of thousands of genes and the detection of their
resultant products-that we are entering an era in which variability can indeed be
artificially created and precisely controlled. This by no means implies that classical
approaches will or even should be forgotten. On the contrary, only integrating molecular
approaches with traditional strategies can lead to the full realization of breeders'
dreams. Moreover, public acceptance of the novel variability will further dictate the
borders within which breeders can advance. I hope that the thoughts and strategies,
presented side by side in this book, of molecular and classical geneticists, which are not
always complementary or even compatible, will serve to spark the imaginations of
breeders as well as students entering the exciting world of state-of-the-art ornamentals.

Alexander Vainstein
EVOLUTION OF SEXUAL REPRODUCTION AND FLORAL DIVERSITY

RJ. GRIESBACH
Floral and Nursery Plants Research Unit
U.S. National Arboretum, USDA, ARS
Beltsville, MD 20705-2350
USA

Effective plant breeding requires a knowledge of several basic principles, one of which is
the structure and function of flowers. Flowers are borne on inflorescences. In some species
(e.g. Narcissus), it is difficult to determine where the inflorescence actually begins and ends. A
great labyrinth of terminology exists on the classification of inflorescences. The most common
inflorescence1ypes aretheraceme,spike,panicle, umbel, cyme, and head (Fig. 1). The parts
of a flower are arranged in successive whorls (Fig. 2). The first whorl consists of the sepals
which together form the calyx. The general function of the sepals is to protect the flower as it
is developing. In many flowers, the sepals are green and very small. The second whorl
contains the petals, which together form the corolla. The general function of the corolla is to
attract pollinators. The petals are usually brightly colored and are the most attractive part of
the flower. In some flowers (e.g. Clematis), however, the corolla is absent and the calyx is the
brightly colored strncture which attracts the pollinators. The calyx and corolla together form
the perianth. In some plants there maybe little difference in the appearance of the petals and
sepals. In those plants, the petals and sepals are referred to as tepals. In wind-pollinated
flowers (e.g. Salix), the entire perianth may be absent. In those species, the flowers only
consist of reproductive organs.
The third and fourth whorls contain the reproductive organs. The third whorl contains a set
of stamens. The stamen is the male organ and consists of the anther on a stalk called a
filament. Within the anther are pollen grains which are the male germ cells. When the anther
is mature it opens and exposes the pollen grains in a process called dehiscence. The number of
stamens in a flower can range from one (Canna) to over 20 (Ranuncu Ius). The size and shape
of the stamens can also vary. Long stamens can be over 5 em in length (Lilium ), while short
stamens can be less than 1 rom (Acacia). They can be round, linear, branched or coiled. In
most plants, adjacent stamens are separated; however, they may be fused at their f:tlaments
(Pisum) or anthers (Calendula). The number of pollen grains within the anther can vary from
as few as 25 (Mirabilis) to as many as 60,000 (Bora go). Pollen grains can also vary greatly in
size and texture. In orchids, the pollen grains adhere to one another to form a solid mass; in
most plants. however. the pollen grains are single and appear as fme powder.
The fourth whorl contains the carpel or pistil. The carpel is the female organ and consists
of the ovary connected to the stigma by a stalk called a style. Within the ovary are the ovules
which are the female germ cells. The number of ovules within the ovary can range from one

A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, !-6.
© 2002 Kluwer Academic Publishers.
2

(Hordeum) to over 500,000 (Cattleya). The number of carpels within a flower can vary from
one in most plants (Nicotiana) to over 25 (Fragaria). The shape of the carpel is also variable.
In some instances, the style can be completely absent (Papaver).

Ft: ~ T,-~
spike raceme panicle head umbel cyme

Figure I. Diagram ofinllorescmcetypes.

Figure 2. Diagram of the parts of a flower.

Flowers can be classified in many ways. One involves the relative position of the ovary. In
hypogynous flowers (Petunia), the floral parts are attached below the ovary; in epigynous
flowers (Hemerocallis), they are attached above the ovary; and in perigynous flowers
(Portulaca), they surround the ovary. Most flowers are bisexual or perfect, containing both
stamens and a carpel. In unisexual or impeifect flowers, the stamens and carpel are found in
separate flowers. In monoecious species (Zea), the imperfect flowers occur on the same plant.
In dioecious species (!lex), the imperfect flowers occur on different plants, creating male and
female plants.
Flowers can also be classified as either regular or irregular depending upon symmetry.
Regular or actinomorphic flowers (Chrysanthemum) are radially symmetrical (capable of
being dissected into mirror images along more than one axis). Irregular or zygomorphic
flowers (Gladiolus) are bilaterally symmetrical (capable of being dissected into mirror images
along only one axis). Regular flowers have petals which are identical, while irregular flowers
have one or more petals that differ in appearance. A peloric mutation is a type ofmutation that
changes the symmetry of a flower. Regular peloria occurs when a normally irregular flower
becomes regular. Similarly, irregular peloria occurs when a normally regular flower becomes
irregular. There are several examples of peloric mutations in ornamental plants, the most
common of which are the splash-petaled orchids.
 3

Peloria is an example of a specialized mutation (homeotic) which controls flower

development. A homeotic gene regulates the expression of a battery of genes that lead to the
development of a specific organ, such as a petal. Homeotic mutations cause one organ to be
replaced by another; however, not all homeotic mutations result in complete replacement. The
degree of replacement depends upon when in development the mutation acts. Ifthe mutation
acts early in development, then more complete replacement occurs. One of the most obvious
homeotic mutations results in a double flower. In a double flower, there are multiple flower
parts. For example, an extra set of petals can be created by the conversion of stamens into
petals (Peonia). Doubleness is not always the result ofa homeotic mutation. Extra floral parts
can be created without sacrificing another organ (hose-in-hose Azalea).
Pollination is the process of placing pollen on the stigma. When placed upon the stigma, a
pollen grain germinates, producing a pollen tube which grows down the style into the ovary.
Upon reaching the ovary, a nucleus within the pollen tube enters the ovule and unites with a
female nucleus in a process called fertilization. Fertilization results in aZ}gote which develops
into an embryo. In flowering plants, double fertilization occurs. In double fertilization, a
second male nucleus enters the ovule and unites with a second female nucleus to create the
endospenn. The endosperm is the food reserve of the embryo. The double-fertilized ovule
then develops into a seed.
The seeds are contained within afrnit. Fruits develop from the carpel and are classified as
either simple, aggregate or multiple. Simple fruits are derived from a single carpel
(Delphinium); aggregate fruits are a collection of simple fruits derived from a corresponding
number of separate carpels from a single flower (Rubus); and multiple fruits arise from the
fused carpels of the multiple flowers on an inflorescence (Morus ). Other parts of the flower
can also contribute to the mature fruit. If these other parts are very conspicuous, the fruit is
classified as an accessory frnit. In the accessory fruits of Fragaria, the main succulent tissue
consists of an enlarged flower stem with numerous small fruits embedded in its surface. In the
accessory fruits of Malus, the main succulent tissue is derived from the bases of the calyx,
corolla, and stamens.
In a plant, vegetative cells (stem. leaf, and root) are usually diploid (2x) having two sets of
chromosomes, one set derived from each parent. Preceding cell division, the DNA of each
chromosome replicates to create a doubled chromosome composed of two chromatids. When
a cell divides, the chromatids separate from one another into two daughter cells. Each daughter
cell is identical to its parent cell. On the other hand, germ cells are haploid (lx), having only a
single set of chromosomes. Haploid germ cells are created from diploid vegetative cells
through the process of meiosis (Fig. 3). During meiosis, a vegetative cell undergoes two cell
divisions which result in four germ cells. During the first cell division, the corresponding or
homologous chromosomes from each parent adhere or pair with one another. After pairing,
DNA segments on the different chromatids can be interchanged. Through this process of
crossing-over, new gene combinations are created. Many environmental factors (temperature,
day length, humidity, etc.) can effect pairing and crossing-over. In addition, there are genes
which control both pairing and crossing-over. Finally, the recombined homologous
chromosomes separate into two daughter cells. Each daughter cell then contains a single set of
chromosomes, each of which has two chromatids. In the second meiotic cell division, the
chromatids separate into two daughter cells, similar to mitosis.
While the vegetative cells are diploid and the germ cells are haploid, the endosperm cells
4

are polyploid, with more than two sets of chromosomes. The exact chromosome number of
the endosperm depends upon the species. For example, the endosperm is triploid (3x or three
sets of chromosomes) in lea and pentaploid (5x or five sets of chromosomes) in Lilium.
Endosperm development and embryo viability depends upon the endospenn balance number
(EBN). Every species has a specific EBN which restricts breeding to other plants with the
same number. This is one of the reasons why many interploidy crosses (2x x 4x) do not result
mprogeny.

Figure 3. Diagram of meiosis. Two homologous dtromosomes are represented, each composed of
two dtromatids. One dtromosome is from the male parart (stippled) and the other from the female
parent (white). During the frrst meiotic division, the homologous dtromosomes pair, exchange
DNA segments and segregate into two daughter cells. During the second meiotic division, the
dtromatids of each chromosome separate, forming four germ cells.

The flowering plants or angiospenns first appeared about 150 million years ago during the
Jurassic period of geological history. Angiosperms are defmed by ovules which are enclosed
within carpels. The enclosure of the ovules protects them from predation and desiccation and
allows pollination and fertilization to occur while they are still immature. Besides enclosed
ovules, double fertilization is also unique to the angiosperms. These characteristics allow
angiosperms to have a more efficient and shorten life style as compared to gymnosperms,
ferns and cycads. For example in Pinus, 2 years lapse between pollination and seed dispersal.
Not only are angiosperms able to produce seeds more quickly, they are also able to reproduce
in less hospitable habitats. Unlike gymnosperms, ferns and cycads, angiosperms do not require
water for fertilization.
The oldest known fossil angiosperm, Archaefructus liaoningensis, was recently
discovered in China (Sun eta/., 1998). The 145 million-year-old fossil of Archaefructus
consists of a branching stem with 48 fruit. The fruits were formed from folded carpels. Each
fruit was -8 mm long and was connected to a leaf-like structure which was -15 mm long. The
stamens were deciduous, leaving 0.5 mm peg-like bases. There were two to four ovules per
fruit.
 5

The fossil record indicates that the first angiosperms produced flowers that were wind-
pollinated and that early in evolution there was an extremely rapid radiation of flower types.
Many believe that this rapid evolution was the result of strong selection pressure favoring
insect pollination. During this time period. insects were also rapidly evolving. By the mid-
Cretaceous period (~95 million years ago), the fossil record shows a great diversity of insect-
pollinated flowers. These flowers were extremely minute (~2 mm in natural spread) with an
undifferentiated perianth of overlapping bracts. The stamens were massive, with little or no
differentiation into either a filament or anther. The number of carpels per flower varied, but a
single ovule was usually present in each carpel. Fossil flowers from this time period have an
affinity to the modern heath, laurel, witch hazel, chloranthus, magnolia and hydrangea
families. These flowers, however, bear little resemblance to modern taxa. By the close of the
Tertiary period (~25 million years ago), all of the modern orders of plants had evolved.
Plants can be propagated by asexual and sexual methods. Increasing plants via division,
cuttings, corms, bulbs, or tissue culture is called asexual or vegetative reproduction and the
resulting plants are called clones. Clones are genetically identical. Many ornamental plants,
such as rose, gladiolus and rhododendron, and fruits, such as apple, raspberry and strawbeny,
are produced as clones.
Increasing plants by seed is called sexual reproduction and the resulting young plants are
called seedlings. In most instances, each seedling is genetically different from its siblings and
parent(s). Most bedding plants, such as marigold, zinnia and cosmos, and nearly all vegetable
and agronomic crops, such as wheat, tomato and com, are produced as seedlings. Ifthe pollen
and ovules arise from different clones, then the seedlings are the result of cross-pollination.
On the other hand, self-pollination results when the pollen and ovules originate :from the same
clone. In many species, mechanisms have evolved to prevent self-pollination. In the most
extreme instances, the plants are self-incompatible. In self-incompatible plants (e.g. Petunia),
the pollen, when deposited on the stigma of the same clone, aborts its development before it
can effect fertilization. Other mechanisms are much less extreme. In some species, the pollen
is shed either before (Impatiens) or after (Ranunculus) the stigma is receptive. In other
species, the position of the stigma relative to the anthers prevents self-pollination (Anemone).
Surprisingly, plants can also be reproduced asexually from seed. Apomixis is the process
by which one of the vegetative cells within the ovary develops into a seed without fertilization.
Usually apomictic and zygotic seeds co-exist in the same seed capsule. The frequency of the
apomictic type can range from less than 1 to 100%. Apomixis has been reported in many
species, such as strawberry, lily, rose, citrus, birch and orchid.
In order to produce a crop of uniform appearance, most sexually reproduced plants are
produced as either an inbred or a hybrid line. An inbred is developed by self-pollinating a
plant and its resulting seedlings for several generations. Through inbreeding, seedlings
become more uniform in appearance. However in some instances (inbreeding depression),
inbred seedlings may exhibit weak growth and are not suitable for commercial production.
Hybrids are then created. A hybrid is produced by crossing two divergent inbred lines.
Hybrids are generally very uniform in appearance and have increased plant health and vigor
(hybrid vigor). The classical example of a hybrid is found in com.
In breeding, it is important to understand how parental germplasm was developed before it
can be appropriately and efficiently used in further plant improvement. For example, in
petunia there are both sexually ('Purple Dream', 'Pink Sur:finia', etc.) and asexually ('Red
6

Magic', 'Blue Fantasy', etc.) reproduced cultivars. Seedlings from a sexually reproduced
cultivar would be expected to be uniform (homozygous) if developed as an inbred line and
variable (heterozygous) if developed as a hybrid line. On the other hand, seedlings from an
asexually reproduced cultivar would be expected to be highly variable. Self-incompatibility
and apomixis also exist in petunia. A different breeding approach is needed to develop a
vegetatively reproduced cultivar vs. sexually reproduced hybrid. Self-incompatibility would
complicate the development of inbred lines, but have little effect on the development of
vegetatively reproduced cultivars. Similarly, virus resistance is generally more important in
the development of vegetatively reproduced cultivars than in sexually reproduced hybrids, for
most viruses are mechanically transmitted, not via seeds. Thus, efficient and effective plant
breeding improvement requires a thorough understanding of the mode of reproduction of the
species.

References and Recommended Textbooks

Allard, R W. (l960)Principles ofPlant Breeding, Wiley, NY.

Crane, MB. and Lawrence, W.J.C. (1934) Genetics ofGarden Plants, MacMillan, Loudon.
Cutter, E.G. (1911)Plant Anatomy: Experiment and Interpretation, Addison-Wesley, Reading, MA
Endress, P.K. and Friis, E.M (1994) Early evolution of flowers, PL Syst. Evol. Suppl. 8.
Esau, K. (1965)PlantAnatomy, Wiley, NY.
Foster, AS. and Gifford, E.M. ( 1974) Comparative Morphology ofVascular Plants, W.H. Freeman, San Fnmcisco.
Hayes, H.K., lmmer, F.R. and Smith, D.C. (1955)Methods ofPlant Breeding, McGraw-Hill, NY.
Sun, G., Dildler, D.L., Zhwg, S., and Zhou, Z. (1998) In search of the first flower: a Jurassic angiospenn,
Ardtaefructus, from northeast China, Science 282, 1692-1695.
Thomas, B.A and Spicer, R.A (1987) The Evolution and Palaeobiology of Land Plants, Dioscorides Press,
Portland, OR
TRANSMISSION GENETICS

AASHRI
Jacob and Rachel Liss Professor of Genetics and Breeding,
The Hebrew University ofJerusalem, Faculty ofAgricultural, Food and
Environmental Quality Sciences
Rehovot 76100, Israel

1. Introduction

Mankind has always been attracted to animals and plants; not only to food or pasture
plants, but also to flowers and ornamentals, observing, collecting and multiplying
different forms. Gardeners have developed, by accumulated experience, a wide range of
varieties with different flavors, colors and shapes, different adaptation ranges, different
leaves, etc. Actually, when Gregor Johann Mendel, the ''Father of Genetics" conducted
his famous studies on the inheritance of various traits in peas (1850s-1860s) he used 22
well-established, true breeding varieties. In making crosses between differing parents
Mendel followed earlier gardeners. However, whereas earlier investigators examined
only hybrid plants (F1), Mendel continued and studied also their progeny (Fz) which
were produced by self-pollination (normal in peas). The publication of his findings and
conclusions in 1866 could mark the beginning of the science of genetics.
Unfortunately, his ideas were not appreciated until they were rediscovered in 1903.
Thus, the science of genetics was founded about 100 years ago. It now has many
aspects and sub-disciplines. This chapter will deal with the way parents transmit their
traits to their offspring, hence its name-transmission genetics.

2. The Physical Basis

The genetic information controlling all aspects of the development of the organisms is
encoded in the DNA. In plants, as in animals, nearly all ofthe DNA is contained in the
chromosomes which are located in the nucleus of the cell. Very small amounts of
DNA, albeit with vital information, are located in the cells' cytoplasmic organelles, in
the mitochondria and in plants also in the plastids.
Each species has a typical chromosome number and chromosome morphology.
During cell multiplication the chromosomes are duplicated and distributed regularly
and equally to the daughter cells in mitosis. The mitochondria and proplastids are also
distributed to the daughter cells during mitosis but in a random fashion-not equally;
thus, more organelles may reach one of the daughter cells. Also, since their distribution
7
A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, 7-23.
© 2002 Kluwer Academic Publishers.
8

is random, if there is heterogeneity in the organelles (e.g. following cell fusion or

mutation) the daughter cells may receive unequal genetic complements (see below-
extranuclear inheritance).
During the meiotic division, which produces the gametes-the pollen grains and
the egg nuclei-the number of chromosomes is halved. Thus, while the number of
chromosomes of the plant (sporophyte) is 2n, the number of the chromosomes in each
of the gametes is n. Upon fertilization, when a pollen grain's generative nucleus (n)
unites with an egg nucleus (n), a zygote with 2n chromosomes is formed. As a rule, the
cytoplasmic organelles of the zygote come from the female parent through the egg.
There are a few exceptions, where some such organelles are contributed to the zygotes
by the male parent also, via the pollen.
Thus, in diploid plants, each chromosome is present in the cell nucleus twice. The
pairs of chromosomes are termed homologous chromosomes. Such chromosomes have
the same morphology, carry the same gene loci and form pairs (bivalents) during the
first meiotic prophase and metaphase.
Thus, the zygote receives one set of paternal chromosomes and genes via the pollen
grain and one maternal set via the egg. As a result, in diploid plants each gene appears
twice. If for a given gene there is the standard (wild-type) DNA sequence (A) and a
mutant one (a), then a plant can be AA (homozygous) or Aa (heterozygous) or aa
(homozygous). The different forms (DNA sequences) of the gene are termed alleles
and the site of the gene in the chromosome is termed locus (plural-loci). An allele
can be dominant, i.e. in a heterozygous plant (Aa) only the A character will be
manifested; the allele that is not manifested in this case (a) is termed recessive. There
can also be intermediate states with nQ--()r partial-dominance.

3. Monohybrids

Monohybrid studies deal with the genetic consequences in offspring of crosses between
two plants which differ in one trait. The parents can be of the same or different
varieties and sometimes different species. The hybrids derived from a cross are termed
Ft (first filial generation) and their offspring through self-pollination or full-sib-
pollination (brother-sister mating) are F 2 and so forth. The expected consequences will
be illustrated for both the no-dominance and the dominance allelic relations.

3.1. NO DOMINANCE

Assume that in snapdragon, two homozygous pure-line varieties, one with white
flowers, the other with red ones, are crossed The expected results are shown in Fig. 1.
The typical (1:2:1) F 2 segregation ratio is derived from the fact that half of the gametes
produced by the F1 hybrid carry one allele (R) and half carry the other allele (r), and
their random zygote formation, as shown below (Fig. 2). In this case every genotype
(genetic constitution) AA, Aa and aa codes for a different phenotype (resulting
manifestation).
 9

p Red(RR) X White (rr)

F1 phenotype is intermediate,
Pink (Rr) hence no dominance between
the alleles.

Self-pollination ®..t..
Fz y,. Red(RR) Yz Pink (Rr) : Y.. White (rr)

Self-pollination ®..t.. ®..t.. ®..t..

F3 All red(RR) y,. Red(RR) All White (rr)
YzPink (Rr)
Y.. White (rr)
Figure 1. The expeded results of a monohybrid cross, with no dominance baween the alleles (P denotes parents).

Female gametes Male gametes (pollen grains)

(eggs) YzR Yzr
YzR Y.. RR (red) y,. Rr (pink)
Yzr y,. Rr (pink) Y.. rr (white)
Figure 2. The expeded F2 progeny in a monohybrid cross with no dominance.

3.2. DOMINANCE

In a large majority of the cases there is dominance between the alleles, which means
that the heterozygous genotype (Aa) has the same phenotype as that of the homozygous
genotype (AA). Such cases usually indicate than one allele, the recessive one (a) does
not code for a product (e.g. polypeptide, enzyme) while the dominant allele (A) codes
for the product and its presence in one dose (Aa) suffices to produce the same
phenotype as the presence of the allele in two doses (AA). With dominance, the
phenotype of the F1, and the F2 phenotypic segregation, will be different from that
shown above. This is illustrated (Fig. 3) with one of the traits studied by Mendel in the
1850s and 1860s in peas: round, smooth seeds vs. wrinkled seeds. In Mendel's
experiments with this trait, the F1 always had round seeds and in the F2 he obtained
5,474 plants with round seeds and 1,850 plants with wrinkled seeds, a ratio of
2.96:1.00, very close to the expected 3:1 ratio.
Homozygous dominant (RR) and heterozygous (Rr) F2 plants can be distinguished
by selfing or test crosses (crossing with homozygous recessive). Upon self-pollination,
10

the homozygous (RR) plants will yield F 3 progeny which will all have round seeds,
while the heterozygous plants (Rr) will give F 3 progeny which will segregate as in the
F2, % of the plants with round seeds: '!.! with wrinkled seeds. Similarly, with test
crosses, the cross of F 2 RR plants with rr will yield uniformly round seeded (Rr)
progeny; the cross of Rr plants with rr will give progeny that will segregate, half of the
plants-round seeded (Rr) and half of the plants with wrinkled seeds (rr).

P Round, RR X Wrinkled, rr

Gametes All~ All 0

F1
"V Round, Rr

Self pollination ® ..1.

Pollen grains
Eggs
R r
R RR, round Rr, round
r Rr, round rr, wrinkled

F2 phenotypic ratio--3/ 4 of the plants will have round seeds et 4 RR + 2/ 4 Rr) and 1/4
of the plants will have wrinkled seeds (rr)

Figure 3. The expected results of a monohybrid cross with complete dominance between the alleles.

3.3. OTHEROOMINANCE RELATIONS

Other relationships between the alleles are also possible. If the dominance is
incomplete (partial), the heterozygotes will be phenotypically close to one parent but
not quite like it, i.e. one dose of the allele is not sufficient to produce the same
phenotype as two such alleles. Another possible situation is codominance. In this case,
both alleles in the heterozygotes code for some product and both are present in the
plant (e.g. isozymes).
It should be emphasized that the dominant vs. recessive designations do not refer to
the selective or evolutionary value of the alleles. Often the recessive alleles are such
because their DNA sequence is flawed and they do not code for a product while the
dominant alleles do. In the pea seed shape example, the round seeds are such because
the R allele codes for the starch branching enzyme l which converts amylose to
amylopectin while the r allele does not produce the enzyme, hence the seeds contain
more sugar which draws more water and upon maturation and drying, the seeds
 11

become wrinkled. It is interesting to note here that the DNA of the r allele contains an
insertion (intron) of 800 bases, which leads to a shorter polypeptide (enzyme), which is
non-functional.

4. Multiple Alleles

As noted earlier, the genes are sequences of DNA, which can be several hundred base
pairs long, or longer. Thus, there are many potential sites for base changes in the
genes, i.e. mutations which can be manifested in a modified product and phenotype. In
view of this, there can be quite a few alleles for any given gene in the population.
A well-known example for this is self-incompatibility, controlled by the locus S.
There are many alleles for this gene in each of the species where it is present, which
are numbered S1, S2, S3, S4, ...... Sn. It should be noted, though, that any given diploid
individual may have only two of the alleles (usually different), e.g. S1S2 or S2S3 or
S1S3 .

p AABB X aabb

Gametes AB ab

~/
AaBb

Gametes

Self-pol1rtnatlon

~
AB Ab aB ab
ggs lf4 lf4 lf4 lf4
ABY.! AABB AABb AaBB AaBb
Ab lf4 AABb AAbb AaBb Aabb
aBY.! AaBB AaBb aaBB aaBb
ab lf4 AaBb Aabb aaBb aabb

Figure 4. The expected results of a dihybrid cross with dominance between the alleles in both loci.
12

5. Dihybrids and Higher Hybrids

The genetic expectations in crosses where the parents differ by two or more genes are
based on the monohybrid. When the loci involved are on different chromosomes (not
linked, see below), the distribution of the alleles of one gene to the gametes is not
related and has no effect on that of the alleles of the second. The alleles assort
independently.
As a general scheme, if the parents differ by two genes, with dominance between
the alleles of each, the F1 and F2 expectations will be as shown in Fig. 4.
The expected proportions of any genotype or phenotype can be calculated by less
laborious methods, based on the typical monohybrid segregations.
Assume a dihybrid cross AAbb x aaBB, or AABB x aabb, resulting in F1 plants
which are heterozygous for both genes (AaBb). The proportion of any given F2
genotype will be the product of the probabilities for each locus. Example:
• What will be the proportion among the F2 progenies of AaBB plants? Based on the
monohybrid expectation for each locus it will be Yz(Aa) x Y-!(BB) = 1/8 .
• What will be the proportion of A-, B- (the- indicates that the second allele can be
either the dominant or the recessive) plants in F2? %(A-) x %(B-) = 9/ 16.
Still another way is to carry and combine the two monohybrids. In the example
above the F1 is AaBb, the alleles and loci will assort independently hence the F2 ratios
expected can be calculated as follows:

f-
X Y-!BB ~ 1h6AABB
Y-!AA X YzBb ~
2/16AABb
1/!6AAbb
X Y4 bb ~

X Y-!BB 2/16AaBB

~
~

YzAa X YzBb ~
4h6AaBb
2/!6 Aabb
X Y4 bb ~

Y-!aa L X Y-!BB
X YzBb
~ 1h6 aaBB
2h6 aaBb
\ X Y4 bb
~

~
1h6aabb

In a test cross, i.e. AaBb x aabb, the resulting offspring will segregate at a ratio of
1: 1:1:1, i.e. Y4 AaBb, Y4 Aabb, Y4 aaBb and Y4 aabb.
The same approaches can be employed in calculating F2 and other segregations in
crosses in which the parents differ by three, four or more independent (unlinked-see
below) genes, with or without dominance between the alleles in some of them. For
example, assume a hybrid between two parents differing by three genes with complete
dominance between the alleles in each locus, AaBbCc.
• In the F2, what will be the proportion of A-Bbcc plants? Based on the monohybrid
expectation for each locus it will be %(A-) x Yz(B-) x Y-!(cc) = 3/32.
 13

6. Goodness of Fit of Segregations

The goodness of fit of a given F2 segregation, i.e. its closeness to the expected, or any
other segregation is assessed using the chi-square (·l) method. Basically, the method
examines the probability that the observed deviations from the expected can occur by
random chance alone. If this probability is lower then 5%, the deviation is considered
significant and the proposed genetic hypothesis is rejected The reader is referred to a
basic genetics or statistics text for detailed explanations. Here it should be stressed that
the actual numbers of individuals in the various phenotypic classes should be used and
not fractions or percentages.
Chi-square tests are a good indication of goodness of fit. However, for proof of a
proposed hypothesis, progeny testing in F3 and/or test crosses are required. Sometimes
further generations are desirable.

7. Gene Interactions

The development of the organism and its activities are controlled by many genes. The
products of one gene are utilized or modified by another. Thus, since there are various
biosynthetic pathways, the final phenotype is a product of gene interactions. Some
modes of interaction will be illustrated using two genes (Table 1), but it should be clear
that more genes may be involved in any activity. The four genotypes shown in Table 1
can be regarded as four building blocks which can be assembled in different
combinations, depending on the modes of gene action. Such interactions are often
encountered in flower colors; where one locus codes for a basic pigment molecule and
other loci affect, add or remove hydroxyl groups, or methylate a position, etc. The main
types of interaction are summarized in Table 1.

TABLE 1. Expeded F2 segregation ratios with various modes of interaction bawew two genes, with
dominance bawew the alleles in both loci.
Modes Genotypes
A-B- A-bb aaB- aabb
Dihybrid ratio 9 3 3 1
Epistasis, recessive 9 3 ---4--
Epistasis, dominant --12--- 3 1
Complementary 9 -----7-------
Duplicate --------15----- 1
Additive 9 ----6-- l

Epistasis describes gene interactions whereby one gene affects the phenotypic
expression of another non-allelic gene or genes. Epistasis of the recessive allele means
that the homozygous recessive condition for one locus (aa in the table) supresses the
manifestations of B vs. b, yielding a 9:3:4 ratio in F2• Epistasis of the dominant allele
means that the presence of one dominant allele (A-) will suppress the expression ofB
14

vs. b, yielding a 12:3:1 ratio in the F 2.

Complementary gene action describes interactions in which the phenotype depends
on the action of the dominant alleles of two loci; if either one is missing the
biosynthetic process cannot be completed, hence the F2 ratio of 9:7. Duplicate action
occurs when two independent loci with the same function are present in the organism,
thus in the F 2 only the homozygous recessive-aabb-will have a different phenotype.
Hence the 15:1 ratio.
Additive action describes cases in which two loci control the same product (e.g.
hormone); genotypes with a dominant allele in one locus only (A-bb or aaB-) produce
the same phenotype, when dominant alleles are present in both loci (A-B-) their
additive affect leads to a different phenotype, hence the F 2 ratio of 9:6:1. The term
additive gene action is often used to describe quantitative gene action (see further on).

8. Linkage and Chromosome Maps

Any given plant contains many thousands of gene loci. At the same time the number of
chromosomes in plants' cells is small, a few tens at the most. Therefore, it is obvious
that each chromosome in the complement holds many loci, i.e. contains linear DNA
which codes for many traits. This is termed linkage and the genes are linked. During
meiosis and chromosome migration to the poles, the linked genes will tend to go
together except where a recombination event, crossing over, has occurred.
Recombination occurs during the first prophase of meiosis when segments of non-sister
chromatids in the paired homologous bivalent are exchanged. The frequency of
recombination between any two loci is affected primarily by the distance between them:
if they are very distant it will be high and if they are very close it will be low. Thus,
recombination can be used to identify linked genes and the frequency can be used as a
yardstick to measure their distance from each other. Generally the map distances
correspond to actual physical length, except for the centromere regions which appear
shorter in the map because recombinations there are very infrequent.
 15

Example: Genes A and B are linked, located on the same chromosome; what is the
distance in map units between them (% recombination, known also as
centimorgans), i.e. what is the frequency of recombinant gametes due to crossing
over events between A and B?

Parents: A B a b

a b

Test Cross ofFt: a b

X

a b a b

Possible gametes: AB } Parental

~
ab types abAll

: } Recombinant

Offspring: Assume AB 40%

ab
ab
40%
ab

Ab
10%
ab

aB 10%
ab

Frequency of recombinant offspring, i.e. frequency of recombination= 0.10 + 0.10 =

20
0.20. The chromosome can be mapped as shown: A < >B
I I

Depending on the arrangement of the genes and the alleles on the parental
chromosomes entering the cross, the parental and recombinant types will differ as
follows:
16

a. The genes are in coupling:

P AB xab
AB ab

J,
AB
ab

Gametes, parental types-AB and ab

Gametes, recombinant types-Ab and aB

b. The genes are in repulsion:

p Ab XaB
Ab aB
-J..
Ab
aB

Gametes, parental types-Ab and aB

Gametes, recombinant types-AB and ab

Linkage studies are used to find which genes are linked and to map them along the
chromosomes. The linear order of the genes along the chromosomes is established by
crosses involving three linked genes ("three-point cross"). In some plants, hundreds of
genes have been mapped, e.g. tomato, peas, maize, rice, wheat, and Arabidopsis. At
present there are also efforts to merge linkage maps with maps of molecular markers
such as RFLP maps.
Linkage affects Fz segregation ratios markedly. This will be illustrated with the
proportions of homozygous recessives for two linked genes in F 2 • In the example above,
where A and B are 20 centimorgans apart (20% recombination), the frequencies of the
different genotypes in the following F 2 will diverge greatly from those expected in a
dihybrid and will be different for the repulsion or coupling phases.
 17

A. Coupling:
p AB ab
-x-
AB ab
,1,
AB
ab

Gametes:
0.40AB}
0.40 ab Parental

0.10 Ab}
0.10 aB Recombinant

Following selfing, the expected frequency of ab/ab F2 offspring will be 0.4 x 0.4 =
0.16 (i.e. about 1/ 6 instead of 1/ 16). The frequencies of the other genotypes and
phenotypes can be calculated and shown to be different from the expected dihybrid
ratio.

B. Repulsion:
Ab aB
p -x-
Ab aB
,1,
Ab
aB

Gametes:
0.40 Ab}
0.40 aB Parental

0.10 AB}
0.10 ab Recombinant

Following selfing, the expected frequency of ab/ab F2 offspring will be 0.1 x 0.1 =
0.01 (i.e. 11100 instead of 1h6).
The frequencies of the other genotypes and phenotypes can also be calculated and
shown to be different from the expected dihybrid ratio.
The term linkage group refers to all the genes that are located on a given
chromosome. In a diploid plant with 2n chromosomes, the number of linkage groups is
n.
18

9. Quantitative Inheritance

Quantitative inheritance refers to situations in which a trait is controlled by many

genes, often called polygenes. Characters which are controlled this way show a wide
range of variation in the F2, which is continuous and more affected by the
environmental conditions. The phenotypic description of the parents, F~. F2, etc.,
requires measurement data. Visual scores such as tall and short do not suffice. Many of
the economically desirable traits such as yield, plant height, length of growing period,
flowering time, and oil content are controlled by polygenes. Because many genes affect
a given trait, the contribution of each gene to the phenotype is small. Also for that
reason, mutations in such genes are difficult to identify.

..--
1:::1

c:a..
c.-..
0

0
.
:z

30 200 em
X~ X P2

Figure 5. Plant height in peas, expect.ed frequency distributions and means of the parents, F, and F2
populations, acoording to the additive model of quantitative inheritance.

The model used to analyze the genetic mode of control of such traits stipulates that
they are controlled by many genes, with equal and additive action, without dominance
and without epistasis (interaction). Consequently, the variation of the F2 population
will span in a normal curve both parents and the F1 as shown in Fig. 5. According to
 19

the model, the mean of the F 2 is expected to be the same as the mean of the F1 and both
will be equal to the mean of the parents (=mid-parent):

Deviations from the above indicate that the model does not hold for the given trait,
i.e. that some major genes may also be involved, or that the genes are not strictly
additive. In recent years, many economic traits have been found to also be controlled by
QTL (quantitative trait loci) that behave as major genes. Molecular markers are used
in more and more cases to map the QTL to specific chromosomes and locations within
them.
The analysis of the genetic control of quantitative traits requires biometrical
approaches that will not be discussed here.

10. Extra-Chromosomal Inheritance

As noted above, the plastids and the mitochondria that are located in the cytoplasm
contain DNA which codes for vital functions of the cells and the plants. The numbers
of plastids and mitochondria per cell vary from a few to many. During cell divisions,
these organelles migrate to the opposite poles at random, and the numbers reaching the
daughter cells may differ. Generally, the egg cells contribute the organelles to the
zygotes; there are a few exceptional species where organelles are also transmitted
through the pollen. As a result of this, extra-chromosomal inheritance (also known as
cytoplasmic inheritance) is characterized by matroclinal inheritance: all the offspring
have the maternal phenotype, generation after generation. Consequently, in cases of
cytoplasmically inherited characters, if the crosses are made in both directions ( A~ x
Bd' and B~ x Ad') the reciprocal F1 hybrids will have different phenotypes.
Furthermore, there will be no Mendelian segregation in the F2 offspring and no
dominance vs. recessive relations. Moreover, in such cases, changing the nuclear
genetic information by backcrosses (or transplantation) will have no phenotypic effect.
There are also traits which are controlled via an interaction between the genes in
the nucleus and the cytoplasm. A very important economic trait with cytoplasmic
control is male sterility, which facilitates efficient large-scale production of commercial
hybrid seeds. In cytoplasmic male sterility (CMS), all the offspring of a male sterile
plant (S cytoplasm) will be male sterile regardless of the pollen parent. Similarly, all
the offspring of a male fertile plant (known as N or F) will be male fertile. Such a
mechanism can be used to produce commercial F1 hybrids where the desired parts of
the plants are vegetative (e.g. lettuce) or male-sterile cut flowers which last longer and
shed no pollen. However, such a mechanism is not suitable for plants producing seeds
and fruit.
A second male sterility type is controlled by both a nuclear gene with two alleles (R
orr) and the cytoplasm, which can be of two types: male fertile (F, known at times as
N) and male sterile (S). The plants are male sterile if their genotype is (S,rr) and male
20

fertile if they are (S,Rr) or (S,RR) and also if they have the F cytoplasm (even if they
are rr). This mechanism is very suitable for the production of F 1 hybrid seeds and is
used extensively in seed-producing crops, e.g. maize, sunflower and sorghum.
Mutations do occur in the DNA in the organelles. However, the recovery of such
mutants is difficult. For instance, if in an F,RR (male fertile) plant a mutation occurs in
the cytoplasmic components and it becomes S,RR, the branch or plant will still be male
fertile. The F to S mutation will go undetected. Furthermore, since there are few to
many plastids or mitochondria in a cell, a mutation which occurs in the DNA of one of
them, and gives heteroplasmon cells, may remain undetected for generations.
Some plastid mutations can lead to variegation, e.g. Mirabilis jalapa. Mixed cells
in the apical meristem can lead to sectors, even branches, which are green, albino or
mixed.

11. Genetic Studies in Polyploids

Polyploids contain several sets of chromosomes. Triploids contain each chromosome

three times in each cell, tetraploids four times and so forth. This situation affects the
breeding behavior of the plants, affects the genetic ratios in F2 and the recovery of
mutations. The implications are illustrated here with tetraploidy.
There are two main types of tetraploids (and polyploids in general):
autotetraploids are obtained by doubling the chromosomes of diploids while
allotetraploids are obtained by doubling the chromosomes of interspecific F1 hybrids.
In autotetraploids, each homologous chromosome appears four times in each cell.
Thus, there are five possible genotypes: AAAA, AAAa, AAaa, Aaaa, aaaa. In this type
of tetrasomic inheritance, the expectations for segregations in the F 2 are different.
Also, the recovery of mutations will be slow and require larger populations. If in a
plant which is AAAA a mutation occurs and it becomes AAAa, none of the offspring
following selfing will be recessive because the gametes will be AA or Aa. Only among
descendants from the Aaaa plant(s)-which will be obtained from AAAa plants two or
more generations later- will some aaaa offspring be recovered.
The allotetraploids are also termed amphidiploids (or amphiploids) because they
have two genomes (chromosome complements)-one from each parent species--and
within each every homologue appears twice. Duplicate genes are often encountered in
such plants. Allelic segregations in one genome may be covered by the segregations in
the other, modifying the expectations in F 2, etc. If a trait is controlled by duplicate loci,
recovery of mutants will be very difficult because the same mutations would have to
occur in both loci. Other aspects of polyploids are dealt with elsewhere in this book.

12. Changes in Chromosome Structure

The nature and cytogenetic implications of changes in chromosome structure are

discussed elsewhere. Here only their effects on genetic behavior are described.
 21

Deletion (=deficiency)-a terminal or interstitial segment of a chromosome is lost.

If it is a small sequence of DNA it may behave as a recessive allele (since there will be
a loss of function). A larger deletion may have the same effect, or be lethal in the
homozygous condition. Deletions also affect linkage and distance between loci.
Duplication-a segment of a chromosome is duplicated, in tandem, or in another
chromosome. Duplications can modify expected genetic ratios, and block or hinder
expression of mutations. They also affect linkage and distance between loci.
Inversion-after two chromosome breaks, a segment within the chromosome can
rejoin but in inverted order. Thus, a chromosome with gene loci A B.C DE.!: in that
linear order ( • denotes break points) may become ABED C F. The genetic
consequences are that the linkage relations of some genes will change. Secondly, the
inversions tend to conserve the existing linear gene order, rendering gametes with
recombinant chromosomes non-viable.
Reciprocal translocation-This change refers to the joining of broken arms or
smaller chromosome parts to non-homologous chromosomes. The main genetic
consequences are changed linkage relations and at times, some aborted gametes
(especially in the pollen).

13. Genes in Populations

In species which are cross-pollinated, i.e. outbreeding, the plants cross-fertilize each
other at random. Thus, any given population will be a heterogeneous mixture of
homozygotes and heterozygotes reflecting the genetic variation which it contains, i.e.
the frequencies of the different alleles in the various loci. A population can have one or
more alleles for a given locus and as long as they have the same selective value their
frequencies in the population will be maintained. In large populations gametes and
zygotes will be formed generation after generation and the alleles will retain their
frequencies, provided all gametes and genotypes have the same viability, the same
reproductive capacity and mutations in both directions are at an equal rate (A~ a).
The equilibrium distribution is known as the Hardy-Weinberg equilibrium. If the
frequency of the A allele in the population is p, and the frequency of the a allele is q,
then p + q = 1. The frequencies of the possible genotypes in the population can be
calculated as follows:

p(A) + q(a) = 1
[p(A) + q(a)f = 12
l(AAJ +2pq(Aa) +i(aa) = 1

These allelic and genotypic equilibrium states are maintained under the conditions
stated above. Such balanced populations are also known as Mendelian populations.
The conditions for the Hardy-Weinberg equilibrium are fully met only in a few
cases. Still, this approach makes it possible to study and compare various populations
22

and to draw conclusions on allelic frequencies from the findings, including deviations
from the model.
This equilibrium can also deal with multiple alleles and be used to calculate allelic
frequencies. The interested reader is referred to any basic genetic text for more details.
In self-pollinated species (inbreeding) the plants are homozygous as a rule, since they
do not intercross. Therefore, the Hardy-Weinberg equilibrium does not hold in such
populations. Furthermore, there will be selection against deleterious recessive alleles;
its severity will depend on the degree of their negative effect in the homozygotes.

14. Heritability (h1)

The final phenotypic expression of any character is the outcome of the interaction of
the genotype and the environment. Some traits may be more affected by the
environment, e.g. yield or plant height, while others are usually unaffected under
common growing conditions, e.g. flower color or leaf shape. Heritability measures the
extent to which the phenotype is affected by the genotype vs. the environment. The
heritability value, h 2, can vary between l (only the genotype is responsible, no
environmental influence) and 0 (no genetic component, environmental effect only). In
many breeding programs the traits have heritability values around 0.50 to 0.70. This
would indicate that there is an appreciable degree of genotype x environment
interaction. Therefore, larger populations are needed, a larger number of promising
plants should be selected (because the phenotype may be misleading) and proper
progeny tests are required.
There are various methods to calculate heritability. Heritability values measuring
only the additive gene effects are known as narrow sense heritability, h~, while values
which deal with all modes of gene action are known as broad sense heritability, h~.
The variance of populations which are genetically uniform (parent varieties-Vr?1,
Vr?2, F1 hybrids-V~I) is environmental. The variance of segregating populations
such as F2 is both genetic and environmental (VF2 = VG + VE). One way to calculate
h~ is thus the following:

h b2 _ Genetic variance
-
Total variance

Genetic variance is obtained by subtracting the environmental variance from the

total variance of the segregating population. Thus:

V0 = VFz - VE (mean ofvalues of parents and F1 )

h2 = VG
b vFz
 23

15. Summary

The study of genetics is in a state of flux with molecular biology creating many
innovative approaches. At the same time, experience in recent years has shown that
knowledge of classical genetics is vital for fuller understanding of the organisms.
Awareness of the classical aspects of genetics is most important for integrated, sound
mobilization of all the "genetic tools" in an efficient manner in breeding programs.

16. Recommended Genetics Textbooks

Brooker, R.J. (1999) Genetics: Analysis and Principles, Benjamin/Cummings, Menlo Park, CA
Griffiths, AJ.F., Gelbart, W.M., Miller, J.H., and Lewontin, R.C. (1999) Modern Genetic Analysis, W.H.
Freeman & Co., NY.
Griffiths, AJ.F., Miller, J.H., Suzuki, D.T., Lewontin, R.C., and Gelbart, W.M. (2000) An Introduction to
Genetic Analysis, 7th Edition, W.H. Freeman & Co., NY.
Klug, W.S. and Cummings, M.R. (1996) Essentials of Genetics, 200 Edition, Prentice Hall Inc., Upper Saddle
River, NJ.
Klug, W.S. and Cummings, M.R. (2000) Concepts of Genetics, 6th Edition, Prentice Hall Inc., Upper Saddle
River.NJ.
MOLECULAR GENETICS: GENE ISOLATION, CHARACTERIZATION AND
MANIPULATION

B.R GLICK, D.M. PENROSE

Department ofBiology
University of Waterloo
Waterloo, Ontario, Canada N2L 3Gl

1. Introduction

The information encoded in genetic material (deoxyribonucleic acid, DNA) is

responsible for establishing and maintaining the cellular and biochemical functions of
an organism. In most organisms, the DNA is an extended double-stranded polymer. The
sequence of units (deoxyribonucleotides) of one DNA strand is complementary to the
deoxyribonucleotides of the other strand. This complementarity enables new DNA
molecules to be synthesized with the same linear array of deoxyribonucleotides in each
strand as an original DNA molecule. The process of DNA synthesis is called
replication. A specific order of deoxyribonucleotides determines the information content
of an individual genetic element (gene). Some genes encode proteins and others RNA
molecules. The protein-coding genes (structural genes) are decoded by two successive
major cellular processes: RNA synthesis (transcription) and protein synthesis
(translation). First, a messenger RNA molecule (mRNA) is synthesized from a
structural gene. Second, an individual mRNA molecule interacts with other components
including ribosomes, transfer RNAs and enzymes to produce a protein molecule. A
protein consists of a precise sequence of amino acids which is essential for its activity.
Recombinant DNA technology, which is also called gene cloning or molecular
cloning, is an umbrella term that encompasses a number of experimental protocols
leading to the transfer of genetic information from one organism to another. There is no
single set of methods that can be used to meet this objective; however, a recombinant
DNA experiment often follows the following format (Fig. 1).

i) The DNA (cloned DNA, insert DNA, target DNA, foreign DNA) from a donor
organism is extracted, enzymatically cleaved (cut, digested) with a restriction
endonuclease, and joined (ligated) to another DNA entity (cloning vector) to form a
new, recombined DNA molecule (cloning vector-insert DNA construct, DNA
construct).
ii) This cloning vector-insert DNA construct is transferred into and maintained within a
host cell. The introduction of DNA into a bacterial host cell is called transformation.
iii) Those host cells that take up the DNA construct (transformed cells) are identified
and selected (separated, isolated) from those that do not.
25
A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, 25-45.
© 2002 Kluwer Academic Publishers.
26

iv) If required, a DNA construct can be prepared to ensure that the protein product that
is encoded by the cloned DNA sequence is produced by the host cell.

PBsmidDNA

l
lsolale
plasrrld
DNA

0
1::""
dgeslion

Figure 1. Recombinant DNA cloning procedure. Chromosomal DNA that bears the target gene is extracted
from a donor organism, cleaved with a restriction endonuclease and ligated into a linearized plasmid cloning
vector. The cloning vector-insert DNA construct is trllnsfurred to a target host cell, in a procedure called
transformation, and the cells that carry the construct are identified and grown.

Recombinant DNA technology was developed from discoveries in molecular

biology, nucleic acid enzymology, and the molecular genetics of both bacterial viruses
and bacterial extrachromosomal DNA elements (plasmids). However, recombinant
DNA technology would not exist without the availability of enzymes (restriction
enzymes, restriction endonucleases) that recognize specific double-stranded DNA
sequences and cleave the DNA in both strands at these sequences.
 27

2. Restriction Endonucleases

For molecular cloning, both the source DNA that contains the target sequence and the
cloning vector must be consistently cut into discrete and reproducible fragments.
Subjecting isolated chromosomal DNA either to passage through a small-bore needle or
to sonication produces double-stranded pieces of DNA that may range from 0.3 to 5 kb
in length. Unfortunately, these simple procedures induce breaks randomly, so each time
a DNA sample is treated, a different set of fragments is generated. It was only after
bacterial enzymes that cut DNA molecules internally at specific base-pair sequences
were discovered that molecular cloning became feasible. These enzymes are called type
II restriction endonucleases.

Figure 2. Symmetrical, staggered cleavage of a short fragment of DNA by the type ll restriction endonuclease
EcoRI. Cleavage of the intemucleotide bond is between the oxygen of the 3'-carbon of the sugar of one
nucleotide and the phosphate group attached to the 5'-carbon of the sugar of the adjacent nucleotide. The
symmetrical staggered cleavage of DNA by EcoRI produces two single-stranded, complementary cut ends,
each with extensions of fuur nucleotides. The arrows show the sites of cleavage in the DNA backbone.
S, deoxynbose sugar; P, phosphate group; OH, hydroxyl group: A, adenine; T, thymine; C, cytosine; G,
guanine. The EcoRI recognition sequence is encircled.

One of the first of these type II restriction endonucleases to be characterized was

from the bacterium Escherichia coli and it was designated EcoRI. This enzyme binds to
a DNA region with a specific palindromic sequence (the two strands are identical in this
region when either is read in the same polarity, i.e., 5' to 3') of 6 bp and cuts between the
guanine and adenine residues on each strand (Fig. 2). It specifically cleaves the
internucleotide bond between the oxygen of the 3'-carbon of the sugar of one nucleotide
and the phosphate group attached to the 5'-carbon of the sugar of the adjacent
nucleotide. The symmetrical staggered cleavage of DNA by EcoRI produces two single-
stranded, complementary cut ends, each with extensions of four nucleotides. In this
case, each single-stranded extension ends in a 5'-phosphate group, and the 3'-hydroxyl
group from the opposite strand is recessed.
In addition to EcoRI, hundreds of other type II restriction endonucleases have been
isolated from various bacteria. The naming protocol for these enzymes is the same as
that for EcoRI; the genus is the capitalized letter and the first two letters of the species
name are in lowercase letters. The strain designation is often omitted from the name and
28

Roman numerals are used to designate the order of characterization of different

restriction endonucleases from the same organism. For example, Hpal and Hpall are the
first and second type II restriction endonucleases that were isolated from Haemophilus
parainfluenzae. The palindromic sequences where most type II restriction
endonucleases bind and cut a DNA molecule are called recognition sites. Some
restriction endonucleases digest (cleave) DNA, leaving 5'-phosphate extensions
(protruding ends, sticky ends); some leave 3'-hydroxyl extensions; and some cut the
backbone of both strands within a recognition site to produce blunt-ended (flush-ended)
DNA molecules. The length of the recognition site for different enzymes can be four,
five, six, eight, or more nucleotide pairs. Because of the frequency with which their
recognition sites occur in DNA, restriction endonucleases that cleave within sites of
four (four-cutters) and six (six-cutters) nucleotide pairs are used for most of the
common experimental protocols for molecular cloning.
The importance of the type II restriction endonucleases for gene cloning cannot be
overstated. When a DNA sample is treated with one of these enzymes, the same set of
fragments is always produced, assuming that all of the recognition sites are cleaved.
Moreover, physical maps that designate the linear order ofrestriction endonuclease sites
on a specific piece of DNA can be constructed by treating the DNA molecule singly
with different restriction endonucleases and then with combinations of restriction
endonucleases. The positions of the cleavage sites can be deduced from an analysis of
fragment sizes, which are determined by agarose gel electrophoresis. After the wells of
a gel are loaded with sample, an electric field is applied across the gel and DNA
molecules migrate through the gel in the direction of the anode. The distance that a band
moves into a gel depends upon the size of the DNA molecules; the smaller molecules
travel further than the larger ones. A restriction map is formulated by comparing the
fragment sizes produced in each single digestion with those from double digestions.
Restriction endonuclease cleavage is also used in another way. When two different
DNA samples are digested with the same restriction endonuclease that produces a
staggered cut (the same 5' or 3' extension or sticky end) and then mixed together, new
DNA combinations can be formed as a result of base pairing between the extension
(overhang) regions (Fig. 3).
Restriction enzymes alone are not sufficient for molecular cloning. First, when the
extended ends that are created by restriction enzyme (e.g., Bamlll) cleavage are aligned,
the hydrogen bonds of the four bases that pair are not strong enough to keep two DNA
molecules together. A means of reforming the internucleotide linkage between the 3'-
hydroxyl group and the 5 '-phosphate group in the backbone at the two broken bond sites
(nicks) is required. This problem can be resolved by using the enzyme DNA ligase,
usually from bacteriophage T4. This enzyme catalyzes the formation ofphosphodiester
bonds at the ends of DNA strands that are already held together by the base pairing of
two extensions. It also joins blunt ends, albeit less efficiently than staggered ends, that
come in contact when they both bind to the enzyme.
Second, the ability to join different DNA molecules together would not by itself be
useful unless the new DNA combination (i.e., recombinant DNA) could be perpetuated
in a host cell. One of the DNA molecules must provide the biological information for
the cellular maintenance of the recombined DNA, and the other must contain the gene
that was targeted for cloning. This problem is solved by using cloning vectors.
 29

Third, digestion of the source DNA with a restriction endonuclease produces a

mixture of various DNA molecules and, after ligation with a cloning vector, a number
of different DNA constructs are formed. There has to be a way to identify the DNA
combination in a host cell that contains the target DNA sequence. Screening procedures
that detect host cells carrying a particular cloning vector-DNA insert construct solve this
problem.

Bam HI
=CCTAG
Bam HI

T4 DNA Ligase

Figure 3. Annealing complementary DNA extensions after staggered cleavage with the type II restriction
endonuclease Bamm. Two different DNA fragments are cut with Bamm, mixed and annealed. A break in the
phosphodiester bond in one strand of duplex DNA is called a nick. The hydrogen bonds of the four base pairs
between nicks on opposite strands are not sufficiently strong to hold DNA molecules together for long periods
in solution. The enzyme T4 DNA ligase is used to reform the phosphodiester bonds by joining 5'-phosphate
and 3'-hydroxyl groups at nicks in the backbone of the double-stranded DNA. A, C, T and G represent
nucleotides.

3. Plasmid Cloning Vectors

Plasmids are self-replicating, double-stranded, circular DNA molecules that are

maintained in bacteria as independent extrachromosomal entities. Virtually all bacterial
genera have plasmids. Some plasmids carry information for their own transfer from one
cell to another (F plasmids); others encode resistance to antibiotics (R plasmids); others
carry specific sets of genes for the utilization of unusual metabolites (degradative
plasmids); and some have no apparent functional coding genes ('cryptic' plasmids).
Plasmids can range in size from less than 1 to more than 500 kb. Each plasmid has a
sequence that functions as an origin of DNA replication; without this site, it cannot
replicate in a host cell.
Some plasm ids are represented by 10 to 100 copies per host cell; these are
designated as high-copy-number plasmids. Others maintain 1 to 4 copies per cell and
are called low-copy-number plasmids. Seldom does the population of plasmids in a
bacterium make up more than approximately 0.1 to 5.0% of the total DNA. When two
30

or more types of plasmids cannot coexist in the same host cell, they are said to belong to
a single incompatibility group. But plasmids from different incompatibility groups can
be maintained together in the same cell. This coexistence is independent of the copy
numbers of the individual plasmids. Some microorganisms have been found to contain
as many as 8 to l 0 different plasmids. In these instances, each plasmid can carry out
different functions and have its own unique copy number, and each belongs to a
different incompatibility group. Some plasmids, because of the specificity of their origin
of replication, can only replicate in one specific species of host cell. Other plasmids
have less specific origins of replication and can replicate in a number of bacterial
species. These plasmids are called narrow- and broad-host-range plasmids, respectively.
As autonomous, self-replicating genetic elements, plasmids have the basic attributes
to make them potential vectors for carrying cloned DNA. However, naturally occurring
(unmodified, unengineered) plasmids often lack several important features that are
required for a high-quality cloning vector. These features are (1) small size, which is
necessary because the efficiency of transfer of exogenous (foreign) DNA into E. coli
decreases significantly with plasmids that are greater than 15 kb; (2) unique (single)
restriction endonuclease recognition sites into which the insert DNA can be cloned; and
(3) one or more selectable genetic markers for identifying recipient cells that carry the
cloning vector-insert DNA construct. Consequently, plasmid cloning vectors have to be
genetically engineered.

4. Cteating and Sereening a Library

4.1. MAKING A GENE LffiRARY

The isolation of genes that encode proteins is often the goal of a biotechnology
experiment. In prokaryotic organisms, these structural genes each have a continuous
coding domain in the genomic DNA; in eukaryotes, however, the coding regions
(exons) of structural genes are separated by noncoding regions (introns). Consequently,
different cloning strategies have to be used for prokaryotic and eukaryotic genes.
In a prokaryote, the desired sequence (target DNA) is frequently a minuscule portion
(0.02%) of the total chromosomal DNA. The problem, then, is how to clone and select
the targeted DNA sequence. To do this, the complete DNA of an organism is cut with a
restriction endonuclease and each fragment is inserted into a vector. Then, the specific
cell line (clone) that carries the target DNA sequence must be identified, isolated,
subcultured, and characterized. This process of subdividing genomic DNA into clonable
elements and inserting them into host cells is called creating a library, a clone bank, or a
gene bank. A complete library of host cells, by definition, contains all of the genomic
DNA of the source organism.
One way to create a DNA library is by treating the DNA from a source organism
with a four-cutter restriction endonuclease (e.g., Sau3AI), which, theoretically, cleaves
the DNA approximately once in every 256 base pairs. The conditions of the digestion
reaction are set to give a partial, not a complete, digestion. In this way, all possible
fragment sizes are generated (Fig. 4). However, because restriction endonuclease sites
are not randomly located, some fragments may be too large to be cloned. In these cases,
 31

an incomplete library is available for selection, so it may be difficult, or even

impossible, to find a specific target DNA sequence.

A
B B B B

8
Increasing timed digestion -----lilt~

--
Kb
23 Direction of
migration
9.8
6.E

4.0

2.3
2.0

Figure 4. (A) Location of restriction endonuclease Bamm (labeled B) and restriction

endonuclease Sau3Al recognition sites on a DNA fragment. Bamm cleaves the DNA at the
sequence GGATCC leaving a GATC 5' extension. Sau3Al cleaves DNA at GATC
sequences, also leaving a GATC 5' extension. Because Sau3Al needs only fuur, rather than
six, specific nucleotides to bind and cleave the DNA, there are many more Sau3Al sites than
Bamm sites. (B) Representation of an agarose gel fullowing separation by electrophoresis of
partial restriction endonuclease digestions of the DNA shown in (A) by Sau3Al. By
increasing the time of digestion, more cleavages occur and more smaller fragments are
generated. The horizontal lines under each of the digestion conditions represent
schematically the locations of the DNA fragments (bands) in the lanes of the gel after
electrophoresis and staining of the DNA with ethidium bromide.

After a library is created, the clone(s) (cell lines) with the target sequence must be
identified. Four popular methods of identification are used: DNA hybridization with a
labeled DNA probe, followed by radiographic screening for the probe label;
immunological screening for the protein product; screening for protein activity, and
screening by mutant complementation.
32

4.2. SCREENING BY DNA HYBRIDIZATION

The presence of a target nucleotide sequence in a DNA sample can be determined with
a DNA probe. This procedure is called DNA hybridization and depends on the
formation of stable base pairs between the probe and the target sequence. DNA
hybridization is feasible because double-stranded DNA can be converted into single-
stranded DNA by heat or alkali treatment. Heating DNA breaks the hydrogen bonds that
hold the bases together (denaturation) but does not affect the phosphodiester bonds of
the DNA backbone. If the heated solution is rapidly cooled, the strands remain single-
stranded. If the temperature of a heated DNA solution is lowered slowly, however, the
double-stranded, helical conformation of DNA can be re-established due to the base
pairing of complementary nucleotides (renaturation). The process of heating and slowly
cooling double-stranded DNA is called annealing. Some of the products of this process
contain molecules of hybrid DNA, that is, double-stranded DNA in which the two
strands come from different DNA molecules.
In general, for a DNA hybridization assay, the target DNA is denatured and the
single strands are irreversibly bound to a matrix (e.g., nitrocellulose or nylon). This
binding process is often carried out at a high temperature. Then, the DNA probe, which
is labeled with either a radioisotope or another tagging system, is incubated with the
bound DNA sample. If the sequence of nucleotides in the DNA probe is complementary
to a nucleotide sequence in the sample, then base pairing (i.e., hybridization) occurs
(Fig. 5). The hybridization can be detected by autoradiography or other visualization
procedures, depending on the nature of the probe label. If the nucleotide sequence of the
probe does not base pair with a DNA sequence in the sample, then no hybridization
occurs and the assay gives a negative result. Generally, probes range in length from 100
to more than 1,000 bp, although both larger and smaller probes can be used. Depending
on the conditions of the hybridization reaction, stable base pairing requires a greater
than 80% match within a segment of 50 bases.
DNA hybridization may also be used to ascertain that a particular organism contains
a DNA sequence that is homologous to the hybridization probe. This is done by
digesting the isolated target cell chromosomal DNA with a restriction enzyme. The
resultant fragments are separated on an agarose gel and then transferred to a membrane
by capillary action. The transfer from the gel and the subsequent localization of the
DNA fragments of interest, i.e., those that bind to the probe, is called Southern
hybridization. When this procedure is used to characterize RNA rather than DNA, the
procedure is called northern hybridization and this procedure may be used for analysis
of gene expression.
There are at least two possible sources of probes for screening a genomic library.
First, cloned DNA from a closely related organism can be used (a heterologous probe).
In this case, the conditions of the hybridization reaction can be adjusted to permit
considerable mismatch between the probe and the target DNA to compensate for the
natural differences between the two sequences. Second, a probe can be produced by
chemical synthesis. The nucleotide sequence of a synthetic probe is based on the
probable nucleotide sequence that is deduced from the known amino acid sequence of
the protein encoded by the target gene.
Genomic DNA libraries are often screened by plating out the transformed cells on
the growth medium of a master plate and then transferring samples of each colony to a
 33

solid matrix such as a nitrocellulose or nylon membrane, lysing (breaking) the cells,
deproteinizing and denaturing the DNA, and binding the DNA to the matrix. At this
stage, a labeled probe is added; if hybridization occurs, signals are observed on an
autoradiograph. The colonies from the master plate that correspond to samples
containing hybridized DNA are then isolated and cultured. Because most libraries are
created from partial digestions, a number of colonies (clones) may give a positive
response to the probe. The next task is to determine which clone, if any, contains the
complete sequence of the target gene. Preliminary analyses that use the results of gel
electrophoresis and restriction endonuclease mapping reveal the length of each insert
and identity those inserts that are the same and those that share overlapping sequences.
By using overlapping sequences, it may be possible to join sections of the gene in
additional cloning experiments. Alternatively, if an insert in any one of the clones is
large enough to include the full gene, then the complete gene can be recognized after
DNA sequencing, because it will have start and stop codons and a contiguous set of
nucleotides that code for the target protein.

l
Double-stranded
1111111111111111111111111111111111111111111111 target DNA

Denaturation and
binding to membrane

1111111111111111111111111111111111111111111111

Labeled
single-stranded
Hybridization probed

1111111111111111111~11ff1111111111111111 Labeled probe

hybridized to
target DNA

Figure. 5. DNA hybridization. The double-stranded target DNA is denatured and the two strands
are kept apart, usually by binding them to a solid matrix such as a nitrocellulose or nylon
membrane. Labeled probe DNA (approximately 100 to 1000 bp) is denatured and the single-
stranded labeled probe is added to the denatured target DNA Hybridization (base pairing)
between the probe and target DNA may occur under these conditions. The membrane is then
washed to remove unhybridized probe DNA, and the membranes are assayed. If the probe
hybridizes with the target DNA, then it can be detected with an assay that identifies its labeled
tag. If the probe does not hybridize, then no label is detected. The symbol (o) denotes the labeled
tag (signal) of the probe DNA
34

Unfortunately, there is no guarantee that the complete sequence of a target gene will
be present in a particular library. If the search for an intact gene fails, then another
library can be created with a different restriction endonuclease and screened with either
the original probe or probes derived from the first library. Alternatively, libraries can be
created that contain DNA fragments larger than the average prokaryotic gene to increase
the chance that some members of the library will carry a complete version of the target
gene.

4.3. SCREENING BY IMMUNOLOGICAL ASSAY

If a DNA probe is not available, alternative methods can be used to screen a library. For
example, if a cloned DNA sequence is transcribed and translated, the presence of the
protein, or even part of it, can be determined by an immunological assay. Technically,
this procedure has much in common with a DNA hybridization assay. All cell lines
(clones) of the library are grown on master plates. A sample of each colony is
transferred to a matrix, where the cells are lysed and the released proteins attached to
the matrix. The matrix with the bound proteins is treated with an antibody (primary
antibody) that specifically binds to the protein encoded by the target gene. Following
the interaction of the primary antibody with the target protein (antigen), any unbound
antibody is washed away, and the matrix is treated with a second antibody that is
specific for the primary antibody. In many assay systems, the secondary antibody has an
enzyme, such as alkaline phosphatase, attached to it. After washing the matrix, a
colorless substrate is added. If the secondary antibody has bound to the primary
antibody, the colorless substrate is hydrolyzed by the attached enzyme and produces a
colored compound that accumulates at the site of the reaction.
The colonies on the master plate that correspond to positive results (colored spots)
on the matrix contain either an intact gene or a portion of the gene that is large enough
to produce a protein product that is recognized by the primary antibody. After detection
by immunoassay of genomic DNA libraries, the positive clones must be characterized
further to determine which, if any, carry a complete gene.
Immunological assays may also be used to characterize expression at the protein
level. When proteins are first separated by polyacrylamide gel electrophoresis and then
transferred to a membrane prior to interaction with a primary antibody the procedure is
called western hybridization. To localize expression within the cell, RNA and protein
hybridization experiments may be performed in situ.

4.4. SCREENING BY PROTEIN ACTIVITY

If the target gene produces an enzyme that is not normally made by the host cell, a plate
assay can be devised to identify members of a library that carry the functional gene
encoding that enzyme. For example, the genes for a-amylase, endoglucanase, and
~glucosidase from various organisms have been isolated by plating the genomic library
in E. coli onto medium supplemented with a specific substrate and then using a selective
stain to identifY those colonies that are capable of utilizing the substrate.
 35

4.5 SCREENING BY MUTANT COMPLEMENTATION

A bacterium that has been mutated and can no longer perform a specific function may
be used as a host cell as part of a strategy to isolate the wild-type gene that encodes the
defective protein (Fig. 6). For example, E. coli can normally synthesize a sufficient
amount of the amino acid histidine to fulfill its own needs. Consequently, wild-type E.
coli cells are able to grow on minimal medium to which histidine has not been added.
On the other hand, either spontaneous or induced E. coli mutants that are no longer able
to grow on minimal medium without histidine may be readily isolated-these mutants
are said to be His-. To clone the wild-type (functional) version of the gene that encodes
the protein altered in the mutant the following steps may be undertaken-this gene
presumably encodes a protein that participates in the biosynthesis of histidine.

i) Construct a gene library of wild-type E. coli DNA

ii) Introduce the library into the E. coli His- mutant
iii) Plate the transformed cells onto minimal medium that does not contain any histidine,
selecting all clones that are able to grow
iv) Isolate the plasmid DNA from all selected clones and characterize

Screening by mutant complementation enables isolation of genes involved in a

function or pathway without prior knowledge of the genes being sought. This approach
is useful in isolating genes whose protein products are a part of complex systems like
nitrogen fixation or antibiotic biosynthesis involving the participation of many genes.

5. Cloning DNA Sequences That Encode Eukaryotic Proteins

Special techniques are required for cloning eukaryotic structural genes. Prokaryotic
hosts cannot remove the introns from transcribed RNA molecules; therefore, this
messenger (m) RNA is not translated correctly in a bacterial host cell. Moreover, a
eukaryotic DNA sequence needs prokaryotic transcriptional and translational control
sequences to be properly expressed. In a functional eukaryotic messenger RNA, which
does not have introns, there is a G cap at the 5' end and, usually, a string of up to 200
adenine residues (poly(A) tail) at the 3' end.
The poly(A) tail can be used to separate the mRNA fraction of a tissue from the
ribosomal and transfer RNAs (rRNA and tRNA, respectively). Extracted cellular
eukaryotic RNA is passed through a column packed with cellulose beads to which are
bound short chains of thymidine residues, each about 15 nucleotides in length (oligo-
dT; dT 15). The poly(A) tails of the messenger RNA molecules bind by base pairing to
the oligo-dT chains. The tRNA and rRNA molecules, which lack poly(A) tails, pass
through the column. The mRNA is removed (eluted) from the column by treatment with
a buffer that breaks the A:T hydrogen bonds, thereby releasing the bound mRNAs.
Before the mRNA molecules can he cloned into a vector, they must be converted to
double-stranded DNA. This is accomplished by using, in succession, two different kinds
of nucleic acid polymerases: reverse transcriptase and the Klenow fragment of DNA
polymerase I (Fig. 7). After the mRNA fraction is purified, short (unbound) sequences
36

of oligo-dT molecules are added to the sample, along with the enzyme reverse
transcriptase and the four deoxyribonucleotides (dATP, dTTP, dGTP, dCTP). The
oligo-dT molecules base pair with the poly(A) tail regions and provide an available 3 '-
hydroxyl group to prime the synthesis of a DNA strand.

~:~poDNAwHh
~-··

r ~0··-
Q ::::.::::-..

Host eel tran~fOJmed with

co,....ment;,g goot

Figure 6. Mutant complementation. A gene library is constructed of wild-type DNA that

contains the normal target gene. The library is transferred to a host cell, with a mutant
form of the target gene, which is unable to grow on minimal medium. The transformed
cells that carry the normal gene are able to grow on minimal medium; these transformants
are identified and grown.

Reverse transcriptase, which is produced by certain RNA viruses, uses an RNA

strand as a template while directing deoxyribonucleotides into the growing chain. Thus,
when an A, G, C, or U residue of the template RNA strand is encountered, the
complementary deoxyribonucleotide (i.e., T, C, G, or A) is incorporated into the
growing DNA strand. Before synthesis ceases, the DNA strand usually turns back on
itself for a few nucleotides (Fig. 7), to form a hairpin loop.
The second DNA strand is synthesized by the addition of the Klenow fragment of E.
coli DNA polymerase I, which uses the first DNA strand as a template and adds
deoxyribonucleotides to the growing strand, starting from the end of the hairpin loop.
After the reaction is complete, the sample is treated with the enzyme RNase H, which
degrades the mRNA molecules, and with S 1 nuclease, which opens the hairpin loops
 37

and degrades single-stranded DNA extensions. At the end of this procedure, the sample
contains a mixture of partial and complete double-stranded complementary DNA
(eDNA) copies of the more prevalent mRNAs in the original sample.

mRNA

~-"'~~-------------~

l==-
G-----------~

rm

l
~--------------------~
-----------rrrr
l<leooNfr11lJ118111
and lou" dNTPs

~-----------~
CI II II 11111111111111111111 II II II II I rrrr

l:=
111111111111111111111111111 II IIIII rrrr
OoiJlle s1randad eDNA _..-/

Figure 7. Synthesis of eDNA Oligo (dT) primer is added to a purified mRNA preparation, and reverse
transcriptase with the four dNTPs is used for the production of DNA from the RNA template. The looping
back of the growing DNA strand produced by the reverse transcriptase provides a 3'-hydroxyl group for the
Klenow ftagment to complete the synthesis of the second strand, using the initial eDNA strand as a template.
After synthesis of the second strand is completed, the mRNA is hydrolyzed with the enzyme RNase H and the
DNA is treated with S I nuclease to produce blunt-ended linear molecules without hairpin loops.

This eDNA population can be cloned by blunt-end ligation or other joining

mechanisms into a plasmid cloning vector to form a eDNA library. A eDNA library can
be screened by either DNA hybridization or immunological assays, as explained earlier,
to identify clones that carry a specific plasmid-eDNA construct. For immunological
screening, the eDNA must be cloned into a site that puts the cloned sequence under the
control of a bacterial promoter, which ensures its transcription. However, with most
cloning vectors, there is no guarantee that, after insertion of the eDNA, its nucleotides
will have the correct reading frame and therefore be able to direct the synthesis of the
true amino acid sequence of the protein. Nevertheless, positive clones that are isolated
by either screening method must be examined further to determine which one(s) carries
the complete coding sequence for the target protein.
38

6. Genetic Transformation of Prokaryotes

6.1. TRANSFERRING DNA INTO E. coli

Transformation is the process of introducing free DNA into a bacterial cell. For E. coli,
which is the main host cell for recombinant DNA research, the uptake of plasmid DNA
is usually achieved by treating cells with ice-cold CaCh, followed by a brief exposure to
high temperature. This method has a maximum transformation frequency, which is
defined as the fraction of the cell population that can be transformed, of about I
transformed cell per 1,000 cells (10-3). For this procedure, the transformation efficiency,
which is defined as the number of transformants per microgram of added DNA, is
approximately 107 to 108 transformed colonies per microgram of intact plasmid DNA.
Although a 100% transformation frequency would be ideal, selection schemes that
enable plasmid-transformed cells to be readily identified overcome the drawback of a
low transformation frequency.
The mechanism of action of the CaCh-heat transformation procedure in E. coli is
not known. It is assumed that the bacterial cell wall is broken down in localized regions,
a condition that allows the plasmid DNA in solution to be taken into the interior of the
bacterial cell. Bacterial cells that are able to take up DNA are said to be competent. For
E. coli, competence must be induced. In some other bacteria, it occurs naturally. For
bacteria that are refractory (resistant) to chemically induced competence or are not
naturally competent, other DNA delivery systems must be used.

6.2. ELECTROPORATION

Uptake of free DNA can be induced by a procedure called electroporation by subjecting

bacteria to a high-strength electric field in the presence of DNA. This treatment yields
transformation efficiencies for small plasmids of 109 transformants per microgram of
DNA, and 106 for large plasm ids. Thus, electroporation is an effective way to transform
E. coli with plasmids with inserts that are> 100 kb.
Very little is known about the mechanism of DNA uptake during electroporation. It
is thought that transient pores are formed in the cell wall as a result of the electroshock
and that, after contact with the cell membrane, the DNA is taken into the cell.

6.3. CONJUGATION

For some bacteria, the natural transmission of plasmid DNA from a donor cell to a
recipient cell has been used to transfer plasmid-insert DNA constructs to a host cell that
is not readily transformed. Some plasmids are genetically equipped to form cell-to-cell
junctions through which plasmid DNA is transferred from one cell to another. Effective
contact between a donor and recipient cell is due to conjugative functions; and the
mechanical transfer of the DNA is the consequence of mobilizing functions. Most of the
plasmids that are used for recombinant DNA research lack conjugative functions and
therefore the DNA of these plasmids cannot be passed to recipient cells by conjugation.
However, some plasmid cloning vectors can be mobilized and transferred if the
conjugative functions are supplied by a second plasmid in the same cell. Thus, by
introducing a plasmid with conjugative functions into a bacterial cell that carries a
 39

mobilizable plasmid cloning vector, it is possible to transfer the plasmid cloning vector
to a recipient cell that is difficult to transform by other means.
The standard experimental protocol for this procedure entails mixing three strains
together (Fig. 8). When the cells are in close proximity, the conjugative plasmid, which
in this case is also mobilizable, can be self-transferred to the cell with the mobilizable
plasmid cloning vector. Then, with the help of the conjugative plasmid, the plasmid
cloning vector is transferred to a targeted recipient cell. All possible combinations of
plasmid transfer occur among the cells, but the genetic features of the strains and
plasmids are designed to select for the targeted recipient cells that receive the cloning
vector. Because the transfer of plasmid DNA requires conjugation among three bacterial
strains, this procedure has been designated tripartite mating.

E. coli E. coli P. putida

0TelA

0 KanR

Helper Donor Recipient

Figure 8. Transferring plasmids from one bacterium to another by conjugation (tripartite mating
procedure). The conjugative plasmid that carries a tetracycline resistance gene (Tef) is self-
transferred from the helper E. coli cell to the E. coli donor cell. The mobilizable plasmid cloning
vector that bears a kanamycin resistance gene (KanR) is transferred, with the help of the conjugative
plasmid, to the P. putida recipient cell. All possible combinations of plasmid transfer occur among
the cells but only the targeted recipient cells that have acquired the plasmid cloning vector can grow
in minimal medium that contains kanamycin. If the targeted recipient cell receives both types of
plasmid this rare event may be detected by replica plating onto minimal medium and selecting those
transconjugants that can grow in the presence of kanamycin but not in the presence of tetracycline.

7. Chemically Synthesized DNA

The ability to chemically synthesize DNA with a specific sequence of nucleotides

easily, inexpensively, and rapidly has contributed to the methodologies ofDNA cloning
and characterization. Readily available, single-stranded DNA oligonucleotides may be
used to assemble whole genes or gene fragments, to amplify specific DNA fragments, to
introduce specific mutations into isolated DNAs, as DNA hybridization probes, as
adjuncts to gene sequencing, or as linkers to facilitate gene cloning.
Machines that automate the chemical reactions involved in DNA synthesis have
made the synthesis of single-stranded oligonucleotides into a routine procedure.
40

Generally, DNA synthesizers consist of a set of valves and pumps that are programmed
to introduce, in the correct order, specified nucleotides and the reagents required for the
coupling of each consecutive nucleotide to the growing chain. Chemical DNA synthesis
does not follow the biological direction of DNA synthesis; rather, in the chemical
process each incoming nucleotide is coupled to the 5'-hydroxyl terminus of the growing
chain. All the reactions are carried out in succession in a single reaction column, and
both the duration of each reaction and the washing steps are computer-controlled.

8. Polymerase Chain Reaction

The polymerase chain reaction (PCR) is an effective procedure for generating large
quantities of a specific DNA sequence in vitro. This amplification, which can be more
than a millionfold, is achieved by a three-step cycling process. The essential
requirements for PCR are i) two synthetic oligonucleotide primers (-20 nucleotides
each) that are complementary to regions on opposite strands that flank the target DNA
sequence and that, after annealing to the source DNA, have their 3'-hydroxyl ends
oriented toward each other; ii) a target sequence in a DNA sample that lies between the
pair of primers which can be from 100 to -35,000 bp in length; iii) a thermostable DNA
polymerase that can withstand heating to 95°C or higher; and iv) the four
deoxyribonucleotides.
A typical PCR process entails a number of cycles for amplifying a specific DNA
sequence; each cycle has three successive steps.

i) Denaturation. The first step in the PCR amplification system is the thermal
denaturation of the DNA sample by raising the temperature within a reaction tube to
95°C. In addition to the source DNA, this reaction tube contains a vast molar excess
of the two oligonucleotide primers, a thermostable DNA polymerase (e.g., Taq DNA
polymerase, isolated from the bacterium Thermus aquaticus), and four
deoxyribonucleotides. The temperature is maintained for about one min.
ii) Renaturation. For the second step, the temperature of the mixture is slowly cooled to
-55°C. During this step, the primers base pair with their complementary sequences
in the source DNA.
iii) Synthesis. In the third step, the temperature is raised to -75°C, which is optimum for
the catalytic functioning of Taq DNA polymerase. DNA synthesis is initiated at the
3'-hydroxyl end of each primer (Fig. 9).

The duration of each step and the temperature changes required during a PCR cycle
are usually carried out in an automated, programmable block heater in which the
reaction tubes are immersed. One cycle generally lasts from 3 to 5 min.
To understand how the PCR protocol succeeds in amplifying a discrete segment of
DNA, it is important to keep in mind the location of each primer sequence and its
complementary sequence within the strands that are synthesized during each cycle.
During the synthesis phase of the first cycle, the new DNA attached to each primer is
extended beyond the end point of the sequence that is complementary to the second
primer. These new strands form 'long templates' that will be used in the second cycle.
 41

During the second cycle, the original DNA strands and the new strands synthesized
in the first cycle (long templates) are denatured again and then hybridized with primer
sequences. A second round of synthesis again produces long templates and also some
DNA strands that have a primer sequence at one end and a sequence complementary to
the other primer at the other end ('short templates').

Target DNA

11111111111111!11111!11111111111111111111
/

P1
1 1. Denature double
stranded DNA
2.Addprimars
P1 andP2

1
P2

Taq DNA polymerase

+fourdNTPs

P1
-------------------------·
·----------~---------~
NMy synlhesized DNA

Figure 9. First cycle of the PCR. The PCR contains the target DNA, two primers (PI and P2), Taq
DNA polymerase and the four deoxyribonucleotides, i.e., deoxyribonucleoside triphosphates
(dNTPs). The mixture is heated to 95"C to denature the target DNA and then slowly lowered to
55 "C. The primers, which are present in excess, base pair to the original DNA strand of the sample
during the renaturation step. The temperature is raised to approximately 75"C, and DNA synthesis
commences from the 3'-hydroxyl ends of the primer sequences and continues past the regions of the
DNA strands that are complementary to the other primer sequence. The products of the reaction are
long strands of DNA which will serve as DNA templates in the second cycle ofthe PCR.

During the third cycle, short templates, long templates and original strands all
hybridize with primer sequences and are replicated. In subsequent cycles, the short
templates preferentially accumulate, and by the thirtieth cycle these strands are about a
million times more frequent than either the original or long template strands.
The PCR has become a pervasive technique. One important PCR application is the
identification of a pathogenic organism that is associated with a disease in humans,
animals or plants. In these cases, purification of the target DNA is unnecessary and,
often, the detection assay can be carried out with small, crude samples. The nucleotide
sequence of the possible pathogenic organism must be known so that a pair of primers
42

that anneal exclusively to sites within the targeted DNA can be synthesized. After PCR
cycling, a DNA fragment of a specific size that is equivalent to the lengths of the two
primers plus the length of the DNA between them will be amplified only if the target
DNA is present in the sample.
PCR protocols have also been devised to detect naturally occurring mutations, to
produce specific mutations in vitro, to assemble whole genes from synthetic
oligonucleotides and for DNA sequencing. For many applications, it is frequently
helpful to clone the PCR product.

9. Expression of Cloned Genes

The primary objective of gene cloning for biotechnological applications is the

expression of the cloned gene in a selected host organism. Unfortunately, the insertion
of a gene into a cloning vector does not necessarily ensure that it will be successfully
expressed. The molecular biological features that have been manipulated to modulate
gene expression include i) the nature of the relevant transcriptional promoter and
terminator sequences; ii) the number of copies of the cloned gene; iii) the final cellular
location of the synthesized foreign protein; and iv) the intrinsic stability of the cloned
gene protein within the host cell.
An effective gene expression system often includes the presence of a strong and
regulatable promoter sequence upstream from a cloned gene. A strong promoter is one
that has a high affinity for RNA polymerase, with the consequence that the adjacent
downstream region is highly (frequently) transcribed. The ability to regulate a promoter
enables the cell (and the researcher) to control the degree/extent oftranscription in a
precise manner.
It might seem that a good way to optimize the expression of a cloned gene would be
to express it under the control of a continuously transcribed strong promoter. However,
a high level of continual expression of a cloned gene is often detrimental to the host
cell, because it creates an energy drain, thereby impairing essential host cell functions.
To overcome this drawback, it is desirable to control transcription in such a way that a
cloned gene is expressed only at a specific stage in the host cell growth cycle and only
for a specified duration or, in plants, only in certain tissues.

10. Eukaryotic Expression Systems

Prokaryotic expression systems are generally useful for producing heterologous

(recombinant) proteins from cloned eukaryotic cDNAs. In some cases, however,
eukaryotic proteins that have been synthesized in bacteria are either unstable or lack
biological activity. In addition, despite careful purification procedures, bacterial
compounds that are toxic or that cause a rise in body temperature in humans and
animals (pyrogens) may contaminate the final product. To avoid these problems,
investigators have developed eukaryotic expression systems for the production of
proteins that can be used as therapeutic agents in either humans or animals. A human
protein intended for medical use is generally required to be identical to the natural
protein in its biochemical, biophysical, and functional properties. The inability of
 43

prokaryotic organisms to produce authentic versions of proteins is, for the most part,
due to the absence of appropriate mechanisms for generating certain post-translational
modifications.
In eukaryotes there are a number of modifications that may occur at the post-
translational stage, after protein synthesis is complete:

i) Correct disulfide bond formation. This reaction is mediated by an enzyme called

disulfide isomerase. An improperly folded protein is unstable and lacks activity.
ii) Proteolytic cleavage of a precursor form. Selected segments of amino acid
sequences are removed to yield a functional protein.
iii) Glycosylation. This reaction is a major modification that endows a protein with
stability and, in some instances, its distinctive properties. The most common protein
glycosylations occur by the addition of specific sugar residues to serine or threonine
(i.e., 0-linked glycosylation) or to asparagine (i.e., N-linked glycosylation).
iv) Additions to amino acids within proteins. Modifications of this type include
phosphorylation, acetylation, sulfation, acylation, y-carboxylation, myristylation,
and palmitylation.

Of these modifications, prokaryotic host cells are least likely to carry out either
proper glycosylation or additions to specific amino acids within the heterologous
protein. Moreover, no single eukaryotic host cell system is capable of performing all of
the possible post-translational modifications for every potential heterologous protein.

11. Modification of Expressed Proteins

In addition to isolating natural genes that encode useful properties, conventional

mutagenesis and selection schemes can be used in an attempt to create and perpetuate a
mutant form of a gene that encodes a protein with the desired properties. However, the
number of mutant protein~ach with a different amino acid change-that are possible
following the alteration of individual nucleotides within a structural gene by
conventional mutagenesis is extremely large. In practice, the mutagenesis-selection
strategy rarely results in any significant beneficial changes to the targeted protein
because most amino acid changes decrease the activity of an enzyme.
It is not a simple matter to produce a new protein with specified predetermined
properties. However, it is quite feasible to modify the existing properties of known
proteins. Unfortunately, it is not always possible to know in advance which individual
amino acids or short sequences of amino acids contribute to a particular physical,
kinetic, or chemical property. For example, a particular property of a protein may be the
consequence of two or more amino acids residues that are far apart from each other in
the linear sequence but are juxtaposed as a result of the folding of the protein. In this
case, two or more amino acid residues may have to be changed to produce a protein
with the desired properties. In the not too distant future, computer programs may be able
to make accurate predictions of protein function on the basis of deduced amino acid
sequences thereby simplifying the task of producing a protein with specific
predetermined properties. At present, although it is relatively straightforward to
44

introduce new coding information into cloned genes, large numbers of novel proteins
must be assayed to determine whether or not a particular property has been created.
The process for generating amino acid coding changes at the DNA level is called
directed mutagenesis. Determining which amino acids of a protein should be changed to
attain a specific property is easier if the three-dimensional structure of the protein has
been well characterized by X-ray crystallographic analysis and other analytical
procedures. But for most proteins, such detailed information is lacking, so directed
mutagenesis becomes a ''trial and error" strategy in which changes are made to those
nucleotides that are most likely to yield a particular change in a protein property. And
then, of course, the protein encoded by each mutated gene has to be tested to ascertain
whether the mutagenesis process has indeed generated the desired change.
A large number of experimental approaches have been devised for the directed
mutagenesis of cloned genes. Some of the methods are designed to mutate specific
nucleotides of a cloned gene. Others introduce mutations randomly over a short segment
of the cloned gene, thereby creating a panel of mutated proteins among which one may
have the desired activity.

12. Genetic Manipulation of Plants

Considerable effort has gone into developing varieties of plants that produce increased
yields and have enhanced nutritional value. Although much of this endeavor has been
directed toward the three major grains-com, wheat, and rice-successful breeding
programs for other food plants and horticultural species have also been established.
Recombinant DNA technology, which has been used extensively with microbial
systems, is also an important tool for the direct genetic manipulation of plants. There are
a number of effective DNA-delivery systems and expression vectors that work in a
range of plant cells. Furthermore, most plant cells are totipotent-meaning that an entire
plant can be regenerated from a single plant cell-so fertile plants that carry an
introduced gene(s) in all cells (i.e., transgenic plants) can often be produced from
genetically engineered cells. If the transgenic plant flowers and produces viable seed,
the desired trait is passed on to successive generations.
There are three major reasons for developing transgenic plants. First, the addition of
a gene(s) often improves the agricultural, horticultural, or ornamental value of a crop
plant. Second, transgenic plants can act as living bioreactors for the inexpensive
production of economically important proteins or metabolites. Third, plant genetic
transformation (transgenesis) provides a powerful means for studying the action of
genes during development and other biological processes.
Some of the genetically determined traits that can be introduced into plants by a
single gene or, possibly, a small cluster of genes include insecticidal activity, protection
against viral infection, resistance to herbicides, delay of senescence, tolerance to
environmental stresses, altered flower pigmentation, improved nutritional quality, and
self-incompatibility. To date, numerous transgenic plants have been generated,
including many crop and forest species. In the future plant biotechnology will not only
have an enormous impact on traditional plant breeding programs because it can
significantly decrease the 10 to 15 years that it currently takes to develop a new variety
 45

using traditional plant breeding techniques, but it will also be used to create plants with
novel characteristics.
When genetic transformation of plants became routine, research efforts were
directed towards introducing a wide range of plant and bacterial genes in plant cells.
The transformed plants were assayed for the production of the foreign protein and
studied physiologically to assess how the presence of an additional protein affected the
whole plant. Many of these early experiments utilized promoters that were expressed
constitutively in a range of plant cells. More recently, additional plant promoters have
been isolated and characterized and used to express foreign proteins in specific cells at
certain times during the growth and development of the plant. For example, instead of
the strong constitutive 35S promoter from cauliflower mosaic virus which is expressed
in all plant tissues and throughout the life of the plant, researchers have used the
promoter for the small subunit of the photosynthetic enzyme ribulosebisphosphate
carboxylase which is active only in photosynthetic tissues such as leaves. Similarly,
plant promoters active only in a specific tissue such as roots or flowers, or only during
periods of environmental stress, e.g. the PR or pathogenesis-related promoters, have
been used to control the expression of some foreign genes.
The presence of antisense RNA or unnaturally high levels of sense RNA leads to a
decrease in the synthesis of the normal gene product. Theoretically, it is possible to
prevent the synthesis of any plant protein using these antisense or sense-suppression
approaches.

13. Recommended Textbooks

Glick, B.R. and Pasternak, J.J. (1998) Molecular Biotechnology: Principles and Applications ofRecombinant
DNA, American Society for Microbiology Press, Washington, D.C.
Glick, B.R. and Thompson, J.E. (1993) Methods in Plant Molecular Biology and Biotechnology, CRC Press,
Boca Raton, FL.
Greene, J.J. and Rao, V.B (1998) Recombinant DNA Principles and Methodologies, Marcel Dekker, Inc., NY.
Lindsey, K. (1998) Transgenic Plant Research, Harwood Academic Publishers, Amsteldijk.
Maliga, P., Klessig, D.F., Cashmore, A.R., Gruissem, W. and Varner, J.E. (1995) Methods in Plant Molecular
Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Sambrook, J. and Russell, D. (2001) Molecular Cloning: A Laboratory Manual, 3nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY.
BREEDING METHODS AND BREEDING RESEARCH

W.HORN
Lehrstuhl fiir Zierpjlanzenbau, Techn. Universitiit Miinchen
D-85350 Freising- Weihenstephan

1. Introduction

In a broad sense, the term "ornamental plants" covers all kinds of plants used for one
ornamental purpose or another in homes, gardens and parks. Ornamental plant breeding,
therefore, covers the breeding of all ornamental plants in the broad sense. On the other
hand, floriculture includes mostly herbaceous ornamental plant species, i.e., bedding
plants, cut flowers, pot plants (annuals, biennials, herbaceous perennials) which, during
their production period in nurseries, are kept in greenhouses or under temporary
protection. In this chapter on ornamental plant breeding, only floriculture crops are
addressed. Ornamental shrubs and trees are not considered, though quite a number of
them have been improved by selection after hybridization and/or polyploidization.
Ornamental plant production has developed rapidly, and still has a high growth potential
for future expansion. In view of the worldwide rising economic importance of
ornamental horticulture, ornamental plant breeding has a promising future.

l.l. FLORICULTURE-A GENERAL REVIEW

Regarding the history of floriculture, certainly, early man appreciated gaily colored
wildflowers. Ornamentals have been associated with all advanced civilizations of the
world. The use of flowers for ritual and religious acts is found in India, East and
Southeast Asia, Egypt, the Near East and Europe, and can be traced back to at least ca.
1000 B.C. For that purpose, and for their aesthetic value, flowers were cultivated in
classic Greece and Rome. After the fall of the Roman Empire, Arabs brought the
appreciation of gardens and flowers back from the Orient to southern Europe in the 71h
century. Plant species from the Mediterranean area enriched European gardens.
Monasteries tended flowers for centuries, and when towns in Central Europe developed
in the 13th century, flower gardens also made their appearance. Botanical gardens, first
mentioned in the 14th century, contributed decisively to floriculture in the gardens of
European kings, princes and prosperous citizens, after plants from all continents had
been brought to Western Europe, in the late 16th century.
Often originating from subtropical or tropical zones, the plants' new environments
deviated considerably from those of their area of origin, so that they had to be cultured
under protection in climatic conditions cooler than their original habitat. Today, the
production of floricultural plants is more controlled with respect to cultural
requirements than any other form of crop production in agriculture. From about 1700
47
A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, 47-83.
© 2002 Kluwer Academic Publishers.
48

onwards, independent commercial floriculturists made their appearance in Europe. The

floriculture trade flourished after railways, starting in about 1865, connected Italy and
southern France with Western, Central and Eastern Europe. Floriculture showed up
early in North America as well, where "A treatise on the cultivation of ornamental
flowers" by R. Green was published in 1828. The beginning of the 19th century saw the
development of horticultural science. In 1889, the first International Horticultural
Congress took place in Paris. Around 1925, horticultural courses were taught at
universities in the USA, Australia and 10 European countries.

1.1.1. Economic Importance of Floriculture

In the catalogue of an ornamental seed house, about 250 plant genera are found which
are sold for their flowers. Taking into account the lists of propagators ofyoung plants,
herbaceous perennials, flower bulbs and corms, certainly, nearly 300 genera have
captured the attention of floricultural plant breeders. There is, furthermore, a large
number of foliage plant genera, where some breeding takes place. Ornamental plant
production is based on sound scientific lines, has developed rapidly, and still has very
high growth potential. It forms an industry of considerable economic importance.
Statistics with regard to floricultural area are not available for all countries. It should be
kept in mind that in several countries outside of Europe and North America, only cut-
flower data are given, and often the traditional local market production is not included.
The data for 26 countries with the largest areas in Table 1, therefore, should be
regarded as rough estimates. Due to the problems of existing and lacking statistics, it is
undoubtedly difficult to estimate the economic value correctly.
In the l 0 most important countries, the production value of pot plants and cut
flowers amounts to about US $31,000 million. The worldwide value of retail
consumption of flower products may be approximately US $50,000 million. Floriculture
crop production in developed countries alone can be valued as high as US $500,000 per
hectare. The worldwide wholesale production value of bedding plants is estimated at US
$2,500 million, nearly half of this in the USA. Economic importance can also be
expressed by comparing retail values of floriculture with those of agricultural products.
In Germany (1996/97, million DM) they amounted to: cereals 6,636; sugar beets
2,549; potatoes 1,516; and floriculture 2,31 0. Data on per capita consumption of
floricultural products are scanty. In Europe (1992), they ranged between SFr 51.9 in the
United Kingdom (US$ 28.5) and SFr 208.6 in the Netherlands (Heinrichs, 1999), and
in South Korea, US $15.3 is reported. World floriculture imports (fresh cut flowers,
plants, fresh cut foliage) had a value of nearly US $7,962 million in 1998 (Floraculture
International 6/2000). Major import markets are Germany, USA, France, UK, and the
Netherlands; major exporting countries are the Netherlands, Colombia, Denmark, Italy,
and Belgium. However, countries like Ecuador, Costa Rica, Kenya, Zimbabwe,
Thailand, Singapore, India, Australia and Malaysia also export considerable amounts of
floricultural products. World exports amount to US $7,438 million.
Important floriculture crops are given in Table 2. Worldwide, the most important cut
flowers are roses, carnations (Dianthus caryophyllus), chrysanthemums
(Dendranthema) and gladioli. But there are many more, such as tulips, freesias,
gerberas, orchids, lilies, alstroemerias, Anthurium, Gypsophila and Aster, which are
significant in one or another country. Species grown for cut flowers in summer in the
 49

open or under light protection are: Aster, Eustoma, Tanacetum, Limonium, Trachelium,
Anemone, Antirrhinum, Matthiola, Delphinium, Callistephus, Cosmos, Dahlia, Zinnia.
Important flowering pot plant species are Begonia Elatior hybrids, Saintpaulia,
Kalanchoe, azalea (Rhododendron simsii), poinsettias (Euphorbia pulcherrima),
Dendranthema (chrysanths), Cyclamen, Hydrangea, Spathiphyllum, Primula spp., and
important foliage plants Ficus, Dracaena, Yucca, Schefflera, Dieffenbachia, Hedera,
palms. The most grown bedding plant species are geraniums (Pelargonium spp.),
pansies (Viola wittrockiana), Fuchsia, Petunia, Impatiens spp., begonias (B.
tuberhybrida, B. semperjlorens hybrids), Tagetes, Salvia, Dianthus chinensis,
Catharanthus, Verbena, Lobelia. There are many more species of more local
significance.

TABLE 1. Woody ornamentals, bulbs, flower and foliage crops, cropped area (ba). Sources:
Heinrichs 1999, Floraculture International1995, 1998, 2000, Dt. Gartenbau 1996,1998
ASIA EUROPE
China 59,500 Netherlands 36,800
India 34,000 Germany 21,300
Japan 10,200 United Kingdom 17,800
Thailand 7,000 France 13,600
South Korea 5,600 Italy 7,700
Australia 3,900 Spain 5,000
Middle East 4,000 Belgium 4,300
Israel 2,200 Denmark 3,600
Malaysia 1,300 Greece 1,000

AMERICA AFRICA
USA 18,300 Kenya 1,300
Mexico 10,000 Zimbabwe 1,100
Colombia 4,200
Costa Rica 3,600 WORLD 250,000
Ecuador 2,100 (Flower & foliage crops)
Brazil 1,100

1.2. BREEDING

Ornamental plant breeding is an ancient craft. It is well documented in European

countries, but has also been practiced on other continents as shown by the early
introductions: chrysanthemums, dahlias and tulips, for example, came to Europe as
well-developed garden types from Asian and American countries. Written reports of the
chrysanthemum cultivated in China date from about 500 B.C.; it reached Japan in the
year 386, and most present-day forms were developed in Japan before 950 (Dowrick,
1953). Initially, ornamental plant breeding in Europe was carried out by plant
enthusiasts and early horticulturists who made the observation that cross-fertilization
frequently resulted in increased size and vigor. The practice began more consciously
50

early in the 18th century (Zirkle, 1932; Stubbe, 1965). In the middle of the 19th century,
private seed growers began breeding on a larger scale, followed by nurserymen at
public institutions, and later at universities and research stations. Subjects of
floricultural plant breeding are species of annuals, biennials, herbaceous perennials and
a few compact-growing woody ornamentals, which can be utilized as bedding plants,
cut flowers or pot plants. Only limited breeding work has been conducted on foliage
plants. At present, there are numerous ornamental plant breeders in all parts of the
world. Besides private amateur breeders, there are many small-scale and specialty
breeders (nurserymen) who engage in breeding as a part-time business.
Furthermore, two professional categories generally exist: seed companies breeding
sexually (by seed) propagated species, and specialty propagators (usually selling rooted
cuttings) breeding asexually (vegetatively) propagated species which are utilized like
the seed-propagated species as bedding plants, cut flowers and pot plants. It is through
the sale of propagules of the cultivars that they develop that these private breeders can
recover their investment in their breeding programs. Formerly, cultivars were often
open to unauthorized propagation and renaming since, for instance, clonal cultivars
could be patented in the USA but not in Europe. After UPOV (International Union for
the protection of new varieties of plants) regulations came into force in the USA, Japan
and Europe in 1968, and after plant breeders' rights were granted for ornamentals as
well in the 1970s, breeders were in a better position to recover their investments in their
breeding programs. This spurred private breeding companies to invest in breeding,
especially of asexually propagated species. Since that time, floriculture breeding has
considerably intensified. In the European Union, nearly 3900 applications for
Community Plant Variety Rights were received between 1995 and 1998, making up
54% of all agricultural and horticultural applications. In July 1998, 1420 rights (45%)
already existed, rose, chrysanthemum and geranium being the most important species,
and the Netherlands, Germany and France being the most actively breeding European
countries. Most applicants from non-European Union countries came from the USA,
Israel, Australia, Switzerland, New Zealand, Japan and South Africa (Kiwiet, 2000).
Besides numerous research and trade papers, there are several books or reviews on
floricultural plant breeding and breeding research: Emsweller eta/. (1937), Crane and
Lawrence (1952), Horn (1968, 1979), Sink (1976), Langton (1987), and Harding eta/.
(1991 ). The Section Ornamentals of the European Association for Research on Plant
Breeding Eucarpia has published proceedings of its 19 meetings from 1971 to 1998.

1.2.1. General Breeding Aims

Before the 19th century, breeding aims were first and foremost set by plant enthusiasts.
Curiosity, unusual features, special beauty, novelty and uniqueness were in demand.
Now, floricultural plant breeders not only produce for a local market, but for all
important production areas of the world, and practical traits of economic significance
have become important. Today, following the enormous changes that have occurred in
the production of ornamentals, floricultural products have to be refined to a very precise
ideotype corresponding to different target markets, such as pot plants, plants finished in
cell packs (for mass-market outlets), garden performance and hanging baskets. Product
differentiation occurs on details of color, habit, etc. Aesthetic appeal, novelty, novel
flower patterns or colors, color range and hue, flower size, foliar characteristics, plants
 51

proportions, tolerance to environmental stress, among others, are major factors in

ornamental cultivar selection.
Uniformity and stability of a cultivar in all important characters are also of the
utmost significance. Generally, yield per area and per cropping period, number of
flowers per plant per unit time, quality, shipping ability, shelf life and ease of growing
play a major role. Regarding temperature and light requirements, tolerance to heat and
low temperature, as well as to low irradiance, are of importance, e.g. for winter
production, since fuel costs remain a major concern. Further objectives are growth type,
production time, post-harvest quality, disease and pest resistance, cutting yield per plant
and period, rooting time of cuttings, and germinating ability of seeds.

1.2.2. Variability and Selection

A general prerequisite for successful selection in plant breeding is the existence of a
sufficiently great store of genetic variability in the original population. If this does not
exist, it has to be developed by introducing new types, through hybridization within or
between species (also termed cross-breeding), through mutagenic treatment or by
changing the ploidy level. In early ornamental plant breeding, only few plants of a
species or small seed lots were available, especially when imported from a different
continent, producing a lack of variability.
On the other hand, under cultivation in gardens and nurseries, a sudden increase in
the frequency of recessive types occurred because of the decrease in the size of the
originally cross-pollinated breeding group, and since in artificial selection, fitness
values are determined by the breeder. Under conditions of reduced competition, deviant
types have a selective advantage due to the flower breeder's special attention. Thus,
cultivation and reduced competition became one of the important factors contributing to
the origin of new traits in floriculture crops. Because often only a few plants were
available to the breeder, intra- and interspecific hybridization (cross-breeding) was
employed on a large scale, followed by inbreeding and/or selection till uniform and
stable cultivars were obtained. Selection in a variable population is the most difficult
part of plant breeding and demands not only experience but also some form of
replicated trials, usually in different environments.

1.2.3. Breeding Strategy and Breeding Methods

Before breeding a new plant species, or before a breeding program is extended, the
question of whether breeding is worthwhile in that particular case has to be addressed.
When minor species or characters are concerned or feasibility ofbreeding for resistance
is being considered, this question is of particlar importance. Often, it is better to
disinfect the growth medium than to breed for resistance to soilborne fungi, or to
propagate by seeds instead of asexually, if the disease agent is not transmitted through
seeds. Ultimately, the answer to that basic question is an economic decision. Likewise,
the extant variability of the characters under consideration is of importance for the
breeder. If the variability is sufficient, one can begin suitable selection immediately. If
the available variability of the characters under consideration is limited, it can be
enhanced by inbreeding, hybridizing or the artificial induction of mutations. If, after
careful consideration, the answer is in the affirmative, decisions concerning breeding
procedures can be made.
52

TABLE 2. Important floriculture crops, their mode of propagation and breeding system.
Predominantly used as C = cut flower, P = pot plant, B = bedding plant; S = seed propagated, V =
vegetatively propagated, PP = polyploidy occurs
Plant species c p B s v pp

Ageratum houstonianum + + + +
Alstroemeria hybrids + + +
Anemone coronaria + +
Anthurium scherzerianum, A. andreanum + + +
Antirrhinum majus + + + +
Argyranthemum frutescens + +
Aster spp. + + + +
Begonia spp. + + + + +
Bromeliads
Guzmannia + + +
Vriesea + + +
Calceolaria integrifulia, C. hybrids + + + +
Callistephus chinensis + + +
Catharanthus roseus + + +
Cosmos bipinnatus, C. sulphureus + + + +
Cyclamen persicum + + +
Dahlia hybrids + + + + + +
Delphinium hybrids + + + + +
Dendranthema grandiflorum + + + +
Dianthus spp. + + + + +
Dieffenbachia spp. + +
Dracaena spp. + +
Euphorbia pulcherrirna, E. fulgens + + + +
Eustoma grandiflorum + + +
Ferns
Nephrolepis + +
Asplenium + +
Ficusspp. + +
Freesia hybrids + + + +
Fuchsia hybrids + + + +
Gazania hybrids + + +
Gerbera jamesonii hybrids + + + +
Gladiolus hybrids + + +
Gypsophila paniculata, G. elegans + +
Hedera helix + + +
Hydrangea macrophylla + +
Impatiens walleriana, I. hawkeri hybrids + + + + +
Iris hollandica hybrids + + +
Kalanchoe blossfeldiana hybrids + + +
 53

TABLE 2. Continued
Plant species c p B s v pp

Lilium hybrids + + +
Limonium spp. + + + +
Lobelia erinus + + +
Matthiola incana + + +
Narcissus spp. + + +
Nicotiana sanderae hybrids + +
Orchids
Cymbidium hybrids + + +
Dendrobium hybrids + + +
Phalaenopsis hybrids + + +
Palms
Chamaedorea spp. + +
Cbrysalidocarpus lutescens + +
Howea + +
Phoenix + +
Pelargonium hybrids + +
Primula spp. + + + +
Petunia hybrids + + + +
Rosa hybrids + + + +
Rhododendron simsii hybrids + + +
Saintpaulia ionantha hybrids + +
Salvia splendens + +
Schefllera spp. + + +
Senecio cruentus hybrids + + + +
Spathiphyllum hybrids + + +
Tagetes spp. + + +
Tanacetum spp. + + +
Trachelium caeruleum + +
Tulipa hybrids + + +
Verbena spp. + + + +
Viola wittrockiana hybrids + + +
Yucca elephantipes + +
Zinnia elegans, Z. angustifulia + + + +

In principle, the breeding procedures that may be used with floriculture crops do not
differ from those in other crop species. They are determined by the mode of
reproduction. Regarding the reproductive or breeding system, most ornamental plant
species are cross-pollinated or preferentially cross-pollinated, i.e. outbreeding. Only a
few are obligatorily or preferentially self-pollinated, i.e. inbreeding. Due to the mode of
propagation, there are clonal cultivars, as well as cultivars from the sexually propagated
population (from open-pollination) and Fl-hybrid cultivars (from controlled crosses of
54

inbred lines). Many floriculture species include all three cultivar types {Table 2). All
these cultivar types are genetically heterozygous, but homogenous regarding the main
characters with the exception of population cultivars, which may vary somewhat within
the cultivars and are, therefore, more heterogenous. In accordance with the different
types of cultivars, one can distinguish three basic breeding methods (Schnell, 1982).
The choice of a particular breeding method depends not only on the cultivar type
and the reproductive system of the species in question, but also on its cost. A breeder
will choose a cost-saving method if the expected financial return from the sale of
propagules of a new cultivar is low. This will be the case for many of the great number
of ornamental species on the market. On the other hand, one could contemplate hand-
pollination for inbreeding and hybrid seed production in a greenhouse, if the expected
financial return is adequate. Since greenhouses are available, floricultural plant breeders
utilize them not only for the culture of exotic species and for hybridization, but also for
overwintering their breeding stock, and to grow more than one generation per calendar
year, thereby accelerating the introduction of a new cultivar.
Before starting breeding, breeding research results are worth considering, since
besides the breeding (reproductive) system, mode of trait inheritance and level of
polyploidy play an important role in choosing a breeding procedure. The breeding
strategy depends decisively upon the mode of inheritance. If a trait is quantitatively
inherited, heritability, i.e. the coefficient between genetic (or its additive component)
and phenotypic variance, is the basis of selection procedures. Heritability estimates are
markedly influenced by the material under study, its interaction with the environment,
and the number of replications within a trial. If major genes control a character,
recombination of genes by crossing is the choice, and replicated trials are often
dispensable. Floriculture breeders have profited from fundamental research conducted
in some flower crops, such as Antirrhinum (see Stubbe, 1966) and Petunia (see Cornu
and Maizonnier, 1983; Sink, 1984).

2. Results of Breeding Research

2.1. POLYPLOIDY

As indicated in Table 2, polyploidy is a widespread phenomenon in floriculture crops.

In many cases, commercial cultivars are polyploids; in others, polyploid and diploid
cultivars are grown. A common effect of autoploidy is to increase the size of flowers
and vegetative portions of the plants, often making autoploids more compact and
vigorous than the corresponding diploids, and their internodes, in inflorescences like
hyacinths, gladioli or primulas, are shorter than in diploids. Flower number per plant
and sexual fertility, however, are reduced in autoploids. Triploidy leads in most cases to
sterility. It is, therefore, used to lengthen the blooming period (Begonia, Ageratum,
Tagetes), and to prevent illegitimate seed increase, but cannot be utilized generally
since crosses between diploids and tetraploids are unsuccessful in several species.
Spontaneous autoploid types have been unconsciously selected for by plant breeders
in the past, when looking for large flowers and strong flower stalks. In hyacinths, in ca.
1850, triploid cultivars appeared on the market; in 1880 tetraploid cultivars appeared,
and after 1915, only polyploid cultivars could be found. In Bearded Iris, from 1900 to
 55

1914, 28 of 42 new cultivars were diploid; in 1935 to 1939, only 5 out of 100 were
(Darlington, 1963). Tetraploid clonal cultivars replaced diploids in Freesia in ca. 1950,
and the proportion of polyploid cultivars steadily increased, as well as in Semperflorens
begonias, cyclamens, Primula malacoides and geraniums, among others. Alloploidy
was welcomed to restore fertility in interspecific crosses by spontaneous doubling of the
chromosome number, for instance in begonias, Impatiens, Kalanchoe, lily and others.
Tagetes patula, for example, is an allotetraploid hybrid between T. erecta and T.
tenuifolia (Towner, 1956), pansies (Viola wittrockiana) are autoallooctoploid (Horn,
1956), and perennial Delphinium hybrids are allohexaploid (2x D. grandiflorum x 4x D.
elatum).
Polyploidy has its natural origin in the rare occurrence of somatic mutations (mitotic
doubling of chromosome number), and in the formation and fusion of unreduced
gametes (i.e. failure of meiotic reduction) in diploids so that tetraploids originate, often
via triploids. Almost att natural polyploids have arisen by way of unreduced gametes.
The doubling of chromosome numbers with the help of colchicine (,colchiploids") is
useful in overcoming interspecific sterility barriers. Colchiploid forms have often been
induced to get large-flowered types, but only a few have been successful (in
Antirrhinum, Cosmos, Dimorphotheca sinuata, Helipterum roseum, Nemesia,
Tanacetum parthenium). The way how autopolyploids or segmental allopolyploids
originate is of significance for their fitness. As among others Skiebe (1966) has
demonstrated in Primula malacoides, autotetraploids from mitotic doubling of
heterozygous genotypes are less fertile than those from fusion of unreduced gametes
(,meiotic polyploidization"). Plant breeders should, therefore, utilize meiotic
polyploidization for instance through crosses between diploids and tetraploids, when
looking for new tetraploids.
Polyploidy may have important effects on plant-breeding procedures.
Allotetraploids can be treated as diploids. Several problems arise in breeding
autopolyploids since genetic segregations as described in textbooks, and response to
inbreeding and selection differ considerably from those in diploid species (Wricke and
Weber 1986). Crosses at the tetraploid level yield very variable progeny since the
occurence of four alleles at one gene locus sets up the possibility of special inter-allelic
interactions, and of diverse combinations. Beside monoattelic and biattelic genotypes as
in diploids, triallelic (e.g. ala2a3a3) and quadriallelic (ala2a3a4) as well as biallelic-
duplex genotypes can occur in autotetraploids. There are examples of dosage effects of
flower color genes in polyploids for instance in Dahlia (Crane and Lawrence 1952), and
of genes controlling flower doubleness in several species. Differing responses to
inbreeding and different results regarding hybrid vigor depend on the presence of two or
more than two atteles at one locus, what may be a result of earlier inbreeding. For
instance Sparnaaij (1979) observed a lower phenotypic variability, i.e. a higher
uniformity in tetraploid progenies of certain floriculture species than in comparable
diploid progenies especiatty regarding polygenic traits. His observations on a more
pronounced hybrid vigor and slower inbreeding depression in tetraploids are in contrast
to e.g. Trang (1979) who in pansies (Viola wittrockiana) found the means of inbred
progenies lower than was to be expected according to the inbreeding coefficient, and
explained that by interactions between more than two alleles. He also found that the
inbreeding depression of the parents cannot be fully removed in single cross hybrids,
56

and that double crosses or synthetics were superior to single crosses. Less inbreeding of
parents, therefore, may result in better performing single-cross hybrids.

2.2. INHERITANCE OF FLOWER COLOR

Flower color holds a central position in floriculture species. It is caused mainly through
the incidence of flavonoids (flavones, flavonols, anthocyanins) and closely related
compounds (chalcones, aurones) as well as carotenoids. Flowers usually contain
mixtures of flavonoids and of flavonoids and carotenoids. The yellow colors in roses,
daffodils, :freesias, Gerbera, lilies, pansies and others come from carotenoids. In a few
plant families (Aizoaceae, Amaranthaceae, Nyctaginacea, Portulacaceae, Cactaceae),
betalains cause the colors. However, genetic studies of betalains and carotenoids in
flowers are scarce. The biosynthetic pathway ofbetalains has been described by Steiner
eta/. (1999). Three genes acting in that pathway have been demonstrated in Portulaca
by Trezzini and Zryd (1990). One (C/c) acts between DOPA (dihydroxyphenylalanine)
and betalamic acid, one (R/r) between DOPA and cyclo-DOPA, and the third (IIi)
inhibits conjugation of amino acid residues with betalamic acids, required for
betaxanthin formation. Red flowers, the result of both betacyanin and betaxanthin
synthesis, are of the genotype C. R. ii, violet flowers of the genotype C. R. I. Those
genes have not yet been correlated to the enzymes known to be active in the pathway.
Regarding carotenoids, the most universal carotenoid biosynthetic pathway is the
sequence leading to the formation of ~-carotene. The first carotene of this pathway is
phytoene. Genes encoding different phytoene desaturases are available from, among
others, higher plants, and their activity leads to lycopene and then to ~-carotene
(Sandmann 1994). In Zinnia, chrysanths (see, among others, Teynor eta/. 1989) and
Gerbera, the presence of carotenoids in flowers is inhibited by a dominant supressor;
another locus is responsible for the synthesis of carotenoids (Boyle and Stimart, 1988;
Tyrach, 1994).
The inheritance of flavonoid pigments is well documented. First inheritance studies
of flower colors due to flavonoids date back to the time ofMendel and the beginning of
the 20th century, and several reviews of Mendelian flower color genetics have been
provided, for instance by Alston (1964). In most cases, the inheritance has been
described in terms of six genes with epistatic interactions. Monogenic segregations are
obtained in character pairs such as cyanic/acyanic, rose/red or yellow/non-yellow. A
first biochemical survey of flavonoid flower color genes was published by Scott-
Moncrieff(1936), who showed that genes influencing flower color control single steps
in flavonoid modification, such as hydroxylation, methoxylation or glycosylation
(Table 3). Harrison and Stickland (1974) showed the action of genes in the biosynthetic
pathway of flavonoids. Later, evidence was produced that certain genes acting in the
pathway control the activity of certain enzymes on a one gene-one enzyme basis (see
reviews: Seyffert, 1982; Forkmann, 1991, 1993) as detailed in another chapter in this
book.
Dominance and epistatic relationships between the different anthocyanidins can be
shown as: delphinidin > cyanidin > pelargonidin, malvidin > peonidin > pelargonidin,
petunidin > peonidin, peonidin > cyanidin. There are numerous examples in floriculture
crops where genes for certain flavonols or anthocyanins can be defined in terms of the
 57

biosynthetic pathway by analogy. Examples can be found in pot azaleas (Heursel and
Horn, 1977), Primula (Horn and Eltorkey, 1989), Pelargonium (Horn, 1994), Gerbera
(Tyrach and Horn, 1997), and cyclamens (Takamura eta/., 2000). Besides, there are
genes that influence color by co-pigmentation, pH of the vacuole, interaction of
flavonoids with metal ions, and by morphological characters such as hairs, papillae, etc.

TABLE 3. Modifications ofthe B-ring ofthe flavonoid molecule

Flavonoid 3' 4' 5' Flavonoid 3' 4' 5'

Anthocyanidins F/avonols
Pelargonidin H OH H Kaempferol H OH H
Cyanidin OH OH H Quercetin OH OH H
Delphinidin OH OH OH Myricetin OH OH OH
Peonidin OCH1 OH H Flavones
Petunidin OCH3 OH OH Apigenin H OH H
Malvidin OCH3 OH OCH3 Luteolin OH OH H

2.3. INHERITANCE OF FLOWER DOUBLENESS

Double flowers are of interest to the floriculture crop breeder not only because of their
aesthetic appeal but also because they may serve as a kind of male sterility which is
sought after for controlled crosses. Usually, in double flowers, stamens are petaloid;
those of Asteraceae contain female ray florets only, but in monoecious Begonia, only
male flowers are double. In nearly all cases investigated, there is one major gene locus
controlling double flower. Doubleness can be dominant or recessive. It is recessive in
Antirrhinum (mut. plena), Callistephus chinensis, Dianthus barbatus, Eschscholtzia
californica, Matthiola incana, Papaver rhoeas and Salpiglossis, and dominant in
Cyclamen persicum, Dianthus caryophyl/us, Gerbera, Pelargonium hortorum, Petunia,
Rosa, Saintpaulia ionantha, Sinningia and Tagetes. The dominance of doubleness can
be incomplete, for instance in Gerbera, Pelargonium hortorum and Petunia, so that
heterozygous individuals can be separated from homozygous ones, and in polyploids a
dosage effect according to the number of alleles is observed.
In Pelargonium, Almouslem and Tilney-Basset (1989) showed the action of three
gene loci controlling flower doubleness. Double Cosmos bipinnatus are dominant at two
loci, one of which is heterozygous (Samata, 1958), and double Begonia semperjlorens
are homozygous recessive at two loci. There are, however, in addition to major genes,
modifYing genes and environmental effects which alter the phenotype (Reimann-
Philipp, 1969). In pot azaleas, flower doubleness appears to be quantitatively inherited
with a large additive component of variance (Heursel and Garretsen, 1989).
Cytoplasmatically controlled doubleness is found in tuberous begonias (Noack, 1962).
In capitulae of Asteraceae, warm temperatures often reduce the degree of doubleness. In
Gerbera, the number of ray florets is quantitatively inherited, the additive component of
variance and heritability being relatively large (De Leo and Ottaviano, 1979; Wricke et
a/., 1982; Harding et al., 1991; Yu et al., 1993).
58

2.4. INHERITANCE OF PLANT HABIT, FLOWER AND FOLIAGE TRAITS

The plant habit is one of the most important traits in floriculture plant breeding. It is the
goal of the plant breeder to determine the genetic form of a plant, in order to suit it to
special needs. Plant form has a specific genetic basis. Especially in bedding and pot
plants, breeders have long looked for compactness, and have succeeded in obtaining
compactly growing cultivars in many species. Most hypotheses associate changes in
plant form with alterations in the hormonal balance, or in the phytochrome dynamics.
There are several examples of monogenic recessive loci controlling compact or dwarf
types. Monogenic inheritance of compactness has been found, for instance, in marigold,
geranium, snapdragon, stock and petunias. Amongst the mutations found in the
snapdragon (Antirrhinum majus) and described by Stubbe (1966), there are seven
compact mutations, including the useful mutant nana. Another mutant, eramosa,
depresses branching, and has been used in breeding unbranched forcing types. In sweet
pea (Lathyrus odoratus), a locus Bib controls branching: bb-genotypes produce at least
twice as many lateral shoots as B (Ross and Murfet, l988).1n China aster (Callistephus
chinensis), three loci control plant habit. Beside the triple dominant, normal habit
(typica), the growth types pyramidalis with erect branches, compacta, and nana, each a
homozygous recessive genotype, as well as the double-recessive typica compacta and
nana pyramidalis, can be found (Wit 1937). Within the genetically determined growth
classes, modifier genes and environment influence the plants' habit.
Flower type in Asteraceae is often monogenically controlled, such as the spider type
in chrysanths and in Gerbera (Tyrach, 1994), the apetalous type in Antirrhinum (mutant
deficiens), Petunia, Tagetes, Zinnia, and Cosmos (two loci) as well as the different
types, e.g. ostrich plume, in the china aster (four loci, Wit, 1937). In Antirrhinum, a
completely new flower type was constructed in 1944 by Knapp (1967). He combined
the mutants Div (dominant) and eye!-', thereby creating a snapdragon with regular
instead of zygomorphic flowers.
Studies of the genetics of flower size, in a botanical and floricultural sense, are rare.
The trait is controlled by major genes and polygenes. In a floricultural sense, a flower
may be single, a floret, or an inflorescence. The large flower size of Petunia is
monogenic dominant (G) but the respective gene is linked to a lethal factor I. Breakage
of that linkage led to the development of homozygous grandiflora petunias (Table 4,
right).

TABLE 4. Genetics oftlower size in Petunia (after Reimann-Philipp, 1969)

Gametes G1 gL GL g1

G1 GI/G1 t Gl!gL 2 grandi:llora : 1 mu1ti:llora GL GUGL GUg1 only grandi:llora

~---- GllgL gUgL (hom~zygous~i!Jo~..!L_ ___ ~ _ _Q.Ygi -~gl t _!13 bomo~ous _

The length of ray florets in china aster crosses (ostrich plume X common type)
shows a continuous distribution in F1 and F2, and an effect of one major gene with
incomplete dominance. Flower size (capitulum diameter) of Gerbera has been found to
be quantitatively inherited with a large additive component of variance (Maurer, 1968;
De Leo and Ottaviano, 1979; Wricke et al., 1982; Harding eta/., l99la; Yu eta/.,
 59

1993). Values of heritability estimated from components of variance ranged around

80% when studying the size of the inflorescence of Phlox paniculata (Morgner and
Horn, 1970).
Inheritance of leaf pattern and leaf zonation was studied in Cyclamen and
Pelargonium hortorum by Seytfert {1955) and Amoatey and Tilney-Bassett (1993), and
found to be controlled by a series of multiple alleles.

2.5. FLOWERING TIME

The transition from vegetative to reproductive meristem is an important stage in plant

development, and time to flowering is of high significance for floriculture crops. The
period from propagation to flowering is also important for the grower because a shorter
crop cycle means conserving energy, less fuel and labor costs, and the possibility of
producing a second crop from the same glasshouse area. For most bedding plant
markets, growers require cultivars that show first flowers when offered to the consumer
at the beginning of spring. There are a number of environmental components
contributing to flowering time, for instance light and temperature, but genes controlling
different physiological steps are also involved.
When investigating the heredity of flowering time one has to differentiate, for
instance, between plant species that are induced to flower by photoperiod or cold
temperatures, and day-neutral species. As early as 1919, in the F2 of a cross between
short-day and day-neutral tobacco, the latter was shown to be monogenic dominant to
short-day sensitivity, and in Salvia splendens, day-neutral was monogenic recessive
with partial dominance of the short-day character (Lai et al., 1974). In Coleus, the short-
day character was dominant to long-day, but the range of responses indicated the
existence of modifier genes. In sweet pea (Lathyrus odoratus), day-neutral cultivars are
winter-flowering, while quantitative long-day types flower in spring or summer. Two
complementary flowering gene loci (one with three alleles) have been identified, and
two dominant alleles (Sp Dnh) must be present to confer the late- or summer-flowering
(wild-type) phenotype (Ross and Murfet, 1988). Sp Dni and sp Dnh are spring-flowering
genotypes, and due to epistasy, all dn genotypes are day-neutral. In Lunaria, biennial
flowering is monogenically dominant over annual flowering, and in Silene armeria, a
triallelic locus confers early (Ef) or late flowering (ef) (Wellensiek, 1973, 1976).
In geranium, photosynthetic photon flux (PPF) is decisive for flowering, and there
exist low-irradiance-requiring early-flowering and high-irradiance-requiring late-
flowering types. Whilst Horn (1974) could successfully select low-irradiance-requiring
strains, the difference between early and late flowering is according with Craig (1993),
a function of two genes, early being dominant. In marigold, which does not react to
photoperiod, Towner (1956) described a monogenic recessive late-flowering mutant.
Also in snapdragon (Anti"hinum majus), PPF and a quantitative long-day reaction play
a role. The seemingly continuously variable character, flowering time {heritability mean
0.55), has been converted into discontinuous segregation with respect to budding time
(ca. 1 week after induction), time from induction to anthesis, and number of leaves to
flowering (Edwards, 1974). One gene with dominance in different environments for
early budding (and no. of leaves) has been found which interacts with temperature, so
that the genotype 'late flowering' at 25°C, is early flowering at l2°C and vice versa.
That makes producing specific early-flowering F1 hybrids with low-temperature
60

tolerance possible. Since within early-flowering cultivar series, the number of days to
flowering still varies for ca. 2 weeks, one has to assume modifYing genes in addition to
that major gene. Combining genes for a short vegetative phase and a short time from
induction to anthesis can lead to very early flowering types (Rabinowitch et al., 1976).

2.6. STRESS TOLERANCE, RESISTANCE TO DISEASES AND PESTS

2.6.1. Tolerance to Abiotic Stress

In greenhouse production of floriculture crops, environmental stresses can be nearly
completely eliminated with the help of heating or cooling systems, or supplemental
artificial lighting. There is, however, the question of cost. Fuel costs are one of the main
limiting factors in floriculture crop production in temperate climates. Ideally, low-
temperature tolerance without an equivalent reduction in growth and quality is looked
for, or alternatively, faster growth at the usual temperatures, so that reduced costs result
from shortening production time. Tolerance to environmental stresses is considered to
be the product of many genes. There are several reports on breeding programs aimed at
adaptation to low temperatures, or to high temperatures in areas with hot summers. De
Jong (1991) presented data on the temperature response of contrasting chrysanthemum
clones, which showed significant differences in optimum temperature for days to
flowering and wide variation regarding the number of days to flowering at optimum
temperature. Equally, considerable genetic variation with regard to temperature exists in
roses, and in African violet (Saintpaulia ionantha, Amberger et al., 1984). Flower
breeders have also been very successful in adapting the flowering process to marginal
light conditions (DeJong, 1991).
During the post-production period, products of floriculture, including cuttings, are
exposed to environmental stress. Mention is given to rain and wind tolerance, which are
important parameters for bedding plants. In petunias, for instance, cultivars developed
in Europe performed better than those from California under the summer conditions of
Central Europe, at least until recently. During shipping, due to chilling or heat stress,
endogenously produced ethylene may rise to deleterious levels; during the retail phase,
and in consumer's homes, light levels are too low. Leaf, flower, and bud drop are the
result of ethylene action. Shelf or vase life is one consequence of tolerance to post-
production stress, and is not easily assessed as it is also affected by growing conditions
prior to harvest and stage of flowering at harvest. Cultivar longevity differences have
been found in several floriculture species (e.g. Borch eta/., 1995), and selection for that
trait has been successful. Recommendations are difficult to make regarding selection
strategy when breeding for tolerance to temperature and light stress. The best approach
is still to test segregating populations and seedling clones at the target locations. Since
that is probably not always the fastest approach, selection experiments have been
conducted in which plants are grown in climatically controlled growth rooms or even in
vitro, making partial use of stress indicators like proline, and promising results have
been obtained. Some of the results, however, were not durable or were contradictory
(see De Jong, 1991) in terms of what may be due to epigenetic causes or special
genotype-environment interactions.
 61

2.6.2. Resistance to Disease Agents

In floriculture crops grown under some kind of coverage, and often in pots or
greenhouse beds in special growth substrates, diseases are controlled relatively easily by
cultural and chemical methods. Moreover, research institutions have rarely been
interested in those crops, so information on the biology of parasites, host-parasite
relationships, genetics of host resistance and parasite virulence, inoculation and
screening procedures, are often missing. Therefore, breeding for resistance on a broader
scale did not start until 1960. Public pressure on growers to reduce chemical disease
control, conditions imposed by the authorities in international flower trade demanding
absolute freedom of disease agents and absence even of cosmetic effects such as leaf
spots demanded by retailers and consumers, have led to integrated control including
genetically determined resistance.
The principles underlying breeding for resistance to diverse disease agents, such as
fungi, viruses, bacteria, insects, and nematodes, are similar. In vertical resistance, not
regarded as durable, there are mostly dominant major genes conferring host resistance
to all known pathotypes (genotypes) of a pathogen or to some special pathotypes only.
The so-called horizontal resistance is polygenically controlled, pathotype-nonspecific,
and conferring some form of resistance, usually not complete, against all pathogen
populations. If a major gene conferring resistance to all pathotypes is available, it will,
of course, be used in breeding, while the use of the other form of vertical resistance is
risky. Vertical resistance is often available in wild species only, sometimes with
different chromosome numbers, and then a long backcross program is required.
Horizontal resistance may evolve by natural selection, which takes time but should be
used in perennials.
Though breeding for resistance in floriculture crops as such began relatively recently
and on a limited scale, flower breeders and growers have long looked for less sensitive
or tolerant genotypes. There are many reports of different grades of susceptibility in
trade and scientific journals. With regard to mildew, for instance, in Elatior begonias
(Microsphaera begoniae), poinsettias (Oidium spp.) and Verbena, such different grades
are known. Uchneat eta/. (1999) evaluated the range of genetic resistance to foliar and
floral infection by Botrytis cinerea (gray mold) in the genus Pelargonium, and found
differential levels of susceptibility, partial resistance, and a genetic component in
species and cultivars. About half of a large number of Saintpaulia cultivars proved to be
fairly resistant to Phytophthora nicotianae var. parasitica, and in pot azaleas
(Rhododendron simsii), several cultivars remain free of Phytophthora citricola.
A few investigations on the biology of disease agents and host-parasite relationships
were already being conducted in the 1930s. One example is China aster wilt caused by
the soilborne Fusarium oxysporum, the study of which began in ca. 1930 in the US (see
Persiel and Lein, 1989). These authors postulated a gene-for-gene-relationship. They
used a differential set of six cultivars for eight pathotypes of the fungus, found strains
resistant to most pathotypes, and observed dominance of resistance after crosses as well
as heterogeneity within cultivars regarding reaction to those pathotypes. A monogenic
dominant resistance to rust (Puccinia anti"him) in Anti"hinum located on the eos-
chromosome, and the existence of pathotypes was demonstrated as early as 1934
(Aitken eta/., 1989). Concerning rust disease in chrysanth (Puccinia horiana), cultivars
are known which show symptoms only when there is heavy infection pressure, while
62

others are susceptible or completely resistant. Resistant cultivars carry one dominant
allele. In roses, breeding for resistance to black spot disease has been conducted since
ca. 1950 (Palmer et al., 1966). This has been a complicated endeavOr because oflack of
resistance in cultivars, the occurrence of different pathotypes, and differing ploidy
levels between species and cultivars (De Vries, 2000). Malek and Debener (1998)
recently described a monogenic dominant resistance. Fusarium resistance in Gladiolus
was introduced from a wild species and found to be polygenically inherited (Straathof et
al., 1997). Concerning insect pests in Pelargonium hortorum, Grazzini et al. (1997)
found heritable resistance to spider mite dependent on a few genes governing trichome
density and phenolic acid content.
Results of research on breeding for resistance against diverse disease agents have
been described by Sparnaaij (1991). Among them are projects regarding leaf miner
resistance (Liriomyza huidobrensis) in chrysanths (Fig. l), different levels of resistance
to the thrips Frankliniella and to aphids, quantitatively inherited resistance to vascular
wilt in carnation caused by the fungus Fusarium oxysporum, and virus resistance in
tulips, where resistance is available in Tulipa fosteriana. There are other cases in which
only wild species may serve as sources of resistance, for example in Cyclamen,
Helianthus, Rosa, and Zinnia.

"survival
100~------------------------------~
I um-,.,.
eo ....... .

81 ....

40 ........................... ..

1 2 3 4 5 8 1 8 9 10 11 12 13 14 15
c:ultivar
Figure I. Survival of larvae of Liriomyza inside the leaves of
15 Dendranthema cultivars (DeJong and Van De Vrie, 1987).

3. Breeding Seed-Propagated Crops

Following the introduction of species from other continents to Europe starting before
1600 (and still continuing), and after first hybridizations by horticulturists in England in
the early 18th century (Zirkle, 1932; Stubbe, 1965), nurserymen utilized the possibility
of vegetative propagation of new types. Hybridization between cultivars, botanical
varieties and species forms, either intentional or accidental, is responsible for the origin
of many of the commercial cultivars. However, also interspecific hybridization has
 63

played a major role in developing new floriculture crops and cultivars. The main
objective of such hybridizations is to improve a genotype by transferring to it one or a
few characters from another genotype of the same or another species or to achieve new
character expressions. Interspecific hybridization is dealt with in another chapter in this
book. Later, ornamental plant breeders tried to stabilize characters of selected plants by
selfing with no consideration of the natural reproductive system till uniform, stable
cultivars for propagation by seed were obtained. Also, nurserymen and plant breeders
used mass selection, first in its simplest form, to improve cross-pollinated ornamentals,
i.e. merely to harvest and bulk open-pollinated seed from selected plants and use it to
establish a cultivar. In that way, it is still used in maintaining genetic identity and purity
of cultivars.

3.1. OPEN-POLLINATED CULTIVARS

3.1.1. Mass Selection

Mass selection (without progeny testing) is a relatively cost-effective procedure for
selection in a source population or after cross-breeding. Generally, individual seed
parents are selected on the basis of phenotype, but selected superior plants are also
pollinated by inferior pollen parents, even if off-type plants are rogued out. Seed from
the remaining open-pollinated individuals is composited, and used to grow the next
generation (Fig. 2). Their number must be large enough to avoid inbreeding depression.
If mass selection is conducted over several cycles, a form of random mating with
selection (Allard, 1960;, Wricke and Weber, 1986) results. It changes gene frequencies,
and shifts the population mean in the direction of selection. The purpose of mass
selection is to increase the proportion of superior genotypes and uniformity. Its
efficiency depends on the number of genes controlling a character and its heritability. It
is rather inefficient in improving characters of low heritability.
Simply inherited and highly heritable characters which are easily seen or measured can
be efficiently improved. Examples of such characters include flower color, internode
length and growth type.
Mass selection is successfully applied in self-pollinated crops, and in cross-
pollinated ones when selecting for a recessive trait. If genotypes homozygous for the
desired recessive allele can be identified phenotypically and before pollination by
inferior pollen parents, one step of selection is fully successful. If a character is
controlled by a dominant allele or if it can be scored only after pollination has taken
place, successful mass selection needs more selection cycles (Table 5). The same is true
when selecting for a continuously varying character such as yield or flowering time. If
mass selection is continued for a number of years, one has to take into account the effect
of natural selection, and the fact that selected genotypes endure only in certain
environments.
Progeny Testing. Mass selection can be conducted with progeny testing: single
plants are selected on the basis of their progeny's performance, i.e. when separate
progeny are grown from phenotypically superior individual plants, and all progeny
containing off-type plants are eliminated. In this case, in addition to the plant's
phenotype, its genotype, as inferred from the progeny, is taken into account. Seeds from
plants in each selected progeny are bulked and used to grow the next generation, or
seeds from selected plants from selected progeny are used to perform a subsequent
64

progeny test. Quantitative traits and characters with lower heritability respond well to
this type of selection, in contrast to mass selection without progeny testing .

Source
• + • +, • • + • • • ,+
population + . • • • +. • + • • + •
• • + ~ ....• · . ~ • • + • •
.,-' .·
-·'l
Improved • • • • • • + • • • .+
population + • • • • • • • • • •
•• • • • + • • + • •
.. l" . ..

New
cultivar
I......:/;{~({(.
.
.
.·.:/// .. ./.• .. .
./ //,~ /

/..' /
·.
·. .// ;
...
:

Figure 2. Mass selection (from Becker, 1993).

A modification of mass selection with progeny testing utilizes remnant seeds of

plants selected in a first step, a procedure called ear-to-row method in cereal breeding.
This method is used for characters which can be assessed only after pollination has
taken place. It attempts to determine the relative breeding value of different selected
plants by planting, in a first step, a portion of their seeds into separate plots, and scoring
the performance of the plots. Remnant seeds from the plants having shown the best plot
performance are sown as the second step. In that way, only plants with superior
breeding value act as pollen parents. Seeds from a second-step plot are bulked and can
be used for a second selection cycle.

3.1.2. Selection in the Glasshouse Environment

Genotypes can differ considerably in their sensitivity to environmental variation and
inter-plant competition. It has always been worthwhile to select directly in the particular
environment in which the target cultivar will be grown. The detection of genes which
are specific to certain temperatures or light levels suggests that selection might be more
effectively carried out in certain carefully chosen environments (Edwards, 1974).
Rabinowitch et al. (1976) showed that the best environment for selecting early-
flowering snapdragons would be to sow during the shortening daylength and light-
intensity conditions of late August and early September. Experiences in the field of
interactions between the glasshouse environment and breeding objectives have also
been reported by Hom (1974). With regard to productivity, e.g. in carnations and roses,
central benches, edge rows, and benches facing west or south give a better yield than
inner rows or benches at the side walls, or those facing north. In addition, in gerberas,
flower yield is affected by glasshouse type and replications within the glasshouse (Table
6). Significant differences could be observed not only between genotypes but also
between location (glasshouses), and with respect to genotype/location interactions.
 65

Those results showed that selection for yield in glasshouse crops is influenced by the
site of planting and that different genotypes may interact differently with a given
environment. When breeding non-branching strains of snapdragons for forcing,
which should be able to tolerate the low-light conditions of winter months, clear-cut
differences were found between strains with respect to the non-branching habit;
however, plants within strains varied with respect to side shoot number and length
according to their position on the greenhouse bench, insofar as edge rows developed
more and longer branches than inner rows. Spike quality was not influenced by planting
site. Generally, all the observed effects could be explained in terms of microclimate, i.e.
light and temperature, indicating that selection under glasshouse conditions must be
conducted at carefully chosen sites.

TABLE 5. Effect of mass selection without (MS) and with progeny testing (PT)
on% of unwanted types in a population (in F2 p(A) = q(a) = 0.5; selection coeff.
s= 1),a=selectionforaa, b=selectionfor A. (after Kappert 1953,expanded)
F3 F4 F5 F6

Self-pollinated crops
MS a 0 0 0 0
b 16.7 10.0 05.5 02.94
PT a 0 0 0 0
b 16.7 0 0 0
Cross-pollinated crops
MS befure pollination a 0 0 0 0
b 11.1 06.3 04.0 02.77
PT befure pollination a 0 0 0 0
b 11.1 02.8 0.7 00.17
Cross-pollinated crops
MS after pollination a 50.0 25.0 12.5 06.25
b 16.7 12.5 09.6 07.76
PT after pollination a 50.0 25.0 12.5 06.25
b 16.7 10.4 06.9 04.51

As already mentioned, some floriculture crops have to be bred for highly specialized
conditions, such as plug and pack production in glasshouses in early spring.
Culturing plugs often includes the utilization of additional lighting to promote
seedling growth. Selection of improved cultivars therefore must take into account the
source of radiation, as different light sources affect plant habit in different ways.
According to Zimmer (1986), elongation of the main axis and branching in Petunia are
strongly affected by a 20-day treatment with supplementary light of different spectral
composition. Differences exist between cultivars regarding the reponse to different light
spectra. Selection under relevant long-day conditions will result in cultivars which
remain compact and branch well, while selection without considering day-length
extension with artificial lighting systems will lead to unsatisfying results. Dwarf
66

genotypes can be selected under far-red light (which promotes elongation), because they
will remain relatively compact under that light regime. On the other hand, the number
of days to flowering in petunias is shortest under long days. When quantity of irradiance
is a major factor for earliness, as in geranium, the selection environment should be kept
at a rather low light level. If, however, day length plays a major role, as in Petunia,
selection under short days is the most discriminating environment (Van Kester, 1986).

TABLE 6. Gerbera, analysis of variance for yield characters

Source of DF MS flower no. MS shoot no. in
variation Jun-Dec Oct

Clones 50 93.4 * 11.46*

Locations I 1176.96** 4.71
Reps.in Joe. 2 180,6 •• 11.79**
Clones X Joe. 50 25.89* 3.57
Error 100 15.4 2.23
Total 203 44,65 4.94

In species responding to photoperiod, the same considerations are valid when

selecting for critical day length (which also depends on temperature), for reaction time
(i.e. the time needed from start of day-length treatment to the beginning of flowering),
or necessary number of day-night cycles for induction or sufficient flower quality. In
chrysanths, for example, the necessary number of day-night cycles for induction of
different cultivars varies between about 10 days and 6 weeks, critical day length
between 11 and J5 h, and reaction time in seedling populations from 43 to 90 days.
Further information on selection for production traits in flower crops is given by
Langton (1991).

3.1.3. Control ofCross-Pollination

The success of mass selection is limited due to the fact that pollination is not controlled,
and selected superior plants, therefore, are also pollinated by inferior pollen parents.
The disadvantages of uncontrolled pollination can be avoided by several advantageous
properties of ornamental plant species. They usually develop many flowers that will
open in succession, and quite a number can be successfully transplanted during
flowering time. In this way, it is possible to use the ftrst flower to score floral quality
and at the same time, for example, growth type. Superior plants are then dug up, and
potted or replanted after open flowers have been removed, and inter-pollinated in an
isolated plot. As a result, open pollination by superior pollen parents is secured. The
same aim is reached if a plant species can be propagated asexually. Fortunately,
development of micropropagation by in-vitro techniques now permits asexual
propagation in most cases. Superior plants are selected, self-pollinated or test-crossed,
and clones are established. After the sexual progeny have been completely assessed, the
clonal progeny of plants with good breeding value are transplanted to an isolated plot
where only the superior genotypes inter-pollinate in the same or following season (Fig.
 67

3). Herbaceous perennials can be handled in a similar way: selection in the ftrst season,
growing the sexual progeny of selected plants in the second, transplanting superior
phenotypes with a good breeding value into an isolated area, and inter-pollination in the
third year.
Source population Clones

00 0 1 11111111111111111111
Selection
0 ·----~~~~------------- 2 111111111111
3 111111111111
4 111111111111
5 11111111111111
6 1111111111111

Progeny 1 • • • • 2 o• oo oo
3 • • 4 • • • • 5 o• oo 6 • • • • 1 11111111111111
. •••• oo•• o••• •••• oeoo ••••
testing • • • • • • 0 • o• oo • • • • • o• • • • • • 2 11-1111 II 1111 111'111
•••• oeeo oeeo •••• ooeo •••• 3 11..1111 11111111
•••• oeoo eoee •••• eoee ••••
4 111111111111111111
5 lUI II 1111 11111
6 1111111111111111

Seed •••••••••••• •••

•••••••••••• ••• •••••••
•••••••
production •••••••••••• •••
•••••••••••• ••• • ••••••
•••••••
•••••••••••• •••
•••••••••••• ••• •• ••••••
••••••
Seed for sale Breeders seed
Figure 3. Improved mass selection through cloning (modified after Reimann-Philipp 1969).

In floricultural plant breeding, emasculation by hand followed by bagging of plants

or individual flowers, to exclude pollen-carrying insects, and hand-pollination are often
used to control pollination. Male sterility has become an important tool for ensuring
cross-pollination, and is genically (gms) or cytoplasmatically (ems) determined. The
separation of male and female flowers in monoecious Begonia species, where they are
on different parts of the same inflorescence, also permits control of cross-pollination. In
species carrying hermaphroditic (including both sexes) flowers, obligate cross-
pollination can be achieved by protandry or protogyny, as well as through self-
incompatibility. In particular, male sterility and self-incompatibility offer an alternative
to hand-emasculation and subsequent hand-pollination. Nevertheless, in Petunia, where
ems occurs (Izhar, 1984) and has been used in breeding, and self-incompatibility is
68

available, breeders prefer manual operations because of other advantages offered in that
species, such as easy flower handling and many seeds per pollinated flower (Ewart,
1984). In pansy, hercogamy (position of male and female organs that prevents self-
pollination) eliminates the need for hand-emasculation.
In self-incompatibility systems, one or more multiallelic S loci impose a
physiological barrier between pollination and fertilization while male and female
gametes are fully functional. Fertilization is prevented whenever an S allele in the
pollen grain matches the allele in the pistil. Self-incompatibility can be surmounted by
bud-pollination, by end-of-season pollination or by a high-temperature treatment. Self-
incompatibility systems in floriculture crops have been described by Ascher (1976), and
occur in many species, such as Ageratum, Bellis, Heliotropium (Reimann-Philipp,
1983), Lilium, Petunia, Nemesia, Oenothera, Chrysanthemum, Nicotiana sanderae,
Cosmos bipinnatus, Iberis amara, and Primula, which show heterostyly, a
heteromorphic self-incompatibility system. That system is inherited differently from the
multiallelic S locus insofar that it is controlled by a single locus with two alleles
designated S/s. The long-styled pin genotype (short stamen) is homozygous recessive,
the thrum genotype (short style, long stamens) heterozygous. Heterostyly also occurs,
inter alia, in several species of Limonium, Pentas and Lythrum.

3.1.4. Synthetic Cultivars

Synthetic cultivars are developed from several open-pollinated clones or inbred lines,
which have been tested for performance and general combining ability. The parental
generation is called syn 0. Synthetic cultivars of floriculture crops are rarely found. In
the amphitetraploid pansy (Viola wittrockiana), a syn 1 varietal series is on the market,
which is produced from intercrossing several clones. Trang (1979) compared the
performance of synthetics and F1 hybrids in Viola, and found, in several characters,
better performance with the synthetic varieties.

3.1.5. Early Use ofMorphological Markers

Self-pollinated stocks (Matthiola incana) have garnered the attention of seedsmen and
plant breeders since at least the middle of the 18th century, when seed growers in Erfurt,
Germany tried to develop strains with a high percentage of plants with double flowers.
Such plants, however, are completely sterile due to peta1oidy, and must be grown from
heterozygous (Ss) single-flowered plants. Besides such normal stocks, an eversporting
type became known, which on selfmg segregated into 1 single : 1 double (Saunders,
1911). Frost (1915) postulated linkage between the allele (S) for single flowers and a
gametic lethal allele (let) which sterilizes the pollen carrying these alleles, S let. Kappert
(1937, 1940) proved that linkage and found an additional linkage, let C /+ c, cc
rendering cotyledons and leaves light green (Table 7). If all seedlings with deep green
cotyledons are discarded, a 100% double-flowering progeny will result. That principle
of selectable stocks has been patented and is widely used in stock breeding. When,
however, the marker was to be transferred to other strains by backcrossing, such strains
had to serve as female parent, otherwise the marker was lost after the ftrst cross due to
the sterility of double flowers. All selectable stock types for greenhouse forcing carry
that marker in Europe.
 69

TABLE 7. Inheritance of double flowers in Matthiola

SletC s+ c

S !etC S let C I s + c single dark green

s+ c s + c I s + c double light green

The slender trisomic (Frost et al., 1959) has conspicuous narrow, long-petioled
leaves. Its extra chromosome fragment is transmitted by as much as 18% of the pollen
and more extensively by the ovules. Thus, slender can be used as a marker, and when
trisomies are eliminated at the seedling stage, the percentage of doubles in the normal
disomic progeny is relatively high (Table 8). Trisomic stocks are available mainly in the
USA.

TABLE 8. Progeny from sel:fing trisomic slender (Sl) Matthiola (from Frost et al., 1959)
Parent %double %double %double % no.
disomic trisomic total slender plants

1 s+s+ISI 94.2 19.0 72.6 28.8 146

2 s+s+IS I 98.1 7.1 67.1 34.1 82
3 s+s+ISI 96.1 25.0 76.6 27.4 175
4 Sls+ls+ 72.9 25.2 51.3 45.3 236

3.1.6. Breeding Cyclamen persicum

From the different species of Cyclamen, only C. persicum has contributed to floriculture
as a pot plant. Indigenous to the east Mediterranean, from the Peloponnesus to Cyprus,
and from southern Turkey to Israel, it turned up in Europe in the early 17th century.
Several flower color types were described in the 18th century. In France and Great
Britain, prior to 1850, selection had already produced new flower colors, but due to
heterozygosity, no true breeding cultivars originated. In the last third of the 19th century,
a new type with large flowers was found, apparently the first autotetraploid,
encouraging breeders in different countries to intensify breeding. According to
Doorenbos {1950), German breeders were the most successful from 1895 to 1940.
Breeding research has since investigated chromosome numbers and the inheritance of
flower and foliage characters, and a number of major genes have been found (Seyffert,
1955, 1971; Wellensiek et al., 1961). Elsherif (2000) described the enzymes of the
flavonoid biosynthetic pathway. Beside different flower colors (cyanidin and
delphinidin derivatives, and flavonoles), petal shape and edge (fimbriatum,
denticulatum, undulatum, each controlled by one dominant allele, and homozygous
recessive marginatum) are of interest. A new recessive yellow flower color, already
mentioned in old records then lost, containing the chalcone isosalipurposide, has
recently been developed in Japan (Miyajima et al., 1991; Takamura et al., 2000).
Many open-pollinated diploid and tetraploid cultivars have been developed through
crossing within species, some self-pollination, and mass selection adjusted to
 70

glasshouse conditions (Fig. 4). For instance, from open-pollinated population A, a

phenotypically superior population B is selected, kept in a separate glasshouse, and
hand-pollinated with pollen from a larger or smaller group C of top phenotypes. The
plants of group C are also inter-pollinated and produce, after bulking, the breeder's
seeds for the next generation, while population B produces the seeds for sale. Breeding
aims concern characters of flowers (color, size, petal number and shape, form of petal
edge), pedicel (length, sturdy, ascending), leaf (pattern, size), and plant habit. Many
new flower colors have been produced, such as deep crimson, blue-mauve, salmon-
scarlet and vivid scarlet. Additionally, uniformity of cultivar, good germination ability,
and the shortest possible production time are sought. There are cultivars bred for use as
cut flowers which should produce at least 40 saleable flowers per pot. Since
uniformity, a high proportion of saleable plants, and fast production are best
obtained in F1 hybrid cultivars, such cultivars have been developed at the diploid level
in the Netherlands since 1970, and more recently in other countries. They hold a
remarkable share of the market. At present, in-vitro technology is utilized to propagate
breeding stock asexually (micropropagation, somatic embryos) (Bach eta/., 1998).

Source population A

0000000000000000000000000
0000000000000000000000000
0000000000000000000000000
0000000000000000000000000

B c0
000000000 000
/ 000000000 000 "
If 000000000 000 ~

Seed for sale Breeders seed

Figure 4. Mass selection under glasshouse conditions (Horn, 1979).

Because of many disease agents, breeding for resistance is of interest, especially to

Fusarium oxysporum f.sp. cyclaminis. No resistance is, however, available in C.
persicum (n = 24, 48) but it is found, for example, in C. purpurascens (n = 34). As no
interspecific crosses between C. persicum and other species have been successful by
conventional means (only apomictic seed is formed), interspecific persicum x
purpurascens hybrids have been produced using ovary culture for transfer of Fusarium
resistance (lshizaka and Uematsu, 1995; Ewald, 1996). Seed from spontaneous
amphidiploid hybrids (2n = 82) as well as from its crosses to tetraploid C. persicum has
been obtained (Ewald et al., 2000).
 71

3.2. BREEDING OF HYBRID CULTIVARS

The term hybrid cultivar designates Fl populations that are used for commercial
plantings. Fl hybrid cultivars of many floriculture species are on the market.
Nevertheless, the proportion ofF1 hybrids to the total number of floriculture species is
much lower than for vegetables, for example. Historically, the first diploid hybrid
cultivar 'Erfordia', resulting from the cross of two Begonia species, was introduced in
Erfurt, Germany, in 1894. The hybrid was nearly sterile, so the cross had to be repeated
yearly to reproduce the cultivar, and was traded until 1964. From about 1950, steady
development of new Fl hybrids began. At present, hybrid cultivars play a major, if not
leading role in Ageratum, Anemone, Antirrhinum, Aquilegia, Begonia (wax begonias,
tuberous-rooted begonias), Brassica (ornamental), Calceolaria, Capsicum (ornamental),
Cyclamen, Dahlia, Dianthus, Eustoma, Godetia, Helianthus, Impatiens walleriana,
Leucanthemum, Lobelia speciosa, Mimulus, Nicotiana, Pelargonium, Petunia, Primula,
Ranunculus, Sinningia, Tagetes, Viola, and Zinnia.

3.2.1. The Basic Scheme

Fl hybrid cultivars can be obtained by crossing clones, open-pollinated varieties, inbred
lines, or other populations that are genetically dissimilar but homozygous in the
important characters. Hybrid cultivars make better use of heterosis than any breeding
procedure. Usually, potential parents are selected in open-pollinated populations, and
selfed through several generations to produce homozygous inbred lines. In the I 1
generation only the least desirable lines are discarded, and from 12 onwards, single plant
selections are made from the best lines only. Inbreeding may be virtually unnecessary,
or one generation of inbreeding adequate in homogeneous cultivars. Inbred lines are
tested for combining ability (e.g. through top-crosses), i.e. the ability to produce
heterozygous but uniformly superior hybrids. All inbred lines with good general
combining ability are then combined in pairs (Fig. 5). Two complementary lines with
the best specific combining ability and capable of satisfactory seed production are then
crossed to produce so-called single-cross hybrids. The parental lines should allow some
kind of pollination control (see 3.1. 3). Usually two or more rows of the male sterile seed
parent are planted to each row of the pollen parent if hybrid seed is produced in the
field.
A review on hybrid vigor and techniques of pollination control in floricultural crops
is given by Reimann-Philipp (1983). Most of the floricultural Fl hybrid cultivars are
produced in glasshouses through hand-emasculation and hand-pollination. Inbred seed
and pollen parents are often vegetatively propagated, mainly through micropropagation.
To eliminate the emasculation step, male sterility (e.g. Antirrhinum, Impatiens
walleriana), heterostyly (Primula), self-incompatibilty (e.g. Ageratum), monoecy
(Begonia) or hercogamy (Viola) are utilized. In California, hybrids of Tagetes and
Zinnia are produced in the field utilizing apetalous female parents. A distinction is made
with regard to seed and pollen parent only if male sterile or apetalous seed parents are
used. In other cases, seed can be produced on either parent. Certain problems arise using
the most common monogenically controlled homozygous recessive male sterile (ms ms)
or apetalous lines. Maintaining such lines requires backcrossing with heterozygous
fertile genotypes (Ms ms) repeated in every generation. Seeds must always be harvested
from ms ms plants and give equal proportions of ms ms and Ms ms plants. For the
72

production of Fl seed, especially in the field, Ms ms male fertiles must be removed

from the rows of the female parent line as soon as identification is possible. For a few
years now, male sterile genotypes have been micropropagated in vitro, making the
repeated backcross to male-fertile types unnecessary.

Source
Population I populations Population II

Hybrid cultivar

Figure 5. Basic scheme for breeding Fl hybrid cultivars (after Becker, 1993).

The first flower species to be used for Fl hybrid production were those which could
be easily inbred, in which spontaneous self-pollination could easily be prevented, and
cross-pollination was easy, and which also gave large numbers of seeds per hand-
pollinated flower. Thus, monoecious begonias and petunias were the first on the market,
followed by species with self-incompatibility or male sterility, because realization of
crossing as well as prevention of self-pollination had to be guaranteed. Breeding of
Petunia cultivars and hybrid seed production has been described by Ewart (1984).
Generally, commercial quantities of hybrid seed of floriculture species are produced in
glasshouses by hand-pollination and if necessary, hand-emasculation, and in suitable
climates also in the field. In Europe, most hybrid seed of flower crops is produced in
glasshouses by hand-pollination, meaning, in addition to the high cost of development,
 73

high cost of seed production, and consequently high seed costs compared to a
conventional cultivar. These high costs are usually prohibitive for floriculture crop
species of limited significance.
The advantages of F1 hybrids can be briefly stated: uniformity in all selected
characters, combination of dominant alleles, greater vigor expressed as flower
production, better germination, and faster growth. An additional advantage is
exclusivity of control by the breeder since, as a consequence of their heterozygosity,
hybrid cultivars segregate in F2. Thus, there is built-in protection against unauthorized
seed propagation, which is welcome since for seed-propagated floriculture species,
breeders' rights do not exist. The original parent inbreds are usually owned exclusively
by the particular seed company. In a few cases, F2 hybrids are on the market, for
instance in Portulaca, Delphinium and Viola, which show only limited segregation due
to homozygosity ofF1 in important characters. They are produced by open-pollination
of hybrid cultivars in an isolated field area, and contribute to reducing the costs ofF1
development.

3.2.2. Production ofHybrid Cultivars in Begonia x semperflorens-cultorum

Wax begonias, sometimes called fibrous-rooted begonias, are a fine example of the
evolution of a bedding-plant species, and the significance of polyploidy and Fl hybrids.
As spontaneous self-pollination can easily be prevented, cross-pollination and
emasculation are made easy in this monoecious species; moreover, as large numbers of
seeds result per hand-pollinated flowers, begonias became the first species in which Fl
hybrid cultivars were developed. This most important bedding-plant species is derived
from the south Brazilian species Begonia cucullata var. hookeri (synon. B.
semperjlorens), and reached Germany purely by chance in 1821, and Great Britain in
1828. After 1870, first selection for true breeding bedding types started in France and
Germany. When B. Schmidtiana, also from southern Brazil was introduced in 1880,
crosses between these species resulted in 1894 in 'Erfordia' (syn. 'Bliitenmeer',
'Rosamunde'), a uniform but nearly sterile F1 hybrid which had to be produced anew
yearly from the original parents. It showed hybrid vigor with regard to flower number
and tolerance to adverse weather conditions. Its further development has been described
by Skiebe (1966a).
Early breeders then tried to select a fertile and true-breeding Fl generation of the
cross, and most probably developed an F2 generation, as well as BlFl progenies from
backcrossing the F1 to both parental species. In each case, polyploids emerged in single
triploid plants originating via the occurrence of non-reduced gametes, and from further
inter-pollination of eventually fully fertile tetraploids in F3 or BlF2, respectively.
Skiebe (1966a) could produce tetraploids directly in BlF2 from a backcross to B.
cucullata. Originally, the fully fertile tetraploids, being very floriferous with a compact
habit and termed gracilis type, appeared for the first time in Germany and France in
about 1895. Original gracilis types produced fertile progeny only after crosses to
Skiebe's B1F2 from the backcross to B. cucullata var. hookeri, while crossing with the
other BlF2 resulted in sterile progeny; thus that it seems justified to assume that the
natural tetraploids evolved via cucullata backcrosses. Tetraploid true breeding cultivars
were then developed after inbreeding and inter-crossing, and in 1909, a tetraploid Fl
hybrid cultivar ('Primadonna') was introduced which surpassed the true breeding
74

cultivars in hybrid vigor, and which was produced from parent clones until 1961
(Skiebe, 1966a).
Another milestone in begonia breeding was the development of a triploid hybrid
cultivar ('Rosa Tausendschon', syn. 'Rosalinde') in 1934, which, due to its sterility, had
an exceptionally long performance season as a bedding plant. At present, triploid
hybrids are produced from inbred lines (inbred and cloned parents)-to that aim
population I (Fig. 5) is tetraploid and population II is diploid-just as some diploid and
tetraploid hybrid cultivars are. In seed parents, male flowers are removed and
pollinations are carried out by hand using male flowers of pollen parents, which are kept
in a different glasshouse. New tetraploids were selected for combining ability and
proved to be superior parents in the production of further improved triploid and
tetraploid hybrid cultivars.
There are a few studies on inheritance of flower and leaf color, flower doubleness,
and habit (see Reimann-Philipp and Lorenz, 1975). The most important breeding aims
in wax begonias are early start of flowering and long performance season from spring to
frost, tolerance to adverse weather conditions, flower color (scarlet, red, rose, white,
bicolored), leaf color (green, bronze) and size, flower size and number, habit (compact,
tall). In recent years, new interspecific hybrids have been developed from crosses
between B. x semperjlorens-cultorum and not yet publicly known species. It should be
mentioned that double-flowering cultivars are vegetatively propagated.

4. Breeding Vegetatively Propagated Crops

4.1. THE BASIC SCHEME

Especially in early times and after interspecific hybridization, it often happened that the
desired flower colors and plant forms did not come true from seed. Then, asexual
propagation resulting in clonal cultivars was, and still is, the only way to maintain a
desired genotype. In the early days of floriculture plant breeding, nearly all new types
were first vegetatively propagated, and at present, a great number of important species
are still increased that way. There are a number of floriculture species which develop
vegetative propagules, such as bulbs and corms. As examples, gladioli, tulips, and
daffodils can be mentioned. Because of high heterozygosity and a mostly polyploid
condition, others are propagated by budding or grafting (e.g. roses), cuttings (e.g.
pelargoniums, carnations) or division (hardy herbaceous perennials). To improve,
however, such species, either crosses followed by one or more cycles of sexual
propagation and selection, or clonal selection are required. The induction of somatic
mutations will be treated in another chapter in this book. The basic scheme for breeding
new cultivars of vegetatively propagated crops is given in Fig. 6.
The first step is to select and cross suitable parents which are taken from existing
cultivars, advanced seedling populations, or (rarely) inbred lines. It is widespread
practice to base that selection on previous experience with the potential parent clone
population or on the performance of the clones themselves. Often some kind of a
limited top-cross is made, i.e. candidate parents are crossed to clones of known merit to
assess their hereditary value, or are inbred to see whether desired traits are inherited,
and rarely, to increase the genetic variation. Heterosis (hybrid vigor) is a major yield
 75

factor in clonal cultivars. To optimize the heterosis effect in the seedling progeny,
which usually contains the new clonal cultivar, unrelated parents should be crossed, and
inbreeding should be avoided. In a second step, superior individuals are selected in the
Fl seedling population and screened in several cycles. Often a backcross might be
advisable so that selection starts in BlFl. Selected plants are asexually propagated.

o o&o o&o o&o o o o o o o Source population

oliE_o o o o o o o o o&,o o&,o

(F 1 or B1F1)

o o o&,o o o o o&,o o o o o o
o&,o o o o o&,o o o o o&,o o

A-Clones

B-Ciones

C-Ciones

D New
Clone cultivar

Figure 6. The basic scheme for breeding clone cultivars (from Horn, 1979).

The first clonal generation of separately kept seedling genotypes (A-clones) is

evaluated, and increased by vegetative propagules or by micropropagation in vitro
which permits rapid propagation to give the next clonal generation (B-clones). B-clones
are tested in replicated trials and superior genotypes propagated to C-clones. Depending
upon the multiplication factor, B- or C-clones are tested at different locations, often on
different continents, to select genotypes suitable to different climates. If the best
genotype for each location is found, it will be vegetatively propagated for marketing as
a new clone cultivar. Before the advent of micropropagation, more clonal generations
and more time were required for breeding a new clonal cultivar.
76

4.2. CORRELATION BETWEEN SEEDLING AND CLONAL GENERATION

Since genes controlling a certain trait are obviously identical in an individual genotype
and its clonal progeny, the genetic correlation coefficient between seedling and clone
should be equal to one. That would considerably facilitate early selection, also called
preselection, for desired characters. Experience, however, has shown that the
correlation between the two generations is not as significant as expected. This may be
due to the fact that other genes act in different ontogenetic phases, to epistatic effects,
and to genotype-environment interactions. Early selection using seedling/clone
correlations is especially useful in crops such as tulips, where several years are required
from the seedling year until first flower (Van Eijk and Legwater, 1975; Weber and
Horn, 1978; Horn et a!., 1979). These authors investigated bulb weight and bulb
number of tulips in several clonal generations and found high genetic correlations
between the second year (first =year of sowing) and the fifth. Thus, selection for bulb
yield can begin in the second year. In Table 9, correlations between seedlings and clonal
progeny of Pelargonium are given. They demonstrate that early selection in the seedling
year is promising in that species.

TABLE 9. Correlation coefficients between seedling and clonal generation in

Pelargonium hortorum. rph, r8: phenotypic and genotypic correlation coefficient,
respectively (from Hom eta/., 1979)
Character Tpb rg

Plant height 0.86 0.89

Days to first flower 0.72 0.68
No. of nodes to first infloresc. 0.64 0.60
No. of nodes between 1" and 2nd infloresc. 0.66 0.88
First inflorescence, size 0.71 0.72
No. of flowers 0.75 0.84
Size of first floret 0.84 0.85

4.3. CLONAL SELECTION

Clones remain stable until somatic mutations (sports) occur spontaneously. Genetic
variability arises frequently in this way, and is often expressed as a chimera (flower
color, leaf pattern). Less obvious but of significance are genetic changes in continuously
varying traits. Selecting spontaneous mutants within a clone is called clonal selection.
The procedure is to select individual plants of a clonal cultivar, and to grow A-clones
and usually B-clones in replicated trials to test their performance, as well as their
distinctness and superiority compared with the original cultivar. Many cultivars of rose,
carnation, chrysanthemum, tulip, hyacinth, begonia and pot azalea arose as sports
(Horn, 1968; Broertjies eta!., 1980;, Heursel, 1999): in fact, 52% of all cultivars in pot
azaleas are sports. Selections for characters other than flower color, such as
photoperiodic reaction time or higher growth rate of pot plants, have been conducted,
 77

e.g. by Bech (1983), Christensen (1983), Ottosen and Christensen (1986) (Table 10),
Bech et al. (1985), and Schaper and Zimmer (1991 ). During selection, cultural practices
and optimal growth conditions must be maintained.

TABLE 10. Results of clonal selection in Crossandra and Kalanchoe (after Bech, 1983; Ottosen and
Christensen, 1986)
Crossandra Kalanchoe
Clone Days to No. of No. of Clone Reaction cv
no. anthesis infloresc. lat. shoots no. time

10 158 7.6 6.9 3 81.3 1.5

12 150 4.7 3.6 28 81.5 1.2
18 152 4.8 3.6 40 84.8 1.9
20 145 5.0 3.1 63 86.3 2.0
28 147 5.1 3.2 82 87.6 1.4
34 151 4.7 3.7
44 144 4.9 2.8
LSD 6 1.1 0.9 0.8

4.4. BREEDING PELARGONIUM HORTORUM

These pelargoniums (so-called geraniums) go back mainly to two South African

species, P. inquinans and P. zonale, first described in Germany, the Netherlands, and
England around 1700 (Wimmer, 1999). The first hybrid between those species was
observed in 1732, but planned hybridizations started around 60 years later (Knuth,
1912) in England and later on the European continent. After first tetraploid clones (2n =
4x = 36) of geraniums had arisen in France around 1865 (probably in a seedling
population), plant breeders in Europe and the USA developed many hundreds of clonal
cultivars at diploid and tetraploid levels. Since diploid and tetraploid genotypes are
cross-incompatible (Badr and Hom, 1971a, 1971b), clonal cultivars of both groups
existed until recently, though tetraploids were in the majority. Also around 1860, flower
doubleness and white flower color were found for the first time. A great number of
spontaneous somatic mutants affecting flower color, leaf color and pattern, as well as
continuously varying characters, have been selected. Examples of clonal selection are
given by Christensen (1983) (Table 11). Two other major events had an impact on
Pelargonium breeding: the occurrence of up to 18 different viruses and of bacterial
blight caused by Xanthomonas pelargonii in the 1950s, and the introduction of sexually
propagated, healthy and true-breeding (inbred) cultivars in Germany (1933) and the
USA (since 1963), which disappeared after some years due to inbreeding depression. In
the USA, improved seed technology (scarification in 1959), however, permitted the
development of Fl hybrid cultivars as well as genetic studies. Bacterial blight and
viruses could be controlled by culture-indexing in clonal cultivars, and by seed
propagation. There are several reviews of breeding research (Craig, 1971; Harney,
1976; Hom, 1994).
78

TABLE II. Clone selection in Pelargonium 'Pink Cloud' (from Christensen, 1983)
Clone Days to Plant Flower No. of No. of Leaf
no. flower height height shoots infloresc. area
(cnh
811 104.5 24.4 21.1 4.8 9.5 129.3
812 110.1 27.2 22.7 4.9 9.4 120.2
821 103.4 24.8 22.9 4.8 9.4 148.2
822 106.1 25.1 22.7 4.5 8.8 131.4
831 108.8 26.8 25.1 5.3 10.2 138.8
832 105.0 25.8 22.4 5.1 10.4 133.5

LSD 3.6 2.2 2.0 n.s. 1.3 16.3

4.4.1. Breeding Aims

Objectives in breeding Pelargonium hortorum are numerous. Concerning the habit, a
semi-dwarf compact, self-branching plant is desired, having a firm texture to withstand
damage by rain and wind-plant height might be taller for use as bedding or container
plants in certain climates-the leaves should not be too large, should have a dark green
color or be marked with a darker zone, should be tolerant to Botrytis (see section 2.6),
and resistant to rust (Puccinia pelargonii). There are special ornamental-leaved types.
Regarding flower characteristics, the individual floret of a clonal cultivar should be
fairly large, semi-double, non-shattering, have a flat, round, open form and a special
color. The inflorescence should be a round umbel borne on strong peduncles standing
well above the foliage, have a long decorative life, and should carry approx. 50 to 60
florets. The plant should carry an inflorescence at each or every second node. An array
of cultivars should be bred for different climates, culturally adapted to various areas.
Stock plants of clonal cultivars for propagation should produce a large number of
cuttings within a limited period of time, which root easily and quickly, which have good
shipping ability, and grow within a short time to saleable prefinished and finished
plants. When breeding sexually propagated cultivars selection for a short development
time from sowing to first and second flower is of prime importance. To date, resistance
to bacterial blight and viruses has not been found in cultivated types.

4.4.2. F1 Hybrid Cultivars

Programs to develop Fl hybrid cultivars at the diploid level started in several countries
(USA, 1958, Germany, 1958). With sexually propagated pelargoniums, a large part of
disease transfer from one generation to the next can be eliminated, and the labor, time
and space to maintain stock plants are saved. Varietal series were marketed first in the
USA in 1966, and later in the Netherlands, France and the United Kingdom. They hold
a remarkable share of the market. Tetraploid hybrid cultivars from Germany (1974)
failed due to insufficient seed yield.

5. Maintenance

Maintenance of the products of plant breeding-the cultivar-has to ensure its genetic

identity and purity. In ornamental plants, it is the responsibility of the plant breeder to
 79

maintain breeder or basic seed, and basic stock for vegetatively propagated species
respectively. In Europe, therefore, it is sometimes called maintenance breeding. Every
cultivar, whether open-pollinated or clonal, as well as the inbred lines for an Fl hybrid,
will deteriorate if not properly maintained. This is due to chance cross-pollinations from
stray pollen, mutations, pests, diseases and mechanical mixing when handling
propagules. In seed-propagated crops, the procedures for maintaining cultivar purity are
simple mass selection, i.e. planting small seed plots each year, roguing out the off-type
plants, and testing progenies of a number of single plants by the plant-to-row method.
The seed from approved plants or rows is then bulked to start a seed increase. Basic
seed lots of inbred lines are maintained by hand-pollination. Careful roguing is required
to remove any off-type plants followed by plant-to-row planting. There are, however,
several species where the inbreds, especially ems strains, are kept in vitro and
micropropagated. In such cases, maintenance follows the same principles as in
asexually propagated crops.
To maintain clonal cultivars, single plants are selected and tested for genetic identity
and health, and propagated for disease-free basic stock. This is an indispensable but
costly activity, and includes clonal selection (Christensen, 1983) as well as culture of
meristem tips to produce culture-virus-indexed nucleus stock plants. In Pelargonium,
for example, culture-indexing started in 1952, for bacterialleafspot {blight) in 1960, and
the first heat treatments for virus eradication began in 1958. The procedure to clean
stock plants takes at least 1 year, and until certified cuttings reach the grower, ca. 2
more years are required for propagation (Oglevee-O'Donovan, 1993; Westerhof and
van Ruiten, 1993).

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INTERSPECIFIC HYBRIDIZATION AND INTROGRESSION

J.M. VAN TUYL, 1 K.B. LIM, 1 M.S. RAMANNA2

1Plant Research International, (WUR)

P.O. Box 16, NL-6700AA Wageningen, The Netherlands

2Laboratory of Plant Breeding

Wageningen University and Research Center (WUR)

P.O. Box 386, NL-6700AJ Wageningen, The Netherlands

1. Introduction

Some of the economically important horticultural crops have two common features.
First, they are of interspecific hybrid origin and, second, they are mostly polyploids.
The histories of the origin of such ornamentals as Rosa, Narcissus, Iris, Crocus and
Chrysanthemum, among others, have been well documented (Stem, 1946; Wylie, 1952;
Darlington, 1976; Brighton eta/., 1980; Brandham, 1987) and they can be traced back
to 18th and 19th centuries or even earlier. Initially, most of these crops were diploids, the
polyploid forms originated spontaneously from interspecific hybrids in the breeders'
nurseries. In those early days, the knowledge regarding the status of the species, the
genomes and polyploidy was nonexistent; nevertheless the horticultural breeders were
successful in creating considerable genetic variation. These efforts were subsequently
responsible for the selection of thousands of horticultural varieties in some of those
crops. For example, in Narcissus, more than 25,000 cultivars have been recorded
(Throckmarton, 1980). In Lilium, which has probably followed a similar path of origin
as other horticultural crops, more than 7000 cultivars have been registered since 1960
alone (Leslie, 1982). The existence of thousands of cultivars in Tulipa is well
documented.
Despite the phenomenal improvement in our knowledge of species, interspecific
hybrids, genomes and polyploidy during the 20th century, breeding of horticultural crops
remained, for the most part, an art rather than a science. This was because, unlike in
most of the commercially important field crops, both professional and amateur breeders
were involved in breeding, such that information regarding the development of cultivars
was, in many cases, subjective at best. Therefore, it is important to elucidate the
cytogenetic composition of many of these crops in order to practice more systematic
breeding. Traditionally, the success or failure of producing interspecific hybrids and
their backcross progeny depended solely on the seed set after normal sexual
hybridization. However, because the prevalence of pre- and post-fertilization barriers to
crossing species, as well as the high degree of sterility of the F 1 hybrids, introgression in
plants has not always been successful and, when successful, was a highly laborious and
frustrating process. Nevertheless, the introduction of ovule culture and embryo-rescue
methods has greatly facilitated hybridization followed by backcrossing in a wide variety
85
A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, 85-103.
© 2002 Kluwer Academic Publishers.
86

of plant species (Hadley and Openshaw, 1980; VanTuyl eta/., 1990; Buitendijk eta/.,
1995; De Jeu and Jacobsen, 1995; Van Tuyl and De Jeu, 1997). Although these
techniques have opened the way to the production of interspecific hybrids and
backcross progeny on a large scale, the subsequent important steps of selecting
genotypes from the backcross derivatives, i.e., selection methods, are required. In this
context, the recently developed molecular techniques are potentially of great value for
improving the efficiency of introgression. These techniques include in situ DNA
hybridization for the identification of genomes and chromosomes, as well as
recombinant segments, and molecular (DNA) marker mapping methods such as RFLP,
RAPDS and AFLP. Some examples of horticultural crops in which in situ hybridization
has been practiced include Alstroemeria (Kamstra et al., 1997, 1999; Kuipers eta/.,
1997), Crocus (0rgaard eta/., 1995), ornamental Allium (Friesen eta/., 1997) and
Lilium (Karlov et a/., 1999; Lim et a/., 2000). Molecular markers have been used in
Alstroemeria (Picton and Hughes, 1997; Han et al., 1999), Allium (Friesen eta/., 1997)
and Lilium (Straathof et a/., 1995).
A combination of in situ hybridization and molecular markers is useful not only for
monitoring the hybrids and backcross progenies for introgression, but also for
elucidating the modes of origin of 2n gametes, the extent of genetic recombination and
the phylogeny of the species and hybrid cultivars. In the following pages, some of our
results on Lilium andAlstroemeria are briefly summarized and some of the literature on
the status of horticultural crops that are relevant in the context of interspecific
hybridization and polyploidy is reviewed.

2. Techniques for Overcoming Pre-Fertilization Barriers

2.1. GENETIC VARIATION IN INTERSPECIFIC CROSS ABILITY

"The statement, that two species are not crossable, is controversial unless a broad genetic
variation of the parental species has been used and the cross combinations have been
carried out on a large scale under a wide range of environmental conditions." This
quotation of Hermsen ( 1984) implies that crossability is determined by both genetic and
environmental factors. It is therefore necessary to test different accessions of both parents
for hybridization programs (Van Eijk et al., 1991). Unilateral incongruity is the
phenomenon whereby a cross is successful in only one direction, whereas the reciprocal
cross fails. In lily, crossing barriers can be overcome by using cut-style pollination, but
mostly in one cross direction only (Van Creij et al., 1993).

2.2. USE OF MIXED AND MENTOR POLLEN

The use of mixed pollen, i.e. a mixture of compatible and incongruous pollen (Kunishige
and Hirata, 1978) and mentor pollen, i.e. compatible pollen genetically inactivated by
irradiation but still capable of pollen-tube growth used together with incongruous pollen,
has been reported to overcome inhibition in the style in many plant species. For lily,
mentor pollen was effective in overcoming self-incompatibility, but not in interspecific
crosses (VanTuyl eta/., 1982).
 87

2.3.INFLUENCE OF ENVIRONMENTAL CONDITIONS

A positive effect of high temperature on overcoming incongruity has been detected and
applied in breeding programs of lily by effecting pollination at high temperatures (Van
Tuyl et a/., 1982; Okazaki and Murakami, 1992). In this case, heat-sensitive inhibitors of
pollen-tube growth are probably inactivated. Comparable effects of floral ageing on
pollen-tube growth have been reported by Ascher and Peloquin (1966).

2.4. STYLE AND OVARY MANIPULATIONS

Inhibition of pollen-tube growth in the style can be overcome using different pollination
techniques in which the style and ovary are manipulated (Fritillaria: Wietsma eta/., 1994;
Lilium: Myodo, 1963; VanTuyl eta/., 1988, 1991; Janson eta/., 1993). One of these
manipulations involves removal of the stigma and part or all of the style, and subsequently
pollination of the cut end. This is referred to as stump pollination, 'cut-style' or 'amputated-
style' pollination. In a comparison of several pollination methods, it was shown that pre-
fertilization barriers in lily could be circumvented by using the cut-style technique (Van
Tuyl eta/., 1991; Janson eta/., 1993). Following this technique, many pollen tubes of, for
example, lily and Friti/laria grow normally into the ovary. In this way, pollen circumvents
stylar and stigmata! barriers. However, a complication associated with this method in lily
is low seed set, probably caused by the premature arrival of pollen tubes in the ovary
(Janson eta/., 1993). Most of the pollen tubes either grew past the inner integument or
grew along, but not into the micropyle after cut-style pollination. Despite the low seed set,
a large number of unique interspecific lily hybrids were obtained using this method
(Asano and Myodo, 1977a,b; Asano, 1980; Okazaki eta/., 1992, 1994; Van Creij eta/.,
1993). In addition, Wietsma eta/. (1994) were able to obtain interspecific hybrids using
the cut-style technique in crosses between Fritillaria imperialis and F. raddeana.
The grafted-style technique successfully improved the cut-style technique (VanTuyl
eta/., 1991). In this method, pollen grains are deposited on a compatible stigma. After 1
day, the style of the pollen donor is cut 1 to 2 mm above the ovary and grafted onto the
ovary of another incongruent plant. Style and stigma are joined in vivo using a section of a
straw filled with, in this case, Lilium longiflorum stigmatic exudates, or are stuck together
with only the exudate. In vitro, a piece of 'water agar' is placed on the style.

2.5. CHEMICAL TREATMENTS

Pollen-tube growth and penetration are essential processes for fertilization between
remote species. In the remote crosses, the pollen on the stigma cannot germinate, or its
germination is inhibited by interspecific cross incompatibility.
L. longiflorum pistils secrete large amounts of exudate. The stylar canal is also
covered with the same exudate, which is composed of a gel-like solution containing
protein, polysaccharide, phenolic compounds and lipids (Labarca eta/., 1970).
Specific proteins and lipids of the exudate from the stigma inhibit or accelerate the
pollen germination (Martin, 1970). Some of specific lipids, such as trilinolein, promote
the growth and penetration of pollen tube into the pistil. These lipids can be used to
overcome the cross incompatibility (Wolters-Arts eta/., 1998).
Application of growth regulators, such as auxins, cytokinins and gibberellins, to the
88

pedicel or ovary at the time of, or soon after pollination, may improve fruit and seed set
after interspecific crosses in lily and tulip (Emsweller and Stuart, 1948; Van Creij eta/..
1999).

3. Techniques for Overcoming Post-Fertilization Barrien

Interspecific hybridization relies heavily on the genotype combination among species.

Fertilization can be classified into embryo and endosperm formation, the so-called
double-fertilization discovered by Navashin (1898). Fertilization failure in interspecific
hybridization is assumed to be due mainly to the genetic discrepancy and chromosome
degeneration during cell division of the zygote. In interspecific hybridization of Lilium,
embryo and endosperm formation and development can be categorized into different
groups. Because embryo development is highly affected by the development of
endosperm, if there is no endosperm formation, the embryo can normally no longer
survive. In this case, very early embryo rescue, such as ovary slice culture, may be
helpful. There are several prerequisites to interspecific hybridization which contribute to
its success. First, fertilization has to be successful with both the embryo and endosperm.
Second, embryo development in each cross combination has to be checked. Third,
endosperm development in each cross combination also has to be monitored. Based on
the results of the cross combination, suitable rescue methods can be applied. In the case
of early embryo degeneration, the ovary slice culture method is preferred. When the
embryos degenerate in more middle stages of development, ovule culture can be
applied.
Making good cross combinations is another method of overcoming incompatibility.
Genetically speaking, a certain cross combination has specific cross incompatibility. In
lily, for example, an LA (Longiflorum x Asiatic hybrid) x A hybrid cross exhibits better
fertilization than LA x L crosses. Similarly, an A x LA cross exhibits higher
fertilization frequencies than an L x LA cross. In these cases, all parents produce normal
female and male gametes; however, the frequency of successful fertilization is
dramatically different.

3.l.OVARY CULTURE AND OVARY SLICE CULTURE

Ovary culture has been applied in many species: Lilium, Nerine and Tulipa (VanTuyl et
a/., 1990; Van Creij eta/., 1999b). Ovary-slice culture was applied by Kanoh eta/. (1988),
and Van Tuyl et a/. ( 1991) for the production of interspecific Lilium hybrids. Ovaries were
harvested 7 to 40 days after cut-style pollination and, after surface-sterilization, sliced into
2.0-mm thick disks. Seed germination occurred 30-150 days after pollination. By this
method, plantlets were obtained from very small embryos.

3.2. OVULE CULTURE

In those crops in which the fruit is aborted before embryo culture can be applied, ovule
culture is an easy and rapid alternative. This technique is applied inA/stroemeria (Bridgen
eta/., 1989, Buitendijk eta/., 1995; De Jeu and Jacobsen, 1995), lily, Nerine (Van Tuylet
 89

al., 1990) and tulip (VanTuyl et al., 1990; Van Creij eta/., 1999b).

3.3. EMBRYO CULTURE

Embryo culture can be applied successfully in crosses in which the pollinated flowers stay
on the plant for a sizeable length of time. In most cases, embryos can be rescued when the
globular stage is reached. This method has been applied in a large number of crops. Some
examples in flower bulbs are: Allium (Nomura and Oosawa, 1990), Alstroemeria
(Buitendijk et al., 1992), Freesia (Reiser and Ziessler, 1989), Hippeastrum (Bell, 1972),
Li/ium (VanTuyl et al., 1991), Tulipa (Custers et al., 1995) and Zantedeschia (Yaoetal.,
1995).

3.4.1N VITRO POLLINATION

In many interspecific and intergeneric crosses, integrated techniques that manipulate both
pre- and post-fertilization barriers have been applied. In vitro pollination and fertilization
is one such technique (Zenkteler, 1990). Unlike other techniques which retain the zone of
inhibition (stigma and style) and manipulate pollen germination and pollen-tube growth to
overcome pre-fertilization barriers, in vitro pollination brings pollen grains in direct
contact with the ovules, and is, therefore, considered more effective.
In Lilium, various combinations of in vitro pollination (cut-style and grafted-style
method) and embryo rescue (ovary, ovule and embryo culture, placental pollination), were
applied to control the whole fertilization process (VanTuyl et al., 1991; Janson, 1993).
This resulted in a range of new interspecific hybrids (VanTuyl et al., 1990; Van Creij et
a/., 1993). Similar results were obtained from interspecific crosses in Tulipa and
intergeneric crosses between Nerine and Amaryllis (VanTuyl eta/., 1990, 1991; Van
Creij et al., 1999b). To date, in vitro fertilization in bulbous crops has not been achieved
using isolated sperm and eggs.

4. Techniques for Overcoming F 1-Sterility

4.1 CHROMOSOME DOUBLING

With only very few exceptions, F 1 hybrids from distantly related plant species are
highly sterile. In most cases, sterility results from a failure in chromosome pairing
during meiosis which leads to the formation of spores with unbalanced chromosome
constitution leading to sterility. The most widely used method of restoring fertility in
interspecific hybrids is to double the chromosome number in F1 to produce an
allopolyploid (also called a disomic polyploid). In such plants, meiosis will be normal
because of the regular chromosome pairing during metaphase I followed by regular
divisions during subsequent stages, and fertility restored. One sizeable drawback of this
approach is, however, that because of the preferential pairing of chromosomes between
the constituent genomes of the hybrid, the possibility for homoeologous chromosome
pairing and crossing-over is minitnal, if not totally nonexistent. Since homoeologous
recombination is a crucial prerequisite to introgressing specific desirable characteristics
into a cultivar, chromosome doubling of the F 1 hybrid is not a desirable method.
90

Figure I. Genomic in situ hybridization of mitotic chromosomes ofF~, BC. and BC2 plants of LR and LA
hybrids. (a) The meiotic chromosomes of BC 1 (LLR) with 12 bivalents (yellow fluorescence) indicating L.
longiflorum and 12 univalents (red fluorescence) representing L. Rubellum; (b) 36 chromosomes of the BC1 of
LLR without any recombinations; L. longiflorum (yellow fluorescence) and L. rubellum (red fluorescence);
(c) aneuploid BC 2 plant from backcrossing the BC 1 (LLR) to 4x L. longiflorum (LLLR); 36 (three sets) of L.
longiflorum (yellow fluorescence) with 8 L. rubellum chromosomes (red fluorescence). (d) The meiotic
chromosomes of LA in metaphase I with two bivalents indicating chromosome association of L. longiflorum
(yellow) and Asiatic (red) (e); 36 chromosomes of the ALA plant with three recombinations; L. longiflorum
(yellow fluorescence) and L rubellum (blue fluorescence); (f) aneuploid BC2 plant (2n=30) from backcrossing
of the BC 1 (ALA) hybrids to diploid Asiatic; five chromosomes of L. longiflorum (green fluorescence) and 25
Asiatic chromosomes including two recombinant chromosomes.

However, whole chromosomes can be added to the backcross progeny, as has been
demonstrated in diploid (2n=2x=24) hybrid Lilium longiflorum x L. rubel/um and their
BC 1 progeny using GISH (Lim et a/., 2000; Fig. 1). However, in this intersectional
 91

hybrid there is evidence of homoeologous chromosome pairing and crossing-over,

although such recombinant products cannot be recovered in the progeny via
chromosome doubling. As described further on for a different hybrid, through the use of
2n gametes homoeologous recombinant products can be recovered in the backcross
progeny.

4.2 APPLICATION OF 2n GAMETES

Occasionally, plant species and interspecific hybrids produce gametes with sporophytic
chromosome number instead of the normally expected haploid, or n gametes. The
former are called unreduced, or 2n gametes. When they occur in interspecific hybrids at
reasonably high frequencies, 2n gametes can be used for the production of sexual
progeny either through crossing or selfing. Progenies in these cases consist of
polyploids and offer an alternative to colchicine doubling. Because the polyploids are
produced in this approach through sexual crossing, the process is called sexual
polyploidization, or meiotic doubling (different from somatic chromosome doubling
through colchicine which is called mitotic doubling). In a cross, if only one of the
parents contributes a 2n gamete it is called unilateral sexual polyploidization. On the
other hand, if both parents of a cross contribute 2n gametes, it is called bilateral sexual
polyploidization (Mendiburu and Peloquin, 1977). Careful scrutiny of the literature on
several horticultural crops indicates that both unilateral and bilateral sexual
polyploidization may have greatly contributed to the spontaneous origin of polyploid
cultivars. Indeed, meiotic doubling has been practiced successfully in some crops, such
as Alstroemeria (Ramanna, 1992; Buitendijk eta/., 1997; Kamstra et al., 1999) and
Lilium (VanTuyl, 1989; Karlov eta/., 1999; Lim eta/., 2001a).
Meiotic doubling is not just an alternative to mitotic doubling. The former offers
substantial advantages over the latter. Unlike mitotic doubling, sexual polyploidization
saves time that would have been spent on colchicine treatment. But the most important
advantage of meiotic doubling is that homoeologous pairing and crossing-over occurs
during meiosis in the diploid interspecific hybrid. This is because, unlike in a
somatically doubled allotetraploid where preferential pairing of homologous
chromosomes is the rule, in a diploid interspecific hybrid, the homoeologous
chromosomes are forced to pair during meiosis. This forms the basis for the occurrence
of intergenomic recombination in the 2n gametes. Such homoeologous recombination
due to crossing-over has been clearly demonstrated in the case of Alstroemeria
(Kamstra eta/., 1999), Lilium (Karlov eta/., 1999; Lim eta/., 2001a) and Gasteria
lutzii xAloe aristata (Takahashi eta/., 1997). The types ofintergenomic recombination
that occur during the origin of 2n gametes depend on the meiotic abnormalities that give
rise to 2n spores. Generally, depending on the meiotic stages at which nuclear division
abnormalities occur, two distinct modes of origin of 2n gametes have been recognized
in plants. These are the so-called first- and second-division restitution (or FDR and
SDR, respectively) mechanisms which are diagrammatically illustrated in Fig. 2.
Because homoeologous chromosome pairing can be highly variable in interspecific
hybrids, ranging from the formation of only univalents, i.e., without any crossing-over,
to complete pairing as bivalents (high degree of crossing-over) in some of the spore
mother cells, several possibilities can be considered. When only univalents are formed
92

Met~ph!'se 1 111 ..-Chromo~ome 1111. . Telophase 11

p a 1r 1n g behav1our'

~@
Ab®
B

c
\
~~
rillr~
r llu
1
IT TI

Figure 2. A schematic representation of three possible types of meiOtic nuclear

restitution in a diploid interspecific hybrid in the case of 2n=2x=4. The homoeologous
pairs of chromosomes are shown as black and white chromosomes. (Aa) FDR without
recombination; (Ab) FDR with recombination. At metaphase I, one bivalent and two
univalents are formed. In the subsequent stage, two half-bivalents and two univalents
align on the equatorial plate and divide equationally. The result is that the
homoeologous chromosomes do not assort independently, and that the centromeres of
both genomes are intact in the 2n gametes; (B) SDR with recombination shows
independent assortment of homoeologous pairs of chromosomes. In this case, both pairs
of homoeologous chromosomes disjoin at anaphase I but restitute subsequently, i.e.,
without the second division. The notable features of SDR are that the homoeologous
pairs assort independently of each other and the number of centromeres of the parental
genomes is not preserved intact in the resulting 2n gametes. Moreover, each centromere
is always represented in pairs; (C) IMR showing unequal distribution of the centromeres
of the parental genomes. At metaphase I, a bivalent and two univalents are formed. The
bivalent disjoins normally as in anaphase I, whereas the two univalents divide
equationally. Consequently, the chromosome constitution of the parental genomes is not
preserved in the 2n gametes and, furthermore, the centromeres of each of the parental
genomes are present in odd numbers.
z In all cases, meiosis is incomplete. Because of this, the different stages of meiosis
cannot be strictly defmed.
 93

at metaphase I, the entire chromosome complement may be oriented on the equatorial

plate and all of them divide equationally (centromeres divide prematurely in relation to
cytokinesis of the first meiotic division) like in mitosis and give rise to two identical
nuclei. After cell-wall formation, and without the second division, a dyad with two 2n
spores is formed. This is a typical case of FOR without homoeologous recombination
(Fig. 2Aa). However, FOR can also occur when both univalents and bivalents are
formed during metaphase I. In this case, the bivalents fall apart (in late metaphase I) as
half-bivalents and these, together with all the univalents, are oriented on the equatorial
plate and divide equationally as described for FOR, giving rise to a dyad. In this case,
recombinant chromatids are present in the 2n gametes and therefore they form FOR
gametes with recombination (Fig. 2Ab). It should be pointed out that in the case of
FOR, with or without recombination, all the chromosomes of the hybrid are represented
only once in the FOR gamete.
At the other extreme, in some of the spore mother cells all the homoeologous
chromosomes may pair in metaphase I and disjoin normally in anaphase I, leading to a
random assortment of homoeologous chromosomes. In these cases, the products of
anaphase I disjunction, i.e., the haploid set, restitute, in the sense that the centromeres
divide but the sister chromatids of each half-bivalent are included in the same nucleus.
Since restitution occurs after disjunctional separation of the half-bivalents, it is SDR
(Fig. 2B). Two salient features of SDR are that not all parental chromosomes are
represented in the 2n gamete and, most importantly, all chromosomes will be invariably
present in pairs. In SDR, genetic recombination due to crossing-over as well as
homoeologous chromosome assortment occur. In the event of complete chromosome
pairing and chiasma formation in a distant hybrid, balanced n, as well as 2n gametes
may occur. Generally, these may not be viable due to the lack of compensation between
homoeologous chromosomes.
Besides the two well-recognized mechanisms of 2n gamete formation, i.e., FOR and
SDR, a novel type of restitution mechanism has been discovered in an interspecific
hybrid between L. longiflorum x Asiatic hybrid (Lim et a/., 200la; Fig. 1). In this
diploid hybrid (2n=2x=24), the 12 chromosomes of L. longiflorum and 12 chromosomes
of the Asiatic hybrid can be clearly distinguished through GISH and FISH. During
microsporogenesis, the chromosome associations vary from 24 univalents to two to five
bivalents in different cells. The unique feature of this restitution mechanism is that all
the bivalents and the univalents are oriented on the equatorial plate in the microspore
mother cell and all of them "divide" simultaneously in the modified anaphase I (Fig.
2C). During this division, the bivalents disjoin as in normal anaphase I so that the half-
bivalents move to the opposite poles. At the same time, all the univalents divide
equationally as in FOR and the chromatids move to the opposite poles. Thus, the half-
bivalents and the chromatids congregate and form a single restitution nucleus at each
pole, giving rise to a dyad and two microspores. This mechanism has been indicated as
indeterminate meiotic restitution or IMR for short. The net result of this mechanism is
that the exact sporophytic chromosome number of 24 is restored but the proportion of
chromosomes from the two parents in the restitution nucleus will be quite different from
that observed in either FOR or SDR. For example, in the case of FOR, the 12
chromosomes from each of the genomes will invariably be represented and they are
present in single copies. In the case of SDR, the proportion of the parental
chromosomes is disrupted in the restitution nucleus because of the random assortment
94

of the homoeologous chromosomes, but all the chromosomes will be represented as

pairs-never in single copies. In the case of IMR, some of the chromosomes will be
represented as single copies (similar to FOR) whereas the others will be present in pairs
as in SDR. The salient feature of IMR. is that genetic recombination due to
homoeologous crossing-over, as well as assortment of homoeologous chromosomes,
can occur. Evidence of the viability of IMR. 2n gametes has been given through analysis
of the F1 progeny using GISH and FISH (Lim eta/., 2001a).
In view of the occurrence of different types of meiotic restitution mechanisms in
distant hybrids, it is possible to predict the potential outcome of using 2n gametes in
breeding provided we have knowledge of the modes of their origin. But two questions
need to be answered regarding the practicability of using 2n gametes in introgression
breeding. These are: 1) Is it possible to select desirable genotypes that produce
reasonably high frequencies of certain types of 2n gametes?, and 2) How does one
proceed with the breeding of polyploids when they have triploid or allotetraploid
constitutions, which are normally expected in uni- or bilateral sexual polyploidization?
From the available literature on the occurrence of 2n gametes in plants, it is clear that
they occur in almost all plants (Harlan and de Wet, 1975) but their frequencies vary
greatly. There are claims that different mechanisms of nuclear restitution are determined
by single genes in crops like potato (Mok and Peloquin, 1975), as well as many others
(reviewed by Britagnolle and Thompson, 1995).
Regardless of their genetic control, it is possible to select genotypes that produce
reasonably high frequencies of 2n gametes so that they can be used in breeding, as in
potato (Ramanna, 1983) and Alstroemeria (Ramanna, 1992). When sterile hybrids
produce even low frequencies of 2n gametes, sexual polyploids can easily be obtained
simply because they are the only viable gametes. On the other hand, when both n and 2n
gametes are produced in a fertile diploid plant, the progeny consists of a mixture of
diploids and polyploids. The selection of po1yp1oids will be laborious in this case.
However, in a considerable number of plant species, a phenomenon called 'triploid
block' operates and in these the selection of polyploids can be highly efficient. For
example, in potato, when a diploid female is crossed to a tetraploid male parent, i.e., a
2x-4x cross, the normally expected triploid progeny are not viable due embryo-
endosperm imbalance. This is triploid block. When the progeny are produced from a 2x-
4x cross, they will be exclusively tetraploids derived from the functioning of the 2n
eggs of the diploid parent. Because triploid block operates in a number of plant species,
including Lilium (VanTuyl et al., 1989), it can be exploited for the selection of 2n eggs.
As regards the second question on how to proceed with triploids and allotetraploids
derived from uni- and bilateral sexual polyploidization, there is considerable literature
on using both triploids and allotetraploids; however certain aspects have been ignored in
the past. Generally, triploids are regarded as sterile and of little interest in breeding. A
cursory survey indicates that triploids have been used successfully as parents in planned
crossings (for reviews, see Brandham, 1982; Kuspira et al., 1986) as well as during
spontaneous polyploidization, e.g., in Narcissus, extensively (Throckmarton, 1980). An
interesting feature of triploids is that it is possible to generate progeny with a near-
diploid (circa diploid) or tetraploid chromosome constitution by making 3x-2x (or
reciprocal) and 3x-4x (or reciprocal), respectively. The progeny with circa diploid
constitution are potentially useful for breeding at the diploid level as has been
demonstrated in the case of analytic breeding in potato and other polysomic polyploids.
 95

Like in other plant species, near-diploid progeny have been obtained by crossing a
triploid Lilium hybrid (2n=3x=24) with diploid (2n=2x=24) genotypes (Lim eta/., in
preparation; Fig. 1). Unlike the triploids, the tetraploid derived from bilateral sexual
polyploidization can be fertile and produce progeny on selfing or crossing with other
genotypes. If an allotetraploid has exclusively originated through the functioning of
FDR 2n gametes without homoeologous recombination, then its meiotic behavior will
not be different from an amphidiploid produced through colchicine doubling. However,
if the FDR 2n gametes with homoeologous recombination have given rise to an
allotetraploid, then its behavior will be quite different from an amphidiploid. This is
because the presence of recombinant segments leads to multivalent formation in an
otherwise allotetraploid and the genetic loci on those pairs that are involved in
homoeologous recombination assort randomly, leading to genetic segregation. This
means that the segregating allotetraploid populations can be potentially useful for
selection. The tetraploid (2n=4x=24) progeny derived from bilateral sexual
polyploidization of an interspecific hybrid of Alstroemeria inodora x A. pelegrina
(2n=2x=16) were shown, through GISH, to possess homoeologous recombinant
segments and form multivalents which assort during meiosis (Ramanna, unpublished).

5. Techniques for Increasing Introgression

5.1 GENOMIC IN SITU HYBRIDIZATION

One of the important requirements for introgression breeding is the availability of

techniques for monitoring the presence or absence of alien chromosomes, or their
recombinant segments, in the backcross progeny. Traditional cytogenetic methods were
inadequate for this purpose because there were no efficient techniques for
discrimination of the alien genomes, chromosomes or recombinant segments from those
of the recurrent parent. In other words, efficient selection methods were not available.
The development of DNA in situ hybridization methods during the last two decades has
enabled the identification of alien genetic material in the hybrids and backcross
derivatives with unprecedented accuracy. Briefly stated, these techniques involve the
direct hybridization of labeled, single-stranded DNA of one species to denatured
metaphase chromosomes of mitotic or meiotic stages that are spread out on a
microscopic slide. Generally, the chromosome preparations are observed under a
fluorescent microscope and analyzed in the same way as in traditional cytogenetic
methods. For the purpose of hybridization, either the total genomic DNA of one of the
parental species or cloned DNA sequences of other origins are used. For the detection of
the hybridization patterns, several so-called fluorochromes have become available
which enable the simultaneous detection of chromosomes from two or more sources in a
hybrid (multicolored FISH or GISH) in the same cell. Because these techniques are
applicable in a wide variety of plant species and their hybrids (review, Gill and Friebe,
1998), they are likely to have a considerable impact on introgression breeding. Besides
being suitable for the detection of alien genetic material in hybrids and their progeny,
these techniques are of great value for elucidating certain basic questions on the
phylogeny (Leitch and Bennett, 1997), meiotic nuclear restitution mechanisms and
genetic recombination (Kamstra eta/., 1999; Lim eta/., 2001a; see Fig. 1).
96

5.2 CHROMOSOME MAPPING

In the past, diagrammatic representation of genetic and cytological markers on

chromosomes was only possible in a few exceptional organisms, such as maize and
Drosophila. But this process was extremely laborious. This situation, however, changed
dramatically soon after the introduction of molecular methods for cloning genes and
specific repetitive DNA sequences, as well as in situ hybridization methods. At present,
it is possible to construct chromosome maps for almost any plant species in a short time
when necessary. These techniques enable the mapping of not only the genes and
repetitive DNA sequences of a particular species on the chromosomes of its genome,
but also the DNA sequences from a totally different alien species can be used for this
purpose. Some examples among horticultural crops are Alstroemeria and Lilium, in
which a beginning has been made for the localization of cloned repetitive DNA
sequences on chromosomes. In Alstroemeria, species-specific repetitive DNA
sequences were cloned in two different species and physically localized on the
chromosomes through GISH and FISH (De Jeu, eta/., 1997; Kamstra eta/., 1997). This
method was useful for estimating the extent and position of homoeologous
recombination in the backcross progeny of interspecific hybrids of Alstroemeria. In
Lilium, the highly conserved rDNA sequences from wheat were used for mapping the
nucleolus-organizing regions in different species (Lim eta/., 200lb). This analysis has
been shown to be useful for the identification of chromosomes, establishing the
phylogeny of the species, as well as the origin of some of the hybrids.

5.3 MOLECULAR MARKER TECHNIQUES

Phenotypically identifiable genetic markers in plants are generally rare and, when
available, it is time-consuming to assign them to their respective linkage maps. During
the last decade, a number of molecular mapping techniques such as restriction fragment
length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and
amplified fragment length polymorphism (AFLP), among others, have emerged (treated
in another chapter, this book). These techniques have enabled the construction high-
density linkage maps for almost any organism. Although such maps have yet to be
constructed in any of the horticultural crops, the efficacy of these techniques has been
demonstrated in some, such asAlstroemeria (Han eta/., 1999) and ornamental Allium
(Friesen et a/., 1997).

6. Examples of Wide Hybridization in Some Ornamental Crops

6.1. HISTORY OF BREEDING ORNAMENTAL PLANTS

The breeding of ornamentals started centuries ago. Reliable data are only available for the
last 50-100 years. Moreover, most of the breeding efforts have been carried out by private
firms or hobbyists. Therefore, it is not known or not certain which interspecific crosses are
the basis of the cultivars which are used nowadays. It is, however, clear that interspecific
crosses are the basis of most cultivated flower crops, e.g. rose (Rowley, 1966; Darlington,
1976), Chrysanthemum (Crane and Lawrence, 1934), carnation (Spamaaij and Koehorst
 97

van Putten, 1990), tulip (Van Eijk eta/., 1991), lily (van Tuyl et al., 2000), Gladiolus
(Ohri and Khoshoo, 1983a,b), Narcissus (Coleman, 1964; Brandham, 1986), Tagetes
(Bolz, 1961), Delphinium (Legro, 1961), Freesia (Goemans, 1979), Hippeastrum (Traub,
1958) andAlstroemeria (De Jeu and Jacobsen, 1995). Except for tulip and lily during the
evolutionary process of cultivating the flower bulbs interspecific hybridization went hand
in hand with polyploidization. The vegetative propagation of flower bulbs is favourable
for the development of a polyploid assortment, because fertility is less important for
reproduction. Recently, some of these processes are more or fully controlled and carried
out in more goal-oriented breeding programs (Tulipa: Van Creij et al., 1999a,b;
Fritillaria: Wietsma et al., 1994; Iris: Eikelboom and Van Eijk, 1990; Li/ium: VanTuyl et
al., 1996;Alstroemeria: Bridgen et al., 1989; Buitendijk et al., 1995; De Jeu eta/., 1992;
Nerine, Amaryllis: Coertze and Louw, 1990; Van Tuyl et al., 1992; Ornithogalum,
Lachena/ia: Ferreira and Hancke, 1986; Zantedeschia: Yao et al., 1995). To trace the
ancestors of cultivars, new techniques are now available. 0rgaard eta/. ( 1995) analyzed
the hybrid origin of two cultivars by molecular techniques including genomic Southern
and in situ hybridization. In lily, section-specific RAPD markers are detected, which can
identify the parental sections of inter-section hybrids (Yamagishi, 1995).

6.2. ALSTROEMERIA

When compared to lily, Alstroemeria is a very new horticultural crop that first appeared
in the early 1960s (Goemans, 1962). To begin with, the first cultivar, Walter Fleming,
was an interspecific diploid hybrid (2n=2x=l6) between two Chilean species, the names
of which were either unknown or undisclosed. Though highly sterile, Walter Fleming
was used successfully as a parent for producing many cultivars. These cultivars were
spontaneous polyploids that originated through the functioning of 2n
gametes.Taxonomists have listed more than 100 species of Alstroemeria, which are
endemic to South America and predominantly distributed in Chile and Brazil. Almost
all are diploids (2n=2x= 16) and interspecific hybridization requires in vitro culture of
ovules or embryos (Buitendijk et al., 1995; De Jeu and Jacobsen, 1995). Reproductive
isolation barriers exist between Chilean species as well as between Chilean and
Brazilian species. The cultivars include hybrids of inter-Chilean species crosses (so-
called Orchid-type) and hybrids between Chilean and Brazilian species that are called
Butterfly-type. Because the genomes of these species in general are highly
differentiated, interspecific hybrids are highly sterile due to chromosome-pairing
abnormalities. Using predominantly Chilean and Brazilian species, Buitendijk et a/.
(1995) produced more than 250 different interspecific hybrids involving 50 different
parental combinations. An important feature of these interspecific hybrids is that they
produce highly variable frequencies of 2n gametes and sexual polyploidization can be
easily achieved in many cases. Generally, the F 1 hybrids between the Chilean and
Brazilian species produce very high frequencies of both 2n pollen and 2n eggs and
bilateral sexual polyploidization is very efficient in these hybrids (Ramanna,
unpublished). Inter-Chilean species hybrids produce low frequencies of2n gametes and,
to date, only unilateral sexual polyploids have been produced (Kamstra et al., 1999).
There are, at present, more than 300 registered cultivars in the Netherlands alone.
98

6.3. IRIS

Different groups of irises can be distinguished. For cut-flower production, the Dutch
irises are most important, derived from the Spanish iris (Iris xiphium, 2n=34). For year-
round flowering, the "large-sized" irises were developed by interspecific hybridization
with /. tingitana (2n=28). These irises (2n=31), with cultivars like Ideal and
Wedgewood, are sterile, but their fertility can be restored by chromosome doubling
(Van Eijk and Eikelboom, 1990). Using these tetraploids, various triploid cultivars are
produced.
Other groups of irises are the English iris (/. xiphoides 2n=42) and the bearded iris.
The last groups are complex hybrids of a number of species(/. chamaeiris, pal/ida and
variegata) with different ploidy levels. Stern (1946) presented data on the years of
introduction of cultivars with different levels. In 1895, about 25 diploid (2n=2x=24)
cultivars and only one triploid (2n=3x=36) were recorded. The first tetraploid forms
(2n=4x=48) appeared in 1900. By about 1943, there were a total of 145 diploid, 23
triploid and 24 7 tetraploid cultivars. While illustrating the pedigree of the cultivar
'Purissima', Stern (1946) pointed out two instances of unilateral sexual
polyploidization: one involving a 4x-2x cross (/. cyprina xI. pal/ida) and the other
being a 2x-4x cross (Janiata x /. mesopotamica).

6.4.UUUM

The history of lily breeding is relatively young. It is an example of how advanced

techniques, in a relatively short period, can exploit the variation of species within one
genus. The Asiatic and Oriental hybrid groups have been developed in the last 50 years
from interspecific crosses within the sections Sinomartagon and Archelirion, respectively,
of the genus Lilium. The development of these hybrid groups resulted in the increase in
lily acreage from 100 ha in 1970 to more than 3500 ha in The Netherlands in 1995. In the
last 15 years, wide interspecific crosses of genotypes of different sections have been made
by applying a range of pollination and embryo-rescue techniques. In particular, L.
longiflorum played an important role in these interspecific crosses (VanTuyl eta/., 1996).
Commercial cultivars from crosses between L. longiflorum and Asiatic hybrids (LA
hybrids) have already been obtained. At the same time breeding research on polyploidy in
lilies was initiated (VanTuyl, 1989). To overcome F 1 sterility and the introgression of
traits in assortment breeding at the polyploid level is essential. In the near future, LO
hybrids (crosses of L. longiflorum x Oriental hybrids) and OA hybrids (crosses of Oriental
x Asiatic hybrids) are expected to completely innovate the lily assortment (van Tuyl eta/.,
2000).

6.5. NARCISSUS

Successful development of cultivars through interspecific hybridization followed by

spontaneous polyploidization is best illustrated in the case of cultivated Narcissus. This
is also one of the crops in which the early history of breeding has been meticulously
documented (Brandham, 1986, 1992). According to Brandham (1986), who has
investigated the chromosome counts and the history of the origin of polyploids in this
genus, before 1885 there were few diploid (2n=2x=14) or triploid (2n=3x=21) cultivars
 99

and the first tetraploid (aneuploid) was introduced in 1887. From about 1920 onwards,
there was an explosive increase in polyploids. At present, triploid and tetraploid
cultivars predominate. From the pedigree records collected by Throckmarton (1980), it
appears that numerous triploids have been used successfully as parents. Both uni- and
bilateral sexual polyploidization appear to be possible in Narcissus. Paradoxically, there
have been very few cytological investigations, if any, on the aspects of meiotic nuclear
restitution mechanisms and 2n gamete formation in this crop. There is evidence that
species with three different basic chromosome numbers, i.e., n=7, n=10 and n=ll, are
present in the genus Narcissus and all have contributed their genomes, although n=7 is
predominant.

6.6. ORCHIDS

The existence of numerous species of orchids is well known in tropical and subtropical
regions of the world. Compared to their numbers, only a fraction of the species and their
hybrids are cultivated as horticultural crops. Some of the genera of interest are Catleya,
Dendrobium, Cymbidium, Vanda, Oncidium and Phalaenopsis, among others. Besides
species or their hybrids within the genus, intergeneric hybrids are common among the
horticultural varieties, some of the examples being Aranda (hybrid between Vanda x
Arachnis), Ascocenda (Ascocentrum x Vanda), Vandaenopsis (Phalaenopsis x Vanda),
Holttumara (Arachnis x Renanthera x Vanda). Because orchids produce hundreds of
thousands of seeds in their fruits, even difficult crosses can be made successfully and
the rare hybrids propagated. The occurrence of sexual polyploids due to the functioning
of 2n gametes has been well recognized and different types of meiotic nuclear
restitution mechanisms have been described by Storey (1956), Lee (1987) and Teo
( 1984) among others.

6.7. TULIPA

The history of tulip breeding goes back to the 12th or 13th century and it is not known
whether or which interspecific crosses were made for what is now called Tulipa
gesneriana. The assortment of Tulipa (2n=2x=24) consists mainly of cultivars from the
'species' T. gesneriana and of (sterile and triploid) Darwin hybrids (2n=3x=36), obtained
after interspecific hybridization between T. gesneriana and T. Josteriana. Within the
subgenus Tulipa (Van Raamsdonk and De Vries, 1992, 1995), the possibilities for
interspecific hybridization are studied by Van Eijk eta/. (1991) and by VanRaamsdonk et
al. (1995). Recently pre- and post-fertilization barriers have been identified by Van Creij
eta/. (1999a). In vitro embryo-rescue methods have been developed and new unique
hybrids, e.g. T. gesneriana x T. praestans and T. gesneriana x T. agenensis created (Van
Creij et a/., 1999b; Custers et a/., 1995). Recently, using in vitro polyploidization
techniques, tulip tetraploid cultivars have also been produced. For future tulip breeding,
these techniques combined with polyploidization methods will undoubtedly play an
important role.
100

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MUTATIONBREEDINGOFVEGETATIVELYPROPAGATEDORNAMENTALS

A.M. VANHARTEN
Department ofPlant Breeding
Agricultural University
Wageningen, The Netherlands

1. Introduction

Throughout the ages, ornamentals have been a source of joy for many. The pleasure
derived from growing and admiring plants, shrubs and trees with various growth habits,
different colors and types of flowers, leaves with aberrant shapes or variegation
patterns, etc., has led to considerable effort by amateurs and professionals alike to
procure or breed new forms or cultivars (which in practice are also often indicated as
varieties) for all kinds of ornamentals. In the late 19th and early 20th century,
commercial companies became more and more interested in this profitable field of
activity. In industrialized countries, applied breeding work is now done for the most part
by commercial companies. In contrast, universities and government institutions are
responsible for most ornamental breeding in less affiuent countries.
Breeding work, i.e. all activities aimed at the (purposeful) genetic improvement of
plant material, can be performed in different ways. The most elementary of these is to
collect as many different types as possible of an interesting ornamental species (genus,
family) and, after careful comparison of those types, commercialize the most attractive
ones. When following this simple approach, breeders often do not care much about the
parentage or genetic background of the observed variation.
Another, and in fact the most important breeding method for most crops, is to
perform crosses within or between different genotypes, followed by selection of the
most attractive types in the segregating progeny. This subject is treated in other chapters
of this book and will not be further discussed here. Apart from general remarks, we will
also not deal here with the application of what nowadays is commonly called plant
biotechnology or genetic engineering. Mutation-breeding techniques, depending on the
source of information, may or may not be classified under this heading. Genetic
engineering, unquestionably, has very significant scientific and practical potential for
modern plant breeding, but in general the costs of applying such methods are (still) very
high. With respect to ornamentals, it appears that for at least the next 10 to 20 years,
costs of genetic transformation methods and the like will turn out to be prohibitive as
standard breeding tools, in particular because the implementation of such methods often
proves to be highly cultivar-dependent. Of course, an exception may be made for a very
few ornamentals of considerable economic importance, such as rose, chrysanthemum
and carnation.
105
A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, 105-127.
© 2002 Kluwer Academic Publishers.
106

A third breeding method, which has demonstrated its value in the past and is often
particularly promising in ornamental crops, concerns mutation breeding. The expression
'mutation' refers to a sudden genetic change not caused by normal genetic segregation
or recombination. The mutation-breeding method is predominantly based on the
possibility of artificially inducing genetic changes in already existing cultivars, for
instance with X-rays or gamma rays, or by exposing plant material to a number of
chemical compounds known for their mutagenic properties. In addition, in nature,
spontaneous mutations for all kinds of traits do occur at rather low frequencies. Such
spontaneous mutants-which are also often called 'sports' or 'bud sports'-may be of
much interest to the breeder. They may be used as starting material for further breeding
work, but in quite a few cases they may even be directly officially registered and
commercialized as new cultivars. In particular, breeders of ornamentals are interested in
'sports' with aberrant flower and leaf types or with a different plant habitus, because
often in such mutants, only one trait of commercial interest has been changed. This
gives customers who are already familiar with a specific type of ornamental a choice
between a range of different flower colors, whereas the fact that those mutants often can
be grown and handled under about the same conditions as the original plant cultivar is
very convenient and economical for growers. One additional remark is that, when
referring to mutation breeding, it is common practice to include both the artificial
induction of mutations and the search for and commercialization of spontaneous
mutants. In this chapter, we will also follow this approach.
As already stated, a very attractive aspect of the mutation-breeding method-and in
fact often the most important advantage-is that it can often yield changes or
improvements without significantly changing the overall genetic make-up of the starting
material. This enables the plant breeder to start his mutation program from the best
available cultivars known in his crop in which he would like to induce genetic changes
for a specific trait of commercial interest. More generally said: in this way, the genetic
variation for a given trait can be increased, and good, already existing cultivars or
promising crossing products can be further improved and perfected. In addition,
mutation breeding requires no expensive laboratory facilities or highly trained or
specialized personnel and, as a result, costs of mutation programs are relatively low.
Plant breeders who are sufficiently familiar with the possibilities offered by mutation
breeding often start their mutation programs with plant material that has high so-called
'utility value' for growers and shopkeepers. This implies that in particular, the presence
of useful resistances, a low light demand (for glass house ornamentals) or a long shelf
life are much favored traits to start with. The application of vegetative propagation
methods in combination with mutation breeding lowers the risk that such traits will be
lost relative to the application of cross breeding. Moreover, mutation breeders know by
experience that the induction of and selection for mutations for resistances, light
demand, cold tolerance, etc. are much more difficult, costly and time-consuming than,
for instance, for flower-color mutants.
An important aspect of many ornamental crops is that they can be vegetatively
propagated, by either natural or artificial means, 'in vivo' or in vitro. This implies that
once an interesting mutant plant has been obtained, it can be further propagated 'true to
type', provided of course that the applied method of vegetative propagation does not
lead to the induction of new (unwanted) mutations.
Mutation breeding by means of human intervention has been applied since the late
 107

1920s. In the next section, we will first discuss some early examples of spontaneous and
artificially induced mutations in ornamentals.

2. Spontaneous 'Sports' and Early-Induced Mutants

Spontaneous mutants in plants undoubtedly made their appearance right from the
beginning of plant life on earth. Early spontaneous mutants in ornamentals were already
being discovered and described several centuries ago in different countries. In many of
the early reports, the information is insufficient to decide beyond a doubt whether the
observed 'off types' did indeed result from 'real' mutations or could be explained
differently as well. A very good illustration is found in a Japanese report from the late
17ili century which describes how the citizens of Edo (now Tokyo) competed with one
another in growing the most beautiful aberrant types and colors of flowers for the
ornamental Ipomoea nil (morning glory) in their gardens. From this report (original not
consulted; for more details and reference see van Harten, 1998), some authors deduced
that most of the observed variation may have resulted from spontaneous mutations, but
as further details are lacking, there is no hard evidence for this opinion.
It is generally agreed that a publication by the botanist Gaspard Bauhin in 1598
concerning an aberrant plant with deeply incised leaves in Chelidonium majus (greater
celandine), which was found in 1590 by the pharmacist Sprenger in his herb garden in
Heidelberg, Germany, represents the first reliable report on the occurrence of a
spontaneous 'sport'. In a recent review on induced mutations in ornamental plants,
Schum and Preil (1998) refer to the so-called moss rose mutant of Rosa centifolia,
which was first observed in 1696. Another reliable example concerns a mutant with
actinomorphic flowers of the wild ornamental Linaria vulgaris (toad flax), which
normally carries two-sided symmetrical (zygomorphic) flowers. This form, commonly
designated as the 'peloria type', was found near Uppsala, Sweden and described in 1744
by the famous botanist Linneaus.
In his book 'The Variation of Animals and Plants under Domestication', Charles
Darwin (1868) defines 'bud variations' (which is identical to 'bud sports') as "all
changes in structure or appearance which occasionally occur in full-grown plants in
their flower-buds or leaf-buds". Examples were given for a range of plant species,
including several ornamentals, but Darwin was unable to give a cause for the observed
'spontaneous variability'. Other early reports about spontaneous bud variations in many
crops, including conifers and other ornamentals, were collected by Carriere (1865) and
Cramer (1907).
In 1901, the Dutch botanist Hugo de Vries coined the word 'mutation' for sudden
spontaneous 'shock-like genetic changes' of common plant traits, and also predicted
that it may become possible in the future to artificially induce such mutations. In 1905,
the same author described various categories of mutations and mutation-like effects in
his book 'Species and Varieties: Their Origin by Mutation'. Remarkably, de Vries
mistook several cases of normal segregation and other rather common genetic events in
nature-such as variations in ploidy level or the presence of aneuploidy-as proof of
the occurrence of spontaneous mutants.
Despite many efforts to artificially induce mutations early in the 20ili century, it took
another 25 year before Muller (1927), who worked with the fruit fly (Drosophila
108

melanogaster), presented definite proof that such mutations could indeed be induced by
X-rays. In the next year Stadler (1928a,b), for the first time, successfully induced
mutations in various crop plants after irradiation with X-rays and radium rays. The first
known commercial mutant in crop plants, after treatment of inflorescences with X-rays,
was produced in the 1930s in the former Dutch East Indies in tobacco (Nicotiana
tabacum) by Tollenaar (see van Harten, 1998).
In ornamentals, the first artificially induced commercial mutant: cv. Faraday, a
flower color mutant in tulip (Tulipa sp.), was released in 1949 in the Netherlands by
W.E. de Mol (or de Mol van Oud Loosdrecht) from X-irradiated bulbs of cv. Fantasy,
following irradiation in 1936. A second flower color mutant cultivar in tulip, cv. Estella
Rijnveld, was released by the same researcher in 1954. Another early and much quoted
example concerns the mutant cv. White Sim no. 1 of carnation (Dianthus caryophyllus),
which resulted from treating rooted cuttings with y-rays and was introduced in 1962 in
the USA by Mehlquist.
The long time span between treatment and year of release of cv. Faraday in tulip
shows that mutation breeding, contrary to what is often claimed, does not always lead to
considerable speeding up of the breeding process. This holds true in particular for crops
which are commonly vegetatively propagated, as is the case for most economically
important ornamentals. The main reasons for this long period may be the time needed to
obtain the next vegetative generation, the mostly low multiplication rates (e.g. for bulb
and tuber crops) and the necessity to test all promising mutants over several generations
before they can be officially released. Plant breeders considering the prospects of
mutation breeding should be aware that, despite many successful efforts in recent years
to shorten propagation cycles and obtain much higher multiplication rates, for instance
by in vitro methods, it still may take quite some time to produce new cultivars.
During the last 30 years, the release of hundreds of commercial mutants in
ornamentals has been reported. Information can be found in specialized books, chapters
and reviews by, for instance, Broertjes and van Harten (1978, 1988), Schum and Preil
(1998), van Harten (1998), and in a range of publications, including detailed lists of
officially released or approved mutant cultivars, from the International Atomic Energy
Agency (IAEA) in Vienna, Austria. Recent information from the IAEA shows that to
date, at least 500 mutant cultivars for about 30 ornamental taxons have been registered.
This number is actually a gross underestimate of the real number of induced mutants in
ornamentals, partly because many ornamentals breeders do not like to reveal all the
details of the origin of their new cultivars. This tendency to 'secrecy' may be further
nourished, for instance, by misleading or unjustified reports in the press in which
mutagenic treatments are associated with "radioactivity" or with the dangers of 'atomic
energy'. In practice, when a whole range (or 'family') of new cultivars, derived from
the same original cultivar, is marketed, the distinction is no longer made between
artificially induced mutants and spontaneously obtained 'sports'. A final reason why
most estimates of valuable induced mutations are too low is that many promising
mutant genotypes are not directly released as new cultivars but used for further crossing
work and, therefore, when finally released, can no longer be traced as such.
 109

3. Some Economic Data on 'Sports' and Mutant Cultivars in Ornamentals

From the previous section it can be concluded that there is ample convincing evidence
demonstrating the practical value of mutation breeding. However, details on the
economic value of early spontaneous 'sports' are virtually absent, whereas for
artificially induced mutant cultivars of ornamentals, only very few data are available.
The large number of plant species involved, the reticence of many plant breeders to
disclose the origin of their cultivars and the fact that the local and international market
for ornamentals is not easy to fathom, may to a large extent account for this.
In earlier times, rich owners of rural estates and larger gardens employed their own
gardeners and sometimes even organized trips abroad to collect new species and rare
forms. Their gardeners often produced their own plant material by performing crossing
work, by multiplying sowing seed and by making cuttings and grafts. Occasionally, they
may have exchanged plant material with colleagues or, during the last centuries, ordered
plant material of particular interest from some commercial growers and merchants of
ornamentals. Undoubtedly, some of the rare and highly favored species in the gardens
had originated as spontaneous 'sports' but evidence for this is not easy to find, except
for some special cases, reported for instance by Carriere (1865). Some old price lists
and brochures of 'seed companies' and commercial growers of ornamentals also contain
interesting information about 'sports' for sale.
Sometimes, exceptional prices had to be paid for novelties or rare varieties. Famous
in this respect has become the speculation with rare tulips (or 'tulip-o-mania') in the
'Golden Age' (17th century) in The Netherlands which bankrupted many merchants.
What proportion of these rare tulip varieties had originated from spontaneous 'sports' is
not known. More recently, the number of 'sports' commercialized or offered for a
particular crop may also give some clue as to their economic significance.
A nice early example showing the practical value of some 'sports', derived from
Bergann (1954), refers to the non-ornamental species Ulex europaeus (common
corse).This originally thorny shrub, a fodder crop growing wild (or semi-domesticated)
along the roads in, for instance, France, became popular as a fodder crop after some
thornless 'sports' were found. The economic importance of these 'sports' must have
been considerable, at least for the poor farmers in regions where the common corse
occurred, but details are not known. In the late 19th century, breeding efforts by the
famous French breeder Vilmorin to obtain seed from the thornless mutants of this crop
remained unsuccessful, most probably because of periclinal chimerism, a rather
common phenomenon in vegetatively propagated plants that will be discussed later in
this chapter.
Information about numbers and relative importance of 'sports' derived from
cultivars of different ornamental species may be derived from scientific reports and
recent brochures of horticultural companies, as well as from cultivar lists produced by
government authorities. In 1933, the aforementioned de Mol van Oud Loosdrecht (see
Broertjes and van Harten, 1988) listed 38 flower color sports for cv. Murillo, a well-
known double and early flowering cultivar of tulip (Tulipa sp.). Unfortunately, again no
data are available about the relative importance of each 'sport'. However, considering
that 'novelty' is an important commercial factor for ornamentals, there can be no doubt
that the release of a number of 'nearly-like' descendants of an already popular cultivar
of tulips which only differ in, for instance, flower color, may stimulate sales for the
110

original cultivar and its descendants and, thus, may be commercially attractive. The
same, of course, applies to many other ornamentals, such as lilies (Lilium sp. ), hyacinths
(Hyacinthus sp.) or chrysanthemums (Chrysanthemum sp. or Dendrathema.
Reliable estimates concerning the contribution of 'sports' to the market for a number
of so-called 'florist' crops' in The Netherlands were given by Wasscher (1956), who
reported that at that time the percentage of 'sports' grown in The Netherlands for
carnation (Dianthus sp.) was 25% vs. 40% for glass-house roses (Rosa sp.) and even
70% for winter flowering begonias (Begonia sp.). Such figures, however, may have
differed considerably over the years and may be much lower for some other
ornamentals. Van Tuyl (personal information), for instance, estimated that for
spontaneous 'sports' of hyacinths in the 1980s the corresponding figure was only about
6%. From 5819 rose cultivars marketed in Germany during the period 1937 to 1976,
about 15% had resulted from spontaneous 'bud mutations' (Schum and Preil, 1998).
As said before, in more recent years data about spontaneous 'sports' and artificially
induced mutants are often combined. Schum and Preil ( 1998), referring to Belgium and
Germany, mention that about 50% of all cultivars for azaleas (Azalea sp. or
Rhododendron sp.) and chrysanthemums have originated from natural 'sports' and
artificially induced mutants. For chrysanthemums, similar figures were reported by
Langton (1986) for England. Detailed information for The Netherlands concerning
several successful cultivars and their 'mutant families' in chrysanthemums can be found
in van Harten (1998). One example is given here. For cv. Reagan and its 'family' with
more than 20 mutant cultivars, 400 million flower stalks, representing 35 to 40% of the
total Dutch market, were sold on an annual basis in 1992 and 1993. In those years, less
than 1.5% of the sales involved the original cultivar. Finally, the 1994 Dutch cultivar
list indicates that for 23 of the total 42 recommended chrysanthemum cultivars, the
original cultivar is accompanied by a number of mutant cultivars.

4. Pros and Cons of Mutation Breeding in Ornamentals

The application of mutation breeding offers good prospects for most ornamentals and
many examples-some of which were mentioned in the previous paragraph-do prove
that this approach can be very rewarding. In fact, it is the combination of various factors
that has made mutation breeding such a valuable tool in ornamentals.
According to Langton (1987), it is 'novelty per se' that, to a large extent, explains
why mutation breeding has such a great impact in ornamental species. Indeed, all parties
involved in the flower trade (breeding companies, growers, merchants, retailers and
shoppers) express a permanent demand for 'novelty', but this point needs some further
clarification. It is the earlier mentioned conflict of interest between commercial growers
and buyers that, to a large extent, explains why the mutation-breeding method is
particular suitable for ornamentals. Whereas most customers like to choose from a wide
selection of flower types and colors, growers of one or a few ornamental crops, on the
other hand, do not like to change their range of cultivars all the time: this applies in
particular when different growing regimes are required for various cultivars of one crop.
It goes without saying that growing plants on a large scale and under uniform conditions
is the most economical system. By applying the mutation method, new (mutant)
cultivars with genetic variation for traits of interest to the customer (flower color, flower
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type, etc.) may be obtained in a relatively easy way, whereas in many cases the growing
conditions of the new cultivars do not differ from those of the original cultivar that was
subjected to mutagenic treatment. This combination of changing only one or a few
commercially important genetic traits in a crop without significantly disturbing the
overall genetic set-up cannot be achieved with the same ease by (other) conventional
breeding methods. And, as was also mentioned in the introduction of this chapter,
although modern techniques like genetic transformation may be (or become in future)
an attractive alternative, such methods are still far too costly for most ornamentals. It is
also difficult to predict whether application of' genetic engineering' methods will garner
wide public support in future.
A second important point in favor of mutation breeding is that most ornamentals can
be vegetatively propagated. This implies that all vegetative (clonal) descendants of one
plant are genetically identical, i.e. have the same genotype. Many ornamental species
have originated from crosses between different species, which leads to a high degree of
heterozygosity of the crossing products. Often, polyploid and aneuploid plants also
occur. Throughout the years, selection has taken place in ornamentals, for instance for
larger flowers and other (vegetative) plant parts. Heterozygotic plants in many cases
show very vigorous growth, whereas polyploids are also often characterized by having
larger vegetative parts. As a consequence, selection for larger vegetative parts may
favor selection of heterozygotic and/or polyploid plants. Further, polyploids often show
decreased sexual fertility and, despite a range of methods which have been developed to
stimulate seed production from 'difficult crosses', generative propagation in such cases
may meet with many difficulties. Triploids are particularly notorious for their irregular
meiotic behavior, which results in a high level of sterility and in the absence of seed
setting, as can be illustrated by triploid varieties of Chrysanthemum or Alstroemeria
(e.g. from a 4x x 2x cross). One additional reason why sexual breeding work in
polyploids is not very attractive, is the fact that very large populations have to be
studied in order to find interesting genotypes.
For some crops which have been vegetatively propagated for many generations, the
ability for sexual propagation may have been (almost) completely lost. These are the so-
called obligate vegetatively propagated crops; however, practically none of the
ornamental crops belong to this group, but are so-called facultative propagated crops.
One exception to this rule (see van Harten, 1998) is the ornamental hybrid species
Zinnia x marylandica, an allopolyploid which, as a result of a very complicated genetic
set-up, displays practically no genetic segregation in subsequent generations.
In conclusion, once an attractive ornamental cultivar has been produced, vegetative
propagation, in most situations, will be the obvious route of propagation. Many systems
of vegetative propagation are already known in nature (e.g. by tubers, bulbs, stolons,
rhizomes, etc.) and in addition, a range of other methods have been developed, for
instance by making cuttings, budding, grafting, layering, by in vitro propagation, etc.
Because today, for almost all ornamental crops, some system of vegetative propagation
is available, we will limit ourselves for the rest of this chapter to mutation breeding for
ornamentals where vegetative propagation is feasible.
One final reason why ornamentals are ideally suited for mutation breeding, also
mentioned by Schum and Preil (1998), is that many economically important traits like
flower characteristics can be easily monitored after mutagenic treatment.
Each breeding method that can be applied in a specific case involves a number of
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specific drawbacks as well. Mutation breeding for (vegetatively propagated)

ornamentals is no exception. Plant breeders should have some understanding of the
most important bottlenecks and pitfalls in order to avoid unnecessary errors, as well as
to maximize their chances of a positive result.
A subject that requires special attention is chimera formation (or chimerism) and the
way to handle this phenomenon in plant breeding programs. Chimerism can be briefly
described as the presence of genetically different cells within the somatic plant tissue
(i.e. all plant parts and cells apart from the gametic plant tissue). Chimerism is a
common feature when mutations occur in multicellular tissues. From the point of view
of plant breeders, chimerism has both positive as well as negative aspects and will be
discussed in the next section.
Another point to bear in mind, again in particular for vegetatively propagated crops,
is that deleterious mutations-which form most of the spontaneous and artificially
induced mutations-may pile up in successive generations of plant populations. This
can be explained by the lack of a so-called 'meiotic sieve' which eliminates most of the
damaged or malfunctioning genes, but which is only active in sexual propagation. Mass
selection against such undesired mutations is only feasible when they can be easily
detected.
Mutations may also express undesirable pleiotropic effects, as for instance, when a
much-liked flower color occurs only in combination with an unappealing flower type or
with delayed flowering. The common way out would be to transfer the pleiotropic
mutant gene to a more favorable 'genetic background' by making crosses, but this
method may not always be easily accessible or even possible to apply to vegetatively
propagated crops. In that case, the only option for the breeder is to repeat his
experiments, preferably on a large scale, trying to re-induce the desired mutation. The
pleiotropic effect observed in the mutant obtained from the first treated series is not
expected to occur again or, more likely, will be expressed in a less pronounced way
(note that it is often very difficult to distinguish between 'real pleiotropic effects' and
the close linkage of two different genes).
A next point of concern in vegetatively propagated crops is the risk of (clonal)
transmission ofviruses. Most breeders of such crops are well aware ofthis threat. Apart
from the fact that viruses may destroy whole plant populations, their symptoms may
sometimes be mistaken for mutations, for instance, when a breeder is looking for
variegated leaves with light-green spots in foliage plants. All possible precautions
should be taken to start from plant material that is free of virus and plant populations
should be permanently screened to limit the risks of viral infection. Needless to say that
this work should be done by specialists who know their crop well and who can
distinguish between mutations and viruses.

5. Chimera Formation and Its Implications

Mutations occur as single-cell events with polyploids, produced by treating plant

tissues, with colchicine or oryzalin, as the proverbial exception. Plants consisting of two
or more genetically different somatic tissues are commonly called chimeras. Thus, the
induction of a mutation in a multicellular tissue automatically results in chimera
formation. The word 'chimera' was introduced by Winkler (1907) for graft-hybrids
 113

which arise when a scion of one plant species is grafted onto a rootstock of another
species. Baur (1910) proposed to apply this word to all situations in which the somatic
tissue of a plant is not genetically homogeneous and this interpretation has been applied
ever since.
Early 20th century research contributed little to a better understanding of chimerism
in plants. Later in that century, a few very good publications, for example by Baur, F.
and L Bergann, Dermen, Pratt, Stewart and Tilney-Bassett, did emerge. References to
these publications, as well an extensive discussion of chimerism and its implications for
mutation breeding can be found in van Harten (1998). Only some main lines are
touched upon here.
Mutation breeders in particular have to be well aware of the fact that, when starting
from multicellular plant material, in nature as well as after successful mutagenic
treatment, each (favorable) mutation, initially, is present in one cell only. An important
prerequisite for successful mutation breeding is to understand the fate of the mutated
cell. Some 40 years ago it was the general opinion that mutations induced in adult plant
cells with a low division rate were of no significance for further breeding work. For
mutations induced in young, meristematic tissues outside the area of the shoot apex, it
was believed that such mutant cells may have some practical value in only a few cases
(Bergann, 1967). Most promising, on the other hand, were considered those (favorable)
mutations that occur in the meristematic cells present in the area of the shoot apex.
Bergann reserved the expression 'chimera' for genetically heterogeneous tissues within
shoot apices and applied the word 'chimeroid' for all other-in his opinion
unfavorable-situations. Nowadays, this distinction has lost its significance and is no
longer used. A main reason for this is that during the last decades for many crops of
economic importance 'in vivo' and in vitro methods of vegetative reproduction have
become available, often starting from plant parts outside the original apical areas.
Meristematic cells (or groups of cells) in shoot apices may retain-at least for a
considerable length of time-their original position in shoot apices and can produce a
whole lineage of cells which are genetically identical to the mother cells and can be
passed on to new tissues. A mutant cell, present among a number of non-mutated cells
in the meristematic area of a shoot apex, may also produce a cell lineage of mutated
cells which, ultimately, could result in, for instance, a side branch of a plant carrying the
mutation. This, of course, would make it much easier to secure favorable mutations,
provided of course that the mutated traits are detected.
In 1868, Hanstein had already presented the idea that angiosperms are characterized
by some kind of stratification, based on the presence of a kind of central core of cells,
surrounded by a few layers which are regularly organized. The central core (which is
also often called a 'layer'), as well as the surrounding layers, to a degree, behave
independently of each other and cover each other mantle-wise. Or, in plain words, a
vegetative shoot apex may be compared with a hand (the core) covered by a few gloves.
Hanstein further stated that in the ultimate shoot tip of each so-called histogenic layer, a
very small number of initial cells must be situated which give rise to all other cells of a
specific layer. In later years, a number of additions and modifications to Hanstein's
concept were suggested, which made the original outline more flexible. Furthermore,
some other theories to explain shoot apex behavior were launched as well. However,
there is general agreement that the basic concept of a layered plant structure with a core,
surrounded by one or two histogenic layers, still holds.
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The most important consequence for mutation breeding is that the cell lineage of
(mutated) cells derived from a mutated initial cell, predominantly, will remain restricted
to the layer from which the initial cell is part; that is to say, as long as vegetative
propagation takes place. So, to stick with the example of gloves around a hand, a
mutation induced in an initial cell of the outer glove will give rise to a cell lineage of
mutated cells within that layer and, eventually, in layers derived from the original layer
at later stages of the plant's development. An example may further clarify this point.
Mutations affecting the expression of chlorophyll, resulting for instance in variegated
leaves or in leaves with light-green or white margins, are relatively common. But
contrary to what is often thought, if such mutations would occur in initial cells that
contribute to the formation of the outer histogenic layer (indicated as L-1 or 1 1) from
which the leaf epidermis originates, no visible changes would be observed! This can be
explained by the fact that-with the exception of the chlorophyll grains in the guard
cells of stomata-no chlorophyll is present in the epidermal layer. lf, on the other hand,
chlorophyll mutations are induced in initial cells of the inner layers (indicated as L-2
and L-3), such mutations may become visible, for instance as a white leaf margin.
Provided that the mutated initial cell remains present in the shoot apical meristem and
continues to produce daughter cells, the trait 'white margin' will remain present in all
plants resulting from vegetative propagation. It may be unnecessary to add here that
new plant forms with an aberrant leaf margin color or variegated leaf patterns in an
originally uniform green cultivar may be commercially very attractive.
One additional remark must be made about the aforementioned 'variegated' leaves.
Variegation in leaves, flowers and stems, often shown as stripes or patches of aberrant
colors, is by no means caused only by effects of histogenic processes. In fact, a whole
range of different mechanisms may be involved, such as specific regulation of gene
expression in some tissues, the presence of maternal inheritance, sorting out of plastids
and the symptoms shown by some viruses. This subject covers an extensive field of
specialized studies and cannot be further discussed here. A good introduction with many
examples of this topic is given by Tilney-Bassett ( 1991 ). Examples and references can
also be found in van Harten (1998).
To give another example, one could also conceive that the genetic constitution for
plant leaves with a 'hairy' or 'thorny' appearance is present only in the L-1, whereas the
inner layers, genetically speaking, do not carry these traits (see also section 3 of this
chapter for Ulex sp.). The chimeric situation of the plants involved would be noticed
only in special situations, for instance when damaged tissue from L-1 origin would be
replaced by the (genetically different) cells from deeper layers, or when special
techniques would be applied to reveal the genetic constitution of the inner layers for the
aforementioned traits.
Mutations for flower color may also serve to illustrate layer effects. The flower color
of a plant depends mainly on the genetic constitution of the L-1 layer of flower petals. A
mutation affecting flower color in an initial cell from which-at a later stage-the outer
layer of a flower petal arises, may be directly observed as an aberrant patch or stripe on
the petal, provided of course that the mutated area is large enough to be noticed. The
size of the mutated area depends on the number of initials present at that time in the L-1
and on the relative 'fitness' of the mutated initial cell to divide and produce daughter
cells when compared with the other initials of that layer. However, in general, genetic
changes for flower color induced in L-2 or L-3 will not be expressed and, hence,
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favorable mutations for flower color which were induced in cells of deeper layers,
normally will be lost. This, in particular, will be the case when plant breeders are not
acquainted with the fact that the aforementioned (desirable) mutations occur as well in
deeper layers; or when no suitable methods are known to isolate and propagate cells or
tissues in which such mutations are present. In reality, the situation may be more
complicated, as it is stated in the literature that the genetic constitution of the inner plant
layers also contributes to the ultimate color of a flower (see van Harten, 1998).
Moreover, in this example we have assumed a mutation in a young initial cell, from
which a whole lineage of mutated cells is produced. If, on the other hand, the mutation
had been induced in an (L-1) cell of a fully developed flower petal, its detection,
mostly, would be much more difficult because of the small size of the mutated area as a
result of the low rate of cell division in more adult plant parts.
In the previous examples we restricted ourselves to visible mutations which can be
observed relatively easily. Obviously, it would be much more laborious and, hence,
expensive, to investigate mutations for traits for which such easy methods are not
available. Again, this explains why ornamentals, with an economic value largely
determined by easily observable traits, are very well suited for mutation breeding.
Because of the foregoing, there can be no doubt about the answer when the question
arises whether to start mutation breeding work from a cultivar with, for instance, good
resistance against certain diseases and pests, a long 'shelf life' or high production
capacity, or, alternatively, from an ornamental with an attractive flower color but
without one or more of the aforementioned traits. The advice, of course, is to start from
cultivars with proven positive characteristics for 'difficult' traits and to apply mutation
breeding for 'easy' traits like new flower colors. As this chapter concentrates on
vegetatively propagated crops, we do not further discuss the pros an cons of cross-
breeding to combine certain traits in that way.
One other comment must be made here. In all aforementioned cases, except for the
original plant epidermis, the word 'layer' should not be taken too strictly, as the current
interpretation of histogenic layers does not necessarily imply that each histogenic layer
refers to a single layer of cells only. This certainly does not apply for the central part of
the outmost apical area (commonly called L-3), which even at a very early stage of
development consists of a large number of irregularly oriented cells.
Further, it must once again be stressed that everything that has been said here about
chimeras only holds when plants are vegetatively propagated. A chimeric situation as a
result of the induction of a mutation will be immediately terminated when seed
propagation takes place. As the gametes trace back to L-2, mutations present in L-1 or
L-3, will be lost in the case of sexual propagation. On the other hand, a mutation in L-2
that normally would remain unobserved (see before), will be fully expressed when seed
propagation is applied and the resulting, mutated plants are solid, i.e. completely
mutated in all their cell layers.
In practice, a distinction is often made between so-called sectorial chimeras,
mericlinal chimeras and periclinal chimeras. The expression 'sector' or sectorial
chimera was first used by Baur (1909) for seedlings of Pelargonium with green leaves
present on one side of the plant axis, white leaves on the other side and, occasionally,
some intermediate types. Sometimes, within one leaf both leaf halves also showed
different colors. Baur explained these results by assuming the presence of different
'sectors' within the shoot apex (or growing point) of the variegated plants. However,
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because of the aforementioned layered structure of shoot apices (and the plant parts
derived from those apices), and the fact that such layers act rather independently of each
other, it can be inferred that in vegetatively propagated (chimeric) plants many, if not all
of the so-called 'sectors' consist of one layer only and, in fact, do not represent sectors
in the true sense of the word. Real sectors, by definition, would have to include tissues
derived from the inner layers of the plant as well. When indeed the mutated area is
limited to a single histogenic layer, as is common in vegetatively propagated plants,
such chimeras should be called 'mericlinal chimeras' as was proposed by Jorgensen and
Crane (1927), but most often the expression 'sectorial chimeras' is still used in such
cases.
Mericlinal chimerism is never a stable situation. In L-1 and L-2, the two outer
histogenic layers, cell divisions take place almost exclusively in an anticlinal direction,
i.e. sideways within the layer itself. Cells ofL-3 origin, on the other hand, may divide in
all directions. As a result, cell exchanges between L-1 and L-2 are an exception. So,
when a meristematic cell carrying a mutation is present in the L-1 layer, this cell
normally will divide only sideways or, in other words, the mutation will remain
restricted to the L-layer. The chimeric situation in mericlinal chimeras will end when
mutated cells are not present in the tissue giving rise to the L-1 of an axillary bud and
the shoot resulting from this bud. In that case, the plant will return to the original-non-
mutated-genotype. When mutated cells are incorporated in an axillary bud, the
original mericlinal chimera-after some cycles of vegetative propagation-
automatically will be changed into a periclinal chimera.
From a practical point of view, periclinal chimeras are the most important group of
chimeras, in particular when mutation breeding is applied in ornamentals. The
expression 'periclinal chimera' refers to the situation in which all cells of, for instance,
L-1, carry a mutation which is absent in L-2 and L-3 and in tissues derived from the
latter two. The expression is used for all situations in which all tissues tracing back to
one or two layers are mutated, whereas the tissues derived from the other layer(s)
remained unchanged (or carry a different mutation). It is easy to realize that various
combinations of mutated and non-mutated (or differently mutated) layers are possible.
Many examples have been described in a range of publications by the Berganns.
References can be found in van Harten (1998).
Periclinal chimerism in general represents a stable situation and when, for instance,
cuttings are made from side shoots, the resulting plants will all be identical and similar
to the original plant. This is explained by the fact that the axillary buds from which the
side shoots arise are organized in a way that is identical to the main apex of the plant.
When a mutant genotype with a periclinal constitution for flower color (e.g. L-1
different from L-2 and L-3) is propagated in the aforedescribed way, almost all plants
will show the flower color determined by the genetic constitution of L-1. In such
situations, it is often assumed that solid genotypes (all layers having the same genetic
constitution for flower color) are involved. However, when root cuttings are made, the
periclinal chimeric situation will be immediately revealed because root cuttings, without
exception, trace back to L-3 tissue. Self-pollination, when possible, provides another
method of distinguishing between solid plants and periclinal chimeras as gametophytic
cells always originate in the L-2 layer.
Many cultivars of vegetatively propagated ornamentals and other plants are, in fact,
periclinal chimeras. Good examples are carnation and chrysanthemum where, from an
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original cultivar-often resulting from previous crossing work-a whole 'family' of

mutant varieties with different flower colors can be obtained relatively easily and
quickly. Most periclinal chimeras are stable enough to be easily accepted by the
authorities responsible for official lists of (recommended) varieties and for granting
breeders' rights. The large majority of the spontaneously arisen or artificially induced
flower color mutants of these crops are periclinal chimeras with an genetically aberrant
L-1.
Most breeders of ornamentals deal with the periclinal constitution of their mutant
cultivars in an unconcerned way, because such mutants are stable and relatively easy to
obtain. However, when they consider using such mutants in further cross-breeding
work, or when, instead of propagation by cuttings from side shoots, alternative methods
of vegetative propagation are preferred, some understanding of periclinal chimerism is
indispensable.

6. The Use of Adventitious Bud Techniques in Mutation Breeding

From previous considerations about the single-cell origin of mutations and the way in
which shoot apices are organized, it may have become clear that only very few
mutations have a chance to survive. This may be a comforting thought when deleterious
or unwanted mutations are involved, but it is rather discouraging to know that many
favorable mutations may have been induced somewhere in the plant that the breeder is
unable to employ afterwards. A way out of this last problem may be the use of the so-
called adventitious bud technique. This method refers to the induction of re-growth
starting from various tissues on locations that were not predetermined for bud
development.
In 1937, Naylor and Johnson carried out botanical studies on the African violet
(Saintpaulia ionantha) and discovered that adventitious shoots which developed at the
base of the petioles of detached leaves most probably were of single-cell origin.
Sparrow et a!. ( 1960) were the first to relate this finding to their observation that, after
irradiation of detached leaflets of African violet, all mutated shoots at the base of the
leaf petioles appeared to be non-chimeric. It was therefore shown that, without a phase
of sexual propagation, a single mutated cell situated in a multicellular tissue could
produce a non-chimeric (solid) mutant plantlet. Also interesting was the observation
that such adventitious shoots, of which easily 10 or 20 can arise rather soon at the base
of a single detached petiole, did originate from epidermal cells. In other words: thanks
to the availability of an adventitious bud method involving epidermal (i.e. L-1) cells, it
was now possible to transfer mutants with a mutated L-1 only (for instance periclinal
chimeric flower color mutants), rapidly and easily into solid mutants. It was in
particular Broertjes in the Netherlands who, starting from the 1960s, developed and
promoted this method for mutation breeding in ornamentals.
Before going further into systems of adventitious bud formation, it must be
reiterated that it is not always necessary and sometimes even undesirable to transfer
periclinal chimeras into solid mutants. This last situation could be imagined when a
(mutated) so-called pleiotropic gene causes changes in two or more genetic traits that
are not obviously related. To give a-hypothetical-example, one could imagine a
(mutated) gene for resistance that is active in L-1 by keeping off insects or fungi, but
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that at the same time is responsible for the biosysnthesis of a chemical substance
negatively affecting plant growth when present in the inner plant parts. In that case, a
periclinal chimeric situation with the resistance gene present only in L-1 should be
preferred.
A literature survey by Broertjes et a/. (1968) showed that development of
adventitious plantlets was already known for over 350 plant species, but also that for
quite a few plant species such propagation systems were either unknown or not
recorded. Broertjes performed detailed studies on 'in vivo ' adventitious bud formation
with a range of ornamentals like Saintpaulia, Streptocarpus, Kalanchoe andAchimenes,
which all belong to the family of the Gesneriaceae. In all these crops, adventitious buds
could be induced. However, for instance for Streptocarpus, it became clear that the
numerous adventitious shoots developing along the edges of detached leaf halves from
which the main vein had been removed, did not trace back to L-1 cells and, moreover,
were not of single-cell origin. It was further observed that considerable differences in
adventitious bud production and general regeneration occur between species and even
between different varieties of one species. Mutagenic treatments of all kinds of plant
material may significantly enhance such effects.
Results collected by Broertjes and others showed that for many ornamentals of
commercial interest, suitable adventitious bud methods were not (yet) available or were
not published. A general piece of advice for plant breeders is that it is always worth
checking the literature for a specific crop to determine whether a single-cell adventitious
bud system is already known. In addition, literature should be studied for reports about
previous unsuccessful efforts. A next step-if such a literature survey turns up
negative-would be to try out various methods, using, of course, several cultivars of the
plant species under examination. In the case of positive reports in the literature, breeders
should try out these methods for their own material.
Next to the successful single-cell regeneration method for Saintpau/ia and some
other taxons, other systems to reduce chimerism are known. Sometimes use can be
made of a 'natural' system, as in the case of Alstroemeria where after irradiation of
actively growing young rhizomes, which in fact are stems growing underground, almost
exclusively non-chimeric mutants were obtained. Several of these mutants were
released as commercial cultivars (Broertjes and Verboom, 1974; Przybyla, 1996, 1998).
The solid nature of these mutants has been explained by the characteristic sympodial
growth of the rhizomes of Alstoemeria. A sympodial growth system, and hence
reduction or dissolution of chimerism, can also sometimes be observed in the higher
zones of aerial stem parts, as is the case for tomato (Lycopersicon escu/entum).
In addition to these 'natural systems', of which many examples can be found in
Broertjes and van Harten ( 1988), much time and energy is being spent on developing
suitable in vitro adventitious bud systems and many publications on this subject can be
found in recent issues of journals on plant breeding, tissue culture and related fields of
research. The enormous interest in this topic can be explained by the urgent need for
reliable regeneration systems when plant transformation and other methods of genetic
engineering are applied. Essential for such regeneration methods is that they can be
applied without much adjustment for each genotype of a species. Equally important is
that they must be genetically stable, because one of the most important advantages
claimed for these modem methods is that in this way a single gene can be replaced by a
more favorable one without affecting the rest of the genome. Therefore, regeneration
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methods must be strictly 'true to type' or, in other words, apart from the 'improved
gene' the transformed plant must be genetically fully identical (at least for all important
traits) to the original plant material. Additional mutations or off-types as a result of the
applied system of regeneration cannot be accepted.
The genetic instability of cell suspensions, single-cell cultures and some other in
vitro regeneration systems is well known and nowadays often denoted 'somaclonal
variation'. Some scientists consider 'somaclonal variation' as a blessing, others as a
disaster. The truth, of course, lies somewhere in between, but the instability of many so-
called 'somaclonal variants' is rather notorious, partly because a considerable part of the
observed phenotypic variation is not genetically determined and non-permanent or
related to differences in gene activity during various stages of plant development (so-
called epigenetic variation). The subjects of 'somaclonal variation' and epigenetic
effects, about which in recent years hundreds of publications have been written, will not
be further discussed here. Many references can be found in recent journals and a critical
discussion of the practical value of 'somaclonal variation' for plant breeding with many
references can be found in van Harten (1998).
A general conclusion may be that the application of, in particular, 'in vivo ' single-
cell adventitious bud methods could be very attractive for applied mutation breeding
with vegetatively propagated ornamentals, but suitable methods in many cases are not
available. In such a case, it is worthwhile, after having checked the literature, to do
some experimenting oneself. There is, however, no guarantee that such efforts will be
successful. And, unfortunately, quite a few plant breeders have learnt the hard way that
application of in vitro adventitious bud methods in combination with mutation breeding
may be even less accessible.

7. Starting a Mutation Breeding Program for Ornamentals

Before embarking upon mutation breeding, a plant breeder should be well aware that
this is just one of a number of breeding methods to choose from. The choice of one
specific method-or of a combination of several methods-largely depends on the
breeding characteristics of the crop and on the specific breeding goals being pursued.
For a good judgement of the pros and cons of mutation breeding vs. other approaches,
various aspects have to be considered. The easiest method is to start from a list of
questions, to be answered as thoroughly as possible. To facilitate the work of the
breeder, a questionnaire was formulated by Broertjes and van Harten (1978, 1988).
Subjects brought up in this list concern the decision-making process itself, type and
amount of plant material required, the way consecutive steps of the breeding program
should be organized, etc. Essential is that breeders are well acquainted with their crops,
that is to say from a genetic, agronomic as well as economic point of view. But even
then it may be difficult, or even impossible, to find an adequate answer to all relevant
questions. A checklist could contain, for instance, the following questions:

1. For which traits is genetic variation desired?

2. What is known about the genetics of the trait(s)?
3. What is known about the crop, its previous breeding history and its cultivars?
4. Why should mutation breeding be applied in relation to the previous points?
120

5. What is known about spontaneous mutations in this crop?

6. What would be, genetically speaking, the best kind of starting material?
7. What kind of plant material should be used for mutagenic treatment?
8. Which mutagenic treatment(s) should be chosen and under which conditions?
9. How should the material be handled after mutagenic treatment?
10. Which selection procedures should be followed?
11. How should the program be finalized to reach the stage of an (approved) cultivar?

It is of course impossible to discuss here all these points extensively, but some
additional comments may help the ornamental breeder to find his way.

7.1. CONCERNING THE TRAITS (QUESTIONS 1, 2, 4, 6)

The genetic traits for which the application of mutation breeding might be an attractive
option can be culled from the literature. Most cases of successful mutation programs for
vegetatively propagated ornamentals until about the mid-1980s, arranged according to
crop, have been reviewed by Broertjes and van Harten (1978, 1988). For later reports, a
range of journals and a very useful review by Schum and Preil ( 1998) can be consulted,
in which mutations are broken down into those for flower color, morphology of flowers
and inflorescences, flower fragrance, leaf characteristics (form, size and pigmentation),
growth habit characteristics (compact, climbing and branching types) and physiological
traits (including photoperiodic response, early flowering, free-flowering, keeping
quality of flowers and tolerance to biotic and antibiotic stresses).
On the basis of the available literature, Schum and Preil ( 1998) conclude that 55%
of the reports refer to changes in flower color and 15% to changes in flower
morphology. Induced mutations for flower color are reported in about 40 ornamental
species and changes in flower morphology in almost 20 crops. Mutations for leaf
characteristics occur in more than 40 species and for growth habit characteristics in
about 30 species. For physiological mutants no figures are mentioned, but several
examples are described and reference is made to an overview on selection for
physiological traits in ornamentals by de Jong (1991). Schum and Preil correctly
mention that the genetic basis of most physiological traits is still poorly understood and,
moreover, that simple Mendelian segregation has been reported in only a few papers.
From the foregoing, it may be clear for which kinds of traits mutation breeding may
be an attractive option. In addition, as a large majority of the mutations go from
dominant to recessive, it would be very uninviting to start from plant material from
which the 'target genes' are known to be present in a recessive condition, because in
order to detect under such circumstances the few dominant mutant genes, very large
plant populations must be studied.
Similarly, plant breeders should not expect too much from mutation programs for
traits in which more than one or a few genes are involved. Polyploidy may also often
complicate mutation breeding work, but several exceptions to this rule are known.
These comments may also help answer question 4 on the list. The general attitude is
that mutation breeding should be applied only when there is reason to do so. Sound
reasons, of course, are that previous work has already demonstrated the value of
mutation breeding in a particular case in the sense that the method proved to be easier,
faster or more profitable than others. Another legitimate reason could be that other
 121

suitable methods simply are not available. However, in practice, still much so-called
'applied' mutation work is done just to try one's hand or out of curiosity because an X-
ray apparatus is handy. But, on occasion, even this haphazard approach may result in a
lucky strike!

7.2. SPECIFICS OF THE CROP AND ITS BREEDING HISTORY (QUESTIONS 3, 5,

6, 9, 10)

Only some short remarks on these subjects should suffice here. As already noted, it may
be anticipated that serious plant breeders will be well acquainted with their crops before
embarking on any breeding program, including mutation breeding. When essential data
are lacking-for instance because some ornamentals have only recently been introduced
in a breeding program-as much relevant information as possible should be collected
on their new crops, starting of course with the sources from which the new material was
obtained. Often, additional research may be necessary as well, not only about the
general breeding features of the crop, but also with respect to choosing which cultivars
and which types of starting material would offer the best prospects from the viewpoint
of mutation breeding. This information may concern, for instance, the ancestry of the
crop, its ploidy level (identical for all accessions?), its degree of heterozygosity, the
available methods of (vegetative) propagation, growth differences between cultivars
under various 'in vivo ' or in vitro conditions, etc. Has mutation breeding been tried
before? Do spontaneous mutations occur more or less regularly? For which traits and in
which direction (e.g. from flower color A---+ color B) do mutations predominantly go?
Do different cultivars produce more mutants than others: are some genotypes, for
instance with different flower colors, more easy to mutate than other cultivars, etc.?

7.3. WHICH KIND OF PLANT MATERIAL TO START FROM AND WHICH

MUTAGENIC TREATMENT? (QUESTIONS 7, 8)

Different methods of vegetative propagation can be employed in mutation breeding.

Some systems have been known for years in nature, whereas other methods have been
developed more recently by man. Several examples have been discussed, and many
more can be found in van Harten (1998). An extensive list of various plant parts,
including tubers, dormant buds, rhizomes, graft-wood, dormant shoots, single-budded
sets, cuttings, nodal stems, and various types of in vitro material like shoot tips, leaf
rachis and petioles, leaf and leaflet blades, segments of pedicels, callus derived from
ovules and pollen mother cells, was published by Micke and Donini ( 1993 ).
The purpose of the mutagenic treatment ultimately determines to a large extent
which kind of starting material is most suitable, provided of course that different
options are available. Breeding work in ornamentals often aims in particular at
increasing the range of genetic variation for a number of important traits in a specific
crop. From the new 'gene pool' obtained in this way, the most attractive genotypes are
then selected. In early phases of their breeding programs, breeders are often rather
indifferent as to the origin of the genetic variation and the genetic stability of the
material. When, during these first breeding steps or at later stages, propagation methods
are applied which bear the risk of producing genetically unstable plant material, as may
be the case with some in vitro methods and certainly when 'somaclonal variation' is
122

involved, the next, and often decisive step in the breeding program must be to select
thoroughly and under a wide range of conditions for genetic stability of the changed
traits. It may be anticipated that after previous in vitro work with unstable structures,
only a few aberrant types will successfully pass this stage. If, on the other hand, a
breeder wishes to induce changes for only one trait (e.g. plant stems without thorns, a
lower degree of branching, dwarfing types, white leaf margins, etc.) without taking
unnecessary risks of changing other useful traits as well, it would be wise to start from
stable structures, such as nodal cuttings, in which axillary buds are already present.
From this point of view, when in vitro methods are applied, care should be taken that
the callus phase be as short as possible.
With respect to suitable mutagenic treatments for ornamentals, only a few realistic
options are available. Most mutation work in (vegetatively propagated) ornamentals is
performed with X-rays or gamma-rays, irrespective of whether 'in vivo' or in vitro
methods are applied. In most cases, acute irradiation is preferred at relatively low doses.
Sometimes UV radiation can be used as well, but this technique can be applied only in
the case of very small objects such as pollen grains, or for in vitro treatment of cell
suspensions, stripped epidermal parts and the like because of the low penetration power
ofUV.
Before any additional details about radiation treatments are given, it should be noted
that treatments with chemical mutagens can be performed as well, in particular in
combination with in vitro methods. This approach is often favored by workers and
research students at universities and institutes. So far, not many results of practical
significance for breeding ornamentals have been achieved and, as a consequence, many
projects have ended in untimely fashion or sunk into oblivion. For vegetatively
propagated crops, Micke eta/. (1990) and Maluszynski eta/. (1992) registered only 14
cases of chemically induced mutant cultivars (amongst which 12 were ornamentals),
whereas for the same group of crops, 509 mutant cultivars resulted from radiation
treatments. The main problem with using chemical mutagens ·in vivo ' is to obtain
uniform penetration of the mutagens in the target meristems when larger plant parts are
treated. This makes dosimetry very difficult and reliable reproduction of experimental
conditions almost impossible. In the past, many authors have stated that the use of
chemical mutagens in combination with in vitro methods should be very promising, but
results have failed to live up to expectations. If, nevertheless, treatments with chemicals
are preferred, the well-known EMS (ethyl methane sulfonate) is the most logical choice.
As alternatives, nitroso-compounds like NEU (nitrosoethyl urea) and NMU
(nitrosomethyl urea), or sodium azide (NaN 3) are used. For sodium azide, it has often
been claimed that most mutations induced by this compound are 'real' gene mutations
and not deletions and the like, but this is certainly not always the case. Moreover, to be
successful, sodium azide treatments require very specific conditions and it appears that
not all crops act favorably to this mutagen. Earlier in this chapter, mention was made of
colchicine and oryzalin, chemicals which can be used to induce polyploidy. For all
aforementioned and other chemical mutagens, many references and essential
information about concentrations used, durations of treatments, etc., can be found in the
most recent edition of the IAEA Manual on Mutation Breeding (Anonymous, 1977), in
other publications from the IAEA and in van Harten (1998).
Returning to the use of treatments with (in most cases) X-rays or gamma-rays, the
doses and dose rates used to date for ornamentals can be found in Broertjes and van
 123

Harten (1988). More recently, Micke and Donini (1993) compiled a table with doses for
the treatment of a range of vegetatively propagated crops. The doses recommended by
different scientists often show large variations, even when they concern the same plant
species. As a result of the enormous variation in radiation sensitivity between and
within different plant species, and also because of different opinions as to what would
be the most appropriate dose, it is always necessary to perform pilot experiments with
several cultivars or genotypes. Before deciding on how to embark upon large-scale
practical mutation work the best start, of course, would be to take a dose recommended
in the literature and to experiment on a relatively small scale (e.g.100 objects per
treatment) with a rather wide range of doses and under various conditions.
A practical problem is how to decide from initial experiments which dose and which
treatment conditions are best. The most reliable method would be to decide on the basis
of mutation frequencies for the trait(s) in which the breeder is interested, but such data
are only very rarely available. As a second choice, information can sometimes be found
in the literature about mutation frequencies for other (often unrelated) traits, such as
chlorophyll changes obtained after various mutagenic treatments. When such data are
lacking as well, one has to fall back on data about survival rate or degree of growth
reduction after mutagenic treatments and accept the fact that this is very poor and
unreliable information, but still better than no information at all. Contrary to what was
often advised in the past, much lower doses are now used. A growth reduction rate
nearer to 30% than to 50% is generally thought to give the best results. High radiation
doses produce mostly higher mutation rates and also lead to a reduction in the number
of surviving cells, which is considered favorable from the point of view of reducing the
amount of chimerism. But, on the other hand, high doses also cause much more
radiation damage and a higher number of unintentional (and mostly unwanted)
mutations for non-target genes. One must be aware that for vegetatively propagated
crops it is very difficult to get rid of chromosomal damage and other negative genetic
side effects because of the absence of a meiotic phase which normally acts as an
effective sieve to catch larger chromosomal aberrations. Experience may be a very
useful guide for deciding at which radiation dose, dose rate and other treatment
conditions a reasonable balance can be found between positive and negative effects.
To give one more illustration of the kind of practical questions that have to be
answered, an example concerning mutation breeding of tulip (Tulipa sp.) in the
Netherlands: Irradiation can either be performed directly after harvest when the bulblets
are at the youngest possible stage of development or some months later. In the latter
case, the treatment is directed at the cells in the bulblets from which the apices for the
secondary bulblets will subsequently arise. The preference for one of these methods also
depends on the goals of the experiment. For instance: irradiation directly after harvest is
to be preferred when the main goal is to test for differences in mutability for a range of
cultivars. However, the size of the bulbs to be treated also plays a role. Early irradiation
is preferred for smaller bulblets, whereas late irradiation seems to give better results for
large bulblets. A much more detailed discussion of this subject can be found in the
chapter on tulips in Broertjes and van Harten (1988).
For 'in vivo' treatment of vegetative plant parts, doses between 20 and 80 Gy (gray),
or in old units 2000 and 8000 rad (or 2 - 8 kilorad), are mentioned in literature. When in
vitro methods are applied, in most cases radiation doses of about 10 to 35 Gy are given,
but sometimes much higher doses are suggested as well.
124

As most discussions in earlier paragraphs referred to 'in vivo ' situations, some
additional comments are made here concerning in vitro work. Although much has been
published about this subject, only a few overviews are found in which various in vitro
options for different types of starting material, treatment conditions, etc., are critically
compared. Methods to apply radiation to in vitro material do not differ much from 'in
vivo' treatments. High doses may drastically affect the regeneration capacity of the
treated material. At present, it is generally advised to allow a survival rate of about 70%
of the irradiated plantlets instead of the 50% suggested earlier.
With respect to irradiation of mericlinal or periclinal chimeras, one special point has
to be briefly mentioned here. Irradiation of buds may result in the occurrence of
anomalous histogenic processes in organized plant tissues and, for instance, cause loss
of integrity of the different histogenic layers in meristems. When shoot apices of
periclinal chimeras in which layers differ in ploidy level are irradiated, the
radiosensitivity of cells of the various layers may differ as well. As a result of such
effects, an exchange of cells may occur, for instance between L-1 and L-2 or,
alternatively, cells of L-3 origin may replace damaged cells from outer layers (which
also happens in nature or as a result of mechanical damage to the outer plant layers).
Bergann (1967) and others have demonstrated that radiation treatments can be used to
transfer a mutation that is present only in the cells of one layer, to another layer. This
finding could be of practical significance in plant breeding, but further details are
outside the scope of this chapter. More information can be found in van Harten (1998).

7.4. FROM MUTATED CELL TO MUTANT CULTIVAR: THE FINAL STEPS

(QUESTIONS 9, 10, 11)

After mutagenic treatment, it is important to have a clear concept of the further strategy.
The way of handling the treated material, of course, also depends on the specifics of the
crop, numbers of plants, bulbs or explants (including untreated control material!), the
intensity of selection for attractive mutant types, the available manpower, glass-house
facilities, etc.
As was explained earlier, it may take quite some time before single-cell mutations
are transformed into a solid mutant or a stable enough periclinal chimera. T~e question
of whether to prefer solid mutants or stable periclinal chimeras, as already discussed,
largely depends on the breeding goals and the ease with which mutants can be induced
and selected. Further procedures are illustrated again by referring to mutation breeding
for tulip and chrysanthemum in The Netherlands. For tulip, in order to allow a mutated
cell to further divide and develop into a larger, visibly mutated (flower) 'sector' that
will be expressed in first- or second-generation bulblets, at least 4 years of selection are
required. After this first step, continued selection for other important traits (e.g. bulblet
production) must take place during further clonal propagation. In comparison, after
irradiation of chrysanthemum cuttings, the growing plants and their outgrowing axillary
shoots are usually cut back two or three times to obtain shoots with larger mutated
areas. As a consecutive step, cuttings are taken for flowering trials. Selection for flower
color mutants is performed on a one-plant basis during the flowering period, preferably
under commercial growing conditions.
It may be clear that the first and most important task for the mutation breeder at this
stage is to optimize chances that mutated cells will be expressed in later plant
 125

generations and-at least for visible traits-to increase the size of the mutated area in
the plant in order to detect useful mutations. Several reasons make it unadvisable to
look already for mutations in vM 1: the first vegetative generation after mutagenic
treatment. The small size of the mutated area in this generation has already been
mentioned. In addition, useful genetic changes may remain masked in this generation as
a result of various kinds of damage to the plants, caused by the mutagenic treatment.
Sometimes damage effects are mistaken for mutations, for instance when leaves show
chlorophyll spots or a crinkled appearance in vM 1 or when the breeder is particularly
focused on selecting compact plant types. Radiation damage, predominantly, is of a
non-genetic nature, and a common feature which normally disappears in later
generations. In vegetatively propagated crops, several cycles of (vegetative) propagation
are often required.
After the presence of useful mutations has been established and confirmed, further
breeding procedures are practically identical to common breeding procedures for
vegetatively propagated crops, bearing in mind, however, the previous remark that
during the first few generations much of the observed aberrations are of a non-genetic
origin. New mutants must be tested over several generations and under various
conditions to establish the stability of the mutated traits and also to ensure that no
negative genetic side effects (as a result of pleiotropic effects or close linkage of
different genes) occur. Sometimes environmental conditions, such as differences in
glass-house temperature or day length, may significantly modify the level of expression
of such unwanted side effects.
To safeguard the financial renumeration for their new attractive mutants by so-called
breeders' rights, breeders must take action to get their mutants officially registered. In
this respect, many countries adhere to the guidelines of the International Union for the
Protection of Varieties (UPOV) and follow the so-called UPOV Convention. In 1991, a
new provision in breeders' rights with significant consequences for mutation breeders
was proposed and accepted, although not all member countries have put this change into
effect yet. The amendment implies the introduction of the principle of so-called
essential derivation which provides that legal owners of an established cultivar in which
only one trait-say flower color-has been changed as a result of a mutagenic treatment
by another person or company can now claim their share of the financial profits of the
new mutant cultivar. It is clear that this new regulation has important economic
consequences for mutation breeders but, so far, the interpretation of the new rules is still
far from easy and not undisputed. Official applications for breeders' rights should be
started as early as possible, because procedures take a long time. Patenting of new
genetically improved plants and other living organisms is a new and rather controversial
option and a snake pit for non-lawyers!
Finally, marketing plans should be made for the new mutant cultivars. For 'big'
crops like chrysanthemum, in which a whole 'family' of mutant cultivars can sometimes
be produced from one original cultivar in a relatively short time (see Broertjes et a/.,
1980), the strategy not to introduce all new mutants at the same time is often followed
in order to maximize profits and secure revenues over a range of years. This example
again emphasizes that in order to be successful, plant breeders should be not only good
technicians who are also able to see beyond today, but also have sound commercial
insight. And this certainly is the case when ornamentals are involved.
126

8. References

Anonymous (1977)Manual on Mutation Breeding (2nd edn), IAEA, Vienna.

Baur, E. (1909) Das Wesen und die Erblichkeitsverhaltnisse der 'Varietatis albomarginatae hort.' von
Pelargonium zonale, Zeitschriftfor induktiveAbstammungs- und Vererbungslehre 1, 330-351.
Baur, E. (1910) Propfbastarde,. Biologisches Zentralblatt 30,497-514.
Bergann, F. (1954) Praktische Konsequenzen der Chimarenforschung fur die Pflanzenzuchtung,
Wiss~nschaftliche Zeitschrift der Karl-Marx-Universitat, Leipzig (Mathematisch-naturwissenschaftliche
Reihe) 4, 281-291.
Bergann, F. (1967) The relative instability of chimerical clones: the basis for further breeding, in H. Stubbe
(ed.), Induzierte Mutationen und ihre Nutzung (Proceedings Erwin-Baur-Ged.1chtnisvorlesungen IV,
Gatersleben, 1966), Akademie-Verlag, Berlin, pp. 287-300.
Broertjes, C. and van Harten, AM. (1978) Application ofMutation Breeding Methods in the Improvement of
Vegetatively Propagated Crops, Elsevier Scientific Publishing Company, Amsterdam.
Broertjes, C. and van Harten, AM. (1987) Application of mutation breeding methods, in AJ. Abbott and R.K.
Atkin (eds.),Improving Vegetatively Propagated Crops,. Academic Press, London, pp. 335-348.
Broertjes, C. and van Harten, AM. (1988) Applied Mutation Breeding for Vegetatively Propagated Crops,
Elsevier, Amsterdam.
Broertjes, C., Koene, P., and van Veen, J.W.H. (1980) A mutant of a mutant of a ... : Irradiation of progressive
radiation-induced mutants in a mutation breeding programme with Chrysanthemum morifolium. Ram,
Euphytica 29, 525-530.
Broertjes, C., Haccius, B., and Weidlich, S. (1968) Adventitious bud formation on isolated leaves and its
significance for mutation breeding, Euphytica 17, 321-344.
Carriere, E.A (1865)Production et Fixation des Varietes dans les Vegetaux, Paris.
Cramer, P.J.S. (1907) Kritische Ubersicht der bekannten Faile von Knospenvariation, Natuurkundige
Verhandelingen der Hollandsche Maatschappij van Wetenschappen 3.6.
Darwin, C. (1868) The Variation ofPlants and Animals under Domestication (lOth impr. of the 2nd ed.) Vol. 1
(1921) Murray, London.
de Jong, J. (1991) Selection for physiological traits, in J. Harding, F. Singh, and L.N.M. Mol (eds.), Genetics
and Breeding ofOrnamental Species, Kluwer Academic Publishers, Dordrecht, pp.l 09-133.
de Vries, H. (1901)DieMutationstheorie I, Veit and Co, Leipzig.
de Vries, H. (1905) Species and Varieties: Their Origin by Mutation, The Open Court Publishing Company,
Chicago.
Jorgensen, C.A and Crane, M.B. (1927) Formation and morphology of Solanum chimeras, J. Genet. 18, 247-
273.
Langton, F.A (1986) Mutation breeding and its role in the improvement and commercialization of
vegetatively propagated crops, in B.T. Styles (ed.), Intraspecific Classification of Wild and Cultivated
Plants (Proceedings Symposium Oxford, U.K., 1984), Clarendon Press, Oxford, pp. 263-276.
Langton, F.A (1987) Breeding for improved ornamental plants, in AJ. Abbott and R.K. Atkin (eds.),
Improving Vegetatively Propagated Crops, Academic Press, London, pp. 159-180.
Maluszynski, M., Sigurbjornsson, B., Amano, E., Sitsch, L., and Kamra, 0. (1992) Mutant varieties-databank,
FAO/IAEA data base, part II, Mutation Breeding Newsletter, 39, 14-33.
Micke, A and Donini, B. (1993) Induced mutations, in M.D. Hayward, N.O. Bosemark, and I. Romagosa
(eds.), Plant Breeding, Principles, Prospects, Chapman and Hall, London, pp. 52-62.
Micke, A, Maluszynski, M., and Donini,B. (1990) Induced mutations for crop improvement, Mutation
Breeding Review 7, 1-41.
Muller, H.J. (1927) Artificial transmutation ofthe gene, Science 66, 84-87.
Naylor, E.E. and Johnson, B. (1937) A histological study of vegetative reproduction in Saintpaulia ionantha,
Amer. J. Bot. 24, 673-678.
Przybyla, A (1996) Studies on the induction of mutations in Alstroemeria L. and evaluation of selected
mutants, Zeszyty Naukowe Inst. Sad. i. Kwiac. Skierniewice. Monografie i. Rozprawy, 1-37.
Przybyla. A ( 1998) Mutagenesis in creation of new Alstroemeria genotypes, Acta H ort. 508, 3 51-353.
Schum, A and Preil,W. (1998) Induced mutations in ornamental plants, in S.M. Jain, D.S. Brar, and B.S.
Ahloowalia (eds.), Somaclonal Variation and Induced Mutations in Crop Improvement, Kluwer
Academic Publishers, Dordrecht, pp. 367-378.
Sparrow, AH., Sparrow, R.C., and Schairer. L.A (1960) The use of X-rays to induce somatic mutations in
Saintpaulia,African Violet Magazine 13,32-37.
Stadler, L.J. (1928a) Genetic effects of X rays in maize, PNAS USA 14,69-75.
 127

Stadler, L.J. ( 1928b) Mutations in barley induced by X-rays and radium, Science 68, 186-187.
Tilney-Bassett, R.AE. (1991) Genetics of variegation and maternal inheritance in ornamentals, in J. Harding,
F. Singh, and J.N.M. Mol (eds.), Genetics and Breeding of Ornamental Species, Kluwer Academic
Publishers, Dordrecht, pp.225-249.
van Harten, A.M. (1998) Mutation Breeding: Theory and Practical Applications, Cambridge University
Press, Cambridge.
Wasscher, J. (1956) The importance of sports in some florist's flowers, Euphytica 5, 163-170.
Winkler, H. (1907) Uber Propfbastarde und pflanzliche Chimaren, Berichte der Deutschen Botanischen
Gesel/schaft 25, 568-576.
INTRODUCTION OF NEW CUT FLOWERS: DOMESTICATION OF NEW
SPECIES AND INTRODUCTION OF NEW TRAITS NOT FOUND IN
COMMERCIAL VARIETIES

D. WEISS
The Kennedy-Leigh Centre for Horticultural Research
Faculty of Agricultural, Food and Environmental Quality Sciences
The Hebrew University ofJerusalem
P.O. Box 12
Rehovot76100
Israel

1. Introduction

The ornamental plant industry is characterized by its great diversity. There are more
ornamental species cultivated today than all other agricultural and horticultural crops
combined. New cut flowers are frequently introduced to the ornamental industry and in
recent years, they have come to represent a large proportion of production. Consumer
demand for cut flowers with new, showy traits, in addition to the desire for a large
choice of flowers, makes the introduction of new species an important task. Scientists,
breeders and plant collectors are continually developing new products to introduce
improved versions of known cut flowers, as well as new species previously unknown as
ornamentals. The introduction of new ornamental crops is easier in some ways than
that of food crops. Neither their nutritional value nor their toxicity to humans need be
considered, as evidenced by plants such as Aconitum, Diffenbachia, Oleander and
many others, which contain toxic ingredients.
The collection of new ornamentals started several centuries ago, taking a leap
forward in the 18th century, when people started to travel more frequently overseas and
to discover new places with novel flora. Sailors, soldiers, botanists, missionaries and
others collected new plants and brought them to their homelands. In many cases, these
collectors were hired and financed by botanical gardens, natural science societies,
trading firms and wealthy garden aficionados. The introduction of new ornamentals
took another, dramatic leap forward in the 1970s, with the increased consumer interest
in ornamentals and new ornamental plants. Concomitantly, new methods for
propagation, growth and storage arose and transportation systems improved (Von
Renting, 1994).
Several sources can serve for the introduction of new cut flowers, including garden
plants, plants grown in botanical gardens or special collections, and native plants. The
129
A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, 129-137.
© 2002 Kluwer Academic Publishers.
130

introduction of new cut flowers is a long process involving a complex procedure, and
normally includes the following steps (Halevy, 1995):
• Searching for potential crops
• Selection
• Developing propagation methods
• Studying the growth and flowering physiology and developing practical means for
their control
• Studying post-harvest physiology and developing practical methods for post-
harvest handling, transport and storage
• Semi-commercial production and shipment to the marketplace
In many cases, the new plant is not suitable for commercial production unless
breeding is performed. Out-crossed plants generally show a high level of variability in
yield, flower quality and color, time of flowering, disease tolerance, etc. (Sedgley,
1998). Such diversity in the wild population enables intraspecific hybridization for the
introduction of improved varieties. Wild germplasm can also be used as a source for
new traits not found in commercial ornamentals. Many species from wild flora are
relatives of cultivated ornamentals (Horovitz and Danin, 1983) and it is therefore
possible to transfer new and important traits by inter/intraspecific hybridization
(Uosukainen, 1992). Since most cut flowers are propagated vegetatively, it is possible
to obtain an improved cultivar by a simple breeding program followed by vegetative
propagation of a selected clone.
Many of today's new cut flowers were already available on the market in the past
for limited periods. Cultivation problems, diseases or low demand caused their
disappearance. Many of these plants were introduced again after further breeding,
improved production techniques, development of new chemicals to control diseases
and pests, or changes in consumer taste (Vonk Noordegraaf, 1991). Since consumer
tastes are constantly changing, the demand for cut flowers with specific colors or
shapes also changes continuously. Collectors and breeders of new cut flowers have to
predict consumer trends. Since many factors affect consumer taste, it is almost
impossible to predict it with any certainty. However, several criteria can guide
collectors and breeders in the domestication and introduction of new cut flowers.

2. Criteria for Early Selection

Several important criteria should be followed when searching or selecting for new
potential cut flowers.

2.1. ESTHETIC QUALITY

The first and most difficult criterion to satisfY is the attractiveness of the cut flower.
This factor is extremely important but not simple to evaluate. It is a subjective criterion
in that what attracts one might not be attractive to another. Furthermore, consumer
tastes change frequently and a desired shape or color at the time of selection or
 131

collection may no longer be considered attractive once the introduced plant is ready for
commercial production. In addition, some flowers, such as Geraldton wax flower
(Chamelaucium uncinatum) and Gypsophila, or cut green shoots, such as Ruscus
hypophillum, are not attractive, but are well-suited as ''fillers" in flower arrangements
and have therefore become very successful commercial products. Finally, not only must
the new flower have an attractive appearance, it should also look unique. Flowers with
unique appearance, i.e. color or shape, are highly desired by consumers and can be sold
at high prices. Moreover, it is difficult for the new flower to compete with well-known
old cut flowers if they appear similar. In recent years, much effort has been vested in
the introduction of wild flowers with unique appearance from Australia and South
Africa. New species such as Protea, Banksia, Gravillea, Leucadendron,
Leucospermum, Sandersonia, Leptospermum, Chamelaucium uncinatum, Anigozanthus
(kangaroo paw), Ornithogalum dubium and others have been successfully introduced to
the cut-flower industry.

2.2. LONGEVITY

One of the most important criteria for a successful cut flower is its vase life. A cut
flower should stay vigorous and attractive for at least 1 week in the final customer's
vase. Since it normally takes 3 days from harvest to final sale, a commercial product
therefore needs to stay fresh for at least 10 days. Various approaches to increasing the
vase life of cut flowers have been developed (Halevy and Mayak, 1981 ). When ethylene
is the decisive factor in the regulation of senescence, ethylene inhibitors such as silver
thiosulfate (STS) can prolong vase life significantly. However, when senescence is
caused by other factors, such as water absorption, the effect of the available treatments
is less significant. In many cases, no available treatment can increase the longevity to
the required period. An attractive flower with short vase life cannot become a
commercial cut flower. For example, iris species from the Oncocyclus subgenus, native
to southwest Asia, have the most attractive flowers of this genus but the flower has a
very short vase life. Similar to other members of the family Iridaceae, senescence in
this group is not regulated by ethylene (Woltering and van Doom, 1988). All efforts to
significantly prolong its vase life and introduce the plant as a major new cut flower
have been only marginally successful. Collectors or breeders of new potential cut
flowers should test the new flower's longevity before initiating the process of
introduction. If vase life is short and cannot be prolonged to the required period by the
available treatments, it is better to abandon this plant. However, if variation in
longevity is found in the wild population, it is possible to breed and select for plants
with the desired appearance and longer vase life.

2.3. INFLORESCENCE ARCHITECTURE

The structure of the flowering stem is an important criterion for selection. A very
attractive flower carried on a short, branched stem is usually not desirable as a cut
flower. Although it can serve as an attractive pot plant, it is not suitable for display in a
vase. Often, the length of the flowering stem contributes to the price of the flower:
132

longer stems command higher prices. If the flowering stem is too short, it is often
possible to increase its length by hormonal treatments, e.g. gibberellin (Michniewicz
and Lang, 1962), or shading (Armitage, 1991). If a variation in stem length is found in
the native populations, it is possible to breed and select for plants with long flowering
stems. In most cases, breeding is preferable, since both hormonal and shading
treatments which cause stem elongation reduce the stem's firmness. In some cases,
interspecific hybridization can be used to improve the architecture of the flowering
stem. Leucospermum lineare, for example, was used in a breeding program with other
Leucospermum species to contribute a slender, light-weight stem with narrow foliage
(Criley, 1998).
A branched flowering stem is also problematic in terms of packaging and shipping.
A branched stem may break during shipping and its shipment is usually more
expensive. Thus, an ideal cut flower should have a long, unbranched stem with a
solitary flower or compact inflorescence. Indeed, most leading cut flower crops, i.e.
rose, tulip, Gerbera, carnation, Chrysanthemum, etc. have such a structure.

3. The Process of Introduction

During the process of introduction, growing conditions, regulation of flowering time,

harvesting methods, post-harvest treatments, storage conditions, and sensitivity to
disease and insects are studied, as well as the ways to control them.

3.1. GROWING CONDITIONS

Climate, soil, water quality and light intensity affect the specific growing procedure
and therefore the introduction of specific plants must be performed in the region of
production. The information gained in one region is not always valid for other regions
with different environmental conditions. When the introduced plant is taken from the
wild, it is very important to get as much information as possible on its natural growing
conditions, including the regional climate, light conditions, type of soil, pH, water
quality, etc. This can help in deciding where to grow the plant and what its optimal
growing conditions will be. Understory plants require shading if grown in sunny
places. Plants from subtropical or tropical regions require additional heating in
temperate zones. Plants from rainy zones cannot grow on saline water. Plants which
normally grow under high humidity will probably fail to develop in dry places and
plants which grow in acidic soil perform poorly in alkaline soil. If the introduced plant
is sensitive to a specific environmental condition, such as low temperature, high pH,
saline water, etc., it is important to search for variation in the wild. If the plant is
naturally found in different geographical regions, there is a good chance for diversity.
A good correlation was found between tolerance to low temperature and the latitude of
origin of 15 wild populations of Leptospermum scoparium (Decourtye and Harris,
1992). Crossing cold-resistant clones with commercial cultivars, in this case, can
increase cold tolerance. In some plants, interspecific hybridization can be performed to
introduce resistance to a specific stress. Many species of Leucospermum are grown as
 133

cut flowers but their production is limited to regions with low soil pH. L. patersonii is
tolerant to high pH but has several disadvantages as a cut flower. When L. patersonii
was crossed with the vigorous but pH-sensitive L. conocarpodendron, the hybrids were
both vigorous and tolerant to high soil pH (Shchori et al., 1995).
Finding the optimal growing conditions during the introduction process is essential
for the plant's success as a commercial crop. It is also important for the evaluation of
the expected yield and quality of the new cut flower before entering commercial
production. Although yield and quality can be roughly evaluated during the early
stages of introduction, both can be improved dramatically under optimal growing
conditions.

3.2. REGULATION OF FLOWERING TIME

The ability to control flowering time is crucial to the success of the new cut flower for
several reasons: a) At certain times of the year, the demand for cut flowers increases
dramatically. Forcing the plant to bloom at this time will meet market demand and can
increase the profitability of the product. b) Manipulation of flowering time enables
year-round production. c) Some plants flower once a year for a very short period. This
usually limits production since the amount of flowers produced is higher than market
demand. The ability to regulate flowering time, in this case, can prolong the
production period and increase sales. d) Certain plants flower sporadically year-round.
Such flowering performance prevents commercial production. Here, the ability to
control flowering can enable the concentration of flowering periods and advance
commercial production.
The most important factor involved in the regulation of flower initiation is day
length. Many plants from temperate zones require long days for flowering, while some
plants from subtropical and tropical regions require short days (Thomas and Vince-
Prue, 1997). If the plant has an obligatory or quantitative requirement for short or long
days, it is relatively easy to control its flowering time by artificially lengthening or
shortening the photoperiod. In this way, summer-blooming plants can be forced to
flower in the winter and vice versa.
Many plants from cold regions require exposure to low temperatures (vernalization)
for flower induction. When these plants are grown in warm, subtropical or tropical
regions, they usually require artificial vernalization treatment for flowering. Since this
treatment requires long exposure to low temperature (0-4°C), it is extremely hard to
apply it to mature green plants. In certain plants, however, this treatment is relatively
easy since it can be given to seeds. Furthermore, in most geophytes the treatment can
be easily performed on bulbs or roots. For example, for winter flowering of Aconitus
napel/us, tubers are cold-stored during the summer. For year-round production of
Achillea filipendulina, the crowns must be stored at low temperature before planting.
To advance flowering in Eryngium planum, the roots should be stored for a few weeks
at 4°C (Ohana and Weiss, 1998).
Temperature is also important for flower development: Flowers of most plants
require moderate or high temperatures for their development and opening. Therefore,
increasing temperature during the flowering period can advance flowering time in
134

most plants. In deciduous plants, low temperature is required to break flower-bud

dormancy. This treatment requires long exposure of the mature plant to low
temperatures and therefore, it is complicated to perform in subtropical regions with
relatively warm winters. Peony (Paeonia lactiflora) has been in cultivation in China
for thousands of years, where it is grown as a garden and outdoor cut-flower variety.
Cut flowers are, however, available only for a few weeks in the late spring. Studies on
the physiology of peony flowering revealed that this plant requires a period of low
temperature to release flower buds from dormancy (Byrne and Halevy, 1986). During
the introduction of this plant as a new cut flower in Israel, practical methods for
extending the flowering season and obtaining production in the winter were developed:
plants are grown under the natural ambient cold temperatures of the early winter. After
sufficient cold units have been accumulated, the plants are covered with polyethylene
in mid-winter, and drenched with gibberellin. Sprouting and flowering soon follow.
Many new plants are abandoned during their introduction since it is either
impossible to control their flowering time or extensive investment is required.
Elimination of the requirement for a flowering signal is therefore an important task. A
natural variation in the requirement for a specific signal in the natural or commercial
population may enable breeding to eliminate or reduce this requirement. Early
carnation varieties required long days for flowering. However, during the 19th century,
selection for a perpetually flowering plant was performed and now, none of the modern
carnation cultivars require a signal for flowering (Holley and Baker, 1991). The statice
Limonium sinuatum Mill. requires vernalization for flower initiation. Since this plant
is a mandatory cross-pollinator, genetic variation in the level of vernalization needed
for flowering exists in the natural population. This variation served as the basis for a
breeding program to select plants with a low requirement for vernalization (Cohen et
al., 1995). Although the demand for a specific environmental flowering signal is
regulated by a number of genes, mutations in a single gene can eliminate or reduce this
requirement (Kinet, 1993). It is therefore possible to induce mutations, and select for
plants with low requirements for environmental flowering signals.
A large group of plants do not require any clear environmental signal for flowering.
These plants are termed autonomous-flowering plants. Most annual plants from this
group flower after producing a certain number of leaves. It is possible to control
flowering time in this group by changing the sowing date or growing conditions:
increasing light intensity or temperature promotes their vegetative growth and
advances flowering time. The situation is more complex in perennial autonomous-
flowering plants. Plants from this group flower either sporadically year-round, or at a
specific time of the year. Since flower induction in these plants is not controlled by
environmental signals, it is usually difficult to manipulate flowering time.
If the wild population of the introduced plant shows variability in flowering time, it
is important to collect various clones. These clones can be propagated vegetatively to
create several commercial cultivars with different flowering times. In this way, the
flowering season can be prolonged and production can be increased. A search for
native Geraldton wax flowers (Chamelaucium uncinatum) in Australia enabled the
establishment of a wide assortment of various plants, which bloom from December to
April (Shillo et al., 1985). The selected plants were propagated via cuttings to form
 135

uniform varieties with different flowering times (Halevy, 1995). Rice flower
(Ozothamnus diosmifolius) is a spring-flowering perennial shrub, native to the eastern
coast of Australia. The natural flowering period of the plant is very short (2-4 weeks).
Commercial cultivation of selected clones with different flowering times was
performed in Australia and several cultivars are now being used in commercial
production to prolong the flowering season (Turnbull and Beal, 1998).

3.3. PROPAGATION

Propagation is a central issue in the introduction of new horticultural products. All

major cut flowers are propagated vegetatively. This implies that inbreeding to obtain
homozygosity and uniformity is not an essential part of breeding programs (Sparnaaij,
1991 ). If the introduced plant can be efficiently propagated by vegetative means, it is
possible to choose a clone with the desired performance and create a new commercial
crop. When conventional vegetative propagation approaches, such as cuttings, root
cuttings, crown division, bulb division, etc., are not efficient, the introduction of the
new commercial product may take longer. This is the case for many rosette plants in
which the elongated stem is the inflorescence and cannot be used for propagation. In
some cases, it is possible to overcome this problem using micropropagation in culture.
When vegetative propagation is difficult or too expensive, seed propagation must be
performed. The commercialization of new products, in this case, can fail or take longer
since seed propagation of many wild germplasms causes variability, and uniform
performance is a prerequisite for commercial cut flowers. Phylica pubescens, a native
of South Africa, was introduced as a new cut flower in Israel. Evaluation of seed-
propagated plants revealed large phenotypical variability. Since all efforts to propagate
selected clones vegetatively failed, the new plant was abandoned (Gili Barel, M.Sc.
thesis, 1996, The Hebrew University).

3.4. YIELD

The yield of the new product is important in terms of its commercial success. If the
new plant has all the desired traits but the yield is too low, it might not be profitable
for commercial growth. Yield should be calculated per field or greenhouse area. Many
plants can produce only one flowering stem, but the plant itself requires very limited
space and the total yield per area is high. In other cases, the plant may produce several
flowering stems but requires a large space and the total yield is low. Growing
conditions can affect the number of flowering stems produced by the plants. Several
agricultural treatments can affect yield, including: pinching to break apical dominance,
forcing the plant for several flowering cycles, delaying flowering to produce additional
vegetative growth, and changing fertilization and irrigation regimes. Campanula
rapunculus, for example, produces three to four inflorescences in the wild, but when
grown in a greenhouse under irrigation and fertilization conditions, it produces 30 to
50 flowering shoots (Halevy et al., 1990). Thus, plants with low yield in the wild
should not be abandoned until they have been tested under optimal growing conditions.
136

Several other studies should be performed during the introduction, before the
commercialization of the new plant. These include an examination of its sensitivity to
diseases and pests and the development of post-harvest treatments. The former is
particularly important in the introduction of new species taken from the wild. Growing
the plant under different environmental conditions with new diseases to which it has
never been exposed may be lethal to it. The development of post-harvest treatments is
the last step of the introduction process. The commercialization of the new flower
should start only after these treatments have been developed and a high-quality product
with satisfactory vase life can be presented to the market. There are many examples of
plants that were released to the market too early, i.e. before the proper growing
conditions had been found, post-harvest methods developed, etc. (Vonk Noordegraaf,
1991). Although many of these flowers obtained high prices when they first appeared,
their sales decreased rapidly as a result of their low quality. In most cases, such plants
fail to become a commercial product. Early release can be disastrous for the new plant
and even if the proper methods are developed later on, customers that experienced the
low-quality product will reject the improved one. It. is therefore crucial to release the
new flower to the market only once it is ready for growth and marketing as a high-
quality product.

4. Concluding Remarks

The introduction of new species as cut flowers is a long process with a low success
rate. Nevertheless, it remains very important for the ornamental industry. While the
market is saturated with "old cut flowers" such as roses and carnations, consumer
demand for new flowers with unique, showy traits is continually on the rise.
Introduction of new species or improved varieties is the only way to increase
production and sales. Most new cut flowers are highly desired and command high
prices when sold in small numbers. However, their mass production usually causes a
decrease in market value (Halevy, 1995). A successful new cut flower is one that keeps
a gainful price, even when sold in large numbers. Only a few ''new cut flowers" have
succeeded in becoming important and leading crops in the last three decades. This
might suggest that most ''new cut flowers" are attractive only when they first appear on
the market and are still exotic. Since the first few years of the ''new flower" are the
most cost-effective, it is important for growers to continually introduce new products.

5. References

Armitage, A.M. (1991) Shade affects yield and stem length of field-grown cut-flower species, Hortscience 26,
ll74-ll76.
Byrne, T.G. and Halevy, A.H. (1986) Forcing herbaceous peonies, J. Am. Soc. Hort. Sci. 111, 379-383.
Cohen, A., Harazy, A., Rabinowitch, H.D., and Stav, R. (1995) Selection for early flowering in statice, Acta
Hort. 420, llS-124.
Criley, R.A. (1998) Leucospermum: botany and horticulture, Hortic. Rev. 22, 27-90.
 137

Decourtye, L. and Harris, W. (1992) Selection fur cold resistance in Leptospermum scoparium, Acta Hort. 320,
39-43.
Halevy, AH. (1995) Introduction ofnew plants as cut-flower crops, Acta Hort. 404, 166-170.
Halevy, AH. and Mayak, S. (1981) Senescence and postharvest physiology of cut flowers-part 2, Hortic. Rev.
3, 59-143.
Halevy, A., Weiss, D., and Frank, S. (1990) Cultivating Campanula rapunculus (rampion), an Israeli wild
flower, Hassadeh Quarterly l, 36-38.
Holley, W.O. and Baker, R. (1991) Carnation Production, Kendall/ Hunt Publishing Company, Dubuque, lA
Horovitz, A. and Danin, A. (1983) Relatives of ornamental plants in the flora oflsrael, lsr. J. Bot. 24, 26.
Kinet, J.M. (1993) Environmental, chemical and genetic control of flowering, Hortic. Rev. 15,279-333.
Michniewicz, M. and Lang, A (1962) Effect of nine difterent gibberellins on stem elongation and flower
furmation in cold-requiring and photoperiodic plants grown under non-inductive conditions. Planta 58, 549-
563.
Ohana, 0. and Weiss, D. (1998) Environmental and physiological :lilctors regulate Eryngium planum flowering.
lsr. J. Plant Sci. 46;47-51.
Sedgley, M. (1998) Banksia: new proteaceous cut flower crop, Hortic. Rev. 22, 1-26.
Shchori, Y., Ben-Jaacov, J., Ackerman, A, Gilad, S., and Metchnik, B. (1995) Horticultural characters of
intraspecific hybrids of Leucospermum patersonii X Leucospermum conocarpodendron. Acta Hort. 420,
135-137.
Shillo, R., Weiner, A., and Halevy, A.H. (1985) Environmental and chemical control of growth and flowering of
Chamelaucium uncinatum Schauer. Sci. Hortic. 25,287-297.
Spamaaij, L.D. (1991) Breeding fur disease and insect resistance in flower crops, in J. Harding, F. Singh, and
J.N.M. Mol (eds.), Genetics and Breeding ofOrnamental Species, Kluwer Academic Publishers, Dordrecht,
pp. 180-211.
Thomas, B. and Vince-Prue, D. (1997) Photoperiodism in Plants, Academic Press, San Diego, CA.
Turnbull, L.V. and Beat, P.R. (1998) Ozothamnus and Cassinia with potential fur commercialization, Acta Hort.
454, 147-156.
Uosukainen, M. (1992) Rhododendron brachycarpum subsp. tigerstedtii nitz.-a transmitter of extreme frost
hardiness, Acta Hort. 320, 77-81.
Von Renting, W.U. (1994) Circular of the wnrking group "New Floricultural Crops" (ISHS), Geisenheim,
Germany.
Vonk Noordegraat; C. (1991) Changes in floricultural crops, Acta Hort. 337,43-52.
Woltering, E.J. and van Doorn, W.G. (1988) Role of ethylene in senescence of petals: morphological and
taxonomical relationships,]. Exp. Bot. 39, 1605-1616.
TISSUE CULTURE FOR ORNAMENTAL BREEDING

A.C. CASSELLS
Department ofPlant Science
National University ofIreland
Cork, Ireland

1. Introduction

Plant cell, tissue and organ culture contributes to ornamental plant breeding in two
ways; first, it can be used to clone and store aseptic, pathogen-free germplasm; and
second, it can provide experimental material and systems for plant genetic
manipulation. These applications are restricted in that some families, species and
genotypes are recalcitrant in vitro (George, 1993, 1996). Further, genotypes, depending
on the explant and protocols used, may be unstable in vitro, giving rise to somaclonal
variation, i.e., genetic and epigenetic variability. Somaclonal variation shares many of
the characteristics of spontaneous mutation but at a higher frequency, and it has been
exploited as a source of variability in plants (Mohan Jain eta/., 1998); however, it can
also be a potential source of cryptic variability in transgenic lines. Adventitious
regeneration may result in chimeral breakdown and rearrangement. De novo chimerism
may result in the instability of selected lines from both mutation breeding (including
somaclonal variants) and transformation.
In exploiting plant tissue culture, there is the problem of establishing and
maintaining pathogen- and contaminant-free cultures (Cassells, 1997). The problem of
eliminating bacterial contaminants from cultures is particularly intractable and this
includes the elimination of Agrobacterium tumefaciens from inoculated cultures and
transgenic plants derived from these. Here, the focus will be on tissue culture systems
for the conservation and cloning of elite lines, for the production of systems for genetic
manipulation, with an emphasis on mutation breeding, in vitro selection, haploidization
and polyploidization.

2. Plant Tissue Culture for Cloning

2.1. IN VITRO CLONING-AN OVERVIEW

There are two prerequisites for the use of tissue culture systems in general, namely, the
ability to establish and maintain pathogen- and contaminant-free cultures (Cassells
, 1997), and the development of multiplication protocols that maintain genetic stability,
i.e. in vitro cloning systems (George, 1993, 1996). The earliest application of plant
tissue culture was the use of meristem explants to eliminate viruses from plants (see
139
A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, 139-153.
© 2002 Kluwer Academic Publishers.
140

George, 1993) and this strategy may also eliminate, subliminally, many endophytic
plant bacteria. The prevention of laboratory contamination by environmental pests, e.g.
micro-arthropods, bacteria and fungi, is dependent on impeccable laboratory practices
and is highly problematic (Cassells, 1997). Equally problematic is the maintenance of
genetic and epigenetic stability and physiological quality during the establishment,
multiplication and re-establishment phases (Cassells eta/., 2000). It is essential that
ornamental plant breeders who use in vitro cultures be aware of these often overlooked
fundamentals if they are to avoid the loss of valuable lines and stocks; and, where
Agrobacterium transformation is concerned, the escape of Agrobacterium-carried
transgenes into the environment (Barrett eta/., 1997).

2.2. ESTABLISHMENT AND MAINTENANCE OF PATHOGEN-FREE ASEPTIC

CULTURES

It is beyond the scope of this chapter to detail the strategies for the elimination of
pathogens and contaminants from in vitro cultures, other than to outline the principles
and to refer to appropriate reviews (Cassells, 1997). Principle 1 is that pathogen escapes
should be sought. Plants that appear pathogen-free should be disease-indexed using
approved indexing strategies, such as those laid down by the European and
Mediterranean Plant Protection Organization (EPPO: www.eppo.org). Ornamental
plants present special problems as they may be infected by a range of uncharacterized
pathogens. Assistance can be sought from crop-specialist plant pathologists and
increasingly, broad-spectrum or quarantine nucleic acid-based diagnostics which are
becoming available, e.g. 16s ribosomal DNA probes to detect fastidious bacterial
endophytes and multiplex DNA probes against conserved non-strain-specific genome
regions to detect members of virus families. Principle 2 is that thermo- or chemotherapy
may be applied to the stock plant to reduce the pathogen titer; then vegetative
propagation from buds is attempted and the resulting plants indexed following the above
guidelines. Principle 3: where such Stage 0 indexing is positive, pathogen or subliminal
pathogen/endophytic contaminant elimination can be attempted using small apical tip
explants. These should preferably be cultured on bacterial expression media and
protocols for in vitro thermo- and chemotherapy have been described. Again, the in
vitro plants should be grown under appropriate conditions to provide suitable tissue
material for disease indexing. Principle 4 is that HACCP (Hazard Analysis and Critical
Control Points) guidelines should be followed for the management of laboratory
contamination (Leifert and Waites, 1994).
The essence of these guidelines is that every effort is made to establish aseptic
cultures and that good laboratory management is exercised. In practice, culture
contamination is a major problem in commercial micropropagation, in slow-growth
germplasm storage, and especially when working with deliberately inoculated cultures,
as in Agrobacterium transformation, where the efficacy of antibiotics may be low. Long
(1997) has proposed transfer to autotrophic ('microhydroponic') culture as a strategy to
reduce bacterial contamination in affected cultures and as a strategy to control bacterial
contamination in micropropagation. In the case of elite lines, whether obtained from
conventional breeding or transformation, the best strategy to eliminate bacterial
contamination remains re-establishment in vitro via meristem tip explants. In the case of
heavily contaminated cultures, transfer to autotrophic culture in vitro to reduce
 141

microbial titer may be attempted prior to microplant establishment and followed by

antibiotic treatment of the established plants prior to meristem excision.

2.3. AXILLARY AND NODAL BUD PROLIFERATION

Plant genomes can show high levels of genetic, epigenetic and physiological instability
reflected in mutations, altered development and hyperhydricity (syn. vitrification)
(Deberg and Zimmerman, 1991 ). This is widely recognized in micropropagation, where
stable cloning strategies that yield plants of good physiological quality are required. The
most stringent conditions apply in the mass clonal propagation of certified virus-free
stock, e.g. of potato and strawberry, and generally a lack of confidence in
micropropagation by the certification authority is reflected in the imposition of
restrictions on the number of subcultures that may be carried out before returning to
new in vivo stock plant selection and re-establishment of fresh aseptic cultures. The
experience of micropropagators is varied, but in general, bud culture is recognized as
the safest propagule to use for in vitro cloning and even using buds, 'nodal' culture is
preferred to proliferation by dividing clusters of precocious axillary buds (Fig. 1;
George, 1993). In strawberry, the latter have been shown to give rise to adventitious
buds and over subculture, the latter increase as a percentage of the buds formed; with
this shift from axillary to adventitious bud formation, genetic variability increases. Even
in the case of nodal culture, media and containers may influence the epigenetic and
physiological quality of the plants, affecting their performance at and post-
establishment. In the case of ornamentals, these in vitro factors have been shown to
affect time to flowering, which may be important in evaluating lines.
Many factors influence the multiplication rate and quality of in vitro plants
(microplants); these include the spectral characteristics and PPF (photosynthetic photon
flux) of growth-room lighting, the temperature, the culture vessel characteristics and the
culture media (George, 1993). Traditionally, plants were introduced into culture via
meristem tip explants and placed on an appropriate basal medium formulation, e.g. that
of Murashige and Skoog for herbaceous plants or that of McCown and Lloyd for woody
plants (see George, 1996 for details). Then the principle of Skoog and Miller was
followed in achieving the appropriate morphogenic responses by altering the ratio of
auxin to cytokinin. Classically, auxins and cytokinins were the principle growth
regulators used in combination with gibberellins and anti-gibberellins to control stem
elongation (George, 1993).
In recent years, the quality of microplants has come under criticism, particularly the
poor weaning and post-weaning performance (Preece and Sutter, 1991; Swartz, 1991 ). It
has been shown that quality improvement may be achieved by inducing transpiration
steam in the culture vessel, by transfer to liquid or autotrophic culture, and by in vitro or
immediately post vitrum inoculation with biocontrol or growth-promoting
microorganisms (Cassells eta!., 2000).

2.4. ADVENTITIOUS REGENERATION

Adventitious regeneration involves bud induction directly in explants lacking pre-

formed buds or meristemoids, termed direct organogenesis, e.g. in Saintpaulia
 N
-"""
Q, !.

1 ·-" · ~::·~
kom
-
--· ,.,---
~- ~7
- " " '" : . culture
l ~ Direct shoot Somolc
= ....,.. ·-"'~~'
~
DIRECT
...,- "'::""
MORPHOGENESIS
F==i'1
..,.. Direct
L...Y ~ ~ L_JJ embryogenesis
/ ......... .........
STOCK PUWT
E><p&or<S•om Indirect shoot
.............. ""' INDIRECT ~ - ~ -
L5 formation
.._. ._
"'"..'="""
- MORPHOGENESIS ~ "---._) - ,..""""

CaM <>"""' ( CaM )""' -·>ous

""""""" I "" ,_.
~ em~;!1~~~~esls
/ ...... --
~
.......
e <!Jp\1
----
(FtOm tingle mil)

Figure I. The strategies used to multiply plants in vitro from axilla')' buds and from adventitious buds (reproduced with permission from George, 1993). The
prefen·ed strategy to maintain genome stability is nodal culture, as apical explant culture may give rise to adventititious bud formation from basal callus. The
genotype-dependent risk of somaclonal variation increases from direct morphogenesis via somatic embryos to adventitious shoots, while indirect morphogenesis,
particularly when it involves callus subcultures, is associated with high- frequency variation.
 143

ionantha; or in callus on, or subcultured from tissues, termed indirect organogenesis,

e.g. in Solanum tuberosum (Fig. 1; George, 1993). In many cases, de-differentiation,
callus induction and adventitious shoot formation can occur on a single basal medium-
one-stage organogenesis--containing auxins and cytokinins, as in tobacco. In other
cases, induction and expression of competence require exposure to different or
sequential hormone combinations-two-stage organogenesis (Christianson and
Warnick, 1985). Organogenesis in some plants, e.g. crucifers, is very sensitive to
ethylene accumulation in the cultnre vessel, which necessitates the inclusion of
ethylene-binding agents such as potassium permanganate in the vessel or the use of
ethylene inhibitors, e.g. AVG (aminoethoxyvinylglycine). There may also be a role for
auxin-interfering compounds in regulating endogenous auxin, e.g. the auxin transport
inhibitor TIBA, in inducing organogenesis (George, 1993).
The main problem in adventitious regeneration is spontaneous mutation or
somaclonal variation which is dependent on genotype, type of explant and cultnre
longevity (Mohan Jain et a/., 1998). Apical meristems have putative stabilizing
mechanisms, e.g. due to diplontic drift (Balkema, 1972), whereas there is evidence that
genetic diversity may occur in the somatic cells of plant tissues (D'Amato, 1978). This
'pre-existing' variability may be expressed in shoots derived from these aberrant cells.
There is also evidence that the plant genome expresses genotype-dependent genetic,
epigenetic and physiological instability in response to putative oxidative stress in vitro
caused by the explant excision, culture media and environmental factors (Fig. 2).
Mutation (D'Amato, 1978) and epigenetic variability (Meins and Biuns, 1978) may
occur at high frequency in vitro and cell line selection may occur on serial subculture
with a loss of competence, or be expressed in somaclonal variability in the progeny. In
some cases, variability observed at the callus level may not be expressed, with
genetically variable callus giving rise to normal progeny. Somaclonal variation has been
comprehensively reviewed recently by Mohan Jain eta/. (1998).
Adventitious regeneration may be from cells of one somatic layer (Broertjes and van
Harten, 1988), which is seen where variegated plants give tissue culture progeny of one
cell lineage, but this is not always the case as chimeral progeny have been reported in
adventitious regeneration (Marcotrigiano and Gouin, 1984). This has implications for
transformation or mutagenesis where adventitious unstable chimeric regenerants may
occur and this may explain the instability of selected progeny in subsequent vegetative
propagation. Arguably, chimerism may be especially prevalent where in vitro selection
for toxin resistance, as in selection for transformants, is employed.

2.5. SOMATIC ENBRYOGENESIS

Like adventitious shoot formation, somatic embryogenesis is an adventitious

regeneration process which can also be direct or indirect (Fig. 1; George, 1993).
Somatic embryos can also be used to obtain secondary somatic embryogenesis (Litz and
Gray, 1995). In general, the protocols to obtain somatic embryogenesis are as for
adventitious shoot induction and in many cases, both adventitious shoots and somatic
embryos occur in the same explant or callus, indicating a possible cell developmental or
hormonal gradient in the tissues. Somatic embryogenesis parallels zygotic
embryogenesis. Somatic embryos are induced on common basal media, either under the
influence of auxins and cytokinins following the principles of Skoog and Miller for
144

adventitious shoot formation (one-step or direct embryogenesis), or after explant culture

on an auxin-containing medium, frequently 2,4-D, when transferred to an auxin-free
medium (two-step or permissive induction) (Litz and Gray, 1995; George, 1996). It
appears from more recent studies that the frequency of somatic embryogenesis can be
increased by incorporating second-generation growth regulators such as thidiazuron
(TDZ) into the medium (Gillet al., 1994). Media for somatic embryogenesis are also
sometimes supplemented with amino acids and other organic supplements (George,
1996).

CALLUS

STOCK PLANT

GENETICALLY-ALTERED
PLANTS

Mulaled part of
callus !issue
originales from
SIOCk plan!

Figure 2. The origins of somaclonal variation (reproduced with permission from George, 1993). Variation
may exist in the cells of the explant or arise de novo during callus formation (D' Amato, 1978). Adventitious
shoots may be chimeric.

The cloning potential of somatic embryogenesis has been discussed extensively,

based on large-scale production of somatic embryos in bioreactors coupled with
encapsulation of the embryos to produce synthetic seed (Redenbaugh eta/., 1991).
There have, however, been many problems, including the recalcitrance of many crops,
asynchronous formation, and precocious development (Litz and Gray, 1995), which
limit cost reduction by automation; moreover, an essential feature of the true seed,
namely, shelf life, can be lacking. A key technical step in artificial seed production is
seen as the embryo's ability to withstand desiccation leading to 'dormancy' or
quiescence-which gives shelf life-and to resume growing following rehydration.
 Water inside cells
Is v1tr1f1ed wilhout
ice lormat.on.

Tissue in a small amount of

liquid and in a CC<ltainer whch
allows ral)id heat ltansfer

~ Vo~ .,,;, f
cooling
•
STARTING
MATERIAL r------------------- ----------------,
I
I
lnlracellular Ice forms w«hin
cells because they still contain
0 Cell structure
I a relatively large amount of water is destroyed

Cytoplasm partially : ca.-30"C to .oo·c -196"C

plasmolysed by I
cryoprotectanrs I '!-
'*" -~ .,.., ,:.!--..
~~~ ,.~. ~r~
'•"' ""
Rapid cooling .:-~~ --~ ,,... 0
---· , ~
...
~
Tissues (cells)
GJ
in liquid medium
*"0
Ice crystals begin lo Cells lose water Into
lorm .., the med1um the medium. This Water inside cells
and cell constiluenls The amount of Ice in equalises the vapour is v•tnfted Without
become sope<cooled. lhe med1um increases. pressure dit1erence. lee fatmation.
ca. -IO"C ca ·20"C ca.-30'C 10 -60'C · 196"C

Slow cooling ~!~ .,,, ,,# ,,, ~!.

' ...., ;i' *~if'
(progressiVe or
'!-'
~~~ ~* ,,,
stepwise)
~ -+ ~ ~ '•'
0 · ~~- :et.. 0
-~
Bocau"' lhe c:yloplasm Is The medium beCOmes The rato or wat81 loss
surr<X.Wlded by a membrane ancl mot"e concantlaled so lhat from cells depends Oil
oont,ajns dtssotved substance:!, thG \I'IJPOLif pi855Ut'O the rate of coohng
Its freezing poW Is around ·IO"C. dtf1erence bOrwoon the
The formation of W'llracellulat k:e cells and tne medium rises.
at this temperatute must be avotded.
......
+:>.
Vl
Figure 3. The stages ofcryopreservation (reproduced with pennission from George, 1993).
146

3. Plant Tissue Culture for Germplasm Conservation

Large gene banks exist in the major geographical regions in National Collections, e.g.
the USDA in the US and the Vavilov Institute in the Russian Federation, and in the
International Research Centres (CGIAR), with an estimated total of approx. 4.5 million
accessions; of these, only approx. 4.5 thousand are of ornamental plants (Villalobos and
Engelmann, 1995). The principle locations for ornamental germp1asm conservation are
in situ, in botanical gardens, in commercial nurseries, in plant breeders' collections and
in some amateur conservation societies. There are two main strategies for germplasm
conservation of vegetatively propagated crops, namely, the maintenance of cultures
under slow growth conditions and cryopreservation (Fig. 3; Villalobos and Engelmann,
1995). The problem with in vivo conservation is the cost of maintaining the material,
errors in labeling and loss and deterioration due to abiotic and biotic stresses. The
problem with in vitro storage is, again, the risk of stock loss due to culture
contamination, technical failure, and genetic instability, which is dependent on the
genotype and protocol. Classically, genetic stability assessment was based on UPOV
(The International Union for the Protection of New Varieties of Plants) guidelines
(www.uoov.org) namely, on morphological and phenotypic character assessment. This
can be applied to in vivo material but is not practical for in vitro material where growing
batches of the material to flowering or crop production for morphological assessment
would be impractical. For both in vitro- and in vivo-conserved stock management, a
reliable genetic fingerprinting screen is desirable.

3.1. SLOW GROWfH

Slow growth (Villalobos and Engelmann, 1995) is based on extending the period
between subcultures; it should also be based on the manipulation of protocols known to
maintain stability in the target genotype (George, 1993) and these manipulations should
be confirmed not to be destabilising. Frequently, slow growth is achieved by reducing
culture temperature and/or light, incorporating osmotic agents such as mannitol in the
medium, or including a chemical growth inhibitor (see review by Villalobos and
Engelmann, 1995). While temperature reduction has the appeal of simplicity, some
tropical crops such as bromeliads may be temperature-sensitive, in which case a
combined strategy of low temperature and the use of osmotic agents, e.g. high sucrose,
may be effective. A strategy which merits further research is growth control based on
induced mineral deficiency.
The risk of microbial and micro-arthropod contamination is high in long-term stored
cultures (3 - 18 months), not only to the cultures being conserved but also to the
growthrooms and sterile areas in general. This risk can be greatly reduced and
contamination contained by culture in sealed gas-permeable vessels, the simplest form
of which is the heat-sealed plastic bag system. This system has been adopted
successfully by some micropropagators to store varieties between demand cycles using
commonly available, heat-sealable food-storage bags.
 147

3.2. CRYOPRESERVATION

Cryopreservation is a long-term. low-maintenance (subculture-free) storage method that

carries a low risk of bacterial contamination (Villalobos and Engelmann, 1995).
Cultures are usually stored in liquid nitrogen (-196°C). Genetic stability is claimed to be
high but again, this depends on the genotype and protocol used. The procedure involves
pre-culture on cryoprotectants such as dimethyl sulfoxide (DMSO) which penetrate the
cell and protect membranes and proteins, and/or osmotic agents such as mannitol. This
is followed by a slow cooling stage at a rate of usually not more than 1°C/min. Cultures
are stored in liquid nitrogen in vials to be retrieved as required. Thawing is rapid, by
immersion in a water bath at approx. 40°C and the tissue is placed on recovery medium
before transfer to multiplication medium (Fig. 3; George, 1993, 1996).
There are a number of variants of the basic technique of cryopreservation. In
encapsulation/dehydration, artificial seed production technology is used to encapsulate
apices or zygotic embryos in alginate beads. These are imbibed with high sucrose
medium and dehydrated in sterile air before rapid freezing to -196°C. Little protocol
development is required compared to the aforedescribed traditional methods and there is
no requirement for specialized freezing equipment. Another variant is vitrification (to be
distinguished from this term used in tissue culture as a synonym of hyperhydricity),
where the tissues are immersed stepwise in increasing concentrations of
cryoprotectant(s) which prevents the formation of harmful ice crystals during the
freezing process. Growth reactivation requires stepwise removal of the cryoprotectant.
Here also there is no need for controlled-temperature freezing equipment, but the high
concentrations of cryprotectants can be toxic to some genotypes and protocol
optimization can be lengthy (Villalobos and Engelmann, 1995).
While recently, simplified cryopreservation techniques such as vitrification have
attracted considerable attention, protocol development for a given genotype may be
slow. If apical buds or somatic embryos are frozen, the probability of maintaining
genetic stability is highest, regardless of genotype, because of the stabilizing influence
of diplontic selection that exists in apical meristems (Balkema, 1972). In contrast to
slow-growth conservation where the recovered cultures can be used directly for mass
multiplication, cultures from cryopreservation are limited in supply, with sometimes
low establishment rates and a slow initial growth response.

4. In Vliro Mutagenesis

In this section we consider both spontaneous in vitro variability and induced mutation
with an emphasis on the contribution that tissue culture per se has made to the increased
efficiency of mutation breeding (Mohan Jain et al., 1998). In essence, tissue culture
systems miniaturize the target tissues for mutagenesis and for selection, thus facilitating
treatments for vegetatively propagated crops. The test tube and hospital X- or gamma-
ray machine has replaced the need to arrange plants in rows in lead-lined bunkers for
exposure to high output cobalt sources (FAO/IAEA, 1977). Furthermore, the problem of
chimerism can be largely, if not completely avoided by direct formation of adventitious
homohistonts ('solid' mutants) from the individual mutated cells, or by serial subculture
in vitro (Broertjes and Van Harten, 1988). It has been argued that 'diplontic selection'
148

(Balkema, 1972), which relies on putative stabilizing mechanisms in the organized apex
to select against debilitating mutations, can also be used in the irradiation of apices in
vitro to increase efficiency in the rejection of worthless off-types (Sonnino et al., 1986).
Mutation breeding is well suited to the alteration of phenotypic characters such as plant
habit, branching and altered pigmentation, based on up- and down-regulation of genes
controlling these traits or on influencing genes with pleiotropic effects on these
characters (Broertjes and Van Harten, 1988).

4.1. SPONTANEOUS MUTATION IN VITRO ('SOMACLONAL VARIATION')

The spontaneous variability arising in tissue culture, termed somaclonal variation by

Larkin and Scowcroft, has been the subject of intensive debate and investigation (see
Mohan Jain et al., 1998). Early suggestions that it generated high-frequency useful
variability have given way to a recognition that the range of variability is similar to that
induced in mutagenesis (Mohan Jain et al., 1998), with the implication that most
somaclonal variants are worthless. A further factor mitigating against its use in plant
improvement is that it is, unlike induced mutation, genotype-dependent (Mohan Jain et
a/., 1998). Both heritable genetic change and epigenetic change have been observed in
plants derived from in vitro culture (Mohan Jain et al., 1998). The underlying
mechanisms are reported to include changes in chromosome number and structure, in
DNA from point mutation to amplification. and in methylation. The latter may be
associated with the juvenility and rejuvenation that can occur in microplants. In general,
bud culture maintains genome stability but can give rise to developmental variation, e.g.
rejuvenation in woody species (George, 1993, 1996) and juvenility in herbaceous plants
such as potato. As discussed earlier, genetic variability in tissue culture is associated
with adventitious pathways of regeneration and is dependent on the genotype, explant
source, media factors and duration in culture (Fig. 1).

4.2. INDUCED MUTATION IN VITRO

Broertjes and Van Harten (1988) were major proponents of tissue culture systems for
mutation breeding emphasizing the advantages of adventitious regeneration from single
mutagenized cells for the production of homohistontic ('solid') mutants. Mutation
breeding in vitro has been used successfully for the creation of variability in ornamental
species,. where the commercial value of mutants is not as constrained by requirements of
high yield and retention of culinary traits as in vegetable crops. Most mutation is
induced by physical rather than chemical mutagens, as the latter are easier to optimize.
However, the effect induced by chemical mutagens, e.g. a high frequency of point
mutations, is different from that induced by physical mutagens and this should be
remembered when designing an experimental program (F AO/IAEA, 1977, 1982).
Protocols for both chemical and physical mutagenesis have been developed by the
International Atomic Energy Agency (IAEA), Vienna whose publications should be
consulted (www.iaea.org). In general, when using physical or chemical mutagens, the
dose rate that reduces growth by approx. 50 to 70% is chosen as a compromise working
dose namely, where there is induction of a practical level of mutation without the risk of
excessive genomic damage (FAO!IEAE, 1977). While induced mutation, like
somaclonal variation. may increase the mutation rate from approx 1 in 500,000 to
 149

percentage levels, it must be recognized that a very low frequency of these mutants will
be of any value. Therefore, the efficiency of the method depends on the early
elimination of worthless variants from trials.

4.3. MUTANT SELECTION

In ornamental breeding, random variability from spontaneous or induced mutation may

yield a novel phenotype of economic value (Broertjes and Van Harten, 1988). If,
however, mutation is to be used to correct character defects, e.g. lack of disease
resistance, or to improve a trait such as flower color, then an efficient selection process
is important in reducing trialing costs (Kowalski and Cassells, 1999). It is assumed in
the latter cases that retention of the characteristics of the parent genotype is required,
that is, that the mutants be near-isogenic with the parent, as in a flower color series.
Critical elements in a mutagenesis program to correct character defects are the use of
strategies that (i) select for cell fitness, e.g. use of bud irradiation (Sonnino et al., 1986);
(ii) facilitate chimeral breakdown, e.g. by serial subculture in vitro; (iii) select for the
target trait (Dix, 1990), e.g. select for resistance to fungal toxins; (iv) select against
gross aberrants, e.g. polyploids and gross aneuploids using flow cytometry, and
morphological aberrants using image analysis, and (v) select for near-isogenicity using
computerized image analysis (Kowalski and Cassells, 1999). A caution against in vitro
selection is that not all traits may be expressed in juvenile (in vitro) tissues. There are,
for example, many constitutive and inducible components of disease resistance, some of
which--e.g. lignification and phytoalexin production-may not be expressed in vitro
(Jones, 1990). A selection strategy has negative and positive elements aimed at reducing
the through-put of worthless mutants to the field (Fig. 4; Kowalski and Cassells, 1999).
This facilitates early field trials, that is, large replicated blocks to assess meaningfully
traits such as plant productivity and disease resistance.

5. Haploidization

Haploid lines have proven to be of value to agricultural crop breeders for the expression
of recessive traits, which can be doubled (see below) to give homozygous lines for crop
improvement. The principal in vitro method for the production of haploids is pollen and
anther culture (Atanassov et al., 1995). While efforts have been made to culture ovules
and ovaries, these, in general, have been more intractable (Atanassov et al., 1995). A
variant of the latter method is exploitation of the bulbosum method with embryo rescue
in vitro. This depends on loss of the chromosomes of one parent in some interspecific
crosses, leading to sterility. If embryos from such crosses are rescued, i.e. cultured in
vitro, haploid plants may be obtained (Atanassov et al., 1995).
A number of factors are reported to influence androgenesis, including genotype,
donor-plant growth conditions, developmental stage of the microspores, pre-treatment
of the anthers, culture media and culture conditions (George, 1993). Nevertheless,
anther culture has been achieved and exploited by breeders of cereals and solanaceous
crops. While it may not be cost-effective to breed for competence in ornamental crops,
breeding lines can be screened for androgenetic competence. Microspore culture, while
having some advantages over anther culture--e.g., in in vitro selection, is considered
150

............. ;~~~;~~;,.............1
Novel Correction of
Phenotype sought character defect
sought
····················T····················'
Grow In vitro selection In vivo selection
plant population

!. . . . . . . . . . . . . . . . . . . . . .
and make
..........................................!
]
visual selection

Use tissue culture

to clonally progagate for
J
Use serial subculture Use flow cytometry
to eliminate
...

t_:.:~··--· ~~~~~;;.~;~
largescale evaluation and
commercialization
....... .......

ry·~-~ JLto eliminate

polyploids and
oss aneuploids
1
Use image analysis
to identify and
disg~ard outliers

. ::1 ...~-
[ Use image analysis
J r-··-·---·---~
! Make selection i
......;~~";...... L.... ~. . . ..J
Make selection Use image analysis
for target to select for isogenicity
character

I I
~~ Use image analysis
to select for isogenicity
! Use tissue culture to
l clonally propagate
j
j for lagescale evaluation!
L. . ! and commercializa~~

Use tissue culture to

clonally propagate I
!.~~~;;s;~;~;~;..
Figure 4. A seledion sdleme to reduce the through-put of off-types in mutation breeding where flow
cytomttry is used early in seledion to eliminate polyploids and gross aneuploids. This can be complemented
by the use of computerized image analysis to eliminate motphological variants dftected as outliers in the
character distribution based on a standard deviation assay. In programs to correct character defects or to alter
specific characters, e.g. flower color, this can be combined with positive selection in vitro or in vivo for target
traits (Dix, 1990). Selected improved lines can be further selected for near isogenicity (lines which are within
character distributions as determined by discriminant analysis) with the parental line based on computerized
image analysis (Kowalski and Cassells, 1999).
 151

more difficult. A limitation on the use of haploids derived from pollen and anther
culture is the phenomenon of gametoclonal variation (analogous to the aforedescribed
somaclonal variation). Some of the variants, e.g. dihaploids, may have breeding
applications, but with the caution that they may contain cryptic background genetic
changes (Mohan Jain eta/., 1998).
Ovule and ovary culture are generally more difficult than anther culture and
consequently tend to be attempted when pollen and anther culture have failed. This
phenomenon also shows strong genotype dependence but is reportedly less dependent
on the stage of ovule development (Atanassov et al., 1995).

6. Polyploidization

Amongst the uses of polyploidy for ornamental plant breeders are restoration of fertility
in interspecific hybrids (Stebbins, 1950) and improved agronomic and morphological
traits e.g., increases in flower size. High-frequency spontaneous polyploidization is a
component of somaclonal variation in some genotypes, but in some species
organogenesis may occur preferentially from diploid cells. Polyploidy has also been
induced by treatment of nodes and microplants in vitro with colchicine. Colchicine has
also been added to anther culture media to produce doubled haploids (Navarro-Alvarez
et al., 1994).

7. Conclusions

Plant tissue culture technology provides the breeder with a technological infrastructure
for producing, maintaining and rapidly multiplying disease- and contaminant-free lines.
Adventitious regeneration is, in principle, to be avoided in clonal multiplication. It is
essential to confirm genome stability in clonal multiplication and in cryopreservation. In
principle, genome stability, aside from epigenetic instability, is maintained where the
strategy involves organized apices; however, there are difficulties in some cases in
obtaining microplants of good physiological quality from bud and nodal culture. A
limitation on the use of tissue culture for plant breeding is genome instability and/or
intractability in some genotypes in adventitious regeneration.
Arguably, spontaneous in vitro mutation (somaclonal variation) and especially in
vitro-induced mutation are relatively underutilized strategies for ornamental plant
breeding. Historically, in vivo mutation for plant breeding was seen to be inefficient,
demanding large-scale facilities and resulting in unstable chimera! progeny. The
efficiency can be significantly increased by using in vitro systems with the exploitation
of diplontic selection, and in vitro selection for improved traits and against worthless
off-types. To overcome the intractability of some genotypes to adventitious
regeneration, nodal culture with physical or chemical mutagenesis of bud cultures may
be used, with the latter selection principles. Anther culture can be used for haploid
production; colchicine has been used in in vitro culture to produce doubled haploids and
polyploids; spontaneous and induced mutation can also be used for polyploid
production.
152

8. References

Atanassov, A, Zagorska, N., Boyadjiev, P., and ~ilianov, D. (1995) In Vitro production of haploid plants,
World J. MicrobioL BiotechnoL 11, 400-408.
Balkema, G.H. (1972) Diplonticdriftin dlimericplants,RadationBot, 12,51-55.
Bamtt, C., Cobb, E., McNicol, R., and Lyon, G. (1997) A risk assessment study of plant genetic
transfonnation using Agrobacterium and implications for analysis of transgenic plants, Plant Cell Tiss.
Org. Cult. 47, 135-144.
Broertjes, C. and van Harten, AM (1988) Applied Mutation Breeding for Vegetatively Propagated Crops,
Elsevier, Amsterdam.
Cassells, AC. (1997) Pathogen and Microbial Contamination Management in Micropropagation, Kluwer,
Dordredtt.
Cassells, AC., Doyle, B.M and Curry, R.F. (2000) Mdhods and markers for quality assurance in
micropropagation, Acta Hort. 530.
Christianson, ML. and Warnick, D.A (1985) Temporal requirements for pbtytohonnone balance in the
control of org;mogenesis in vitro, Dev. Bioi. 112, 494-497.
D' Amato, F. (1978) Chromosome number variation in cultured cells and regenerated plants, in T.A Th01pe,
(ed.), Frontiers ofPlant Tissue Culture IAPTC, U. Calgary Printing Services, Calgary, pp. 287-295.
Deberg, P.C. and Zinunennan, R.H. (1991) Micropropagation: Technolagy and Application, Kluwer,
Dordredit.
Dix, P.J. (1990)PlantCellLine Selection, VCH, NY.
FAOIIAEA(1971)Manual on Mutation Breeding ,2nded, IAEA, Vienna.
FAO/IAEA (1982) Induced Mutations in Vegetatively Propagated Plants II, IAEA, Vienna.
George, E.F. (1993)Plant Propagation by Tissue Culture: The Technology, Exegetics, Basingstoke.
George, E.F. (1996) Plant Propagation by Tissue Culture: In Practice, Exegetics, Basingstoke.
Gill, R., Senaratna, T., and Saxena, P.K (1994) Thidiazuron-induced somatic embryogenesis enhances
viability ofhydrogel-encapsulated somatic embryos of geranium, J. Plant Physiol. 143, 726-729.
Jones, P.W. (1990) In Vitro selection for disease resistance, in P.J. Dix (ed.), Plant Cell Line Selection ,VCH,
Weinheirn, pp. ll3-150.
Kowalski, B. and Cassells, AC. (1999) Mutation breeding for yield and Phytophthora infostans (Mont.) de
Bary foliar resistance in potato (Solanum tuberosum L. cv. Golden Wonder) using computerised image
analysis in selection, Potato Res. 42, 121-130.
Leifert, C. and Waites, W.M (1994) Dealing with microbial contaminations in plant tissue and cell culture:
hazard analysis and critical control points, in P.J. Lumsden, J.R. Nicholas,. and W.J. Davies (eds. ),
Physiology, Growth and Development ofPlants in Culture, Kluwer, Dordrecht, pp. 363-378.
Litz, R.E. and Gray, D.J. (1995) Somatic embryogenesis for agricultural improvement, World J. Microbiol.
Biotechnol.ll: 416-425.
Long, R.D. (1997) Photoautotrophic micropropagation-a strategy for contamination control?, in AC.
Cassells (ed.), Pathogen and Microbial Contamination Management in Micropropagation, Kluwer,
Dordredrt, pp. 267-278.
Marcotrigiano, M and Gouin, F.R. (1984) Experimentally synthesised plant dlimeras. l. In Vitro recovery of
Nicotiana tabacum L. chimeras from mixed callus cultures, Ann. Bot. 54, 503-511.
Meins, F. Jr. and Binns, AN. (1978) Epigenetic clonal variation in the requirement of plant cells for
cytokinins, Symp. Sac. Dev. Biol. 36, 188-201.
Mohan Jain, S., Brar, D.S., and Ahloowalia, B.S. (1998) Somaclonal Variation and Induced Mutations in
Crop Improvement, Kluwer, Dordredtt.
Navarro-Alvarez, W., Baenziger, P.S., Eskridge, K.M, Hugo, M., and Gustafson, V.D. (1994) Addition of
colchicines to wheat anther culture media to increase doubled haploid plant production, Plant Breed. 112,
192-198.
Preece, J.E. and Sutter, E.G. (1991) Acclimatization of micropropagated plants to greenhouse and field, in
P.C. DeberrJt and R.H. Zinunennan (eds.), Micropropagation: Technology and Application, Kluwer,
Dordredrt, pp. 71-94.
Redenbaur]t, K., Fujii, J.O., and Slade, D. (1991) Synthetic seed technology, in I.K. Vasil (ed.), Scale-Up and
Automation in Plant Propagation, Academic Press, NY, pp. 36-75.
Sonnino, A, Ancora, G., and Locardi, C. (1986) in Nuclear Techniques and In Vitro Culture for Plant
Improvement, IAEA, Vienna, pp. 385-394.
Stebbins, G.L. (1950) Variation and Evolution in Plants, Ed Arnold, London.
 153

Swartz, H.J. (1991) Post cuhure hmaviour: genetic and q~igenetic effects and related problems, in P.C.
Debergh and R.H. Zinunerman (eds.), Micropropagation: Technology and Application, Kluwer,
Dord:redrt, pp. 95-122.
Villalobos, V.M and Engelmann, F. (1995) Ex situ conservation of plant gennplasm using biotechnology,
World J. Microbial. Biotechnol. 11. 375-382.
GENE TRANSFER TO PLANTS

S.C. DEROLES, M.R. BOASE, C.E. LEE, T.A. PETERS

Crop & Food Research
Private Bag 11,600
Palmerston North, New Zealand

1. Introduction

Worldwide exports of floricultural products in 1999 totaled over US $7 billion and have
been steadily increasing over the past 10 years (http://www.pathfastpublishing.com).
The major portion of this production was cut flowers at 47%, followed by whole plants
(35%), bulbs and tubers (10%) and cut foliage (8%). One of the keys to a successful
ornamentals industry is the constant production of new crops, either by the introduction
of new species (eg. Zantedeschia and Sandersonia-Brundell and Reyngoud, 1985;
Funnell, 1993) or by creating new cultivars in existing crops through the alteration of
characteristics such as flower color and plant form. In addition, alteration of agronomic
characteristics such as growth stature, vase life, pest and disease resistance is important
for production efficiency.
To date, traditional breeding methods have been the principal source of new
products and their success is evident in the diverse range of crops currently available.
Breeders have been able to provide new variants in flower color and form, foliage color,
scent, plant stature and vigor, leaf form, and pest and disease resistance. However,
traditional breeding is limited to variations contained in the gene pools of the parent
species. The first attempt to expand the available gene pool was made by the
development of in vitro breeding techniques such as embryo rescue, in vitro pollination,
protoplast fusion and mutagenesis (George, 1993). Incompatibility between species has
been overcome by the use of non-stigmatic pollination and embryo rescue (Janson,
1993; Kishi et a/., 1994). In crosses that do not yield a surviving embryo, protoplast
fusion of adult cells can be used to generate new hybrids. Several new ornamental
cultivars, for example in Gypsophila, Lilium and Sandersonia, have been generated
using such techniques (Janson, 1993; Kishi eta/., 1994; Morgan eta/., 2001). However
a common disadvantage of both traditional and in vitro breeding techniques is the need
to carry out several backcrosses to isolate the new characteristic in a desirable
background. This can often take many years and has limited the number of
commercially viable cultivars produced to date.
Even with the advent of modem breeding techniques, in some mature crops fewer
new cultivars are being produced. In addition, some more recent crops (e.g.
Sandersonia) are based on a single species, and to date, breeding efforts have yielded
few new cultivars. Furthermore, despite many decades of breeding, specific traits, such
155
A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, 155-196.
© 2002 Kluwer Academic Publishers.
156

as blue flowers in rose, have proven elusive. The key reason for these problems remains
the limitation in the available gene pool and hence the variability available to breeders.
To continue producing new crops and cultivars, breeders and researchers are now
turning to the new techniques of direct genetic modification to introduce new
characteristics such as flower color, and disease and pest resistance. Genetic
transformation overcomes both the limited gene pool and the necessity for successive
backcrossing. Since the DNA used in the transformation event can be isolated from any
living organism, gene pool barriers are eliminated. Genetic transformation involves the
transfer into the host plant of, at most, a few genes. The result is a progeny that is
genetically identical to its parent with the addition of one or two additional characters.
As a result, the desirable horticultural properties of the host plant are preserved and no
backcrossing is necessary to purify the desired phenotype.
New genetically modified (GM) commercial cultivars have been generated in
established crops such as carnation and chrysanthemum. As well as generating new
cultivars, the new GM varieties provide additional material for use in more traditional
breeding techniques.
Ornamental crops are ideal candidates for genetic engineering. Purely ornamental
GM crops may be more acceptable to the public than GM food. In addition, many of the
characteristics that would be targeted by genetic modification techniques (e.g. flower
color) are the product of well-characterized, biochemical pathways whose biosynthetic
genes have already been isolated (e.g. anthocyanin biosynthesis). The result is a
worldwide effort to genetically modify ornamental crops.
The genetic transformation of a plant relies on the ability to transfer genetic material
to a single plant cell followed by stable incorporation of the gene(s) into the plant
genome and finally regeneration of an intact plant from that cell. The critical points in
this process are the ability to deliver the DNA to a single cell and regeneration of a plant
from that cell. Currently available transformation techniques fall into two classes:
controlled DNA transfer using Agrobacterium and direct DNA transfer using a variety
of techniques, such as microprojectile bombardment, electrophoresis, electroporation
and microinjection. Agrobacterium-mediated gene transfer remains the most popular
technique for the generation of new ornamentals. Its major drawback, however, is its
inability to deliver DNA to most monocotyledonous plants. Most of the other techniques
described in this review aim to provide a gene transfer system as simple and reliable as
Agrobacterium but without the host -range limitation.
There have been several recent reviews of plant transformation methods and the
transformation of ornamental crops (Burchi eta/., 1996; Birch, 1997; Deroles eta/.,
1997; Tanaka eta/., 1998; Zuker eta/., 1998; Hansen and Wright, 1999; Mol eta/.,
1999; Zupan et a/., 2000). Deroles et a/. (1997) describe a range of different
transformation techniques used to genetically modify ornamental crops until 1995.
More recent techniques have been developed in an attempt to combine the DNA
transfer/integration characteristics of Agrobacterium with the host-range independence
of direct DNA transfer (Hansen eta/., 1997). In addition, microrganisms have been used
as the projectile (Kikkert et a/., 1999) to avoid having to isolate and purify large DNA
constructs. In this review, we concentrate on publications from 1995 to the present on
ornamental crop transformation. Table 1 lists all the ornamental species from which
transgenic plants have been generated, along with the transformation methods used.
Most of the ornamental crops that have been transformed are cut-flower crops, which
 157

reflects the dominance of cut flowers in the ornamentals industry. Agrobacterium-

mediated transfonnation remains the most popular technique.

2. Ornamental Crops and Transformation Methods

2.1. ALSTROEMERIA (Aistroemeria sp.)

Alstroemeria is an important monocotyledonous ornamental. Many different varieties

have been regenerated through traditional and interspecific crosses. However, delayed
senescence and virus resistance are two desirable characteristics that are not available in
the alstroemeria gene pool. Genetic transformation offers an alternative method for the
development of these characteristics.
To our knowledge, there is only one report on the successful transformation of
alstroemeria (Lin eta/., 2000). Embryogenic callus from the cultivars 'W024C' and
'BT207C' were bombarded with plasmids containing the luciferase reporter gene (/uc)
or GUS and the bar gene. Intact transgenic plants were obtained using both plasmids;
however the process, requiring the isolation of stable, unifonn transgenic plants via use
of the /uc gene on its own, was lengthy and labor-intensive. The bar gene proved to be a
very effective selection agent. The authors also report that the most effective promoter
for expression in alstroemeria is the ubil promoter from maize. Several transgenic lines
were recovered from both cultivars.

2.2. ANTHURIUM (Anthurium andraeanum)

Anthurium hybrids are popular worldwide as cut flowers and potted plants. One of the
principal difficulties in the cultivation of this crop is its high susceptibility to bacterial
blight caused by Xanthamonas campestris. Transformation protocols are being
developed for anthurium with the aim of inserting antibacterial genes.
Chen and Kuehnle report the production of transgenic anthurium through A.
tumefaciens-mediated transformation (Chen and Kuehnle, 1996). Etiolated internode
explants were co-cultivated with A. tumefaciens strain LBA4404 carrying the binary
vector pCa2Att. This vector contains the antibacterial gene attacin, the kanamycin
resistance gene (nptll) for selection of transgenic events and the GUS reporter gene.
Two anthurium cultivars, 'Rudolph' and 'UH1060', were used and both produced
transgenic plants. Chen et a/. (1997) also generated transgenic plants via the
transformation of root cuttings using the strain/vector LBA4404/pCa2Att. Five cultivars
were tested ('UH1003', 'UH1060', 'Anuenue', 'Rudolph' and 'Mauna Kea') with only
the cultivar 'Anuenue' producing transgenic shoots at an efficiency of 1.3%. These are
the first reports of the production of transgenic anthurium plants.
A second transformation system using A. tumefaciens has also been developed and
is being used to generate plants with disease resistance and altered flower color (Dr P
Umaharan, The University of the West Indies, St. Augustine, Trinidad & Tobago, pers.
comm.).
158

TABLE 1. Ornamental species from which transgenic plants have been generated.
Common name Plant Species Transformation Reference
Method

Alstroemeria Alstroemeria sp. Microprojectile (Lin et al., 2000)

Anthurium Anthurium andraeanum A. tumefaciens (Chen and Kuehnle, 1996)
(Chen et al., 1997)
Antirrhinum Antirrhinum majus A. rhizogenes (Senior et al., 1995)
(Hoshino and Mii, 1998)
(Hoshino et al., 1998)
A. tumefaciens (Heidmann et al., 1998)
(Cui et al., 2000)
(Cui et al., 2001)
Begonia Begoniasp. A. tumefaciens (Einset et al., 1995)
(Kiyokawa et al., 1996)
(Kishimoto et al., 2000)
Carnation Dianthus caryophyllus L. A. tumefaciens (Firoozabady et al., 1995)
(van Altvorst et al., 1995)
(van Altvorst et al., 1996)
(Zuker et al., 1999)
(Miroshnichenko and Dolgov,
2000)
Microprojectile (Zuker et al., 1995)
Chrysanthemum Dendranthema sp. A. tumefaciens (Ledgeretal., 1991)
(Boase et al., 1998a)
(Robinson and Firoozabady, 1993)
(Dolgov eta/., 1997)
(Yepes et al., 1999)
(Sherman et al., 1998a)
(Takatsu et al., 1999)
(Seiichi et al., 1995)
Microprojectile (Yepes et al., 1995)
Cyclamen Cyclamen persicum Mill. A. tumefaciens (Boase et al., 2000)
(Aida et al., 1999)
(Sironi et al., 2000)
Datura Daturasp. A. tumefaciens (Ducrocq et al., 1994)
(Curtis et al., 1999)
A. rhizogenes (Giovannini et al., 1997)
Daylily Hemerocallis sp. Microprojectile (Ling et al., 1998)
Delphinium Delphinium sp. A. tumefaciens (Hirose et al., 1998)
Forsythia Forsythia x intermedia A. tumefaciens (Rosati et al., 1996)
Gentian Gentianasp. A. rhizogenes (Hosokawa et al., 1997)
(Momcilovic et al., 1997)
(Vinterhalter et al., 1999)
Microprojectile (Hosokawa et al., 2000)
 159

TABLE 1. Continued
Common name Plant Species Transfonnation Reference
Method

Gerbera Gerbera hybrida A. tumefaciens (Elomaa et al., 1996)

(Robinson and Firoozabady,
1993)
(Orlikowska et al., 1997)
(Nagaraju et al., 1998)
(Reynoird et al., 2000)
Gladiolus Gladiolus grandiflorus A. tumefaciens (Babu and Chawla, 2000)
Microprojectile (Karno et al., l995a)
(Karno et al., l995b)
Hyacinth Microprojectile (Langeveld et al., 2000)
Iris Iris germanica A. tumefaciens (Jeknic et al., 1999)
Kalanchoe Kalanchoe sp. A. tumefaciens (Aida and Shibata, 1996)
Lavatera Lavatera sp. A. tumefaciens (Vazquez-Thello et aL, 1996)
Linum Linumsp. A. tumefaciens (Ling and Binding, 1997)
Lily Lilium longiflorum Microprojectile (van der Leede-Plegt et al., 1997)
(Langeveld et al., 1997)
(Watad et al., 1998)
Lisianthus Eustoma grandiflorum A. tumefaciens (Ledger et aL, 1997)
(Hecht et al., 1997)
(Semeria et al., 1996)
A. rhizogenes (Handa, 1996)
(Giovannini et al., 1996)
Microprojectile (Takahashi et al., 1998)
(Semeria et al., 1995)
Orchid Orchidsp. A. tumefaciens (Belarmino and Mii, 2000)
Microprojectile (Kuehnle and Sugi, 1992)
(Nan and Kuehnle, 1995)
(Yu et al., 1999)
(Yang et al., 1999)
(Knapp et aL, 2000)
Ornithogalum Ornithogalum sp. Microprojectile (De Villiers et al., 2000)
Osteospermum Osteospermum sp. A. tumefaciens (Allavena et al., 2000)
Pelargonium/ Pelargonium sp. A. tumefaciens (Robichon et aL, 1995)
Geranium (Krishnaraj et al., 1997)
(Boase et al., 1996)
(Boase et al., 1998b)
A. rhizogenes (Pellegrineschi et al., 1994)
(Pellegrineschi and
Davoliomariani, 1996)
Petunia Petunia hybrida A. tumefaciens (van der Meer, 1999)
Vacuum Infltn (Tjokrokusumo et al., 2000)
160

TABLE 1. Continued
Common name Plant Species Transfonnation Reference
Method

Poinsettia Euphorbia pulcherrima Electrophoresis (Vik et al., 2000)

Rhododendron Rhododendron sp. A. tumefaciens (Pavingerova eta/., 1997)
(Ueno et al., 1996)
Microprojectile (Hsia and Korban, 1998)
Rose Rosasp. A. tumefaciens (Firoozabady et al., 1994)
(van der Salm et al., 1997)
(Souq et al., 1996)
Microprojectile (Marchant et al., 1998b)
Torenia Torenia fournieri A. tumefaciens (Aida and Shibata, 1995)
Verticordia Verticordia grandis A. tumefaciens (Stununer et aL, 1995)
A. rhizogenes (Stununer et al., 1995)

2.3. ANTIRRlllNUM (Antirrhinum majus)

Antirrhinum is both an important ornamental crop and frequently used as a model crop
for the study of flower development, color and form (Jordan and Anthony, 1993; Martin
and Gerats, 1993). One impediment to its use as a model system has been the absence of
an efficient transformation system, which would enable scientists to confirm gene
identity and function through complementation and antisense studies. Early reports of
transformation of antirrhinum describe the production of transgenic tissue and plants
using A. tumefaciens and A. rhizogenes (Reviewed in Dero1es eta/., 1997; Tanaka eta/.,
1998). However, none of these techniques were efficient enough to be used for the
routine generation of transgenic plants. More recent reports have described
improvements on these techniques. Two groups have recent publications on the
generation of transgenic antirrhinum using A. rhizogenes. GUS-positive transgenic
plants were produced from the variety 'Golden Monarch' after transformation of
hypocotyls with the A. rhizogenes strain LBA9402 canying the binary vector pBI121
(Senior et a/., 1995). Hairy root clones were obtained from four other varieties, but no
transgenic plants were recovered. The transgenic plants from 'Golden Monarch' all
showed dwarf phenotypes typical of hairy root-derived transgenics. Attempts to separate
the GUS and rol phenotypes through outcrossing failed. However progeny produced by
propagating side shoots showed reduced dwarfing. The genetic basis of the reduced
abnormalities in side shoot-derived progeny remains unknown. Hoshino eta/. generated
transgenic plants from hairy roots (Hoshino and Mii, 1998). Young leaves were
inoculated using the wild-type A. rhizogenes strain Al3 to produce transgenic hairy
roots from which shoots spontaneously regenerated. They found that the frequency of
shoot regeneration from transgenic root tissue increased fivefold when the
phosphinothricin-containing herbicide Bialaphos (0.5 mgll) was added to the growth
medium. The effect was specific to transgenic tissue with no improvement in shoot
production observed from non-transgenic roots. Hoshino and co-workers suggested that
the stress induced by the presence of the herbicide somehow interacts with the rol
 161

transgenes to promote shoot production. The resultant transgenic plants exhibited

altered phenotypes consistent with the presence of rol genes (e.g. reduced apical
dominance, highly branched stems, and short internodes). In a later publication, Hoshino
and co-workers used the same technique to produce transgenic plants resistant to the
herbicide phosphinothricin through insertion of the bar gene (Hoshino eta/., 1998). As
in their previous experiments, Hoshino et al. used the wild-type strain Al3. Just over
half (54.5%) of the hairy root-transgenic clones isolated also carried the bar gene from
the binary vector pARKS. The transgenic plants exhibited dwarfing phenotypes
consistent with the presence of rol genes and were also resistant to phosphinothricin at
typical field concentrations. Herbicide resistance may prove beneficial to growers for
weed control, and altered morphologies such as dwarfing may lead to new antirrhinum
cultivars.
More recently, techniques have been published on the use of A. tumefaciens for the
generation of transgenic antirrhinum. These techniques allow the production of
morphologically normal plants without interference from rol genes. Heidmann et al.
transformed highly regenerative hypocotyls withA. tumefaciens strain GV3101 carrying
the pMP90RK helper plasmid (Heidmann eta/., 1998). This is the first report of a
system that is able to transform several (four) different varieties. The efficiency of
transformation is low and varies between varieties but may be improved with individual
culture conditions for each line. All plants produced were phenotypically normal and
passed on the GUS gene through both male and female gametes.
A recent development in vector technology called the multi-auto-transformation
(MAT) vector system has also been used to transform antirrhinum (Cui eta/., 2000,
2001 ). This system uses the morphological changes caused by the oncogenes of
Agrobacterium (ipt or rol genes) as the selectable marker. The vector is designed to
excise the oncogenes through the use of removal elements after the transformation
event, thus producing a morphologically normal transgenic plant carrying only the gene
of interest. This system may also do away with the need for additional selective
markers, such as antibiotic or herbicide resistance (Sugita eta/., 1999, 2000a,b). Leaf
pieces from young plants of two antirrhinum varieties were inoculated with the A.
tumefaciens strain GV2260 carrying the MAT vector pNP1702. This vector contains the
GUS gene and a removal element containing the rol genes A, B, C, and D. Hairy root
cultures were isolated from leaf pieces of both varieties and 11 independent lines gave
rise to shoots, resulting in the production of morphologically normal GUS-positive
transgenics. The MAT vector system promises to be a valuable tool in the
transformation of recalcitrant species.

2.4. BEGONIA (Begonia sp.)

Begonia species are popular as garden and potted plants. There are three recent reports
on the transformation of Begonia. Einset eta/. (1995) produced transgenic plants from
the cultivar Begonia x hiemalis cv. 'Hanne' (Christmas Begonia). This cultivar is
popular in Scandinavian countries but suffers from short-lived flowers and excessive
elongation in the spring. Einset and co-workers inoculated young leaf pieces with A.
tumefaciens LBA4404 carrying the binary vector pBASC (nptll, antisense tomato ACC
oxidase) to reduce ethylene-mediated flower senescence. Their transformation method is
based on that of Horsch eta/. (1985). In the transgenics tested, antisense ACC oxidase
162

gene reduced the activity of the endogenous enzyme by 20 to 35%. However, this was
not enough to significantly inhibit ethylene production, and hence no change in flower
longevity was observed. Critical steps in this transfonnation protocol are the selection of
succulent healthy leaf explants and the necessity for continuous antibiotic selection over
a period of 6 months.
Kiyokawa eta/. (1996) also used A. tumefaciens LBA4404 to transform leaf and
petiole explants from three Begonia species: Begonia semperjlorens 'Link et Otto',
Begonia x hiemalis (Fotsh.) cv. 'Schwabenland Red' and Begonia tuberhybrida cv.
'Perfecta'. They used the binary vector pBI121E15 containing the nptll resistance gene,
GUS reporter gene and the rol A, B, and C genes from the Ri plasmid pRiA4.
Transgenic, kanamycin-resistant calli were regenerated from all three Begonia species.
However, only one species, Begonia tuberhybrida, regenerated shoots under selection. It
is possible that the level of selection prevented shoot formation in the other Begonia
species. Nineteen transgenic plants were regenerated from a total of 80 explants, giving
a transfonnation efficiency of24%. Both leaf and petiole cuttings gave rise to transgenic
plants. All of the transgenics were GUS-positive and had phenotypes associated with the
rol genes, namely dwarfing, delayed or inhibited flowering, and wrinkled leaves. The
variability in the level of rol-type phenotypes was not associated with transgene copy
number and may have been caused by positional effects or transgene inactivation.
The most recent report (Kishimoto et a/., 2000) describes the transfonnation of
Begonia x hiemalis via Agrobacterium carrying the binary vector piG121Hm
(nptll/intron GUS/hpt). No further details of this work are available.

2.5. CARNATION (Dianthus caryophyllus L.)

Carnation is one of the world's top four cut-flower crops. As a result, much effort has
been put into developing a transfonnation system for this crop (reviewed in Deroles et
a/., 1997). Two primary targets of this work are the extension of vase life through the
modification of ethylene biosynthesis and perception, and the generation of new colors.
Carnation is one of three ornamental crops (carnation, chrysanthemum and petunia) in
which GM cultivars with commercial potential have been produced. In chrysanthemum,
a white form of the cultivar 'Moneymaker' was produced (Courtney-Gutterson eta/.,
1994), and in petunia new varieties with orange flowers were generated (Meyer eta/.,
1987). Neither of these cultivars are currently in commercial production. In carnation,
new mauve colors (cv. Moondust and cv. Moonshadow, see www.florigene.com.au)
have been produced. Both are now in commercial production and being sold in
Australia, Japan and the USA. Carnation is the first ornamental crop to have GM
cultivars in commercial production.
Five research groups have developed transfonnation systems for carnation, using A.
tumefaciens and microprojectile bombardment. Van Altvorst and co-workers generated
transgenic carnations via A. tumefaciens-mediated transfonnation of leaf (van Altvorst
et a/., 1995) and petal (van Altvorst et a/., 1996) explants using the A. tumefaciens
strain AGLO carrying one of two binary vectors, p35SGUSint or pCGN7001. Both
vectors contain the GUS reporter gene and nptll selective marker gene. Using leaf
explants, van Altvorst et a/. (1995) generated transgenic plants from five cultivars
(CPRO clone numbers 89073, 89074, 89077, 89086, and 89172). Transformation
efficiencies ranged from 0.1 to 1.5%. The binary vector pCGN7001 produced higher
 163

transformation efficiencies with all cultivars tested, with petal explants producing
transformation efficiencies of 1.3 to 2.5%. However, most of the transgenics derived
from petal explants were hyperhydric, grew slowly and did not survive transfer to the
glasshouse. The authors concluded that leaf explants, whilst giving a reduced
transformation efficiency, were more suitable for the generation of transgenic plants.
These authors used the leaf-based transformation system to generate transgenic
carnations carrying the Arabidopsis thaliana etrl-1 gene to inhibit the ethylene
response. Several transgenics were produced in which flower senescence was
significantly delayed (Bovy eta/., 1999).
Zuker et al. (1995, 1999, 2000) have used both microprojectile bombardment and A.
tumefaciens-mediated transformation to produce transgenic carnation plants. For
microprojectile bombardment-mediated transformation, stem segments of the cultivar
'White Sim' were bombarded with tungsten M25 (1.7 urn) at 1500 psi shot pressure
from a distance of 6 em using the PDS 1000He system (Zuker et al., 1995). The
particles were coated with 5 Jlg of DNA/0. 75 mg particles with 1 Jlg DNA being used
per shot. The plasmid (pDP532) carried both the GUS reporter gene and the selectable
marker gene bar: 70% of the explants produced plantlets under Bialaphos selection. Of
these, 3% expressed GUS in all tissues and a further 6% expressed GUS in localized
areas, indicating that they may be chimeric in nature.
For A. tumefaciens-mediated transformation, Zuker et al. (1999) describe a system
using the strain AGLO containing the binary vector pCGN7001, which is the same
strain/vector combination used by van Altvorst et a!. (1995, 1996). Zuker and co-
workers inoculated stem explants that had been pre-wounded using the particle gun
(PDS1000). Explants were bombarded twice with 100 Jll of tungsten particles with a
pressure of 10 Mpa at a distance of 9 em. They determined that the combination of pre-
wounding followed by co-cultivation in the dark produces a 10-fold increase in transient
GUS expression. Stably transformed plants with no chimerism were generated by two
rounds of shoot regeneration in which shoots were regenerated under selection from
leaves of the primary transgenic lines. An overall transformation frequency of 10 to
20% was achieved using three cultivars, 'White Sim', 'Desio' and 'Eilat'. The
procedure is consistently efficient and several hundred independent transgenic lines
have been produced, including transgenic plants with altered flower color and plant
form (Ovadis eta/., 2000; Zuker eta/., 2000, 2001 ).
Miroshnichenko and Dolgov (1999, 2000) developed a transformation system for
the cultivar 'Dial2' using A. tumefaciens strain EHA105 and the binary vector
p35SGUSint (hpt/intron-GUS). The transformation efficiency was most affected by
explant source with leaves from in vitro-grown plants giving a transformation efficiency
of 11.5% compared to 0.5% for greenhouse-grown plants.
Firoozabady et al. (1995) developed an efficient transformation method using leaves
from in vitro shoot cultures. The explants were co-cultivated for 5 days with A.
tumefaciens then transferred to shoot regeneration medium. The selective agent used in
these experiments was chlorsulfuron. Resistant shoots were subjected to two rounds of
selection, after which all the resistant shoots were vitrified. However, all of these shoots
were normalized, rooted and successfully transferred to the glasshouse. Kanamycin and
geneticin were also tested as selective agents but were not found as effective as
chlorsulfuron. All of the plants transferred to the glasshouse were transgenic.
The biotechnology company, Florigene, which uses an Agrobacterium-mediated
164

transformation method with stem explants (C. Lu eta/., Florigene, Australia, pers.
comm.), has also produced transgenic carnations. Their system has been modified and
extended to over 16 cultivars with over 2000 transgenic plants being produced
(reviewed in Deroles et a/., 1997). However, details of the system have not been
published. Florigene is the first organization to commercially produce a GM ornamental
(http://www.florigene.com.au). They now have two GM carnation cultivars on sale,
Moondust and Moonshadow. These cultivars have been modified through the insertion
of the 3'5'-hydroxylase gene to generate novel mauve and purple flower colors.

2.6. CHRYSANTHEMUM (Dendranthema sp.)

Chrysanthemum is one of the most popular cut flowers worldwide and is available in a
wide variety of flower forms and colors. However, several desirable characteristics,
such as blue colors and disease resistance, are not available to chrysanthemum breeders.
Because of the importance of this crop, considerable effort has been invested in
developing transformation systems. Results to date have shown that Agrobacterium-
mediated transformation of chrysanthemum is very cultivar-specific, both in
susceptibility to Agrobacterium infection and in in vitro plant regeneration methods
(reviewed in Deroles eta/., 1997). Our laboratory has produced transgenic plants from
four cultivars (D. indicum cv. 'Korean K4', D. morifolium cvs. 'Yellow Lucondra',
'Peach Margaret' and 'Horizon'), using A. tumefaciens-mediated transformation ofleaf
pieces (Ledger et a/., 1991; Deroles eta/., 1997; Boase eta/., 1998a,c). Many other
cultivars have also been transformed using A. tumefaciens (reviewed in Deroles eta/.,
1997).
Perhaps the most cultivar-independent system is the one used by Florigene
(www.florigene.com.au) which has been successful with 15 cultivars (Robinson and
Firoozabady, 1993, C. Lu et al., Florigene, Australia, pers. comm.). Recent references
on transformation systems for chrysanthemum include Dolgov et al. (1997) who tested
five A. tumefaciens strains (A281 wild type, A281 disarmed, GV3101, C58, and
CBE21). Small numbers of transgenic plants were produced, with the most efficient A.
tumefaciens strain being the wild type A281 (4%). Using this system, plants were
produced carrying the ro/C gene and showed dwarfing characteristics (Mitiouchkina
and Dolgov, 2000). In addition, plants transformed with the antisense CHS from
Antirrhinum majus showed loss of color in petals which increased with flower age
(Mitiouchkina et a/., 2000).
Microprojectile bombardment has also been used to transform chrysanthemum in an
effort to overcome the limitations in Agrobacterium susceptibility. Yepes eta/. (1995)
developed a shoot regeneration system from leaf and stem explants harvested from in
vitro shoot tip cultures for four cultivars, 'Iridon', 'Tara', 'Blush' and 'Dark Bronze
Charm'. To transform these cultivars, the explants were bombarded with the plasmid
pBIN19fTSWV-BL (nptii, nucleocapsid gene from tomato spotted wilt virus) carried on
1 J.Ufi tungsten particles. The highest regeneration efficiency was obtained using stem
segments and occurred from the basal end. Kanamycin selection was delayed for 10
days then gradually increased over several subcultures. This procedure dramatically
increased the number of stable transgenics and lowered the number of escapes. A total
of 82 transgenic plants were produced from all four cultivars. This is the first report of
transgenic chrysanthemum generated using the particle gun.
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In a more recent paper, Yepes eta/. (1999) went on to compare the efficiency of
Agrobacterium- and biolistic-mediated transformation methods. Three commercially
important cultivars were used in this study, the cultivars 'Polaris' and 'Golden Polaris',
and the highly regenerable cultivar 'Iridon' used in their previous study (Yepes eta/.,
1995). The explants and biolistic transformation system used were the same as in the
previous work (Yepes eta/., 1995). For Agrobacterium-mediated transformation they
used the bacterial strains LBA4404, C58sZ707 and EHA105 carrying either the binary
vector pBIN19 (nptii) or pGA482GG (nptii, GUS). In addition, each binary vector
contained the nucleocapsid protein genes from one of three tospoviruses, a significant
disease in the cultivars 'Polaris' and 'Golden Polaris'. Explants (stem and leaf) were co-
cultivated overnight. Selection for transgenes was performed using a stepwise increase
in kanamycin concentration. A total of 497 transgenic plants were recovered from the
three cultivars. The efficiency of transformation was influenced most significantly by
the viral gene and cultivar identity. Leaf explants were more readily transformed using
Agrobacterium while stem explants gave similar efficiencies from both techniques. The
type of antibiotic used to stop Agrobacterium overgrowth dramatically affected
regeneration efficiencies. Cefotaxime and the tablet form of carbenicillin both produced
significant reductions in regeneration efficiencies when compared to the liquid form of
carbenicillin. Overall transformation efficiencies from these experiments were 3 to 6%.
From the number of separate experiments and the number of transgenic plants
produced, it is clear that these techniques are a reliable way of producing transformed
plants from the three cultivars tested.
The cultivars 'Polaris' and 'Iridon', along with 'Hekla', were also transformed by
Sherman eta/. (1998a). Unlike the method of Yepes eta/. (1995), using direct shoot
formation from leaf and stem explants, Sherman and co-workers developed a callus-
based regeneration system from leaf explants. They produced transgenic plants from all
three cultivars using the Agrobacterium strain EHA105 carrying pBI121 (nptii, GUS).
Using this system, Sherman eta/. (1998b) produced transgenic plants from the cultivar
'Polaris' carrying sense and antisense genes of the nucleocapsid protein from tomato
spotted wilt virus. Over 20 transgenic plants were produced and screened for virus
resistance, resulting in the selection of three lines that appeared totally virus resistant.
Both sense and antisense strategies produced virus resistance. This is the first example
of the generation of disease resistance in a major ornamental crop.
Chrysanthemum transgenics have also been generated carrying genes for Botrytis
resistance (Takatsu et a/., 1999). Two Agrobacterium strains, C58 and MP90 carrying
pBI121 (containing the rice chitinase gene), were used to transform in vitro stem
segments of the spray cultivar 'Yamabiko' using the method ofTakatsu eta/. (Takatsu
eta/., 1998). Sixteen transgenic lines were recovered with a transformation efficiency of
0.8 to 0.9%. Three lines showed significant resistance to Botrytis in the lab and are now
being evaluated in the field.
Seiichi eta/. (1995) developed a regeneration system for the cultivar '1581' using
stem explants. Using the Agrobacterium strain AGLO carrying the vector pMOG410
(nptii, GUS) they produced stable transgenic plants. Transgenic plants carrying the
phyA and phyB genes from rice andArabidopsis, respectively, were produced using this
transformation system. However, expression levels in the transgenics were not high
enough to produce dwarf phenotypes (Petty eta/., 2000).
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The number of groups working on the transformation of chrysanthemum has

increased markedly in the last 5 to 6 years and reflects the importance of this
ornamental crop. Most of the successful transformation systems use Agrobacterium-
mediated transformation of stem and leaf explants. It appears that the transformation of
new chrysanthemum cultivars is only limited by the development of new and efficient
regeneration systems. For cultivars not susceptible to Agrobacterium infection, particle
bombardment remains a possibility, as demonstrated by Yepes et al. (1995, 1999). The
main focus to date is the generation of viral and fungal resistance, with some effort also
going into the modification of flower color (Courtney-Gutterson et al., 1994) and plant
form (Petty et al., 2000). Judging by the results obtained to date, it will not be long
before the first transgenic chrysanthemum cultivars are released onto the market.

2.7. CYCLAMEN (Cyclamen persicum Mill.)

Cyclamen is one of the most popular pot plants in northern Europe. Both diploids and
polyploids have been developed as commercial cultivars as well as F1 hybrids. The first
report on the generation of transgenic cyclamen tissue described the A. tumefaciens-
mediated transformation of in vitro seedlings of the Fl hybrid of Cyclamen persicum
cv. 'Sierra Rose' (Boase and Borst, 1995). Stable transgenic loci were recovered after
infection by theA. tumefaciens strains A281 and LBA4404, both containing the binary
vector pKIWillO (nptll, GUS). Boase and Borst (1997) reported transient GUS
expression results from experiments in which four strains of A. tumefaciens (A281,
A722, EHA105 and LBA4404) containing either pMOG410 or pTOK233 were used to
infect etiolated hypocotyls of cv. 'Sierra Rose'. The disarmed strain LBA4404 gave the
highest gene transfer frequencies. Using the Agrobacterium strain/vector combination
LBA4404/pMOG410, Boase et a/. (2000) generated stable transgenic shoots from
etiolated hypocotyls of cv 'Sierra Rose' with a transformation efficiency of 9 to 14%.
Aida et al. (1999) reported the production of transgenic cyclamen from etiolated
petiole segments of cv. 'Anneke' by using A. tumefaciens strain AGLO with the binary
vector piG 121Hm. Transgenics were generated under selection with 5 mg/1 hygromycin
in adventitious shoot regeneration medium containing 1 mg/1 TDZ and 1 mg/12,4-D.
Hvoslef-Eide eta/. (2000b) report the transient expression of the GUS reporter gene
in suspension cells of the cv. 'Purple Flame' 2 days after bombardment with plasmid
p1515 coated on gold particles. They are seeking shoot regeneration through somatic
embryogenesis.
Sironi et a/. (2000) regenerated kanamycin-resistant plantlets that expressed the
GUS gene via transformation with A. tumefaciens strain EHA105 carrying pKIWI105
or strain GV2260 carrying pGUSint. They co-cultivated etiolated petiole explants for 2
days with A. tumefaciens and then selected in the dark for 4 months on media
containing 100 mg/1 kanamycin. Transgenic plantlets were regenerated from calli via
somatic embryogenesis, 2 months after transfer to hormone-free selective media.
Successful transformation of cyclamen has been achieved but only on a limited
number of cultivars. The primary targets in the generation of new GM cultivars are
disease resistance, particularly against Fusarium, and new flower colors. It remains to
be seen whether existing systems are able to transform a wider range of cultivars.
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2.8. DATURA (Datura sp.)

The genus Datura includes several species of ornamental shrubs and small trees. Like
other members of the Solanaceae, Datura is amenable to genetic transformation. The
first report of the transformation of Datura was in the medicinal species Datura innoxia
(Sangwan et al., 1991). Improvements on this technique were reported in Ducrocq et al.
(1994). Zygotic embryos were co-cultivated with A. tumefaciens and placed on media to
induce somatic embryogenesis with kanamycin as the selective marker. A
transformation rate of 76% was achieved. All the transgenics were morphologically
normal and passed on the transgenes to progeny in a Mendelian fashion. Recently, two
groups have reported the transformation of three ornamental Datura species
(Giovannini et al., 1997; Curtis et al., 1999).
Giovannini et a/. (1997) transformed the ornamental species D. arborea and D.
sanguinea using A. rhizogenes strain NCPPB1855 on young leaf explants. D. arborea
produced a higher number of hairy root clones and a greater proportion of them
spontaneously regenerated shoots. Five independent transgenic lines of D. arborea and
one line of D. sanguinea were produced. All the transgenic plants showed increased
rooting potential and a range of altered phenotypes typical of plants transformed with A.
rhizogenes, such as reduced height, shorter and more numerous internodes, increased
branching and altered leaf shape. Some of these plants show potential as new
ornamental cultivars.
Curtis et a/. (1999) transformed D. meteloides cv. 'Evening Fragrance', one of the
most attractive ornamental Datura species. They transformed leaf pieces with A.
tumefaciens strain 1065, a derivative ofLBA4404 carrying the binary vector pVDH65
and the super virulence vector pTOK47. Shoots were selected using kanamycin
resistance and tested for GUS expression. They found enhanced transformation
frequencies with a 2- to 3-day pre-culture ofthe leaf pieces and a 1:10 to 1:20 dilution
of the bacterial inoculum. Different inoculation periods had no significant effect. Pre-
wounding the leaf pieces caused the production of excess phenolics, resulting in a loss
of transformation efficiency through tissue necrosis. All transgenic plants produced,
over 40 independent transgenic lines, were phenotypically identical to the non-
transgenic controls.
For the purposes of this review the species names for Datura are used as described
in the individual references. However, several species in the tribe Datureae have
recently been reclassified. For example, the species Datura arborea and D. sanguinea
have been renamed Brugmansia arborea and B. sanguine a. A recent description of the
tribe Datureae can be found in Mace et al. (1999). In the medicinal Datura species (eg
D. innoxia), transformation studies have focused on the in vitro production of medicinal
compounds through hairy root cultures. Currently, the primary aim of the
transformation work on ornamental species is the alteration of plant form through
dwarfing, via use of the rot genes from A. rhizogenes.

2.9. DAYLILY (Hemerocallis sp.)

The daylily is a popular ornamental monocot. However, its principal drawback for cut-
flower use is its very short vase life. Ling et a/. (1998) developed a transformation
system for this plant with a view to extending vase life. They bombarded cell
168

suspensions from the cultivar Hemerocallis x 'Stella de Oro' with a vector carrying the
phosphinothricin acetlytransferase gene. They began selection using phosphinothricin 2
to 3 weeks after bombardment and successfully isolated transgenic plants.

2.10. DELPlllNIUM (Delphinium sp.)

Delphinium has been successfully transformed by Hirose et a/. (1998) using A.

tumefaciens LBA4404 carrying the binary vector piG 121 (nptiiJ GUSint/ hpt). Several
transgenic plants were recovered, all showing the presence of the GUS gene based on an
activity assay and PCR. No further details are available on this work and to our
knowledge, this is the only report of the successful transformation of this species.

2.11. FORSYTHIA (Forsythia x intermedia)

Forsythia is a widely cultivated ornamental shrub noted for its abundant yellow flowers.
Rosati eta/. (1996, 2000) have developed a transformation system for this shrub with a
view to producing new flower colors through alteration of the flavonoid and carotenoid
biosynthetic pathways (Rosati eta/., 1997, 2000). Stem internodes from in vitro-grown
plantlets were inoculated with the A. tumefaciens strain EHA101 (GUS, nptll).
Transgenic shoots were selected via kanamycin resistance. Although the transformation
frequency was only 1%, a consistent number of plants were obtained from each
experiment. The authors suggest that the low transformation frequency may have been
due to disparity between the preferred sites for shoot organogenesis and for
Agrobacterium infection.

2.12. GENTIAN (Gentiana sp.)

Hosokawa eta/. (1997, 2000) have transformed gentian using both A. rhizogenes and
particle bombardment. Both techniques used stem segments from in vitro-grown
plantlets of the gentian cv. 'Polarno White' (G. triflore x G. scabra). In the A.
rhizogenes system (Hosokawa et at., 1997), the stem segments were infected with the
wild-type strain A4 and hairy root cultures produced. Shoots were recovered from 17%
of the hairy root cultures and plants transferred to the glasshouse. All of the transgenic
plants showed some degree of altered morphology typical of changes induced by the
presence of rot genes, such as dwarfing. To overcome this problem, the authors
developed a second transformation system, based on the bombardment of cell
suspension cultures (Hosokawa eta/., 2000). Cell suspension cultures were established
from leaf pieces, spread onto a filter disk and bombarded using an automatic pneumatic
particle gun (Morikawa et at., 1994). The cells were transformed with the plasmids
pBI221 (GUS) or pCH (hpt). The highest rates of GUS expression were achieved after
the cells had been pre-cultured for 5 days and shot three times with gold particles, with
approximately 1300 GUS-expressing loci being observed per plate. Stable hygromycin-
resistant transformants were recovered after bombardment with the plasmid pCH.
However, only two independent clones were produced from 12 plates of bombarded
tissue. The authors also report that attempts to transform the same cultivar with a
disarmed strain of A. tumefaciens were unsuccessful. We also found that disarmed A.
tumefaciens strains were unable to transform another Gentianaceae, lisianthus (Eustoma
 169

grandiflorum}, but that successful transformation could be obtained by using the wild-
type strain A722. Such a strategy may also work for other gentians.
A second group (Momcilovic et a/., 1997) transformed four medicinal gentian
species using A. rhizogenes. In vitro shoots were inoculated using a hypodermic needle
and hairy root cultures recovered. G. purpurea and G. lutea were transformed with A.
rhizogenes strain ATCC15834 with 5% and 20% of shoots, respectively, producing
hairy root cultures. G. cruciata and G. acaulis were transformed with A4M70GUS, a
wild-type strain haiboring a co-integrative plasmid carrying the GUS reporter gene. G.
cruciata and G. acaulis, respectively, produced transformed roots in 63% and 44% of
the shoots inoculated. Transgenic plants were recovered from G. purpurea and G.
cruciata and all had phenotypes typical of plants transformed with A. rhizogenes, with
short internodes and rolled leaves.
The medicinal plant G. punctata has also been transformed with A rhizogenes
carrying the plasmid A4M70GUS (Vinterhalter eta/., 1999). Internodes on in vitro
shoots were inoculated, with 17% producing hairy roots. Two hairy root cultures
spontaneously produced shoots and the resultant plants had short internodes, leaf
epinasty, formation of callus on the cut surface and a tendency to become hyperhydric.
These results show that the production of transgenic gentian plants is possible but a
routine system for the generation of morphologically normal transgenic plants remains
to be developed.

2.13. GERBERA (Gerbera hybrida)

Gerbera is one of the top cut-flower crops worldwide and as a result is a prime target for
genetic manipulation to develop new cultivars. Currently, five groups have published
techniques for gerbera transformation. Elomaa eta/. (1993, 1996) produced transgenic
plants from the cultivar 'Terra Regina' carrying the antisense chalcone synthase gene
(gchs) for the alteration of flower color. Pre-cultured petioles were transformed using A.
tumefaciens strain C58 carrying the disarmed Ti plasmid pGV2260. Two binary vectors
were used, pHTT370 and pHTT377, carrying antisense gchs1 and 2, respectively. Four
transgenics were generated in the first transformation experiment and 17 in the second.
The reported efficiency for this technique is low (four plants from 4800 explants) but
they have demonstrated the consistent production of transgenics.
The biotechnology company Florigene is also able to transform gerbera using A.
tumefaciens strain LBA4404 (Robinson and Firoozabady, 1993). They observed that
transformation efficiency is dependent on the choice of cultivar and explant. No details
of this technique are available.
Orlikowska eta/. (1997) have studied a range of A. tumefaciens strains (LBA4404,
EHA101, and EHA105) with binary vectors containing the GUS and either the nptll or
bar selective marker genes for the transformation of the gerbera cultivar 'Mariola'. They
found very young petioles to be the most effective explant. The highest transformation
efficiencies were achieved using EHA101 and the bar gene for selection of
transformants. A total of 10 stable transgenics were regenerated from 200 explants (5%
efficiency).
Nagaraju eta/. (1998) used LBA4404 carrying the binary vector pBI121 (GUS and
nptll) to generate transgenic plants from the cultivar RCGH164. They tested petiole,
leaf and shoot tip explants harvested from in vitro-grown shoots and obtained
170

transformation efficiencies of 3%, 0.5% and 17%, respectively. All the transgenics
produced were morphologically normal.
Reynoird et a/. (2000) studied the effect of endogenous phytohormone levels on the
regeneration ability of gerbera cultivars, and the consequential effect on transformation
efficiency. They transformed mature leaf pieces from two cultivars of gerbera (clones 10
and 11) with the two wild-type A. tumefaciens strains 82139 and Ach5. Clone 10
produced more tumors (from 50% of shoots) than clone 11 (10%) when inoculated with
82139. Spontaneous regeneration occurred in 10% of the tumors arising from clone 10.
Strain Ach5 produced a similar frequency of tumor formation for both clones but no
shoots were regenerated. This group showed that tumors that produced shoots had
enhanced levels of zeatin riboside prior to the formation of shoot buds. Since the tumors
were cultured on hormone-free media, the only source of enhanced cytokinin was from
the inserted oncogenes. The authors suggest that the use of such oncogenes could extend
the range of gerbera cultivars that can be transformed. These findings indicate that the
newly developed MAT vector system (Sugita et a/., 2000a) using the shooty gene from
Agrobacterium for enhanced regeneration may be very useful in gerbera transformation.

2.14. GLADIOLUS (Gladiolus grandijlorus)

Gladiolus is one of the top 10 cut-flower crops worldwide. Considerable effort has been
put into transforming this monocotyledonous crop for the generation of viral and fungal
resistance. Chauvin eta/. (1997) performed some preliminary studies on the suitability
of selective marker genes. They found gladiolus to be relatively insensitive to
kanamycin; hygromycin and phosphinothricin were more effective.
The bulk of the work on the transformation of this crop has been done by Kamo et
a/., who used the gene gun andAgrobacterium to transform several explant types. Kamo
eta/. (1995b) produced transgenic plants after co-bombardment with both p35Sac (bar
(phosphinothricin acetlytransferase)) and the GUS-containing vector pAct1-F4. Callus
and cells from the cultivars 'Jenny Lee' and 'Peter Pears' were bombarded and placed
on selection medium (PPT or Bialaphos) immediately or 1 week after bombardment.
Seventy percent of the PPT-resistant transgenic plant lines that were recovered also
expressed the GUS gene, showing a high frequency of co-transformation. Transgenic
plants were regenerated from transformed callus and cell suspension from both
cultivars. A total of 33 plates of callus and 18 plates of cell suspensions yielded 110
transformed plants. Recovery of more plants was possible so this figure represents a
minimum transformation efficiency.
This technique is very efficient and has the potential to be used on different
cultivars. However, it does depend on the ability to regenerate plants from callus tissue,
which is not possible for all gladiolus cultivars. As a result, Kamo eta/. (l995a) have
also generated transgenic plants from cormels-small corms formed from a mature
corm or at the base of an in vitro plant which has become dormant. Cormels were
harvested from in vitro shoots of three cultivars: 'Jenny Lee', 'Peter Pears' and 'Florida
Flame'. The cormels were sliced transversely, pre-cultured for 10 to 14 days then
bombarded using the protocol and vectors described for callus and cell suspensions
(Kamo eta/., 1995b). Two rounds of selection on PPT produced PPT-resistant, GUS-
positive plantlets from the cultivars 'Jenny Lee' and 'Peter Pears'. The importance of a
second round of selection to remove escaped lines was emphasized. The frequency of
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transformation was lower than when using callus or cell suspensions. Furthermore, GUS
expression was not uniform in all tissues of the transgenics with many showing striped
patterns of gene expression in the leaves. The pattern of GUS expression could be the
result of tissue specific expression driven by the actll promoter (as observed in rice-
Zhang et at., 1991) or it could be due to chimerism. However, the chimeric nature of the
transgenics has not yet been established. It is interesting to note that transgenics derived
from cell cultures or callus did not show a variable pattern of GUS expression in leaf
tissue. This indicates that cellular tissue is a more desirable target than cormels for
stable and uniform gene expression in the resulting transgenics (Kamo, 1997; Kamo et
at., 1997a).
Kamo et at. also studied the efficiency of co-transformation with the gene gun, of
two vectors (bar and GUS) compared to the use of a bar-GUS gene fusion (Kamo et at.,
2000b). Co-bombardment produced six times the number of transgenic cell lines
compared to the use of the fusion gene. However, the fusion gene produced enhanced
GUS expression in the resultant transgenic lines. They also reported that the optimum
concentration of DNA for producing stable transformants was 10 times less than that for
optimum GUS expression.
Using the GUS gene, Kamo et al. (1995b, 2000a) and Kamo and Blowers (1999)
extensively analyzed different promoter activities in transgenic tissue. Callus and cell
suspensions were initiated from cormel slices or in vitro plants from two cultivars,
'Jenny Lee' and 'Peter Pears', and shot with the PDSlOOO/He system using MlO
tungsten particles (Kamo et al., 1995b). The activity of different promoters in gladiolus
tissue was tested by cloning them upstream of the GUS gene in pUC-based vectors. The
following promoters were tested: actl (actin), 35S, ubil (ubiquitin), mas2 (mannopine
synthase), and raiD. For the analysis of promoter activity, the cultures were assayed for
GUS expression 48 h after bombardment.
Results from the promoter analysis revealed that promoters normally most active in
monocot tissue (ubil, actl) were the least active in gladiolus tissue. When studying
transient expression it appears that gladiolus tissue responds more favorably to
promoters active in dicot tissue (35S, mas2, raiD), with the mas2 promoter being the
most active and 35S and raiD having similar activities. This result was repeated in the
work of Loffier eta/. (2000), who also found that the 35S promoter produces higher
levels of GUS expression in the callus after bombardment than the monocot promoters,
actl and ubil. Further work by Kamo and Blowers (1999) and Kamo et al. (2000a)
confirmed this result for stable expression in transgenic plants. They studied the
performance of the bar-GUS fusion gene under the control of the promoters 35S,
double 35S, mas2, raiD, EF-la. (translation elongation factor 1 subunit a.), actl, and
ubq3 (Arabidopsis ubiquitin) using the same transformation method. The raiD and the
two 35S promoter constructs produced the greatest number of transgenic plants,
followed by ubq3 and mas2, then the actl and EF-la. constructs. The level of GUS
expression in leaf tissue varied greatly in individual transgenics plants. Callus tissue
from the same plant lines had a significantly lower level of variation. GUS expression
under the actl, mas2, EF-la., raiD and 35S promoters followed the expression patterns
seen in other monocots. This work continued with the analysis of GUS expression in
tissues from 28 independent transgenic plant lines after three seasons of dormancy
(Kamo, 2000). No loss of GUS expression was observed under the 35S, raiD, mas2, and
172

ubq3 promoters, with the 35S promoter producing the highest level of activity. In
addition, there was no correlation between gene copy number and the level of
expression. This result clearly demonstrates that these promoters remain consistently
active in gladiolus tissue over several growing seasons and are thus suitable for use in
the generation of transgenic cultivars.
The technique of Kamo et al. (1995b) has been used to transfer disease-resistance
genes into gladiolus (Kamo et al., 1997a,b). Transgenic plants have been produced
carrying the bean yellow mosaic virus (BYMV) coat protein gene (Kamo et al., 1997a).
Expression of the gene varied widely across the 31 transgenic plant lines produced. The
transgenic plants carrying an active copy of the gene grew with reduced vigor both in
vitro and in the greenhouse compared with the non-transformed controls.
Kamo and co-workers also explored the possibility of transforming gladiolus tissue
using Agrobacterium (Kamo, 1997). Five tumorigenic Agrobacterium strains (A208,
A281, A348, B6, and C58) were used to inoculate in vitro plants, corms and seedling
tissue from five gladiolus cultivars. In each experiment, all five strains produced gall
tissue on all cultivars. The Agrobacterium strains At644, 654, and 646 carrying binary
vectors all produced transient GUS expression when used to inoculate explants from the
cultivar 'Peter Pears' and 'Jenny Lee'. Seedling tissue from the wild species G. carneus
was also inoculated with these strains. GUS expression was observed only when the
seedling tissue was grown in the dark. Although no transgenic plants were regenerated
from this work, the results indicate that A. tumefaciens-mediated transformation of
seedling tissue with octopine- or nopaline-type strains may lead to the production of
stable transgenic plants.
Babu and Chawla (2000) also observed transient expression of GUS after
Agrobacterium-mediated transformation of gladiolus tissue. They used the
Agrobacterium strain LBA4404 carrying either the binary vector pBI 141 (act 1-GUS) or
pTOK233 (35S-GUS). Cormel slices or in vitro shoot tip explants from two cultivars,
'American Beauty' and 'Yellow Topaz', were inoculated after wounding the tissue with
a scalpel or by bombardment with 1.6-Jlm gold particles. The highest levels of GUS
expression were obtained on both cultivars after wounding with gold particles and
inoculation with LBA4404/pTOK233, although LBA4404/pBI141 also gave good
results. Antibiotic-resistant shoots were recovered and were still GUS-positive 3 weeks
after inoculation. This result strongly indicates that Agrobacterium-mediated
transformation is also a viable method for producing transgenic gladiolus plants.

2.15. HIBISCUS (Hibiscus rosa-sinensis)

Hibiscus is a popular ornamental shrub, but it has limited use as an outdoor plant in
temperate regions due its sensivity to temperatures below 11 °C. Traditional breeding in
this crop has not produced any cold-tolerant varieties. Production of transgenic tissue
from hibiscus has been achieved by Vazquez-Thello et al. (1996). Cell suspension
cultures of hibiscus were co-cultivated with A. tumefaciens LAB4404 carrying the
binary vector pBI121 (GUS, nptii). After co-cultivation for 2 h, the cells were placed on
solid media and transformed loci were selected via kanamycin resistance. Unfortunately
the authors were unable to regenerate plants from callus tissue. To overcome this they
fused protoplasts from the transgenic tissue with protoplasts from Lavatera thuringiaca,
which is readily regenerated from callus tissue. However, in all the fusion products
 173

generated, no regeneration was possible indicating that the block to regeneration in

hibiscus may be a dominant character. To our knowledge, no transgenic hibiscus plants
have been produced.

2.16. HYACINTH

Hyacinth is an attractive ornamental spring flowering bulb but commercial production is

hampered by its susceptibility to viral infection. Transgenic hyacinth plantlets have
been generated via particle bombardment of embryogenic callus derived from leaf
material. Hygromycin-resistant plantlets were produced carrying the coat protein gene
from hyacinth mosaic virus in an effort to generate virus-resistant plants. Analysis of
these plants is in progress (Langeveld eta!., 2000).

2.17. IRIS (Iris germanica)

Iris germanica is one of the dominant tall bearded irises. Because of the high level of
incompatibility between species, most breeding efforts have been intraspecific.
Regeneration of I. germanica in in vitro culture is difficult, with low frequencies of
shoot production. Jeknic eta/. (1999) developed efficient plant regeneration from cell
suspensions of the cultivar 'Skating Party' and have used this system to produce
transgenic plants. Five antibiotics (methotrexate, hygromycin, geneticin, gentamycin
and phleomycin), three herbicides (glyphosate, chlorsulfuron and basta) and one amino
acid analogue (4-methyl-tryptophan) were tested as agents for the isolation of transgenic
tissue. Although the herbicides were very effective at inhibiting growth of non-
transgenic tissue, they also inhibited the regeneration potential of resistant tissue. The
most suitable selection agents were hygromycin and geneticin.
Three A. tumefaciens strain/vector combinations were tested (LBA4044/pTOK233,
LAB4404/pCAMBIA1201 and EHA105/pCAMBIA1201) with LBA4404/pTOK233
giving the highest level of transient GUS expression in cell suspensions.
Cell suspensions were co-cultivated with Agrobacterium for 5 min then spread onto
solid media for 3 days, after which they were transferred to media containing selection
agents. pTOK233 carries resistance genes for both hygromycin and geneticin so both
agents were used in the selection process. After two rounds of selection, antibiotic-
resistant calli were regenerated into intact plants. A total of 23 independent transgenic
lines produced plants that expressed both the nptll and GUS genes in their leaf tissue.
This is an efficient transformation system for iris. However, a prerequisite for the
system is the ability to efficiently regenerate plants from cell suspension cultures. It is
unclear how cultivar-independent this system is.

2.18. KALANCHOE (Kalanchoe sp.)

Several species of Kalanchoe (e.g. K. diagremontiana and K. /aciniata) have been

transformed using Agrobacterium. K. blossftldiana is the most important kalanchoe
species for ornamental use and Aida and Shibata (1996) have reported transformation of
this species. They transformed the cv. 'Tetra Vulcan' using a transformation system
based on that of Horsch et a/. (1985). Leaf pieces from in vitro-grown plants were co-
cultivated with A. tumefaciens LBA4404 carrying one of three binary vectors,
174

piG121Hm (nptll, GUS, hpt), pBE2113 (nptll, GUS) or pBI121 (nptll, GUS).
Transgenic plants were isolated using kanamycin resistance as the selective marker.
Transformation frequencies from the three binary vectors were: piG121Hm-2.9%,
pBE2113-2.0%, and pBI121-1.4%. All the transformants were transferred to the
greenhouse where they grew normally, flowered and set seed. GUS expression varied
widely across the transgenic lines with a high level of gene silencing observed. In some
lines, this silencing was reversed in the selfed progeny. GUS activity was highest in
plants transformed using piG 121 followed by plants from pBE2113 then pBI121. There
was no correlation between gene copy number and level of expression in mature tissues.
This transformation system yields low but consistent efficiencies with different binary
vectors and will be useful in the generation of new cultivars in kalanchoe.

2.19. LAVATERA (Lavatera sp.)

The Lavatera genus contains about 25 species, many of which are used as ornamental
plants. To date, only one species has been transformed, Lavatera thuringiaca, which is a
popular ornamental shrub. Vazquez-Thello et a/. (1996) produced transgenic plants
from the transformation of cell suspensions with A. tumefaciens LBA4404 carrying the
binary vector pMM454 (Nos-Hpt). After co-cultivation for 2 h, the cells were placed on
solid media and transformed loci were selected via hygromycin resistance. Transgenic
plants were produced via somatic embryogenesis under selection. As reported for
hibiscus (see above), protoplasts from a transgenic line of Lavatera thuringiaca were
fused with transgenic hibiscus protoplasts in an attempt to transfer the cold tolerance of
lavatera into hibiscus. Selecting for resistance to both kanamycin (from the transgenic
hibiscus protoplasts) and hygromycin (from the transgenic lavatera) isolated somatic
hybrids. Unfortunately the inability of hibiscus to regenerate from callus was also
transferred, meaning no transgenic hybrids were produced. However, the technique
developed for Lavatera thuringiaca could be used to develop new cultivars for this
species.

2.20. LIMONIUM (Limonium sinuatum)

Limonium, a bushy upright perennial, is a popular dried flower. Kimizu et a/. (2000)
reported on progress towards developing a transformation system for this crop. They
have developed a shoot regeneration system in six cultivars, primarily from petiole
tissue. They have also shown transient GUS expression and the isolation of
hygromycin-resistant callus tissue from the transformation of petioles with A.
tumefaciens EHA101 carrying the binary vector piG121Hm. To date no shoot
regeneration has been observed from the transgenic callus. The authors indicate that
improvements in the efficiency of regeneration from petiole tissue are needed for a
successful transformation method.

2.21. UNUM (Linum sp.)

Ling and Binding (1997) described the transformation of two species of linum. Linum
usitatissimum was transformed via the co-cultivation ofplastocytes with A. tumefaciens
GV2260 carrying the binary vector pBinl9-35SGUS. Five cultivars were tested with cv.
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'Bionaa' (five transgenic lines) and cv. 'Langeland B' (one line) producing kanamycin-
resistant, GUS-positive shoots. L. suffruticosum protoplasts were transformed via PEG-
mediated direct gene transfer using the vector pGL2 (hpt resistance) and via A.
tumefaciens-mediated transformation of plastocytes. The latter proved more efficient.
Whilst these techniques do consistently produce transgenic plants they suffer from the
length of time taken to recover plants from protoplasts and the greater chance of
somaclonal variation. The species described here are mainly grown for fibre production
but are also used as ornamental plants. These techniques may also be applicable to
linum species bred more for their use as ornamentals.

2.22. LILY (Lilium longiflorum)

Lily is one of the three most important ornamental bulb crops. As a result there is much
research into the development of a transformation system for this crop with an initial
aim to confer viral resistance.
Miyoshi eta/. (1995) have used electroporation to transfer the GUS gene into lily
pollen protoplasts. They tested several promoters, 35S, Lat52 (tomato pollen-specific
promoter) and Zml3 (maize pollen-specific promoter). The promoter Zm13 produced
the highest level of expression, followed by Lat52. The 35S promoter produced little or
no GUS activity and none of the promoters were able to produce GUS activity in
uninucleate pollen protoplasts. Thus, GUS expression was only possible in protoplasts
from bicellular pollen. Using Zml3, up to 70% of the surviving protoplasts were GUS-
positive. This is in contrast to the transformation efficiencies (0.1- 4.0%) obtained from
bombardment of intact pollen. This system will be useful for the analysis of promoter
activity and, if plant regeneration from protoplasts is achieved, as a novel route for the
generation of transgenic lilies.
Tsuchiya eta/. (1996) tested the activity of promoters in bulb scales and immature
embryos of three Lilium species (L. xformolongi, L. dauricum, L. japonicum). They
studied the promoters 35S, adh (maize), actl (rice), and ubi (maize). They also studied
the effect of the intron from the maize adh gene and bean catalase gene. Gene delivery
was via particle bombardment using 1.6-Jl.IIl gold particles, as 1.0-Jl.IIl particles were
significantly less efficient. Bulb scales from the three species were shot with the GUS
gene under the control of the 35S, adh (plus adh intron), 35S (plus bean intron) and actl
promoters. In L. xformolongi, the actl promoter produced the highest transient GUS
expression. In addition, a 10-day pre-culture enhanced GUS expression for all
constructs. In L. dauricum, promoter type and pre-culture had no significant effect. In L.
japonicum, the overall level of GUS expression was low and not influenced by
promoter type but was significantly influenced by pre-culture period, with 2 and 10
days pre-culture showing significant improvement. These results show that promoter
activity and the efficiency of transformation methods are species-specific. Immature
embryos of L. x formolongi were shot with the GUS gene under the actl promoter.
Embryo age and pre-culture length and conditions all significantly affected GUS
expression levels. Thirty-five-day-old embryos pre-cultured for 10 days in the presence
of 2,4-D gave the best results. In a subsequent experiment, different promoters were
compared in bulb and embryo tissue from L. xformolongi. The promoters act I and 35S
(with the bean intron and an enhancer sequence) produced the best results in both
tissues. These results show that individual promoter activity is species-specific. In
176

comparison to the pollen expression work above, they also indicate the significance of
tissue specificity.
Constitutive promoters have also been studied in lily leaf pieces (Wilmink et a/.,
1995b). The following promoters were used to express the GUS gene in leaf pieces
transformed by particle bombardment: 35S, 35Si (35S plus maize adh 1 intron), emu
(maize), act1 (rice) and ubi (maize). The presence of the maize intron in the 35S-GUS
construct inhibited GUS expression in lily leaf pieces. The highest expression levels
were obtained from the rice act1 and maize ubi promoters. This result is in contrast to
the results for embryos and bulb tissue.
Transgenic lilies have been recovered from the bombardment of lily pollen (van der
Leede-Plegt et a/., 1997). Mature pollen from Lilium longiflorum cv. 'Gelria' was
bombarded with the vector pCP01.2gus carrying the GUS reporter gene and kanamycin
resistance gene aphii. GUS expression was observed in germinating pollen. Bombarded
pollen was then used to pollinate flowers of the cultivar 'Indian Summer' and the
harvested seeds were germinated in the presence of kanamycin. Three transgenic
seedlings from 65,000 seeds tested were able to grow and form bulbs in the presence of
kanamycin. Transgenic plants have been produced by this method but the frequency is
very low (3/65000). However, because of the relative ease of testing large numbers of
seeds and the absence of a regeneration step(s) this technique is still useful in the
production of transgenic lily.
Microprojectile bombardment has also been used to transform lily via the
bombardment of callus (Watad et a/., 1998) and bulb scale tissue (Langeveld eta/.,
1997). Langeveld and co-workers bombarded bulb scales with a vector containing the
hpt resistance marker, GUS reporter gene and the coat protein from the LSV virus.
Hygromycin-resistant callus was isolated that also expressed the GUS gene. Shoots and
bulblets have been regenerated from these callus lines. To our knowledge, no further
information is available on the virus resistance of the transgenic plants. Watad eta/.
(1998) bombarded callus derived from in vitro bulblets from the cv. 'Snow Queen' with
plasmids carrying the GUS reporter gene and the selectable marker bar. The callus was
shot with tungsten particles using the PDS1000/He system and the herbicide Bialaphos
was used to select transgenic tissue and subsequently shoots. Bombardment pressure
and shot distance were the most influential factors on transient expression levels.
Osmotic treatment had no effect on the level of GUS expression. A total of 1800 callus
samples were bombarded yielding 72 herbicide-resistant loci. A substantial number of
the shoots arising from the calli were highly sectored. Subsequent rounds of
regeneration under selection yielded 19 plants from independent transformation events
that carried the bar gene (PCR analysis), representing a 1% transformation efficiency.
Preliminary studies have been carried out into the development of an
Agrobacterium-mediated transformation system for lily (Langeveld et a/., 1995). In
vitro plantlets of cv. 'Harmony' produced gall tissue after infection with wild-type A.
tumefaciens strain C58. Stem segments were then inoculated with five Agrobacterium
strains, A. tumefaciens strains C58, C58-C1 (disarmed C58), C58-C9 (cured negative
control), and A. rhizogenes strains 1855 and ATCC 15834. All strains carried the binary
vector p35SGUSINT containing the GUS reporter gene. The A. tumefaciens strain C58
produced the best results with C58-C1 while the A. rhizogenes strains produced few or
no transgenic loci. This demonstrates that lily is susceptible toAgrobacterium infection.
However, to date no further progress has been reported.
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2.23. LISIANTHUS (Eustoma grandiflorum)

Lisianthus has become a popular cut flower in recent years and there is much interest in
developing new cultivars via genetic modification of flower color, plant form and
disease resistance. To date five groups have developed transformation systems for this
crop.
Handa et al. (1995) and Handa (1996) inoculated seedling stems with the wild-type
A. rhizogenes strains Al3 and A4, with the latter carrying the binary vector pBI121. In
both experiments, transgenic plants were recovered from the spontaneous regeneration
of shoots from hairy root clones. All the transgenics had phenotypes typical of plants
carrying ro/ genes, such as short internodes and small leaves. GUS expression was also
observed in mature tissues from plants transformed with pBI121. This method is a rapid
way of producing transgenic plants with intact plants being formed only 3 months after
the initial inoculation. The disadvantage is the altered phenotypes caused by the ro/
genes. However, it is possible that the undesirable phenotypes can be bred out using
traditional backcrossing methods.
Hecht et a/. (1997) produced transgenic shoots via direct organogenesis from the
edge ofleafpieces transformed with A. tumefaciens carrying the binary vector pBI121.
Shoots were GUS-positive 15 weeks after transformation. ·
Stable transformation of lisianthus by particle bombardment has been reported by
Takahashi et a/. (1998). Stem and root explants from etiolated seedlings were
bombarded with 1.1-Jlm gold particles carrying either plasmid pBI221 (GUS) or
pARK22 (bar). Transient GUS expression showed a high level of DNA transfer.
Explants shot with pARK22 were subjected to selection for Bialaphos resistance and
yielded a transformation efficiency of approximately 0.1 %. Since particle bombardment
is not host-specific, this technique may be applicable to any lisianthus cultivars capable
of regenerating from seedling explants.
Semeria et a/. (1995, 1996) developed transformation systems using both
microprojectile bombardment and Agrobacterium. The microprojectile method used a
homemade particle inflow gun based on the design of Finer et a/. (1992) to shoot
suspension cultures of the lisianthus cv. 'Jodel Blue'. The vector used was pKIWI105
carrying the GUS reporter and kanamycin-resistance genes. Approximately four
kanamycin-resistant loci were recovered from each shot event. This translated into a
transformation efficiency of 4.1 plants per 1675 loci. All resistant loci regenerated intact
transgenic plants.
Semeria eta/. (1996) also developed a transformation system using A. tumefaciens.
They tested two strains, A281 and its disarmed derivative EHA105, both containing the
binary vectors pKIWI105 or pSWP (carrying the nucleoprotein gene ofTSWV for viral
resistance). The wild-type strain A281 produced the highest number of transgenic calli
and tumors. However it was difficult to regenerate intact plants from these clones.
Semeria et a/. concluded that their system's primary bottleneck for transformation
efficiency was the regeneration of shoots from transgenic callus or tumors. Co-workers
in the same group also transformed lisianthus with A. rhizogenes strain NCPPB1855
(Giovannini eta/., 1996). They used 16 lisianthus genotypes and were able to produce
hairy root clones from 15 of them. Nine genotypes spontaneously produced transgenic
plants, which were transferred to the glasshouse. Like previous workers who have used
178

A. rhizogenes, the resultant transgenics exhibited varying degrees of altered morphology

due to the presence of the rol genes.
Our laboratory has also developed a transformation system for this crop using the
wild-type A. tumefaciens strain A722 (Ledger eta/., 1997) to transform leaf pieces. We
observed results similar to those of Semeria eta/. (1996), in that the disarmed strain
LBA4404 produced significantly fewer transformants than its wild-type relative did.
However, unlike Semeria et a/. (1996), we have had no problem regenerating
morphologically normal transgenic shoots. It is possible that the A. tumefaciens
strain/vector combinations that we used produce a greater proportion of non-
tumorigenic callus that can produce shoots. This technique was first reported in Deroles
eta/. (1993) and has been used to produce transgenic plants carrying the GUS gene and
the UFGT gene for the modification of flower color (Schwinn et a/., 1997). This
technique had a low efficiency of transformation, with approximately 1. 9 transgenic
plants produced per 100 explants. We have now improved it to give a transformation
efficiency of 10 to 15% when tested on eight different cultivars (author's unpublished
results). Using this system, we have produced transgenic lisianthus plants carrying a
variety of flavonoid biosynthetic and regulatory genes for the modification of flower
color: CHS antisense (Deroles et a/., 1998); Lc (Bradley et a/., 1999); FLS (Davies et
a/., 1997); F3'5'H, DFR, CHR, and CHI (unpublished data). To date, we have produced
over 400 independent transgenic plants from eight different cultivars using a variety of
binary vectors and gene constructs. We have not observed any phenotypic variations
that could be attributed to the presence of A. tumefaciens-derived oncogenes.
It is clear that lisianthus is readily transformed by a number of methods and that the
transformation methods are not as cultivar-specific as some other species (e.g.
chrysanthemum). To date, the most successful method for producing high numbers of
plants with phenotypes unaltered by the transformation process other than the gene of
interest is via the use of A. tumefaciens. It is interesting to note that disarmed strains of
both A. rhizogenes and A. tumefaciens are not as effective as the wild-type strains. In
addition, the use of a wild-type A. tumefaciens strain does not lead to altered
morphologies through the expression of the wild-type T -DNA genes. Transformation of
this crop is more fully summarized in Handa and Deroles (2001).

2.24. ORCHID (Orchid sp.)

A number of orchid species have been successfully transformed. The most common
technique used is microprojectile bombardment. However, one species has also been
successfully transformed using Agrobacterium.
Kuehnle and Sugi (1992) were the first to report the production of transgenic
Dendrobium orchids using microprojectile bombardment. Three-month-old protocorms
were bombarded with tungsten particles (Sylvania MlO) coated with DNA from the
plasmid pGA482GG/cpPRV4 (GUS, nptll). The particle gun used was that described by
Klein eta/. (1987) and transformed tissue was selected via kanamycin resistance. After
17 months of culture, 13 plants were still resistant to kanamycin and contained copies of
the kanamycin resistance gene as shown by Southern analysis. Kuehnle and Sugi found
that the use of kanamycin as a selective marker does not completely halt the growth of
non-transformed tissue, and recommended the use of hygromycin as the selective agent.
In addition, some problems were experienced with chimerism in the transformed
 179

population. This may be overcome by using callus or embryogenic cell suspension

cultures as the target tissue. In a subsequent publication (Nan and Kuehnle, 1995),
different shooting parameters were tested with gold particles, the use of protocorm-like
bodies (PLBs) as the target tissue and enhanced promoters, all giving improved
transformation efficiency.
Yu eta/. (1999) also recovered transgenic Dendrobium orchids using hygromycin
selection after microprojectile bombardment of protocorms. Protocorms were subjected
to osmotic treatment using 0.25 M sorbitol 24 h prior to bombardment. After
bombardment using the PDSlOOO/He system, the protocorms were cultured without
selection for 2 to 3 months then transferred to medium containing hygromycin for
further proliferation and plant regeneration. They obtained a transformation efficiency
of5 to 10%.
Transgenic Cymbidium orchids have been produced by Yang eta/. (1999). PLBs
that had been pre-cultured for 7 days were bombarded with gold particles carrying the
vector pKN200 (nptlll GUS-INT) using the PDSlOOO/He device. Bombarded PLBs
were proliferated in liquid medium without selection. Regenerating shoots were then
further screened by rooting in the presence of kanamycin. Both the pre-culture and
selection free post-culture periods significantly improved the transformation frequency.
Using this technique, the authors report a transformation efficiency of over 15%. The
technique is being used to alter flowering time and morphology as well as to develop
viral resistance.
Phalaenopsis orchids have been transformed by both microprojectile bombardment
andAgrobacterium-based methods. Chan eta/. (2000) studied transient GUS expression
in PLBs using both transformation techniques. Using Agrobacterium, transformation
efficiencies were significantly improved by wounding the PLBs in the presence of the
bacteria through sonication for 150 s. Transgenic Phalaenopsis plants have been
generated by Belarmino and Mii (2000) using Agrobacterium strains LBA4404 and
EHAlOl carrying binary vectors containing the GUS and hpt genes. This is the only
report of the transformation of orchid species using cell clumps from a suspension
culture rather than protocorms or PLBs as the target explant. Co-cultivation of the cells
was performed in two steps. The cells were initially co-cultivated for 10 h in liquid
culture then transferred to solid medium for a further 3 days of co-cultivation. The cells
were then transferred to hygromycin selection for proliferation, the development of
PLBs and finally, plant formation. Acetosyringone added to both the Agrobacterium
culture medium and the 3-day co-cultivation medium increased transformation
efficiency. Using this technique the authors were able to recover 10 to 24 plants per
gram fresh weight of the starting suspension culture.
Knapp et a/. (2000) generated transgenic plants from three species of orchid,
Brassia, Cattleya and Doritaenopsis. PLBs were bombarded with the bar gene and then
proliferated on selection medium containing the herbicide Bialaphos. Proliferating PLBs
were minced and cultured on selection medium. This procedure was repeated two more
times, then plants were regenerated in the absence of Bialaphos. Using this technique,
transgenic plants were recovered from all three species. Ten plates of target tissue were
bombarded for each species, yielding 11 Brassia, 3 Cattleya and 18 Doritaenopsis
transgenic plants. All the transgenic plants proved resistant to the herbicide when
applied in the greenhouse at concentrations sufficient to kill the non-transgenic controls.
180

These reports show that a number of important orchid species can be successfully
transformed. If the protocols are reliable, new transgenic cultivars cannot be far away.

2.25. ORNITHOGALUM (Ornithogalum sp.)

Ornithogalum is a popular cut flower or pot plant but commercial development of this
crop is hampered by its susceptibility to disease, particularly to Omithogalum Mosaic
Virus. De Villiers et a/. (2000) reported the generation of a single transgenic
ornithogalum plant via microprojectile bombardment. Callus tissue generated from
leaves of the hybrid 0. thyrisodies x 0. dubium were bombarded using the pDS1000/He
gun with 1. 6-!Jlll gold particles carrying vectors containing the GUS reporter gene and a
bar-GUS fusion gene. Transient expression studies were used to assess promoter
activity. The enhanced 35S promoter proved the most effective when compared to other
promoters, such as the rice actin and potato ubiquitin promoters. These results are in
agreement with those of other workers who showed that monocots such as gladiolus
(Kamo eta/., 1995b) and lily (Watad eta/., 1998) also perform best with promoters
more commonly preferred in dicots. Using plasmid p35SAC carrying the bar gene,
many herbicide-resistant calli were produced, but no stable transgenic plants were
recovered. In a variation of this technique, called the Taxi protocol (Chen eta!., 1998),
single-stranded DNA from the plasmid was coated with histone Hl protein prior to
precipitation onto the particles. Using this technique, a single transgenic plant was
recovered that was consistently herbicide-resistant and contained the bar gene. This is
an encouraging result but the efficiency of transformation needs to be improved for it to
become a useful technique for the generation of new omithogalum cultivars.

2.26. OSTEOSPERMUM (Osteospermum sp.)

Osteospermum is a ground-cover ornamental of which most varieties are interspecific

hybrids. Recently it has gained in popularity as a pot plant. Current targets for new
cultivars include new flower colors, new plant forms, vase life and disease resistance.
Many of these attributes can be addressed through genetic modification, and tissue
culture systems using leaf tissue as the explant have been established for this crop.
Allavena et a!. (2000) produced transgenic osteospermum using a leaf piece
transformation method similar to that of Horsch et a/. (1985). Leaf pieces from the
hybrid cultivar 0. eck/onis were co-cultivated with four A. tumefaciens strains,
GV2260, GV3101, EHA105 and LBA4404. All but LBA4404 produced kanamycin-
resistant transgenic plants. Hygromycin and phosphinothricin were also tested as
selective markers but produced no transgenic plants. Transgenic plants were then
produced carrying the ro!AB, ro!ABC and rotC genes from A. rhizogenes using the A.
tumefaciens strain GV3101 to transform the Osteospermum genotype DM005
(Giovannini eta!., 1999; Allavena eta!., 2000). All trans genies showed varying degrees
of compact habit, earlier flowering and increased flower numbers. In addition they
produced transgenic plants from two cultivars carrying the nucleoprotein N gene from
tomato spotted wilt virus. The transgenics showed varying levels of viral resistance in
the laboratory with higher levels of resistance correlated with multiple copies and very
 181

low levels of transgene expression. This indicates that the mechanism of resistance may
be co-suppression. These plants are now being tested under field conditions. Thus
Allavena and co-workers have developed an efficient transformation system and
demonstrated its value in the development of new osteospermum cultivars.

2.27. PELARGONIUM (Geranium, Pe/argonium sp.)

Pelargoniums are the most popular potted plant worldwide and are also very popular as
a garden plant. There is considerable interest in the development of a transformation
system for various pelargonium species to improve disease resistance and to alter flower
color and plant form. In addition, genetic modification to eliminate petal shatter may
enable pelargonium species to be used as cut flowers. Boase and Smith ( 1994) reported
transient GUS expression in 11 pelargonium cultivars, including eight regals and four
zonals, after transformation with the A. tumefaciens strains LBA4404 and A722. In a
later publication, Boase eta/. (1996) reported the generation of transgenic plants from
the regal cultivar Pe/argonium X domesticum cv. 'Dubonnet'. Leaf pieces from
glasshouse plants were co-cultivated with A. tumefaciens strain LBA4404 containing
binary vectors carrying the GUS and nptii genes. Transgenic shoots were regenerated
under selection via adventitious organogenesis. This transformation system has now
been improved and is based on A. tumefaciens and leaf explants derived from plantlets
grown in vitro, giving a transformation efficiency of 19.3% (Boase eta/., 1998b). Using
this technique, GM pelargoniums with dwarf phenotypes such as reduced plant height,
leaf area and petal area have been produced through the action of the ro/C gene (Boase
eta/., 1998d). In addition, transgenic plants have been produced carrying the oatphyA
gene (Boase eta/., 1998b), and the maize Lc gene (Bradley eta/., 1999). Boase eta/.
have also generated stable, kanamycin-resistant calli from zonal cultivars, after
transformation by A. tumefaciens strains LBA4404, A722 and A281 (reviewed in
Deroles et at., 1997).
Genetic transformation systems have also been reported for four scented
pelargonium species (Pellegrineschi eta/., 1994; Pellegrineschi, 1996; Pellegrineschi
and Davo1iomariani, 1996). P. graveolensi petiole fragments were inoculated with three
A. rhizogenes strains (A4RSII, 15834 and LBA9402). Hairy roots were cultured and
transgenic shoots spontaneously regenerated from several transgenic clones. The
resultant transgenic plants showed reduced growth stature, typical of plants carrying ro/
genes, and thus improving the stature of the parent cultivar, which is known for its
chaotic, ungainly growth (Pellegrineschi eta/., 1994). P. fragrans, P. odoratissimus, and
P. quercifoliai transgenic plants were produced using the same technique and all had
reduced growth stature (Pellegrineschi and Davoliomariani, 1996). Transformation of
these species with A. rhizogenes has resulted in plants with improved stature and
rooting efficiency.
A fifth species of scented geranium (Petargonium sp. 'Frensham') has been
transformed by Krishnaraj et at. (1997). Leaf petioles from greenhouse-grown young
leaves were co-cultivated with A. tumefaciens strains LBA4404 carrying the binary
vector pBI121 (nptii! GUS) or LBG66 carrying pJQ418 (nptiiJ Int-GUS) for 2 days
then transferred to selective media (kanamycin) for the development of embryos.
Embryos that developed in the presence of kanamycin were germinated without
selection and the resulting plants transferred to the glasshouse. A transformation
182

efficiency of up to 14% was achieved for both strain vector combinations and, unlike
techniques using A. rhizogenes, all the progeny were morphologically identical to the
parent cultivar. Bi et a/. (1999) used this system to produce transgenic geraniums
carrying the antimicrobial protein (Ace-AMP) from onion for resistance to Botrytis
cinerea. All the transgenics expressing the gene showed increased bacterial resistance
with the level of resistance correlated to the amount of antibacterial protein being
produced.
Some of the most popular pelargoniums are the zonal varieties (Pelargonium X
hortorum). Robichon eta/. (1995) developed anA. tumefaciens-mediated transformation
system for the zonal cultivar 'Alain' using the strain EHA 10 1 carrying the binary vector
pKHG3. This vector carries the GUS gene and resistance genes for both kanamycin and
hygromycin. Cotyledon and hypocotyl explants were cultured for 15 days then
inoculated with Agrobacterium for 20 min. No co-cultivation step was included to
reduce the pathogenic damage from bacterial overgrowth. Unlike the aforedescribed
transformation methods for pelargonium, kanamycin was unable to effectively select for
transgenic tissue and hygromycin resistance was used instead. Shoot and root formation
was carried out under selective pressure. A transformation efficiency of 20% was
achieved and all the progeny were morphologically identical to the parent cultivar.
Using the technique of Robichon et al. (1995), transgenic plants were generated
from P. hortorum and P. peltatum carrying the cecropin gene for resistance to the
bacterial pathogen Xanthamonas campestris. Some resistance to the pathogen has been
determined in the laboratory and the plants are now under field evaluation (Renou et al.,
2000).
Both major varieties of pelargonium (zonal and regal) have been successfully
transformed and the techniques are now being used to develop new commercial
cultivars through disease resistance and alteration of flower color and plant form.
However, as with many other transformation systems, there is a degree of cultivar
specificity to be overcome before these techniques become universally applicable for
pelargonium as a crop.

2.28. PETUNIA (Petunia hybrida)

Numerous cultivars of petunia have been routinely transformed and are used extensively
in plant genetic research. Recent references to transformation in petunia are too
numerous to mention here. Petunia is easily transformed by a variety of methods and is
also used to develop new transformation methods. Recently, transgenic petunias were
generated via vacuum infiltration of pollen with the A. tumefaciens strain AGLO
carrying the binary vector pCGP1258 (Basta resistance). The infiltrated pollen was then
used for pollination, leading to a transformation frequency of 55% in the T 2 population
(Tjokrokusumo eta/., 2000). The authors also generated transgenic plants by applying
Agrobacterium to the stigma just prior to pollination. This led to the generation of
transgenic seedlings but at a lower efficiency (Tjokrokusumo et a/., 2000). The most
common method for producing transgenic petunia is via A. tumefaciens-mediated
transformation of leaf pieces (van der Meer, 1999). Selection can be via a number of
compounds, including antibiotic or herbicide resistance. Recently, transgenic petunias
have been used to study several characteristics such as: the genes of the flavonoid
biosynthetic pathway (Oud et a/., 1995; Bradley et a/., 1998; Davies et al., 1998;
 183

Tanaka eta/., 1998), fungal resistance (Esposito eta/., 2000), plant form (Winefield et
a/., 1999), ethylene perception (Gubrium eta/., 2000), self incompatibility (Dowd eta/.,
2000), and alkaloid production (Thomas et a/., 1999). Despite of the large number of
transgenic petunias that have been produced, we are unaware of any transgenic cultivar
in commercial production. This probably reflects the predominant use of this plant as a
model species rather than as a target for commercial modification. However, red-leafed
transgenic petunia lines from our laboratory (Bradley eta/., 1998) are currently under
commercial evaluation in field trials.

2.29. POINSETTIA (Euphorbia pulcherrima)

Poinsettia is a popular ornamental plant, particularly during the European Christmas

period and is a major floricultural crop in Norway. Breeders have explored wild
populations extensively in order to generate new varieties and now one of the only
potential sources of new variations is via genetic modification. Hvoslef-Eide et a/.
(2000a) and Vik eta/. (2000) used a novel technique to transform this plant. The authors
compared three techniques, A. tumefaciens, the particle gun, and DNA electrophoresis
of genes into the apical meristem.A. tumefaciens and the particle gun produced GUS-
expressing loci in 4 to 10% of treated meristems and DNA electrophoresis, 26%. For
DNA electrophoresis the apical meristem(s) of a plant in the glasshouse is exposed and
gently blotted of any excess moisture. DNA is dissolved in agarose (1 mg/ml) and
loaded into a 1 ml pipette tip (150 !il). The pipette tip is lowered to cover the exposed
meristem and a negative electrode is placed in the tip with the positive electrode in the
soil. The plant is then subjected to a weak current (0.5 rnA at 55-75 V) for 10 min, then
left to grow. GUS activity was measured in the new leaves 8 to 9 weeks later. Of 34
shoots recovered from 27 meristems, nine tested positive for GUS expression. The
electrophoresis technique has two important advantages: no requirement for
adventitious shoot formation or embryogenesis, and no in vitro steps. As a result, this
method has the potential to be used on many species recalcitrant to traditional
transformation techniques. DNA electrophoresis has also been used to transfer DNA
into meristems of carnation, chrysanthemum, lisianthus (Burchi eta/., 1995) and orchid
protocorms (Griesbach, 1994).

2.30. RHODODENDRON (Rhododendron sp.)

Transformation of rhododendron using A. tumefaciens and microprojectile

bombardment has been reported by three groups. Pavingerova eta/. (1997) generated
transgenic plants from five cultivars, 'America', 'Catawbiense Grandiflorum roseum',
'Madame Carvalho', 'Mars', and 'Nova Zembla'. Stem explants were co-cultivated with
A. tumefaciens strain LBA4404 carrying the binary vector p35SGUSINT (GUS, nptll)
for 24 h. Transgenic plants were selected using kanamycin resistance and were
recovered from all cultivars. The transformation efficiency varied widely between
cultivars.
Ueno et al. (1996) also used A. tumefaciens strain LBA4404 carrying a binary vector
containing the GUS and nptll genes (pBI121). In this method, the explants used were
segments of in vitro shoots (cultivar 'Percy Wiseman') that had become thickened after
culture on a high zeatin medium. Subsequent culture of the thickened segments leads to
184

adventitious bud formation. After co-cultivation for 4 days the segments were placed
under kanamycin selection. Six independent transformed plants were recovered from
120 initial explants, giving a transformation efficiency of 5%. All the plants were
phenotypically normal.
Hsia and Korban. ( 1998) produced transgenic plants from the two azalea cultivars,
'Hino-Crimson' and 'Fuchsia' using microprojectile bombardment. Hygromycin was
used as the selective marker.
These techniques show that Agrobacterium and microprojectile bombardment can
readily transform several cultivars of rhododendron, opening the way to the generation
of novel colors and plant forms in this popular garden ornamental.

2.31.ROSE (Rosa sp.)

Rose is one of the most important ornamental crops worldwide with a wide variety of
flower colors and plant forms readily available. Because of the size of the rose market,
both in garden plants and cut flowers, considerable effort has gone into the development
of a transformation system to allow the introduction of characteristics not obtainable via
traditional breeding. Recent reports in this field come from four research groups.
Firoozabady et a/. (1994) developed a transformation system using the somatic
embryogenic callus line isolated by Noriega and Sondhal (1991) from the hybrid tea
rose cultivar 'Royalty'. They co-cultivated the callus with A. tumefaciens
LBA4404/pJJ3931 (nptiiJ LUC) or A. rhizogenes 15834/pJJ3499 (nptiiJ GUS).
Selection for transformed calluses was performed using kanamycin. Transformation
efficiencies were high with between 40 to 60 kanamycin-resistant calluses per gram of
inoculated rose callus. Transgenic plants regenerated from the independent transformed
calluses were shown by PCR to contain the transgenes. All plants produced were
reported to be morphologically normal. This is fortunate, since there is an extended
callus phase in the protocol, increasing the risk of somaclonal variation.
The biotechnology company Florigene developed a transformation system for six
different cultivars using Agrobacterium-mediated gene transfer. No specific details are
available for the method, which is now being used to generate varieties with new colors
(see http://www.florigene.com.au).
Marchant et a/. (1998b) reported the successful transformation of rose via
microprojectile bombardment. Somatic embryogenic callus from the cultivar Rosa
hybrida 'Glad Tidings' was bombarded with 0.4- to 1.2-J.Ull gold particles using a
PDS1000/He device. For transient expression studies, the vector MJD67 carrying the
GUS-reporter gene was used. The vector pBI101 carrying only the nptll gene was used
for the generation of stable transgenics. Transgenic tissue was selected via kanamycin
resistance at all stages of plant development. Marchant and co-workers tested several
parameters and determined optimum firing distance (70 mm) and pressure (9000 kpa).
Furthermore, the addition of osmoticum (myoinositol) before and after shooting
significantly increased the level of transient expression. After shooting, the calli were
placed on kanamycin-containing medium and subcultured fortnightly until homogenous
lines of resistant calli were recovered. These lines went on to produce transgenic
plantlets under selection. Twenty independent transgenic plants were produced from a
total of 90 bombardments. Each transgenic line contained between one and five copies
 185

of the kanamycin resistance gene. The level of transgene expression did not correlate
with copy number.
Marchant et a/. (1998a) have now used their transformation system to generate
transgenic roses carrying the chitinase gene in an attempt to induce blackspot resistance.
Twenty transgenic plants were produced. Chitinase activity in all the transgenics was
four to fifteen times greater than in the non-transformed control and, like the transgenics
in their previous paper (Marchant eta/., 1998b), the level of expression did not correlate
with transgene copy number. Bioassays showed the severity of blackspot development
to have been reduced by 13 to 43% and the level of suppression to be correlated with
the level of transgene expression. Marchant and co-workers are currently developing
transformation systems for rose root stocks with the aim of introducing genes for
disease resistance in the non-transformed scion. In addition, they aim to produce
transgenic roses with increased branching and hence flower number.
Marchant and co-workers have demonstrated that their gene transfer system is
efficient, reliable and suitable for the genetic improvement of rose. It will be interesting
to see comparative results in other rose cultivars using the same technique.
Derks eta/. (1995) tested the transformability of 15 rose cultivars in order to transfer
bacterial resistance for the enhancement of vase life. Somatic embryogenesis from root
tissue was established for six of the cultivars. The three most efficient of these
('Melody', 'Tineke', and 'Sonia') were used for transformation studies. Embryogenic
callus was co-cultivated with two A. tumefaciens strains, C58Cl/pMP90, and AGLO
carrying the binary vectors p35SGUSint, pMOG410 or pMOG410 (CecB). All the
vectors contained the GUS and nptll genes and pMOG410 (CecB) also carried the
cecropin B antibacterial gene. The calli were co-cultivated for 3 days then subjected to
kanamycin selection. To date, kanamycin-resistant calli have been recovered from all
three cultivars and somatic embryos have been recovered from the cv. 'Sonia'. To our
knowledge, no transgenic plants from this work have been reported.
Van der Salm et a/. (1996, 1997) used a variation of the technique described by
Derks eta/. (1995). Stem slices were co-cultivated with A. tumefaciens and adventitious
roots were produced under kanamycin selection. Callus was then produced from the
transgenic roots, followed by somatic embryogenesis and eventually plant formation.
Van der Salm and co-workers used the rootstock cultivar 'Moneyway' to test the effect
of rol gene combinations on rooting ability. Stem sections were cultivated with the A.
tumefaciens strain GV30101 carrying the vectors p35sGUSint (GUS), pPCV002-B1100
(ro/B) or pMRKE15 (rolA,B.C). The vectors also contained the nptll gene for
kanamycin resistance. All the stem slices produced either one or two transgenic root
cultures from all of the vectors, leading to a very efficient method of producing
transgenic lines. Transgenic roots carrying the rolA,B,C construct showed increased
lateral root formation, whereas the ro/B and 35SGUSint transgenics had no increased
root formation. Only 2 to 3% of the root lines produced shoots via embryogenesis,
yielding 19 independent transgenic plant lines. Plants carrying the ro/A,B, C construct
showed reduced apical dominance, decreased shoot length and, in one line, wrinkled
leaves. Cuttings from these plants produced roots much faster and had more vigorous
root systems than non-transgenic controls. The increased rooting ability of these lines
may lead to more vigorous rootstocks (van der Salm et a/., 1996, 1997).
This research was continued by the study of grafted roses using the rolA.B.C
transgenic rootstock (van der Salm eta/., 1998). The transgenic line ABC1 was chosen
186

due to the moderate effect the transgenes had on the plant phenotype while still
significantly increasing rooting ability. Grafted plants were generated by grafting the cut
rose cultivar 'Madelon' onto the transgenic 'Moneyway' rootstock ABC I. As expected,
the grafted plant produced significantly more roots than the non-transformed rootstock.
Axillary bud release at the base of the non-transformed scion was also stimulated,
indicating that there is some trans gene-related signal transferred between the transgenic
rootstock and non-transformed scion. Leaf area in the transgenic hybrids was also
increased, leading to an overall increase in vigor. Since the number of axillary shoots at
the base of a rose plant is related to flower production, the transgenic hybrids also
produce more flowers. Vander Salm eta/. (1998) showed that the genetic enhancement
of rootstocks can produce plants with increased vigor and flowering. Both of these
attributes are of considerable benefit to commercial growers.
Souq et a/. (1996) have used A. tumefaciens-mediated transformation of
embryogenic callus to produce transgenic hybrid tea roses. They used A. tumefaciens
strain GV3101 carrying two binary vectors, pPCV002 (GUS/ nptll) with either the rotC
or the antisense rose chalcone synthase (CHS) gene. Four transgenic plants were
generated from the cultivar 'Madame G. Delbard deladel®' carrying the rotC gene.
These plants showed two kinds of altered morphology, dwarfed plants with wrinkled
leaves and plants with multiple stems. In a second round of transgenic production, rotC-
containing transgenics had reduced height, smaller, more numerous thorns and chlorotic
foliage. In a third group the flower morphology was modified to smaller flowers with
fewer petals and a non-functional pistil. The transgenics all had weaker root systems
and, after pruning, the new stems failed to flower. Two transgenic lines were generated
carrying the CHS antisense gene, leading to alterations in color intensity but no white
flowers. Greater transformation efficiencies need to be achieved before this
transformation system can be used to produce new rose cultivars.
Current progress indicates that transformation of rose remains at a low frequency
and is still cultivar-specific. However, in several cases, the methods are able to produce
sufficient plants with altered phenotype for the production of new cultivars (van der
Salm et al., 1997, 1998; Marchant et a/., 1998a,b and Florigene [J. Mason, pers.
comm.]).

2.32. TORENIA (Torenia fournieri)

Torenia, or wishbone flower, is a popular summer bedding plant. Transformation of this

plant was first reported by Aida and Shibata (1995) who used a system based on shoot
regeneration from leaf pieces. Leaf pieces (3 mm square) from in vitro plants were co-
cultivated with A. tumefaciens for 7 days in the presence of acetosyringone, and
transgenic calli and shoots were selected using either kanamycin or hygromycin.
Transformation efficiency measured over several experiments was about 10%.
Using this transformation system, Aida and co-workers have evaluated promoters
via GUS expression (Aida eta/., 1995). Transgenic plants were generated carrying three
constructs: 35S-GUSint (with the bean catalase intron), El2n/GUS (35S promoter
joined to the Omega arrangement from tobacco mosaic virus), or PRla-GUS
(pathogenesis-related la protein inducible promoter of tobacco). Overall, GUS activity
was higher in plants carrying the E12n/GUS construct compared to the 35S-GUSint
 187

construct. The PRla promoter was successfully induced by treatment of plant tissue
with salicylic acid.
Aida et al. (1998) reported the production of transgenic plants carrying a fragment
of the torenia ACC oxidase gene in both sense and antisense orientations. Both sense
and antisense constructs increased flower longevity with the sense construct having the
greatest effect. Aida et a/. (2000) also generated transgenic plants with modified flower
color through the insertion of the torenia CHS or DFR genes in both antisense and sense
orientations. All the constructs produced altered flower color with the CHS and DFR
genes producing flowers with loss of color patterns ranging from uniform loss to pale
tubes with colored lips. Suzuki et at. (2000) also reported the generation of pink flowers
from a blue parent via the co-suppression of the flavonoid 3 '5 '-hydroxylase gene. In
other publications, Aida and co-workers have reported the production of transgenics
with modified flower color (Aida et al., 2001) and transgene silencing in stable
transgenic lines and their progeny (Aida, 1998; Aida and Shibata, 1998). A summary of
the transformation of torenia can be found in Aida and Shibata (2001). Clearly, the
transformation system developed by Aida and co-workers is easy to use, efficient, and
capable of producing many new cultivars for this crop.

2.33. TULIP (Tulip gesneriana)

The development of novel characteristics in tulip is significantly hampered by its

heterozygosity, which leads to a wide distribution of characteristics on the progeny.
Genetic transformation offers a more direct route to the development of new cultivars.
No transgenic tulip plants have been produced. However, preliminary research into
selective agents, reporter genes and promoters has been reported. Chauvin eta!. (1997)
and Wilmink et at. (1995a) have studied selective and reporter genes in tulip. Both
reports show that kanamycin is not effective as a selection agent with the preferred
agent being PPT (bar gene). The activity of monocot-derived promoters and 35S
promoter in tulip was compared in Wilmink et at. (1995b). Tulip tissue behaved in a
similar manner to Nicotiana tissue with all the promoters tested generating a high level
of expression (35S, emu, actl, emu). In addition, the inclusion ofthe adhl intron with
the 35S promoter (35Si) significantly reduced promoter activity. Thus, it seems that
tulip tissue behaves more similarly to dicot tissue than to other monocots, such as rice.
To our knowledge no complete transformation system for tulip has been reported to
date.

2.34. VERTICORDIA (Verticordia grandis; Myrtaceae)

Verticordia gran dis is a native Australian plant of the Myrtaceae family, much prized
for its bright red flowers. Stummer et a!. (1995) reported the development of a
transformation method for this crop with the aim of modifying flower color and root
development. The regeneration system used in this method is based on shoot production
from the meristematic regions of in vitro-grown leaf petioles. Several Agrobacterium
strains were tested, four wild-type A. rhizogenes strains, K565, K568, K596 and K597,
and two disarmed A. tumefaciens strains, LBA440 and AGLl. To generate transgenic
plants, the binary vector pBI121 (GUS, nptll) was transferred to all strains except
AGLl, which carried the vector pKIWI105 (GUS and nptll). All strains except AGLl
188

produced transgenic plants, with the wild-type A. rhizogenes strain K596 being the most
efficient (6%). However, some plants produced from the A. rhizogenes strains exhibited
altered morphology associated with the presence of the Ri T-DNA. This report shows
that transgenic plants can be produced from V. grandis using either A. tumefaciens or A.
rhizogenes.

3. Conclusions

The predominant methods for transforming ornamentals remain A. tumefaciens and

microprojectile bombardment. However, A. rhizogenes has been used where dwarfing
and increased root production are desirable characteristics (e.g. pelargonium and rose).
Much progress has been made in the transformation of ornamentals, with transgenic
plants being generated from new species and cultivars. Despite this progress, many
obstacles to the production of new varieties still exist. Perhaps the greatest is the
continuing lack of cultivar-independent transformation systems. One of the most
interesting new techniques is direct DNA electrophoresis into plant meristems, which is
being used to generate transgenic poinsettias. This technique does not require an in vitro
regeneration system and is not host-specific.
The field of ornamental transformation is rapidly expanding, with methods being
developed for many new species and cultivars. However there is still only one GM
ornamental in commercial production (carnation, www.florigene.com.au). Judging by
the level of activity in this field, many new cultivars will be produced in the next few
years. The biggest impediment to the introduction of GM ornamentals may prove to be
public perception and regulations regarding GM products in individual countries.
However, ornamentals may encounter less public resistance than food and medicinal
crops.

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MOLECULAR APPROACHES FOR INCREASING PLANT RESISTANCE TO
BIOTIC AND ABIOTIC STRESSES

M. LORITO, G. DEL SORBO, F. SCALA

Dipartimento di Ar.Bo.Pa. Ve. - sezione di Pato/ogia Vegetale
Universita di Napoli Federico II
Via Universita, 100, 80055 Portici, Italy

1. Introduction

The level of understanding reached in the genetics and molecular mechanisms of plant-
pathogen interaction and stress response, as well as the variety of new approaches tested
and biotechnologies discovered, have made the past 20 years of research and
development a very exciting period in the field of plant biotic and abiotic stress control.
It is during this time that we have seen the establishment, with all of its associated
controversy, of transgenic crops resistant to insects and chemicals, the cloning of
resistance genes and the exploitation of new breeding techniques, which together serve
as the basis for a gradual change from chemical- to biotechnological-based stress
control in agriculture. Success stories like the production and commercialization of Bt
plants, and today's availability of powerful molecular techniques, such as genomics and
proteomics, have elicited studies that pursue their final objective of increasing plant
stress resistance by investigating the plant stress response. Most of this research has
been performed on important food crops (potato, tomato, rice) or model plants
(arabidopsis and tobacco), while very little work has been done on ornamentals, the
latter having been used almost exclusively as a gene source. However, the stress control
methods which have been investigated or postulated to date are, in most cases, of
general significance and could be readily applied to ornamental plants in the near future.
In fact, in terms of commercial agriculture, non-food crops may prove to be an ideal
target for establishing transgenic biotechnologies involving theoretical safety issues that
cannot be fully addressed without large-scale testing. This chapter briefly reviews some
of the most interesting molecular approaches to controlling biotic and abiotic stresses,
with particular attention to the use of transgenes for increasing plant resistance.

2. Control of Biotic Stresses Caused by Fungi, Bacteria and Viruses

A variety of molecular approaches have been followed, with varying degrees of success,
to control diseases caused by microbes and viruses. Since the discovery that chitinases
and glucanases are produced in response to pathogen attack, one of the most tested
methods for improving resistance to fungi has been the overexpression of the plant
genes encoding these hydrolytic enzymes in a variety of crops (Schickler and Chet,
197
A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, 197-218.
© 2002 Kluwer Academic Publishers.
198

1997) (Table lA). Several successful cases have been reported in the literature, starting
with the work of Broglie et al. (1991), but a high level of resistance was usually
obtained only by combining the expression of different enzymes or other antimicrobial
proteins (Zhu et al. , 1994; Jach et al., 1995). More recently, the work ofLorito et al.
(1998), subsequently confirmed by other groups (Bolar et al., 2000), has provided
convincing evidence that microbial rather than plant chitinases are an effective tool for
establishing broad-range resistance to fungal diseases. This has induced several other
groups to use fungal chitinases for transforming a variety of plants, including tobacco,
tomato, apple, rice, wheat, lemon, broccoli, cabbage, and petunia (Fig. I).

Figure I. Resistance of transgenic petunia expressing the ech42 endochitinase-encoding

gene from Trichoderma harzianum, to the soil-borne pathogen Rhizoctonia so/ani. A =
transgenic lines, B = non-transfurmed or vector-transformed controls. For details see
Esposito et al., 2000 and Lorito et al. , 1998.

Although none of these new varieties has reached the market, the use of cell wall-
degrading enzymes as molecular weapons against fungi remains an attractive strategy
because of the safety and substrate selectivity of the transgenic protein (Lorito, 1997,
1998; Lorito and Scala, 1999). This approach has also been attempted with lysozymes
from various sources, including human, to achieve control of bacterial pathogens
(Nakajima et al., 1997). Since many plant chitinases also display lysozyme activity, the
attractive perspective is to reach a very broad spectrum of resistance against unrelated
microbes. Other transgenes encoding antimicrobial enzymes and tested with some
success are bacterial and yeast ribonucleases, which selectively degrade the fungal RNA
or the viral dsRNA genome (Strittmatter et al., 1995; Watanabe et al., 1995). In this
case, the choice of the right inducible promoter appears to be important for limiting the
expression of the transgene to the infection site.
A few genes encoding enzymes that are not directly inhibitory, but that generate
antifungal and antibacterial compounds, have been expressed in tobacco and potato
 199

(Table 1A). The two most interesting cases involve the transgenic expression of a
glucose oxidase from the fungal pathogen Aspergillus niger, and two phytoalexin
synthases from plant or fungi. High levels of glucose oxidase in the plant caused rapid
accumulation of reactive oxygen species, which are important factors in the defense
response to pathogens, and increased resistance to both fungi and bacteria (Wu eta/.,
1995). Equally innovative has been the transfer of genes encoding enzymes with a key
role in the synthetic pathways of the phytoalexins resveratrol and trichodiene from a
fungus or grape to tobacco, which produced progeny resistant to Botrytis cinerea and
other microbes (Hohn and Ohlrogge, 1991; Hain eta/., 1993). Finally, a recent attempt
was made to induce SAR against Phytophthora infestans in potato by transgenic
expression of an exogenous catalase (Yu eta/., 1999).
A popular approach has been the transfer to plants of genes encoding toxic, non-
enzymatic compounds, originating from plant, viral, bacterial, insect and even human
sources (Table 1A). Among the plant-derived toxins, there are the pathogenesis-related
(PR) proteins: osmotins and osmotin-like, ribosome-inactivating proteins (RIP), PR-1,
and various other antimicrobial compounds involved in the defense against fungi and
bacteria. The overexpression of osmotins produced partial resistance against the
Oomycetes Phytophthora and Pythium (Liu eta/., 1994; Li eta/., 1999), while the
barley and maize RIPs enhanced resistance against Rhizoctonia so/ani (Logemann eta/.,
1992; Jach eta/., 1995). Significant enhancement of the resistance to important fungal
(Fusarium and Alternaria) and bacterial (Pseudomonas) pathogens has also been
obtained by using genes encoding the so-called thionins and lipid-transfer proteins
(Carmona et a/., 1993; Epple et al., 1997), as well as other defense-involved toxic
peptides (named defensins, germin-like, etc.) transferred from radish, wheat and other
plants into tobacco, wheat, com and apple (Norelli eta/., 1996; Schweizer et al., 1999).
More recently, Schaffrath et a/. (2000) reduced rice susceptibility to Magnaporthe
grisea by overexpressing a protein, the mechanism of action of which is still unknown,
which accumulates extracellularly after pathogen attack. Further, there have been a
variety of antibacterial lytic peptides from insect, crustaceous and human sources which
have been expressed, with positive and negative effects on disease resistance, mainly in
potato and tobacco (Table lA). These include the well-known case of the cecropin-
encoding genes, plus a battery of other compounds capable of increasing plant
resistance to Pseudomonas spp. and Erwinia spp. (Huang eta/., 1997, see During, 1997,
and Amick Dempsey et a/., 1998, for reviews). In a couple of more "exotic"
approaches, the disease incidence of Pseudomonas syringae on tobacco was reduced by
expressing subunit A of the cholera toxin which blocks bacterial GTPase activity (Beffa
et a/., 1995), whereas the genes obtained from a mycovirus were able to increase
resistance against killer toxin-susceptible strains of the smut fungi Ustilago maydis and
Tilletia tritici in tabacco and wheat (Clausen et at., 2000). Obviously, the limiting
factors and most common causes of failure in using transgenically toxic peptides for
pathogen control are the undesirable effects of the gene product on the plant and,
possibly, on the consumer. Since ornamentals often contain many toxic substances per
se, these plants may be acceptably used as receptors of those potentially "health-
threatening" transgenes.
200

TABLE 1A Transgenes directly expressed in plants to enhance resistance against pathogens, by improving
the strength of the defense res~nse
Gene and product Source Activity I effect Transgenic Reference
plant

Antimicrobial
enzymes

Chitinase or Various plants, Degradation of fungal cell Tobacco Borkowska et a/.

glucanase including wall. Resistance to potato 1998; Broglie et
petunia Rhizoctonia so/ani, rice al., 1991; Orison
Cylindrosporium oilseed eta/., 1996;
concentricum, Phoma lingam, rape Schickler and
Sclerotinia sclerotiorum, carrot etc. Chet, 1997
Botrytis cinerea, Sclerotium
ro/fsii, Phytophthora infestans
Chitinase + Rice, alfalfa, Synergistic interaction. Tobacco Zhu eta/., 1994,
glucanase tobacco Resistance to Cercospora tomato
nicotianae, Fusarium
oxysporum f.sp. lycopersici
Chitinase and/or Barley Synergistic interaction. Tobacco Jach eta/., 1995
glucanase + RIP Resistance toR. so/ani,
Alternaria alternata, B.
cinerea
Chitinase (chiA) Serratia Antifungal enzyme. Tobacco Howie et al., 1994
marcescens Resistance to Alternaria
longipes and
R. so/ani
Endochitinase Trichoderma Strongly antifungal chitinase. Tobacco Bolar et a/., 2000;
(chit42) (chil) harzianum Resistance to A. alternata, A. potato Esposito et al.,
Rhizopus so/ani, B. cinerea, R. so/ani, apple 2000; Lorito et
oligosporus Venturia inequalis, petunia a/., 1998;
S. sclerotiorum grape Terakawa et al.,
etc. 1997
Lysozyme Human, egg Lytic activity on pathogen Tobacco During, 1996;
white, virus cells. Resistance to Erysiphe potato Nakajima et al.,
and other cichoracearum and 1997
sources Pseudomonas syringae pv.
tabaci, Erwinia carotovora
ssp. atroseptica
Ribonuclease Bacillus amy- Ribonuclease localized at Potato Strittmatter eta/.,
(bacterial bamase ), loliquefaciens infection site. Degradation of tobacco 1995; Watanabe
(yeast pacl) Schizosaccharo dsRN A Reduction of P. et al., 1995
mycespombe infestans sporulation.
Resistance to viruses
Enzymes
generating
antimicrobial
compounds

Glucose oxidase Aspergillus Generate reactive oxygen and Potato Wu eta/., 1995
niger HR. Broad-spectrum tomato
resistance to fungi and
bacteria (E. carotovora and P.
infestans)
 201

TABLE 1A Continued
Stilbene synthase Grapevine, Phytoalexin synthesis. Tobacco Hain et al., 1993;
(Vstl), trichodiene Fusarium spo- Resistance to B. cinerea, other Hohnand
synthase (Tri5) rotrichioides microbes and insects Ohlrogge, 1991
Catalase (Cat2St) Tobacco Induce expression of Potato Yuetal., 1999
endogenous catalase and elicit
SAR. Resistance toP.
infestans
Toxins and defense
proteins
Osmotin, osmotin- Tobacco Resistance toP. infestans, Potato Li et al., 1999;
like Pythium ultimum Liu et al., 1994
Ribosome- Barley, maize Resistance toR. solani Tobacco Jach et al., 1995;
inactivating Logemann et al.,
proteins (RIP) 1992
Thionins, lipid Arabidopsis, Resistance to F. oxysporum, Arabidop. Carmona et al.,
transfer -proteins, barley P. syringae and other bacteria. tobacco 1993; Epple et al.,
1997
Defensins (Rs- Radish, wheat, Antimicrobial peptides. Tobacco Norelli et al.,
AFP2) and germin- etc. Resistance to Alternaria com 1996; Schweizer
like proteins longipes, Erysiphe graminis, apple et al., 1999
other fungi and bacterial wheat
diseases
Rir1b Rice Extracellular defense protein. Rice Schaffiath et al.,
Resistance to Magnaporthe 2000
grisea
Cecropin (Shiva-1, Insects (silk Antibacterial lytic peptides. Potato Huang et al.,
SB-37. MB39), moth), crab, Resistance to Pseudomonas tobacco 1997, see Amick
attacin E, human solanacearum, P. syringae pv. apple Dempsey et al.,
lactoferrin tabaci, Erwinia amylovora, E. 1998 and During,
tachyplesin, etc. carotovora ssp. atroseptica 1997 for review
Killer toxin Ustilago maydis Toxic to closely related Tobacco Clausen et al.,
(mycovirus) Ustilago and Tilletia species. wheat 2000
Resistance to U. maydis and T.
tritici
Cholera toxin, Vibrio cholerae Antibacterial: blocks GTPase. Tobacco Beffa et al., 1995
subunit A Resistance toP. syringae

Probably the most exciting molecular-based strategies for enhancing disease

resistance against fungi and bacteria involve the use of the recently cloned avirulence
and resistance genes, to artificially induce programmed cell death at the site of infection
and/or activate a SAR status in the plant (Jones, 2001; Staskawicz, 2001) (Table lB).
Theories on how to use the Flor gene-for-gene concept to conveniently alter plant
sensitivity to pathogens have now been realized with the application of pathogen-
derived (avirulence) genes that allow transgenic plant lines to respond and resist races
and pathovars that are highly virulent on the wild type. The list of avirulence genes
applied transgenically to date include avr9 and inffrom Cladosporium fulvum and P.
infestans, respectively, and avrB, avrPto, avrRpt2 from Pseudomonas syringae (see
Table lB for references). In all these cases, elicitation of the plants' own defense system
and enhanced resistance to fungi, bacteria and/or viruses have been reported. Even more
202

TABLE lB. Transgenes directly expressed in plants to enhance resistance against pathogens by activating
elant defense responses
Transfonned
Gene and product Source Activity I effect Reference
plant

Elicitors,
avirulence proteins,
resistance genes
andHR/SAR
activating factors
Avirulence factor Cladosporium Elicit HR Resistance to Tomato, Kamoun et al.,
(avr9, infl) folvum, fungi and viruses tobacco 1999; Honee et
Phytophthora al., 1997;
infestans
Avirulence factor Pseudomonas Elicit genotype-specific Arabidopsis Gopalan et al.,
(avrB) (avrPto) syringae, P. HR or general defense tomato 1996; McNellis et
(avrRpt2) syringae pv. response. Resistance to al., 1998; Tobias
tomato bacteria and viruses etal., 1999
Resistance factor Tomato Signaling component of Tobacco see Amick
(Ptil) defense pathway. Enhance Dempsey et al.,
HR against Pseudomonas 1998 for review
carrying A vrPto
Resistance genes Tomato, rice, Activate defense response. Tobacco, Cao et al., 1998;
(Pto, PrJ, Xa21, arabidopsis, Resistance to powdery rice, Oldroyd and
RPWB, NPRJ, Cf9, pepper mildew pathogens, Arabidopsis Staskawicz, 1998;
Bs2) Pseudomonas syringae pv. pepper, Tai et al., 1999;
tabaci and pv. maculicola, tomato Thilmony et al.,
Xanthomonas oryzae pv. 1995; Wanget
oryzae, and other bacterial al., 1996; Xiao et
pathogens al.,2001

ElicitQr (3- P. cryptogea Elicit defense response. Potato, Keller et al.,

cryptogein Resistance to tobacco 1999; Tepfer et
Phytophthora parasitica, al., 1998
Thielaviopsis basicola,
Erysiphe cychoracearum,
Botrytis cinerea
Bacterio-opsin Halobacterium Activate defense response. Tobacco, Abad et al., 1997
proton pump (bO) halobium Resistance to viruses, P. potato
syringae pv. tabaci, P.
infestans
Allene oxide flax Promote jasmonic acid Tobacco Wang et al., 1999
synthase biosynthesis and enhance
expression of defense
enes
Enzymes that
produce elicitors or
aher plant-pathogen
signaling

Pectate lyase Erwinia Activate disease response Potato Wegener et al.,

(PL3) carotovora by releasing elicitors from 1996
plant. Resistance to E.
carotovora
 203

TABLE 1B. Continued

Chitinases Various Release elicitors from Various Lorita et al.,
glucanases sources pathogen or plant cell crops 1998; Schickler
walls. Resistance to fungi and Chet, 1997
and bacteria
Oligogalacturonide E. carotovora Degrade sugar oligomers Potato Weber et al.,
lyase into inactive products, 1994
interfere with signaling,
suppress pathogenicit)l of
E. carotovora

intriguing is the possibility of combining, in the same plant, pathogen-inducible

expression of both avirulence and resistance genes, as described by Joosten and de Wit
( 1999). Indeed, recently cloned resistance genes have been transferred from one plant to
another and functionally overexpressed, resulting in activation of the defense system
and reduced susceptibility to Pseudomonas, Xanthomonas and powdery mildew
pathogens (see Table lB for references). Interestingly, highly similar homologues of
some of these resistance genes have been found in petunia, snapdragon and other
ornamentals, and these may soon be tested as transgenes (Johal and Briggs, 1992). The
combined use of resistance and avirulence genes, the availability of more tightly
controlled inducible promoters, and a sorely needed understanding of the role and
mechanisms of interaction with the pathogen of the resistance gene products, should
fully reveal the potential of this approach for disease control.
Another intriguing but less explored strategy is based on modification of the
signaling pathways that regulate the defense response, by overproducing, for instance,
resistance factors capable of inducing the hypersensitive response (see Amick Dempsey
et al., 1998 for a review). Alternatively, a gene encoding a well-known elicitor from a
bacterial pathogen has been transferred to tobacco and potato, and has provided
significant disease resistance against four different species of fungal pathogens (Tepfer
et a/., 1998; Keller et al., 1999). Control of a large set of pathogens also seems
achievable by using a bacterial proton pump to activate local and systemic responses
against viruses, bacteria and fungi (Abad eta/., 1997). In a variation on this theme, the
plant's ability to "sense" the pathogen could be improved by applying transgenic
enzymes that release elicitors from plant (Wegener eta/., 1996) or pathogen (Lorito et
a/., 1998) tissues, as done with a pectate lyase or a mycoparasitic chitinase. In addition,
transgenic enzymes can break down molecules used by the pathogen to activate its own
genes (Weber et al., 1994), as demonstrated with an oligalatturonide lyase to reduce
Erwinia carotovora pathogenicity on potato. The possibilities of successfully applying
all of these HR/SAR-inducing genes are endless, and depend on the ability to control
the plant's reaction and to induce a response sufficiently effective and durable under
field conditions.
Less recently, plant susceptibility to Pseudomonas syringae was specifically reduced
in tobacco and bean by expressing genes derived from this same pathogen, which either
inactivate the main fungal virulence factor (the toxin tabtoxin) (Anzai eta!., 1989) or
accumulate a toxin-resistant version of the metabolic target of the pathogen toxin (De la
Puente-Martinez eta/., 1992). Similarly, genes such as the com resistance Hml, which
204

specifically degrades the Coch/iobolus carbonum HC toxin, could be transgenically

expressed to provide genetic resistance against this particular pathogen (Multani eta/.,
1998). Although the obvious limits of this approach lay in the specificity and narrow
range of the resistance obtained, it may represent an effective method to control
important pathogens that act via one or two well-known pathogenicity factors.

TABLE 1C. Transgenes directly expressed in plants to enhance resistance against viruses (only a few
si~ificant or recent cases are shown}

Gene and product Source Activity I effect Transgenic plant Reference

Coat protein eDNA Many viruses Inhibit assembly or other Tobacco potato see Baulcombe
(wild type or (> 15 taxa) mechanisms/phases of etc. 1996, Beachy,
mutated) the cycle. Resistance to 1997, 1999,
many viruses and Harms,
1992 for
review
Movement protein TMV Inhibit viral movement. Tobacco Cooper et a/.,
defective mutant Resistance to a variety of 1995
viruses
PLRVORF4 PLRV Mutated version of a Potato Tacke eta/.,
(pr17) movement protein. 1996
Enhanced resistance to
PLRV, PVX and PVY
Sense, antisense PVY Post-transcriptional gene Tobacco rice Waterhouse et
gene (Pro) silencing. Resistance to al., 1998
PVY
Ribosome Pokeweed Antiviral protein. Tobacco potato Wang eta/.,
inactivating protein Resistance to TMV, 1998
(PAP, PAPII) PVX, other viruses, and
R. so/ani
Resistance gene obacco potato Resistance to TMV and Tomato tobacco Bendahmane et
(N,Rx) PVX potato a/., 1999;
Whitham eta/.,
1996
Single-chain Fv Resistance to specific Tobacco Tavladoraki et
antibody (scFv) viruses a/., 1993
RNAse, Mammals RNA degrading system. Tobacco Mitra eta/.,
oligoadenylate Resistance to TMV, 1996
s~thetase CMV and other viruses

Transgenic plants of agronomic importance that are resistant to viruses are

becoming a reality. Modified lines of squash, papaya and other crops have already been
approved for commercial release or are in the final phase of development. Since the first
reports on the use of pathogen-derived genes to enhance virus resistance (Powel Abel et
a/., 1986), an increasing number of strategies have been investigated or suggested
(Table 1C), with the expression of viral coat proteins (CP) and the use of gene
silencing-based mechanisms remaining as the two main approaches. In contrast to gene
silencing, which is seen as very effective against specific viral strains, CP-mediated
resistance may affect a wider range of viruses. Today, CP genes from perhaps 40 or
more different viruses have been expressed, either as the wild-type or mutated (more
 205

effective) form, in a great variety of model plants or food crops (see Harms, 1992 and
Beachy, 1997, 1999 for reviews), but not ornamentals. Despite the solid evidence
accumulated in favor of this technology, the mechanisms that activate CP-mediated
resistance in plants are still debated and may involve, depending on the virus and the
host plant, blocking of the early infection phases, coating of the viral genome, as well as
replication, translation or assembly into virions of the viral RNA (Reimann-Philipp,
1998; Maki-Valkama eta/., 2000). Other methods, not based on gene silencing, to
increase resistance include expression of mutated viral movement proteins (MP), or
direct inhibition of the viral cycle by a transgene or its RNA transcript. In the first case,
resistance could theoretically be obtained against a wider range of viruses as compared
to other techniques (Cooper eta/., 1995; Hou eta/., 2000). In the second case, the
transgene or its RNA exerts a decoy action and produces a factor that interferes with the
expression of the viral genome (Baulcombe, 1996). There are several examples of virus-
derived resistance in which a post-transcriptional mechanism of gene silencing
operating at the RNA level appears to act by suppressing the accumulation of viral RNA
that has some sequence identity with the transgene (Jan et a/., 1999). A detailed
description of this homology-dependent gene silencing can be found in several
dedicated reviews (Baulcombe 1996, 1999; Smyth, 1999). The list of less-explored
pathogen-derived methods for transgenically increasing resistance to viruses includes
the expression of viral replicase genes or the entire viral genome, which apparently have
the potential to confer nearly full immunity to the source virus or related strains
(Baulcombe, 1996; Suzuki eta/., 1996; Beachy, 1997), and the use ofavirulence genes
cloned from fungi (Kamoun eta/., 1999; Tobias et al., 1999). Finally, other transgenic
strategies not involving the application of pathogen genes include: the use of protein
synthesis-inhibiting RIPs (Wang eta/., 1998) which may also provide cross-resistance
to fungal pathogens; plant resistance genes (i.e. the tobacco N gene or the potato Rx
gene) (Whitham et a/., 1996; Bendahmane et a/., 1999); monoclonal antibodies
(Tavladoraki et a/., 1993); and proteins that cause the degradation of the dsRNA
intermediates produced by most plant viruses (Mitra eta/., 1996).

3. Control of Biotic Stresses Caused by Insects and Nematodes

The insecticidal crystal proteins (ICP) produced by the soil Gram positive bacterium
Bacillus thuringiensis (= Bt), also known as 8-endotoxins or Bt toxins, have been
exploited for over 30 years to control insect and nematode pests. This large family of
proteins is encoded by the cry genes, which count, at present, over 130 members among
the various strains of the bacterium. According to the current classification (see
(http://www.epunix.biols.susx.ac. uk/Home/Neil_Crickmore/Bt/index.html for updated
nomenclature of cry genes), the cry genes are grouped into 32 primary groups. cry I and
cry9 groups encode proteins (Cry1 and Cry9, respectively) which are selectively toxic
on Lepidoptera, whereas Cry3, Cry7 and Cry 8 are toxic on Coleoptera; Cry2, Cry4,
CrylO, Cryll, Cry21 and Cyt proteins (the latter having hemolytic activity) are toxic to
Diptera, whereas Cry5, Cry12 and Cryl3 are nematocidal. Direct use of the Bt toxins as
a spray treatment has some drawbacks because of the photochemical instability of the
protein, high production costs and difficulties in reaching certain plant sites. This, and
the need to have insect-free crops over the whole growing season, prompted researchers
206

to engineer Cry-expressing plants producing Bt toxins (Table 2) (Wallimann, 2000). In

1995, potato varieties expressing a high level ofBt protein and resistant to the Colorado
potato beetle (Leptinotarsa decemlineata) were introduced for large-scale cultivation.
Since then, the acreage of plants expressing Bt toxin has steadily increased: Bt-
protected com, cotton, and potato were grown on approximately 10 million acres in
1997, 20 million acres in 1998, and 29 million acres in 1999. These cultivations were
protected from some of their major pests, such as the European com borer (Ostrinia
nubilalis), southwestern com borer, tobacco budworm (Heliothis virescens), cotton
bollworm (Helicoverpa zea), pink bollworm, and Colorado potato beetle, reducing the
need for spraying with conventional chemical pesticides (Betz eta/., 2000).
The development of resistance towards Bt toxins represents, at the moment, a major
threat to both companies producing Bt-engineered seeds and farmers. Strategies based
on the use of plants containing a high dosage of Bt toxin in combination with the
adoption of a "separate" refuge (i.e. an area cultivated with non-transgenic plants) has
proven to be effective in maintaining a low frequency of homozygotic resistant insects
within the field populations (Shelton eta/., 2000). Recently, a significant improvement
in the methods to overcome Bt toxin resistance has been described (Kota eta/., 1999).
The development of vectors able to target foreign gene integration as well as knowledge
of chloroplast genome and identification of its spacer regions, have allowed the
production of tobacco plants having chloroplasts transformed with a member of the cry2
gene family (cry24a2). Feeding several species of lepidopteran pests, including
resistant populations of tobacco budworm, cotton bollworm and beet armyworm
(Spodoptera littoralis) killed 100% of both sensitive and Bt-resistant populations. The
extension of Bt technology to other plants, including a variety of flowers and
ornamentals, could greatly reduce the use of chemical insecticides, especially for those
species, such as carnation, whose major insect pests (Cacoecimorpha pronubana and
Epichoristodes acerbella) are susceptible to the Bt toxin.
Other strategies to obtain insect-resistant plants have been based on the expression
of genes encoding inhibitors of insect digestive enzymes, primarily proteases and a.-
amylases (Table 2). Hilder eta/. (1987) expressed the gene encoding a cowpea trypsin
inhibitor in tobacco, enhancing resistance to the tobacco budworm. The same gene was
also used to engineer rice and strawberry plants resistant to Lepidoptera larvae (Graham
eta/., 1995; Xu eta/., 1996). Similarly, a barley trypsin inhibitor was used to enhance
the resistance of tobacco to Agrotis ipsilon, the beet armyworm (Spodoptera littoralis),
and of wheat against the grain moth (Sitotroga cerealella) (Altpeter et a/., 1999).
Inhibition of trypsin-like enzymes and subsequent nutrient deprivation can result in
reduced ovary development and oogenesis. A 10-residue peptide designated DEMOP
(=digestive enzyme modulating and oostatic peptide), causes inhibition oflarval growth
and increased mortality. Tobacco plants transformed with a synthetic DNA sequence
carrying multiple copies of the gene encoding DEMOP were markedly toxic to the
tobacco budworm and substantially reduced average larval weight (Tortiglione eta/.,
1999).
Inhibitors of a.-amylases are a widely diffuse class of small protein molecules which
have also been used to produce transgenic plants resistant to Lepidoptera and
Coleoptera larvae. For instance, expression of an a.-amylase inhibitor from wheat in
tobacco plants resulted in enhanced protection againstAgrotis ipsilon (Carbonero eta/.,
 207

1991). Pea and azuki seeds produced by plants expressing genes encoding selected a.-
amylase inhibitors from bean displayed resistance towards several bruchid beetles
(Shade et al., 1994; Ishimoto et al., 1996; Morton et al., 2000). A recent review reports
that at least 16 different genes encoding proteinase and a.-amylase inhibitors have been
introduced in 20 crop species to create or increase resistance towards insects (Schuler et
al., 1998).
The potential of chitinases as biopesticides relies on their direct interference with
insect growth. Normally, the endogenous production of these enzymes only occurs
during molting, and serves to degrade the peritrophic membrane. It has been postulated
that exposure of larvae to chitinases during non-molting periods can have a negative
effect on insect growth. In fact, constitutive expression of a gene encoding a chitinase
from tobacco hornworm (Manduca sexta) in tobacco plants yielded plants which are
less damaged by the budworm (Ding et al., 1998) (Table 2).
Besides Bt toxin and protease/amylase inhibitors, novel classes of proteins and
peptides are being evaluated for their utility as insect-resistance molecules (Masler et
al., 1993; Ffrench-Constant and Bowen, 2000). The sequences of several members of
peptide families that block ecdysone (growth-blocking peptides)juvenile hormone
synthesis (allatostatins) or gut motility (proctolin), or stimulate the activity of the
cardiac muscle (p-casomorphis) or visceral muscles (pyrokinins) have been determined
(Masler et a/., 1993). In general, very low concentrations of these compounds are
sufficient to exert their insecticidal activity, which seems not to be influenced by pH
(Petrilli et al., 1994). However, much more knowledge on the eventual side effects and
risks deriving from the use of neuropeptides and the expression in plants of their
encoding genes is needed, before they can become of practical use in insect pest control.

TABLE 2. Transgenes expressed in plant to enhance resistance against insect pests

Action I
Gene and product Source Transgenic plant Reference
mechanism

Bt crystal proteins Bacillus Formation of pores Tobacco, see Schuler et a/., 1998
(over 130 thuringiensis, in midgut epithelial tomato, for a review
members) B. popilliae cells soybean, corn,
cotton, etc.
Cowpea trypsin Cowpea Inhibition of Tobacco, rice, Hilder eta/., 1987;
inhibitor digestive enzymes strawberry Graham eta/., 1995;
Xu eta/., 1996
Barley trypsin Barley Inhibition of Tobacco, wheat Altpeter eta/. 1999
inhibitor digestive enzymes
DEMOP (see text) Inhibition of Tobacco Tortiglione eta/., 1999
digestive enzymes
a.-amylase Wheat, Inhibition of Tobacco and Carbonero eta/., 1991;
barley, bean, digestive enzymes other plants Shade et al., 1994;
etc. Morton et al., 2000
Chitinase Manduca Lysis of internal Tobacco Ding eta/., 1998
sexta chitin structures

For control of plant nematodes, besides the utilization of plants expressing some
classes of Cry proteins (see earlier), the expression of genes encoding some anti-
208

feedants has been tested transgenically. For instance, cystatin, expressed in arabidopsis
singly or in combination with a cowpea trypsin inhibitor, produced a high level of
resistance to both the beet cyst nematode (Heterodera schachtii) and root-knot
nematode (Meloidogyne incognita) (Urwin et al., 1997, 1998).

4. Control of Biotic Stresses Caused by Weeds (Plant Herbicide Resistance)

During the past decades, the use of crop-selective and non-selective herbicides has
become a worldwide cultural practice. Herbicides of the last generation (namely
glyphosate, phosphinothricin, the sulfonylureas chlorsolfuron and sulfometuron methyl,
and the imidazolinones) are non-selective molecules which interfere with the
biosynthesis of essential amino acids. The isolation of genes conferring resistance to the
mentioned herbicides has allowed production of genetically engineered plants with
improved or even complete resistance to these herbicides. A milestone achievement was
the generation, followed by commercial release in 1996, of soybean tolerant to the
herbicide glyphosate. These plants were obtained by transformation with a plant- or
bacterial-derived gene encoding EPSPS variants resistant to the action of glyphosate
(Shah et al., 1986; Della Cioppa eta/., 1987). Plants resistant to bialaphos, which is
cleaved in vivo to release its active derivative phosphinothricin or glufosinate
ammonium, have also been created. Successful production of tobacco, tomato and rice
plants tolerant to bialaphos has been achieved by transformation with a bacterial gene
encoding a tranferase able to detoxify phosphinothricin (Oard et al., 2000).
The sulfonylurea herbicides chlorsolfuron and sulfometuron methyl are potent
inhibitors of acetolactate synthase (ALS), an enzyme required for the biosynthesis of
branched amino acids (Chalef and Mauvais, 1984; Ray, 1984). In tobacco, two genes
encoding ALS versions insensitive to the herbicide have been identified (Chalef and
Ray, 1984) and used for engineering plants resistant to ALS inhibitors (Beetham eta/.,
1999). Imidazolinones are non-selective herbicides whose mode of action consists of
the inhibition of acetohxydroxyacid synthase (AHAS) another enzyme involved in
branched amino acid biosynthesis. In mutant maize cell lines showing a very high level
of tolerance to this herbicide, a mutation in the AHAS-encoding gene which conferred
resistance by reducing binding of the chemical, was identified (Satashivan eta/., 1991).
Maize plants resistant to the herbicides were engineered by using a newly developed
technique, based on self-complementary chimeric RNA/DNA oligonucleotides, to
obtain a targeted mutation at the AHAS locus (Zhu et a/, 2000). Applying the same
method, tobacco plants resistant to sulfonylurea herbicides have also been constructed
(Beetham eta/., 1999).
Transgenic research on enhancing plant resistance to traditional herbicides has also
yielded some important results. Expression of a bacterial monooxygenase gene from the
Alcaligenes eutrophus resulted in tobacco plants resistant to 2,4 dichlorophenoxyacetic
acid (Lyon et at., 1989). Expression of a yeast lanosterol-14-demethylase in tobacco
enhanced resistance to gamma ketotriazole (Grausem et al., 1995). Selectivity of the
dinitroaniline herbicides (trifluralin, ethalfluralin, oryzalin and pendimethalin) was
strongly improved by transforming tobacco with a mutant herbicide-resistant a./~
tubulin gene (Anthony et a/., 1999). Interestingly, the concerns about spreading
herbicide-resistance genes through pollen of engineered plants to wild weedy relatives
 209

have found an adequate answer in the application of chloroplast genome transformation

techniques, which result in herbicide-resistant plants whose pollen does not carry the
transgene (Daniell eta/., 1998).

5. Control of Abiotic Stresses

Much effort has been directed towards the identification and cloning of genes capable of
increasing tolerance to environmental stresses, such as drought, freezing, heat, salinity,
metal toxicity, hypoxia and UV-B radiation (Gelvin, 1998; Holmberg and Bulow, 1998;
Smimoff, 1998; Leone eta/., 1999; Nuccio eta/., 1999; Zhu, 1999, 2000; Cushman and
Bohnert, 2000). The partial elucidation of some biochemical pathways important for
stress resistance has supported the search for stress genes. For instance, having found
that specific compounds, such as glycine betaine, protect stressed cells from damage,
the gene encoding the enzyme that synthesizes this substance has been cloned and
transferred to plants (Holmstrom et al., 2000; Sakamoto and Murata, 2000). Important
genes have also been isolated through comparisons of gene expression profiles under
stressed and non-stressed conditions, as done for salt stress tolerance with halophytes
and glycophytes. Such comparative studies of gene expression have gotten a strong
push via the application of large-scale methods based on DNA microarray and gene
chips, which permit the analysis of thousands of DNA sequences of all kinds (genomic
clones, ESTs, ORFs, etc.). These global analyses of plant gene expression, together with
functional analyses based on complementation of stress-sensitive mutants or
overexpression/underexpression experiments, may be used to discover new genes with a
role in stress response. Some of the genes cloned from plants or other organisms, and
overexpressed in plants to improve resistance to abiotic stresses are reported in Table 3.
One or more of these genes are transferred under the control of a strong constitutive
promoter, and their products include: protective proteins such as LEA (late
embryogenesis abundant) which have been shown to accumulate in plants during seed
maturation or in response to environmental stresses (Dure, 1993); enzymes
(desaturases) that maintain membrane stability in response to low temperature by
increasing the degree of unsaturated fatty acids (Ishizaki-Nishizawa et a/., 1996;
Murukami et a/., 2000); proteins that accumulate during drought stress
(osmoprotectants) which seem to stabilize macromolecules and preserve the integrity of
the cells under stress (Nuccio et a/.,1999; see Table 3); enzymes that act as scavengers
of reactive oxygen species and protect against oxidative stresses (see Table 3 for
references). It should be noted that stress tolerance in the resulting transgenic plants has
only rarely been evaluated in field experiments, and negative pleiotropic effects due to
gene introgression in plants have not been adequately considered. So far, no commercial
transgenic cultivars resistant to abiotic stresses have been released, unlike cultivars with
resistance to some biotic stresses.
Since plants respond to abiotic stresses by activating complex mechanisms of
adaptation that involve numerous genes, it is possible, for instance, to achieve plant
protection through the expression of components of signaling pathways, genes for
stress-induced transcription factors, genes encoding products involved inABA-signaling
cascade, genes controlling cellular ion homeostasis, etc. (Luan, 1998; Snedden and
Fromm, 1998; Leone eta/., 1999; Hasegawa eta/., 2000). A number of genes encoding
210

TABLE 3. Genes used to engineer elants for resistance to abiotic stresses

Transgenic
Genes and product Source Tolerance Reference
plant

Protective proteins

LEA(HVAJ) Plant High salt Rice Xu eta/., 1996

Antifreeze (Ajp) Fish Low temperature Potato Wallis et a/.,1997

Protectant osmolytes

Glycine dehydroge- E. coli Salinity, Tobacco Holmstrom et a/.,

nases (betA) low temperature 2000
Choline dehydroge- E. coli Salinity, Tobacco Lilius et a/., 1996
nases (betA) low temperature
Choline oxidase (coda) Arthrobacter Light, salinity, Arabidopsis Alia et al., 1999
g/obiformis low temperature rice Sakamoto et a/.,
1998
Trehalose-6-phosphate Yeast Drought Potato Yeo eta/., 2000
synthase (TPSJ)
Pyrroline-5-carboxy- Plant Drought Tobacco Kavi-Kishor et
!ate synthetase (P5CS) al., 1995
D-ononitol (Imtl) Plant Salinity, drought Tobacco Sheveleva et al.,
1997
Mannitol1-P (MtlD) E. coli Salinity Tobacco Thomas et al.,
1995
Proline dehydrogenase Plant Salinity, Arabidopsis Nanjo eta/., 1999
(AtProDH) low temperature
Scavenging enzymes

Fe-superoxide Arabidopsis Low temperature Maize Van Breusegem et

dismutase (FeSOD) al., 1999
Glutamine synthetase Rice Salinity Rice Hoshida et al.,
(GS2) 2000

Aldose/aldehyde Alfalfa Drought, paraquat, Tobacco Oberschall et al.,

reductase heavy metals 2000
Mn-superoxide plant Oxidative stress Alfalfa McKersie et a/.,
dismutase (MnSOD) 1996
Glutathione transferase plant Oxidative stress Tobacco Roxas et al., 1997
glutathione peroxidase
(Gst!Gpx)
Enzymes that increase
membrane stability

Desaturases Arabidopsis Low temperature Tobacco Murukami et al.,

(fad7,fad8) 2000
L'.-9--desaturase (des9) A. nidulans Low temperature Tobacco Ishizaki-
Nishizawa et al.,
1996
 211

TABLE 3. Continued
Ca++ dependent
compounds
Calcineurin Yeast Salinity Tobacco Pardo et al.,
(CNAtr CNBJ) 1998
Kinase (OsCDPK7) Rice Salinity, drought, low Rice Saijo et al., 2000
temperature
Transcription factors
alfinl Alfalfa Salinity Alfalfa Winicov,2000
DREBJA Plant Salinity, drought, low Kasuga et al.,
temperature 1999
Ion tranporters
HALl Yeast Salinity Tomato Gisbert et al.,
melon 2000
Bordas et al.,
1997
Na+llr antiport Arabidopsis Salinity Arabidopsis Apse et al., 1999

enzymes involved in signal transduction have been identified. These include kinases
and phosphatases, transcription factors, ion transporters, calcium-dependent proteins,
etc.
Salt-tolerant tobacco plants have been obtained by expressing the activated form of
calcineurin, a Ca++/calmodulin-dependent protein phosphatase involved in the yeast salt
stress signal transduction pathway that regulates Na+ influx and efflux (Pardo eta/.,
1998). Transgenic rice plants with overexpressed levels of a calcium-dependent protein
kinase that is induced by cold and high salt, showed enhanced tolerance to these stresses
(Saijo eta/., 2000).
Salt tolerance has been achieved in alfalfa by overexpressing the transcription factor
Alfinl, a zinc finger protein that binds DNA in a sequence-specific manner and
increases expression of the salt-inducible gene MsPRP2 in roots (Winicov, 2000).
Another interesting use of transcription factors has been made by Kasuga eta/. (1999).
The dehydration response element (ORE) plays an important role in regulating gene
expression induced by drought, salt loading and freezing, while the transcription factor
DREBlA specifically binds to ORE and activates the expression of stress genes.
Transgenic overexpression of the gene encoding DREBlA induced the activation of
many stress genes and increased resistance to drought, salt and freezing. In another
approach, the possibility of increasing tolerance to salinity by compartmentalizing
sodium ions away from the cytosol was considered (Apse et a/., 1999) and the
overexpression of a vacuolar Na+m+ antiport has been applied to enhance salt tolerance
in Arabidopsis.
HALl, a yeast halotolerance gene expressed constitutively at high levels increased
internal K+ concentration and decreased intracellular Na+ concentration under high-salt
conditions. When introduced in tomato or melon, HALl improved salt tolerance in two
transgenic lines bearing one and four copies of this gene (Bordas eta/., 1997; Gisbert et
a/., 2000). The insertion and overexpression in tobacco of a glyoxalase-encoding gene
212

(Giy I) from Brassica juncea, which is upregulated in response to salt, water and heavy
metal stresses, also improved tolerance to these stresses (Veena et a/., 1999). An
intriguing strategy to obtain plant resistance to different abiotic stresses has been
followed by Nanjo eta/. (1999). Since proline levels in plants appear to be correlated to
environmental stress responses, antisense Arabidopsis plants were produced with the
AtProDH gene encoding a proline dehydrogenase (ProDH) that catalyzes proline
degradation. Several transgenic lines accumulated proline and showed increased
tolerance to freezing and salinity.

6. Acknowledgements

The authors are grateful to Sheri Woo and Anna Tuck for the critical revision of the
manuscript.

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MOLECULAR CONTROL OF LIGHT SENSING IN PLANT DEVELOPMENT

A. SAMACH, 1 M. PINEIR02
1Institute ofPlant Sciences and Genetics in Agriculture

The Hebrew University ofJerusalem

Faculty ofAgricultural, Food and Environmental Quality Sciences
P.O.Box 12, Rehovot 76100, Israel
2Centro Nacional de Biotecnologia

Campus de Ia Universidad Autonoma de Madrid

Cantoblanco, 28049 Madrid, Spain

1. Introduction

Light affects virtually all aspects of plant life. Plant growth and development are
adjusted to local, diurnal and seasonal changes in light quality and intensity. Among
other things, light can phase circadian rhythms, affect the formation, expansion and
content of leaves, change the rate and direction of stem elongation, and modulate
flowering time. Different plant species respond to similar light conditions in diverse
ways. Modifying light response in ornamental plants could prove extremely useful in
increasing variation and yield by changing both plant form and the timing of certain
processes such as flowering. During the past decade, molecular genetic approaches in
model plant systems have introduced us to an impressive array of potential molecular
tools for breeding. The aim of this chapter is to present examples of recent progress
made in revealing molecular components involved in light sensing in plants. The reader
will be introduced to a few light-controlled processes in plants, provided with examples
of molecular components involved in these processes-including the circadian clock,
and presented with current theories on the molecular mechanism of photoperiodic
control of flowering. Much of our current knowledge is based on many years of
excellent research performed by hundreds of researchers studying several plant species.
For lack of space and knowledge, this chapter will focus for the most part on recent
achievements in the model plant species Arabidopsis thaliana.

1.1. INTRODUCTION TO LIGHT-CONTROLLED PROCESSES IN PLANTS

Young seedlings of dicot plants germinated in the dark (skotomorphogenic) develop

rapidly elongating hypocotyls to push unopened cotyledons above the soil surface.
Upon exposure to light (photomorphogenesis), hypocotyl elongation is inhibited and the
cotyledons start to expand and to become photosynthetically competent. These
developmental changes are collectively referred to as de-etiolation. The orientation of
plant growth is controlled by two main factors: gravity and light. To ensure that leaves
receive optimal sunlight for photosynthesis, plants respond to local changes in the
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A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, 219-238.
© 2002 Kluwer Academic Publishers.
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direction of the light source by curving towards the light (phototropism). A plant needs
to assess the proximity of potentially competing neighbors in order to decide upon
changing its growth strategy. A shade-avoiding plant will elongate in a way that will
project its leaves into unshaded regions of daylight. If unsuccessful, the plant will try
and enhance the probability of survival by accelerated flowering and early production of
seeds. Shade avoidance is a strategy employed by most angiosperms, ranging from
small herbs to large trees. The switch from a vegetative to a reproductive phase
represents perhaps the most dramatic developmental change in the angiosperm life
cycle. Many plants are adapted to flowering at particular times of year, which ensures
optimal pollination and seed maturation. In those plants, flowering is controlled by
environmental signals that reflect the changing seasons, particularly day length and
temperature. The response to day length (photoperiodism) varies among and within
species growing at different latitudes. The ability to respond to photoperiod requires a
mechanism capable of measuring the duration of the dark and light periods during the
24-h cycle. The circadian clock controls daily rhythms and has been proposed to act as
the timer in photoperiodic responses.

2. Light Receptors

Plants detect light through photoreceptors that consist of a light-absorbing pigment, or

chromophore, bound to a protein effector molecule, or apoprotein. Absorption of light
by the chromophore induces a chemical or conformational change in the receptor
apoprotein that is transmitted to downstream signaling molecules. In plants, light
receptors have been divided into groups based on their action spectrum-red/far red
(RIFR) or blue.

2.1. PHYTOCHROMES

Phytochromes are RIFR-light-absorbing photoreceptors that mediate a variety of

cellular and developmental responses to light. Phytochromes are present in all plants
examined, as well as in some algae. The first clues regarding the role of RIFR-light-
absorbing photoreceptors in plant development came from studies initiated in the 1930s
on red-light-induced morphological responses (Flint, 1936). In 1952 it was suggested
that a single pigment could exist in two interconvertible forms, a red-light-absorbing
form (Pr) and a far-red-light-absorbing form (Pfr) (Borthwick eta/., 1952). The Pr form
can be converted to Pfr by exposing plants to red light, and the reverse reaction occurs
in far-red light. Light with a reduced RIFR ratio that mimics light reflected from
neighboring vegetation causes the conversion of phytochrome from the active Pfr form
to the inactive Pr form, and results in a shade-avoidance response. The phytochrome
molecule was identified as a unique chemical species in 1959 (Butler eta/., 1959). The
first phytochrome sequences cloned were from oat (Hershey et a/., 1985). In
Arabidopsis, five genes encode phytochromes (PHYA to PHYE; Sharrock and Quail,
1989; Quail et a/., 1995). Physiological studies of plants that either have mutations in
phytochrome genes or overexpress these genes have revealed that different family
members have distinct yet overlapping functions. Analysis of phyA mutants has
revealed that PHY A is the primary, if not the only, phytochrome responsible for de-
 221

etiolation in continuous far-red light (Dehesh et al., 1993; Nagatani eta/., 1993). On the
other hand, the PHYB gene encodes a protein that is the primary phytochrome
responsible for de-etiolation in response to red light and is a major contributor to the
shade-avoidance response of plants (Reed eta/., 1993; Whitelam and Devlin, 1998).

2.1.1. Structure
Phytochromes are water-soluble chromoproteins. They consist of a tetrapyrrole pigment
molecule (which traps light photons) covalently attached to a protein backbone
comprised of a polypeptide dimer. Each phytochrome polypeptide has two major
domains. The amino-terminal domain mediates light absorption and photo-reversibility
through the tetrapyrrole chromophore. The carboxy-terminal effector domain was
recently shown to have light-regulated serine-threonine kinase and autophosphorylation
activities. It is also important for dimerization and protein/protein interactions. Different
family members share considerable sequence similarity within the amino-terminal
chromophore-binding domain and greater sequence divergence at the carboxy-termini.
Despite the apparent divergence in physiological functions of PRYA and PHYB,
domain swap experiments demonstrate that the carboxy-terminal domains of different
phytochromes are fully functionally interchangeable. The biochemical mode of action
and immediate reaction partners of the many phytochrome gene family members,
therefore, are likely to be identical.

2.1.2. Mode ofAction

Phytochromes are synthesized in the dark in the Pr form. Upon exposure to red light,
they convert to the Pfr form. Since most phytochrome-mediated responses are induced
by red light, Pfr is thought to be the active form. Upon exposure to far-red light, Pfr
reverts back to the inactive Pr form. PHYB is the most abundant phytochrome in light-
grown plants. The PHYB Pfr form is highly stable, whereas the PRY A Pfr form
undergoes rapid degradation. Unlike other phytochromes, PRY A mediates far-red light
responses. Therefore, the Pr form of PRY A may be active. However, there is a paradox
in the ability of PRYA to mediate responses to far-red light: exposure to far-red will
induce the conversion of PRY A to the Pr form, the same form in which it was
synthesized in darkness. Since dark-grown seedlings do not show a constitutive light
response, the Pr form created from Pfr cannot be the same as that made in the dark. It
has been proposed recently that the Pr form generated upon exposure to far-red light is
somehow different from its newly synthesized counterpart. In fact, the
autophosphorylation activity of the Pr form is higher if it has cycled through the Pfr
form (Yeh and Lagarias, 1998). Therefore, this cycled Pr form may be the active form
of PRY A (Shinomura eta/., 2000).
Individual phytochromes may have at least two separate mechanisms of action: one
that rapidly and reversibly operates to modulate cellular ionic balances, and another that
results in selective expression of target genes. Phytochrome regulation of gene
expression might require interaction with specific partners that are directly involved in
gene regulation or, alternatively, act through multiple substrates, thereby regulating
gene expression differentially.

Phytochromes in Signal Transduction Events. Based on a series of pharmacological

studies (Neuhaus et a/. 1993; Bowler et a/. 1994), a biochemical model involving
222

distinct signal-transduction components was developed regarding PHYA signaling. The

proposed pathway involves heterotrimeric G proteins and downstream signaling
components divided into cyclic-GMP-dependent and calcium/calmodulin-dependent
pathways.

Light-Regulated Nuclear Translocation. Based on physiological, biochemical,

immunocytochemical and molecular analyses, phytochromes have long been considered
cytosolic light receptors. Light plays a major role in modulating transcription of many
genes. How are light-dependent changes in phytochrome communicated to the nucleus?
Recent studies using green fluorescent protein (GFP) fusions have proven that
phytochromes move to the nucleus (Sakamoto and Nagatani, 1996; Kircher eta/., 1999;
Yamaguchi et al., 1999). Both PHYA and PHYB proteins localize to the nucleus under
light conditions in which they mediate light responses, whereas they remain cytosolic in
the dark. This suggests that nuclear localization may be important for phytochrome
signaling.

Phytochromes as Kinases. A recent breakthrough in determining the mode of action of

phytochromes was the discovery of light-regulated kinase activity associated with
phytochrome (Yeh and Lagarias, 1998; Frankhauser et al., 1999). Analysis of
recombinant oat PHYA expressed in yeast demonstrated serine-threonine kinase activity
in vitro. Phosphorylation activity was dependent on proper chromophore assembly and
was stimulated by red light. This observation was supported by sequence analysis of the
carboxy-terminal domains of several higher plant phytochromes, which revealed
significant homology to histidine kinase transmitter modules, found on bacterial sensor
proteins. The possibility that plant phytochromes evolved from prokaryotic histidine
kinase two-component regulators gained credibility from a recently characterized
prokaryotic phytochrome from Synechocystis PCC6803 (Yeh et al., 1997). When
expressed in a heterologous yeast system, it demonstrated histidine kinase activity in
vitro. The activity was inducible by far-red light and reversible by red light, in contrast
to higher plants where red light is inductive.
A substrate of phytochrome was identified in a yeast two-hybrid screen and named
PHYTOCHROME KINASE SUBSTRATE 1 (PKSl; Frankhauser et al., 1999). PKSJ
encodes a novel protein detected in plants in the phosphorylated form, but red light or
PHYB overexpression increase the level of its phosphorylation in vivo. It was also
shown to be phosphorylated in a light-regulated fashion by oat PHYA in vitro. It
interacts with both PHYA and PHYB, and localizes to the cytoplasm. Since
overexpression ofPKSl results in reduced sensitivity to red light, it may be involved in
repressing phytochrome function. No phenotype was seen in plants with reduced levels
ofPKSl (using antisense).
There is evidence that the NUCLEOSIDE DIPHOSPHATE KINASE 2 (NDPK2;
Choi et al., 1999) gene is also involved in mediating phytochrome signaling. A
mutation in this gene causes a reduced response to both red and far-red light, including
cotyledon opening and greening. The activity of recombinant NDPK2 was increased
due to red-light-dependent phytochrome interaction in vitro. NDPK acts as a tumor-
suppressor in animal cells.
PIF3 Provides a Link Between Phytochromes and Gene Expression. Recent exciting
findings suggest that plants have evolved an extremely simple and efficient way of
 223

modifying gene expression in response to changes in light. A potential mechanism for

how light might affect transcription emerges from work on the PIF3 gene
(PHYTOCHROME-INTERACTING FACTOR 3; Ni eta/., 1998; 1999; Halliday eta/.,
1999; Martinez-Garcia et al., 2000; Zhu et al., 2000). PIF3 encodes a nuclear-localized
transcription factor containing a DNA-binding basic helix-loop-helix (blll.H) motif and
the protein-protein interaction PAS domain. The protein was first identified in a yeast-
two-hybrid screen for proteins that interact with the carboxy terminal domain of
phytochrome. Genetic evidence clearly suggests that it is involved in phytochrome
signaling responses. Antisense-imposed reductions in PIF3 mRNA levels cause reduced
photo-responsiveness, suggesting that PIF3 is necessary for normal PHYA and PHYB
signal transduction in the cell. An allele ofPIF3 causing overexpression of the gene was
identified independently in a genetic screen for Arabidopsis seedlings hypersensitive to
red light.
Red-light-dependent binding of phytochrome (with a ligated chromophore) to PIF3
not bound to DNA has been demonstrated in vitro. PIF3 binds specifically to a G-box
motif found in the promoters of several light-regulated genes. The resulting complex
formed by PIF3 and its target DNA is also susceptible to interaction with phytochromes:
phytochromes A and B were able to bind a G-box-bound PIF3, but only upon light-
triggered conversion of the photoreceptor to its biologically active conformation. In
addition, this light-dependent binding of phytochrome to PIF3 is reversible, since a
pulse of far-red light released the activated phytochrome from the complex. PHY B
bound with greater affinity than PHY A. The authors investigated whether PIF3 is
necessary for the phytochrome-regulated expression of light-activated genes using
antisense strategy. Of the genes examined, the induction of the clock-associated CCAJ
and LHY (section 3.2) genes was reduced in transgenic plants expressing PIF3 antisense
mRNA. Thus, these two genes are probably direct targets of the phytochrome-PIF3
pathway. The emerging picture is unexpectedly simple: Phytochromes perceive a light
stimulus, move into the nucleus, and interact with PIF3, which is bound to the G-box
motif of a light-activated gene, and that gene is switched on (Fig. 1).

2.1.3. A Genetic Approach to IdentifYing Downstream Components

Other recent work has aimed to identify downstream targets of phytochromes. Several
mutations lead to phenotypes similar to those caused by mutations in phytochrome
genes (Whitelam et al., 1993; Ahmad and Cashmore, 1996; Barnes et al., 1996; Lin and
Cheng, 1997; Wagner eta/., 1997; Soh eta/., 1998; Hudson et al., 1999) or confer
hypersensitive red and/or far-red light responses (Genoud eta/., 1998; Hoecker eta/.,
1998, 1999). These mutations may affect genes encoding immediate targets of
phytochrome action or downstream regulators of phytochrome signaling. For example,
genetic screens for downstream components in the PHYA signaling pathway identified
the HFRJ (also known as REPJ) locus (Fairchild eta/., 2000; Soh et al., 2000). HFRJ
encodes a nuclear protein containing a bHLH motif. Phenotypic analysis revealed that
the hfr 1 mutation abrogated only a subset of PHY A-mediated responses, such as
inhibition of hypocotyls elongation, gravitropic modulation of hypocotyl growth, and
late, sustained induction of a light-induced gene. HFRl could not be shown to interact
directly with phytochromes, but did heterodimerize with PIF3. This result suggests that
a network of bHLH proteins is involved in the regulation of plant development by
phytochromes.
224

Nucleus

Far C to lasm
Red
Fig. I. A schematic diagram depicting a possible mode of phytochrome action.
Upon red-light radiation, the Pfr torm of Phytochrome B enters the nucleus. The Pfr form
can interact with CRY2 in the nucleus or with PIF3, which is bound to the G-box motif of
a light-activated gene, such as LHY, causing the gene to be switched on.

2.2. CRYPTOCHROMES

2.2. 1. Introduction to Blue-Light Receptors

Since the time of Darwin we have been aware that plants respond specifically to blue
light and that distinct blue-light-absorbing photoreceptors must exist. Phototropism,
briefly described in section l.l, is elicited preferentially or exclusively by blue light
(wavelengths lower than 500 nm) and not by red light, eliminating phytochrome as a
possible photoreceptor. Other blue-light responses include de-etiolation, regulation of
flowering time, circadian clock entrainment and regulation of gene expression.
Molecular genetic studies using Arabidopsis as a model system have identified three
blue-light receptors. Unlike phytochromes, these receptors appear to be derived rrom
more than one evolutionary lineage. The first of these, cryptochromes, are described in
this section, and the other, NPHl, is described in section 2.3.

2.2.2. CRYPTOCHROME Genes in Arabidopsis

The long sought-after blue-light photoreceptor was first cloned in Arabidopsis (Ahmad
and Cashmore, 1993). The hy4 mutation, which lacks blue-light-stimulated inhibition of
hypocotyl elongation, turned out to be a mutation in this photoreceptor, designated
CRYPTOCHROMEJ (CRYJ). The hy4 mutant is not completely blind to blue light,
suggesting the existence of additional blue-light photoreceptors. A second
cryptochrome, with considerable homology to CRYl, was identified and named CRY2.
Mutations in the CRY2 gene were shown to be allelic to thejha mutations, which cause
late-flowering under long days.
 225

2.2.3. Structure
Plant cryptochromes contain a conserved amino-terminal chromophore-binding domain.
This domain shares significant homology to DNA PHOTOLY ASES, microbial proteins
that catalyze the blue-light-dependent repair of UV-damaged DNA. Recombinant
CRYl protein purified from an E. coli expression system bound two blue-light-
absorbing chromophores, a flavin and a pterin. The carboxy-terminal domains ofCRY1
and CRY2 show almost no sequence relatedness although they seem to provide similar
functions. Domain swap experiments showed that all of the domains of CRY 1 and
CRY2 proteins are functionally interchangeable in chimeric photoreceptors (Ahmad et
a/., 1998a). This would indicate that the reaction partners of both CRY1 and CRY2
photoreceptors may also be identical. Genes encoding cryptochromes have been
identified in numerous plant species,including ferns and algae. All cryptochromes share
considerable sequence similarity in their chromophore-binding domains but the
carboxy-terminal domain is divergent.

2.2.4. Function
Several studies have provided insight into Arabidopsis cryptochrome function (Ahmad
and Cashmore, 1993; Lin eta/., 1995a, 1995b, 1996, 1998; Guo eta/., 1998, 1999;
Cashmore eta/., 1999; Kleiner et al., 1999; Mas eta/., 2000). CRYJ encodes a soluble
protein expressed at similar levels in dark- and light-grown seedlings. Loss-of-function
mutants show reduced sensitivity to blue light and transgenic gain-of-function mutants
confer hypersensitivity to blue light. CRY2 overexpression studies in transgenic plants,
as well as the identification of null-mutant alleles in CRY2, revealed that CRY2 function
is essentially redundant to that of CRYl. Unlike CRY1, CRY2 protein is rapidly
degraded in seedlings upon irradiation by wavelengths of light (UV, blue, green) that
activate this receptor. It is conceivable that the absorption of blue light by CRY2
changes its conformation and triggers its own degradation. Thus, CRY2 protein does
not accumulate to high levels in bright white light and may, therefore, play less of a role
in many high-irradiance responses. Similar to phytochromes, CRY1 and CRY2 enter the
nucleus, although no light-regulated transport to the nucleus has been reported.

2.3. PHOTOTROPIN NPH1

A blue-light photoreceptor involved in phototropism has recently been cloned and

characterized (Khurana and Poff, 1989; Reymond et a/., 1992; Liscum and Briggs,
1995; Huala eta/., 1997; Christie eta/., 1998, 1999; Nozue eta/., 1998; Baum eta/.,
1999; Motchoulski and Liscum, 1999; Salomon et a/., 2000). A screen for
phototropism-deficient mutants in Arabidopsis identified several loci named non-
phototropic hypocotyl (nph). One of the genes, NPHJ, has all the characteristics of the
blue-light receptor for phototropism, and therefore was named PHOTOTROPIN. NPHJ
encodes a 116-kDa flavoprotein previously shown to be associated with the plasma
membrane. NPH1 expressed and purified from a baculovirus system is a soluble protein
that binds the chromophore flavin mononucleotide (FMN) via two LOY (for light,
oxygen and voltage) domains. It contains a serine-threonine-kinase domain at the
carboxy-terminal (falling into the PVPK1 family within the protein kinase C group). It
has been shown to undergo blue-light-dependent auto-phosphorylation within minutes
after irradiation of etiolated seedlings in vivo or microsomal extracts in vitro.
226

2.4. INTERACTIONS AMONG PHOTORECEPTORS

Having at least two distinct sets of photoreceptors that respond preferentially to red and
blue light enables the plant to maximize its response throughout the visible spectrum.
Do these different visual pathways function independently of each other, or is there
communication between them? There is growing evidence suggesting the existence of
interactions among different photoreceptors (Ahmad and Cashmore, 1997; Neff and
Chory, 1998). In vitro phosphorylation experiments with purified recombinant
photoreceptors indeed showed that CRYl serves as a substrate for phytochrome-
dependent kinase activity (Ahmad eta/., 1998b). Recently, CRY2 was shown to interact
in planta with PHYB within nuclear speckles (Mas et a/., 2000). The authors suggested
that these speckles represent "transcriptosomes": sites of RNA transcription and
processing. There is evidence that during evolution chimeric photoreceptors were also
developed. A fern gene (PHY3) with strong sequence homology to the entire NPHJ
gene contains an additional amino-terminal domain with sequence similarity to the
chromophore-binding domain of phytochrome. The PHY3 protein expressed and
purified from yeast showed the typical phytochrome photochromic reaction, as assayed
by the RIFR differential spectrum (Nozue eta/., 1998). The LOY domain of PHY3
expressed in E. coli bound to the chromophore FMN. Therefore, fern PHY3 might be a
dual photoreceptor that can mediate RIFR-light and blue-light responses.

3. The Circadian Clock

The relative position of the sun subjects all inhabitants of our planet to a 24-h cycle of
variation in light intensity. The behavior of plants and animals is often synchronized
with this daily cycle of light and dark, for example, overt rhythms in plant leaf
movement or human sleep habits. These rhythms in behavior are a consequence of
rhythms in gene expression. Features of such rhythms are that the duration of one cycle
is approximately 24 h, that they are entrained to (or synchronized with) the day/night
cycle by environmental rhythms in light/dark or in temperature, and that the rhythm
persists when organisms are shifted from light/dark cycles to continuous conditions of
light or dark. These circadian rhythms are driven by an endogenous biological clock.
The foundation of any circadian clock, the central oscillator, is expected to be a self-
sustaining, 24-h rhythm in the expression of certain pacemaker genes. Input pathways to
the oscillator synchronize the oscillation to the day/night cycle and outputs from the
oscillator are overt rhythms in gene expression and behavior.
Output pathways in Arabidopsis control the expression of a wide range of clock-
controlled genes that peak in expression in different phases (Harmer eta/., 2000). These
pathways also control overt rhythms in leaf movement, stomatal opening, hypocotyl
elongation and the photoperiodic control of flowering (Millar and Kay, 1991; Hicks et
a/., 1996; Dowson-Day and Millar, 1999; Samach and Coupland, 2000).

3.1. PHOTORECEPTORS IN CIRCADIAN CLOCK ENTRAINMENT

Light signals affect the pace of the clock by activating different photoreceptors.
Circadian output in Arabidopsis is often followed using a fusion of the promoter of the
 227

circadian clock-controlled gene CAB2 to the LUCIFERASE marker gene (CAB:LUC).

In response to increasing light intensity, the period length of CAB.·LUC expression
decreases. The role of individual receptors in entrainment of the circadian clock has
been investigated by analyzing the effect of mutations on circadian output (Millar eta/.,
1995a; Anderson eta/., 1997; Somerset a/., 1998a). Mutations in any of the four light
receptors: CRY1, CRY2, PHYA andPHYB, influenced circadian clock entrainment under
particular conditions. A quadruple mutant lacking all four of these photoreceptors still
retained robust circadian rhythmicity measured through leaf movement (Yanovsky et
al., 2000). CRYl and PHYB were the most important light receptors in circadian clock
entrainment under high-fluence conditions: PHYA and CRY2 had no effect on clock
entrainment. However, period changes were observed under specialized low-fluence
conditions. These results indicate that cryptochromes and phytochromes are required for
light entrainment but are not essential components of the central oscillator in
Arabidopsis. PHYB, CRY1, CRY2 and NPH1 transcripts are also regulated by the
circadian clock (Kozma-Bognar eta/., 1999; Harmer eta/., 2000). Since phytochromes
and cryptochromes mediate light input to the clock and are also output components
(clock regulation of transcript), they create an outer feedback loop. Plants might possess
additional photoreceptors specifically involved in photo-entrainment. The ZTL 1 and
FKFl proteins mentioned further on could provide such a function.

3.2. GENES REQUIRED FOR NORMAL CLOCK FUNCTION

Several loss-of-function mutations and overexpression alleles that disrupt circadian

regulation have been described inArabidopsis. The phenotypes ofloss-of-function early
flowering 3 (elj3) mutants suggest that ELF3 is required to maintain circadian output
under certain conditions (Hicks eta/., 1996). e/j3 mutant plants entrained in light/dark
cycles and moved to continuous white light showed arrhythmic expression of circadian-
regulated genes as well as arrhythmic leaf movements (Hicks eta/., 1996; Schaffer et
a/., 1998 Dowson-Day and Millar, 1999). However, in constant darkness e/j3-1 plants
retained a circadian rhythm, and it was proposed that ELF3 mediates an interaction
between light and the circadian clock, rather than being a component of the clock itself.
The genes LATE ELONGATED HYPOCOTYL (LHY; Schaffer eta/., 1998) and
CIRCADIAN CLOCK ASSOCIATED 1 (CCA1; Wang and Tobin, 1998) encode very
similar proteins containing a single DNA-binding MYB repeat. Indeed, the CCA1
protein binds to specific DNA sequences (Wang eta/., 1997). The mRNAs encoded by
both genes show a daily rhythm of expression in light/dark cycles with peak levels at
dawn, and a similar pattern of expression has been shown for the CCA 1 protein. When
moved to constant light conditions plants express both genes in a circadian rhythm, and
LHY clearly maintains a circadian rhythm of expression in constant darkness. Circadian
patterns of expression of LHY and CCA 1 may be required to maintain all circadian
outputs. Constant high levels of either transcript in transgenic plants caused disruption
of all rhythmic outputs examined, both in constant light and in constant dark.
Furthermore, overexpression of either gene caused a loss of rhythmicity (with
expression at close to trough levels) of their own and each other's transcript (Schaffer et
a/., 1998; Wang and Tobin, 1998; Fowler eta/., 1999). A CCA1loss-of-function mutant
demonstrated that CCA 1 is required for circadian clock regulation with a normal period
(Green and Tobin, 1999). The cca1 knockout allele may have a less severe effect on
228

circadian-clock control than CCA 1 overexpression because of the presence of genes

such as LHY that may be able to partially compensate for the loss of CCAI function.
The CKB3 regulatory subunit of the protein kinase CK2 can interact with and
phosphorylate CCAI in vitro, and overexpressing CK2 shortens period (Sugano eta/.,
1998, 1999). Interaction with CKB3 stimulated the binding of recombinant CCAI to
DNA in vitro, but phosphorylation did not. Physiologically, the CCAI-CK2 interaction
might either permit a kinase-substrate interaction or sequester one of the partners.
The semi-dominant timing of CAB expression 1 (TOC1) mutant was recovered in a
screen for period-length mutants. In continuous light or dark, the semi-dominant toc1-1
mutant has a shorter period for several markers (Millar eta/., 1995b; Somerset a/.,
1998b; Strayer et a/., 2000). The predicted TOCI protein contains several notable
features. It has a carboxy-terminal CCT motif common to CONSTANS protein (see
further on), a receiver-like domain at the amino-terminus, and two direct 47-amino-acid
repeats in the center of the protein. TOC1 mRNA levels cycle robustly in light/dark
cycles. This cycling persists in the absence of entraining signals (continuous light).
The genes FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1; Nelson eta/.,
2000) and ZEITLUPE (ZTLJ; Somerset a/., 2000; also known as LKP1; Kiyosue and
Wada, 2000) encode proteins containing an amino-terminal PAS motif similar to the
flavin-binding LOV domains of NPH 1 (see earlier). In addition, these proteins contain
an F-box motif found in targeting ubiquitin-mediated degradation, and six tandem kelch
motifs predicted to mediate protein-protein interactions. Anfkflloss-of-function mutant
is late-flowering in long days (but not photoperiod-insensitive), and has a slightly
hypersensitive de-etiolation response at certain wavelengths. In continuous light, two
markers show a slight change in circadian expression in this mutant. The FKF1
transcript is diurnally regulated and this oscillation persists in continuous light. The zt/J-
1 and ztll-2 alleles (probably not complete loss of function) cause alterations in a wide
range of clock-controlled processes (long period, 3-4 h phase delay). Mutants have a
hypersensitive de-etiolation response under red light. Unlike FKFI, light/dark cycles or
the circadian clock do not affect the ZTLJ transcript. The increase in period length in
the mutant is more pronounced at low fluence rates. The fact that fluence rate affects the
degree of period disruption suggests that similar to the phytochromes and
cryptochromes, ZTLI is placed within a light signaling pathway to the clock, while
TOC 1 is either at the terminus of a light input chain or within the central oscillator
itself.
The GIGANTEA (GI) gene encodes a very large protein with no familiar domains
(Fowler eta/., 1999; Park eta/., 1999). Although computer-based predictions suggested
that GI would be an integral plasma-membrane-localized polypeptide, the protein has
been shown to reside in the nucleus (Huq eta/., 2000). The amplitude of several clock-
regulated genes is reduced in a gi mutant. The gi mutant also affects light signaling to
the clock. GI mutants show reduced sensitivity to red light, as seen in reduced inhibition
of hypocotyl elongation (Huq eta/., 2000).

4. The Shade Avoidance Response

The shade-avoidance response, described in the introduction, requires a means of

perceiving changes in light quality. Overall reduction in light intensity caused by
 229

shading is not a good estimate of shade caused by plants. Daylight contains roughly
equal proportions of red and far-red light (R:FR 1.2). Phytochromes are used as
sensitive estimators of the spectral changes that occur within plant communities when
daylight interacts with photosynthetic structures. Photosynthetic pigments absorb red
light, causing a change in the ratio towards the far-red. The absorption maximum of the
phytochrome Pr form is close to that of the chlorophylls (red light), but the Pfr form
absorbs at a longer wavelength (far-red light). Genetic analysis suggests that
phytochromes B, D and E act collectively in mediating between plant proximity and the
shade-avoidance response.

5. Photoperiodism

In annuals-plants that flower only once-induction of flowering represents not only

the onset of reproduction but also the start of senescence. Many of these plants are
adapted to flowering at particularly favorable times of the year to ensure optimal
pollination and seed maturation. In these plants, flowering is controlled by
environmental signals that reflect the changing seasons, particularly day length and
temperature. The response to day length varies, so that plants isolated at higher latitudes
tend to flower in response to the long days of spring and summer, while plants from
lower latitudes avoid the extreme heat of the summer by responding to short days.
Gamer and Allard (1920) first proposed a role for day length in controlling the initiation
of flowering. Since then, extensive research in many plant species has led to detailed
physiological models regarding photoperiodism (reviewed in Thomas and Vince-Prue,
1997). Arabidopsis plants grown under long day (LD) conditions flower earlier and with
fewer leaves than those grown under short days (SDs). A response to LDs can be
detected soon after germination; seedlings grown in LDs and shifted to SDs when they
are 8 to 10 days old will flower at around the same time as plants grown continuously
under LDs. Exposure to a single LD can be sufficient to induce flowering in older
Arabidopsis plants. Short exposures to light in an otherwise non-inductive long dark
period can also, under certain conditions, promote flowering ofArabidopsis (Brown and
Klein, 1971; Goto eta/., 1991; Hempel and Feldman, 1995; Mozley and Thomas, 1995;
Corbesier et al., 1996; Hempel et al., 1998).

5.1. THE CIRCADIAN CLOCK ACTS AS THE TIMER IN DAY-LENGTH

MEASUREMENT

The photoperiodic control of flowering requires a mechanism which can measure the
relative lengths of the light and dark periods. The circadian clock has been implicated as
this timer since 1936 (Bunning, 1936; Hamner, 1960; Thomas and Vince-Prue, 1997).
Recent advances in Arabi dopsis have revealed the central role of the circadian clock in
photoperiodic control of flowering. Most mutations that perturb clock function result in
photoperiodic phenotypes. Indeed, most of the genes described in section 3.2 were
originally identified as genes involved in flowering time. elj3 mutants are early-
flowering and the phenotype is stronger under SD conditions (Zagotta et al., 1996).
Constant overexpression of the LATE ELONGATED HYPOCOTYL (LHY) (Schaffer et
al., 1998) and CCAJ (Wang and Tobin, 1998) genes caused late flowering under LDs,
230

and LHY overexpression caused plants to become day-length-insensitive with respect to

flowering time. The photoperiodic response of the semi-dominant timing of CAB
expression 1 (TOCJ) mutant is perturbed in such a way that plants flower much earlier
under non-inductive SD conditions. Correct photoperiod response was restored in tocl-
1 plants grown in a light/dark regime that matched its 21-h internal clock (Strayer eta/.,
2000). An fk/1 loss-of-function mutant is late-flowering under LDs (but not
photoperiod-insensitive), and has a slightly hypersensitive de-etiolation response at
certain wavelengths. The ztll-1 and ztll-2 alleles (probably not complete loss of
function) are also late under LDs (Somers et al., 2000). ZTLJ overexpression caused
late-flowering under LDs as well (Kiyosue and Wada, 2000). A gi loss-of-function
mutant is late flowering under long days (Koornneef et al., 1991). It has been suggested
that GI participates in a feedback loop of the plant circadian system (Park eta/., 1999),
in the PHYB-signaling pathway and in photoperiodic control of flowering. While it is
not yet clear how GI is involved in all these processes, it seems that the delay in
flowering time observed in gi mutants could be mediated by a reduction in the
amplitude of cycling of clock-regulated genes (see earlier).

5.2. A ROLE FOR PHOTORECEPTORS IN PHOTOPERIODIC CONTROL OF

FLOWERING

Mutations in genes encoding photoreceptors have revealed the involvement of these

molecules in the regulation of flowering time. Genetically, phytochrome B acts as a
floral repressor (Halliday et a/., 1994; Mockler et a/., 1999) since loss-of-function
mutants are early flowering. Other phytochromes, such as PHYE, also play a role in the
control of flowering (Devlin eta/., 1998). Yet CRY2 and PHYA seem to be specifically
required for LD promotion of flowering in Arabidopsis. Loss-of-function alleles of
CRY2 (fha) have no flowering-time phenotype under SDs and are late-flowering under
LDs (Mozley and Thomas, 1995), although they still respond slightly to changes in
photoperiod (Bagnall et al., 1996). The flowering phenotype is more pronounced in
some ecotypes (e.g. Columbia) than others (e.g. Landsberg erecta). When grown in
constant light, the late-flowering phenotype of cry2 was no longer apparent under a low
R:FR ratio. This suggests that the cry2 phenotype is dependent on the active Pfr form of
phytochrome (Mas eta/., 2000). However, under certain light conditions, CRY2 is only
partially dependent on PHYB to promote flowering. Since the flowering time of the
double mutant cry1cry2 is later than either single parent, it has been proposed that the
PHYB-independent CRY2 activity is redundant with that of CRY I. On the other hand,
transgenic plants overexpressing CRY2 flowered slightly early under SDs but not under
LDs. Therefore, both the mutation and the overexpression of the CRY2 gene caused
reduced sensitivity to photoperiods.
The phyA-1 mutant is late-flowering under extended SD conditions and exhibits a
reduced response to night-break treatment (Johnson eta/., 1994; Reed eta/., 1994).
Overexpression of PHYA caused early flowering under SDs and photoperiod
insensitivity. As with cry2, the effect of phyA on photoperiod response is probably not
mediated by an effect on the input to the clock; it is more likely a result of an
independent PHYA signaling pathway (Somerset al., 1998). It is interesting that both
PHYA and CRY2 are light-labile: the absence of either causes late flowering under
 231

LDs, and neither seems to be required for normal period length of the circadian clock
under conditions of high irradiance.

5.3. PHOTOPERIODIC CONTROL OF FLOWERING IN ARABIDOPSIS IS

MEDIATED BY CONSTANS

The CONSTANS (CO) gene (Putterill eta/., 1995) encodes a nuclear-localized B-box
protein with a conserved carboxy-terminal region also found in the clock-associated
TOC1 protein. Mutations in CO delay flowering under LDs but have only slight or no
effect under SDs (Koornneef et a/., 1991). Transgenic plants in which CO is
overexpressed are severely early flowering, under both SDs and LDs, and the plants are
insensitive to day length (Simon et a/., 1996; Onouchi et al., 2000). Similar to
previously described clock-associated genes, the abundance of CO transcript is
circadian-regulated and the peak pattern of CO expression is affected by day length
(Suarez-Lopez eta/., 2001). Still, the lack of any clock-oriented phenotypes in the loss-
of-function mutant, aside from photoperiodic control of flowering, suggests that
CONSTANS is not involved in clock function. More likely CONSTANS mediates the
flowering response to day length. Recent evidence shows that the CO product is directly
involved in transcriptional regulation of flower-promoting genes (Samach et a/., 2000).
These are FLOWERING LOCUS T (FI) and SUPPRESSOR OF OVEREXPRESSJON
OF CO 1 (SOCJ). TheFT gene encodes a protein with similarity to the Raf kinase
inhibitor protein (Kardailsky et al., 1999; Kobayashi eta/., 2000). Several other genes
in Arabidopsis and other plants code for proteins similar to FT. The SOCJ gene (also
known asAGL20) encodes a MADS-box transcription factor. Loss-of-function mutants
in either gene are late-flowering. socl mutants flower late under the influence of both
long and short days, whereas ft mutants flower late in response to LDs. Overexpression
of either gene causes very early flowering (Kardailsky et al., 1999; Bonhomme eta/.,
2000; Kobayashi eta!., 2000; Lee eta/., 2000; Samach et al., 2000).

5.4. REPRESSION OF FLOWERING MEDIATED BY FLOWERING LOCUS C

In certain plant species and in specific Arabidopsis genetic backgrounds, flowering is

promoted by preliminary exposure to extended periods of low temperature
(vernalization). This response can be considered a form of biological timer that ensures
that flowering is repressed until the end of the winter. Vernalization has been analyzed
genetically in Arabidopsis by comparing naturally occurring varieties that either do or
do not respond to low-temperature treatments (Koornneef eta/., 1991). Those that show
a vernalization response flower late if they are not exposed to low temperatures, but
flower much earlier if given a low-temperature treatment. In Arabidopsis, the
requirement for vernalization depends on the locus FRJGIDA (FRJ; Johanson et al.,
2000). Arabidopsis ecotypes containing active alleles of FRI require vernalization
treatments for early flowering. These active alleles function by enhancing the
expression of a flowering repressor encoded by the FLOWERING LOCUS C (FLC; also
known as FLF) gene (Sanda and Amasino, 1996; Michaels and Amasino, 1999;
Sheldon et a/., 1999, 2000). FLC also codes for a MADS-box transcription factor.
Vernalization treatment reduces the expression of FLC, and this is correlated with early
flowering. Constitutive overexpression of FLC, however, renders late-flowering plants
232

unable to respond to chilling treatment (see Michaels and Amasino, 2000 for a review).
Surprisingly, mutations in FLC shorten the circadian period by 1 h (at most). This might
suggest a feedback mechanism between flowering-time genes and the circadian clock
(Swamp et al., 1999).

5.5 Ff AND SOCJ INTEGRATE DIVERSE FLORAL STIMULI TO REGULATE

FLOWERING TIME

SOCJ and Ff transcript levels are regulated by photoperiod via CONSTANS activity.
But the levels of expression of both genes also increase with age, independently of
photoperiod (Samach et a/., 2000). In addition, both genes are common components of
other flowering pathways as well: there is growing evidence suggesting that FLC
represses Ff and SOCJ expression (Lee eta/., 2000; Samach eta/., 2000). Thus, the
decision to flower in Arabidopsis seems to be based on the relative levels of Ff and
SOCJ that are repressed by FLC, and induced by age, photoperiod, vernalization and
possibly other environmental and internal stimuli (Fig. 2; Menzel et a/., 1996;
Bonhomme eta/., 2000; Lee eta/., 2000; Samach eta/., 2000).

Fig. 2. Convergence of flower-promoting pathways in Arabidopsis. CO promotes flowering of

Arabidopsis in response to long photoperiods. CO can directly increase expression of FT and SOC1.
FT and SOC1 promote the transition to flowering via activation of floral meristem identity genes.
FLC encodes a floral repressor whose transcript levels increase in plants carrying active FR1 alleles
and decrease after vernalization. FLC seems to have an antagonistic effect on the expression of SOC1
and FT. The abundance of theFT and SOC1 transcript increases with age independently of CO.

6. Prospects for Molecular Breeding

Improving crop growth and introducing new varieties of ornamentals using molecular
 233

breeding is becoming increasingly attractive. Exploitation of genes involved in light

perception and photomorphogenesis will undoubtedly become a useful approach.
Already, manipulation of PHYA expression has led to major changes in plant
architecture in the field, leading to predictions of substantial yield increases (Robson et
a/., 1996). Other applications include those that modify plant responses to photoperiod;
particularly interesting will be those allowing tight control of flowering time (Olsen et
al., 1997).

7. Acknowledgments

Work in the AS lab is supported by THE ISRAEL SCIENCE FOUNDATION-The

Charles H. Revson Foundation (grant no. 436/00-1), and by the Chief Scientist of the
Ministry of Agriculture & Rural Development. MP is funded by CICYT (project
AGF98-0206).

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MOLECULAR CONTROL OF FLOWER DEVELOPMENT

M. VISHNEVETSKY, E.M. MEYEROWITZ

Division ofBiology 156-29
California Institute of Technology
Pasadena, CA 91125, USA

1. Introduction

The last decade has been an exciting period in plant molecular biology in general and in
molecular studies of flower development in particular. The isolation of the first floral
meristem identity and floral homeotic genes of Arabidopsis in the late 1980s-early
'90s opened the way to in-depth studies of molecular aspects of floral development
(Bowman eta/., 1989; Coen eta/., 1990; Sommer eta/., 1990; Yanofsky eta/., 1990).
These investigations have led to insights into inflorescence and flower development in
higher eudicotyledonous flowering plants, using mainly the predominant model of
Arabidopsis thaliana (thale cress). The abundance of mutants, a relatively small
genome, and easy transformation procedures have made this small plant a primary tool
of modem plant biology. Among the ornamentals, Antirrhinum majus (snapdragon) and
Petunia hybrida (petunia) are the best-characterized plants at the molecular level. As far
as is known, flower development in these species follows genetic principles and
mechanisms similar to those in Arabidopsis, although some differences exist in the
details at the molecular level, and will be discussed later.

2. Current Model of Flower Development

All of the above-ground parts of a plant (except the cotyledons) are derived from the
shoot apical meristem (SAM), which is a group of dividing and undifferentiated cells.
During the growth of a plant, small groups of these stem cells are set apart from the
apical meristem, forming different types of primordia or meristems according to the
different phases of the plant's life cycle. lnArabidopsis, during the vegetative phase, the
SAM produces leaf primordia that develop into rosettes ofleaves. After floral induction,
the SAM is converted into an inflorescence meristem that, during the reproductive
phase, gives rise to a spiral of floral meristems on its flanks. In contrast to the
indeterminate SAM, which essentially gives rise to flower primordia indefinitely,
flowers are determinate structures that produce a defined number of organ primordia.
Each floral meristem produces a set of floral organ primordia in a defined spatial and
temporal pattern, whose cells divide and differentiate into the different organs that,
arranged in four concentric whorls or rings, make up the Arabidopsis flower: four
sepals, four petals, six stamens, and two fused carpels (from the outermost to the
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A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, 239-252.
© 2002 Kluwer Academic Publishers.
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innermost whorl).
Numerous genes are required for the initiation and development of flowers; for
simplicity, they can be divided into four distinct classes:
The first class consists of "flowering-time genes", mutations in which cause early or
late flowering. Flowering-time genes can themselves be divided into distinct classes,
based on their differential responses to a number of environmental conditions, such as
day length and vernalization.
The second class specifies meristem identity, and includes genes such as LEAFY
(LFY), APETALAJ (API), APETALA2 (AP2), and CAUUFLOWER (CAL), which
specify flower meristem identity, as well as TERMINAL FLOWERJ (TFLJ) that
maintains inflorescence meristem identity.
A third class includes the flower organ identity genes, which determine the fate of
organ primordia and are incorporated into the "ABC" model of flower development,
which was established by the study of homeotic mutants in Arabidopsis and
Antirrhinum (Bowman et al., 1991; Coen and Meyerowitz, 1991; Meyerowitz et al.,
1991). Examples of organ identity genes include AP1 and AP2 (which are involved in
both meristem and organ identity), APETALA3 (AP3), PISTILLATA (PI) and
AGAMOUS (AG).
A fourth class includes late-acting genes that control ovule development, and
extensive genetic and recent molecular studies have begun to uncover the complex array
of interactions among genes in this class.
This review concentrates mainly on meristem and floral organ identity genes and on
the ABC model of flower development inArabidopsis and ornamental species. Reviews
on flowering-time and late-acting genes can be found elsewhere (Gasser et al., 1998;
Levy and Dean, 1998; Schneitz, 1999; Reeves and Coupland, 2000).

3. Floral Meristem Identity Genes

Initially, the Arabidopsis SAM produces leaves with axillary, second-order shoot
meristems. At the transition to the reproductive phase, the primary shoot switches to the
production of flowers. Two meristem identity transcription factors, LFY and AP 1, have
been found to be necessary and sufficient for this transition (Mandel et a/., 1992a;
Weigel eta/., 1992; Weigel and Nilsson, 1995; Wagner eta/., 1999). The orthologs
from Antirrhinum are FLORlCAULA (FLO) and SQUAMOSA (SQUA), respectively
(Coen et al., 1990; Huijser eta/., 1992). The function of these genes is indicated by the
phenotype of loss-of-function mutants. Severe disruption of the onset of reproduction is
observed in the loss-of-function l.fy-6 mutant, where leaves and second-order shoots
replace most flowers (Weigel et al., 1992). In other species where mutants in LFY
homologs are available, flower formation is similarly disrupted (Coen et al., 1990;
Hofer eta/., 1997; Souer et al., 1998; Shu et al., 2000). Thus, despite species-to-species
variation in their expression (see Coen et al., 1990; Weigel et al., 1992; Weigel and
Nilsson, 1995; Hofer eta/., 1997; Kyozuka et al., 1998; Pouteauet al., 1998; Souer et
al., 1998), LFY homologs appear to play a conserved role in the regulation offlower
meristem formation (Weigel and Nilsson, 1995). In the strong apl-1 mutant, flowers
have partial shoot character (Bowman et al., 1993). Furthermore, the nearly complete
conversion of flowers into shoots observed in lfy ap1 double mutants reveals that they
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act redundantly to specify meristem fate (Irish and Sussex, 1990; Huala and Sussex,
1992; Weigel et a/., 1992; Bowman et a/., 1993). The gain-of-function phenotype
produced by constitutive expression of either LFY or AP 1 results in the formation of
flowers, or leaves and flowers in positions normally occupied by leaves and second-
order meristems (Mandel and Yanofsky, 1995; Weigel and Nilsson, 1995; Souer et
a/., 1998).
Models of LFY function (e.g., Schultz and Haughn, 1991; Weigel et a/., 1992;
Weigel and Nilson, 1995; Parcy eta/., 1998) suggest that its transcription is activated by
exogenous factors (e.g., day length, vernalization) and endogenous factors (a
developmental clock) via hormones such as gibberellic acid (Blazquez eta/., 1998) and
several flowering-time genes (as reviewed in Levy and Dean, 1998; Reeves and
Coupland, 2000). LFY protein then binds to the promoters of target genes (especially
API, AP3, and AG) and, in the presence of necessary coregulators, causes shoot
meristems to differentiate into flowers (Lee eta/., 1997; Parcy eta/., 1998; Busch eta/.,
1999; Wagner eta/., 1999). Most recently, LFY was shown to participate in cell-cell
signaling between and within different layers of the floral meristem. Its nonautonomous
action was accompanied by movement of the LFY protein to adjacent cells, where it
directly activated homeotic target genes (Sessions eta/., 2000). In contrast, API had
only limited nonautonomous effects, apparently mediated by downstream genes,
because activation of early target genes by AP1 was cell-autonomous (Sessions et a/.,
2000).
Two other floral meristem identity genes, AP2 and CAL, have little effect on
meristem identity as single mutations, but the ap2 and cal mutations enhance effects of
lfy and apl mutations on floral meristem identity. It has been suggested that the
overlapping expression domains of LFY and TFLJ in emerging lateral primordia cause
the meristematic proliferation of apl cal inflorescences (Ratcliffe eta/., 1999). Support
for this idea comes from genetic studies, which show that this proliferation does not
occur when lfy or tj/1 mutations are introduced into the apl cal double mutant. Thus,
the role for CAL in an apl mutant background is to activate LFY and repress TFLJ
expression in lateral meristems, thus preventing their overlapping activities (Bowman et
a/., 1993; Ratcliffe eta/., 1999).
Another gene that may play a role during transition to flowering is FR UJTFULL
(FUL) from Arabidopsis. The FUL gene encodes a MADS-box proteinthat has
previously been shown to be required for carpel and fruit development (Mandel and
Yanofsky, 1995; Gu eta/., 1998). However, in addition to its expression domain during
carpel and fruit development, the FUL gene is upregulated in the SAM at around the
transition to flowering, suggesting that it may also play a role during this transition
(Mandel and Yanofsky, 1995; Hempel eta/., 1997). FUL is closely related to API and
CAL, and along with them has been recently shown to play a redundant role in LFY
upregulation, thus promoting floral meristem specification (Ferrandiz et a/., 2000).
Some data indicate that it is also involved in phase transition during development via a
pathway that is independent of LFY. This was clearly illustrated by the observation that
the delay in flowering caused by mutations in LFY is further enhanced infu/ lfy double
mutants (Ferrandiz eta/., 2000).
In the apical inflorescence meristem of Arabidopsis, the action of the floral
meristem identity genes is antagonized by the TFLJ gene (Shannon and Meeks-Wagner,
1991; Alvarez et al., 1992; Bradley eta/., 1997). The activities of TFLJ and floral
 242

meristem identity genes are maintained in separate regions of the shoot apex by a
distinct mechanism of mutual inhibition (Ratcliffe et a/., 1999). TFLJ expression is
prevented at the periphery of the apex through the action of LFY or AP1 and CAL. TFLJ
inhibition of occurs at the level of the RNA transcript, when either transcription of the
TFL1 gene or the accumulation of its message is prevented. Conversely, TFLJ acts to
inhibit the activity of floral meristem identity genes at the center of the shoot apex
(Ratcliffe et al., 1999). Moreover, while TFLJ expression becomes ectopic in lfy or ap1
cal mutants, and is inhibited when these genes are constitutively expressed, TFLJ loss
of function results in ectopic expression of LFY and AP1 in the inflorescence meristem,
thus transforming it into a floral meristem, which generates a terminal flower (Ratcliffe
et a/., 1999). The ortholog and functional equivalent of TFLJ from Antirrhinum is
CENTRORADIAUS (CEN) (Bradley eta/., 1996). TFLJ and CEN encode membrane-
associated proteins and, therefore, may be involved in a signal transduction chain
required to repress the expression of the floral meristem identity genes in the
inflorescence meristem. In contrast, all known meristem identity genes that promote
floral fate encode transcription factors. AP1, CAL, and SQUA belong to the family of
MADS-box genes (for reviews, see Riechmann and Meyerowitz, 1997; Theissen eta/.,
2000); LFY and FLO are members of a small family termed FLO-like genes (Theissen
et al., 2000); and AP2 is the founder of a novel gene family called AP21EREBP-like
genes (Riechmann and Meyerowitz, 1998).

4. Floral Organ Identity Genes

4.1. ABC MODEL OF FLOWER DEVELOPMENT

Arabidopsis genes providing the three homeotic activities A, B and C are known. The A
function is contributed to by AP1 and AP2, the B function by AP3 and PI, and the C
function by AG. Based on studies in Petunia, the ABC model was recently extended by
aD function (ovule development) (Angenent and Colombo, 1996), but while sequence
similarity exists between proposed D-function genes FBP7 and FBP 11 from Petunia
andArabidopsis geneAGLJ 1 (Angenent and Colombo, 1996), the mutation analysis has
yet to be performed. Except for AP2, all of these genes share a highly conserved, ca.
180-bp long DNA sequence, called the MADS box. MADS is an acronym for the four
founder proteins MCM1 (from Saccharomyces cerevisiae), AG, DEFICIENS and SRF
(a human protein), on which the definition of this gene family is based (Schwarz-
Sommer et a/., 1990). It encodes a conserved DNA-bindingldimerization domain
present in a variety of transcription factors from different kingdoms. MADS-box genes
represent a large multigene family in vascular plants. In angiosperms, many of the genes
of the MADS family are involved in different steps of flower development, most
notably in the determination of floral meristem and organ identity.
From genetic and molecular studies of flower development in a number of species,
Antirrhinum in particular, it appears that the basic genetic mechanisms determining
floral organ identity are the same as in Arabidopsis. The organ identity genes from one
species have orthologs in the other, and the phenotypes of the corresponding
Antirrhinum mutants (Carpenter and Coen, 1990; Schwarz-Sommer eta/., 1990) are
also explained by the ABC model (Coen and Meyerowitz, 1991). Pairs ofArabidopsis
 243

and Antirrhinum orthologous genes are: AP 1 and SQUA, AP3 and DEFICIENS (DEF;
Sommer eta/., 1990), PI and GLOBOSA (GLO; Trobner eta/., 1992), and AG and
PLENA (PLE; Bradley eta/., 1993). Moreover, the phenotypic changes that result from
ectopic expression of PLE inAntirrhinum (which is caused by a transposon insertion in
one of its introns; Bradley eta/., 1993) parallel those caused by the ectopic expression
ofAG inArabidopsis (Mizukami and Ma, 1997). AP 1, AP3, PI, andAG homologs have
also been cloned from a number of other species (see further on; and Theissen eta/.,
2000). For some of these homologs, the functional analogy to the corresponding
Arabidopsis genes has been confirmed in ectopic expression experiments or by
generating transgenic plants expressing antisense RNA that phenocopy the mutant
phenotypes of the correspondingArabidopsis genes (Kempin et al., 1993; Pnueli eta/.,
1994). Additional evidence showing that homeotic gene function has been conserved
across the angiosperms was obtained by expressing organ identity genes from one
species in a different one. Examples include the partial rescue of a mutant phenotype by
introduction of the corresponding orthologous gene from a different species (partial
complementation of Arabidopsis AP3 alleles by Antirrhinum DEF; Irish and
Yamamoto, 1995; Samach et a/., 1997), as well as the ectopic expression in tobacco
plants of AG homologs from Brassica napus (BAG1; Mandel eta/., 1992b), Petunia
hybrida (pMADS3; Tsuchimoto eta/., 1993), rice (OSMADS3; Kang eta!., 1995), and
Antirrhinum (PLE; Davies et at., 1996), and of Antirrhinum DEF and GLO (Davies et
a/., 1996).

4.2. REGULATION OF ABC GENES

Little is known about how the initial pattern of ABC gene expression is generated. Since
mutations in the floral meristem identity gene LFY affect floral organ development, it
became a candidate for upstream regulator of the Arabidopsis ABC genes. To find out
whether this is the case, Parcy eta/. (1998) generated a LFY version with constitutive
transcriptional activation potential. In the new LFY allele, termed VP 16, a fusion of LFY
to the strong activation domain from the viral transcription factor VP16 was expressed
under the control of the normal LFY promoter. The rationale behind this experiment was
that if LFY plays a role in regulating floral homeotic gene expression that is separable
from the function of LFY in specifying flower meristem identity, then LFY:: VP 16 might
modify the expression of individual ABC genes and thus affect flower morphology
(Parcy eta!., 1998). Indeed, the analysis of plants transgenic for LFY:: VP16 indicated
that LFY alone could induce expression of the A-function gene AP1. As mentioned
earlier, it was confirmed recently that AP 1 is a direct downstream target of LFY
(Wagner eta!., 1999). In contrast, the B-function gene AP3 was activated by LFY in
region-specific patterns within flowers, depending on other factors such as UNUSUAL
FLORAL ORGANS (UFO) (Parcy et at., 1998). Analysis of a LFY-responsiveenhancer
in the A G indicated that direct interaction of LFY with this enhancer is required for its
activity in plants (Busch et al., 1999). A recent study based on the PJ::GUS expression
patterns in the loss- and gain-of-function alleles of meristem or organ identity genes
showed that both LFY and UFO induce PI expression in a flower-independent manner
via a distal promoter, and that PI and AP3 maintain PI expression (autoregulation) in
the later stages of flower development via a proximal promoter (Honma and Goto,
2000). Furthermore, these authors have shown that de novo protein synthesis is required
244

for the PIIAP3 complex to upregulate PI transcription via a proximal promoter. These
results, together with the finding that the constitutive expression of both PI and AP3
cannot give rise to the expression of PI in non-floral tissues, suggest that a still
unknown flower-specific factor may be necessary for maintaining PI expression
(Honma and Goto, 2000).
The action of the C-function gene A G appears to be under very complex regulation
through different factors. The spatial restriction of A G to third and fourth whorls is
controlled by several partially redundant factors, including AP2, LEUNIG (LUG),
CURLY LEAF (CLF), and STERILE APETALA (SAP). Mutations in these genes result in
various amounts of A G misexpression in different regions of the plant. The major
repressor of AG in first and second whorl cells is AP2. Loss of AP2 function results in
ectopic A G expression in the outer two whorls and the corresponding homeotic
transformations of these organs to carpels and stamens, respectively (Drews et a/.,
1991). lug mutants show similar but weaker homeotic transformation in the outer two
whorls and have been isolated as enhancers of the weak ap2-1 allele (Liu and
Meyerowitz, 1995). In addition to the spatial expansion of the A G expression domain in
both ap2 and lug mutants, AG expression is initiated slightly earlier in both of these
mutants. The recent analyses of the 4-kb intragenic control region of A G allowed
definition of two regions of the A G second intron capable of activating expression in a
stage 3 floral meristem (5' and 3' activation domains). In addition, three major
expression patterns conferred by 19 AG::GUS constructs have been identified: the
normalAG pattern, a stamen-specific pattern, and a predominantly carpel pattern. After
analyzing GUS staining patterns in Arabidopsis plants homozygous for strong ap2 and
lug mutations, Deyholos and Sieburth (2000) concluded that the stamen-specific pattern
was independent of AP2 but dependent on LUG; conversely, the carpel-specific pattern
was independent of LUG but dependent on AP2 (Deyholos and Sieburth, 2000).
Recently, LUG was cloned and shown to encode a glutamine-rich protein, which
belongs to a class of functionally related transcriptional corepressors from yeast and
Drosophila (Conner and Liu, 2000).
A third gene that acts to repressAG in whorls 1 and 2 is SAP. Early sap flowers
produce sepals and small petals, whereas later flowers produce carpelloid sepals and
lack second whorl organs (Byzova eta/., 1999). AG mis-expression is observed in all
floral whorls and in the inflorescence meristem of sap mutants. On the other hand, CLF
acts primarily to negatively regulate A G expression in vegetative tissues and to maintain
repression of AG in flowers at older developmental stages (Goodrich eta/., 1997). The
predominant phenotype of elf mutants is the curling of leaf margins toward the midrib
because of ectopicAG expression in leaves.
Another gene that is apparently involved inAG regulation and may even function as
a class A gene is AINTEGUMENTA (AN7) (Krizek et al., 2000). ANT, which is a
member of the AP2 family of transcription factors, has been previously shown to be
involved in ovule development and in the initiation and growth of floral organs (Elliott
et a/., 1996; Klucher et a/., 1996). Although ant mutants show no homeotic
transformation of organ identity, ap2-2 ant-9 double mutants show a severe phenotype
that resembles ap2-9 /ug-1 double mutants (Elliott eta/., 1996). Krizek eta/. (2000)
showed that the role of ANT in A G repression is entirely redundant with that of AP2. In
addition, ANT appears to act as a second whorl-specific repressor of AG. Unlike AP2
and LUG, ANTis found to not be involved in controlling the initiation ofA G expression,
 245

because it is initiated in a normal pattern in stage 3 floral meristems of ap2-1 ant-6

plants (Krizek eta/., 2000).

4.3. VARIATION ON THE ABC MODEL

Despite its general applicability, some variations in the way an ABC genetic mechanism
may be implemented in disparate species are already known. These variations may be
manifested as differences in the phenotypes caused by loss-of-function mutations in
orthologous genes, or in the phenotypic alterations caused upon their ectopic
expression. Their cause can be that the number of organ identity genes involved in a
particular class of function (A, B, or C) may be different, and/or that their expression
pattern may vary among species.
Recent data from Arabidopsis suggest that the ABC model, while providing a
conceptual framework for explaining flower organ identity, does not cover all the genes
involved in organ identity function. Pelaz and coworkers identified the function of three
functionally redundant genes, SEPALLATA 11213 (SEPJ, SEP2, SEP3), whose activities
are required for the development of petals, stamens and carpels (Pelaz eta/., 2000). All
three are MADS-box genes, previously called AGAMOUS-LJKE2 (AGL2), AGL4 and
AGL9 (Ma eta/., 1991; Mandel and Yanofsky, 1998). To discover the roles of SEP 11213
during flower development, the authors used a reverse-genetics polymerase chain
reaction (PCR)-based approach to identify mutant alleles. They found that while single-
gene mutations produced only subtle phenotypes, a striking phenotype occurred in sep1
sep2 sep3 triple mutants, in which all flower organs resembled sepals (Pelaz et a/.,
2000). The sep11213 triple mutant phenotype was very similar to be (ap3 ag, pi ag)
double mutants, indicating that SEP 11213 are required to activate the B- and C-function
genes, or that the B-and C-function gene products require at least one of the SEPl/2/3
proteins for activity. While the initial patterns of Band C expression were not altered in
the SEP triple mutant and SEP 1, SEP2 and SEP3 were still expressed in b and c organ
identity mutants, the question remains as to how these genes or their products interact
with other ABC genes. Candidate orthologs of SEP genes with similar expression
domains exist in distantly related eudicots (Angenent eta/., 1994; Pnueli eta/., 1994;
Davis et a/., 1996) and in monocots (Kang and An, 1997), indicating that the
conclusions from studies of the SEP genes in Arabidopsis will be applicable to diverse
species. Indeed, antisense and cosuppressed lines of candidate SEP3 orthologs in
petunia (FBP2) and tomato (TM5) have phenotypes resembling the SEP triple mutant
(Pnueli et a/., 1994). It was recently proposed that these type of genes should be called
intermediate or "identity-mediating" (Im) genes, as they supposedly belong to a new
class of MADS-box genes and have a separate function within the ABC model
(Gutierrez-Cortines and Davies, 2000). The question of these genes' mechanism of
action in flower development remains-most probably they encode factors that form
ternary complexes (multimers) with MADS-box ABC proteins (Egea-Cortines eta!.,
1999; Gutierrez-Cortines and Davies, 2000), thus not acting independently or as
downstream targets of ABC genes, but rather providing regulatory function (Pelaz eta/.,
2000). At this point, it is still not clear whether this type of regulatory activity has a
place in the ABC model as a separate Im function.
Mutants that are primarily caused by loss of A function are only known from
Arabidopsis. Antirrhinum mutants with a similar phenotype are due to ectopic
246

expression of a C-function gene in whorls 1 and 2 of the flowers (Bradley et al., 1993).
Searches for A-function genes in Petunia using the candidate approach inspired by
results from Arabidopsis, also remain negative (Maes et a/., 1999). The authors
introduced the Arabidopsis AP2 gene into Petunia. The original reason was to
investigate whether AP2 could complement the blind (bl) mutation of petunia. bl
flowers develop carpelloid sepals and stamenoid petals and thus resemble mutants in the
floral homeotic A-function or the cadastral function, preventing the C function from
expression in whorls 1 and 2. Therefore, BL has been considered an AP2 ortholog
(Tsuchimoto eta!., 1993). However,AP2 did not complement the bl mutant, andBL did
not map at three petuniaAP2-like genes, suggesting that BL is not anAP2-like gene and
that AP2-1ike genes do not exert a floral homeotic A function or a respective cadastral
function in the petunia genome (Maes et al., 1999). However, petunia plants transgenic
for AP2 exhibit altered inflorescence architecture, suggesting that AP2 in the petunia
background plays a role in the determination of inflorescence meristem identity (Maes
eta/., 1999).
There is evidence that Petunia also differs from Arabidopsis in the organization of
its B function. B function inArabidopsis is orchestrated by AP3 and PI (inAntirrhinum,
by the orthologous genes DEF and GLO), which have similar mutant phenotypes,
having sepals occupying whorl 2 and only carpels in whorls 3 and 4. In Petunia,
however, the absence of function of the AP3 homolog, GREEN PETAL (GP; also
known as pMADSJ), affects petal but not stamen development (i.e., causes homeotic
conversion in one whorl only; van der Krol eta!., 1993). In addition, two petunia PI
homologs have been cloned, FBPJ andpMADS2 (Angenent et al., 1992, 1994; Kush et
a/., 1993 ). Consistent with it being a B-function gene, FBP 1 is required for petal and
stamen development (Angenent et al., 1992, 1993), whereas the functional role of
pMADS2 is still unknown (it may be redundant; Angenent et al., 1995). The lack of
requirement of GP activity for stamen development may suggest that petunia has
another, as yet unidentified, AP3 homolog involved in this process (Angenent et al.,
1995). Alternately, the activity of FBP l/pMADS2 in the third whorl may differ at the
molecular level from that of PI by not requiring the interaction with anAP3 ortholog. In
summary, B function in petunia differs from that of Arabidopsis in the number of genes
involved and in the distribution of functional roles among them. Interestingly, this could
be a recent evolutionary innovation, because petunia is more closely related to
Antirrhinum than to Arabidopsis, and these two plant species have two B-function genes
(Xue and Ingram, 1993; Weigel and Meyerowitz, 1994; Theissen and Saedler, 1999).
Recently, a double-mutant line homozygous for both bl and gp mutations has been
constructed (Tsuchimoto et a!., 2000). The bl gp double mutant flower displays
homeotic conversions of sepals into the stigmatic tissue in the first whorl and of the
corolla limb into antheroid structures with stigmatic tips in the second whorl. In the
third and fourth whorls of the mutant flower, organs remained unchanged. In the gp
flower, the petunia B-type gene FBP 1 is expressed strongly in the third whorl organs,
but much more weakly in the second whorl organs. In the bl gp flower, FBP 1 was found
to be expressed strongly in the second whorl organs as well as in the third whorl organs.
The conclusion was drawn that Petunia has a class B gene other than GP that
determines organ identities, both in the second and third whorls of the double mutant
flower, and the action of the postulated class B gene (called PhBX) is prevented by the
BL gene in the second whorl of the gp flower. PhBX appears to be a gene that
 247

specifically interacts with the FBPJ gene, and is involved in the latter's upregulation
(Tsuchimoto et a/., 2000).
Differences in the expression patterns of B-function genes from the Arabidopsis
model have also been described. AP3 inArabidopsis is expressed in whorls 2 and 3, and
also in some cells of the first whorl (at the base of the sepals) (Jacket a/., 1992; Weigel
and Meyerowitz, 1993; Goto and Meyerowitz, 1994). In contrast, DEF in Antirrhinum
is expressed in whorls 2 and 3 and, at reduced levels, in whorl 4 (Schwarz-Sommer et
al., 1992), whereas RNA of the tobacco homolog, NTDEF, is detected in whorl 4 and
weakly in whorl 1 (Davies et al., 1996). In all three cases, the expression of
AP3/DEFINTDEF overlaps with the expression of Pl/GLO/NTGLO in whorls 2 and 3,
where the B function (which requires both genes' activities) controls organ identity.
C-function genes have undergone diversification in Antirrhinum, from which the
second AG homolog, in addition to the ortholog PLE, has been cloned (Davies et al.,
1996), and in tomato, because ectopic expression in these plants of the corresponding
AG homolog, TAGJ, does not provoke first and second whorl homeotic conversions as
complete as those observed upon ectopic expression ofAG inArabidopsis (Pnueli et al.,
1994). From the two petuniaAG homologspMADS3 andFBP6, only pMADS3 was able
to induce homeotic transformations of sepals and petals. Ectopic expression of both
pMADS3 and FBP6, as occurs in the bl mutant, phenocopies the pMADS3 single
overexpressed plants, indicating that there is no additive effect of concertedexpression
(Kater et al., 1998).
While most of the information on MADS-box genes in angiosperms is available
from studies of eudicots like Arabidopsis, Antirrhinum and Petunia, studies of MADS-
box genes in monocots like Zea mays (maize), Oryza sativa (rice), Triticum aestivum
(wheat), Lilium regale (lily), Tulipa gesneriana (tulip) and an orchid species (Aranda x
deborah), have been recently accelerated (McSteen eta/., 2000; Theissen eta/., 2000).
Liliaceae flowers look, superficially, very different from the flowers of grasses. For
example, they are often very large and have a simple, yet showy perianth (perigon)
composed of two whorls of organs, each containing three petaloid tepals. Floral
structures like these could be explained by a modified ABC model in which the
expression of the B function has expanded to whorl 1 (van Tunen eta/., 1993). Tulip
mutants are known to strongly support this hypothesis. A putative loss-of-function
mutant is called 'Viridiflora'. It has flowers where the tepals in whorls 1 and 2 are
homeotically transformed into sepaloid or leaf-like structures. The six stamens of the
mutant tulip flower are transformed into carpel-like structures. A putative loss-of-C-
function mutant is also known. It has tepal-like structures in whorls 1, 2 and 3, and from
the center of the flower a new flower structure arises, which again has tepal-like
structures in all three outer whorls (van Tunen et al., 1993). It does not seem unlikely
that an AG-like gene is affected in the loss-of-C-function mutant. Similarly, a DEF- or
GLO-like gene may be mutated in the loss-of-B-function mutant (Theissen et al., 2000).
However, one should take into consideration that the perianths of monocots and
eudicots may have evolved independently (Kramer and Irish, 1999; Becker eta/., 2000).
But even then, recruitment of DEF- and GLO-like genes for the specification ofperianth
organ identity-independent of a very similar event in eudicots-would seem the likely
scenario to explain the petaloid character of perianth organs in Liliaceae (Theissen et
a/., 2000). Expression of a DEF-Iike gene in both perianth whorls and stamen whorls of
lily support the hypothesis that the petaloid character of all tepals is due to the
248

expression of B-function genes in perianth whorls 1 and 2. Recent cloning of a number

of MADS-box genes from Lilium regale and respective orthologs from tulip (Theissen
eta/., 2000) would help test this hypothesis.

5. Double-Flowered Mutants

Another still not very well understood phenomenon is double-flowered mutants of many
dioecious plants, which have attracted human attention for centuries and are highly
valued by the horticultural industry. In many cases, they are homeotic mutants that
show conversion of stamens to petals. Other phenotypes frequently found in double-
flowered plants include an increase in floral organ number and floral indeterminacy or
secondary flower production. We still know too little about the molecular nature of
doubleness in flowers. The most comprehensive study of double-flowered plants was
performed by Reynolds and Tampion in 1983, before the beginning of the molecular era
in flower development. Double-flowered plants, such as Aquilegia vulgaris, Begonia,
Calendula, Chamelaucium ucinatum, have been characterized anatomically, but not
genetically (Reynolds and Tampion, 1983; Lehmann and Sattler, 1996; McComb et al.,
1996). None of the genes responsible for double-flowered mutants in the above
examples, or in Camellia, Narcissus, Primula, Rosa, and many other horticulturally
important plants, have yet been identified at the molecular level. Yet some progress has
been made in recent years. The double and semi-double forms of Dianthus caryophyllus
(carnation) have been shown to be caused by a dominant mutation at a single locus
(Scovel eta/., 1998) that shows meristic effects and homeotic conversion of stamens to
petals. By contrast, a single recessive allele causes stamens to become petaloid in a
Nicotiana alata mutant (Zainol eta!., 1998) and three recessive alleles are known to
cause double-flowered phenotypes in Papaver somniferum {opium poppy) (Belyaeva,
1995). Molecular analyses of the double-flowered mutant of Si/ene /atifolia (white
campion) indicated that most orthologs of ABC genes are still expressed at the same
level in the mutant as in the wild type (Scutt eta/., 1999). The major difference between
these varieties and the ag mutant inArabidopsis is that most of them retain carpels. So it
does not seem likely that all classes of double-flowered phenotypes can be accounted
for by loss-of-C-function mutations, though dominant negative C-function mutations
may also be involved in certain cases (Scutt eta/., 1999).

6. Concluding Remarks

In spite of the remarkable progress in understanding the fundamental steps in flower

development, only a small part of the overall picture has emerged. Most of the available
data comes from Arabidopsis and Antirrhinum, while even within higher eudicots,
including many ornamentals, there might be more variability in the specification of
floral organ identity than the common ABC model suggests. It is also a matter of great
importance to understand the mechanism of action of MADS-box genes as they control
flowering time, meristem identity and key components of the ABC organ identity
model. Thus, a comprehensive effort in establishing new plant models and obtaining
mutants should be welcomed. An understanding of the molecular nature of double-
 249

flowered mutants and the isolation and subsequent use of flower development genes
from horticulturally important plants in genetic engineering can make current breeding
procedures more effective and become a real alternative to the classic methods.

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 MOLECULAR CONTROL OF FLORAL PIGMENTATION: ANTHOCYANINS

H. BEN-MEIR, A. ZUKER, D. WEISS, A. VAINS1EIN

The Kennedy Leigh Centre for Horticultural Research and The Otto
Warburg Center for Biotechnology in Agriculture
Faculty ofAgricultural, Food and Environmental Quality Sciences
The Hebrew University ofJerusalem, Rehovot 76100, Israel

1. Introduction

Flower color is one of the most important traits in ornamental plants, dictating
consumer interest and attraction. As such, it is a critical factor for the commercial
success of plants on the market. Flower color in a variety of plants is determined by
flavonoids, which also play an important role in both plant reproduction and
development, i.e. in the attraction of pollinators, protection against damage from UV
irradiation, resistance to pathogens and symbiotic plant-microbe interactions.
Flavonoids are a large group of secondary metabolites, over 3000 of which have been
chemically characterized. The biosynthetic pathway of flavonoid has been extensively
studied: first in the 19th century, then with significant breakthroughs in the middle of the
20th century with the development of chromatography and NMR spectroscopy
techniques. The studies of flavonoid genetics started with phenotypic analyses
(Mendelian inheritance), ultimately leading to the isolation, characterization and
utilization of the genes in the pathway.
The most conspicuous group among the flavonoids is the anthocyanins. These are
water-soluble plant pigments, accumulating in the vacuoles of epidermal or sub-
epidermal cells, that are visible to the human eye. In addition to being the major flower
pigment in higher plants, anthocyanins can also occur in roots, stems and leaves. In this
chapter, we will outline the biochemistry and genetics of the pathway leading to
anthocyanin production, closing with an overview on application of the generated
knowledge toward molecular breeding of ornamentals.

2. The Flavonoid Biosynthetic Pathway-Overview

The basic structure offlavonoids consists of two aromatic rings (A and B) connected by
a heterocyclic one (C) (Fig. 1). On this basic Cl5 skeleton several oxidations, additions
and rearrangements occur leading to the formation of aurones, flavanones, flavones,
isoflavonoids, flavonols and anthocyanins. Flavonoids are synthesized from the
phenylpropanoid pathway in several enzymatic steps (Fig. 2). The first committed step
of the pathway is the condensation of three acetate units from malonyl-CoA with 4-
coumaroyl-CoA by the enzyme chalcone synthase (CHS), leading to the formation of
253
A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, 253-272.
© 2002 Kluwer Academic Publishers.
254

yellow chalcone (naringenin chalcone/tetrahydroxychalcone). In the next step, chalcone

isomerase (Cill) catalyzes the isomerization of chalcone to the colorless flavanone
naringenin, a reaction that can also occur spontaneously. Flavanones are the precursors
of flavones, flavan-4-ols and dihydroflavonols. Flavones are synthesized from
flavanones by the introduction of a double bond between C-2 and C-3 by two types of
enzymes: a dioxygenase, flavone synthase I (FNS I), and a mono-oxygenase, flavone
synthase II (FNS II). Hydroxylation of flavanones at position 3 by the enzyme
flavanone 3-hydroxylase (F3H) leads to the formation of dihydroflavonols. The
dihydroflavonols are biosynthetic intermediates in the formation of flavonols, catechins
and anthocyanidins. The first of these are formed from dihydroflavonols by the
introduction of a double bond between C-2 and C-3, a reaction that is catalyzed by
flavonol synthase (FLS).

6'

Figure 1. The basic structure offlavonoids.

The color spectrum of flowers is the result of three basic anthocyanidins, which
differ only in their B-ring hydroxyl groups (Forkmann, 1993b). The brick red
anthocyanidin derives from dihydrokaempferol. Hydroxylation of the B-ring of
dihydrokaempferol (or naringenin), catalyzed by flavonoid 3'-hydroxylase (F3'H) and
flavonoid 3',5'-hydroxylase (F3'5'H) provides the precursors of magenta
(dihydroquercetin) and blue (dihydromyricetin) anthocyanidins, respectively.
Flavonoids hydroxylated at the B-ring can also be produced, albeit at low levels, from
hydroxycinnamic acid derivatives like caffeic acid during the synthesis of the Cl5
skeleton by CHS (Van Tunen and Moll991).
Reduction of dihydroflavonols at position 4, a reaction which is catalyzed by
dihydroflavonol 4-reductase (DFR), leads to the formation of flavan-2,3-trans-3,4-cis-
diols (leucoanthocyanidins). Colorless leucoanthocyanidins are transformed into the
colored unstable anthocyanidin by oxidation reaction catalyzed by anthocyanin synthase
(ANS) (Saito et a/., 1999). Glycosylation of the different anthocyanidins by UDPG-
flavonoid-glucosyltransferase (UFGT) produces the corresponding anthocyanins-brick
red pelargonidin, magenta cyanidin and blue delphinidin. Depending on the species,
anthocyanidin-3-glucosides are further modified by glycosylation, methylation and
acylation. These latter modification steps actually determine the type of anthocyanins
accumulating in a given species or even variety.
It was long accepted that anthocyanin biosynthetic enzymes act independently in the
cytosol. Recent studies, however, support a model in which flavonoid biosynthetic
 255

enzymes are organized in complexes localized to the ER and function in concert

(Burbulis and Winkel-Shirley, 1999).

Phenylalanine ;At Clnnamate~ 4-Cumarateri 4 - C o u p - 3 (Malon~CjaiTeoyi-CoA

l CHS

Cha/cones

cml cml ·
Naringenin chalcone 3, 4, 2', 4', 6'- Penthahydroxychalcone

Flavanones Penthahydroxyflavanon 4 F3'S'H Naringenln F3'H ~ Erlodlctyol

Flavones
F3H
1 '"'~. 1"'':.-
F3'S'H
F3H
F3'H
F3H 1'"' :!-
Dihydrojlavonols

j j
Dlhydromyrlcetin ~ Dlbydrokaempferol Dlhydroquercetin
~FLS "{LS "{Ls
Flavonols
DFR j Myrlcetln
DFR
Kaempferol
DFR
Quercetin

Leucoanthocyanidins leucodelphlnidin leucopelargonidin leucoeyanidin

ANS 1 ANS1 ANS1

Anthocyan/dins Delpbinidin Pelargonidin Cyanidin

GT 1 GT 1 GT1
Anthocyanins Delphinidin 3-Giu Pelargonidin 3-Giu Cyanidin 3-Giu

Figure 2. Flavonoid biosynthetic pathway.

Abbreviations: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA
ligase; CHS, chalcone synthase; CHI, chalcone isomerase; FJ'H, flavonoid )'-hydroxylase; F3'5'H, flavonoid
3',5'-hydroxylase; FNS, flavone synthase; FJH, flavanone 3-hydroxylase; FLS, flavonol synthase; DFR,
dihydroflavonol 4-reductase; ANS, anthocyanin synthase; GT, glucosyltransferases.

3. Flavonoid Biosynthesis Genes/Enzymes

CBS is a key enzyme in flavonoid biosynthesis. All vascular plants are believed to
contain CHS since the products of this enzyme, e.g. aurones, flavanones, flavones, etc.
are effective UV-light protectants. CHS, in general, uses 4-coumaroyl-CoA as a
substrate. However, it has been found in some flower species-such as Dianthus
caryophyllus, Verbena hybrida and Callistephus chinensis-to use caffeoyl-CoA as a
substrate, albeit less efficiently. It can also use feruloyl-CoA, as shown for petals of
Cosmos and tulip anthers (Heller and Forkmann, 1993). In Gerbera hybrida, a CHS-like
protein was shown to use benzoyl-CoA as a substrate (Helariutta et al., 1996). The first
isolated flavonoid biosynthetic gene was Chs from parsley (Kreuzaler eta/., 1983) and
to date, over 100 highly conserved Chs sequences from over 40 plants have been
cloned. In most plant species, the Chs constitutes a multi-gene family, of which some
members are actively transcribed in floral tissue (Table 1).
256

TABLE I. Flavonoid genes from ornamentals.

Enzyme Species Gene Locus Reference

CHS Antirrhinum Chs Nivea Sommer and Saedler, 1986
Chrysanthemum Chs Courtney-Gutterson et al., 1994
Callistephus Chs Forkmann, 1991
Dianthus Chs y Forkmannetal., 1995
Eustoma Chs Davies et al., 1993
Forsythia Chs Rosati et al., 1997
Gerbera Gchsl, Gchs3 Helariutta et al., 1995
Ipomoea ChsA-C, A Durbin et al., 1995
ChsD; ChsE Fukada-Tanaka et al., 1997
Matthiola Chs F Epping et al., 1990
Orchid Ochs3,4,8 Liew et al., 1998a
Perilla Chs Gongetal., 1997
Petunia ChsA; Chs.J Koes et al., 1989
Ranunculus Chs Niesbach-Kiosgen et al., 1987
Rosa Chs Gutterson, 1995
Torenia Chs Suzuki et al., 2000b
CHI Antirrhinum Chi Martin et al., 1991
Dianthus Chi 1 Forkmannetal., 1995
Eustoma Chi Davies et a/.,1993
Petunia ChiA, ChiB Po Van Tunen et al., 1988
F3H Antirrhinum F3h Jncolorata Martinet al., 1991
Callistephus F3h Britsch et al., 1993
Chrysanthemum F3h Park and Kim, 1997
Dianthus F3h Britsch et al., 1993; Dedio et al.,
1995
Ipomoea F3h Hoshino et al., 1997
Matthiola F3h Britsch et al., 1993
Orchid F3h Liew et al., 1995
Petunia F3h An3 Britsch et al., 1992
F3'H Antirrhinum F3'h" Eosina Forkmann, 1993a
Dianthus F3'h" R Spribille and Forkmann, 1982
Matthiola B Forkmann et al., 1980
Petunia F3'h Htl Brugliera et al., 1999
F3'5'H Callistephus R Forkmann,l993a
Catharanthus F3'5'h Kaltenbach et al., 1999
Eustoma F3'5'h Nielsen and Podivinsky, 1997
Gentiana F3'5'h Tanakaetal., 1996
Petunia F3'5'h Hfl;Hf2 Holton et al., 1993a
Torenia F3'5'h Suzuki et al., 2000b
FLS Petunia F1s FL Holton et al., 1993c
Rosa F1s Suzuki et al., 2000a
FNS Antirrhinum Afnsii Akashi et al., 1999
Gerbera Fnsii Martens and Forkmann, 1998,1999
DFR Antirrhinum Dfr Palida Martin et al., 1985
Callistephus F Ruhnau and Forkmann, 1988
Gentiana Dfr Tanaka et al., 1996
Gerbera Dfr Helariutta et al., 1993
Dianthus Dfr A Forkmann, 199l;Stichetal., 1992a
Eustoma Dfr Davies et a/., 1993
Forsythia Dfr Rosati et al., 1997
Ipomoea Dfr Inagaki et al., 1999
Orchid Dfr Liew et al., 1998b
Perilla Dfr Gong et al., 1997
Petunia Dfr An6 Beld et al., 1989; Huits et al., 1994
 257

Table I. Continued
DFR Rosa Dfr Tanaka et al., 1995
Torenia Dfr Suzuki et al., 2000b
ANS Antirrhinum Ans Candi Martin et al., 1991
Callistephus A Forkmann, 1993a
Forsythia Ans Rosati et al., 1999
Perilla Ans Saito et al., 1999
Petunia Ans Antl7 Weiss et al., 1993
Torenia Ans Nakajima et al., 2000
GT Antirrhinum Ufgt Martinet al., 1991
Gentiana 3Gt Tanaka et al., 1996
Forsythia 3Gt Rosati et al., 1999
Perilla 3Gt/5Gt Gon~ et al., 1997
GST Petunia Gst An9 Alfenito et al., 1998
'Genes that have been isolated and patented but unpublished.

em catalyzes an intramolecular reaction to close the C-ring. Two types of CHI

enzymes are known with respect to substrate specificity: one that converts both
trihydroxy- and tetrahydroxy-chalcones to the corresponding flavanones (i.e. Phaseolus
vulgaris, Glycine max and Medicago sativa); and the other that uses only tetrahydroxy-
chalcone as a substrate (i.e. Petunia hybrida, Callistephus chinensis and Dianthus
caryophyllus) (Dixon eta/., 1988).
The accumulation of chalcones in plants is scarce as they are rapidly isomerized by
CHI to form naringenin. Furthermore, even in the absence of CHI activity, chalcone can
isomerize spontaneously to form naringenin, albeit at a lower rate. Nevertheless, chi
mutants accumulating chalcones have been found and characterized. For example,
corollas of Callistephus chinensis and Dianthus caryophyllus varieties that exhibit no
CHI activity are yellow-colored due to the accumulation of chalcone pigments (Kuhn et
al., 1978; Forkmann and Dangelmayr, 1980). Anthers of Petunia and Tulip po mutants,
which lack CHI activity, also accumulate chalcones and are yellow (Forkmann and
Kuhn, 1979; Ebel and Hahlbrock, 1982; Van Tunen and Mol, 1987). The first Chi
eDNA was isolated from Phaseolus vulgaris using antibodies against the purified
enzyme (Mehdy and Lamb, 1987), and today Chi homologues have been cloned and
characterized in a wide variety of plants, including Petunia hybrida, Antirrhinum majus,
Dianthus caryophyllus and Lisianthus (Table 1).
FJH catalyzes the hydroxylation of flavanones at the 3 position of the C-ring. This
soluble enzyme is classified as a 2-oxoglutarate-dependent dioxygenase according to
the co-factor requirements of 2-oxoglutarate, ferrous irons and ascorbate (Britsch and
Grisebach, 1986). In most cases, naringenin serves as the substrate for the F3H enzyme
to form dihydrokaempferol, although eriodictyol can also be used as a substrate
(Forkmann et al., 1980). F3H activity has been detailed in flowers of Matthiola,
Dianthus, Chrysanthemum and Petunia (Britsch and Grisebach, 1986; Heller and
Forkmann, 1993). A eDNA encoding F3h was isolated from petals of Antirrhinum
majus (Martinet a/., 1991) and Petunia (Britsch et al., 1992), as well as from numerous
other ornamentals (Britsch et al., 1993) (Table 1). In all analyzed plants, it was shown
to be a single-copy gene rather than a gene family (Britch eta/., 1992; Dedio et al.,
1995). F3h appears to be evolutionarily older than anthocyanin-specific genes (such as
Dfr or Ans) as chalcones, flavanones and flavonols, but not anthocyanins, occur in
258

primitive plant species. While the evolutionary significance is not yet fully understood,
it is interesting to note that depending on the plant system, regulation of F3h gene
expression is coordinated with either early genes of the pathway (i.e. Chs and Chi in
Arabidopsis and Petunia) or with late genes (i.e. D.fr, Ans, etc. in snapdragon and
maize).
FNS catalyzes the conversion of flavanones to flavones via the introduction of a
double bond between C-2 and C-3 of the flavanones (Heller and Forkmann, 1993). Two
different enzymes with similar activity have been characterized. One (FNS I) is a rare
soluble 2-oxoglutarate-dependent dioxygenase that was first isolated and purified to
near-homogeneity from UV-irradiated Petroselinum crispum cell suspension cultures
(Britsch et al., 1981; Britsch, 1990). In flowers of several flavone-producing plants
however, this reaction is catalyzed by FNS II, a NADPH-dependent microsomal
enzyme (Heller and Forkmann. 1988). FNS II appears to be widespread in plants and
has been characterized in flowers of Antirrhinum majus (Stotz and Forkmann, 1981;
Akashi et al., 1999), Sinningia cardinalis (Stich and Forkmann, l988a), Columnea
hybrida (Stich and Forkmann, l988b), Chrysanthemum morifolium (Stich eta/., 1988),
Gerbera hybrida (Martens and Forkmann, 1998), Torenia (Akashi eta/., 1999) and
from osmotically stressed Glycine max cell suspension cultures (Kochs and Grisebach,
1987). Isolation of the corresponding eDNA has been reported from Antirrhinum and
Torenia petals (Akashi eta/., 1999) and from Gerbera (Martens and Forkmann, 1999)
(Table 1).
FJ'H and F3'5'H are responsible for hydroxylation of the B-ring. These enzymes
hydroxylate both naringenin and dihydrokaempferol in positions 3' or 3', 5',
respectively. The hydroxylated dihydroflavonols serve as direct precursors for
anthocyanins. Hence, the type of anthocyanin, and thus flower color, ultimately
produced is already determined at this stage by the specific dihydroflavonol. If the B-
ring is hydroxylated at the 3' and 5' positions, dihydromyricetin is produced, which is a
direct precursor for the formation of the blue/purple delphinidins. When the
hydroxylation occurs only at the 3' position, dihydroquercetin (a precursor for the
red/pink cyanidins) is formed. When neither of the enzymes are active, i.e. no
hydroxylation occurs at 3' or 3',5' positions, dihydrokaempferol (a precursor for the
brick red pelargonidins) is produced. The activities of both enzymes are localized in the
microsomal fraction and the reaction requires NADPH and 0 2 (Forkmann, 1991 ). It was
first demonstrated in microsomal preparations from Haplopappus cell cultures (Fritsch
and Grisebach, 1975). The cytochrome P450 nature of the enzymes was further
confirmed by the observation that the cytochrome P450 inhibitors, tetcyclacis and keto-
conazole, strongly inhibit F3'H activity (Stich et al., 1988). Measurements ofF3'H and
F3'5'H activity have been reported from several plant systems producing cyanidin (for
F3'H) and delphinidin (for F3'5'H) (for details see Heller and Forkmann, 1988). A
number of F3'h and F3'5'h clones have been identified and characterized in the last
decade (Table 1) and due to their importance to the ornamental industry, some of them
have been patented (Holton eta/., 1993b). Recently, a novel gene (DijF) coding for a
cytochrome b5 protein that enhances F3'5'H activity was identified in Petunia (De
Vetten eta/., 1999).
FLS introduces a double bond between C-2 and C-3 in dihydroflavonols to form
flavonols. Dihydrokaempferol, dihydromyricetin and dihydroquercetin are substrates for
the production of kaempferol, myricetin and quercetin, respectively. The co-factor
 259

requirement of the enzyme is similar to that of the 2-oxoglutarate-dependent

dioxygenase (Britsch et a/., 1981). FLS has been characterized in Petroselinum cell
culture and flower extracts of Matthiola incana (Spribille and Forkmann, 1984),
Petunia hybrida (Forkmann eta/., 1986), Dianthus caryophyllus (Stich eta/., 1992b)
and tulip (Beerhues et al., 1989). Recently, cDNAs encoding FLS have been cloned
from Eustoma (GenBank accession no. AF240764), Matthiola (GenBank accession no.
AAB58800), Petunia (Holton eta/., 1993c), Chrysanthemum (patent no. US5859329)
and Dianthus (patent no. US5859329). Flavonols are crucial affectors of flower color
due to their role in co-pigmentation (Mol et al., 1998). Furthermore, as was shown in
several plant systems, flavonol production is inversely coordinated with anthocyanin
production. For example, high FLS enzyme activity and flavonol accumulation have
been observed in young flower buds of Dianthus, Matthiola and Petunia, which rapidly
decline with the onset of anthocyanin accumulation (Heller and Forkmann, 1993).
DFR catalyzes the reduction of dihydroflavonols to leucoanthocyanidins. Its activity
was first demonstrated in an enzyme preparation from Pseudostuga cell suspension
cultures, where the enzyme was related to catechin and proanthocyanidin formation
(Heller and Forkmann, 1988). Dependence of the reaction on NADPH was established
using extracts from flowers and other tissues of a number of plants, among them
Matthiola, Petunia, Callistephus, Dahlia, Dianthus and Chrysanthemum (for details see
Heller and Forkmann, 1993). In some plant systems, DFR activity has been shown to be
highly substrate-specific. For example, Petunia, Nicotiana and Lycopersicon (members
of the Solanaceae) DFRs do not reduce dihydrokaempferol. Dihydroquercetin, and even
more effectively, dihydromyricetin, are used as substrates for DFR in these plant
species and as a result, only delphinidin and cyanidin accumulate. When 3' and 5' B-ring
hydroxylations do not occur in these plants, dihydrokaempferol accumulates (Gerats et
al., 1982; Forkmann and Ruhnau, 1987; Holton and Cornish, 1995). On the other hand,
the DFR enzymes from Matthiola, Dahlia, Dianthus and Zea mays accept
dihydrokaempferol and dihydroquercetin as substrates, leading to the production of
pelargonidin and cyanidin, respectively. DFR from these plants can also reduce
dihydromyricetin (delphinidin precursor), even though delphinidins do not occur
naturally, probably due to the lack of F3'5'H activity. The first Dfr was cloned from
Antirrhinum majus (Martinet al., 1985). In recent years, several other Dfr genes have
been cloned and studied (Table 1).
ANS converts leucoanthocyanidins to anthocyanidins, the first step in the formation
of colored pigments. Until recently, the biochemistry of the conversion was ambiguous.
The 2-oxoglutarate-dependent character of ANS was suggested, mainly due to the high
homology of the Ans sequences from numerous plants (see Table 1) to 2-oxoglutarate-
dependent dioxygenases such as F3H and FLS (Menssen et al., 1990; Britsch et al.,
1992; Martin and Gerats, 1993). Saito eta/. (1999) showed the first in vitro activity of
ANS (from Perilla frutescens), producing colored anthocyanidins from
leucoanthocyanidins by 2-oxoglutarate- dependent oxidation of the precursor with
subsequent acidification. In this way it was shown, at least in vitro, that only one
enzyme is responsible for the conversion of leucoanthocyanidins to anthocyanidins. In
vivo, the acidification step may be performed by a specific dehydratase (Gerats and
Martin, 1992), or alternatively, it may take place in a non-enzymatic manner. Ans
eDNA has been isolated fromAntirrhinum majus (Martinet al., 1991), Petunia (Weiss
et a/., 1993), Forsythia (Rosati et a/., 1999), Perilla frutescens (Saito et a/., 1999),
260

Torenia (Nakajima eta/., 2000), Callistephus (GenBank accession no. AF015885) and
Dianthus (GenBank accession no. DCU82432).
UFGT-UDP-glucose:3-0-flavonoid glucosyltransferase-catalyzes the
glycosylation of the 3-hydroxy position of the anthocyanidin molecule. The most
commonly found sugar residue in flavonoid compounds is glucose (Vogt and Jones,
2000). Glycosylation of flavonoids is an important modification as it increases their
water solubility. This reaction is also required for acylation of the carbohydrate moiety
with carboxylic acid. The latter modification further increases the water solubility of the
molecule and may also serve as a recognition signal for trans-membrane transport
(Heller and Forkmann, 1988). Enzymatic formation of anthocyanidin 3-0- glucosides
was first demonstrated in pollen of Zea mays (Larson and Lonergan, 1972) and
seedlings of red cabbage (Saleh eta/., 1976). Further characterization of a glycosylating
enzyme came from feeding experiments combined with genetic analyses in numerous
plant systems, i.e. Matthiola incana (Teusch et a/., 1986), Chrysanthemum segetum
(Stich eta/., 1997), Petunia hybrida (Kho eta/., 1978), Tulipa (Kleinehollenhorst eta/.,
1982), Silene dioica (Kamsteeg et a/., 1978), Dianthus, Cal/istephus, Antirrhinum
(Heller and Forkmann, 1988), and in cell cultures of Haplopappus gracilis (Saleh eta/.,
1978). Glycosylation of anthocyanidins can also occur at the 5-hydroxy position,
leading to the generation of 5-0-glycosylated anthocyanins in a variety of plant species,
such as Perilla frutescens, Verbena hybrida and Petunia hybrida (Heller and Forkmann,
1988). The anthocyanins can be further modified by additional glycosylations. Indeed,
such a modifying gene encoding 3-glucoside rhamnusyltransferase (3RT) was
characterized in Petunia (Brugliera eta/., 1994; Kroon eta/., 1994).
In addition to glycosylation, anthocyanins can be further acylated and methylated.
These modifications increase water solubility and protect the glycosides against
hydrolyzing enzymes. Evidence has been presented that acylation is also vital for
further modifications, such as methylation of the B-ring hydroxyl and for vacuolar
uptake of the anthocyanins (Heller and Forkmann, 1993). Anthocyanin 3-
acyltransferase activities have been reported for several flowers. For example, a purified
anthocyanin 5-aromatic acyltransferase (5AT) from Gentiana trijlora flowers and
anthocyanin 3-aromatic acyltransferase (3AT) from Perilla frutescens red leaves have
been characterized (Fujiwara eta/., 1997, 1998a). Moreover, several genes responsible
for the acylation and methylation of anthocyanins have been isolated (Fujiwara eta/.,
1998b; Holton and Cornish, 1995).
The resumed last step of the flavonoid biosynthetic pathway involves the transport
of anthocyanin to the vacuole. Glutathionation is a prerequisite for transport through a
glutathione pump in the tonoplast membrane (Marrs, 1996). Glutathione S-transferase
(GST) catalyzes conjugations of the tripeptide glutathione to a variety of substrates,
among them anthocyanins (Marrs, 1996). The maize Bronze2 (Bz2) gene was the first
characterized functional GST related to anthocyanin accumulation. In a bz2 mutant,
anthocyanins accumulate in the cytosol, leading to a bronze phenotype due to pigment
oxidation (Marrs eta/., 1995). TheAn9 gene from petunia was first identified as a type I
GST based on sequence homology and functional experiments (Alfenito et a/.. 1998).
However, further characterization of An9 revealed that this gene product does not act
directly as a GST but rather as a flavonoid- binding protein that acts as a cytoplasmic
flavonoid carrier protein (Mueller eta/., 2000). Research into the function of GSTs has
raised the question of whether anthocyanin glutathionation is indeed the last step of the
 261

flavonoid biosynthetic pathway, or if it is further modified (Alfenito eta/., 1998).

4. Transcription Factors Controlling Flavonoid Biosynthetic Genes

Numerous genes have been identified that influence the type, intensity and pattern of
flavonoid accumulation but do not encode flavonoid enzymes. Among these are
transcription factors that are directly involved in the control of the expression pattern of
structural flavonoid genes. It should be noted, however, that involvement of these
transcription factors in pigmentation is not necessarily limited to the regulation of genes
from the anthocyanin biosynthetic pathway (Mol eta/., 1998). Studies on the regulation
of pigment accumulation in maize identified several loci controlling tissue-specific
anthocyanin production. These comprise mainly two families of regulatory proteins,
MYB (i.e. C1 and PI) and MYC (i.e. Rand B) -like (Table 2) transcriptional regulators
(Ludwig and Wessler, 1990; Dooner eta/., 1991). The latter gene family, also referred
to as basic helix-loop-helix (bHLH) type, contains a stretch of basic amino acids
involved in DNA binding and a region that forms two a-helixes separated by a loop, a
feature thought to be involved in protein-protein interactions. The maize CI and PI
encode transcription factors related to c-Myb from chicken and contain a structurally
highly conserved N-terminal DNA-binding domain comprised of two repeats of ca. 50
amino acids that form helix-tum-helix structures. An a-helix region in the C-terminus of
the protein functions as a trans-activation domain (Martin and Paz-Ares, 1997).
Expression of all anthocyanin biosynthetic genes in maize have been shown to be
controlled by these MYB/MYC proteins (Table 2).
InAntirrhinum majus flowers, in contrast to maize, regulatory genes-De lila (Del),
Eluta and Rosea (Table 2)-activate the late (from F3h downstream) but not early
biosynthesis genes (Goodrich et a/., 1992; Jackson et al., 1992; Martin and Gerats,
1992). Interestingly, Rosea, identified as a Myb gene, homologous to CI from maize,
appears to interact with Delila, a MYC-type transcription factor similar to RIB from
maize (Martin and Gerats, 1993; Moyano eta/., 1996). Transcription factors regulating
early steps of the flavonoid biosynthetic pathway have also been identified in
Antirrhinum. Floral-specific expression was revealed for two of them, Myb305 and
Myb340 (Jackson eta/., 1991; Moyano eta/., 1996). Both genes were shown to activate
transcription of not only Chi but also a gene from a very early stage of the
phenylpropanoid pathway encoding phenylalanine ammonia-lyase (PAL).
Several regulatory genes affecting flower pigmentation (Ani, An2, An4, and Jafi 3)
were also cloned from Petunia hyhrida (Table 2). While Ani andJafi 3 contain a bHLH
domain, and are homologous to the R protein from maize and DEL from Antirrhinum
(Quattrocchio eta/., 1998; Spelt et al., 2000), AN2 and AN4 belong to the MYB-like
transcriptional activators with similarity to Cl from maize (Quattrocchio eta/., 1998,
1999). Ani, An2 and Jaf13 are required for transcription of the late structural genes
(from Dfr downstream) (Huits et al., 1994; Kroon et al., 1994; Alfenito et at., 1998;
Quattrocchio et al., 1998), but not for the expression of early genes (Chs, Chi or F3h)
(Quattrocchio et al., 1993). Activation of at least one early gene, Chs, was attributed to
PHMYB3, a MYB-like regulator expressed specifically in the petal epidermis (Solano
et at.. 1995). Furthermore, Ani andAn2 are also involved in controlling intracellular pH
in flowers (Quattrocchio et al., 1993).
262

TABLE 2. Regulatory genes controlling anthocyanin biosynthesis/accumulation.

Plants Genes
Myb MyclbHLH WD40 Others
Antirrhinum Rosea, Myb305, Myb340,Mixta Delila Eluta
Arabidopsis Papl,Pap2
Gerbera Gmycl
Maize Cl,Pl,P R, Lc, B, Sn, lnl pacl, vpl
Perilla Mybpl
Petunia Phmyb3, An2, An4 Jaj13,Anl Anll

Additional information on regulation of the anthocyanin biosynthetic pathway

comes from studies with additional plant systems (Table 2). For example, a Myc-type
gene (Gmycl) isolated from Gerbera was shown to play a role in regulating Dfr activity
in the corolla and carpel but not in other floral tissues (Elomaa et a/., 1998). On the
other hand, regulation of the Dfr promoter in Perillafrutescens (Gong eta/., 1999) was
ascribed to theMyb-like geneMybpl.
Recently, dominant-negative regulation of the anthocyanin biosynthetic pathway by
a transcription factor was demonstrated in maize. The Intensifier (Inl) locus encodes a
blll..H protein with high similarity to the R protein. Due to mis-splicing, the in
transcripts encode truncated proteins that act as dominant inhibitors (Burr eta/., 1996).
On the other hand, a dominant mutation that leads to strong activation of the pathway in
vegetative and floral tissues was characterized in Arabidopsis. The mutant was
generated through activation tagging and the gene(s) (Papl and Pap2) responsible for
the phenotype was identified as aMyb-like transcription factor (Borevitz eta/., 2000).
Little is known about the control of the aforementioned Myb and Myc regulators.
The Cl gene in maize is regulated by Viviparous 1 (Vpl) and the pigmentation block in
vpl kernels is due to lack of Cl expression (Carson eta/., 1997). The Pale Aleurone
Color 1 (Pacl), which when mutated causes a reduction in anthocyanin pigmentation in
maize seeds, was recently identified as a regulatory gene controlling activation of B/Cl
(Table 2) or P (Myb-type regulator of the phlobaphenes pathway) transcription factors
(Selinger and Chandler, 1999). The Anll locus, known to affect pigmentation in
petunia, was characterized and found to encode a cytosolic WD40-type protein known
to be involved in protein-protein interactions. AN11 was shown to post-translationally
regulate either the activity or the nuclear localization of MYB and/or blll..H-type
transcription factors (De Vetten eta/., 1997). Nevertheless, the expression of Anll in
non-pigmented tissues might suggest that AN11 has a broader function than only
control of anthocyanin biosynthesis (Mol eta/., 1998).
In several cases, regulatory genes have been shown to function in heterologous plant
systems, suggesting conserved function. For example, maize R-like genes were shown
to enhance the expression of flavonoid genes and the level of pigmentation in
Arabidopsis, tobacco and petunia (Lloyd et al., 1992; Goldsbrough eta/., 1996; Bradley
eta/., 1998). Nevertheless, it should be noted that expression of, for example, Lc eDNA
in Lisianthus and Pelargonium, fails to affect pigmentation (Bradley eta/., 1999). The
ability of Del to override control of pigmentation in heterologous systems seems to
depend, like Lc, on the plant system. While transformation of tomato with De/leads to
increased anthocyanin synthesis in flower and vegetative tissues, Del-transgenic
tobacco only accumulated increased levels of anthocyanins in flowers, whereas in
 263

Arabidopsis, Del had no strong phenotypic affect (Mooney et al., 1995). Hence, it is
apparent that a more detailed characterization of the transcription factors' mode of
action and of their interplay is needed for a better understanding of evolutionarily
determined functional diversity.

5. Determination of Final Color

While an active flavonoid biosynthetic pathway is a prerequisite for anthocyanin

pigmentation, the final flower color is affected by various cues. Light, temperature,
hormones, biotic and abiotic stresses, etc., are widely studied affectors of the expression
of flavonoid biosynthetic genes (Dixon eta/., 1994; Mol eta/., 1996; Winkel-Shirley,
1996; Weiss, 2000). In some cases, regulatory genes have also been found to be
controlled by these environmental/developmental signals (Cone et a/., 1993; Weiss,
2000). Other factors, such as vacuolar pH, metal ions, co-pigmentation or even optical
properties of the cell are also strong modulators of final color. For example, an increase
in vacuolar pH during senescence leads to a blue shift in color in many flowers. In
petunia, several loci affecting vacuolar pH were characterized and one of them (Ph6
gene) was isolated (Mol et al., 1998). Furthermore, the petunia Ani andAn2, regulating
expression of late biosynthetic genes, are also involved in controlling intracellular pH in
flowers (Quattrocchio et al., 1993; Mur, 1995). Interestingly, Ani and Ph6 were found
to be alleles of the same locus (Mol et al., 1998). A blue shift in flower color can also
occur due to the presence of metal ions in the vacuole or due to formation of stacked
complexes (co-pigmentation) between flavonols and anthocyanins (Brouillard and
Dangles, 1993). With respect to optical properties of the cell, mutations were
characterized (Mixta in Antirrhinum and Shp in Petunia) that affect cell shape and lead
to drastic changes in flower color with no effect on the leveVcomposition of
accumulated pigments (Noda et al., 1994; Van Houwelingen et a/., 1998). Substrate
availability is yet another factor that affects flower pigment composition. For example,
competition between FNS I and F3H for flavanones or between FLS and DFR for
dihydroflavonols can result in a shift in the ratio of anthocyanins to flavones and
flavonols, respectively, thereby changing the final color (Holton and Cornish, 1995;
Mol et a/., 1998). This brief overview of the interactions between different factors
regulating biosynthesis and those affecting the end product-anthocyanin, further
emphasizes the complexity of the processes that determine final flower color.

6. Genetic Engineering of Flower Color

To date, new ornamental varieties have been produced mainly by classical breeding.
Although classical breeding has led to great achievements in generating flowers in an
array of colors, it has several limitations, chief among them being the limited gene pool.
Indeed blue rose, carnation, chrysanthemum, tulip and gerbera flowers are still missing
from the marketplace, there is a lack of yellow color in pelargonium and lisianthus
flowers, and gypsophila flowers are mainly white.
264

TABLE 3. Genetic engineering of flower color in ornamentals

Plant Modification technique Flower color Reference
Antisense Sense Wild !IE Trans!!enic
Carnation Chs Pink White Gutterson, 199 5
Fht Orange White Zuker et al., 1999
Chrysanthemum Chs Pink White Courtney-Gutterson et
al., 1994
Chs Pink White Courtney-Gutterson et
al., 1994
Gerbera Chs Red Pink/cream Elomaa et al., 1993
Dfr Red Pink Elomaa et al., 1993
Del Red No change Elomaa and Teeri, 2001
Lisianthus Chs Purple White Deroles et al., 1998
Lc Various No change Bradley et al., 1999
colors
Pelargonium Lc Various No change Bradley et al., 1999
colors
Petunia Dfr Pale pink Orange Meyer et al., 1987
Chs Red White van der Krol et al., 1988
Chs Purple White van der Krol et al., 1990
F3'h Lilac Pink Brugliera et al., 1999
F3'5'h Pale pink Magenta Holton et al., 1993a
Chr White Pale yellow Davies et al., 1998
Fls Purple Red Holton et al., 1993c
3Rt Purple Pink/red sectors Brugliera et al., 1994
Lc White Reddish Bradley et al., 1998
Rose Chs Red Light red Firoozabady et al., 1994
Torenia Chs Violet Light violet Aida et al., 2000
Dfr Violet Light violet Aida et al., 2000
Chs Violet Light violet Aida et al., 2000
Dfr Violet Light violet Aida et al., 2000
Dfr Blue White Suzuki et al., 2000b
F3'5'h Blue Pink Suzuki et al., 2000b

The knowledge gained from biochemical and genetic studies of flavanoids (Elomaa
and Holton, 1994; Holton and Cornish, 1995; Mol eta/., 1998), together with great
progress in the application of genetic engineering techniques in many ornamentals
species (Zuker et a/., 1998), have enabled the production of transgenic flowers with
novel colors (Table 3). Two main strategies for altering flower color have been
successfully used. One is based on modulating (down- or up-regulating) the expression
of the native gene(s) involved in the anthocyanin biosynthetic pathway. The second is
based on introducing foreign genes encoding enzyme activities missing in the target
plant and thus allowing new branching of the pathway. The usefulness of the latter
strategy was demonstrated via expression of the maize Dfr in transgenic petunia (Meyer
et a/., 1987). This pioneering work led to the generation of pelargonidin-containing
brick-red petunia flowers. Recently, the F3'5'h missing in carnation was introduced into
this species leading to the production of carnation flowers with bluish hues (Tanaka et
a/., 1998). Similarly, introduction of Medicago sativa Chr, encoding chalcone
reductase, into petunia led to the generation of yellowish flowers due to accumulation of
a novel stable chalcone derivative not native to petunia (Davies eta/., 1998). Alteration
of flower color via modulation of native gene expression was demonstrated for the first
time by van der Krol eta/. (1988). Chs in antisense orientation was introduced into
 265

petunia and as a result, CHS activity was blocked and transgenes with white flowers
were obtained. To date, similar approaches have been successfully applied in numerous
ornamentals, including major cut flowers (Table 3). Interestingly, in carnation, blocked
F3h expression led to the production of flowers with not only novel colors but also
strongly increased fragrance. Analyses of fragrance compounds in the transgenic plants
revealed increased levels of benzoic acid derivatives, further emphasizing the
importance of understanding the interplay between different biosynthetic pathways
(Zuker eta/., 2001).
With the aim of modulating flower colors of ornamentals, not only flavonoid
structural genes but also regulatory genes have been employed. Bradley eta/. (1998)
generated Lc-transgenic petunias with red flowers from the white wild type. The red
color was mainly expressed in the petal veins and while intense in young flower buds,
the level of pigmentation tended to decline as the flower matured. Intense red
pigmentation was also observed in young vegetative parts of the transgenic petunia
plants. Nevertheless, the usefulness of Lc for flower color modification in additional
plant systems needs to be further assessed, as introduction of Lc into either pelargonium
or lisianthus failed to affect pigmentation (Bradley eta/., 1999). Applicability of Del
was tested in gerbera by Elomaa and Teeri (2001) and while flower color was not
intensified in Del-expressing transgenes, strong enhancement in the pigmentation ofleaf
and flower scapes was observed. Borevitz eta/. (2000) showed that overexpressing the
Papl gene in Arabidopsis and tobacco plants actives the production of anthocyanin,
leading to the development of intense red color in both vegetative and floral tissues. The
applicability of this gene to molecular breeding of flower color in ornamentals remains
to be evaluated.
In summary, the field of biotechnology has made a great step forward in recent years
with respect to both the functional analyses of genes and genetic engineering
techniques. Nevertheless, the flavonoid pathway, which has been studied for several
centuries, still holds many puzzles. Clearly, for fine control of pigmentation to
manipulate toward a specific hue, one needs to thoroughly understand the interplay
between the various outlined processes, as they can "make or break" flower appeal for
the consumer. First steps in this direction based on genetic engineering approaches have
proven the value of this undertaking from the point of view of both applied and basic
science.

7. Acknowledgements

The work in the authors, laboratory is supported by BARD, by the Israeli Ministry of
Agriculture and by the Israeli Ministry of Science.

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Zuker, A, Tzfira, T., and Vainstein, A (1998) Cut-flower improvement using genetic engineering,
Biotechnol. Adv. 16, 33-79.
Zuker, A, Tzfrra, T., Shklarman, E., Scovel, G., Ovadis, M., Itzhaki, H., Ben-Meir, H., Ahroni, A, and
Vainstein, A (1999) Transgenic carnation plants, FOCUS S-6.
Zuker, A, Tzfrra, T., Ben-Meir, H., Ovadis, M., Shklarman, E., Itzhaki, H., Forkmann, G., Martens, S., Neta-
Sharir, I., Weiss, D., and Vainstein, A (2001) Modification of flower color and fragrance by antisense
suppression of the flavanone 3-hydroxylase gene, Mol. Breed. (in press)
MOLECULAR CONTROL OF FLORAL PIGMENTATION: CAROTENOIDS

F.X. CUNNINGHAM, JR., E. GANTT

Department ofCell Biology and Molecular Genetics
University ofMaryland, College Park, MD 20742, USA

1. Introduction

To attract pollinators, plants have evolved flowers of elaborate architecture, alluring fragrance,
and captivating natural beauty. Hwnans have selected and bred a great nwnber ofplants solely
for the vibrant colors and distinctive patterns of pigmentation of their flowers. The yellow,
orange and red colors displayed by many flowers, fruits, roots and other non-green plant
tissues and organs quite often result from the synthesis and accwnulation, in specialized
plastids known as chromoplasts, of a class of isoprenoid compounds referred to as the
carotenoids.
The quite versatile carotenoids fulfill an extraordinary nwnber of primary and secondary
functions in plants. They are essential constituents of the photosynthetic reaction centers in the
thylakoid membranes where they protect in several ways against photo-oxidative damage
(Frank and Cogdell, 1996; Tracewell eta/., 2001). Carotenoids associated with the light-
harvesting chlorophyll-protein complexes absorb visible light and contribute energy to the
photosynthetic process (Yamamoto and Bassi, 1996). Other carotenoids in the membranes
participate in the dissipation of excess light energy absorbed by the light-harvesting antennae
(Niyogi, 2000), and these lipophilic compounds also influence the structural properties of
membranes (Britton, 1995; Young and Lowe, 200 1), provide substrate for the biosynthesis of
the plant growth regulator abscisic acid (Koomeef et a/., 1998), and are metabolized to
produce volatile compounds that are components of fruit aromas and floral fragrances
(Winterhalter, 1996; Leffingwell, 1999). Not least of the functions of carotenoids in plants is
the subject of this article: the provision of color in flowers and fruits for the attraction of
pollinators and agents of seed dispersal.
Carotenoid biosynthesis in the chloroplasts of plants is tightly coordinated with the
synthesis and assembly of other components of the photosynthetic apparatus. A handful of
carotenoids, typically including lutein, (3-carotene, violaxanthin, neoxanthin, and zeaxanthin,
comprise the preponderance of the pigment in many higher plant and algal species (Young,
1993a). The unique structures of these carotenoids enable them to fulfill specific
photochemical functions in the thylakoid membranes (Britton, 1995), and the relative amounts
of these individual carotenoids respond in predictable fashion to changes in environmental
conditions that affect the ability of the plant to utilize absorbed light for photosynthesis
(Young, 1993b).
Carotenoid synthesis and accwnulation in chromoplasts, where the function ofthe pigment
273
A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, 273-293.
© 2002 Kluwer Academic Publishers.
274

may be no more than to provide color, is typically not subject to the same regulation or
constraints that so carefully maintain and adjust the content and composition in chloroplasts.
Pigmentation in non-green portions of flowers and fruits can vary from little or none (e.g.
white petals) to massive amounts, many times more than is present in green tissues. Depending
on the plant species, carotenoids in chromoplasts may include one or more of those
characteristic of the photosynthetic tissues (e.g. lutein in marigold flower petals), consist of
intermediates earlier in the pathway (e.g. lycopene in tomato fruit), or be comprised of one or
more of the many species- or genus-specific carotenoids that have been identified in plants
(e.g. capsanthin and capsorubin in red pepper fruits). These latter pigments are commonly
more oxidized derivatives of carotenoids present in the photosynthetic tissues (Goodwin,
1980), and quite often the carotenoids in chromoplasts are esterified to fatty acids (as is lutein
in marigold flower petals; Moehs eta/., 2001 ).

2. Supply of Substrates for Carotenoid Synthesis

2.1. TWO ISOPRENOID PATHWAYS EXIST IN PLANT CELLS

Carotenoids are isoprenoids. That is, they are assembled from the five carbon building blocks
that are the precursors for all isoprenoid compounds: isopentenyl diphosphate (IPP) and its
allylic isomer dimethylallyl diphosphate (DMAPP). IPP and DMAPP are produced in at least
two different compartments and by two different pathways in plant cells (Fig. 1). A pathway in
the cytosoVendoplasmic reticulum that begins with acetyl CoA and proceeds via
hydroxymethylglutaryl CoA (HMG-CoA) and mevalonate (MVA) is akin to that employed by
animals and fungi (Bach et al., 1999). This linear MVA pathway leads directly to IPP. An IPP
isomerase (IPI) then reversibly converts IPP into DMAPP (reviewed in Ramos-Valdivia eta/.,
1997), which serves as the initial activated primer for the synthesis oflong-chain isoprenoids
(Wang and Ohnuma, 2000).
An entirely distinct pathway leading to IPP and DMAPP resides in plant plastids and in
many bacteria (Lichtenthaler, 1999; Rohmer, 1999). This pathway (Fig. 1) utilizes pyruvate
and glyceraldehyde-3-phosphate (GAP) as the initial substrates. The first step is a thiamine-
dependent, transketolase-like reaction (Sprenger et al., 1997) that yields the five-carbon
compound deoxyxylulose-5-phosphate (DOXP), also a precursor for the B vitamins, thiamine
and pyridoxal, which are synthesized in the plastids of plants (Julliard and Douce, 1991;
Julliard, 1992). Deoxyxylulose-5-phosphate synthase (DXS) and enzymes that catalyze several
subsequent steps in the DOXP pathway in the bacterium Escherichia coli have been
characterized (see Eisenreich eta/., 2001 for a recent review). However, the later and terminal
reactions of this pathway have not been elucidated.
Genetic evidence indicates that, at least for E. coli, IPP and DMAPP are separately
produced via a branching of the DOXP pathway (Rodriguez-Conception eta/., 2000).
Whether the DOXP pathway in plant plastids also branches has not been established. The
isotope labeling patterns of lutein and phytol extracted from cell cultures of Catharanthus
roseus supplied with deoxyxylulose labeled in different positions (Arigoni eta/., 1999) were
taken to indicate that IPP is a direct product of the DOXP pathway in this plant. It could not be
ascertained whether DMAPP is made directly or produced by isomerization of IPP.
 275

MVA pathway sterols, triterpenes, '\

acetyi-CoA Cytosol hopanoids, quinones,

prenylated proteins

;
HMG-CoA ....:H~M::.G::;R~..._ MVA
IPI 21PP
.. .. .. IPP IIIII .. DMAPP ~ FPP(C 15)
t
/
Plastid
DOXP pathway
t!!i.m]

lmt
pyruvate+ GAP IIIII 1,3-diPG
______J pyridoxal ~CH20PP
GGPP

Et :~
DOXP~
thiamine
methylerythritoi-5-P
~OH
YgbP,
HO zeaxanthin (orange yellow)

nX
YchB,
diterpenes OH
YgbB
~ l. .;. ~ ~ ~ ~
t
fragrances 'X_,._ l.
GcpE
HO,().:-~;;~;:;;:;r·'"''T''"
llmiJ'
? ? GPP(C1o) ~
OH
~q 4 "'
.,. HO v1olaxanthm (yellow)
IPP ~ DMAPP -.rubber
I I
f
11m . ~ HO'(YOH OH
• iSOprene ~·~ I I 01Y
r.:r.:r.r.t!
~
DMAPP
+ 3 IPP
cytokinins ~
neoxanthin (yellow)
chlorophylls, tocopherols, taxol, abscisic acid
GGPP (C2o) __... gibberellins, prenylated proteins, J
IB I/GGPP quinones, dolichols, phytoalexins Stroma ,

t
mmJ ZDS LCYb Membrane NCED
I
il!i'll
phytoene ., ..,. ..,. ..,. lycopene _..._....,~-carotene (9-cisl

(C4o) (CRTI In bacteria) CHYb lneoxatnthinl

LCYb NSY
CHYb CHYe ZEP
Ilutein I IIIII Izeaxanthin I ~ lviolaxanthin I
*
4 a-carotene

Haem~~occus CRTO VDE CCS lcap~~cum

astaxanthin capsanthin

Figure 1. Carotenoid biosynthesis in the context of isoprenoid biosynthesis in a plant cell. Enzymes for which there is
evidence of an influence on pathway flux are in white text within black boxes. Carotenoids common in green plants
are boxed. Structures of these five carotenoids and of the isoprenoid precursors IPP, DMAPP, and GGPP are
displayed, with numbering ofthe carbons shown for one of the rings of~-carotene.
276

The IPP isomerase enzyme (IPI) that catalyzes the tenninal step of the cytosolic MVA
pathway (see earlier), has been reported to also be present in plant chloroplasts (Fraser et al.,
2000), chromoplasts (Spurgeon et al., 1984; L11tzow and Beyer, 1988; Camara, 1993), and
etioplasts (Albrecht and Sandmann, 1994 ). Deduced polypeptide sequences for IPI of many
other plants have N-terminal extensions predictive of plastid targeting (Cunningham and
Gantt, 2000). If!PI is ubiquitous in plant plastids, then the DOXP pathway in these organelles
need not have retained both of the ancestral branches, leading separately to IPP and DMAPP,
that must exist in those organisms that lack an isomerase to interconvert the two.

2.2. WHICH PATHWAY SUPPLIES IPP AND DMAPP TO THE CHROMOPLASTS?

Chloroplasts are demonstrably self-sufficient in isoprenoid biosynthesis: newly fixed 14C02 is

rapidly incorporated into carotenoids and other isoprenoids in isolated intact chloroplasts (e.g.
Schulze-Siebert and Schultz, 1987). Unless sufficient carbon stores exist to supply the
endogenous DOXP pathway, the non-photosynthetic chromoplasts must import metabolites for
isoprenoid synthesis from the cytosol. Do these imported compounds feed into the plastid
DOXP pathway, or are IPP and/or DMAPP, synthesized by the cytosolic MVA pathway,
imported directly?
There is evidence to indicate that plastids can import IPP (Beyer et al., 1980;
Schulze-Siebert and Schultz, 1987; Soler et al., 1993; Fellermeier et al., 1999), and that this
capability may vary with the stage of chloroplast development (Heintze et al., 1990). There are
some indications, as well, of movement of longer chain isoprenoid precursors between plastids
and the cytosol (e.g. Adam eta/., 1999). However, a considerable number oflabeling studies
in a variety of plants (reviewed by Lichtenthaler, 1999) indicate that isoprenoids synthesized in
both chloroplasts and chromoplasts are derived predominantly from IPP and DMAPP
produced via the plastid DOXP pathway rather than via the cytosolic MVA pathway.
Additional support for a predominantly DOXP pathway origin for plastid isoprenoids is
provided by reports that chemical inhibition of the MVA pathway in ripening tomato fruit does
not diminish lycopene production (Rodriguez-Conception and Gruissem, 1999), but chemical
inhibition of the DOXP pathway does (Zeidler et al., 1998). Moreover, the nearly albino
phenotype exhibited by Arabidopsis mutants defective in a gene (CIAJ) that encodes DXS
(Araki et al., 2000; Estevez et al., 2000; also see Estevez et al., 200 I) would seem to indicate
a minimal import of products of the cytosolic MVA pathway by developing Arabidopsis
chloroplasts. Quite strikingly, these mutants could be rescued by application or inclusion in the
medium of deoxyxylulose, which is presumably phosphorylated in the plastids to yield DOXP.

2.3. DOES AVAILABILITY OF IPP/DMAPP LIMIT CAROTENOID BIOSYNTHESIS?

The biosynthesis of one molecule of a carotenoid requires two molecules ofDMAPP and six of
IPP (Fig. I), each of which is also utilized to produce such other plastid isoprenoids as the side
chains of quinones and the phytol tail of chlorophyll (Fig. I). Does the availability of IPP or
DMAPP limit carotenoid biosynthesis in plant plastids? If so, which step(s) might be limiting
or regulatory for flux through the DOXP pathway?
Most of what is known of regulation of the DOXP pathway comes from attempts to
enhance the production of carotenoids in strains of E. coli that have been engineered to
 277

produce these pigments. That the reaction catalyzed by DXS, the first step of the DOXP
pathway (the subsequent step, catalyzed by deoxyxylulose-5-phosphate reductoisomerase
(DXR), is actually the first step specific to isoprenoids as far as we know; Fig. 1), is a
significant bottleneck for isoprenoid synthesis in E. coli was first reported by Harker and
Bramley (1999). In plants, several studies (Bouvier et al., 1998; Lois et al., 2000; Veau et al.,
2000; Moehs et al., 2001) have reported an increase in or a high level of mRNA for DXS in
apparent anticipation of a subsequent rise in carotenoid synthesis. Such correlations do no
more than identify DXS as a plausible candidate for a rate-determining enzyme of isoprenoid
biosynthesis. However, there is now experimental evidence to indicate that this enzyme may, in
fact, limit isoprenoid synthesis in plants. Overexpression of a gene encoding DXS was found
to significantly increase the amounts of several isoprenoids (including carotenoids,
chlorophylls and a-tocopherol) in Arabidopsis (Estevez et al., 2001), while an antisense
construct for this gene yielded plants with lesser amounts of these same isoprenoids. Also,
injection of deoxyxylulose into mature green tomato fiuits was found to markedly accelerate
the accumulation of carotenoids and enhance the level of transcripts for DXS as well (Lois et
al., 2000).
A limitation of carotenoid biosynthesis in E. coli by the low activity of IPP isomerase in
this organism was discovered while screening plant and algal eDNA libraries for genes of
carotenoid biosynthesis (Kajiwara et al., 1997; Cunningham and Gantt, 1998). This finding is
at first glance surprising, inasmuch as the E. coli ipi gene is dispensable (Hahn et al., 1999),
and the DOXP pathway in this bacterium leads separately to IPP and DMAPP (see earlier).
We can only surmise that E. coli is not particularly adept at adjusting the relative amounts of
IPP and DMAPP produced via the DOXP pathway.
There are, as yet, no reports that increasing expression of ipi will increase or accelerate
accumulation of carotenoids or other isoprenoids in plant tissues. A recent analysis by Moehs
et al. (200 1) found that the mRNA level for IPI increases in anticipation of carotenoid
synthesis in marigold flowers, but not nearly to the degree observed for DXS transcripts.
Increase of a specific IPI transcript (one of two observed in RNA gel blots) was found to
correlate well with stress-induced synthesis of carotenoids in the green algaHaematococcus
pluvialis (Sun eta/., 1998). A threefold increase in IPI activity in developing etioplasts of
maize led Albrecht and Sandmann ( 1994) to speculate that this enzyme might be limiting for
carotenoid biosynthesis in these plastids.
We recently reported that plant and cyanobacterial homologues of the E. coli lytB gene
significantly increase carotenoid accumulation in E. coli, and proposed that the product of this
gene catalyzes a later step of the DOXP pathway (Cunningham et al., 2000). Information
relevant to the control of flux through the DOXP pathway in plants by LytB is not available,
but the observation that mRNA for this polypeptide is especially abundant in mint secretory
cells specialized for isoprenoid biosynthesis (Cunningham et at., 2000) is certainly suggestive.
Other enzymes for which increased expression has been shown to enhance carotenoid
accumulation in E. coli include DXR (Kim and Keasling, 2001; but see Miller et al., 2000 for
contrasting results), GGPP synthase (Wang et al., 1999), and glyceraldehyde-3-phosphate
dehydrogenase (GAPD; F.X. Cunningham, Jr. and E. Gantt, unpublished data).
278

3. The Pathway of Carotenoid Biosynthesis and Its Regulation

3.1. THECAROTENOIDPATIIWAY AND COLOR

Those requiring a more detailed account of the carotenoid pathway in plants should consult
one of several recent reviews (Britton, 1998; Cunningham and Gantt, 1998; Hirschberg, 1998;
Sandmann, 2001 ). The description contained herein is somewhat abbreviated and concerned
most particularly with how structural modifications influence carotenoid color.
The carotenoid pathway in plants can be considered as comprised of four stages. In the :first
stage the basic C40 carotenoid carbon skeleton is assembled. Three molecules of lPP are
sequentially added to one molecule of the activated primer molecule, DMAPP, to form the
linear C20 geranylgeranyl diphosphate (GGPP; Fig. 1). These additions are catalyzed by the
enzyme GGPP synthase (GGPS). Plant GGPS can also use allylic C10 (GPP, geranyl
diphosphate) and C15 (FPP, famesyl diphosphate) intermediates as primers (Wang and
Ohnuma, 2000). A head-to-head condensation of two GGPP, catalyzed by phytoene synthase
(PSY), then yields the C40 carotenoid phytoene (Fig. 1).
The second stage of the carotenoid pathway entails a series of four desaturation reactions
that are catalyzed by two related enzymes in plants: phytoene desaturase (PDS) and s -carotene
desaturase (ZDS). These reactions serve to extend the chain of alternating or conjugated
double bonds that largely determines the light absorption properties of the carotenoids.
Phytoene has three conjugated double bonds at the center of the molecule (Fig. 2E) and
therefore absorbs only in the UV region (Fig. 2A). Subsequent desaturations yield
intermediates with 5 (phytofluene), 7 (s-carotene), 9 (neurosporene) and 11 (lycopene)
conjugated double bonds, and progressively shift the absorption toward longer wavelengths
and, eventually, into the visible region of the spectrum (Fig. 2A). Thus phytofluene is
colorless, s-carotene is pale yellow, neurosporene is orange-yellow and lycopene, the
predominant pigment in ripe tomato fruits, is pink to red in color.
Linear to this point, the carotenoid pathway branches in the third stage of the pathway:
cyclization. Two types of rings are common in carotenoids of plants: 13- and a-rings.
Introduction of 13-rings at both ends of the linear and symmetricallycopene, reactions catalyzed
by lycopene 13-cyclase (LCYb ), leads to the orange-yellow 13-carotene, an essential constituent
of the photosynthetic membrane and the precursor for several other carotenoids with roles in
photosynthesis (Fig. l ). Though both compounds have 11 conjugated double bonds,
constraints on the double bonds in the rings of 13-carotene shift the absorption to shorter
wavelengths and reduce the spectral persistence (i.e. the peaks are not well defined) for this
carotenoid relative to lycopene (Fig. 2B). Introduction of an a-ring removes a double bond
from conjugation and shifts the absorption to shorter wavelengths but does not reduce the
spectral persistence. Thus the bicyclic a-carotene, with two a-rings and nine conjugated double
bonds (Fig. 2E), has an absorption spectrum (Fig. 2B) and yellow color much like that of the
linear neurosporene (not shown). The absorption spectrum and color of a-carotene (Fig. 2E),
with one 13- and one a-ring, are intermediate to those of 13-carotene and a-carotene. Because
lycopene a-cyclase (LCYe) of most plants adds only one a-ring to lycopene (Cunningham et
a/., 1996; Cunningham and Gantt, 2001 ), the pathway in plants typically proceeds only along
branches leading to carotenoids with one 13- and one a-ring (a-carotene and derivatives
thereof) or two 13-rings (13-carotene and derivatives).
 279

A B

Q)
0
c:
cu
.c
....
0
U)
.c
<(

0
250 300 350 400 450 500 550 echinenone
c
Wavelength (nm) + canthaxanthin
/
~
c:
E cu
.c
....
0
U)
.c
<(

/;;-carotene (pale yellow) 0

X 25 D
lycopene (red)
x5

~ Q)
0
c:
cu
.c
....
lutein

:~ 0
U)
.0
<(

350 400 450 500 550

Wavelength (nm)

Figure 2. Influence ofcarotenoid structure and concentration on the absorption oflight A Extending the conjugated
chain of double bonds from 3 (phytoene) to 7 (I;;-carotene) to 11 (lycopene). B. Addition oftwo P- or two £-rings to
lycopene. C. Addition of 4-keto groups to p-carotene. D. Increasing the concentration oflutein. E. Structures of
some carotenoids (also see Fig. 1). Carbon numbering is shown for phytoene and for one ring of £-carotene. Spectra
are for pigments in an HPLC mobile phase (Cunningham and Gantt, 2001 ).
280

The fourth stage of carotenoid biosynthesis in plants involves the addition of various
oxygen functions and other modifications to the basic bicyclic carotene structures (a- and f3-
carotene). Among the more common reactions in green plants are those leading to a hydroxyl
at the number 3 carbon of the f3- ande-rings (e.g. zeaxanthin and lutein; Fig. 1), anepoxide at
carbons 5 and 6 of the f3-ring (e.g. violaxanthin; Fig. 1), and the alleneofneoxanthin (Fig. 1).
Addition of a 3-hydroxyl has little effect on absorption: the spectra oflutein and zeaxanthin are
much like those of their respective precursors, a-carotene and f3-carotene. A 5-6 epoxide
removes a double bond from the conjugated chain, and the absorption is thereby shifted to
shorter wavelengths (not shown).
A variety of oxidation reactions that lead to a plethora of other carotenoids in non-green
plant tissues further expand the carotenoid color possibilities. Orange and red colors, atypical
of carotenoids in the photosynthetic membranes, may be obtained by modifications that extend
the conjugated chain of double bonds. For instance, introduction of a keto group at the number
4 carbon of the f3-ring of f3-carotene (as catalyzed by the H aematococcus pluvialis CrtO gene
product; Lotan and Hirschberg, 1995) yields the orange carotenoid echinenone with 12
conjugated double bonds (Fig. 2E). The absorption spectrum of echinenone is both red-shifted
and less well defined, compared to that of f3-carotene (Fig. 2C). Addition of a second keto
group to form the orange red canthaxanthin (Fig. 2E) shifts the absorption even further toward
the red (Fig. 2C). Capsanthin, the red pigment of pepper fruits (not shown) is produced from
violaxanthin in a reaction catalyzed by the enzyme capsanthin-capsorubin synthase (CCS;
Bouvier eta/., 1994). An orange to reddish color can also be achieved by accumulating a large
quantity of a yellow carotenoid. In marigold, for example, varieties with flowers ranging in
color from pale yellow to red owe their color to an accumulation of the yellow lutein (Moehs et
a/., 2001 ). Increasing the concentration oflutein effectively broadens the absorption (Fig. 2D),
enabling a more complete capture of longer wavelengths and thereby shifting the perceived
color towards the red.
With the exception of the &-ring hydroxylase (CHYe; Fig. 1), genes for each ofthe enzymes
of the carotenoid pathway in plants have been identified. Table 1 lists known genes and
enzymes of the pathway, some other isoprenoid genes of interest, and their incidence in the
completely sequenced genome ofArabidops is. Surprisingly, a homologue of genes reported to
encode neoxanthin synthase (NSY) in tomato (Bouvier eta/., 2000) and potato (Al-Babili et
a/., 2000) is lacking inArabidopsis. Polypeptides predicted by the tomato and potato Nsy are
similar in sequence to LCYb, and the tomato NSY has been reported by another group (Ronen
et al., 2000) to have f3-cyclase activity and account for the greatly enhanced accumulation of
f3-carotene in the yellow fruits of the tomato B mutant. It may be that theArabidopsis LCYb
functions as both f3-cyclase and NSY. Otherwise, an as yet unidentified gene inArabidopsis
must encode an enzyme with neoxanthin synthase activity.
The functioning of the carotenoid pathway requires more than the enzymes that catalyze the
biochemical reactions. A plastid terminal oxidase (PTOX) is needed for the desaturation
reactions catalyzed by PDS and WS in non-green plastids. A null mutation in Ptox results in
the accumulation of phytoene in tomato fruits (the ghost mutant; Josse et a/., 2000) and a
variegated green and white-sectored phenotype inArabidopsis (the immutans mutant; Carol et
a/., 1999; Wu et a/., 1999). Green sectors are thought to derive from cells in which plastid
development proceeds sufficiently to produce a redox chain that can accept electrons from
PDS and ZDS. White sectors presumably arise where irreversible damage from chlorophyll-
 281

sensitized photooxidation occurs before the photoprotective carotenoids can be produced.

TABLE 1. Genes of isoprenoid and carotenoid biosynthesis in the Arabidopsis genome

Gene Enzyme or product Family members

dxs deoxyxylulose-5-phosphate synthase 3
dxr deoxyxylulose-5-phosphate reductoisomerase I
lytB LytB protein
ipi isopentenyl diphosphate isomerase 2
gps geranyl diphosphate synthase 3
fps famesyl diphosphate synthase 2
ggps geranylgeranyl diphosphate synthase 11
Psy phytoene synthase 1
Pds phytoene desaturase 1
Zds 1;-carotene desaturase
Lcy-b lycopene ~-cyclase
Lcy-e lycopene s-cyclase
Chy-b ~-ring hydroxylase 2
Chy-e s-ring hydroxylase not yet identified
Zep zeaxanthin epoxidase 5
Vde violaxanthin de-epoxidase 2 ( + 1 distant relative)
Nsy neoxanthin synthase no homologue found
Ccs capsanthin-capsorubin synthase none
crtS carotenoid isomerase 1 (+ 2-3 distant relatives)
Ptox plastid terminal oxidase 1 (+ 6 distant relatives)
Pap plastoglobule-associated protein (fibrillin) 3 (+ 4-6 distant relatives)

Phytoene produced by the carotenoid pathway in plants is in the form of the central cis
geometrical isomer ( 15, 15'-cis; Fig. 2E), whereas ~-caroteneformed later in the pathway is in
the all-trans configuration (Fig. 1; Goodwin, 1980). It has long been thought that an enzyme
must exist to carry out a cis-to-trans isomerization reaction at some point in the pathway. The
situation is actually more complicated than it first appears. A tetra-cis isomer of lycopene
referred to as prolycopene (7 ,9, 7' ,9'-tetra-cis-lycopene) accumulates in fruits of the tangerine
tomato mutant (Qureshi et al., 1974), in a dark-grown mutant of Scenedesmus obliquus
(Romer et al., 1991 ), in a cell-free carotenogenic system from daffodil chromoplasts (Beyer et
al., 1989), and in a phytoene-accumulating E. coli strain that expresses theArabidopsis Pds
and Zds (Bartley et al., 1999). In the daffodil cell-free system, prolycopene could be converted
to ~-carotene only after conversion to the all-trans form, a reaction that required NADPH
(Beyer eta!., 1991 ). A gene (crtS) encoding an isomerase enzyme inArabidopsis has recently
been identified (Barry Pogson, personal communication). Quite unexpectedly, it encodes a
polypeptide distantly related to bacterial-type phytoene desaturases (crti).

3.2. REGULATION OF CAROTENOID BIOSYNTHESIS IN PLANT PLASTIDS

What determines the composition and the amounts of carotenoids accumulated in

chromoplasts? Which enzyme(s) controls or limits flux through the pathway in plants? We will
consider what is known for each enzyme of the pathway in turn.
282

3.2.1. GGPPSynthase
As many as 11 genes in Arabidopsis encode GGPS (Table 1). The large gene family is
explained in part by a need for isoforms targeted to the endoplasmic reticulum, mitochondria
and plastids (Okada eta/., 2000), but stands in conspicuous contrast to the mere two genes
that encode IPI, the enzyme that precedes GGPS in the pathway (Fig. 1). An important
question is whether one or more of the plastid-targeted GGPS isoforms is dedicated to
carotenoid synthesis. This question is of particular interest because of reports that GGPS can
combine with PSY and IPI to form a complex that is associated with the internal membranes of
the plastids (Spurgeon eta/., 1984; Camara, 1993; Schledz eta/., 1996; Fraser eta/., 2000)
or, in etiolated plastids, with the prolamellar body (Welsch eta/., 2000). Availability of any of
these three enzymes could determine the abundance of the PSY complex and thereby influence
the rate of carotenoid synthesis.
The few measurements that have been made for GGPS transcript abundance in
carotenogenic plant tissues are consistent with transcriptional regulation. An anticipatory
increase in mRNA for GGPS has been found for marigold flowers (Moehs eta/., 2001 ), and
steady-state mRNA for a pepper chromoplast GGPS is substantially increased during the
chloroplast-to-chromoplast transition in pepper fruits (Kuntz eta/., 1992). More importantly,
an increase in the GGPS activity in pepper chromoplasts was also found.
There is not yet any direct experimental proof that the amount or activity of GGPS limits
carotenoid synthesis in chromoplasts, but there are indications that it could be so in tomato. In
addition to carotenoids, a number of other compounds made in the plastid require GGPP as a
precursor, among them the gibberellins (GA). In transgenic tomato constitutively
overexpressing PSY, amounts of carotenoids in the fruits were increased and GA levels greatly
reduced (Table 2; Fray eta/., 1995). Dwarf plants resulted, arguably as a consequence ofGA
deficiency. For tomato with an antisense PSY construct, the fruit carotenoids were drastically
reduced and GA levels were much higher than in the wild type (Table 2; Fraser eta/., 1995).1t
would seem, therefore, that the carotenoid and GA pathways are in direct competition for
substrate, and the GA pathway, at least, is limited by its availability. The competition,
however, could just as well be for GGPS as for substrate, with sequestration in the PSY
complex rendering this enzyme inaccessible to other pathways that might require it.

3.2.2. Phytoene Synthase

PSY catalyzes the first step specific to the carotenoid pathway and, moreover, catalyzes a
reaction that converts a soluble compound (GGPP) to a lipophilic one (phytoene). It is the
most logical candidate for a rate-determining or regulatory enzyme of the carotenoid pathway
and several lines of evidence indicate that it is so. As already mentioned, constitutive, high-
level expression ofPSY in tomato is accompanied by an increase in carotenoid accumulation
in the fruits (Fray et al., 1995). Transgenic canola (Brassica napus) expressing a bacterial
phytoene synthase gene (criB) under control of an endosperm-specific promoter (Table 2;
Shewmaker et a/., 1999) provides an even more striking demonstration of the influence of
phytoene synthase on flux into and through the carotenoid pathway. Carotenoids in wild-type
canola seeds are in low abundance and of a composition characteristic ofchloroplast pigments.
Carotenoids in seeds of the transgenic plants were up to 50-fold greater in amount, consisted
predominantly of a.- and f3-carotene, and were accumulated in chromoplast-like plastids.
That transcription of phytoene synthase is not the sole mechanism for regulation ofpathway
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flux is made quite evident by results of attempts to produce carotenoids in rice seeds. With a
strategy similar to that used for canola, but employing a plant Psy rather than crtB, Burkhardt
et a/. (1997) obtained only a small amount of phytoene in rice endospenn (Table 2;
carotenoids were not detected in seeds of the wild type). Co-expression of Psy with a bacterial
desaturase gene (crt/) enabled synthesis of lycopene and cyclic carotenoids, but the total
pigment remained quite low (Table 2; Ye eta/., 2000). The poor yield of carotenoids in the
starchy rice endospenn vs. the high yield in the oil-rich canola seeds may reflect the need for a
depot or "sink" to sequester the carotenoids (see below).

TABLE 2. Metabolic engineering of carotenoid biosynthesis in non-green plant tissues

Plant Gene(s) and promoter(s) Consequence(s) Reference(s)

tomato Psy-1 (fruit-specific isofonn) Almost complete absence of carotenoids in Bird et al., 1991
antisense, constitutive flowers and fruit Gibberellins enhanced. Bramley et al., 1992
Fraser et al., 1995
Psy-1 (fruit-specific isofonn) Dwarf plants; up to twofold increase in fruit Fray et al., 1995
constitutive carotenoids. Gibberellin deficiency.
crtl (bacterial desaturase) More 13-ring carotenoids at expense of ROmer et al., 2000
constitutive lycopene and lower total pigment in fruit.
Lcy-b sense fruit-specific Significant increase in l3-carotene and a Rosati et al., 2000
Lcy-b antisense fruit-specific slight increase in lycopene in the fruit.
tobacco crtO flower-specific Red ketocarotenoid esters accumulate in the Mann et al., 2000
(tomato Pds promoter) nectary (vs. yellow lutein in wild type).
canola crtB endosperm-specific High concentration of phytoene, a- and 13- Shewmaker et al., 1999
(napin promoter) carotene in seeds (vs. low and mostly lutein
in wild type). Altered plastid morphology.
rice Psy Small amount of phytoene in the seed. Burkhardt et al., 1997
Psy + crtl (± Lcy-b ). For Psy, Some phytoene, and small amounts of 13- Ye eta/., 2000
constitutive; others are carotene and xanthophylls in seeds. Lcy-b
endosperm-specific (glutelin) increases 13-carotene and xanthophylls.
Notes: crtl is a bacterial gene encoding a desaturase that catalyzes four desaturations, leading from phytoene to
lycopene, that otherwise require the two plant enzymes PDS and ZDS. crtB is a bacterial phytoene synthase gene.

There is evidence of post-translational regulation of PSY activity in plants. In daffodil

flowers an inactive, soluble pool of PSY becomes functional only after association with
chromoplast membranes, a process dependent upon ATP (Bonk eta/., 1997) and requiring
galactolipid to yield an active enzyme (Schledz eta/., 1996). Similarly, a cryptic pool ofPSY
in the prolamellar body of Sinapis alba etioplasts is activated only when chloroplast
development is initiated by illumination, and the enzyme becomes associated with membranes
(Welsch eta/., 2000).
There appear to be feedback mechanisms that affect both transcription of Psy and activity
of the enzyme. Chemical inhibition of the pathway in daffodil flowers by the cyclase inhibitor
CPTA resulted not only in the accumulation oflycopene but also in a doubling of the overall
carotenoid content (Al-Babili eta/., 1999). Transcripts for and amounts of PSY, PDS and
LCYb increased, with PSY most affected. Similarly, treatment of pepper leaves with the
PDS/ZDS inhibitor J852 inhibited fonnation of colored carotenoids and caused an increase in
284

total carotenoids; however, message levels for PSY, PDS, LCY (and PTOX) did not change
significantly (Simkin et al., 2000). The increases in carotenoid accumulation induced by
CPTA and J852 could be due to relaxation of feedback inhibition mediated by ~-carotene or a
subsequent product of the pathway. Consistent with this hypothesis, Fraser et al. (2000) found
that the activity of the tomato chloroplast phytoene synthase (PSY-2) is inhibited by ~­
carotene (and by chlorophyll) but not by linear carotenoids (phytoene, l;;-carotene and
lycopene) in vitro. Surprisingly, little is otherwise known regarding allosteric or feedback
regulation of carotenogenic enzymes in plants.

3.2.3. Phytoene and (-Carotene Desaturases

As for PSY, and indeed for most enzymes of the pathway, transcripts for PDS have been found
to increase in advance of carotenoid synthesis in chromoplasts of a number ofplants, including
tomato (Peeker et al., 1992; Giuliano et al., 1993) and marigold (Moehs et al., 200 l ). An
increase in enzyme protein level with induction of carotenogenesis was demonstrated for the
green alga Haematococcus pluvialis (Grunewald et al., 2000). Inhibition of carotenoid
synthesis by the PDS inhibitor norflurazon resulted in increased transcription from a tomato
Pds promoter in transgenic tobacco plants (Corona et al., 1996), but an increase in PDS
transcript was not observed in norflurazon-treatedArabidopsis (Wetzel and Rodermel, 1998).
An electron acceptor is required for PDS and ZDS function. It has been reported that the
enzymatic activity ofPDS in daffodil flowers depends on the redox state of quinones in the
chromoplast membranes (Nievelstein et al., 1995). This observation reveals what could be an
important mechanism for the control of pathway flux, but it remains to be demonstrated that it
is actually employed for regulation of the pathway in vivo.

3.2.4. Cyclases and Enzymes ofXanthophyll Biosynthesis

The hypothesis that relative activities of the lycopene ~-and s-cyclases determine flux into the
pathway branches leading to ~.~-and ~.s-carotenoids (Cunningham et al., 1996; Peeker et al.,
1996) now has substantial support (Ronen et al., 1999, 2000; Pogson and Rissler, 2000;
Moehs et al., 2001). For example, transgenic Arabidopsis plants overexpressing Lcy-e
accumulate nearly twofold the lutein found in the wild type (Pogson and Rissler, 2000). This is
a major alteration since lutein is the predominant pigment in chloroplasts.
In transgenic Arabidopsis constitutively expressing an antisense Chy-b, the ~.~-ring
xanthophylls (violaxanthin, neoxanthin and zeaxanthin; see Fig. 1) were very much reduced
but the amount of their precursor, ~-carotene, was elevated so that the total carotenoid content
was only slightly lower (Pogson and Rissler, 2000; Rissler and Pogson, 2001). Several
mutations that affect xanthophyll biosynthesis inArabidopsis give much the same result: the
loss of a specific carotenoid is compensated for by increases in others so that total pigment
content remains at about the same level (DellaPenna, 1999). These results make clear that
robust feedback mechanisms exert control over pathway flux in chloroplasts.

3.2.5. Global Regulatory Control

An understanding of the regulatory mechanisms that maintain and adjust the levels and
composition of the carotenoid pigments in chloroplasts and chromoplasts will not be achieved
by analysis only of genes that encode enzymes of the pathway. An alternative strategy that has
begun to reveal some of the regulatory factors involved in control of carotenogenesis in
 285

Arabidopsis (DellaPenna, 1999), tomato (Mustilli et a/., 1999; Ronen et a/., 2000) and
cauliflower (Li eta/., 200 I), is the selection and analysis of pigment mutants. A cauliflower
mutant (Or) that accumulates very high levels of ~-carotene in tissues normally lacking in
carotenoids is particularly intriguing. Steady-state mRNA levels in these tissues for an
extensive list of isoprenoid and carotenoid pathway enzymes (including those encoding DXS,
IPI, GGPS, PSY, and PDS) were found not to differ between the wild-type and mutant strain
(Li et a/., 200 I). The identification of the genetic basis for this precocious pigment
accumulation, hypothesized to be a mutation affecting a regulatory factor, is certain to be
revealing.
Another strategy that may soon shed light on regulation of the carotenoid pathway makes
use of the natural variation in pigmentation in chromoplast tissues of related species or of
varieties of the same species. High-resolution mapping of what are called quantitative trait loci
(QTLs) for pigment accumulation has been carried out for tomato (e. g. Bemacchi eta/., 1998;
Chen et a/., 1999) and pepper (Thorup et a/., 2000). One of three QTLs for carotenoid
accumulation in pepper fruit was found to co-segregate with Psy. Identification of genes or
regulatory regions responsible for the quantitative differences associated with QTLs is likely to
yield new insights into the control of carotenoid biosynthesis in plants.

4. Sites of Accumulation of Carotenoids in Chromoplasts

4.1. STRUCTURES AND POLYPEPTIDES

A number of specialized structures are associated with carotenoid accumulation in

chromoplasts, including those referred to as fibrils and tubules (reviewed in Camara et a/.,
1995). In some plants the internal chromoplast membranes proliferate to accommodate the
carotenoids (e.g. in daffodil), and in others there are no specific structures: the pigment simply
forms crystals (as for lycopene in tomato fruits). Tubules and fibrils and other such structures
appear to derive or originate from plastoglobules (PG), which themselves may be the most
common repositories of carotenoids in chromoplasts (Camara eta/., 1995). PG, however, are
neither restricted to chromoplasts nor dedicated to carotenoid accumulation.
Associated with PG (and fibrils and tubules) in chromoplasts, chloroplasts, and non-
pigmented plastids are members of a family of polypeptides variously referred to as
carotenoid-associated proteins, fibrillins, Chr proteins, plastoglobule-associated proteins
(PAP), and plastid lipid-associated proteins (reviewed in Vishnevetsky eta/., 1999a). Models
attributing a structural role to these proteins have been presented (Deruere et a/., 1994;
Vishnevetsky eta/., 1999a), but whether or not there is a specific interaction betweenPAP and
carotenoids remains unknown. The functions of PG in plant metabolism are not well
understood. Several recent studies (Giuamet eta/., 1999; Monte eta/., 1999; Rey eta/., 2000;
Smith eta/., 2000) suggest roles for PG and PAP in a repair process whereby damaged
components of the photosynthetic apparatus are removed from the thylakoid membrane and
recycled. The formation of specialized plastids dedicated to the accumulation of carotenoid
pigments (i.e. chromoplasts) may be a recent adaptation, for the purpose of visual display, of
this apparently ancient (cyanobacteria contain PAP genes) repair process.
286

4.2. IS A DEPOT OR "SINK" REQUIRED FOR CAROTENOID ACCUMULATION?

The assembly of the more specialized chromoplast structures in which carotenoids reside
appears to depend directly on the availability of the pigments. Chemical inhibition of
carotenoid synthesis greatly reduces the number of tubules or fibrils in developing
chromoplasts of pepper (Simpson and Lee, 1977; Deruere eta/., 1994) and other plants (e.g.
Emter et al., 1990; Wrischer et al., 1998). Amounts of specific PAP proteins in chromoplasts
are likewise very much diminished when carotenoid synthesis is inhibited (Vishnevetsky eta/.,
1999b). The inhibitors that were used in these various studies block the cyclization or
desaturation reactions and result in accumulation of either lycopene or phytoene, (_;;-carotene
and other acyclic carotenoid precursors. The consequent lack of tubules or fibrils in
chromoplasts of the treated plants therefore implies that cyclic carotenoids are required for
stable assembly of these specialized structures. The structural requirements for the carotenoid
are likely to be even more specific than this. Fibrils and tubules commonly contain
xanthophylls with fatty acids esterified to one or both rings.
Are PG, fibrils, tubules or some other form of "sink" required for accumulation of the
carotenoids? The answer in many cases appears to be yes. Perhaps the best evidence comes
from a study on the alga Dunaliella salina which can be induced to accumulate a prodigious
amount ofj3-carotene in PG in the chloroplast. "Minimally inhibitory concentrations" of two
compounds that block the synthesis of the triacylglycerols that largely constitute the PG were
found to drastically reduce both the number ofPG and the amount of 13-carotene with little
effect on photosynthesis and other cell parameters (Rabbani et a/., 1998). In contrast, the
accumulation oflycopene in tomato fruits is not accompanied by formation ofPG, fibrils or
tubules. Likewise, in pepper (Simpson and Lee, 1977) and daffodil (Al-Babili eta/., 1999)
treated with the cyclase inhibitor CPTA, the quite substantial amounts of lycopene that
accumulate are in crystals rather than in the fibrils and membranes that normally serve as the
carotenoid depots in chromoplasts of these plants. Such observations are consistent with the
idea (Rabbani eta/., 1998) that sequestration of the carotenoids serves as much or more to
relieve the end-product inhibition that tightly controls the pathway in green tissues as to
prevent destabilization of the membrane by these compounds at the site of their synthesis.

5. Manipulation of Floral Pigmentation by Metabolic Engineering

Alteration of flower color by metabolic engineering is already well established. However, most
of this work has involved the manipulation of structural genes in pathways leading to the other
major class of pigments exploited by plants to provide floral pigmentation: the anthocyanins
(Mol eta/., 1996, 1999; Ben-Meir eta/., 2001). What are the prospects for manipulation of
flower color by engineering the carotenoid pathway?
There are no major technical obstacles to alteration of flower color by manipulation of the
carotenoid pathway for a plant species that can be stably transformed and regenerated. Genes
for essentially all of the enzymes in the pathway leading to the carotenoids of the
photosynthetic apparatus have now been identified (Fig.1 and Table 1). These, together with
genes for enzymes leading to "secondary" carotenoids such as canthaxanthin and capsanthin,
provide a broad color palette that ranges from a pale yellow to orange to a deep dark red, with
 287

a range of subtle tonal variations throughout. An expanding list of genes available from
bacteria and photosynthetic bacteria (e.g. see Sandmann et al., 1999) extends the color palette
into the purple, and these could be used to produce flowers with unusual colors that are not in
the plant repertoire. DNA shufiling to create enzymes with novel activities promises a further
expansion of the color possibilities (Schmidt-Dannert, 2000), quite possibly to blue pigments.
A blue color is, in a sense, already available through the carotenoid-binding proteins of the
lobster carapace (Britton et al., 1997), which bind the red ketocarotenoid astaxanthin and shift
the absorption so substantially that the pigment-protein appears blue (until boiling of the
lobster denatures the protein).
Earlier attempts to manipulate carotenogenesis in plants have been concerned largely with
improving nutrition (Table 2; reviewed in van den Berget al., 2000). Though in many ways
irrelevant to the engineering of floral pigmentation, where color is the primary concern and the
constraints are fewer, these earlier experiments teach certain valuable lessons. Foremost
among these is the advisability of using promoters that restrict expression to the relevant time
and tissue. Promoters that confer patterns of gene expression useful for floral pigmentation are
available from earlier work on the metabolic engineering of the anthocyanin pathway (e.g.
Goodrich et al., 1992; Kobayashi et al., 1998; see also Mol et al., 1996 and 1999) and
elsewhere. Transgenic tobacco plants that express an algal ketolase (CrtO gene) and thereby
accumulate the red carotenoid astaxanthin in the normally yellow chromoplast-containing
tissue of the flower nectary (Table 2; Mann et al., 2000), are the first of what should soon be
an extensive list of plants wherein the visual appearance of the flower has been purposefully
altered by genetic manipulation of the carotenoid pathway.

Notes Added in Proof

A substantial increase in isoprenoid accumulation in Mentha piperita (mint) has been obtained
by constitutive, high-level expression of a eDNA encoding DXR (Mahmoud and Croteau,
2001).
The participation of the lytB gene product in the DOXP pathway of isoprenoid biosynthesis
has been confirmed in E. coli (Altincicek et al., 2001).
The carotenoid isomerase gene (crtS; see Table 1) has been identified inArabidopsis (Park
et al., 2001) and tomato (Isaacson et al., 2001 ). Prolamellar bodies are absent in etioplasts of
Arabidopsis crtS mutants, indicating a requirement for all-trans carotenoids in the assembly or
stability of these structures.

6. Acknowledgements

We thank Dean DellaPenna (Michigan State University, East Lansing), David F. Garvin
(USDA-ARS, Plant, Soil, and Nutrition Laboratory, Ithaca, NY), and Barry Pogson
(Australian National University, Canberra) for access to manuscripts and data prior to
publication. We are grateful to the NSF and the DOE for funding our research.
288

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MOLECULAR CONTROL OF FLORAL FRAGRANCE

N. DUDAREVA
Department of Horticulture and Landscape Architecture
Purdue University
West Lafayette, IN 47907, USA

1. Introduction

Flowers of many plants attract pollinators by producing and emitting volatile

compounds. The scent emitted by such flowers is typically a complex mixture of low-
molecular-mass compounds (100-200 Da) which gives the flower its unique,
characteristic fragrance. The chemical composition of flower fragrances varies widely
among species in terms of the number, identity, and relative amounts of constituent
volatile compounds (Knudsen and Tollsten, 1993; Knudsen et al., 1993). Such species-
specific floral odors can enhance pollinator specificity, which in turn can reduce the
chance of pollen loss and inappropriate interspecific pollination. Moreover, closely
related plant species that rely on different types of insects for pollination produce
different odors, reflecting the olfactory sensitivities or preferences of the pollinators
(Henderson, 1986; Raguso and Pichersky, 1995). Characteristic floral odors are
correlated with the type of pollinators. Species pollinated by bees and flies have sweet
scents, whereas those pollinated by beetles have strong musty, spicy, or fruity odors
(Dobson, 1994).
Investigations ofthe chemical compositions of floral scents began hundreds ofyears
ago as a result of the commercial value of floral volatiles in perfumery. Since that time,
the perfume industry has amassed a vast catalogue of floral scent compounds, most of
which can be synthetically produced. More recently, several physiological and
ecological studies have examined the functions of floral scent compounds in plant
biology. In addition to improving the efficiency of pollination, some of the volatile
compounds found in floral scent have important functions in vegetative processes. They
may function as chemical signals to attract natural enemies of herbivores to the
damaged plants (Pare and Tumlinson, 1997), or as airborne signals that activate defense
responses in the healthy tissues of infected plants (Farmer and Rayan, 1990; Shulaev et
al., 1997; Seskar et al., 1998). Furthermore, volatiles emitted from flowers and
vegetative parts may serve to repel herbivores (Pellmyr et al., 1987; Gershenzon and
Croteau, 1991). Despite the importance of floral scent compounds in plant biology,
floral scent research has, until recently, remained largely descriptive. More than 700
floral scent compounds have been described from 60 families of plants (Knudsen et al.,
1993), yet most of the biochemical pathways and enzymes involved in the biosynthesis
of these compounds have not yet been elucidated.
295
A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, 295-309.
© 2002 Kluwer Academic Publishers.
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The existence of scent biosynthetic enzymes in flower tissue was demonstrated for
the first time in 1994 by Pichersky and co-workers (Pichersky et al., 1994). Two years
later in the same laboratory, a gene encoding the enzyme linalool synthase, which
catalyzes the formation of the acyclic monoterpene linalool, a common floral scent
compound, was isolated from the sweet-smelling moth-pollinated flowers of Clarkia
breweri, a wild plant native to California (Dudareva et al., 1996). For several years, C.
breweri was the only model system in which enzymes and genes responsible for the
formation of scent volatiles in the flower had been isolated (in total, four genes). The
more recent investigation into the biogenesis of scent production in snapdragon flowers
has resulted in the isolation of an additional scent gene and has also shown that common
regulatory mechanisms are involved in floral scent production in different plant species
(Dudareva eta/., 2000). In this chapter, I summarize and update our knowledge of
biochemical pathways leading to scent biosynthesis, with a focus on the genes and
enzymes controlling these pathways, as well as on the mechanisms responsible for floral
scent production.

2. Biosynthetic Pathways

Floral fragrances belong to a broad category of secondary metabolites and are

dominated by terpenoids (monoterpenes and sesquiterpenes), phenylpropanoid, and
benzenoid compounds. Fatty acid derivatives and a range of other chemicals, especially
those containing nitrogen or sulfur, are also sometimes present (Croteau and Karp,
1991; Knudsen and Tollsten, 1993; Knudsen et al., 1993). Many of these floral scent
volatiles are also synthesized in vegetative tissue under specific conditions (mostly for
defense purposes), and some information concerning their biosynthesis is also available.
Terpenes, especially monoterpenes such as linalool, limonene, myrcene, and trans-B-
ocimene, but also some sesquiterpenes, such as farnesene, nerolidol and caryophyllene,
are very common constituents of floral scent (Fig. 1). Terpenes are also often found in
vegetative tissues, where they serve mostly as defense compounds. In work done mainly
with vegetative tissue, but also with daffodil petals, monoterpenes were found to be
synthesized in the plastidic compartment. In this cellular compartment, isopentenyl
pyrophosphate (IPP) is derived from the mevalonate-independent "Rohmer" pathway
(Lichtenthaler et al., 1997). IPP can be isomerized to dimethylallyl diphosphate
(DMAPP), and one molecule of IPP is condensed with one molecule of DMAPP in a
reaction catalyzed by the enzyme geranyl pyrophosphate synthase (GPPS) to form GPP,
the universal precursor of all the monoterpenes (Fig. IB).
Similar work with vegetative tissue has revealed that in the cytosol, IPP is derived
from the mevalonic acid pathway (McCaskill and Croteau, 1995), and that two
molecules of IPP and one molecule of DMAPP are condensed in a reaction catalyzed by
the enzyme farnesyl pyrophosphate synthase (FPPS) to form FPP, the universal
precursor of all the sesquiterpenes (Fig. 1A) (McGarvey and Groteau, 1995).
In the past few years, genes encoding the enzymes responsible for the synthesis of
many monoterpenes and sesquiterpenes have been identified and characterized
(Bohlmann et al., 1998) (Fig. 1). However to date, only the enzyme that catalyzes the
formation of the acyclic monoterpene linalool has been characterized in floral tissue.
 297

E-f3- farnesene

FPP

farnesol

\s
!}pinene \ PI linalool linalool oxide

limonene myrcene

trans.P,ocimene
1,8- cineole geraniol

Figure l. Pathways leading to volatile terpenes. Volatile compounds are shown with a gray background.
Enzymes that have been identified in the synthesis of volatile compounds in vegetative tissues are shown in
black, enzyme identified in floral tissues is shown in gray. Not all reactions or enzymes have been identified.
A. Pathways leading to sesquiterpenes synthesized in the cytosol. B. Biosynthesis of monoterpenes that occurs
in the plastids, although the location of the reactions leading to further modifications (e.g., linalool oxide) is
not yet clear.
298

The phenylpropanoids, derived from phenylalanine, constitute a large class of

secondary metabolites in plants. Many are intermediates in the synthesis of structural
cell components (e.g., lignin), pigments (e.g., anthocyanins), and defense compounds.
These intermediates are not usually volatile. However, several phenylpropanoids whose
carboxyl group at C9 is reduced (to either the aldehyde, alcohol, or alkane/alkene)
and/or which contain alkyl additions to the hydroxyl groups of the benzyl ring or to the
carboxyl group (i.e., ethers and esters) are volatiles (Fig. 2). Recent work with C.
breweri flowers has resulted in the identification and characterization of three enzymes
that catalyze the formation of floral volatiles from this group: (iso)methyleugenol,
benzylacetate, and methylsalicylate. The enzymes are, respectively, S-adenosyl-L-
methionine:(iso)eugenol 0-methyltransferase (IEMT), acetyl-CoA:benzylalcohol
acetyltransferase (BEAT), and S-adenosyl-L-methionine:salicylic acid carboxyl
methyltransferase (SAMT) (Wang et al., 1997; Dudareva et al., 1998a,b; Wang and
Pichersky, 1998; Ross et al., 1999). In addition, we have identified and characterized
the enzyme S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase
(BAMT), which catalyzes the formation of methylbenzoate in snapdragon flowers
(Dudareva et al., 2000). cDNAs encoding all these enzymes have also been
characterized.

3. Genes Involved in Floral Scent Production

3.1. LINALOOL SYNTHASE

The first scent biosynthetic gene isolated was the linalool synthase (LIS) gene from C.
breweri. Linalool, an acyclic monoterpene alcohol with a sweet, pleasant fragrance, is
one of the most frequently encountered floral scent compounds (Knudsen et al., 1993).
LIS was purified to homogeneity from stigmata of C. breweri flowers by employing
several chromatographic techniques (Pichersky et al., 1995). A protein-based cloning
strategy was used to obtain a eDNA encoding this protein from a C. breweri flower
eDNA library (Dudareva eta/., 1996). This enzyme produces exclusively S-linalool
from geranyl pyrophosphate in a one-step reaction (Fig. lB) (Pichersky et al., 1994). It
is a monomer with apparent molecular mass of76 kDa (determined by gel-permeation
chromatography and polyacrylamide gel electrophoresis), and has a strict requirement
for a divalent metal cofactor, preferentially Mn2+ (Pichersky et al., 1995). The LIS gene
is unique in the C. breweri genome and its coding region of 870 amino acids is
interrupted by 11 introns (Dudareva et al., 1996; Cseke et al., 1998). The complete
amino acid sequence of LIS, derived from the eDNA clone, showed that it is related to
several enzymes involved in terpene synthesis. LIS contains a metal-cofactor-binding
DDXXD motif in the second part of the protein, and the extensive sequence identity
with other terpene cyclases centered around this motif suggests that the active site of
LIS is present in this part of the protein (Dudareva et al., 1996; Cseke et al., 1998).
LIS was also isolated from a linalool-scented relative of C. breweri, Oenothera
arizonica, and from non-scented Clarkia concinna (Cseke eta/., 1998), the proposed
progenitor of C. breweri. Both sequence comparisons and comparisons of the location
of introns in the genes suggest that LIS is actually a composite gene. It includes a
 OOCHJ
~

~ .;7 IEMT .;7 I methyl-

.JSoeugenol
! _ : : :,. . I ----.. : : :,. .

COOH COOH COOH

J !

OH
p-coumaric acid
\
~.ru-=9
COOH CllzOH
.rCOCHJ
?

b· ? • ~ o~ v
benzylalcohol benzylacetate
benzoic acid" '
6
benzaldehyde

BAMT ~ ""' COOH

COOCHJ &
~ O H SAMT _____.
:::::,... ~·
salicylic acid
6
methylbenzoate
methylsalicylate

Figure 2. Pathways leading to volatile phenylpropanoids. Volatile compounds are shown with a gray background. The location of
synthesis of most phenolpropanoidslbenzenoids is not yet known, but is likely to be the cytosol and possibly the peroxisomes. IEMT,
SAMT and BAMT are methyltransferases that use SAM (not shown) as the methyl donor. BEAT is an acetyltransferase that uses acetyl- N
\0
CoA (not shown) as the acetyl donor. Since all these enzymes were identified in floral tissues, they are shown in gray. \0
300

portion of a copalyl pyrophosphate synthase-like sequence at its N-terminus coding

region, and a portion of another terpene synthase (limonene synthase-like) in most of its
second half(Cseke et al., 1998).

3.2. S-ADENOSYL-L-METIDONlNE:{ISO)EUGENOL 0-METHYLTRANSFERASE

Two volatile phenylpropanoids, methyleugenol and isomethyleugenol, are important

components of the floral scent of various species, including C. breweri (Ragusa and
Pichersky, 1995; Wang et al., 1997). They are produced in C. breweri flowers by the
action of a single enzyme, S-adenosyl-L-methionine:(iso)eugenol 0-methyltransferase
(IEMT), which catalyzes the transfer of a methyl group to the 4-0H group of the benzyl
ring of both eugenol and isoeugenol, using S-adenosyl-L-methionine {SAM) as a donor
of the methyl group (Fig. 2) (Wang et a/., 1997). IEMT is encoded by a single-copy
gene in the C. breweri genome. IEMT was copurified with caffeic acid 0-
methyltransferase (COM'lj from C. breweri petals and purified to homogeneity from a
bacterial expression system (Wang and Pichersky, 1998). A eDNA clone encoding
IEMT was isolated from a C. breweri flower eDNA library and its sequence was highly
homologous to that of COMT (83% sequence identity at the amino acid level) (Wang
and Pichersky, 1999). IEMT is active as a homodimer with a subunit molecular mass of
40 kDa and does not require any cofactors for enzymatic activity (Wang and Pichersky,
1998). TheN-terminal halfofiEMT has been shown to be involved in substrate binding
and also to determine its methylation regiospecificity (meta vs. para) (Wang and
Pichersky, 1998). Moreover, only seven amino acid residues account for most of the
substrate discrimination and methylation regiospecificity of both IEMT and COMT
(Wang and Pichersky, 1999).

3.3. ACETYL-CoA:BENZYLALCOHOL ACETYLTRANSFERASE

In many plant species, volatile esters contribute significantly to the total floral scent
output and are important in attracting insect pollinators. Benzylacetate in particular is
one of the most commonly found esters in moth-pollinated flowers (Knudsen and
Tollsten, 1993) and is often found in the aromas of other flowers as well (Knudsen et
a/., 1993; Van Dort et a/., 1993; Watanabe et a/., 1993). In C. breweri flowers,
benzylacetate is made from benzylalcohol and acetyl-CoA in a reaction catalyzed by the
enzyme acetyl-CoA:benzylalcohol acetyltransferase (BEAT) (Fig. 2) (Dudareva et al.,
l998b). The enzyme BEAT has been purified from C. breweri petals, a eDNA encoding
this enzyme has been isolated and characterized, and enzymatically active protein has
been expressed in Escherichia coli (Dudareva et a/., 1998a). Southern blot analysis
showed that there is a single copy of the BEAT gene in the C. breweri genome.
The protein encoded by BEAT eDNA has 433 amino acids, and its sequence shows
little overall similarity to any previously characterized proteins. However, a short
segment of 35 amino acids in the middle of the protein has some similarity to other
proteins believed to bind an acyl-CoA substrate. BEAT has recently been shown to be a
member of a large family of plant acyltransferases (Dudareva et a/., 1998a; St-Pierre et
al., 1998). A few enzymes belonging to this family, which catalyze the formation of
various non-volatile (with the exception ofbenzylacetate) secondary compounds (e.g.,
anthocyanins, phytoalexins), have been identified. However, based on the presence in
 301

the Arabidopsis genome of perhaps up to 70 such related genes, it is clear that the
functions of most of the members of this family remain to be determined (Dudareva and
Pichersky, 2000). Such a large family represents a rich source of enzymes that may be
involved in scent biosynthesis or that may serve as a pool for the evolution of new scent
enzymes.

3.4. S-ADENOSYL-L-METHIONINE:SALICYLIC ACID CARBOXYL

METHYL TRANSFERASE

Methylsalicylate is a benzenoid ester that is widespread in the plant world. It is not only
a common ingredient of floral scent, but is also a part of the plant defense response
mediated by salicylic acid (Knudsen and Tollsten, 1993; Shulaev et al., 1997; Seskar et
al., 1998). Methylsalicylate is made from salicylic acid and SAM as the methyl donor in
a reaction catalyzed by S-adenosyl-L-methionine:salicylic acid carboxyl
methyltransferase (SAMT) (Fig. 2) (Dudareva et a/., 1998a). SAMT was partially
purified from C. breweri petals (Ross eta/., 1999). The enzyme is highly specific for
salicylic acid; however, it does methylate benzoic acid, although its Km value for the
latter is much higher (Ross et al., 1999). A eDNA encoding SAMT was isolated from a
C. breweri flower eDNA library. The protein encoded by SAMT contains 359 amino
acids and shows no significant sequence similarity to any protein in the data bank
whose biochemical function is known. When C. breweri SAMT eDNA was expressed
in E. coli, the bacterial cells synthesized a functional SAMT protein with properties
nearly identical to those of the plant-purified enzyme. The bacterial cells were also able
to produce methylsalicylate when the culture medium was supplemented with salicylic
acid (Ross et a/., 1999).

3.5. S-ADENOSYL-L-METHIONINE:BENZOIC ACID CARBOXYL

METHYLTRANSFERASE

S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT) catalyzes

the transfer of the methyl group of SAM to the carboxyl group of benzoic acid to make
the volatile ester methylbenzoate (Fig. 2), one of the most abundant scent compounds of
snapdragon, Antirrhinum majus. The enzyme was purified from upper and lower petal
lobes of 5- to 10-day-old snapdragon flowers (floral tissue with the highest BAMT-
specific activity) using DE 53 anion exchange, Phenyl Sepharose 6 FF and Mono Q
chromatography (Murfitt et a/., 2000). Enzymatically active enzyme exists as a
homodimer and is highly specific for benzoic acid with no activity toward several other
naturally occurring substrates such as salicylic acid, cinnamic acid and their derivatives
(3-hydroxybenzoic acid, 4-hydroxybenzoic acid, benzylalcohol, 2-coumaric, 3-coumaric
and 4-coumaric acids) (Dudareva et al., 2000). A eDNA encoding BAMT was isolated
from a snapdragon petal-specific eDNA library using a protein-based cloning strategy
(Dudareva et al., 2000). The function of the clone was verified by comparing the
encoded amino acid sequence with the sequence obtained from purified plant protein
and by prokaryotic expression, which yielded an enzymatically active carboxyl
methyltransferase (Dudareva et al., 2000). The E. coli-expressed BAMT protein showed
the same substrate specificity as the native enzyme. Moreover, the culture medium of
the E. coli cells expressing BAMT contained a small amount of methylbenzoate, which
302

increased when the growing culture was supplemented with benzoic acid (Dudareva et
a/., 2000).
The deduced amino acid sequence of BAMT does not share any sequence similarity
to previously characterized proteins, including 0-methyltransferases, with the exception
ofSAMT from C. breweri, to which it is 400/o identical (Dudareva eta/., 2000). BAMT
and SAMT thus define a new class of plant carboxyl methyltransferases. They have in
common the ability to transfer the methyl group of SAM to a free carboxyl group on
either benzoic or salicylic acid, leading to the formation of methylbenzoate and
methylsalicylate, respectively. Arabidopsis has perhaps as many as 30 related genes of
this family, and several ESTs that are derived from genes of this family have been
reported from Arabidopsis and other species; however, none of the reactions catalyzed
by these proteins (with the exception of SAMT and BAMT) have been determined
(Dudareva and Pichersky, 2000). This family is also likely to include scent enzymes
such as the ones catalyzing the formation of methyljasmonate and methylcinnamate.

4. Regulation of Floral Scent Production

Despite the fact that production of volatile scent compounds appears to be very
widespread in the plant kingdom, our knowledge of the processes controlling their
biosynthesis is still limited and based, to date, on the analysis of two model systems -
moth-pollinated C. breweri and bee-pollinated snapdragon. In the last few years, the
production of floral scent compounds in plants has been shown to be under both spatial
and temporal control. These results are discussed below.

4.1. SPATIAL REGULATION- SPECIALIZED ORGANS AND TISSUES

In the flowers of many plant species, the petals are the principal emitters of volatiles,
although various other parts of the flower may also participate in fragrance emission
(Dobson, 1994; Dudareva eta/., 1999). While the same floral scent components are
often emitted from all parts of the flower (although not necessarily in the same amount
or rate), specific compounds may sometimes be emitted from only a subset of the floral
organs (Dobson et a/., 1990, 1996; Pichersky et a/., 1994; Mactavish and Menary,
1997). In addition, in some species, orchids for example, floral volatiles are emitted
from highly specialized "scent glands" (i.e., osmophores) within the flower (Stern et al.,
1986; Curry, 1987). It is not yet clear how prevalent scent glands are among other
scented flowers. So far, investigations of scent glands have been conducted mostly at
the anatomical level, and the question of whether such glands represent only sites of
scent-volatile emission or also of synthesis has not yet been addressed.
In both C. breweri and snapdragon flowers, most volatile emission occurs from the
petals. Identification of the enzymes responsible for the formation of these volatile
compounds allows a determination of how the levels of enzymatic activity are
distributed in different floral parts. When the activity levels of four enzymes (LIS,
IEMT, BEAT and SAMT) are calculated per total weight of each organ of C. breweri
flowers, the highest levels of activity of all these enzymes are found in the petals
(Dudareva et al., 1999). Other parts of the flower, however, also contain detectable
 303

levels of activity. In the case of LIS, the stigma actually contains higher levels of LIS-
specific activity than the petals. However, because the mass of the stigma of C. breweri
is so small compared to the mass of the petals, LIS activity in the petals still comprises
most of the activity present in the flower (Pichersky et al., 1994).
In snapdragon flowers, most of the total BAMT activity is found in the upper and
lower lobes of the petals, with much less activity present in the tube and anthers. None
of the remaining floral organs (pistils, sepals and ovaries) or leaves contain BAMT
activity. These results show that the production of volatile compounds in snapdragon
flowers is limited mostly to the upper and lower lobes, which appear to make almost
equal contributions to the whole-flower fragrance (Dudareva et al., 2000).
One unique aspect of scent production is that small organic molecules, produced
inside the cell and usually water-insoluble, have to move to the exterior of the cell and
evaporate. However, it was not known whether these compounds were synthesized at
the surface itself or whether they were transported there from adjacent cells. Use of in
situ hybridization and immunolocalization techniques has demonstrated that the
biosynthesis of volatile compounds is restricted to specific tissues at the site of
emission. LIS and IEMT genes have been shown to be expressed uniformly and almost
exclusively in cells of the epidermal layer of petals and other parts of C. breweri flowers
(Dudareva et al., 1996; Dudareva and Pichersky, 2000). Similar results were obtained
with snapdragon flowers, but the two epidermal layers of the petal, the inner layer lining
the throat of the tube and contiguous surface of the lobe, and the outer layer lining the
exterior surface of the tube and lobe, were differentially involved in floral scent
biosynthesis (Kolosova et al., 2001). The BAMT gene was expressed at a higher level
in the conical cells of the inner epidermal layer than in the cells of the outer epidermis
lining the surface exterior of the lobe (Fig. 3). By producing scent on the surfaces of
flower petals, which come into contact with a bee's body during landing, plants can
make scent production more concentrated. This can help to intensify bee odor delivery
to the nest thereby increasing the pollinator recruitment rate. When considered together,
these results strongly suggest that scent enzymes are active in epidermal cells of floral
organs, and that scent volatiles that are synthesized de novo there can easily escape into
the atmosphere. There is no information about the subcellular sites of biosynthesis of
scent compounds in C. breweri, although we have been able to show that
methylbenzoate is made in the cytosol in snapdragon flowers (Kolosova et al., 2001).

4.2. TEMPORAL REGULATION

A comparative analysis of volatiles emitted during the life span of C. breweri and
snapdragon flowers showed the developmental regulation of floral scent emission. Both
types of flowers follow a long-term pattern in which emission peaks within a few days
after anthesis and then declines gradually. The composition of the emitted odor
determines the attractiveness of the flowers to specific insects, and is therefore
responsible for pollinator selectivity. Indeed, newly opened young flowers of
snapdragon are not ready to function as pollen donors because their anthers have not yet
dehisced, and they produce fewer odors, which makes them less attractive to pollinators
than older flowers (Dudareva eta/., 2000). Moreover, investigations into the frequency
and duration of bumblebee visits to snapdragon flowers revealed that 1- and 2-day-old
304

flowers received fewer and shorter visits than 4-day-old and older flowers (Jones et a/.,
1998).

Figure 3. In situ localization of the BAMT protein in snapdragon petals. A. Flowers of Antirrhinum
majus. B. Scanning electron micrograph of conical cells from the inner epidermis of petal lobes. C. A
cross section of a petal of 7-day-old flower. Samples were hybridized with BAMT antibodies and
visualized by fluorescent FITC-conjugated secondary antibodies.

In C. breweri flowers, the activities of four enzymes involved in floral scent

production follow two different developmental patterns (Dudareva and Pichersky,
2000). The activities of the first group of enzymes, represented by LIS and SAMT,
increase in maturing buds and young flowers, peaking about 12 to 24 h ahead of peak
emission. LIS and SAMT activities then decline in old (5-day) C. breweri flowers, but
remain relatively high (40 to 50% of the maximum level) even though emission of
linalool and methylsalicylate has practically ceased. The activities of the second group
 305

of enzymes, represented by IEMT and BEAT, show little or no decline at the end of the
life span of the flower, although again, emission of corresponding volatile compounds
declines substantially. A minor difference in the developmental profiles of the latter two
enzymes is that IEMT levels peak on day 1 of anthesis and remains stable (Wang et al.,
1997), whereas BEAT activity does not peak until the fourth day after anthesis
(Dudareva et al., 1998a). The BAMT enzyme from snapdragon flowers appears to
belong to the first group, because its activity declines at the end of the life span of the
flower (9 to 12 days after anthesis; snapdragon flowers are longer-lived than those of C.
brewerz) to 46% of the maximum level, without concomitant emission of
methylbenzoate (Dudareva et al., 2000). The causes and consequences of high levels of
activity of biosynthetic enzymes in old flowers, without concomitant emission of the
volatile products, found in both model organisms, were until recently unknown. In the
case of snapdragon, it has been shown that the total amount of substrate in the cell is
involved in regulating biosynthesis and emission of flower volatiles, and that the low
emission of methylbenzoate in old flowers is due to low levels of benzoic acid in petal
tissue (Dudareva et al., 2000). The low level ofbenzoic acid in old flower petals (9-12
days after anthesis) may indicate that the earlier biochemical steps in the pathway are
blocked as the flower ages or that synthesized benzoic acid is required for some other
processes in the cells.
Expression of genes encoding scent biosynthetic enzymes in the C. breweri flower is
temporally regulated during flower development. The mRNAs encoding LIS, IEMT and
BEAT are first detected in petal cells just before the flower opens and their levels
increase until they peak at or around anthesis, and afterwards begin to decline
(Dudareva et al., 1996, 1998a; Wang et al., 1997). For all three genes, peak levels of
the mRNAs occurred 1 to 2 days ahead of enzyme activity and emission of the
corresponding compound. Similar results were found for these mRNAs in other parts of
the flower.
These data show a good positive correlation between the amount of mRNA, the
amount of protein and enzymatic activity for each of these three enzymes, and the
emission of the corresponding volatile component up to the second or third day post-
anthesis. Beyond that point, the levels of scent enzymes remain relatively high, despite
declining levels of the corresponding mRNA, and also without the concomitant
emission of volatiles (Dudareva et al., 1996, 1999).
Similar to C. breweri, the steady-state levels of BAMT mRNA in snapdragon petals
are developmentally regulated, being highest on day 4 after anthesis (Dudareva et al.,
2000). The amounts of methylbenzoate emission, BAMT activity, and mRNA in petals
all rise and fall simultaneously until the end of the life span of the flower, with mRNA
values peaking one day ahead of enzyme activity and emission. A positive correlation
between levels of emission, enzyme activities, and mRNAs indicates that, similar to C.
breweri, the activities of scent biosynthetic enzymes in snapdragon are regulated by
transcription of the corresponding genes in the flower. In addition, in snapdragon
flowers the earlier biochemical steps in the pathway are also involved in regulation of
volatile production and emission by controlling the supply of the substrate of the
reaction (Dudareva et al., 2000).
In summary, these results suggest that common regulatory mechanisms are involved
in floral scent production in different plant species. The volatile scent compounds are
synthesized de novo in the epidermal cells of organs from which they are emitted
306

(primarily the petals). The activities of enzymes involved in their formation, and
indirectly their emission, are regulated at the transcriptional level. Quantitative and/or
qualitative changes of floral scent chemistry during flower development may be part of
a mechanism to differentiate attraction cues as flowers age.

5. Genetic Engineering of Floral Scent

Many decades of classical breeding of ornamentals have led to a reduction in the scent
of cut flowers due to a focus on maximizing post-harvest shelf life, shipping
characteristics, and visual esthetic values (i.e., color, shape) of flowers (Zuker et al.,
1998; Dudareva et al., 1999). Lack of scent has long been recognized as a major
problem in floriculture, and recent progress in the molecular biology of floral scents
offers tools for modifying the scent composition of flowering plants. Also in recent
years, successful transformation protocols have been developed for several
commercially important cut flowers, such as rose, chrysanthemum, carnation, and
snapdragon (Heidmann et al., 1998; summarized in Zuker et al., 1998). Bioengineering
of the metabolic pathways responsible for the production of volatile compounds in
plants can involve either the modification of existing pathways and/or the introduction
of new enzymes to produce novel products not normally found in the plant. An
increasing number of cloned genes encoding scent biosynthetic enzymes are now
available for use in engineering transgenic plants with modified volatile composition.
The metabolic engineering of floral scent, however, has also required the use of flower-
specific promoters for correct temporal and spatial expression. Promoter regions of a
number of genes that encode enzymes of scent biosynthesis are currently being analyzed
in search of regulatory elements that provide the desired cell and tissue specificity.
Knowledge of the genes encoding scent biosynthetic enzymes and the mechanisms
underlying their transcriptional regulation, as well as the kinetic properties of these
enzymes, is in itself insufficient to guarantee success. A major consideration is the
availability of substrates in the same cell, and their location (or compartmentalization)
within the cell. Indeed, our recent investigations of floral scent emission in snapdragon
clearly demonstrate that the size of the substrate pool in the cell is involved in the
regulation of biosynthesis and emission of flower volatiles (Dudareva et al., 2000).
Scent biosynthetic enzymes characterized to date produce volatile compounds in a
single step from common metabolic intermediates. The channeling of metabolic
intermediates through multienzyme complexes without their release into general
metabolic pools (Hrazdina and Jensen, 1992) may cause a problem when new floral
scent compounds are engineered through expression of transgenes encoding enzymes of
novel pathways that utilize components of existing pathways as substrates. There may
also be deleterious effects on a plant if these substrates are intermediates in the
biosynthesis of essential plant compounds such as acetyl-CoA. To date, for most
substrates required for the synthesis of scent compounds, little information is available
about their own biosynthesis, cellular, and subcellular localization. In the future, to
achieve the production of noticeable amounts of new floral scent compounds, it could
be necessary to insert several genes encoding the biosynthetic enzymes of a given
pathway into the plant genome under the control of promoters that are active in
epidermal cells. In such a way we not only introduce the new scent gene, but may also
 307

increase the cellular concentration of substrate required for production of the

corresponding volatile compound.
Development of plants with modified composition of volatiles and new introduced
aroma will not only increase the value of ornamentals, but may also benefit agriculture
by increasing crop productivity and pest resistance. Modified floral scent composition
can increase flower attraction to pollinators and thereby increase reproduction efficiency
and the yield of important agricultural crops. In addition, floral scent modification could
be manipulated as a means of attracting beneficial insects and predators, and perhaps
deter harmful ones. However, to date there are no examples of the production of
noticeable amounts of new floral scent compounds in plants.

6. Acknowledgements

Work in the author's laboratory is supported by National Science Foundation grant

IBN-9904910 and by grants from The Fred C. Gloeckner Foundation, Inc.

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MOLECULAR GENETICS OF FLOWER SENESCENCE

J. E. THOMPSON, T.-W. WANG

Department of Biology
University of Waterloo
Waterloo, Ontario, Canada N2L 3Gl

1. Introduction

Senescence is the terminal phase in the development of a plant, plant organ or plant
tissue. Flower senescence, for example, occurs after pollination when the petals no
longer serve a useful purpose, and for certain flowers is accompanied by a climacteric
rise in ethylene production. In carnation, this climacteric surge in ethylene formation is
temporally coincident with inrolling of the petals, which is the first morphological
manifestation of petal senescence (Thompson et al., 1982). Petal inrolling is thought to
reflect loss of cell turgor attributable to membrane leakiness. Indeed, there is growing
evidence that a progressive decline in membrane selective permeability and the ensuing
loss of intracellular compartmentation are characteristic features of both flower and leaf
senescence (Thompson et al., 1998).
A further feature of senescence that distinguishes it from other types of programmed
cell death, including necrosis and apoptosis, is that there is extensive salvaging of
nutrients. Thus, not only do the cells of a senescing tissue die as newly synthesized
hydrolytic enzymes take their toll, but there is also provision through senescence-
specific gene expression for the conversion of macromoleucles to phloem-mobile
nutrients, which are translocated out of the senescing tissue (Quirino et al., 2000).
Indeed, senescence is an active process requiring gene expression and the synthesis of
new proteins. The various facets of this genetic regulation, together with its impact on
the physiology of senescing flowers and the prospect of delaying flower senescence
through genetic manipulation, are examined in this chapter.

2. The Molecular Basis for Membrane Deterioration During Senescence

Leakiness, reflecting loss of membrane structural integrity, is one of the earliest

manifestations of senescence. Indeed, leakage of pigments, sugars and electrolytes has
been observed for a variety of senescing tissues and organs including flowers, leaves,
cotyledons and fruit (Hanson and Kende, 1975; Suttle and Kende, 1978). Moreover, it is
a defining feature of both natural senescence and that which is induced prematurely by
episodes of environmental stress or pathogen ingression (Thompson et al., 1998).
Senescence-induced leakiness is commonly detected by measurements of the
conductivity of tissue diffusates. For example, as carnation flowers open and begin to
311
A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, 311-326.
© 2002 Kluwer Academic Publishers.
312

senesce, the conductivity of petal diffusates increases steeply, coincident with initiation
of the climacteric-like rise in ethylene production and well before the appearance of
petal inrolling (Eze et a/., 1986). The rise in conductivity is attributable to increased
levels of electrolytes in the diffusate, and reflects leakiness of cellular membranes and a
consequent loss of intracellular compartmentation within the senescing tissue. Of
particular interest is the finding that the increase in conductivity of carnation petal
diffusates precedes petal inrolling, for it indicates that the onset of membrane leakiness
is an early event in the senescence cascade. Moreover, there is growing evidence that
this is attributable to up-regulated expression of genes responsible for loss of membrane
structural integrity (Hong et al., 2000). That the onset of membrane leakiness is pivotal
to senescence is apparent from experiments in which it has been shown that
programmed cell death can be initiated prematurely by the addition of Ca++ ionophores
to plant tissues, which disrupts the Ca++gradient across the plasmalemma (Leshem et
al., 1984). It is apparent, therefore, that loss of metabolite and ion gradients engendered
by the onset of membrane leakiness disrupts cell function, and ultimately results in cell
and tissue death.

2.1. SENESCENCE-RELA1ED CHANGES IN 1HE MOLECULAR ORGANIZATION

OF MEMBRANE LIPID BILAYERS

2.1.1. Catabolism ofMembrane Lipids

Prior to senescence, cellular membranes exhibit selective permeability, a property that
reflects the impermeable nature of the phospholipid bilayer. Not surprisingly, there is
now growing evidence that the onset of membrane leakiness during the early stages of
senescence reflects molecular disorder in the lipid bilayers brought about by catabolism
of membrane lipids. By far the most conspicuous change in the lipid composition of
senescing membranes is a dramatic decline in membrane phospholipid, reflecting de-
esterification of membrane fatty acids. This has been demonstrated for senescing
flowers of Ipomoea (Beutelmann and Kende, 1977), senescing rose flowers (Borochov
et a/., 1978), senescing carnation flowers (Fig. 1) as well as senescing leaves and fruit
(Thompson et al., 1998). Moreover, de-esterification of membrane fatty acids induces
peroxidation and lipid phase separations, both of which perturb the structure ofbilayers
and increase their permeability (Hong et al., 2000).
These observations imply that there is extensive catabolism of membrane lipids
during senescence. Four lipid-degrading enyzmes, phospholipase D, phosphatidic acid
phosphatase, lipase and lipoxygenase, have been found to be associated with
membranes from senescing tissues (Paliyath and Thompson, 1987). Phospholipase D
cleaves the polar head groups from phospholipids to generate phosphatidic acid, which
in turn is acted upon by phosphatidic acid phosphatase. This latter enzyme removes the
phosphate group from phosphatidic acid to form diacylglycerols. By virtue of their
conformation, diacylglycerols destabilize membranes and promote microvesiculation
(Allan et al., 1976). Diacylglycerols are also readily deacylated by lipase, an enzyme
with a broad substrate specificity that also deacylates phospholipids (Galliard, 1980).
The polyunsaturated fatty acids, linolenic acid and linoleic acid, released by lipase serve
as substrates for membranous lipoxygenase, which in turn can initiate lipid peroxidation
and form activated oxygen (Lynch and Thompson, 1984; Lynch eta/., 1985).
It is also apparent that the phospholipase D associated with senescing membranes
 313

exhibits molecular species preferences whereby highly perturbed phospholipids

containing polyunsaturated fatty acids are broken down more readily than desaturated
molecular species. This has been established by measuring the catabolism of
radiolabeled exogenous molecular species of phosphatidylcholine by senescing
microsomal membranes, and also by assessing changes in the endogenous levels of
molecular species of phosphatidylcholine in naturally senescing membranes (Brown et
al., 1987). These observations suggest that the provision of unique molecular species of
phospholipids, specifically those that contain two polyunsaturated fatty acids, may be a
prerequisite for phospholipid catabolism in senescing membranes.

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Figure!. Changes in the fluidity (measured as polarization) and chemical composition of microsomal
membranes during senescence of carnation flower petals. (Adapted from Fobel eta/., 1987).

There are also both soluble and membrane-associated forms of phospholipase D, and
there is evidence that the soluble form of the enzyme becomes associated with
membranes as senescence progresses (Dryer et al., 1994). This finding suggests that the
distribution of phospholipase D between the cytosolic and membranous compartments
is under stringent developmental control and becomes altered with the onset of
senescence. Indeed, the activity of membrane-associated phospholipase D is positively
314

correlated with the rate of senescence and loss of membrane phospholipid, whereas the
activity of the soluble form of the enzyme is not (Ryu and Wang, 1995). Moreover, a
number of phospholipase D genes have been cloned from Arabidopsis, indicating that
there are various isoforms of the enzyme, and one of these, a.-phospholipase D, is
expressed in senescing leaves (Wang et al., 1994; Ryu and Wang, 1995). However,
since free fatty acids, the product of the action of lipase, rather than diacylglycerol and
phosphatidic acid, the products of phospholipase D and phosphatidic acid phosphatase
action, respectively, accumulate in senescing membranes (Fig. 1), it would appear that
phospholipase D and phosphatidic acid phosphatase serve to prime the lipid bilayer of
senescing membranes for lipase-mediated catabolism.

2.1.2. Changes in the Molecular Organization of Membranes Attributable to Lipid

Catabolism

2.1.2.1. Lipid Phase Separations. Leakiness of cellular membranes and the consequent
loss of intracellular compartmentation are among the earliest symptoms of flower
senescence, and there is growing evidence that for all senescing tissues this reflects
lateral phase separations within the plane of membrane bilayers attributable to lipase-
mediated fatty acid de-esterification. Specifically, in the membranes of non-senescing
tissue, the lipid bilayer is exclusively liquid-crystalline, but with the onset of
senescence, de-esterified fatty acids form small domains of gel-phase lipid {Leshem et
al., 1984; Senarata et al., 1987). The resulting mixture of lipid phases causes the
membranes to become leaky because of packing imperfections at the phase boundaries
(Barber and Thompson, 1980, 1983).
Lipid phase separations in senescing membranes have been demonstrated by wide-
angle X-ray diffi'action studies of isolated membrane fractions from rose petals (Legge
et al., 1982). Diffraction patterns recorded for membranes from young tissue feature a
broad diffuse X-ray reflection corresponding to liquid-crystalline phase lipid. By
contrast, diffraction patterns for membranes from senescing rose petals feature a sharp
X-ray reflection corresponding to gel-phase domains of free fatty acid in addition to the
diffuse reflection representing liquid-crystalline phase phospholipid. This means that
there is a mixture of lipid phases in the bilayers of senescing rose petal membranes.
That the gel phase domains are comprised of free fatty acids de-esterified from
phospholipid has been demonstrated by isolating free fatty acids from senescing
membranes and reconstituting them into liposomes of pure phospholipid. This results in
the same mixture of liquid-crystalline and gel-phase domains seen in naturally
senescing membranes (Yao et al., 1991).
The formation of gel-phase lipid in senescing membranes is also detectable by
freeze-fracture electron microscopy (Platt-Aloia and Thomson, 1985). Membranes from
young tissue exhibit a smooth fracture plane with evenly distributed intramembranous
particles corresponding to membrane proteins. As senescence progresses, the
intramembranous particles are squeezed out of the forming gel-phase domains into
adjacent liquid-crystalline domains, as evidenced by the presence of intramembranous
particle-free areas in the fracture plane. These particle-free areas correspond to
separated domains of free fatty acids. Freeze-fracture electron microscopy, unlike X-ray
diffraction, allows identification of the membranes sustaining lipid-phase separations,
and in senescing cotyledons of cowpea, intramembranous particle-free regions
 315

corresponding to domains of gel-phase :free fatty acids have been noted in the plasma
membrane, endoplasmic reticulum and vacuolar membrane (Platt-Aloia and Thomson,
1985). In senescing carnation petals, gel-phase lipid has been discerned in the
membranes of the endoplasmic reticulum at a very early stage of senescence, prior to
the climacteric-like rise in ethylene production and the onset of petal inrolling (Paliyath
and Thompson, 1990). The early appearance of lipid phase separations in the
membranes of the endoplasmic reticulum may be particularly significant since Ca2+
compartmentalized within the endoplasmic reticulum will leak into the cytosol, an event
that is known to initiate cell death. Indeed, treatment of plant tissues with a Ca2+
ionophore, which makes the plasma membrane leaky to Ca2+ and results in high
cytosolic concentrations of the cation, has been shown to result in premature cell death
(Leshem ef al., 1984).

2.1.2.2. Bulk Lipid Fluidity. Two techniques, fluorescence polarization and electron spin
resonance, have been used to demonstrate that there is a decrease in bulk lipid fluidity
of membranes :from senescing flowers attributable to catabolism of membrane lipids
(Legge et al., 1982). The membranes are labeled with lipid-soluble fluorescent or
paramagnetic probes, and measurements of the rotational motion of these probes in the
bilayer can be related to lipid fluidity (Thompson et al., 1982). The decrease in lipid
fluidity of senescing membranes reflects an increase in the sterol-to-fatty acid ratio (Fig.
1), attributable to selective depletion of fatty acids :from the bilayer by the action of
senescence-induced lipase. Sterols are known to restrict the mobility of phospholipid
fatty-acid chains (Shinitzky and lnbar, 1976), and this enrichment of :free sterols in the
bilayer relative to fatty acid appears to be one of the major factors contributing to the
decrease in bulk lipid fluidity in membranes with advancing senescence.

2.1.2.3. Effects on Membrane Proteins.· Changes in bilayer fluidity are known to affect
membrane proteins. For example, an increase in the sterol-to-phospholipid ratio has
been correlated with altered transport (Grunze and Deuticke, 1974), altered protein
conformation and receptor function (Kirby and Green, 1980) and changes in membrane
enzyme activity (Kramer et al., 1982). Vertical displacement of membrane proteins
towards membrane surfaces has also been demonstrated in response to a decrease in
bilayer fluidity (Shinitzky et al., 1979). This exposes portions of membrane proteins to
a more hydrophilic environment and could result in conformational changes. In light of
this, the decrease in bulk lipid fluidity of membranes during senescence is likely to alter
the properties of membrane proteins. Indeed, spin-labeling studies of isolated
membranes :from senescing bean cotyledons have shown that the decrease in bilayer
fluidity accompanying senescence is sufficient to alter the conformation of membrane
proteins (Duxbury et al., l991b).
Large sterol concentrations such as those inherent in senescing membranes may also
affect membrane proteins in a manner quite independent of the effect of sterols on
bilayer fluidity. This possibility arises from the limited capability of phospholipid
bilayers to fully solvate sterols. At concentrations of up to 33 mol%, sterols reduce
bilayer fluidity by associating with phospholipid at a stoichiometry of 1:2 sterol-to-
phospholipid molecules (McKersie and Thompson, 1979). At higher concentrations,
sterol-sterol interactions begin to occur because there is insufficient phospholipid to
fully solvate the sterols. For senescing plant membranes, sterol concentrations as high
316

as 55 mol% have been reported (Duxbury et al., 199la). At such high sterol
concentrations, all phospholipid molecules will be associated with sterol unless their
affinity for proteins exceeds that for sterols. This is likely to induce changes in protein
conformation because membrane proteins usually require a close association with the
phospholipids that support their function and tertiary structure, and sterols are normally
excluded from the phospholipid annulus surrounding proteins (Warren et al., 1975).
Indeed, it has been documented by freeze-fracture electron microscopy that sterol
concentrations of 27 mol% and higher induce protein aggregation (Legge and Brown,
1988).
There is significant evidence for net catabolism of membrane proteins with
advancing flower senescence. For example, decreased levels of plasmalemma proteins
have been reported during senescence of carnation petals (Borochov et al., 1994),
daylilytepals (Lay-Yee et al., 1992) and iris tepals (Celikel and Van Doorn, 1995). The
accelerated catabolism of membrane proteins during senescence apparently does not
reflect ubiquitin-dependent proteolysis, but there is evidence that changes in the
conformation of membrane proteins, prompted by the enrichment of free sterols in the
bilayer and also by the reaction of activated oxygen with amino acid residues, induce
proteolysis (Duxbury et al., 1991a, b).

3. Increased Production of Free Radicals Attributable to Membrane Lipid

Catabolism

Various studies demonstrating that the vase life of flowers can be modulated by radical
scavengers suggest that free radicals are involved in flower senescence (Baker et al.,
1977, 1985). Of particular interest is the finding that isolated membranes from carnation
flowers produce increased levels of superoxide radical with advancing senescence,
which is indirectly attributable to lipase-mediated fatty acid de-esterification. Indeed,
levels of superoxide radical produced by microsomal membranes from carnation petals
rise by >2-fold as the flowers senesce (Mayak et al., 1983). Several lines of evidence
indicate that superoxide radical is formed as an intermediate during the conversion of
polyunsaturated fatty acids, deesterified by the senescence-induced lipase, to their
conjugated hydroperoxydiene derivatives by lipoxygenase. Specifically, there is a
strong temporal correlation during senescence between changes in microsomal
lipoxygenase activity and changes in superoxide radical production by the membranes,
the formation of superoxide being dependent upon the availability of unesterified
polyunsaturated fatty acid substrate for lipoxygenase, and superoxide radical formation
and lipoxygenase activity being affected in a parallel fashion by specific inhibitors of
lipoxygenase. Finally, Baker et al. (1985) have reported that specific inhibitors of
lipoxygenase delay senescence of cut carnation flowers.

4. Nutrient Mobilization

During senescence, the plant salvages nutrients from the deteriorating cells before
necrotic death of the tissue ensues. Thus, nitrogen, phosphorus, carbon and minerals are
mobilized out of the dying tissue to other growing parts of the plant (Quirino et al.,
 317

2000). The catabolism of proteins to phloem-mobile elements is achieved by

senescence-induced proteases, and salvage of lipid carbon appears to involve the
combined action of lipase and P-oxidation of fatty acids. This latter function is carried
out within peroxisomes. Indeed, as leaves senesce, galactolipids disappear without a
commensurate accumulation of free fatty acids, and this has prompted the view that
membrane fatty acids are metabolized through P-oxidation and the glyoxylate cycle, and
subsequently converted through gluconeogenesis to phloem-mobile sucrose (Landolt
and Matile, 1990).
Higher plant peroxisomes have been classified as glyoxysomes, leaf peroxisomes
and unspecialized peroxisomes, and all plant peroxisomes are capable of carrying out P-
oxidation of fatty acids (Pistelli et a/., 1989). Glyoxysomes are involved in the
mobilization of triacylglycerol in seeds, and contain the enzymes required for fatty acid
p-oxidation, glyoxylate cycle enzymes and peroxisomal enzymes (De Bellis et al.,
1990). Non-specialized peroxisomes are found in achlorophyllous tissues and, although
they contain p-oxidation enzymes, their function is not well defined (Gerhardt, 1986).
Leaf peroxisomes are present in leaves and green cotyledons, and contain the enzymes
of the glycolate cycle, in keeping with the fact that they participate in photorespiration,
as well as p-oxidation enzymes (Gerbling and Gerhardt, 1987). However, leaf
peroxisomes do not contain the enzymes of the glyoxylate cycle (De Bellis et al., 1990).
Of particular interest is the finding that during greening of oil seed cotyledons there is a
functional transition of glyoxysomes to leaf peroxisomes (Titus and Becker, 1985).
Moreover, there is growing evidence that as leaves begin to senesce, the key enzymes of
the glyoxylate cycle, namely malate synthase and isocitrate lyase, reappear in leaf
peroxisomes, indicating that there is a reverse transition of peroxisomes to glyoxysomes
in the event of senescence. Direct evidence supporting this has come from immunogold-
labeling experiments in which it has been demonstrated that levels of glycolate oxidase,
a peroxisomal marker, in microbodies decrease during leaf senescence coincident with
an increase in levels of the glyoxylate cycle enzymes, malate synthase and isocitrate
lyase. Moreover, this reflects senescence-specific upregulation of the genes encoding
malate synthase and isocitrate lyase (Nishimura eta/., 1993).
The presence of senescence-induced glyoxysomal activity is best documented for
senescing leaves, but there is some evidence that this is also a feature of nutrient
salvaging during flower senescence. For example, Droillard and Paulin (1990) have
reported, on the basis of electron microscopic studies, that the proportion of
microbodies to mitochondria increases with advancing senescence of carnation petals.
In addition, malate synthase mRNA has been shown to accumulate in senescing petals
of cucumber and petunia (Graham et a/., 1990). Furthermore, both transcripts and
protein for isocitrate lyase and malate synthase, the key enzymes of the glyoxylate
cycle, are detectable in senescing pumpkin flowers, but not in their non-senescing
counterparts (De Bellis et al., 1991).

5. Role of Ethylene

Ethylene has a profound effect on plant growth and development. These effects include
involvement in seed germination, seedling growth, leaf and root growth, abscission,
318

fruit ripening, stress responses and senescence (Abeles, 1973). Perhaps the best
characterized effect of ethylene is its ability to promote fruit ripening. Fruits have been
classified as climacteric or non-climacteric based on their pattern of respiration and
ethylene production during ripening (Biale, 1960). For non-climacteric fruit such as
citrus, respiratory activity is low when ripening is initiated and progressively decreases
as ripening progresses (Sacher, 1973). Moreover, there is no increase in ethylene
production during ripening. In contrast, for climacteric fruit such as banana, there is an
increase in respiration during ripening which is accompanied by a surge in ethylene
production known as the ethylene climacteric. Depending upon the fruit, the ethylene
climacteric precedes (banana), coincides with (pear, avocado), or follows (plums) the
rise in respiration (Biale and Young, 1981 ).
By analogy, flowers are also classified as climacteric-like or non-climacteric-like,
depending upon whether their senescence is sensitive or insensitive, respectively, to
ethylene (Woltering and Van Doorn, 1988). For example, petal senescence of
Chrysanthemum, Narcissus and Zinnia appears not to be affected by ethylene (Nichols,
1966; Stimart et al., 1983), whereas the senescence of carnation, Tradescantia, Ipomoea
and Hibiscus petals is strongly influenced by ethylene (Kende and Baumgartner, 1974;
Mayak et al., 1977; Suttle and Kende, 1978; Woodson et al., 1985). For these latter
climacteric-like flowers, the formation of ethylene is low as the flower opens, but a rise
to a climacteric-like peak in its production ensues, followed by a decline (Fig. 2).
Moreover, this climacteric peak in ethylene production often coincides temporally with
the onset of petal inrolling signifying the initiation of senescence (Kende and
Baumgartner, 1974). In addition, treatment of pre-climacteric flowers with exogenous
ethylene accelerates the onset of petal inrolling, as well as changes in membrane
microviscosity (Fig. 2), and competitive inhibitors of ethylene action delay the
appearance of these morphological symptoms of senescence as well as the ethylene
climacteric (Kende and Baumgartner, 1974). Indeed, treatment of cut climacteric
flowers such as carnation with silver thiosulfate, an inhibitor of the ethylene signal
transduction pathway, has been used commercially for many years as a means of
increasing their shelf life. Petal inrolling in cut carnation flowers can also be delayed by
applying inhibitors of ethylene biosynthesis such as amino-oxyacetic acid (Fujino et al.,
1980). More recent evidence from experiments with Arabidopsis suggests that ethylene
may simply modulate the progress of senescence rather than actually induce
programmed cell death. Specifically, leaves of Arabidopsis mutants that are insensitive
to ethylene still senesce, but the actual onset of leaf senescence is delayed (Grbic and
Bleecker, 1995).
During senescence, ethylene is produced by floral organs other than petals. The
pistil in particular produces large amounts of ethylene (Nichols, 1977). Petal senescence
is initiated in the event of pollination, and this is accompanied by rapid mobilization of
nutrients out of the senescing petals into the developing ovary. Following pollination,
ethylene is first produced by the styles, and within a few hours there is a burst of
ethylene production by the petals and ensuing flower senescence (Nichols, 1977). In
contrast, when senescence occurs in the absence of pollination, the increases in ethylene
production by the various floral organs are temporally coincident. For example, in
carnation flowers that have not been pollinated, petal senescence still occurs, and it is
accompanied by a climacteric-like rise in ethylene production that is a composite of
ethylene produced by several floral organs including petals, ovary, styles and receptacle.
 319
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';;] 3
.;
0

2
8
~
0
5
~

i,e. 4

3
'§ -Q- Ehtylene
2
·i! --control
~
:i
0
0 2 4 6 7 8
Age (day after cutting)

D
I II III IV II liE

-----
__ ....,. _

Figure 2. Temporal relationships among the ethylene climacteric, expression of the senescence lipase, and an
increase in membrane microviscosity in developing and senescing carnation flowers. In C (0), preclimacteric
flowers were treated with 0.5 ppm ethylene for 10 h. In D, a northern blot of the senescence-induced lipase
and the corresponding agarose gel of the fractionated RNA are illustrated. Stage I, tight-bud flower; stage II,
flower open with outer petals about 60° from vertical; stage III, flower fully open with petals about 90° from
vertical; stage IV, slight petal inrolling; stage liE, stage II flowers that were treated with 0.5 ppm ethylene for
I 0 h (Adapted from Hong eta/., 2000 and Thompson eta!., 1982).

6. Senescence-Induced Gene Expression

Senescence is an active developmental process that requires gene expression and the
synthesis of new proteins. A number of senescence-induced genes have been identified
320

for leaves. These include genes encoding a cysteine protease, ferritin, aspartate protease,
vacuolar processing protease, glutamine synthase, metallothionein and RNase (Blank
and McKeon, 1991; Buchanan-Wollaston, 1997). In senescing carnation flowers, new
mRNA species and their cognate proteins are formed during the ethylene climacteric,
and many of these senescence-specific transcripts are also produced upon treatment of
pre-climacteric flowers with exogenous ethylene (Woodson, 1987). Flower senescence
entails a series of highly coordinated biochemical events, including degradation of
starch and chlorophyll, increased respiration, proteolysis and catabolism of membrane
lipids. Much of this biochemical activity is mediated by newly synthesized hydrolytic
enzymes, which is in turn a reflection of senescence-specific gene expression (Borochov
and Woodson, 1989). Among the genes induced in senescing carnation flowers are
those designated pSR5, pSR8 and pSR12, for which no function has yet been annotated
(Lawton eta/., 1990)

6.1. SENESCENCE-INDUCED LIPASE

Inasmuch as de-esterification of membrane fatty acids is a pronounced feature of

senescence, lipase would appear to be a key agent of senescence-related membrane
deterioration. Thus it is of particular interest that a senescence-induced lipase gene has
been isolated from carnation petals that is strongly expressed coincident with the onset
of membrane leakiness during the early stages of carnation flower senescence (Hong et
al., 2000). Expression of this gene is also induced in young carnation flowers by
treatments with ethylene that induce premature petal senescence. Although lipase genes
encoding proteins that de-esterit)r complex lipids have been cloned previously from
plants, animals and bacteria (e.g.GenBank accession nos. AL021710, AL126316 and
AL126333), the carnation lipase is the first ethylene- and senescence-induced lipase
gene to be identified. Indeed, the timing of this lipase gene's expression during
development of the carnation flower suggests that the hydrolytic activity of its cognate
protein initiates the membrane deterioration that leads to senescence. Of particular
significance is the fmding that expression of this lipase gene is strongly induced by
ethylene, for it has been demonstrated that manifestations of carnation petal senescence
thought to be attributable to fatty acid de-esterification, including membrane leakiness
and consequent petal inrolling, are also induced by ethylene.
The carnation lipase is capable of de-esterifying fatty acids from both the sn-1 and
sn-2 positions of phospholipid, which is a defining feature of phospholipid catabolism
in senescing membranes (Hong eta/., 2000). It can also be inferred from the sequence
of the lipase eDNA ORF that the carnation lipase is a cytosolic protein. Specifically, the
ORF does not have a recognizable signal sequence or transit peptide. It does, however,
feature three putative phosphorylation sites, and in this sense resembles animallipases,
which are normally soluble enzymes that in some instances have been shown to be
regulated by phosphorylation (Yeaman, 1990). The fmding that the carnation lipase is a
cytoslic lipolytic acyl hydrolase means that it is presumably capable of hydrolyzing
complex lipids in all cellular membranes with exposure to the cytosol, and is thus able
to mediate the extensive hydrolysis of membrane lipids that characterizes senescence. It
is also worth noting that the carnation lipase contains the 10-aa consensus sequence,
[LIV]-X-[LIVAFY]-[LIAMVST]-G-[HYWV]-S-X-G-[GSTAC], that has been shown
to be a common feature oflipases cloned from animals (including humans), bacteria and
 321

fungi.

6.2. INDUCTION OF ETHYLENE BIOSYNTHESIS GENES

The ethylene biosynthesis pathway has been fully elucidated. It entails the conversion of
methionine to S-adenosylmethionine (SAM) by SAM synthetase, the conversion of
SAM to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase and, finally,
the conversion ACC to ethylene by ACC oxidase. The reaction catalyzed by ACC
synthase is generally considered to be the rate-limiting step in the pathway, although
there are some reports of ethylene formation being limited by the ACC oxidase-
mediated reaction (Yang and Hoffinan, 1984).
Senescence-induced eDNA clones encoding ACC oxidase and ACC synthase have
been isolated from carnation flowers, and both genes have been shown to be strongly
expressed during the ethylene climacteric (Wang and Woodson, 1991; Park et al., 1992;
Woodson et al., 1992). Moreover, it is now clear that the expression of both of these
genes can be induced by ethylene, and that this is probably the basis for the
autocatalytic production of ethylene in senescing carnation petals. Specifically, northern
blot analyses have indicated that transcript levels for these genes are low in the petals of
pre-climacteric carnation flowers, and that treatment of these flowers with exogenous
ethylene induces a strong and rapid increase in their transcript levels (Woodson et al.,
1992). Moreover, in the presence of 2,S-norbornadiene, a competitive inhibitor of
ethylene binding to its receptor, ETR-1, there is no accumulation of transcript for these
genes, which indicates that their expression is a response to ethylene (Woodson et al.,
1992). Thus, during natural flower senescence, the expression of ACC synthase and
ACC oxidase in petals could be activated by ethylene originating from other parts of the
flower (Overbeek and Woltering, 1990). It is worth noting that the ethylene climacteric
in senescing carnation petals is accompanied by a decrease in the level of SAM synthase
transcript, for this indicates that the availability of a methyl donor is not rate-limiting
(Woodson et al., 1992).

7. Regulation of Senescence

Senescence appears to be controlled by multiple regulatory pathways involving a

complex network of transcription circuitry (Weaver, 1998). This contention is supported
in part by the fmding that, at least within a given species, the promoters of senescence-
induced genes do not appear to have conserved regulatory elements. Nor do they
contain consensus sequences for previously characterized DNA-binding proteins.
(Quirino et al., 2000) There do, however, appear to be conserved response elements in
promoters of the same senescence-induced gene across species. This is the case, for
example, for the promoter oftheArabidopsis senescence-induced gene, SAG12, which
encodes a protease (Noh and Amasino, 1999a and b).
Generally speaking, there is a paucity of information concerning the regulatory
genes that control the various signaling pathways able to initiate senescence. Recently,
however, eDNA clones encoding deoxyhypusine synthase (DHS) (GenBank accession
no. AF296079) and eukaryotic translation initiation factor-SA (elF-SA) have been
shown to be upregulated in senescing tomato blossoms (Wang et al., 2001). DHS
322

catalyzes the first reaction in the two-step conversion of inactive eiF-5A to its activated
form. The DRS-mediated reaction entails the transfer of a butylamine residue from
spermidine to a conserved residue of inactive eiF-5A, forming the unusual amino acid,
deoxyhypusine, which is then converted to hypusine by deoxyhypusine hydroxylase
(Park et al., 1997). Hypusine-modified e1F-5A, the active form of the protein, is the
only cellular protein known to contain hypusine. It is present in all eukaryotic cells and
appears to facilitate mRNA translation (Park et al., 1997). It was initially designated as
a translation initiation factor based on in vitro experiments indicating that it simulates
the formation of methionyl-puromycin, a dipeptide analogue (Kemper et al., 1976).
However, more recent experiments have demonstrated that eiF-5A is not involved in the
initiation of global protein synthesis. Indeed, it appears to function as a
nucleocytoplasmic shuttle protein that facilitates the translation of specific subsets of
mRNA by mediating their translocation from the nucleus to the cytoplasm (Rosorius et
al., 1999). The fmding that DHS and eiF-5A are upregulated in parallel in senescing
tomato flowers (Wang et al., 2001) raises the possibility that they facilitate the
translation of that population of mRNAs required for senescence, and hence are
involved in its regulation.

8. Genetic Engineering of Increased Vase Life

Enhanced vase life is a valuable attribute for cut flowers and has been an objective of
breeding programs for many flower species. Additionally, the vase life of some flowers,
particularly those that exhibit an ethylene climacteric during senescence, can be
significantly increased by the use of chemicals that inhibit either synthesis of the
phytohormone or its perception by flower petals. For example, the vase life of cut
carnation flowers can be extended by treatment with aminooxyacetic acid and
aminoethoxyvinylglycine, which are inhibitors of ACC synthase, as well as by silver
thiosulfate, an inhibitor of ethylene binding to its receptor (Wang and Baker, 1980;
Woodson et al., 1992; Table 1).

TABLE I. Increase in carnation vase life. Adapted from Wang and

Baker, 1980; Woodson et al., 1992; Savin et al., 1995; Bovy et al.,
1999

Treatment Percentage of increase

Aminoethoxyvinylglycine 71 to 80
Silver thiosulfate 71
etrl-1 105 to 189
Antisense ACC oxidase 60 to 80

More recently, the isolation and characterization of senescence-induced genes,

particularly those encoding enzymes of the ethylene biosynthetic pathway, has enabled
the testing of genetic engineering strategies for increasing the vase life of cut flowers.
One such strategy involves suppressing the expression of the gene encoding ACC
 323

oxidase. For example, transgenic carnation flowers expressing antisense eDNA for ACC
oxidase under the regulation of a constitutive promoter have reduced levels of ACC
oxidase transcript and a vase life of 8 to 9 days, compared with ~5 days for non-
transformed control flowers (Savin et al., 1995; Table 1). However, of particular note is
the finding that transgenic flowers with suppressed ACC oxidase expression did not
display typical petal inrolling symptoms with advancing senescence. Rather, they
gradually became desiccated and discolored (Savin et al., 1995). Extended vase life of
carnations has also been observed when ACC oxidase expression is reduced by co-
suppression (Bovy et al., 1999).
Another genetic engineering strategy for increasing the vase life of flowers that are
ethylene-sensitive involves disrupting the ethylene signal transduction pathway. It is
now well established that the physiological effects of ethylene are mediated through a
signal transduction pathway that is activated by the binding of ethylene to a receptor
encoded by ETRl (Ecker, 1995; Smalle and van der Straeten, 1997). Moreover, since
mutated ETR1 is dominant, it can be used to engender insensitivity to ethylene in
transgenic plants. For example, transgenic Arabidopsis plants containing a mutant allele
(etrl-1) of Arabidopsis ETR1 show greatly reduced ability to bind ethylene and a
corresponding reduction in the characteristic physiological responses to ethylene
(Chang et al., 1993). In addition, transgenic carnation flowers expressing Arabidopsis
etrl-1 under the regulation of a constitutive promoter exhibit an almost two-fold
increase in vase life (Bovy et al., 1999; Table 1). It is clear, therefore, that at least for
ethylene-sensitive flowers, this strategy for increasing shelflife has significant potential,
which may need to be realized if the use of silver thiosulfate for inhibiting ethylene
perception and extending vase life becomes banned by regulatory agencies. Moreover,
disruption of the ethylene transduction pathway with etrl-1 can be rendered flower-
specific by expressing the transgene under the regulation of flower-specific promoters,
thereby precluding deleterious effects that might arise from the elimination of ethylene
perception in all parts of the plant. Delayed flower senescence has also been observed in
transgenic petunia expressing a transgene encoding the dominant mutant ethylene
receptor (Wilkinson et al., 1997). This, together with corresponding results for
carnation, suggests that etr1-1 is a powerful tool for engineering increased vase life in a
variety of ethylene-sensitive flowers.

9. Conclusions

Flower senescence entails a regulation phase, during which there is activation of genes
and synthesis of new proteins, and an execution phase leading to nutrient salvaging and
cell death. There is growing evidence that the regulation of senescence is complex,
particularly in that it can be initiated by a number of seemingly independent
transcription circuits. For example, senescence occurs naturally, but it can also be
induced prematurely in response to various types of environmental stress through gene
activation pathways that increasingly appear to be distinct. Recent evidence also
indicates that there is overlap between transcription circuits invoking response to
pathogens and those that regulate senescence. In particular, certain pathogen defense-
related genes have been found to be expressed in senescing leaves, and it has been
proposed that this serves to combat opportunistic infections that might disrupt nutrient
324

salvaging (Quirino et al., 2000). The actual execution of senescence is carried out by
newly synthesized proteins, in particular the hydrolytic enzymes and polypeptides
required for nutrient salvaging, and in contrast to its regulation, there is growing
evidence that the execution of senescence has common elements irrespective of how it
is initiated. For example, membrane fatty acid de-esterification and lipid phase
separation leading to leakiness appear to be a common feature of both natural and
prematurely induced senescence. As more of the genes controlling both the regulation
and execution of flower senescence are isolated and annotated, it seems reasonable to
expect that new and improved technologies for delaying flower senescence will evolve.
This will undoubtedly have a profound influence on the flower industry.

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MOLECULAR MARKERS AS A TOOL FOR ANALYSES OF GENETIC
RELATEDNESS AND SELECTION IN ORNAMENTALS

T.DEBENER
Federal Centre For Breeding Research on Cultivated Plants
Institute for Ornamental Plant Breeding
Bornkampsweg 31
D-22926Ahrensburg, Germany

1. Introduction

The analysis of genetic diversity among populations, between individuals and varieties,
and even between species has been a major task in modem plant breeding (Allard,
1988). However, during the past two decades, selection methods based mainly on the
analysis of morphological and physiological parameters have been increasingly
complemented, and to some extent replaced by the so-called "molecular markers".
In contrast to morphological markers, mostly analyzed as visible mutations which
are easy to score, molecular markers detect genetic differences at the organism's DNA
level. As isozymes reflect the enzyme phenotype of a given individual and because the
amount of genetic variation uncovered by isozymes is very limited, they will not be
considered in the present review.
In the early 1980s, the advent of molecular markers provided scientists with
valuable new tools for genetic and molecular analyses and allowed much more detailed
analyses of the structure of plant genomes (Paterson et a!., 1991; Gebhardt and
Salamini, 1992; Mohan eta!., 1997). Their number is virtually unlimited, their only
limitation being the genome size of the organism and the resources which can be
devoted to the analyses. Their resolution can be higl1 enough to detect single base pair
differences between genomes.

2. Types of Molecular Markers

After first applications in the field of human genetics (Botstein eta!., 1980), molecular
markers were subsequently adopted by plant geneticists and plant breeders (Paterson et
al., 1991; Gebhardt and Salamini, 1992).
The most common molecular markers applied in the field of plant genetics and
breeding are RFLPs (Restriction ~ragment 1ength ~olymorphism), RAPDs (Random
Amplified ~olymorphic _!!NA), AFLPs (Amplified ~ragment 1ength ~olymorphism),
and microsatellites, as well as a number of PCR-based single-locus marker types such
329
A. Vainstein (ed. ), Breeding for Ornamentals: Classical and Molecular Approaches, 329-345.
© 2002 Kluwer Academic Publishers.
330

as SCARs <§.equence !;,haracterized Amplified J!egion), CAPS (!;,leaved Amplified

~olymorphic ~equence), SSCPs @.ingle ~trand !;_onfonnation ~olymorphism) and
related techniques. General descriptions of different marker types and their application
to plant genetics including concise literature references can be found in Weising et al.
(1995) and Staub et al. (1996), whereas applications to problems of genetic diversity
and evolutionary relationships are covered by Avise (1994) and Karp et al. (1998).
In the following, the most important marker types will be described along with
major advantages and disadvantages.
The ideal marker system should fulfill several criteria:
i) it should be selectively neutral and evenly distributed throughout the genome;
ii) it should detect highly polymorphic loci;
iii) it should be highly reproducible;
iv) its generation should be easy, cheap and fast.
As will be explained below, the available marker systems differ with respect to
these criteria but none of them can be considered optimal in fulfilling all of them.

2.1. RFLP

RFLPs are based on the detection of DNA polymorphisms that result in changes of
restriction fragment patterns of a distinct region in the genome (Gebhardt and
Salamini, 1992). For this purpose, the DNA of the genotypes under investigation is
digested with a restriction endonuclease and the resulting complex mixture of DNA
fragments is separated on agarose or polyacrylamide gels. As genomes, except viral
genomes, are too complex to allow the resolution of restriction patterns, the separated
restriction fragments are transferred to nitrocellulose or nylon membranes and
hybridized against a labeled probe. Polymorphisms between individual genotypes are
either the result of point mutations in restriction sites or of deletions, insertions or
inversions within the detection range of the probe. The nature of the probes varies with
the application of this technique. For most applications in genetic analyses, the probes
should represent single or low-copy sequences of the nuclear genome as multilocus
probes will obscure the assignment of restriction fragments to individual alleles. For
this reason, probes have to be pre-selected, mostly in hybridization experiments
(Gebhardt and Salamini, 1992). For some applications, as for example fingerprints of
genomes with mini- or microsatellites, multicopy sequences are used as probes as well
(Weising et al., 1995).
For applications which require the detection of polymorphisms in more conserved
stretches of DNA, RFLPs can also be generated from the plastid (cp) genome. This can
be achieved by either digesting isolated plastid DNA or by detecting cp sequences in
Southern blots of total plant DNA with cp probes. The same strategy also applies for
the mitochondrial (mt) genome though the lower conservation, especially of the mt
genome structure, makes this type of marker less useful in plants as compared to
animals (Avise, 1994).
An advantage of the RFLP technique is its high degree of reproducibility. Its
robustness against experimental artefacts is hardly reached by any other of the current
marker techniques. Therefore the results can be reproduced easily by scientists in
 331

different labs.
RFLPs have three major disadvantages. First, they require large amounts of DNA
of high purity to achieve complete digestion with a variety of restriction enzymes. This
requires large amounts of plant material and laborious and time-consuming techniques
for DNA extraction. Second, RFLPs require several experimental steps before the
polymorphisms can be detected. These include the transfer of restricted DNA to
membranes and DNA-DNA hybridizations. Third, labeled probes are needed which
have to be generated by either the incorporation of radioisotopes or the use of
fluorescent labels.

2.2.RAPD

In the early 1990s, several research groups independently suggested the use of PCR
strategies based on random primers for the detection of polymorphisms which do not
require prior knowledge of target sequences (Welsh and McClelland, 1990; Williams
et al., 1990; Caetano-Anolles et al., 1991; Caetano-Anolles, 1994). Though the term
RAPD is the most widely used, DAF Q!NA Amplification !:ingerprinting), MAAP
(Multiple Arbitrary Amplicon Profiling) and AP-PCR (Arbitrarily Primed PCR) are
preferred by some authors. All strategies rely on the amplification of target sequences
with single random primers of 8 to 30 base pairs. As the shorter primers have a high
probability of detecting complementary sequences in the genome and low annealing
temperatures of down to 35°C additionally favor non-specific binding of primers, a
large number of primers bind closely enough to each other on opposite strands of the
DNA molecule to allow DNA amplification.
As a result. fragment patterns of 5 to 15 fragments in the size range below 3000 bp
are typically observed. Polymorphisms between different genotypes will be detected if
point mutations or inversions in the primer binding site or insertions or deletions
within an amplified fragment occur. Surprisingly, longer primers analyzed at higher
annealing temperatures have led to fragment patterns of similar complexity. This may
be explained as a result of mismatched primer-template hybrids (Welsh and
McClelland, 1990).
The enormous success and wide application of RAPD markers in plant and animal
genetics is based on the fact that no prior knowledge of the target DNA is required.
Furthermore, as the use of primer combinations within the same PCR results in new
amplification products, even a small number of oligonucleotide primers is sufficient to
analyze a large range of different, completely unrelated organisms (Welsh and
McClelland, 1991; Debener and Mattiesch, 1998). As very low amounts of DNA are
required for a single analysis (mostly about 20 ng per PCR) and the purity of DNA is
less critical than for RFLP and AFLP analyses, small amounts of plant material and
relatively crude extractions are sufficient for most plant species (Williams et al., 1990;
Karp et al., 1998).
The major disadvantage of the RAPD technique is its low reproducibility. Though
the procedures can be standardized and lead to highly reproducible results within a
laboratory, it has often been found to be difficult to reproduce amplified fragment
patterns in other labs (Weeden et al., 1992; Ellsworth et al., 1993; Karp et al., 1998).
332

Main factors seem to be the type of thermocycler used, the type of DNA polymerase
and the purity of the DNA Another disadvantage is the dominance of the marker
fragments, which limits the amount of genetic information in mapping experiments
(Hunt, 1997).

2.3. AFLP

As a combination of restriction digestion and amplification of DNA fragments, the

AFLP technique (Vos eta/., 1995) is becoming the most popular marker technique in
plant genetics. DNA is digested with a combination of two restriction endonucleases,
usually one "six-cutter" and a frequently cutting "four-cutter". The digested DNA is
ligated to two different double-stranded sequences (adaptors), each fitting the
appropriate restriction site of the DNA fragments. As a result, three different types of
ligation products are obtained: fragments flanked by two six-cutter sites/adaptors,
fragments flanked by two four-cutter sites/adaptors, and fragments flanked by one of
each type of site/adaptor. The ligation is followed by one or two amplification
reactions. The primers used for the amplification steps are complementary to the
adaptor sequences with an extension of usually one to three additional "selective" bases
reaching into the plant DNA fragment. As the amplification conditions are set to be
highly specific, only those fragments carrying the flanking adaptor sequences and the
selective bases will be amplified. This results in the amplification of only a subset of all
fragments ligated. Usually, a two-step procedure with one selective base in the first
amplification and two to three selective bases in the second round is applied. The
primers of the six-cutter adaptor are labeled either radioactively or with fluorescent
dyes. Fragments of more than 1000 bp are excluded from the analysis by the PCR
conditions and the separation on polyacrylamide gels. Most of the six-cutter fragments
are excluded because of their large average size and the four-cutter fragments will not
be visible on the gels because they are not labeled. Therefore, the analysis is based
merely on fragments with both a six- and a four-cutter site/adaptor. The detection of
the fragments is achieved either by autoradiography with fluorescently labeled primers
on automatic sequencers or by silver staining (Vos et al., 1995; Karp et al., 1998).
The advantages of the AFLP technique over other techniques are threefold. First, as
with the RAPD technique, no prior knowledge about the target genome is required.
Second, the reproducibility is almost as high as for the RFLP technique because the
conditions for the selective amplification are set to be highly specific. Third, the
number of fragments amplified for each pair of selective primers is higher than for any
other technique, usually ranging between 50 and 100 fragments. It is generally
assumed that these fragments result from the amplification of mostly unlinked loci,
although clustering of markers has been observed (Alonso-Blanco et al., 1998).
Disadvantages are the larger number of experimental steps in comparison to
RAPDs, the high technical input which, in part, is necessary to make full use of the
advantages provided by the technique (e.g. automated sequencers), and the fact that in
most cases the marker fragments have to be treated as dominant markers.
 333

2.4. MICROSATELLITES

Microsatellites are short tandem repeats of di-, tri- or tetra-nucleotides dispersed in

large copy numbers throughout the genome of higher organisms (Morgante and
Oliveri, 1993; Wang et al., 1994; Weising eta/., 1995). The name "satellite DNA"
stems from early observations that this fraction of eukaryotic genomes has a buoyant
density different to the non-repetitive fraction and therefore forms a separate band in
cesium chloride gradients during DNA isolation.
Microsatellite loci display an extremely high mutation rate due to different
processes, among which unequal crossover and replication slippage are discussed
(Morgante and Oliveri, 1993; Rafalski and Tingey, 1993). As a result, the number of
repeats per haplotype displays a large allelic variation which can be utilized for genetic
fingerprinting of genotypes (Weising eta/., 1998).
First analyses made use of mini- and microsatellites as hybridization probes in RFLP
experiments. It was shown that the resulting complex banding patterns have a high
discriminating power for variety and genotype identification experiments (Tzuri et al.,
1991; Vainstein et al., 1993; Wolff et al., 1995). Since then, several variations of this
technique have been introduced, among them the hybridization of microsatellite repeat
probes to filters onto which the size-separated products of RAPD reactions have been
blotted. This technique called RAMPO (Random Amplified Microsatellite
~olymorphisms, Weising et al., 1998), detected a new source ofpolymorphisms so far
unexploited by conventional RAPDs.
However, the major breakthrough of microsatellites came with their detection via
PCR-based strategies. Here, primers in conserved DNA regions flanking the highly
repetitive block of the microsatellite sequence are used to specifically amplify this
locus. The resulting amplification products are separated on highly resolving
polyacrylamide gels and length variation based on differences in the number of repeats
can easily be visualized (Morgante and Oliveri, 1993; Weising et a/., 1998).
Apart from genomic microsatellite loci, plastid sequences were also found to contain
microsatellite motifs that can be used to discriminate genotypes within different plant
species (Weising and Gardner, 1999).
The major advantages of microsatellites is the high average degree of
polymorphism per locus that can be detected, and the high reproducibility which is
comparable to the AFLP technique. Also, microsatellites require only small amounts of
DNA and once the markers have been developed, the experimental procedures are
relatively simple.
The major disadvantage is the large investment of time and labor for the
development of appropriate markers which include the cloning, selection and
sequencing of microsatellite loci, as well as the generation and testing of specific
primer pairs (Becher eta!., 2000).

2.5. SCAR, CAPS, SSCP

A group of markers obtained by cloning and sequencing DNA fragments from other
marker types are the SCAR (Paran and Michelmore, 1993), CAPS (Konieczyn and
334

Ausubel, 1993) and SSCP (Jordan eta/., 1998) markers. The principle of this marker
type relies on previously characterized DNA sequences, which ideally, but not
necessarily, have also been mapped in the genome of the organism under investigation.
The sequence information can be obtained from any cloned or amplified DNA
fragment (RFLP, RAPD or AFLP markers or any other cloned DNA fragment). Based
on the sequence, primers are designed that specifically amplify the region
characterized. lf a given set of genotypes displays length variation between the
amplified fragments, the marker is called a SCAR marker. lf no length polymorphism
is visible, the fragment may be digested with restriction endonucleases resulting in
polymorphisms if the pattern of restriction sites within one of the fragments is
different. In this case the markers are called CAPS markers. lfa small (<500 bp) DNA
fragment is denatured and separated on a non-denaturing polyacrylamide gel,
differences in the base composition of single strands may result in different migration
through the gel, thereby separating not only single strands of the same allele but also
different alleles. These markers are called SSCPs. SCAR, CAPS and SSCP markers
may be dominant or codominant. Their main advantage over RFLP markers is their
ease of application once the markers have been generated. In addition, SSCP markers
detect a high level of polymorphism which may not be detected by mere length
differences or the presence or absence of restriction sites within the amplified
fragment. In relation to RAPD markers they are usually more reproducible because the
amplification conditions can be set to be highly specific.
Disadvantages are the large amount of work necessary for the generation of specific
primer pairs (e.g. cloning and sequencing of DNA fragments) and the relatively low
informational content of SCAR and CAPS markers in comparison to AFLPs and
RAPDs. Furthermore, in most cases the detection of polymorphisms for SCAR and
SSCP markers requires more sophisticated conditions for gel separation of the DNA
fragments as compared to RAPD and RFLP markers in varietal fingerprint analyses.

3. Application of Molecular Markers to Ornamental Plant Breeding

The application of molecular markers to ornamental plant breeding occurred later than
in the major agricultural crops, thus reflecting the general delay in the adoption of
advanced breeding strategies (Anls, 2000).
Reasons for this are numerous. Apart from the huge diversity of different species
cultivated for ornamental use, resulting in relatively limited economic importance of
the individual crop, the genetic structure of many ornamentals, some of which are
polyploids, obligate outcrossers, flowering only once a year, etc. often hampers genetic
analyses and planned manipulations of important traits in the breeding process.
Even in the major ornamental crops, for example, roses and carnations, breeding
has been performed mostly without knowledge of the inheritance of major
horticulturally important traits (Scovel eta/., 1998; Gudin, 2000). Therefore, molecular
markers are mostly used for the genetic differentiation of varieties and the analyses of
phylogenetic and genetic relatedness between species and varieties. In only a few cases
have markers been applied to genetic analyses, map construction or targeting of
 335

horticulturally important genes for marker-assisted selection.

3.1. VARIETY AND GENOTYPE IDENTIFICATION

The vegetative multiplication of many ornamental crops, among them the most
economically important ones, such as rose, carnation, tulip, chrysanthemum, etc., and
the relatively high prices for the individual flower/plant lead to frequent infringements
of protected varieties and as a consequence, a considerable amount of money is lost due
to fraudulent propagation.
As the conventional testing of varieties is a time-consuming process which also
cannot rule out errors (Loscher, 1992), molecular markers provide clear advantages
both in accuracy and speed to uncover those illegal practices (Becher et al., 2000).
Therefore, the use of molecular markers for genotype and variety identification has
been the most prominent application, with more than 20 different species having been
investigated to date.
In the most simple experimental set-up, marker profiles are produced for a certain
set of genotypes and the identity or distinctness of the marker patterns is analyzed
manually (Rajapakse eta/., 1992; Jianhua et al., 1997). Plants belonging to the same
clone are expected to display the same marker profile, whereas the profiles of
genotypes resulting from sexual reproduction are expected to be different An example
of studies on variety identification in ornamentals is given in Table 1. For sexually
propagated crops, the picture is more complicated. Here, each variety consists of a
population of genetically distinct individuals. In these cases, markers can either be
used to differentiate varieties via distinctive marker allele combinations (Iqbal and
Rayburn, 1994) or the differentiation of populations has to be expressed as genetic
distances based on allele frequencies (Huff et al., 1993; Yu and Pauls, 1993).This,
however, cannot rule out misinterpretations for small sample sizes. In each case, the
discriminative power of molecular markers is much smaller than in vegetatively
propagated plants.
An interesting question connected to the problem of variety identification is
whether sports can be distinguished from their ancestral variety at the DNA level.
Recent changes in breeders' rights within the EU define sport varieties as "essentially
derived" varieties and concede part of the royalties from these to the breeders of the
ancestral variety (EU-Regulation No. 2100/94). Therefore, the clear assigmnent of
sports to their ancestral varieties is of immense economic importance for breeders of
vegetatively propagated ornamentals.
Sports and their ancestral varieties have been investigated, for example, in
Alstroemeria (Anastassopolous and Keil, 1996), chrysanthemum (Wolff et al., 1995;
Scott eta/., 1996; Trigiano eta/., 1998), Pelargonium (Becher et al., 2000) and rose
(Debener eta/., 2000; Zhang eta/., 2000b). Whereas in most cases sports could not be
differentiated from their ancestral varieties, differences were detected in
chrysanthemum (Trigiano et al., 1998), poinsettia (Starman et al., 1999), rose
(Debener eta/., 2000) andAlstroemeria (Anastassopolous and Keil, 1996). However, in
all examples, the low level of polymorphism in the sports still allowed the assignment
to the ancestral variety as compared to sexually propagated progeny (Becher et a/.,
336

2000; Debener eta/., 2000). This demonstrates the value of fingerprinting techniques
with molecular markers as a new strategy for breeders to claim their rights on these
"derived varieties".

TABLE 1. Ornamental plant species used for variety identification with molecular markers

Genus Marker type Reference

Alstroemeria RAPD Beyermarm et al., 1992; Anastassopolous and

Keil, 1996; Han et al., 1999
Calladium AFLP Loh et al., 1999
Cephalotaxus AFLP Zhang et al., 2000a
Cymbidium RAPD Obara-Okeyo and Kako, 1998
Dahlia AFLP SUdbeck and Debener, 2001
Dendrathema RAPD/DAF Wolff et al., 1993; Wolff and Peters-Van Rijn,
(drrysanthemum) 1993; Wolff et al., 1995; Scott et al., 1996; Wolff,
1996
Dianthus Microsatellite RFLPs Vainstein et al., 1991, 1993
Euphorbia (poinsettia) RAPD/DAF Ling et al., 1997; Starman et al., 1999
Gerbera RFLP!Micros. RFLPs Tzuri et al., 1991; Vainstein et al., 1993
Heliconia RAPD Goh et al., 1995; Kumar et al., 1998
Juniperus RAPD Adams and Demeke, 1993
Lilium RAPD Yamagishi, 1995
Osteospermum RAPD Faccioli et al., 2000
Ozothamnus RAPD Ko et al., 1996
Pelargonium RAPD/DAF Rmou et al., 1997; Starman and Abbit, 1997;
Barcaccia et al., 1999; Becher et al., 2000; Lesur
etal., 2000,
Petunia RAPD/DAF Cerny et al., 1996; Jianhua et al., 1997
Rhododendron RAPD, Microsatellite Dunemarm and Kahnau, 1998; De Riek et al.,
RFLPs 1999 ; Naito et al., 1999
Rosa Microsatellite RFLPs Tzuri et al., 1991; Hubbard et al., 1992;
IRAPD/RFLP Rajapakse eta/., 1992; Torres eta/., 1993;
Vainstein et al., 1993;Vainstein and Ben Meir,
1994; ; Dehmer et al., 1996; Gallego and
Martinez, 1996; Matsumoto and Fukuri, 1996;
Millan et al., 1996 Dehmer et al., 2000
Scaevola RAPD Swoboda and Balla, 1997
Syringa RAPD Marsolais et al., 1993

3.2. PHYLOGENETIC ANALYSES AND ANALYSES OF GENETIC

RELATEDNESS

Markers may not only be used to distinguish genotypes, they may also provide
information about the genetic relatedness between genotypes, and even phylogenetic
 337

relationships between species and higher taxa.

Assuming that the closer the genetic relationship between a given pair of genotypes
is, the larger the number of shared markers will be, different strategies may be
employed to infer the genetic similarities or phylogenetic relationships (Staub et al.,
1996, Avise, 1994). In most cases, markers are treated as individual characters and
their mere presence or absence is recorded without weighing the different marker
fragments. Under the assumption that markers of identical size (e.g. RFLP or AFLP
fragments) represent homologous DNA fragments and that the genetic relationships of
the taxa under investigation are not disturbed by processes summarized under the term
"reticulate evolution" (e.g. sexual hybridization of taxa or "horizontal gene flow"), two
basic strategies are widely applied.
The so-called "maximum parsimony" method uses the marker fragment matrix
directly to compute a dendrogram with a minimum of character differences between
the individual fragment patterns. This method is supported by the "cladistic school" of
phylogenetic inference (Felsenstein, 1988).
For the second method, fragment matrices are first converted into a matrix of
pairwise values for genetic distance or similarity. This can be done by a variety of
different algorithms (Rohlf, 1989). From the similarity matrix, a dendrogram can be
constructed. Here also different strategies may be employed, with UPGMA
(Felsenstein, 1988) and neighbor-joining (Saitou and Nei, 1987) being among the most
popular methods. As an alternative to dendrograms, the genetic distance between
genotypes may also be displayed in three-dimensional representations using principal
component analyses (PCA; Rohlf, 1989).
For both general strategies and numerous available methods, conditions are rarely
met with most real sets of data (discussed in Nei, 1987; Felsenstein, 1988 and Avise,
1994). A major violation of the underlying conditions for all methods is the presence of
hybrid genotypes in the data sets. However, pairwise distances inferred from fragment
data and dendrogram computations with UPGMA clustering turn out to be relatively
robust against inconsistencies in the data sets and are therefore the strategy most
widely applied for the inference of genetic relationships between ornamental plant
varieties and species. A selection of studies on genetic distances between ornamental
plant varieties and species is listed in Table 2.
A variety of computer programs are available for processing marker data and for
computing dendrograms and phylogenetic trees (Rohlf, 1989, Felsenstein, 1995). A
useful overview of available software packages can be found at the Web sites:
http://evolution. genetics. washington.edu/phy1ip.html,
http://corba.ebi.ac.uk/Biocatalog!Phylogeny.html or
http://linkage.rockefeller.edu/softllist.html.
The information about the genetic distance between ornamental plant genotypes or
species may be applied in several ways: In the case of phylogenetic information about
the relationships between different ornamental taxa the data may be used to infer the
evolutionary history of the cultivated varieties. Apart from the increase in basic
knowledge, this information may be useful for the exploitation of exotic germplasm in
that either hybrid ornamental species may be re-synthesized or in selecting most
closely related species for the introgression of horticulturally interesting traits.
338

Information on the genetic diversity within the genepool of cultivated varieties may be
used to select the most distantly related parents in order to minimize inbreeding
depression. This may be of particular importance for groups of ornamental varieties
which, over the last decades, have been bred only within a limited genepool (e.g. cut
roses, carnations, etc.).

TABLE 2. Selection of ornamentals for which genrtic distances or phylogenrtic relationships have been
investigated with molecular markers

Genus Marker type Computational Reference

mrthod
Dendrathema DAF UPGMA/PCA Scott et al., 1996
(chrysanthemum)
Euphorbia (poinsrttia) RAPD UPGMA/PCA Linget al., 1997
Geranium DAF UPGMA/PCA Starman and Abbit, 1997
Juniperus RAPD PCA Adams and Demecke, 1993; Le Due
et al., 1999
Petunia DAF maxunum Cerny et al., 1996
parsimony
Rhododendron various UPGMA Chamberlain and Hyam, 1998;
techniques DeRiek et al., 1999
Rosa RAPD UPGMA Ben-Meir and Vainstein, 1994;
Debener et al., 1996; Janet al.,
1998
Viola RAPD UPGMA Ko et al., 1998

3.3. GENETIC ANALYSES AND MARKER-ASSISTED SELECTION

Apart from the analysis of genetic diversity among genotypes and populations, markers
may also be used for genetic analyses and for marker-assisted selection (MAS).
Preconditions for all these applications are the availability of clearly defined
segregating populations and thorough genetic analyses of traits relevant for MAS.
Molecular marker maps may serve as a general tool for analyses of the genetic
structure of plant genomes (Staub et al., 1996). Maps can be constructed with
segregating F2, backcross, recombinant inbred and "double-pseudo-testcross"
populations. In the latter, two highly heterozygous, sometimes unrelated genotypes are
crossed. In diploids, up to four different alleles may segregate in the resulting
population. This strategy has turned out to be very useful for ornamentals, especially
for some woody ornamentals for which homozygous lines cannot be obtained
(Grattapaglia and Sederoff, 1994). If dominant markers (e.g. RAPDs and AFLPs) are
used, a disadvantage of this strategy is the necessity to start map construction with
individual maps for each parent which have to be merged via common markers with
relatively low informational content.
A number of computer packages are available that are capable of dealing with most
population types. Among these, Joinmap'™ (Starn and VanOijen, 1995) and Mapmaker
(Lander et al., 1987) are the most widely used. Further information about software for
genetic analyses and map construction may also be found at the Web site
 339

http://linkage.rockefeller.edu/softllist.html.
The limited information available on the genetics of ornamental crops is reflected
in the paucity of reports on molecular marker segregation (Table 3). In ornamentals,
general molecular marker maps have been constructed for Petunia (Gerats eta/., 1995),
Rhododendron (Dunemann et al., 1999) and Rosa (Debener and Mattiesch, 1999;
Debener et al., 2001). Whereas an F2 population and RFLP markers were used for the
construction of the Petunia map, AFLP and RAPD markers, and so-called "double-
pseudo-testcross" populations were used to build the maps in Rosa and Rhododendron.
Along with the molecular markers, a quantitative trait locus (QTL) for lime tolerance
was located on the Rhododendron map (Dunemann eta/., 1999) and genes for double
flowers and pink flower colour were located on the rose map (Debener and Mattiesch,
1999).

TABLE 3. Ornamentals for which genetic analyses with molecular markers have been performed

Genus Type of analysis Reference

Dianthus Mapping of a gene for double flowers with Scovel et al., 1998
RAPDs and RFLPs
Lilium QTL mapping of flower longevity van der Meulen Muisers et al.,
1995
Petunia Mapping of actin genes by RFLP analysis, McLean et al., 1990; Pehier et al.,
segregation analyses, construction of a marker 1994; Gerats et al., 1995
map
Rhododendron Construction of a molecular marker map and Dunemann et al., 1999
mapping of Qtls for lime tolerance
Rosa Segregation analyses with markers, Construction Debener and Mattiesch, 1998;
of a marker map, Mapping oftarga genes relative Debener and Mattiesch, 1999;
to molecular markers Malek and Debener, 2000

The combination of marker systems which simultaneously detect a large number of

loci (e.g. AFLPs) with strategies like the so-called "bulked segregant" analysis allows
the identification of markers closely linked to a target gene, even in large genome
species without genetic maps (Michelmore et at., 1991).
Studies in which markers linked to genes of interest were detected without
constructing a whole chromosome map were conducted for carnation (mapping of a
major dominant gene for double flowers, Scovel et at., 1998), Petunia (mapping of
individual genes of an actin gene family, McLean eta/., 1990), Lilium (QTL mapping
of flower longevity, van der Meulen-Muisers eta/., 1995) and Rosa (mapping of a
major dominant gene for blackspot resistance, Malek and Debener, 2000).
Where large segregating populations and "large insert libraries" (e.g. YAC and
BAC libraries) are available, this approach can also be used to isolate the target genes
via positional cloning (Cnops et al., 1996). However, as the construction of complete
YAC and BAC libraries is still a technically demanding enterprise, this strategy for the
isolation of genes is limited to very few of the most economically important
ornamentals (Kaufmann and Debener, 2000).
340

MAS may either focus on single genes (Onion et al., 1998), on QTLs (Paterson et
al., 1991; Lawson et al., 1997), or on the reduction of unwanted genetic backgrounds
(Visscher eta/., 1996).
Although the allelic status of some genes, for example genes for flower
morphology, may easily be screened visually, closely linked markers may be useful in
MAS. For polyploid crops, markers may unravel the allelic status of the particular
locus, thereby enabling the breeder to manipulate complex genetic backgrounds more
effectively. Other traits may be very difficult to screen, for example recessive genes like
the gene for recurrent flowering in roses or certain resistance genes where assays
require controlled laboratory conditions (Fridman eta/., 2000; Malek and Debener,
2000).
Furthermore, the problem of combining several single genes into a particular
genetic background may be significantly enhanced by the application of markers. This
may be of particular importance for pyramiding resistance genes (Kelly and Miklas,
1998; Ordon et al., 1998). Here, markers may be a useful alternative tool in the
selection process. However, as a prerequisite for practical applications, the technical
input for marker analyses has to be reduced. First strategies for the detection of allele-
specific PCR products omitting gel separation have been published (Tyagi and Kramer,
1996, Alcala et al., 1997) but are still too resource-demanding for practical
applications in ornamental plant breeding.

4. Future Trends

Future trends in the application of molecular markers to ornamental plant breeding

will be strongly influenced by two areas of research.
i) The vast amount of information about the genetic architecture of plant genomes
which is currently generated by several plant genome projects will also provide
valuable information for research on ornamental plants (Bennetzen, 1998; Lin et
al., 1999). Well-characterized genes with a high degree of conservation between
taxa may also be isolated from ornamentals and may either be used as markers in
the breeding process or for genetically engineering ornamental crops. Further genes
may be tagged in those cases where the synteny between model organisms and
ornamental taxa is high enough (Ku eta/., 2000; Paterson et al., 2000). In the near
future, this exponentially growing information resource will significantly increase
the speed with which genes from ornamental species can be isolated, characterized
and utilized in the breeding process.
ii) In the area of human genetic diagnostics, strong economic pressure has led to the
development of marker systems with higher resolution, which can be applied with
lower investments in time and money and which are suitable for large-scale
automation (Schafer and Hawkins, 1998). Some of these new tools are already
available for the analysis of genetic variation in plants.
One of the developments already in use for basic research in plant molecular
biology are the so-called "microarrays" (Hoheisel, 1997). Here different sources of
DNA can be covalently fixed to solid supports at very high density so that several
 341

thousand different sequences per square centimeter may be bound in an ordered array.
Through hybridization with fluorescently labeled probes and devices for the automated
and quantitative detection of the resulting signals, these arrays can be used for the
analysis of gene expression, sequencing and also for genotyping via analysis of single
nucleotide polymorphisms (SNPs).
Another strategy also recently developed for the high throughput analysis of SNPs
is MALDI-TOF (Matrix-Assisted Y.ser _!!esorption-!onization !ime-Of-EJ.ight) mass
spectrometry. This technique allows the highly efficient and quick detection of single
base pair differences in short DNA fragments generated by PCR amplification (Crain
and McCloskey, 1998; Griffin and Smith, 2000). With automated sample preparation,
more than 100,000 samples may be processed per machine per day.
These developments, once they become available to laboratories working on
ornamental plants, will lead to significant advances in the areas of both genotype
fingerprinting and MAS.

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Wolfl: K., Schoen, E.D., and Peters-Van Rijn, J. (1993) Optimising the generation of random amplified
polym01phic DNA in chrysanthemum, Theor. Appl. Genet. 86, 1033-1037.
Wolfl: K., Zietkiewitz, and Hofstra, H. (1995) Identification of chrysanthemum cuhivars and stability of DNA
fingerprint patterns, Theor. Appl. Genet. 91, 439-447.
Yamagishi, M. (1995) Detection of section-specific random amplified polym01phic DNA (RAPD) markers in
Lilium, Theor. Appl. Genet. 91,830-835.
Yu, K. and Pauls, K.P. (1993) Rapid estimation of genetic relatedness among heterogeneous populations of
alfalfa by random amplification ofbulked genomic DNA samples, Theor. Appl. Genet. 86, 788-794
Zhang, D., Dirr, M.A, and Price, R.A (2000a) Discrimination and genetic diversity of Cephalotaxus accessions
using AFLP markers, J. Amer. Soc. Hort. Sci. 125, 404-412.
Zhang, D., Germain, E., Reynders-Aloisi, S., and Gandelin, M.H. (2000b) Development of amplified fragment
length polym01phism markers for variety identification in rose, Acta Hort. 508, 113-120.
PLANT-SPECIFIC INTELLECTUAL PROPERTY RIGHTS

S. BERMAN, JUDGE (ret.,) District Court

Hebrew University ofJerusalem
Faculty ofAgricultural, Food and Environmental Quality Sciences
P.O.Box 12
Rehovot 76100, Israel

1. Introduction

Intellectual property rights are legally designed to protect the non-physical (intangible)
creative expressions of the human mind in all fields of human endeavor, be it artistic,
literary, scientific, technological, industrial, agricultural, horticultural, or any other
similar field of intellectual activity. The traditional principles of property law protect
physical objects via the possession doctrine which recognizes the rights of the possessor
of a physical object to enforce his proprietary rights over the object and prevent others
from using it without his consent. Intangible products of the human mind, once publicly
disclosed, cannot be protected under traditional principles of property law. There is no
physical possession of an intellectual creation, distinguished from the tangible product
in which it may be embodied. Once the intellectual creation has left the secret domain
of the creative mind or inventive genius, the author, creator or inventor of said
intellectual product cannot implement his proprietary rights within the legal framework
of property law. 1
Intellectual Property Law was developed with the aim of providing a statutory
framework for assuring property rights, and under some legal systems also moral
rights, in intellectual creations. It is a legislative construction empowering governments
to accord exclusionary economic rights as well as moral rights to the non-physical
results of intellectual creativity, scientific research, industrial and agricultural
innovation or any other similar intellectual activity, for the protection of the integrity
and economic rights of the creators in their intellectual creations. 2 This legalistic
concept was aptly expressed in the Constitution of the United States by empowering
congress "to promote the progress of science and useful arts, by securing, for limited
times, to authors and inventors, the exclusive right to their respective writings and
discoveries ... " 3 The rationale embodied in this concept lies within the public interest by
encouraging intellectual creativity, research efforts and inventiveness as vehicles of
progress and inspiration in arts and sciences, and as important means for industrial
development and economic growth. By granting exclusive rights for limited periods to
authors, artists, scientists, inventors, and other "creators" of intellectual products, the
law enables them to exercise control over their intellectual creations and to prevent
others from making any unauthorized use of said creations within the protected periods.
In return, upon expiry of the legally protected periods, the public gains free access to
347
A. Vainstein (ed.), Breeding for Ornamentals: Classical and Molecular Approaches, 347-379.
© 2002 Kluwer Academic Publishers.
348

new "works," products and processes, which become part of the public domain.
In concentrating on industrial inventions, for the purpose of this survey, it should be
stressed that by assigning exclusive proprietary rights for limited periods, via patents or
via new plant varieties rights, the law grants the inventor or the breeder a restricted
monopoly to commercialize his intellectual creation, and enables him to ensure the
economic benefits of future investments in the development of his product. In return for
the provided incentives for inventiveness and productivity, the inventor is obliged by
law to disclose his invention in a clear and complete manner, enabling it to become
accessible for public use and dissemination upon expiry of his limited exclusive rights.
Generally, under patent laws and breeders' rights laws, the protection is for a period of
20 years.
The granting of a temporary "monopoly" in industrial inventions strikes a balance
between the interests of the individual inventor in obtaining maximal economic benefits
from his intellectual creation and the public interest in encouraging scientific and
technological innovation and promptly gaining free access to its disclosed results, thus
enriching the stock of human knowledge and promoting further research and
development.
The protection of intellectual property rights (IPRs) is, in principle, a matter
regulated by the national laws of each individual country and implemented within its
territorial boundaries. Thus each country establishes its own legal apparatus to strike a
balance between conflicting interests, in accordance with its cultural, political, social
and economic principles. Nevertheless, it should be stressed that already in the late 19th
century, the international community recognized the importance of cross-border
protection of IPRs and found it necessary to create international frameworks, designed
to provide harmonized and effective protection. By signing international or regional
conventions or agreements, the signatory or later adhering states undertook obligations
to protect IPRs within their national legislation, in compliance with the norms
prescribed by such legal instruments.

2. The Relevant International and Regional Infrastructure

In 1883, upon signing the Paris Convention for the Protection of Industrial Property
("Paris Convention"), its member states established the Paris Union for the protection of
intellectual creations having industrial applications. 4
In 1886, the Berne Convention for the Protection of Literary and Artistic Works was
signed, establishing the Berne Union for the protection of copyright and moral rights in
literary and artistic creations. 5
In 1961, the International Convention for the Protection of New Varieties of Plants
(UPOV Convention) was signed, establishing a union with the aim of providing a
harmonized international framework for the protection of breeders' rights in new plant
varieties. In its re-enacted version, the 1991 UPOV Act is the most comprehensive
international legal instrument for dealing with IPRs in new plant varieties, as will be
further discussed. 6
In 1967, with the aim of facilitating efficient protection of intellectual property
throughout the world, the World Intellectual Property Organization (WIPO) was
established. 7 In defining IPRs, the WIPO Convention includes rights in inventions "in
 349

all fields of human endeavor. .. resulting from intellectual activity in the industrial,
scientific, literary or artistic fields." 8
In 1973, the European Patent Convention (the EPC) was signed in Munich,
establishing and empowering the European Patent Organization (the EPO), acting as
from Oct 7, 1977, to grant European patents applicable in any designated European
state. 9 A proposal for granting Community patents, to be valid in all the EU states, is
pending under an ongoing discussion. The EPC provides that European patents shall be
granted "for any inventions which are susceptible of industrial application, which are
new and which involve an inventive step" but it explicitly excludes from patentability:
"plant...varieties or essentially biological processes for the production of plants ... ," not
being "microbiological processes or the products thereof."
In 1993, the North American Free Trade Agreement (NAFTA) was signed between
the governments of Canada, the Mexican states and the USA, establishing a "free trade
area" and stating its aim "to foster creativity and innovation." 10 Within their obligation
under GATT, they pronounced their commitment to provide effective protection and
enforcement of IPRs in their respective territories. 11 NAFT A obliges each of its parties
to accord patents, but allows exclusion from patentability of "plants and animals and
essentially biological processes for their production." However, it requires that each
party to the agreement "provide for the protection of plant varieties through patents or
an effective scheme of sui generis protection, or both."
In 1994, with the aim of strengthening and harmonizing the national treatment of
IPRs on a global scale, an agreement on Trade-Related Aspects of IPRs (TRIPS) was
signed, following the GATT Uruguay Round, which established the World Trade
Organization (WTO) as a common framework for governing international trade and
commerce. 12 The TRIPS agreement, annexed to the multilateral WTO agreement, which
entered into force on Jan 1, 1995, is binding for all members of the WTO. It is the most
comprehensive international instrument dealing with the legal protection of IPRs at
large. 13 Moreover, TRIPS allows its members to exclude from patentability "plants and
animals and essentially biological processes for their production" but explicitly provides
that the exception should not be applied to "microorganisms and non-biological and
micro-biological processes." Without setting any precise standards for the protection of
plant breeders' rights, the TRIPS agreement obliges its member states to provide
protection for plant varieties "either by patents or by an effective sui generis system or
by any combination thereof."
In 1995, with the aim of improving protection ofPVRs within the EU, a Community
Plant Variety Right (CPVR) was created, under an EC Regulation. The community
plant variety office (CPVO), having its seat in Angers (France), is an independent body
of the EC. In co-existence with the European national regimes, the CPVO is empowered
to grant a PVR, which will be valid throughout the entire European Community. 13"
In 1998, with the aim of encouraging investment in the field of biotechnology, the
European Parliament and Council signed a special directive on "The Legal Protection of
Biotechnological Inventions" which entered into force on Sep I, 1999. In recognizing
the increasing importance of biotechnology in a broad range of industries, requiring
high-risk investments, especially in the field of genetic engineering, the EC was of the
opinion that only adequate, clarified and harmonized legal protection of
biotechnological inventions could make them profitable. The directive obliges the EU
member states to protect biotechnological inventions under their national patent laws in
350

conformity with the provisions of the directive, as will be further discussed. 13b

3. The General Framework for Patent Protection of Industrial Property

The basic standards for harmonized patent protection of industrial property at large are
prescribed by the major multi-national legal instruments: the Paris Convention, the
TRIPS agreement and NAFfA, and the EPC.

3.1. THE PARIS CONVENTION

The Paris Convention is designed to secure protection of industrial property via

"patents, utility models, industrial design, trademarks, trade names, indications of
source or appellations of origin and the repression of unfair competition." 14 It provides,
as its main feature, that nationals of any member state of the Union shall enjoy
protection of their industrial property rights in any other member state, in the same
manner as granted to its nationals, subject to prescribed conditions and formalities under
its nationallaw. 15 With regards to patents, the inventor has a right to have his name
mentioned as inventor. 16 When a product is imported into a country of the Union where
a process patent was granted, the patentee may implement his patent rights with respect
to said product. 17
The Convention provides a right of priority to any person who has duly filed an
application for a patent, in any of the member states of the Union. For patents, this right
lasts 12 months, starting from the date of filing the first application. 18 Its member states
are obliged, in conformity with their domestic laws, to grant temporary protection (no
more than 12 months) to patentable inventions "in respect of goods exhibited at
officially recognized international exhibitions held in the territory of any member
state." 19
Members of the Union are bound to ensure effective protection against unfair
competition or any other act contrary to honest practices in industrial or commercial
matters, such as creating confusion, false allegations, misleading information as to the
manufacturing process, the characteristics of a product and its suitability for a named
purpose. 20 Each country of the Union has a right (through legislative measures) to grant
compulsory licenses to prevent abuses.
In securing protection of industrial property, the Paris Convention applies not only
to industry and commerce proper (in the narrow sense) but also to: "agricultural and
extractive industries and to all manufactured or natural products, for example wines,
grain, tobacco leafs, fruit, cattle, mineral, mineral waters, bees, flowers and flour." 21 In
general, these also include-if not otherwise provided-the "horticultural industry,"
covering its products, such as "cut flowers and decorative foliage; pot plants, bedding
plants and herbaceous plants, shrubs and flowering trees; fruit trees, fruit bushes and
fruit plants and bulbs, corms and tubers. " 22

3.2. THE TRIPS AGREEMENT

The TRIPS agreement provides the most comprehensive legal framework for the
protection of intellectual property at large. Tailored to embrace all categories of
 351

intellectual creativity, including products and processes of the most advanced

technologies, it already binds close to 150 adhering member states, with the prospect of
becoming universally binding. In broadening the scope of protection provided by the
Paris Convention, TRIPS requires that all its parties provide protection, via their
national laws, for inventions in "all fields of technology." 23' 24 In establishing a multi-
lateral framework, the parties to the agreement prescribe rules, norms and measures
concerning the availability, scope, use and effective enforcement of trade-related IPRs,
entitling each party to expand the minimal standards, if not in contradiction with the
agreement. 25 Subject to exceptions, the mutuality principle obliges each of the parties to
accord protection of IPRs to nationals of the other parties, as it accords to its own
nationals.
Attentive to new developments in the global economy, TRIPS pronounces that IPRs
"are private rights," thereby asserting a very important legal concept. In listing its basic
principles, TRIPS pronounces the traditional raison d'etre of intellectual property law
by stating that protection and enforcement of IPRs contributes to the promotion of
technological innovation, its transfer and dissemination. 26
In defining patentability, it provides that "patents shall be available for any
inventions, whether products or processes, in all fields of technology, provided that they
are: 1) new; 2) involve an inventive step (non-obvious); and 3) are capable of industrial
application ("useful")-without discrimination as to the place of invention, the field of
technology and whether imported or locally produced. " 27
TRIPS stipulates that exclusions from the general patent availability are allowed
only within the explicitly prescribed exceptions. Parties to the agreement may exclude
inventions whose exploitation may be against the "ordre public" or "morality", or may
affect human, animal or plant life or health, or be harmful to the environment.
Albeit its proclaimed aim to provide standards for patent protection in all fields of
technology, the TRIPS agreement leaves out the special field of plant breeders' rights,
in providing that its members may exclude from patentability: "plants and animals other
than microorganisms, and essentially biological processes for the production of plants
or animals, other than non-biological and micro-biological processes. " 28
In obliging its members to protect plant varieties "either by patents or by an
effective sui generis system or by any combination thereof' it stipulates that this
provision is subject to review, 4 years after entry into force of the WTO agreement. 29
A patent confers on its proprietor the exclusive right (subject to exhaustion rights) to
prevent third parties, not having his consent, from making or using the J>rotected
product or process, or the directly obtained product of a protected process. 3 Limited
exceptions are permitted provided these do not "unreasonably prejudice the legitimate
interests of the patent owner [and] ... of third parties. " 31 The exclusive rights are subject
to several recognized uses without authorization. 32
An applicant for a patent has "to disclose the invention in a manner sufficiently clear
and complete for the invention to be carried out by a person skilled in the art." He may
be requested "to indicate the best mode for carrying out the invention" as known to him
at the time of applying for a patent or on the priority date, if claimed.
The term of protection shall not expire before the end of 20 years from the filing
date. 33
352

3.3. THE NAFTA

By signing NAFTA, the signatory parties (USA, Canada and Mexico) undertook to
provide effective protection and enforcement of IPRs. 34 In adopting the "mutuality
principle," NAFTA prescribes a set of requirements almost identical to that in TRIPS. 35
In obliging each party to make patents available "for any inventions, whether products
or processes in all fields of technology," it sets criteria for patentability and permits
exclusions in the same manner as provided by TRIPS. 36 In setting the scope of patent
protection, it provides that the signatory parties shall confer the exclusive rights, as
provided under TRIPS, subject to the same limited exceptions. 37 Each~ shall permit
assignment, licensing and transfer by succession of patent rights. 8 The protection
should last for at least 20 years from the date of filing or 17 years from the date of
granting the patent. 39
Notwithstanding the permitted exclusion from patentability of plants and animals
and essentially biological processes for their production, NAFfA requires that its
parties shall provide protection for plant varieties "through patents or an effective
scheme of sui generis protection, or both" 40 (emphasis added). Each signatory party is
obliged to give effect to the substantive provisions of the Paris Convention and the
UPOV Conventions (1978, 1991) for the protection of plant varieties. 41 Each party is
obliged to ensure that the measures for enforcing IPRs should not, themselves, become
barriers to legitimate trade.

3.3.1. Special Provision as to Pharmaceutical or Agricultural Subject Matter

NAFT A provides that if a Party has not made available product patent protection for
"subject matter" that relates to naturally occurring substances, prepared or produced by,
or significantly derived from, microbiological processes "intended for food or
medicine" (as of Jan 1, 1992) or "for any other subject matter" (as of Jul1, 1991), said
Party shall provide the means to obtain patent protection for such products, for the non-
expired term of the product patent obtained in another Party. Such protection is to be
given to the inventor of such product or his assignee, provided there was "a timely
request" and the product has not been marketed in the Party providing protection. 42

3.4. THEEPC

With a desire to strengthen cooperation between the European states with respect to
protection of industrial property rights, the EPC provides a comprehensive system of
law for granting European patents, under a "single procedure." The EPO, having its seat
in Munich (Germany), is a legal entity empowered to grant European patents which
have the same effect as the national patents in each of the member states. 43 The
applicant is entitled to decide in which of the European states he is interested in
applying his patent. 44 The procedures prescribed by the Convention are implemented
through its administrative and professional divisions. The decisions of these divisions
are subject to appeal to the Boards of Appeal consisting of technically and legally
qualified members. Points of law are referred to the Enlarged Board of Appeal, all as
provided by the convention. 45 The Administrative Council is empowered to enact rules
of procedure and regulation of implemention. 46 In promoting applications for European
patents, the EPO states that its main task is to grant patents. In underlining the
 353

advantages of obtaining European patents, the EPO stresses cost-effective, time-saving

methods of application, the substantive examination of each application, the search for
alternatives, access to the latest technology and its strength for enforcement. 47

3.4.1. Patentable "Subject Matter"

Inventions which are "new, non-obvious and susceptible to industrial application" are
eligible for a European patent. 48 However "discoveries, scientific theories and
mathematical methods" are not considered as patentable "subject matter. " 49
An invention is considered to be new if it does not form part of the state of the art,
meaning "everything that is already known or made available to the public in whatever
way, before the filing date of the European patent application." 50 However if disclosure
of an invention took place in the framework of an official international exhibition,
within a period of not earlier than 6 months preceding the filing, the disclosure shall not
be taken into consideration, subject to a series of prescribed rigid requirements. 50"
An invention shall be considered "as involving an inventive step" if, having regard
to the state of the art, it is not obvious to a person skilled in the art. 51 An invention shall
be considered as "susceptible of industrial application" if it can be made or used in any
kind of industry, including agriculture. 52
It is explicitly provided under Art. 53(a) of the EPC that "European patents shall not
be granted in respect of inventions the publication or exploitation of which would be
contrary to ordre public or morality." Neither shall European patents be granted for
"plant or animal varieties or essentially biological processes for the production of plants
or animals" not being "microbiological processes or the products thereof," as Art. 53 (b)
provides.
Notwithstanding the fact that "scientific discoveries" 53 are listed among the various
types of intellectual property, the framers of the EPC piously observed the traditional
doctrine of non-patentability of "products of nature." Courts have repeatedly
pronounced that subject matter which occurs naturally and discoveries of "natural
phenomena" are not patentable. These are "free to all men and reserved exclusively to
none." 53• "Inventions," differ from "discoveries," even if they rely on properties of the
material universe, involve an inventive step or demonstrate new uses of natural
properties, whether known before or discovered simultaneously during the invention.
However, it should be stressed that within the last two decades, with the vast
development of new technologies, the sharp distinction between discovery and invention
has been somewhat blurred, as will be further discussed, especially with respect to
biotechnological inventions.

3.4.2. The Application for a European Patent-an Object of Property

The application "must disclose the invention in a manner sufficiently clear and
complete for it to be carried out by a person skilled in the art." 54 It should relate to one
invention only or to a single general inventive concept. 55 It should specify the claims in
a clear and concise manner, defining the matter for which protection is sought. 56 A
search is performed to establish the relevant "prior art." The application and the search
report are to be published upon expiry of 18 months from the filing date, containing the
description, the claims and any drawings, as filed. 57
Subject to some formalities, publication of the application confers on the applicant
interim protection against competitive use of the invention, in all the designated
354

countries, as chosen by the applicant. 57 a

A European patent application "is an object of property" subject to the law of the
designated contracting state. It may be transferred or give rise to rights. It may be
licensed wholly or in part. Assignment of any right in the patent application is to be in
writing, signed by the parties to the contract. 58

3.4.3. Rights Conferred by a European Patent and the Term ofProtection

If, following a requested substantive examination, the invention answers to the triple
criteria of patentability: novelty, inventive step, industrial applicability, the EPO will
grant a patent. Once granted, it confers on its proprietor, from the date of its publication,
the same rights as accorded by a national patent of the designated EU country. If the
subject matter of the European patent is "a process," the protection extends "to the
products directly obtained by such process." Any infringement of a European patent is
to be dealt with by the national law in the respective country. 59 The extent of protection
is determined by the terms of the claims. The description and drawings shall be used as
interpretive tools for the claims. The scope of protection will be defined "with a
reasonable degree of certainty for third parties. " 60
The term of the European patent shall be 20 years from the date of filing the
application. 61 Contracting states are entitled to extend the term of protection or grant
special protection immediately upon expiry of the term, but only if justified by a state of
war or emergency or if "a product or process ... has to undergo an administrative
authorization procedure before it can be put on the market," thus substantially reducing
its commercial exploitation. 62

3.4.4. Requirements Relating to Microbiological Processei3

A series of provisions govern applications for patents relating to a "micro-biological
process or the product thereof," which involve the use of a microorganism which was
"previously unknown or unavailable to the public." If the MO cannot be described in
the patent application, the description requirement may be satisfied "by a deposit." The
invention will be considered as disclosed, sufficiently clear and complete, "if a culture
of the microorganism has been deposited with a recognized depository institution" not
later than the filing date of the application, in compliance with other prescribed
requirements. Relevant information on the characteristics of the MO, as known to the
applicant, has to be given in the application and details of the depository institution and
file number of the culture deposit have to be given within a prescribed period.
Such information constitutes "the unreserved and irrevocable consent of the
applicant" for availability of his deposited culture to the public, subject to prescribed
stipulations. 64 The requester of a sample is subject to a series of cumulative obligations
"vis-a-vis the applicant or proprietor of the patent." He should not make available the
deposited or derived culture to any third party before the application has been refused or
withdrawn and, if after the patent has been granted, not before the last expiry date of the
patent. He may use the deposited or derived culture "for experimental purposes only"
subject to prescribed restrictions. These obligations do not apply if the requester is using
the culture under a compulsory license, including an ex officio license to .use it in the
public interest. 65 Under certain circumstances, the applicant may stipulate that a sample
is to be issued only "to an expert nominated by the requester," meeting the EPO
requirements for such a purpose. 66 The cumulative obligations of the requester "shall
 355

not impede a deposit of derived biological material necessary for the purpose of patent
procedure." This "shall mean any material which still exhibits those characteristics of
the deposited material, which are essential to carrying out the invention." "Biological
material" shall mean "any material containing genetic information and capable of
reproducing itself or being reproduced in a biological system. " 67 Notice of the request
for a sample of the MO shall be given to the applicant or the proprietor. 68 If a deposited
MO ceases to be available from the depository institution, because it "is no longer
viable" or "for any other reason," the applicant may make a new deposit of the same
originally deposited MO, subject to prescribed requirements. 69
If a European patent application discloses nucleotide or amino acid sequences, the
description has to contain a sequence listing (in writing or on a data carrier) as required
by the EPO, for standardized representation of nucleotide and amino acid sequences. 70

3.4.5. The Budapest Treaty

The Budapest Treaty is designed to govern the deposit of "living organisms" to be
stored and maintained in appropriate facilities. It provides that a culture collection in
any of the contracting states to the Budapest Union may be recognized as an
International Depository Authority (IDA) for MOs, for the purpose of patent
applications. The deposit of MOs with one of the affirmed depositories, in whatever
member state, is considered sufficient disclosure of the invention, substituting or
accompanying the written description. The availability of the deposited culture, its
release or issue of samples to third parties, is governed by, and subject to the differences
in the national patent laws of the member states. 70•

4. Breeders' Rights in New Plant Varieties

In principle, economic rights in industrial inventions are most effectively protected by

patent laws. However, until very recently, most of the countries in the world explicitly
excluded "new plant varieties" from patent protection. Although "agricultural
inventions" and "horticultural products" were embraced by the Paris Convention within
the definition of "industrial property" to be protected via patents, the Convention did
not define the "subject matter" for patentability, leaving it to the national discretion of
the adhering states. Although the more recent multi-national legal instruments have
imposed on its member states a duty to protect plant varieties, either by patents or a sui
generis system, or both, as already discussed, these very instruments have also
explicitly permitted exclusion of plants from patentability. A historical survey shows
that most of the adhering states implemented their right of exclusion in adopting the
traditional concepts of non-patentability of plant and animal varieties. The formal
reasons were anchored in the "product of nature" doctrine, and the doubts about
stability of reproduction, precision of description and clarity in formulation of claims.
This later developed into a resistance against patenting "living matter" and a series of
delicate ethical issues of public concern. Although a different route was taken by the
USA, where in 1930 a Plant Patent Act had already been enacted, the almost commonly
accepted protection of breeders' rights was mainly provided under a sui generis system,
governed by the UPOV Convention.
356

4.1. THE 1991 UPOV CONVENTION

The UPOV Convention, as originally signed in 1961, recognized the importance of

securing proprietary rights for plant breeders, in the process of discovering and
inventing new plant varieties. It prescribed uniform principles for the protection of plant
breeders' rights, to be incorporated in the domestic laws of the adhering states.
Although it pronounced that breeders' rights can be protected by either a patent or the
specific right for protection of new plant varieties, the 1961 Convention explicitly
stipulated that "no double protection" shall be accorded "for one and the same botanical
genus or species," even if the national law admits protection under both systems. 71
The UPOV Convention was revised in 1978 but still retained the ban on double
protection. In line with technological developments in the breeding industry however, it
was re-enacted in 1991, obliging each of the previously bound contracting parties to
apply its new version. In this new version, the 1991 UPOV Convention abolished the
ban on double protection. It obliges each contracting party to "grant and protect
breeders' rights," but refrains from stipulating what type of protection is to be accorded.
Each contracting party is free to decide, within its national law, what type of protection
it prefers to accord to plant varieties, whether by "a breeder's right, or by a patent or any
other right, or by any combination of such rights."
The 1991 UPOV Convention provides a comprehensive international framework for
the protection of breeders' rights in new plant varieties. The UPOV is an inter-
governmental independent legal entity, receiving financial and administrative services
from the WIPO.
In listing the genera and species to be protected, the 1991 UPOV Act provides that
each adherent Party shall apply its provisions "to all plant genera and species to which it
applies, on the date of becoming bound by the convention and at the latest by the
expiration of 5 years after the said date." Special implementation provisions apply to
new adhering parties. 72 The 1991 Act conforms with the principle of "national
treatment." 73 Aiming to ensure economic exploitation of plant-based inventions and to
provide investment incentives for further research and development of new plant
varieties, it is designed to become globally bindin~. The 1991 UPOV Act entered into
force on Apr 28, 1998, open for further adherence. 4

4.1.1. A Plant Breeder's Right in a New Plant Variety

The 1991 UPOV Act provides that "a breeder's right" in a new plant variety be granted
"where the variety is new; distinct; uniform and stable," provided "it is designated by a
denomination," as prescribed. The applicant for a "certified variety" must comply with
the legal formalities of the designated party and has to pay the required fees. 75 A variety
in general, whether protectable or non-protectable, as defined by the 1991 Act is: "a
plant grouping within a single botanical taxon of the lowest rank, which grouping,
irrespective whether the conditions for the grant of a Breeder's Right are fully met, can
be:
defined by the expression of the characteristics resulting from a given genotype or
combination of genotypes;
distinguished from any other plant grouping by the expression of at least one of the
said characteristics;
considered as a unit with regard to its suitability for being propagated unchanged."
 357

A breeder (as defined) is: "the person who bred, or discovered and developed, a
variety; the person who is [his] employer or who has commissioned [his] work, where
the laws of the relevant contracting Party so provide; or the successor in title [of any of
the above] as the case may be." 76
The UPOV requirement of novelty differs from the patent law requirement: it
depends upon "the marketing profile of the variety" and not upon its technological or
scientific novelty. The variety "shall be deemed to be new" if at the filing date of the
application "propagating or harvested material of the variety has not been sold or
otherwise disposed of, to others, by or with the consent of the breeder, for purposes of
exploitation of the variety," within the prescribed time limits. Within the territory of the
UPOV country where the application was filed, the variety "may not have been sold,"
earlier than 1 year before the filing date. Within the territory of any other UPOV State,
the variety may not have been sold earlier than 4 years before the filing date, 6 years
with respect to trees or wines. 77 Trials not involving marketing or sale shall not affect
the right to protection.
The UPOV requirement of distinctness is met if, at the time of filing the application
for a breeder's right, the variety "is clearly distinguishable from any other variety whose
existence is a matter of common knowledge." The filing of an application for granting a
breeder's right, in any UPOV country, brings the said variety into the ambit of
"common knowledge," from the date of the application, provided it resulted in granting
a breeder's right. The same rule applies to an application for the entry of a new variety
in an official register of varieties. 78 The distinctness is judged by the relevant
characteristics of the new variety and determined, in many countries, through field
trials. A written description and a deposit of a viable sample of the variety shall
demonstrate the distinctive characteristics. Actual cultivation, marketing, inclusion in a
reference collection, or precise description in a publication may establish "common
knowledge."
The requirement of uniformity is met if, "subject to the variation that may be
expected from the particular features of its propagation, it [the variety] is sufficiently
uniform in its relevant characteristics." 79
The requirement of stability is met if the relevant characteristics of the variety
"remain unchanged after repeated propagation or, in the case of a particular cycle of
propagation, at the end of each such cycle. " 80
A decision to grant a breeder's right should be subject to examination for
compliance with all the prescribed conditions. "In the course of examination the
authority may grow the variety or carry out other necessary tests" or may take into
account the results of other, already carried out, growing tests or trials. soa
In requiring "designation" of a new plant variety "by a denomination," to become its
generic designation, the Convention aims to facilitate identification and classification of
plant varieties by using principles of plant taxonomy and agreed-upon nomenclature. By
precluding misleading information or confusion with respect to the characteristics of the
new variety, its identity or the identity of the breeder, the Convention strives to restrain
unfair competition among plant breeders. The denomination must be different from
every other denomination designating "an existing variety of the same plant species or
of a closely related species," in the territory of any contracting party. "It must not
consist solely of figures, except where this is an established practice for designating
varieties." Upon granting a breeder's right, the denomination is to be registered by the
358

authority.
Each contracting party has to ensure "the free use of the denomination in connection
with the variety even after the expiration of the breeder's right." 80h

4.1.2. Scope ofProtection Conferred by a Plant Breeder's Right

In defining the scope of protection with respect to the propagating material of the
protected variety, the 1991 Convention enumerates a series of acts that are subject to the
breeder's prior authorization. Subject to several compulsory exceptions and exhaustion
of rights, any of the following acts requires the breeder's authorization:
"production and reproduction (multiplication); conditioning for the purpose of
propagation; offering for sale; selling or other marketing; exporting; importing; and
stocking for any of [said purposes]." The breeder may make his authorization subject to
conditions and limitations. Breeders' rights relating to ornamental plants extend to the
whole plant or parts of it, in cases where these "are used commercially as propagating
material in the production of ornamental plants or cut flowers."
The UPOV promotes protection of breeders' rights with respect to the reproductive
or vegetative material, as well as to the whole plant, of the new varieties. These include
cultivated varieties, clones, lines, strains and hybrids which can be clearly distinguished
from other varieties and remain sufficiently stable and homogeneous in their
characteristics within each propagating cycle.
The 1991 UPOV Act provides that in cases where the breeder had no reasonable
opportunity for exercising his right with respect to the propagating material, "his rights
should be extended to the harvested material," including whole plants and parts of
plants, if these were obtained through unauthorized use of the propagating material of
the protected variety. Each contracting party may (optionally) extend the right even
further by providing the breeder with rights in the products made directly from
harvested material of the protected variety, unless he has had a reasonable opportunity
to exercise his right with respect to the harvested material.
In extending the scope of breeders' rights, the 1991 Act also accords protection to
"essentially derived" and certain other varieties. A variety essentially derived from the
protected variety can be protected, provided that the initial protected variety was not
itself an "essentially derived variety." A variety is considered essentially derived from
the initial variety when: i) it is predominantly derived from the initial variety, retaining
the expression of its essential characteristics; ii) it is clearly distinguishable from the
initial variety; iii) it conforms to the initial variety in the expression of its essential
characteristics (except for differences resulting from the act of derivation). Examples for
obtaining essentially derived varieties as listed in the Convention are: "selection of a
natural or induced mutant, or of a somaclonal variant, the selection of a variant
individual from plants of the initial variety, backcrossing, or transformation by genetic
engineering. " 81

4.1.3. The Duration of a Plant Breeder's Right

The breeder may choose in which country he wants to file his first application and he
may apply simultaneously for the same right in another UPOV country. 82
A 1-year priority period is to be granted from the filing date of the first application
for a breeder's right, in whatever UPOV country. Each contracting party has to provide
measures for safeguarding the breeder's interests during the period between the filing
 359

date and the date on which the right is granted. He should at least be entitled to
equitable remuneration from any person who, during that period, has carried out acts
which, upon granting of the right, would be subject to his authorization. 83
The duration of a plant breeder's right should be for a minimum of 20 years from the
date it was granted and with respect to trees and wines, 25 years. 84

4.1.4. The Research Exemption and "Farmer's Privilege"

As stated, the scope of protection of a breeder's right is narrower than the protection
provided by a patent. The breeder's right should not be extended to acts performed for
private and non-commercial uses, or to acts performed for experimental purposes. The
research exemption enables the use of a protected variety as a source for creating new
varieties, subject to the restriction of not repeating the use of that same protected
variety. A compulsory license provides for the widespread distribution of new varieties.
The 1991 UPOV Act permits optional exceptions to breeders' rights. Each
contracting party may" restrict the breeder's right in relation to any variety, in order to
permit farmers to use, for propagating purposes, on their own holdings, the product of
the harvest which they have obtained by planting on their own holdings, the protected
variety, or an essentially derived variety or a variety not clearly distinguishable from the
protected variety. " 85

4.2. EUROPEAN COMMUNITY PLANT VARIETY RIGHT (CPVR)

Along, and in co-existence with, the different domestic regimes in the EU countries, a
CommunityPlant Variety Right (CPVR) was created under EC Reg. No. 2100/94. It
provides breeders with an IPR in new plant varieties, applicable throughout the entire
European Community. 86 In recognizing the modem developments in plant breeding
techniques, the EC Regulation declared that the Community system for protection of
"all botanical genera & species" must provide improved protection and must have
regard to new technologies, including biotechnology. It was pronounced that the CPVR
conforms with the internationally accepted canon of "free access to protected varieties,
for stimulating and furthering development of new varieties." In certain cases "where a
new variety, although distinct, is essentially derived from the initial variety" there
should be created "a certain form of dependency from the holder of the initial variety"
subject "to the use of the prescribed denomination."
It was pronounced that the CPVR must be subjected to restrictions protecting the
public interest. Compulsory licensing should be provided under certain circumstances
for the benefit of the public. This may include the need to supply the market with
material offering specified features, or the need to maintain the incentives for continued
breeding of improved varieties. 87 The safeguarding of agricultural production should be
provided, subject to an authorization for farmers to use the product of the harvest for
propagation under certain conditions, laid down at the Community level. (The
traditional farmer's privilege enables a farmer to retain seeds from a protected variety in
a quantity needed for repeated growing in subsequent seasons.)
In recognizing the importance of providing a common definition for a plant
variety-without altering the already established definitions and not interfering in the
protectability of products, plants, plant material or processes-the EC Regulation
provides that "varieties of all botanical genera and species, including inter alia, hybrids
360

between genera or species" may form the subject matter of a CPVR.

The meaning of a variety is almost identical to the meaning provided by the UPOV
Convention. 88 A plant grouping is defined as consisting of entire plants or parts of
plants, as far as such parts are capable of producing entire plants, both referred to as
''variety constituents. " 89
In discussing "the expression of the characteristics" of a variety, the EC Regulation
provides that it "may be either invariable or variable between variety constituents of the
same kind, provided that also the level of variation results from the genotype or
combination of genotypes. " 90 In stressing the significance of assessing of the important
characteristics relating to the variety, the regulation stipulates that these need not
necessarily relate to their economic importance.
A protectable variety seeking a CPVR should comply with the four basic
requirements of: distinctness, uniformity, stability and novelty. It also "must be
designated by a suitable denomination," meaning that it should not be identical or
confusing with another denomination "of the same or close related species or other
species." The denomination must not be "liable to give offence ... or be contrary to
public policy or to mislead or to cause confusion ... " 91 The requirements for:
distinctness from other varieties that are "in common knowled~e," 92 uniformity in the
expression of characteristics, 93 stability in remaining unchanged, 4 novelty in the market,
are all defined in wording that is almost identical to that used by the 1991 UPOV Act.
A CPVR co-exists with a domestic PVR but they are mutually exclusive. An
application for a CPVR pre-empts an application for a national PVR for the same
variety.

5. Plant-Specific Intellectual Property Rights in the USA

Under the US legal system, new plants, plant varieties and plant-based inventions enjoy
wide-embracing legal protection. Asexually reproduced new plant varieties are
protected under the 1930 Plant Patent Act (1930 PPA). 95•96 Sexually reproduced new
varieties of plants are protected under the 1970 Plant Variety Protection Act (1970
PVPA). 97
Living things. whether genetically engineered or conventionally bred, even if
sexually reproduced, "like seeds and seed-grown plants and parts thereof," may obtain
patent protection as "patentable sub~ect matter" under the general provisions of the 1952
Utility Patents Act (1952 UPA). In line with the US Supreme Court's famous
Chakrabarty (1980) decision, pronouncing that "patentable subject matter" as defined
by sec. 101 of the UPA "is intended to include anything under the sun that is made by
man," 99 the USCA. Federal Circuit has recently ruled, in the Pioneer Hi-Bred (2000)
decision, as will be further discussed, that there is no basis in law for excluding living
things from patentable subject matter. 100
In comparing the liberal principles of plant-specific protection as applied in the USA
with the more rigid principles applied under the European legal systems, two
conceptually codified differences should be stressed, a priori. The UP A differs from the
EPC in that it does not contain an exclusion from patentability with respect to "plant
varieties or essentially biological processes for the production of plants." The scope of
patentable subject matter is broader in that it embraces "whoever invents or discovers
 361

any new and useful process, machine, manufacture or composition of matter, or any
new and useful improvement thereof... " 101

5.1. PLANT PATENT PROTECTION FOR ASEXUALLY REPRODUCED NEW

PLANT VARIETIES

Asexually reproduced, distinct and new plant varieties enjoy patent protection under the
PPA, which was originally enacted in 1930 and is considered to be the first legislation
in the world granting patent rights to plant breeders. It was based on the understanding
that in order to place agriculture on an equal economic base with industry, it was
appropriate to grant plant breeders the same legal status for their work "in aid of nature"
as is granted to mechanical and chemical inventors. Part of this success is ascribed to
Luther Burbank 102 for his intervention: that without legal protection for plants,
"research and breeding would be discouraged to the great detriment of horticulture." 103
The PPA is incorporated, as the separate title 15, in the 1952 UPA. 104
Prior to its enactment it was thought that plants, even those bred by man, are
products of nature, that cannot be a subject matter for patent protection. It was also
believed that plants cannot meet the requirement for differentiation by clear and
complete description, since some new plants (especially flowers) differ from existing
ones by mere color or scent, which is impossible to describe. In enacting the PPA,
Congress removed the product of nature obstacle by pronouncing that "a plant
discovery resulting from cultivation is unique, isolated and is not repeated by nature,
nor can it be reproduced by nature, unaided by man." It also "relaxed" the strictness of
the written description by substituting a more flexible requirement, in providing that
with respect to plant patents the description should be "as complete as reasonably
possible." 105
The PPA provides that a plant patent may be obtained by anyone who "invents or
discovers and asexually reproduces any distinct and new variety of plant, including
cultivated sports, mutants, hybrids, and newly found seedlings, other than a tuber-
propagated plant or a plant found in an uncultivated state." 106 The basic requirement for
"a com~lete and clear written description of the invention," as applicable to utility
patents, 07 can be met by giving a lesser description "as is reasonably possible." The
claims should be specified "in formal terms to the plant shown and described." 108 There
are references specifying the disclosure details for flowers and plants in relation to
blooming habits, buds, bloom, petals, sexual organs, foliage, thorns, color, fragrance,
etc. The specification in a plant patent application must contain a disclosure, as full and
complete as possible, of the plant and the characteristics that distinguish it from related
known varieties. In particular, the description must point out where and how the said
variety was asexually reproduced. 109
Asexual reproduction has been pronounced as being "the heart of the plant patent
system." It has been stated that "the key to the invention of a [patentable] new plant is
the discovery of new traits plus the foresight and appreciation to take the step of asexual
reproduction." 110 Although the asexual reproduction prerequisite narrows the scope of
the plant patent's protection, it has been declared necessary "for ensuring that the
characteristics of the patented plant be maintained." 111
In discussing the various methods of asexual reproduction ( the "semi-sacred word
in the field of plant patents"), it was stressed, by the USCA (Federal Circuit), in the
362

landmark Imazio case, that "each one of these methods consists of the isolation of a
group or mass of vegetative cells from the parent plant, that are capable of reproducing
a plant that is genetically an exact duplication of its parent plant. .. Whether the new
variety is a hybrid, mutant or sport, there is never more than one specimen of it
produced, except through asexual reproduction." It was pronounced and ruled in the
Imazio decision that plants seeking plant patent protection, "must be asexually
reproduced in order to have their identity preserved ... ; due to the asexual reproduction
prerequisite, plant patents cover only a single plant and its asexually reproduced
progeny ... ; accordingly the term variety for the purpose of plant patent protection,
cannot be read as affording plant patent protection to a range of plants," as was argued
in that case. 112 The court was of the opinion that "the characteristics that may
distinguish a new variety from existing varieties would include, among others, those of
habit; immunity from disease; resistance to cold; drought; heat; wind; or soil conditions;
color of flower, leaf, fruit, or stems; flavor; productivity, storage qualities, perfume;
form and ease of asexual reproduction," all subject to degree. 113
It was decided that in an infringement action of a plant patent, the patentee must
prove that the allegedly infringing plant was "asexually reproduced" from his patented
plant. Some sort of physical appropriation from the patented plant has to be proven.
Mere demonstration of similarity between the characteristics of the allegedly infringing
and infringed-upon plant is not sufficient proof of infringement. As in granting a plant
patent, so also in an infringement of it, the sine qua non is the "asexual, reproduction"
of substantially the same plant. "Independent creation" is a legitimate defense against an
infringement action of an "asexually reproduced" plant variety. 114
In disagreement with the District Court's opinion that it is enough to show that the
infringing variety has the same essential characteristics as the patented variety, whether
or not originally cloned from it, the USCA (Federal Circuit) was of the opinion that no
infringement can occur without an actual, physical taking from the plant discovered by
the patentee. Furthermore, the defendant may assert that the alleged infringing plant is
not an asexual reproduction of the patented plant. Part of this proof could be ... that the
defendant "independently developed" the allegedly infringing plant. 115

5.1.2. The Scope and Term of Plant Patent Protection

The granting of a plant patent confers on the patentee "the right to exclude others from
asexually reproducing the plant or selling or using the plant so reproduced" during the
limited term of protection. 116 It is a "blocking" right, in contrast to the positive
"exclusive" right originally accorded.
A plant patent is granted on the entire plant. The narrow confined protection
accorded by the PP A is limited to the exact variety as described in the specifications.
The scope of a plant patent is the asexual progeny of the patented plant variety.
Newly discovered plants which were existing in nature are not considered patentable
subject matter, even if previously not catalogued by botanists. But this is restricted to
plants that are wholly created or reproduced by nature, being a "product of nature,"
unassisted by man. Tissue cultures are not within the scope of plant patent protection,
because these do not fall within the common meaning of the term "plants," which are
things "having roots, stems, leaves and flowers or fruits." 117 The term of protection
provided by a plant patent is for 20 years, the same as for a utility patent.
 363

5.2. PVPA PROTECTION FOR SEXUALLY REPRODUCED PLANT VARIETIES

The restricted protection by the PPA to only asexually reproduced plants was based on
the belief that new varieties could not be reproduced "true-to-type," through seedlings.
It was believed that seedlings, either by chance or by self-pollenization, would not
preserve the characteristics of the individual original plant through generations. Thus,
prior to the enactment of the 1970 PVPA, there were no IPRs for sexually reproduced
plants. In enacting the PVPA, congress recognized the fact that "true-to-type"
reproduction was also possible through other means and accorded "quasi-patent"
protection to new varieties of sexually reproduced plants, grown from seeds.
The PVPA provides that a "breeder of any sexually reproduced or tuber-propagated
plant variety (other than fungi or bacteria), who has so reproduced the variety, or the
successor in interest of the breeder, shall be entitled to plant variety protection for the
variety, subject to the conditions and requirements," prescribed by the Act. 118
The term "breeder" means "the person who directs the final breeding, creating a
variety or who discovers and develops a variety." If the breeding is conducted on behalf
of "a principal" or "an employer," he shall be considered the "breeder." The term
"breeder" does not include a person who redevelops or rediscovers a' variety, the
existence of which is "publicly known or a matter of common knowledge." A variety
may become a matter of common knowledge if its distinguishing characteristics were
disclosed in a technical publication or by other means. An application for protection, in
any countJy, renders the variety "public knowledge" from the filing date, provided it
leads to granting protection. 1188
"Sexual reproduction," as defined by the PVPA, includes any production of a variety
by seed, but does not include the production of a variety by tuber propagation. A "tuber-
propagated variety" means propagation by a tuber or part of a tuber.
The definition of ''variety" is, in general terms, in conformity with the definition
provided by the 1991 UPOV Act. "A variety may be represented by seed, transplants,
plants, tubers, tissue culture, plantlets and other matter." 119
Plants that are "true-to-type," although different in a strict genetic sense, are
protectable under the PVPA, which embraces a broader range of plants than the PPA. 120
The right to protection under the PVPA is determined upon compliance with the
statutory requirements that the ''variety" is to be: new, distinct, uniform and stable.
The novelty requirement is tested upon the marketing history of the "propagating or
harvested material of the variety," for purposes of"its exploitation," within the area of
the USA or outside it, as specifically prescribed by the Act. 121 Disposition of such
material within a program for experimentation or testing in order to ascertain the
characteristics of the variety or increase the variety, on behalf of the breeder, shall not
be considered a disposition for exploitation of the variety. 122 The sale of thus-produced
harvested material shall also not be considered as "sale for exploitation," provided it is
sold "for other than reproductive purposes." 123 The disposition of hybrid seed is to be
considered disposition of harvested material of the varieties from which the seed was
produced. 124
The distinctness of a variety from any other publicly known variety may be based on
one or more identifiable morphological, physiological, or other characteristics
(including characteristics evidenced by processing, or product characteristics), with
respect to which a difference in genealogy may contribute evidence. 125
364

The uniformity of a variety requires that any variations be describable, predictable,

and commercially acceptable. The stability requirement is satisfied if"the variety, when
reproduced, will remain unchanged with regard to the essential and distinctive
characteristics ... " 126

5.2.1. The Scope and Term ofProtection Under the PVPA

A certificate of plant variety protection, issued by the Secretary of the PVP office,
entitles the breeder (or his successor, heir or assignee), during the term of protection, "to
exclude others ... " from using the protected variety or committing any of the specifically
prohibited acts with respect to that variety, to the prescribed extent This does not affect
the right of others to use the variety for developing another variety. Waiver of certain
rights is permissible. 127 The owner of a protected variety may authorize its use subject
to his conditions. A series of authorized acts are prescribed by the law for contractual
relationship between a seed producer and the owner.
The PVPA provides that "plant variety protection shall have the attributes of
personal property." Any interest in a variety or in the application for a plant variety may
be "assignable by an instrument in writing." The owner of a protected variety is entitled
to license or grant and convey an exclusive right to use the variety in the whole USA or
any part of it. 128 In releasing seed or other sexually reproducible or tuber-propagating
plant material for testing only, the owner retains ownership subject to giving notice.
Unauthorized retention of such material violates the property rights of the owner. 129
The term of plant variety protection is for 20 years, and in the case of a tree or vine
25 years, "from the date of issue of the certificate in the United States." The protection
may expire before that term is over if the owner fails to comply with the regulations
relating to "replenishing seed in public repository" or to "requiring submission of a
different name for the variety." 130' 132

5.2.2. Infringement of a Protected New Plant Variety

The rights of the owner of a novel variety are infringed upon if, without authority,
anyone commits, within the protected period, any of the specifically prohibited acts, as
provided by the Act (e.g., use, sell, market, import, export, sexually multiply, or
propagate, etc.). Protection is also accorded to any variety whose production requires
the repeated use of a protected variety; to harvested material including entire plants and
parts of plants (unless the owner has had an opportunity to exercise his rights with
respect to the propagating material). Any variety "that is essentially derived from a
protected variety" is also protected against infringement, provided the protected variety
is not in itself a derived variety. 133 The definition of an "essentially derived variety" and
of the methods for obtaining it is in full compliance with the UPOV provisions. 134•135

5.2.3. Exemptions and Remedies

Any act done privately and for non-commercial purposes shall not be considered as
infringement of the owner's rights in a protected variety. 136 The "use and reproduction
of a protected variety for plant breeding or other bona fide research, does not constitute
an infringement." Neither shall transportation or advertisinr by a person, in the ordinary
course of that business, constitute an infringement. 13 In compliance with the
"Grandfathers' clause," nothing "shall abridge the right of any person to reproduce or
sell a variety developed and produced by (him) more than one year prior to the effective
 365

filing date of an adverse application." The "Fanner's privilege clause" provides that
(subject to some specific restrictions), it shall be no infringement "if a person will save
seed produced by him from seed obtained (or descended) ... , by authority of the owner
of the variety for seeding purposes and use such saved seed in the production of a crop
for use on (his) farm, or for sale." 138 The term "seed" with respect to a tuber-propagated
variety means "the tuber or part (of it) used for propagation."
An owner of a protected variety shall have remedy by a civil action for
infringement. The burden for establishing invalidity of a plant variety protection rests
on the party asserting it. The name of a variety shown on a PVR certificate, presumed to
be valid, is a prima facie presumption that it is the same variety. 139 The courts, having
jurisdiction to decide in these matters, may grant injunctions to prevent violation of
rights or their continuation and may compensate for the infringement by awarding
. d.tscretton.
damages, costs and attorney fiees, at t hetr . 140

5.3. PATENTABILITY OF "LIVING MATTER" UNDER THE UTILITY PATENT

ACT

Until it was challenged, the traditional legal approach regarded the two plant-specific
laws (PPA and PVP A), as the exclusive source for providing IPRs in new plants and
plant-based inventions. It was argued, as expressed in the USA case law, that by
enacting these two plant-specific acts, dealing with "living matter" in the form of
sexually and asexually reproduced plants, Congress intended to exclude "plant
varieties" from "patentable subject matter," which are eligible for patent protection
under the general Utility Patent Act (UP A).
This policy was seriously challenged in the famous Chakrabarty (1980) case. It
concerned microorganisms that were engineered for breaking down or degrading
components of oil spills. In its landmark decision, the US Supreme Court paved the way
for a flexible and broad interpretation of "patentable subject matter," qualifying for
protection under sec 101 of the general UPA. In relating to Chakrabarty's invention as
being in the "highly complex field of cellular or genetic engineering or microbial
genetics," the Supreme Court stated that the general terms of the UPA are drawn to
encompass non-foreseeable future developments in all fields of technology. It stressed
that "the relevant distinction is not between living and inanimate things, but between
products of nature, whether living or not, and the human made inventions." It observed
that "there is nothing in the language or history of any of the two plant-specific laws to
suggest that a patentable invention is to be excluded from patentability, just because it is
alive? ... It is because it is alive, that it is useful!" 141
The US Supreme Court pronounced that there is "no sound reason to refuse patent
protection to microorganisms themselves, or to pure microorganism cultures ... [if ] new
and unobvious." In relating to Chakrabarty's invention, the Court was of the opinion
that the discovery of "a new bacterium ... having potential for significant utility" which
has "markedly different characteristics from any found in nature" is "a product of
human ingenuity" and is patentable under sec. 101 of the UPA.
In following and extending the Chakrabarty decision, the US Court of Appeals for
the Federal Circuit (the sole App. Div. in patent matters) ruled, on Jan 19, 2000, in its
Pioneer Hi-Bred clarifying decision, that also "seeds and seed-grown plants" are to be
regarded as "patentable subject matter" qualifying for patent protection under sec. 101
366

of the UPA. 142 An attempt by the US Commissioner of Patents to draw a distinction

between genetically engineered MO's and seed-grown plants was proven abortive.
In reference to the traditional reasons for the previous non-patentability of plants,
the USCA (Federal Circuit) adopted the District Court's reasoning that since mankind
has learned to modify plants in ways unknown to nature, "precision of description is no
longer a non-surmountable obstacle, due ... to rules authorizing the deposit of new
species in publicly available depositories, and advances in botanical understanding and
analysis."
The description requirement of a new plant "may be met by a color photograph,
identification of the origin or parentage of the plant and a detailed description of the
plants' distinguishing characteristics." Neither Congress nor the PVP A, and also not the
courts, have excluded or removed genetically engineered organisms or new plant
varieties from the patent statute." 143
The USCA (Federal Circuit) ruled that there is no basis in law for excluding "seeds
and seed-grown plants and parts thereof," from patentable subject matter under sec. 101.
The policy underlying the patent system fosters its application to all areas of
technology-based commerce, subject to the traditional non-patentable categories. In
conclusion it affirmed the District Court's ruling "that a person who develops a new
plant variety may have recourse either to patenting under Title 35 of the Utility Patent
Act, or to registration under the PVPA, or to both which provide different rights." 144

6. The European Approach to Contemporary Plant Technologies

The rapid development of modern plant-breeding technologies, biotechnology and

genetic engineering, which has provided significant improvements in the traditional
plant-breeding processes in agricultural and horticultural industries-has seriously
challenged the adequacy of the European traditional protection of proprietary rights in
plant-based inventions. A series of legal questions not previously known were raised in
the field of intellectual property law, which is characterized by its slow pace of reform,
and adaptation Voices were pronounced and legal academic commentaries were
massively published, already in the early '80s, arguing that the sui generis scheme of
protection under PVR laws is not satisfactory, especially in as far as it concerns
transgenic plants. In their learned energetic writings, the proponents for incorporating
plants within patentable subject matter argued that patenting "living matter" has been
recognized by various patent systems, since the 19th century. Attention was drawn to
the wide-embracing protection accorded to plant varieties by the US statutory law and
the flexible judicial interpretation of its provisions. 145
It should be remember that in contrast to the US law which has no exclusion clause
from patentability on plant varieties, the European Patent Convention explicitly
prescribes [in Art. 53 (b)], that a European patent shall not be granted for "plant
varieties and essentially biological processes for the production of plants," not being
"microbiological processes or the products thereof."
However in practice, constant attempts were made to narrow the specific exclusion
by implementing it only in cases of a clear "plant variety" or in reliance on the
exclusion from the exclusion of"microbiological processes or the products thereof." 146
 367

Judged upon the established policy of the EPO, as expressed in case-law of the
competent Boards of Appeal, especially in the further discussed PGS decision rendered
in 1995 and Novartis II decision rendered on Dec 20, 1999, it can be noted that the
European concept is gradually approaching the American concept. In adding the
specific provisions of the further described Directive 98/44/EC on the Legal Protection
of Biotechnological Inventions, embracing also plant-based inventions, it can be stated
that the European patent law is trying to close or at least minimize the existing gap
between the differing concepts in respect of patentability of plants.

6.1. PLANT CELLS/PLANT GENETIC SYSTEMS (PGS) DECISION (1995)

In rendering its Plant Genetic Systems (PGS) decision, which discusses the patentability
of genetically engineered plants and seeds, the Tech. Board of App. of the EPO (TBA)
summed up its general narrowing principles in applying the specific exclusion with
regards to plants. The discussed invention disclosed the use of modem biotechnological
techniques for the production of plants and seeds resistant to a particular class of
herbicides and selectively protected against weeds and fungal diseases. A patent was
sought for the plants, the seeds characterized by the same features and the plant cells, as
well as for the processes of controlling said action in the plants and cells and of
producing plants or reproduction material from said plants. 147
In the opposition/revocation proceeding, it was argued that such an invention should
be excluded from patentability since it is within the realm of a "plant variety" or a
"process for the production of a plant variety." Being a "living organism," it should also
be excluded from patentability under Art. 53(a), since it offends the "ordre public" and
"morality" and is prejudicial to the environment.
In conformity with the existing case law, the TBA stressed that it was never stated
that the exclusion under Art. 53 applies to the entire field of "animate nature." The
exclusion must be construed narrowly. In principle, patents may be granted in respect of
inventions concerning plants, which do not qualify for protection as a "plant variety".
The same is to apply to inventions relating to "processes of a technical nature" for the
production of plants, not being varieties. The legal meaning of a "plant" is not equated
with a "plant variety." The exclusion under Art. 53(b) "prohibits the patenting of plants
or their propagating material, only in the genetically fixed form of a plant variety."
Seeds and plants per se, should not be excluded from patentability under Art. 53(a)
EPC, merely because they represent "living matter." Nor should they be excluded on the
grounds that plant genetic resources should remain the "common heritage of mankind,"
as long as the invention does not relate to "wild-type plant resources," used as starting
material. An innovation disclosing a treatment that could be carried out on propagating
material which does not meet the essential criteria of homogeneity or stability
characteristics of a plant variety should not be excluded from patentability. 148
"Plant cells as such," which modem technology allows to culture much like bacteria
and yeasts, cannot be defined as a plant or a plant variety. The term "microorganism"
includes not only bacteria and yeasts, but also fungi, algae, protozoa, animal and plant
cells, and also plasmids, viruses and all generally unicellular organisms with dimensions
beneath the limits of vision, which can be propagated and manipulated in a labaratory."
"Technical activities" for producing plants, making direct use of microorganisms,
include not only traditional processes, but also "manipulation of MO's by genetic
368

engineering or fusion techniques, the production or modification of products in

recombinant systems, etc., briefly, all activities in which integrated use is made of
biochemical and microbiological techniques, in order to exploit the capacities of
microbes and cultured cells." All these are within the realm of microbiological
processes. 149
"Microbiological processes" for the purpose of Art. 53(b) refer to processes "in
which microorganisms ... or their parts, are used to make or to modify products, or in
which new microorganisms are developed for specific uses ... " Genetic engineering
processes carried out on vegetable cells may be defined as microbiological processes,
and their products, the genetically modified vegetable cells and their cultures, may be
defined as the "products thereof." r50
The question of whether a non-microbiological process is to be considered as
"essentially biological" has to be judged upon the essence of the invention, considering
"the totality of human intervention and its impact on the achieved result." A "process
for the production of plants comprising at least one essential technical step, which
cannot be carried out without human intervention and which has a decisive impact on
the final result-does not fall under the exceptions to patentability under Art. 53(b)." 151
In assessing the "PGS process invention," as was described in its claims, TBA stated
that "the step of transforming the plant cells or tissue with a recombinant DNA,
regardless of whether its performance is dependent on chance or not, is an essential
technical step which has a decisive impact on the desired final result." Thus, "although
the subsequent steps of regenerating and replicating the plants or seeds make use of the
'natural' machinery, the decisive step [the insertion], could not occur without human
intervention." Such a process as a whole cannot be considered as an "essentially
biological process for the production of plants" and is not to be excluded from
patentability. 152
However, in assessing whether the plants can be regarded as "a product of a
microbiological process," the Board stressed that although "the initial microbiological
process step has a decisive impact on the final result," it is important to note that within
this multi-step process, "the subsequent steps of regenerating and reproducing the plants
have an important added value and contribute to the final result. It is by virtue of these
steps that the plant acquires its characterizing feature that is transmitted throughout
generations ... " It is the controlled performance and/or successful occurrence of all of
these phases and events which will then allow the "imprinted plant cells" or "tissue," to
develop into a whole plant. Such a plant is not identical to the "initial starting product,"
"in spite of the fact that it contains the same characterizing genetic information. A
whole plant cannot be assimilated to a plant cell or tissue, for the sole reason that it has
acquired its characterizing feature durin~ the initial 'microbiological' step of
transforming the plant cell or tissue." 53 "Technical processes including a
microbiological step may not simply be equated with microbiological processes."
Accordingly such a plant shall not be considered as a "product of a microbiological
process," covered by the counter-exclusion under Art. 53(b).
In conclusion, it was decided that a product claim "which embraces within its
subject-matter plant varieties," should be excluded from patentability. 154
In discussing concepts of "morality" and "ordre public," the Board affirmed its
general statement that inventions, the exploitation of which may seriously prejudice the
environment, or may not be in conformity with the accepted European standards of
 369

conduct and morality, should be excluded from patentability. However, in deciding on

this matter, the Board was not willing to rely on public opinion polls claiming a bar on
patentability of plants and animals in general and of genetically engineered "super
crops" in particular, in stating that these results do not necessarily reflect "public
opinion" and can fluctuate easily. The Board pronounced that a bar to patentability is to
be considered in each particular case, on its merits.
In recognizing the fact that the use of plant genetic engineering inevitably allows
control of natural phenomena linked to plants, TBA stressed that "this does not render
activities in this technical field, intrinsically wrong." In its opinion "plant biotechnology
per se cannot be regarded as being more contrary to morality than traditional selective
breeding because both: traditional breeders and molecular biologists, are guided by the
same motivation, to change the property of a plant."
The Board observed that "objection to a dominion gained by man over the natural
world," as was expressed in the opposition by Greenpeace, is understandable, because
"the power of science for good and evil has always troubled man's mind." Thus "plant
genetic engineering techniques, like any other tool, can be used for constructive and
destructive purposes. In case of misuse or a destructive use, no patent should be granted
for an invention directed to such a use." In assessing the merits of the specific PGS
case, the Board was of the opinion that none of the claims refer to a misuse or
destructive use of plant biotechnological techniques and cannot be against
conventionally accepted standards of conduct in European culture. 155

6.2. TRANSGENIC PLANT/NOVARTIS AG (NOVARTIS II) DECISION (1999)

EPO's established practice, although gradually narrowing the bar on patentability of

plant varieties, did not satisfy the plant biotechnological industries. The final results, as
expressed in the PGS decision, were challenged several times, reaching a peak in the
Transgenic Plant/Novartis II decision, rendered by the Enlarged Board of Appeals
(EBA) of the EPO. The case related to an invention for controlling plant pathogens in
agricultural crops. Patent protection was sought for transgenic plants containing foreign
genes and for the method of producing such new plants, able to kill or inhibit the growth
of pathogens.
In compliance with the PGS decision, the TBA decided that said plants are not
patentable as a product, since the claim covered "plants which might or might not
belong to a plant variety." In accordance with the established policy it pronounced that
"if a potential embodiment of the invention was a 'variety', it is not patentable." TBA
was not willing to accept an argument that if a patent claim is comprised of more than a
single variety, it is permissible. It was argued that "a claim not specifically relating to
plant varieties, but to transgenic plants having certain features" should be patentable, if
"the technical feasibility of the invention is not confined to a particular plant variety."
TBA pronounced that such an interpretation, suggesting "that a patent should not be
granted for a single plant variety, but might be granted if its claim covered more than
one variety," is not in accordance "with normal rules of logic." An argument that
transgenic plant varieties are not falling under the bar on patentability, because of being
"products of microbiological processes," was also not accepted. TBA pronounced that
"the mere fact that a plant variety was obtained by means of genetic engineering, was
no reason to give the producer of such a variety a privileged position." Furthermore, it
370

would be inconsistent with the practice of protecting such plant varieties within the
scope of UPOV and CPVR provisions. In its opinion, it is the task of the European
legislator and not ofEPO to reconsider the matter of whether "to extend the scope of the
EPC beyond its original intention." 156
The case was referred to the EBA of the EPO on four points of law, including the
general question "to what extent should the EPO examine claims under the exclusion
prescribed by art. 53(b) EPC?"
In addition, the EBA was posed with the following two questions:
i) "Does a claim which relates to plants but wherein specific plant varieties are not
individually claimed ipso facto avoid the prohibition on patenting under Art. 53(b),
even though it embraces plant varieties?"
ii) "Does a plant variety in which each individual plant of that variety contains at least
one specific gene introduced into an ancestral plant by recombinant gene
technology, fall outside the provisions of Art. 53(b) EPC?"
In the NOVARTIS II decision, rendered by EBA on Dec 20,1999, the general
exclusion as prescribed by Art. 53(b) EPC was upheld but its applicability was
narrowed even further. The bar on patentability of plant varieties was minimized in
restricting the exclusion only to such plant varieties which qualify for protection under
plant breeders' rights. Inventions which are "ineligible for protection under the plant
breeders' rights," should be patentable under the EPC, subject to the remaining
patentability requirements. 157 It was stressed that the exclusion "defines the borderline
between patent protection and plant variety protection" in light of the previously
existing UPOV ban on double protection. If there is "no identification of a specific plant
variety in a product claim, the claimed invention is not directed to a plant variety or
varieties ... " EBA explicitly ruled that: "claims wherein specific plant varieties are not
individually claimed, are not to be excluded from patentability, even if one or more
plant varieties are embraced or may be embraced by the claims." 158
Relying upon the UPOV's definition of "variety," it was stated that reference to the
expression of characteristics resulting from a given genotype (or combination of such)
is "a reference to the entire constitution of a plant or a set of generic information." In
contrast, "a plant defined by single recombinant DNA sequences is not an individual
plant grouping to which an entire constitution can be attributed. It is not a concrete
living being or grouping of concrete living beings, but an abstract and open definition
embracing an indefinite number of individual entities defined by a part of its genotype
or by a property bestowed on it by that part." 159 In stressing the limitations of breeders'
rights it was stated that these are granted only "for specific plant varieties and not for
technical teachings which can be implemented in an indefinite number of plant
varieties." It was noted that the inventor in the genetic engineering field would not
obtain appropriate protection if he were restricted to specific varieties, even though he
had provided the means for inserting the gene into all appropriate plants.
In assessing the claimed transgenic plants, defined by certain characteristics
allowing them to inhibit the growth of plant pathogens, EBA stressed the absence of
further characteristics "necessary to assess the homogenuity and stability of varieties
within a given species" and the lack of a "taxonomic category within the traditional
classification of the plant kingdom to which the claimed plants belong." In finding that
"the claimed invention neither expressly nor implicitly defined a single variety ... nor a
multiplicity of plant varieties" as required by UPOV, EBA overruled the decision of
 371

TBA in deciding that "the claimed invention is neither limited nor even directed to a
variety or varieties." Such an invention cannot be protected by a plant breeder's right
"concerned with plant groupings defined by their whole genome," and is thus not
excluded from patentability. 160
Nevertheless, it is important to note that EBA has reaffirmed the principle that
"processes of genetic engineering are not identical with microbiological processes."
Even if under modem biotechnology "cells are comparable to unicellular organisms," it
"does not mean that genetically modified plants are to be treated (ipso facto) as products
of micro-biological processes." EBA was not willing to accept the argument that the
legislator of the EPC did not envisage the possibility of genetically-modified plant
varieties, thus could not have intended to exclude them from patentability. Laws are not
restricted in their application to situations known to the legislator.
EBA stressed the fact that the general exception of plant varieties from patentability
under Art. 53(b), "applies to plant varieties, irrespective of the way in which they were
produced." It also pronounced that "plant varieties per se, even if containing genes
introduced into an ancestral plant by recombinant gene technology, are excluded from
patentability." A producer of a plant variety obtained by genetic engineering does not
acquire a better position than a breeder of plant varieties resulting from traditional
breeding. 161
But in relating to the question of whether process claims can be allowed if the
product directly obtained by the claimed process is or covers a plant variety-EBA
ruled that "protection conferred by a process patent is extended to the products obtained
directly by the process, even if the products are not patentable per se. ... The fact that
Art. 64(2) extends protection to products produced within a protected process does not
affect the examination of claims for the manufacture of plants." So, "when a claim to a
process for the production of a plant variety is examined, Art. 64(2) is not to be taken
into consideration." 162
In discussing the opposition of Greenpeace under the "morality" exclusion clause (in
Art. 53(a)EPC), EBA pronounced that "although the position adopted in society on
genetic engineering is controversial-there is no consensus in the contracting States,
condemning genetic engineering in the development of plants." On the contrary, it
stressed that "promotion of innovation in this field is considered necessary in Europe"
and this is even furthered by the following described Directive. 163

6.3. PROTECTION OF BIOTECHNOLOGICAL INVENTIONS DIR.98/44EC (1998)

Cognizant of the EPO's established policy with respect to patentability and non-
patentability of biotechnological inventions, which required clarification, the European
Parliament and Council, after long years of deliberations, signed said Directive, on July
6, 1998. 164 Considering the fundamental importance of protecting biotechnological
inventions for the Community's industrial development, it was pronounced that the
patent laws must be adapted to take adequate account of technological developments
involving biological material. It was stressed that the patent system should be used to
encourage research and application of biotechnological processes and products, for the
benefit of the environment, in applying new methods of cultivation, less polluting and
more economical in their use of ground. It was noted that the exclusion from
patentability of "plant and animal varieties and of essentially biological processes for
372

their production" created uncertainties, especially with regard to the protection of

biotechnological and microbiological inventions. 165
Generally, it was stated that the Directive is "without prejudice" to the exclusion of
plant and animal varieties from patentability, but it was specifically pronounced that
plant-based inventions, in general, are patentable, provided their application is "not
technically confined to a single plant variety." In noting that the concept "plant variety"
as defined by laws protecting new varieties, refers to its whole genome (possessing
individuality and distinctness), it was stressed that "plant groupings" which are
characterized by a particular gene (not its whole genome) are not covered within the
protection of new varieties. Such plants are not excluded from patentability, even if
comprising new varieties.
However, "if an invention consists only of genetically modifying a particular plant
variety, and a new plant variety is bred, it will be excluded from patentability, even if
the genetic modification is the result of a biotechnological process." 166
Considering the function of a patent, it was stipulated that "the holder of a patent
should be entitled to prohibit use of patented self-reproducing material... for the
production of the patented product itself." In recognizing the "farmer's privilege" in
plant varieties, it was pronounced that in derogation of the patentee's rights, if the
protected "propagating material is sold to a farmer for farming purposes" he is to be
authorized "to use the product of his harvest for further multiplication or propagation on
his farm" subject to conditions set out by the CPVR EC regulation and payment of a
required fee. In relating to the use or exploitation of new plant characteristics resulting
from genetic engineering, it was stipulated that access must be granted by compulsory
license "where, in relation to the genus or species concerned, the plant variety
represents significant technical progress of considerable economic interest compared to
the invention claimed in the patent." 167
The Directive obliges its Member states to protect biotechnological inventions
subject to provisions of the Directive and without prejudice to their obligations under
other international instruments, including the Convention on Biological Diversity.
In laying down certain legal principles, the Directive tries to determine and
minimize the difference between "inventions" and "discoveries." Generally, it provides
that inventions which are new, involve an inventive step and are susceptible of
industrial application, shall be patentable, "even if they concern a product consisting of
or containing biological material or a process by means of which biological material is
produced, processed or used." 168 Although in adherence to the traditionally established
principles, it provides that plant varieties and essentially biological processes for the
production of plants "shall not be patentable," it specifically stipulates that "inventions
which concern plants ... shall be patentable if the technical feasibility of the invention is
not confined to a particular plant variety." Further, it also broadens the counter-
exclusion by stipulating that the general exclusion "shall be without prejudice to the
patentability of inventions which concern a microbiological or other technical process
or a product obtained by means of such a process." 169
In defining the meaning of a "microbiological process," the Directive stipulates that
it means "any process involving or performed upon or resulting in microbiological
material." 170
In trying to minimize the "invention-discovery" dichotomy and broaden the subject
matter of an "invention," the Directive specifically provides that "biological material
 373

which was isolated from its natural environment or produced by means of a technical
process, may be the subject of an invention, even if it previously occurred in nature." 171
"Biological material" as defined means: "any material containing genetic information
and capable of reproducing itself or being reproduced in a biological system."
In defining an "essentially biological process" for the production of plants, the
Directive stipulates that this is a process which "consists entirely of natural phenomena
such as crossing or selection." 172
In extending the protection of a biotechnological invention, the Directive stipulates
that a patent "on a biological material possessing specific characteristics... shall extend
to any biological material derived from [it] through propagation or multiplication in an
identical or divergent form and possessing those same characteristics." The same
applies where the patent is granted "on a process that enables a biological material to be
produced possessing specific characteristics as a result of the invention." The rights
conferred by such a patent shall extend also "to any other biological material derived
from the directly obtained biological material through propagation or multiplication"
performed and resulting in the described manner. 173 It further provides that a patent
granted "on a product containing or consisting of genetic information, shall extend to all
materials .. .in which the product is incorporated and in which the genetic information is
contained and performs its function. " 174 In preventing patent applications for undefined
gene sequences, it provides that "the industrial application of a sequence or a partial
sequence of a gene must be disclosed in the patent application." 175
However, the Directive stipulates that said extension of patent protection shall not
apply "to biological material obtained from the propagation or multiplication of
biological material placed on the market ... where the multiplication or propagation,
necessarily results from the application for which the biological material was marketed,
provided that the material obtained is not subsequently used for other propagation or
multiplication." 176
In derogation from said extensions, the Directive provides that "the sale or other
form of commercialization of plant propagating material (by the patentee or with his
consent) to a farmer for agricultural use, implies authorization for the farmer to use the
product of his harvest for propagation or multiplication by him on his own farm,"
subject to the EC Regulation on CPVR177 [emphasis added].
Taking into account the interests of plant breeders who cannot acquire or exploit a
PVR without infringing a prior patent, the Directive relaxes the patent restriction of
access to important breeding material. It entitles the breeder to apply "for a compulsory
license for non-exclusive use" of the protected invention, subject to payment of an
appropriate royalty "inasmuch as the license is necessary for the exploitation" of his
new variety. The Directive obliges its Member States to provide, under their domestic
laws, that where such a compulsory license will be granted the patentee should be
entitled to a cross license, on reasonable terms, to use the protected variety. 178 The right
to apply for a compulsory license, under the same conditions, is also accorded to a
patentee in a biotechnological invention if he "cannot exploit it without infringing a
prior PVR." 179 In each of such cases it is important to demonstrate that "the plant
variety or the invention constitutes significant technical progress of considerable
economic interest, compared with the invention claimed in the patent or in the protected
plant variety"180[emphasis added].
A series of provisions prescribe the procedure for depositing "biological material" in
374

compliance with the required description, to be "in such a manner as to enable the
invention to be reproduced by a person skilled in the art" and in accordance with the
Budapest Treaty. 181
The Directive excludes from patentability, inventions "where their commercial
exploitation would be contrary to ordre public or morality"; however it provides that
"exploitation shall be not deemed to be so contrary, merely because it is prohibited by
law or regulation." 182 In this context, it should be noted that the "ordre public" and
"morality" exclusion under Art. 53(a), as discussed, has been very seldom invoked by
the EPO. Under its established policy, the EPO repeatedly stressed that it is to be
invoked only in rare and extreme cases, only where the invention may universally be
regarded as "outrageous."
The Directive obliges each EU Member State to adapt its national laws in
accordance with the Directive not later than July 30, 2000. 183 The European
Commission is under an obligation to report to the European Parliament and Council
every 5 years (from July 30, 2000) "on any problems encountered with regard to the
relationship between the Directive and international agreements on the protection of
human rights." With the aim to assess the practical implications of the specific legal
provisions with respect to biotechnological inventions as prescribed by the Directive,
the Commission is required to submit a report (within 2 years from entry) assessing its
implications for basic genetic engineering research of failure to publish, or late
publication of, papers on subjects which could be patentable. The Commission is to
report annually "on the development and implications of patent law in the field of
biotechnology and genetic engineering." 184

7. Conclusions

In studying the diverse interpretations of the specific bar on patentability of "plant

varieties and essentially biological processes for the production of plants," as partially
described in this paper, one becomes responsive to an often-pronounced call for a
harmonized comprehensive system of industrial property protection for plant
innovations. This should apply equally to the European and American systems of
protection. Notwithstanding the fact that the American system of protection is much
broader, it permits overlap or gaps in the triple protection with its blurred borders with
respect to eligible subject matter.
Such a harmonization is especially necessary when taking into account the WTO-
TRIPS Agreement and the 1991 UPOV Convention, providing the possibility for
double-protection or any other combination of protection for "plant varieties." The
specific problems in protecting ornamental plants, as to fragrance and color, deserve
special attention for clarifying and simplifying such protection. Incorporation of certain
provisions taken from trademark law, which recognizes registration of single colors or
fragrances having distinctive characteristics for specific goods, should also be
considered for providing a partial solution.
Although the Directive on the Protection of Biotechnological Inventions aims to
provide clarification of uncertainties in the respected field, by incorporating basic
principles of case law, it leaves many unresolved disparities for future controversial
interpretation of its provisions.
 375

8. Acknowledgements

I would like to extend my gratitude to Prof. Gerhard Schricker, the Director of the Max
Planck Institute for Foreign and International Patent, Copyright and Competition Law in
Munich, for allowing me to use the Institutes Library and facilities, in preparing this
paper.
My special thanks are given to Prof. Joseph Straus of the MPI and Dr. Reiner
Moufang of the EPO, for their guidance, help and reference to their enlightening
writings. The late Prof. F.K. Beier is hereby remembered for acquainting me with the
discussed subject

9. Notes

1. " ... ideas should freely spread from one to another over the globe, for the moral and mutual instruction
of man, .. .like the air in which we breath, move, and have our physical being ... " Thomas Jefferson's
statement (1813), in a letter to a Baltimore inventor.
2. "Inventions cannot... be a subject of property. Society may give an exclusive right to the profits arising
from them, as an encouragement to men to pursue ideas which may produce utility." (ibid.).
3. U.S. Constitution, Art. 1, Sec. 8.
4. The "Paris Convention," first signed on Mar 20, 1883, revised on Dec 14, 1900 (Brussels); on Jun 2,
1911 (Washington); Nov 6, 1925 (Hague); Jun 2, 1934 (London); Oct 31, 1958 (Lisbon)'; Jul14, 1967
(Stockholm) and amended in London on Sep 28, 1979.
5. The "Berne Convention," first signed in 1886; revised in 1896 (Paris); in 1908 (Berlin); in 1914
(Berne); in 1928 (Rome); in 1948 (Brussels); in 1967 (Stockholm); in 1971 (Paris) and amended in
1979. By Apr 1999 there were 13 8 states party to the Convention.
6. The International Convention for the Protection of New Varieties of Plants (UPOV), signed on Dec 2,
1961; revised in Geneva on Nov 10, 1972 and Oct 23, 1978 and re-enacted in 1991 (The 1991 Act).
7. The World Intellectual Property (WIPO) Convention signed in Stockholm on Jul 14, 1967; amended
Sep 28, 1979.
8. See Art. 1-4 of the WIPO Convention.
9. See the European Patent Convention (EPC) signed in 1973, and entering into force on Oct 7, 1977.
10. See the North American Free Trade Agreement (NAFTA) signed on Dec 8, 1993, in force from Jan 1,
1994.
11. See Art. 102, Chap. 1 (general part), NAFTA
12. The World Trade Organization (WTO) Agreement signed on Apr 15, 1994 at the Ministerial
conference in Marrak.esh, entering into force on Jan 1, 1995.
13. The "TRIPS" Agreement, signed on Apr 15, !994, as annex 1C to the WTO.
13a EC No. 2100/94 of Jul27,1994, enforced since Apr 27, 1995.
13b. Directive 98/44/EC of Jul6, 1998, OJ EPO 2/99, p.!Ol.
14. see Art. 1(1 and 2) ofthe Paris Convention.
15. see Art. 2(1) ofthe Paris Convention.
16. see Art. 4 quarter and Art. 4ter of the Paris Convention.
17. see Art. 5A of the Paris Convention.
18. see Art. 4A(l) and 4C(I and 2) ofthe Paris Convention.
19. See Art. 11(1) of the Paris Convention and Art. 11(2) in providing that: "If later the right of priority is
invoked, the authorities of any country may provide that the (priority) period shall start from the date of
introduction of the goods into the exhibition."
20. See Art. 10 BIS of the Paris Convention.
21. See Art. 1(3) ofthe Paris Convention.
22. See Agriculture and Horticulture Act 1964 (UK) PT. Iii (ssii-24) and par. 8 (58-865).
23. See Art. I Sec. 2, TRIPS.
24. See Art. 27( I), TRIPS.
25. See Art. I Sec. I, TRIPS.
26. See Art. 7 Part 1, TRIPS and introduction (above).
376

27. See Art. 27(1) Part II Sec. 5, TRIPS.

28. See Art. 27(2) and 3(a) Part II Sec. 5, TRIPS, emphasized by the author.
29. See Art. 27(3b) Part II Sec. 5, TRIPS.
30. See Part II Sec. 5 Art. 28 (1)(a and b), TRIPS.
31. See Part II Sec. 5 Art. 30, TRIPS.
32. See Part II Sec. 5 Art. 31, TRIPS.
33. See Part II Sec. 5 Art. 33, TRIPS.
34. See Preamble and General Part Art. 102, Chap. 1, NAFTA
35. See Chap. 17 Part 6 Art. 1703, NAFTA
36. See Chap. 17 Part6 Art. 1709(1 and 2), NAFTA
37. See Chap. 17 Part 6 Art. 1709(5), NAFTA
38. See Chap. 17 Part 6 Art. 1709(9).
39. See Chap. 17 Part 6 Art. 1709(12).
40. See Chap. 17 Part 6 Art. 1709(3b and c) NAFTA and compare with TRIPS, requiring review.
41. See Chap. 17 Part 6 Art. 170 1(2c and d), NAFTA
42. See Chap. 17 Part 6 Art. 1709(4).
43. See Preamble and Art. 1-4 ofEPC.
44. The Member states of the European Union and also Switzerland, Cyprus and some of the Eastern
European states, e.g. former Yugoslavia, Slovenia, Roumania, etc.
45. See Art. 21 and 22 Part I Chap. III ofthe EPC.
46. See Art. 33 Chap. IV Part I of the EPC.
47. See the European Patent Office Information Guide, Jun I, 2000.
48. See Art. 52(1) Chap. I Part ofthe EPC.
49. See Art. 52(2a) and 52(4) Chap. I Part II ofthe EPC.
50. See Art. 54(1 and 2) Chap. I Part I of the EPC.
50a. See Art. 55 ibid., and Rule 23 of the Implementing Regulations.
51. See Art. 56 Chap. I Part II ofthe EPC.
52. See Art. 57 Chap. I Part II of the EPC.
53. Art. l(I)I of the Geneva Treaty on the International Recording of Scientific Discoveries, 1978.
53a. See Amgen Inc. v Chugai Pharmaceutical Co., Ltd. (Federal Circuit, 1991) 18 USPQ 2d 1016.
54. See Art. 83 Chap. I Part III of the EPC.
55. See Art. 82 Chap. I Part III of the EPC.
56. See Art. 81, 80 and 78 Chap. I Part III of the EPC and rules 26-36 Chap. II Part III ofthe Implementing
Regulations to Part III of the Convention.
57. See Art. 93 Part IV of the EPC.
57a. See Art. 67(1) Chap. III Part II ofthe EPC.
58. See Art. 71-74 Chap. IV Part II of the EPC.
59. See Art. 62 Chap. III Part II, and 64(1) and 65 Chap. III Part II of the EPC.
60. See Art. 69 ibid., and the protocol on its interpretation.
61. See Art. 63(1) Chap. III Part II of the EPC.
62. See Art. 63(2 a and b) as amended on Dec 17, 1991, entered into force on Jul4, 1997.
63. Patentable under the counter-exception of Art. 53(b) of the EPC.
64. See Rule 28(1) and (2) Chap. II Part III of the Implementing Regulations to the EPC.
65. See Rule 28(3a and b) Chap. II Part III of the Implementing Regulations (OJ EPO 1979, 447).
66. See Rule 28(4) and (5), ibid.
67. See Rule 23b(3) and Rule 28(6) as amended on Jun 16, 1999 following Directive 98/44/EC from Jul6,
1998 on the Legal Protection of Biotechnological Inventions, substituting the term "derived culture"
with "derived biotechnological material."
68. See Rule 28(7, 8 and 9), ibid.
69. Rule 28a ibid., inserted by decision of Adm. Council, in force from Jun 1, 1980 see [64a], p.449.
70. Rule 27a ibid., inserted by decision of Adm. Council, in force from Jan 1, 1993, OJ EPO 1992, 342.
70a. See art. 3 and 4 of the Budapest Treaty on the International Recognition of the Deposit of Micro-
Organisms for the Purposes of Patent Procedure, signed in 1977, enforced since 1980.
71. The ban on double protection did not apply to the USA, the Plant Patent Act 1930 preceded UPOV.
72. See Chap. II Art. 3(1 and 2) 1991 UPOV Act broadening protection to "all plant genera."
73. See Chap. II Art. 4, ibid.
74. Among the states already adhering to the 1991 UPOV Act, as of Jan 1999, are: Japan, Russian
Federation, Australia, USA, Canada, Mexico, UK, Germany, France, Italy, Spain, Israel, etc.
 377

75. See Chap. III Art. 5(1 and 2), ibid

76. See Chap. 1 Art. 1 vi. and iv (definitions) 1991 UPOV Act; compare with the defmition of''variety" in
the 1961 text as: "any cultivar, clone, line, stock or hybrid which is capable of cultivation" and note
that the 1978 Act lacks a definition of''variety."
77. See Chap. III Art. 6(li and ii), the 1991 UPOV Act.
78. See Chap. III Art. 7, ibid
79. See Chap. III Art. 8, ibid.
80. See Chap. III Art. 9, ibid.
80a. See Chap. IV Art. 12, ibid.
80b See Chap. VI Art. 20, ibid.
81. See Chap. V Art. 14(1-5), ibid.
82. See Chap. IV Art. 10, ibid.
83. See Chap. IV Art. 13, ibid.
84. See Chap. V Art. 19, ibid.
85. See Chap. V Art. 15, ibid.
86. SeeECReg. No. 2100/94ofJul27,1994onComm. PVR,OJECNoL227/1 (Sep 1, 1994).
87. See Preamble, ibid.
88. See Art. 5(2), 7(1 and 2), 8 and 9 of Chap. 1 EC Reg. 2100/94.
89. See Art. 5(3) Chap. 1, ibid.
90. See Art. 5(4) Chap. 1, ibid. (Art. 63 ).
91. See Art. 63 for the specific provisions, EC Reg. 2100/94.
92. See Art. 7(1) and Art. 7(2a and b) Chap. 1, ibid.
93. See Art. 8 Chap. 1 (EC Reg. 2100/94).
94. See Art. 9 Chap. 1, ibid.
95. Known as the Townsend-Purnell Act passed by Congress in May 1930.
96. See 35 USC Sec. 161-164 (as revised in 1952 and 1988).
97. See 7 USC Sec. 2321 et seq.
98. See 35 USC Sec. 101 et seq.
99. See Diamond v Chakrabarty, 206 USPQ 193, 197 (1980).
100. See Pioneer Hi-Bred Int., Inc. v J.E.M. Ag Supply Inc. (USCA Federal Circuit 2000) 53 USPQ 2d.
1440 and 49 USPQ 2d. 1813 (N.D. Iowa 1998).
101. See 3 5 USC Sec. 101 in its full wording.
102. Luther Burbank, a distinguished plant breeder and developer of new plant varieties and flowers, esp.
lilies and the Shasta daisy, as cited in Imazio, infra [114], p. 1675.
103. See citation from his letter in the S.R. (105 infra).
104. See [96] supra.
105. Diamond v Chakrabarty citing from S.R. 315 and H.R. Rep. 129, [99] supra.
106. See 35 USC Section 161.
107. See 35 USC Sec. 112 (1988).
108. See 35 USC Sec. 162.
109. Reproduction by "grafting, budding, cuttings, layering, division and the like, but not by seeds."
110. Yoder Bros., Inc. v California Florida Plant Corp. USCA Fifth Circuit 1976 537 F.2d 1347. Compare:
ex parte Tanksley, 26 USPQ 2d 1384 (1387-88) Bd. Pat. App. and INt'f 1991.
111. In reliance on Senate Report 71 Congress. 2d Session (1930).
112. Imazio Nursery Inc. v Dania Greenhouses USCA (Federal Circuit Nov. 3, 1995) 36 USPQ 2d., p. 1673,
1678-9, appeal from (SW) DC (ND. California Dec. 16, 1992) 29 USPQ 2d. p, 1217; see also ex parte
Moore 115 USPQ 145 (1957 POBA).
113. See Imazio USCA (Federal Circuit 1995) 36 USPQ2d, ibid., 1680-1681.
114. See Pan-Am Plant Co, v Matsui (N.D. Calif. 1977), 198 USPQ 462 [in Imazio ibid., at 1680].
115. The D.C. was of the opinion that "independent creation" is not a legitimate defense to a plant patent
infringement action. See also Ex parte Allen 2USPQ2d 1425 and Amgen Inc v Chugai Pharm. Co., Ltd.
13 USPQ2d 1737 and 53a, supra.
116. See 35 USC Sec. 163.
117. See Diamond v Chakrabarty, supra [100].
118. See 7 USC Sec. 2402(a) amm. 1994, PVPA
118a. See 7 USC Sec. 2401(6A, Band C).
119. See 7 USC Sec. 2401(a1, 2, 5, 6, 7 and 9) as amm. In 1994, 1970 PVPA.
120. Imazio supra [112] at 1678.
 378

121. See 7 USC Sec. 2402(a1, A, I, ii) PVPA

122. See 7 USC Sec. 2401(b2) PVPA
123. See 7 USC Sec. 2401(b1) PVPA
124. See 7 USC Sec. 2401(b3) PVPA
125. See 7 USC Sec. 2402(a2) PVPA
126. See 7 USC Sec. 2402(a3 and 4) PVPA
127. See 7 USC Sec. 2483(a1, 2A, Band 3).
128. See 7 USC Sec. 2531(a and b).
129. See 7 USC Sec. 2532.
130. See 7 USC Sec. 2483(b and c) term.
131. See 7 U.S.C Sec. 2422(1 and 4).
132. See 7 USC Sec. 2404.
133. See 7 USC Sec. 2541.
134. See fin. (81] supra.
135. See 7 USC Sec. 2541(d).
136. See 7 USC Sec. 2541(e).
137. See 7 USC Sec. 2544 and 2545.
138. See 7 USC Sec. 2542, 2543 and 2567.
139. See 7 USC Sec. 2561 and 2562.
140. See 7 USC Sec. 2563-2565, 1970 PVPA
141. See Diamond v Chakrabarty (1980), 206 USPQ 193 (197-198).
142. See Pioneer HI-Bred International v J.E.M. Ag Supply Inc. 53 USPQ2d, p. 1440 (1443).
143. See Diamond v Chakrabarty 206 USPQ 193 (198).
144. Pioneer Hi-Bred supra 142 at 1441-2 and see also ex parte Hibberd 1985, 227 USPQ 443, compare
with Pioneer Hi-Bred Ltd. v Canada Comm. of Patents (1987) 3 F.C.R.
145. See the writings of Prof. F. K. Beier, Prof. Joseph Straus, Dr. Reiner Moufang.
146. See first half-sentence and second half-sentence of Art. 53(b) EPC.
147. See T 356/93 Plant Cells/Plant Genetic Systems (PGS) (plants and seeds resistant to glutamine
synthetase inhibitors [GSis]), OJ EPO 1995, p. 545.
148. ibid., p. 571.
149. ibid., T 356/93 PGS, p. 571 (point 23 of the reasons).
150. ibid., T 356/93 PGS, p. 575-576 (point 34-36 of the reasons).
151. See T 49/83, Propagating materiai!Ciba-Geigy 1984 OJ EPO 112; and T 320/87, Hybrid
plants!Lubrizol1990 OJ EPO 71.
152. See T 356/93 PGS OJ EPO 1995 p 545, p. 578 (point 40.1 of the reasons).
153. ibid., p. 578 (points 39; 40.1 and 40.9 ofthe reasons).
154. ibid., p. 581 (point 40.11 of the reasons).
155. ibid., p. 569 (points 18.8-19); for general discussion see points 3-18.7.
156. See T 1054/96 Transgenic Plant!Novartis AG, OJ EPO 1998, p. 511.
157. See G 1/98 Transgenic Plant!Novartis II, O.J.2000, p. 112; 31 IIC No.4, p. 431(437).
158. ibid., 31 IIC No.4, p. 439 (point 3.10 of the reasons).
159. ibid., 31 IIC No.4, p. 432 (point 3.1 ibid.).
160. ibid., 31 IIC No.4, p. 433 (point 3.1 ibid.).
161. ibid, 31 IIC No.4, p. 441 (point 5.1-5.3 ibid.).
162. ibid., 31 IIC No.4, p. 439-440 (point 4 of the reasons).
163. ibid., 31 IIC No.4, p. 438 (point 3.9 ibid.).
164. Directive 98/44EC, OJ EPO 2/1999, p. 101.
165. ibid., Preamble (recitals 8-14).
166. ibid., Preamble (recitals 27-33).
167. ibid., Preamble (recitals 46-53).
168. ibid., Art. 3( 1).
169. ibid., Art. 4(1, 2 and 3).
170. ibid., Art. 2(1b and a).
171. ibid., Art. 3(2).
172. ibid., Art. 2(2).
173. ibid., Art. 8(1 and 2).
174. ibid., Art. 9.
175. ibid., Art. 5(3).
 379

176. ibid., Art. 10.

177. ibid., Art. 11 ( 1).
178. ibid., Art. 12(1) and Novartis 1131 IIC, p. 438-9 point 3.9.
179. ibid., Art. 12(2).
180. ibid, Art. 12(3).
181. ibid, Art. 13(1-5) and Art. 14(1-2).
182. ibid., Art. 6(1).
183. ibid., Art. 15(1).
184. ibid., Art. 16(a, band c).
INDEX
ABC model 240,242,245,247,248
Abiotic stress 60, 197, 209, 210, 212, 263
Accessory fruit 3
Acetyl CoA 274, 299, 300, 306
Acetyl-CoA:benzylalcohol
acetyltransferase 298, 300
Actinomorphic flower 2, 107
Additive action 14, 19
Adventitious bud 117, 118, 119, 141,
142, 183
Adventitious regeneration 139,141, 143,
148, 151
AFLP 86, 96,329, 331,332,333, 334,
336,338,339
AGAMOUS (AG) 240-245, 247, 248,
369
Agarose gel electrophoresis 28
Aggregate fruit 3
Agrobacterium 139, 140, 156, 161, 163-
166, 168, 170-173, 176-179, 181-184,
187
Allele 8-15, 20-22, 55, 57, 59, 62, 63, 68,
69,73,223,225,227,228,230-232,
243-245,248,263,323
Allotetraploid 20, 55, 91, 94, 95
ALS inhibitor 208
Alstroemeria 48, 52, 86, 88-91, 94-97,
111, 118, 167, 158,335
Amphidiploid 20, 70, 95
a-Amylase inhibitor 206,207
Aneuploidy 207
Angiosperm 4, 5, 113, 220, 242, 243, 247
Anther 1, 5, 149, 151, 255, 257, 303
Anthocyanin 56, 156,253,254,257-265,
287,298,300
Anthocyanin biosynthetic gene 261
Anthocyanin synthase 254, 255
Anthurium andraeanum 157, 158
Antirrhinum majus 52, 58, 59, 160, 164,
239,257-259,261,301,304
APETALA1 (AP1) 240
Apomixis 5, 6
381
Apoptosis 311
Arabidopsis thaliana 163, 219, 239
Article 53(a) EPC 353, 367, 371
Asexual propagation 66,74
Asexual reproduction 36, 362
Astaxanthin 287
Automation 14, 340
Autonomous-flowering plant 134
Autoploid 54
Autopolyploid 55
Autotetraploid 20, 55, 69
A virulence gene 201,203,205
Azalea3,49,57,61, 76,110,183
B. sanguinea 167
Bacillus thuringiensis 205
Bacterial contamination 140, 147
Basic seed 79
Basic stock 79
Bedding plant species 49, 73
Begonia 49, 52, 54, 55, 57, 61, 67,71-74,
76,110,158,161,163,248
Begonia semperflorens 57, 162
Begonia tuberhybrida 162
Begonia x hiemalis 161, 162
Benzenoid 296, 299,301
Betalain 56
bHLH-type transcription factor 262
Bialaphos 160, 163, 170, 176, 177, 179,
208
Bilateral91, 94, 95, 97, 99
Bilayer fluidity 315
Biological material355, 371-373
Biotechnology 30, 44, 105, 163, 169,
184,197,253,265,349,359,366,
369,371,374
Biotic stress 146, 197, 205, 208, 209
Botanical genera and species 359
Botrytis 61, 78, 176, 181, 199,200,202
Botrytis cinerea 61, 181, 199, 200, 202
Brassica 71, 212, 243, 282
Breeders' Rights 50, 73, 117
Breeding aim 58, 70, 74, 78
382
Breeding method 47, 51, 54, 105, 106,
110, 111, 119, 155
Breeding strategy 51, 54
Breeding system 52, 53
Brugmansia arborea 167
Bt toxin 205-207
Budapest Treaty 355, 374
Bulk lipid fluidity 315
Bulked segregant analysis 339
CaC}z-heat transformation 38
Calyx 1, 3
Canola 282, 283
Canthaxanthin 280, 287
Capsanthin 274, 280, 287
Capsanthin-capsorubin synthase (CCS)
280,281
Capsorubin 27 4
Carnation 48, 62, 64, 74, 76, 96, 105,
108, 110, 116, 132, 134-136, 156, 158,
162,163,183,187,206,248,263-265,
306,311-313,315-323,337,339
~-Carotene 56, 273, 275, 278-281, 283-
286
~-Carotene 278, 279, 284, 286
£-Carotene 278, 280
a.-Carotene 278,280
~-Carotene desaturase 278
Carotenoid 56, 168, 273-287
Carotenoid-associated protein 285
Carotenoid biosynthesis 273, 275-278,
280,281,283,285
Carotenoid color 278, 280
Carpel!, 5, 27, 239, 241, 244-246, 248,
262
Catabolism of membrane protein 316
Catharanthus roseus 52,274
Cattleya 2, 179
Cauliflower mutant 285
Cecropin 182, 189, 199, 201
Cell lineage 113, 114, 143
Chalcone isomerase 254
Chalcone synthase 169, 185, 253
Chemical mutagen 122, 148
Chimerism 109, 112, 113, 116-118, 123,
139, 143, 147, 163, 170, 178
Chi-square 13
Chitinase 165, 184, 197, 198, 200, 203,
207
Chlorophyl1144, 123, 125, 229, 273,
276,277,281,284,320
Chloroplast 206, 209, 273, 274, 276, 282-
285
Chlorsulfuron 163, 173
Chromatid 3, 4, 14,93
Chromophore 220-223, 225, 226
Chromoplast 273, 274, 276, 281-286
Chromoplast membrane 284, 285
Chromosomal damage 123
Chromosome pairing 89, 91, 93, 97
Chr protein 285
Chrysanthemum 2, 49, 50, 60, 68, 76, 85,
96, 105, 110, 111, 116, 124, 125, 132,
156,162,164-166,178,183,256-260,
263,264,306,318,334-336,338
Chrysanthemum (Dendranthema) 48,
110, 158, 164
CHY 280, 281, 284
Circadian clock 219,220,224,226-229,
231,232
Circa diploid 94
Cis to trans isomerization reaction 281
CLA1276
Climacteric fruit 318
Climacteric-like flower 318
Clonal cultivar 50, 53, 55, 74-79
Clonal propagation 124,141
Clonal selection 74, 76, 77, 79
Clone 5, 25, 26, 30-35, 37 42, 44, 60, 66,
68, 71,74-78,95,96,130,132,134,
135, 139, 160, 161, 162, 167-169, 176,
177,181,201,203,205,209,212,
220,224,225,243,244,246,247,
255,257-259,261,298,300,301,306,
314,320,321,333,335,358,362
Cloning vector 25-30, 37-39,42
Coat protein 171, 172, 176, 204
Codominance 10
Colchiploid 55
Cold-resistant 132
Color 7,13, 22, 50, 55-57, 63, 69, 70, 74,
76-78, 105-110, 112, 114-117, 120,
121, 124, 125, 130, 131, 149, 150,
155-157, 160, 162-166, 168, 169, 176,
177,180,182-184,186,187,253,254,
258,259,263-265,273,278,280,286,

287,306,361,362,366,374
Common knowledge 357, 360, 363
Compactness 58
Competence 38, 143, 149
Complement cell
Complementary DNA 29, 37
Complementary gene action 14
Compulsory license 350, 354, 359, 372,
373
Conjugated double bond 278, 280
Conjugative plasmid 39
CONSTANS 228,231,232
Consumer trend 130
Contaminant elimination 140
Contrary to public policy 360
Co-pigmentation 57, 259, 263
Corolla 1, 3, 246, 257, 262
Cost-effective 63, 136, 149, 353
Coupling 16, 17, 40
CPTA 284, 286
CPVR (Community Plant Variety Right)
349,359,360,370,372,373
Crossability 86
Crossing over 3, 14, 15, 89, 91, 93, 94
Cross-pollination 5, 66, 67, 72, 73, 79
CrtB 282, 283
Crt/ 281, 283
CrtO 280, 283, 287
CrtS 281, 287
Cryopreservation 145-147, 151
Cryptochrome 224, 225,227, 228
Crystal147, 285, 286
Crystal protein 205, 207
Cultivar type 54
Culture indexing 77, 79
Cut flower 19, 47, 48, 50, 52, 70, 98,
129-136, 155-157, 162, 164, 169, 170,
176, 179,180, 183, 187,265,306,
322,350,358
Cut style 86-89
Cyclamen persicum 52, 57, 69, 158, 166
Cyclase inhibitor 284, 286
Cyclization 278, 286
Cymbidium orchid 179
Cyme1
Cytoplasmic inheritance 19
383
DAF 331, 336, 338
Daffodil 56, 74, 281, 283-286, 296
Datura innoxia 166
D. arborea 167
D. indicum 164
D. meteloides 167
D. sanguinea 167
Day length 3, 65, 66, 125, 133, 220, 229,
230,231,240,241
Daylily 158, 167, 316
De-epoxidase 281
De-etiolation 219, 221, 224,228, 230
Dehiscence 1
Dehydration 147, 211
Deleterious mutation 112
Deletion 21, 122, 330, 331
Delphinium 3, 49, 52, 55, 73, 97, 158,
167
Dendranthema morifolium 164
Dendrobium 53, 99, 178
Dendrogram 337
Denomination 356-360
Deoxyribonucleotide 25, 36, 40, 41,
Deoxyxylulose 274, 276, 277, 281
Deoxyxylulose-5-phosphate 274, 277,
281
Depository 354, 355
Derived biological material 355
Desaturation reaction 278, 280, 286
Designation 10, 27, 357
Developmental signal 263
Dianthus caryophyllus 48, 57, 108, 158,
162,248,255,257,259
Dihybrid 11-13, 16, 17
Dihydroflavonol254,258,259,263
Dihydroflavonol 4-reductase 254, 255
Dimethylallyl diphosphate 274, 296
Dioecious plant 248
Dioecious species 2
Diploid 3, 4, 8, 11, 17, 20, 54, 55, 69-71,
74, 77, 78,85,90-95,97,98, 151,166,
338
Diplontic selection 147, 151
Directed mutagenesis 44
Disease agent 51, 61, 62, 70,
Distinct 91, 220, 222, 224, 226, 240, 242,
274,323,330,335,356,359,361,363
Distinctness 76, 335, 357, 360, 363, 372
384
Diversity 1, 5, 129, 130, 132, 143, 263,
329,330,334,337,338,372
DMAPP 274, 275-278, 296
DNA hybridization 31, 32, 34, 37, 39,
331
DNA ligase 28, 29
DNA polymorphism 330
Dominance 8-13, 19,56-59, 61, 135, 156,
160,185,331
Dominant 8-10, 12-14,52,56-59, 61-63,
69, 73,120,172,248,323,332,334,
338,339
Doritaenopsis 179
Dormancy 133, 134, 144, 171
Double fertilization 3, 4, 88
Double flower 3, 57, 68, 69, 248,339
Doubleness 3, 55, 57, 74, 77, 248
Double protection 356, 370, 374
DOXP pathway 274,276,277, 287
Dunaliella salina 286
Duplicate action 14
Duplication 21, 362
DXR 277,281,287
DXS 274, 276,277,281, 285
Dyad 93
Early selection 76, 130
Economic data 109
Economic Rights 347, 355
Electrophoresis 28, 31, 33, 34, 156, 160,
182,183,187,298
Electroporation 38, 156, 174
ELF3 227, 229
Elicitor 202, 203
Eligibility 374
Embryo 85, 88, 89, 98, 99, 149, 155
Embryo rescue 85, 88, 89, 98, 99, 149,
155
Encapsulation 144, 147
Endoplasmic reticulum 274, 282, 315
Environmental selection
Environmental signal 134, 220, 229, 263
Environmental stress 44, 45, 51, 60, 209,
212,311, 323
Environmental variation 64
EPC (European Patent Convention) 349,
350,352,353,360,367,370,371
Epigenetic effect 119
Epigenetic stability 140
Epigynous 2
Epistasis 13, 19,
EPO (European Patent Office) 349, 352-
355,367,369-371,374,375
Epoxide 280
Escherichia coli 27,274,300
Essential biological process 349, 351-
353,360,366,368,371-374
Essential characteristic 358, 362, 364
Essential derivation 125
Essentially derived variety 335, 358, 359,
364
Esterified 274, 286, 314
Ethylene 60, 143, 161-163, 182, 311,
312,315,317-323
Ethylene signal-transduction pathway
318
Etioplast 276, 277, 283, 287
Eudicot 245, 247, 248
Exclusion 349, 351, 352, 355, 360, 366-
368, 370-372, 374
Exclusive rights 347, 348, 351, 352
Exemption 359, 364
Exhaustion of rights 351, 358
Extra-chromosomal inheritance 19
F 1 hybrid 8, 19, 20, 22, 53, 59, 68,70-73,
77-79,85,89,96,97,166
F 1-sterility 89
Farmer's privilege 359, 365, 372
Farnesyl diphosphate synthase 281
Feeding experiment 260
Fertilization 3-5, 8, 49, 68, 85, 87-89,
135
Fibril285, 286
Fibrillin 281, 285
Filament 1, 5
Filing date 351
Fingerprint analysis 334
First-division restitution (FDR) 91-95
FISH 93-96
Flavanone 3-hydroxylase 254, 255
Flavone synthase 254, 255
Flavonoid 56, 57, 169, 253-255, 260,
261,263,265
Flavonoid gene 255,256,261-263,265
Flavonoid 3'-hydroxylase 254, 255
Flavonoid 3',5'-hydroxylase 186, 254,
255

Flavonol synthase 254, 255
Floral pigmentation 253, 274, 286, 287
Floral volatile 295, 287, 302
Floriculture 47, 48,51-57,61,63, 65, 68,
69, 71-74, 306
Floriculture crop 47, 48,51-54,56,57,
59-61, 63, 68, 73
Flower color 13, 22, 55, 56, 63, 69, 70,
74, 76, 77, 106, 108-110, 112, 114-
117, 120, 121, 124, 125, 149, 150,
155-157, 163-166, 168, 169, 176, 177,
180,182,183,186,187,253,258,
259,263-265,286
Flower development 3, 133, 160, 239,
240,243,245,248,249,305,306
Flowering 3, 4, 18, 49, 59, 60, 63, 64, 66,
68, 74,98, 109,110,112,120,124,
130-135, 141, 146, 162, 172, 179, 180,
185,219,220,224,226-233,239-241,
248,306,334,339,350
Flower size 50, 58, 74, 151
Flowering stem 131, 132, 135
Flowering time 179,219,224,229,230,
232,233,240,241,248
Flower type 5, 58, 110, 112
Forsythia x intermedia 158, 168
Fossil4, 5
FPP, farnesyl diphosphate 278
Fragrance 120, 167, 265, 273, 295, 296,
298,303,361,374
Fragrance emission 302
Free fatty acid 314,315,317
Free radical 316
FRUITFUL (FUL) 241
Fusarium 61, 62, 70, 166, 199-201
Galactolipid 283, 317,
Gamete 8-12, 15-17, 20, 21, 55, 58, 68,
73, 86, 88, 91-94, 97,99, 115, 161
Gametoclonal variation 151
Gene bank 30, 146
Gene expression 32, 42, 114, 170,209,
211,221-224,226,258,264,287,311,
319,320,340
Gene interaction 13
Gene isolation 25
Gene library 30, 35, 36
Genetic distance 335, 337
Genetic fingerprint 146, 333
385
Genetic instability 119, 146
Genetic stability 121, 122, 139, 146, 147
Genetic variability 51, 76, 141, 148
Genome 20, 85, 86, 89, 92, 93,95-97,99,
118,140-143,148,151,156,198,205,
206,209,329,246,280,281,298,
300, 301, 306, 329-333, 338-340, 371,
372
Genotype 8, 9, 12-14, 16, 17,20-22,55,
58-61, 63-66, 68, 71, 72, 74-77, 86,
88, 94, 95, 98, 105, 108, 111, 116,
118, 121, 123, 139, 142, 143, 146-149,
151,177,180,202,330,331,333-336,
338,341,356,360,370
Genotype x environment 22
Genotype-environment interaction 60, 76
Genotype/location interaction 64
Gentianaceae 168
Gentian (Gentiana) 158, 168, 169, 256,
257,260
G. acaulis 168
G. lutea 168
G. punctata 169
G. purpurea 168
G. triflore x G. scabra 168
Geranium 49, 50, 55, 58, 59, 66, 77, 159,
180,181,338
Geranyl diphosphate (GPP) 278, 296
Geranylgeranyl diphosphate (GGPP) 275,
277,278,282,296
Gerbera hybrida 159, 169, 255, 258
Germplasm conservation 146
Gibberellin (GA) 282
GIGANTEA 228
GISH 90, 93-96
Gladiolus 2, 5, 52, 62, 97, 159, 170-172,
180
Gladiolus grandijlorus 159, 170
Glucanase 200
Glucose oxidase 199, 200
Glutathione S-transferase 260
Glyceraldehyde-3-phosphate 27 4
Glyceraldehyde-3-phosphate
dehydrogenase 277
Glyphosate 173, 208
Grafted style 87, 89
Graft-hybrid 112
Grandfather's Clause 364
Green fluorescent protein (GFP) 222
386
GREEN PETAL (GP) 246
Growth habit 105, 120
Gypsophila 48, 52, 130, 155, 263
Haematococcus pluvialis 277,280,284
Half-bivalent 92, 93
Halotolerance 211
Haploid 3, 4, 91, 93, 149, 151
Haploidization 139, 149
Hardy-Weinberg equilibrium 21, 22
Harvested material 357, 358, 363, 364
Head 1, 278, 312
Hemerocallis 2, 158, 167
Heritable resistance 62
Heritability 22, 54, 57, 59, 63, 64
Heteroplasmon 20
Heterostyly 68, 71
Heterozygosity 69, 73, 74, 111, 121, 186
Heterozygous 6, 8-10, 12, 54, 55, 57, 68,
71,338
Hibiscus rosa-sinesis 172
Histogenic 113-116, 124
History 4, 47, 96, 98, 99, 119, 121, 337,
363,365
Homeotic gene 3, 239, 243
Homoeologous recombination 89, 91, 93,
95,96
Homogeneity 258, 298, 300, 367
Homologous chromosome 3, 4, 8, 20, 21,
91
Homozygous 6, 8-10, 13, 14, 16, 21, 22,
57,58,63,68, 71,149,244,246,338
Horizontal resistance 61
Human intervention 106, 368,
Hyacinth 54, 76, 110, 159, 172
Hybridization 31-34, 37, 39, 47, 51, 54,
62,63, 74, 77,85,86,88,95,96-99,
130,132,330,331,333,337,340
Hybrid line 5, 6
Hybrid vigor 5, 55, 71, 73, 74
Hydroxymethylglutaryl CoA 274
Hyperhydricity 141, 147
Hypersensitive response 203
Hypogynous 2
Imidazolinone 208
Immutans mutant 280
Imperfect flower 2
Inbred line 5, 6, 54, 68, 71, 74, 79
Inbreeding 5, 22, 51, 54-56, 71, 73, 75,
135
Inbreeding depression 5, 55, 63, 77, 337
Independent creation 362
Indeterminate meiotic restitution (IMR)
92-94
Induced mutation 107, 108, 112, 120,
147, 148
Industrial property 348, 350, 352, 355
Inflorescence 1-3,54, 58, 59, 67, 76, 78,
108,120,131,132,135,239-242,244,
246
Infiingement234,354,362,364,365
Initial cell113-115
Insecticidal crystal protein 205
In situ 34, 86, 146, 304
In situ hybridization 86, 90, 95-97, 303
Intellectual Property Rights 347, 348,
360
Interaction 13, 14, 19, 2, 34, 54-57, 60,
64, 76,197,200,203,221-223,226-
228,240,243,246,253,261-263,285,
315
International convention and agreements
348
Interspecific hybridization 51, 62, 63, 74,
85,86,88,97-99,132
Intraspecific hybridization 130
Introduction of new species 129, 135,
136, 155
lntrogression 85, 86, 94, 95, 98, 209, 337
Invention 348-356, 360, 361, 365, 367-
374
Inversion 21, 330, 331
In vitro 40, 42, 60, 66, 70, 72, 75, 79, 87,
89, 97, 99, 106, 108, 111, 113, 118,
119, 121-124, 139-143, 146-151, 155,
163-173,176,181,186,187,222,223,
225,226,228,259,284,322
In vitro selection 139, 143, 149, 151
Iris52,54,85,97,98, 131,159,172,
173,316
Iris germanica 159, 172
Irradiance 51, 66, 231
Irradiation 86, 108, 117, 118, 122, 123,
148,149,225,253
Irregular peloria 2
Isopentenyl diphosphate 274
 387

Isopentenyl diphosphate isomerase 281 Lobster 287

Isoprenoid 273-277, 280, 281, 285, 287 Longevity 60, 131, 143, 161, 186, 339
Isoprenoid biosynthesis 275-277, 287 Luciferase 157, 227
Lutein 273, 274,279,280, 283,284
Kalanchoe 49, 52, 55, 77, 118, 159, 173 Lycopene 56, 274, 278, 279, 281, 283-
K. blossfeldiana 173 286
K. diagremontiana 173 Lycopene ~-cyclase (LCYb) 278, 281,
K. laciniata 173 284
Ketocarotenoid astaxanthin 287 Lycopene £-cyclase (LCYe) 278, 281,
Ketocarotenoid ester 283 284
Keto group 280 LytB 277,281,287
4-Keto group 279
Ketolase 287 MAAP331
Kinase 211,221,222, 225, 226,228,231 MADS- box 231,241,242,245,247,248
Maize 16, 20, 96, 157, 174, 175, 181,
Lavatera thuringiaca 172-174 199,201,208,210,247,258,260-262,
LCYb 278, 280, 284 264,277
LEAFY (LFY) 240 Male sterility 19, 20, 57, 67, 71, 72
Leucospermum 131, 132 Map construction 334, 338
Light49,51,58,60,64-66, 106,132, Mapping 33, 86, 96,285,331,339
134,146,210,219-230,233,263,264, Marigold 5, 58, 59,274,277,280,282,
273,278,279,283,285 284
Light-sensing 219 Marker-assisted selection 334, 338
Light source 65,220 Marker segregation 338
Lilium 1, 4, 53, 68, 85-89, 91, 94-98, 110, Mass selection 63-67, 69, 70, 79, 112
155,175,247,248,336,339 Meiosis 3, 4, 14, 89, 91, 92, 95
L. dauricum 175 Meiotic division 4, 8, 93
L. japonicum 175 Meiotic nuclear restitution 92, 95, 99
L. longiflorum 87, 90, 93, 98, 159, 174, Meiotic sieve 4, 8, 93
175 Membrane leakiness 311, 312,320
L. xformolongi 175 Membrane lipid catabolism 316
Lily 5, 55, 86-88,97,98, 159, 174-176, Membrane proteins 314-316
180,247 Mentha piperita 287
Limonium sinuatum 134, 174 Mentor pollen 86
Linalool synthase 296, 298 Mericlinal chimera 115, 116
Linkage 14, 16, 21, 28, 58, 68, 96, 112, Meristem identity 232, 239-243, 248
125 Meristematic cell113, 116
Linkage group 296, 298 Metabolic engineering 283, 285, 306
Linum 159, 174 Mevalonate 27 4, 296
L. suffruticosum 174 Microarray 209,340
L. ustiatissimum 174 Microbiological process 349, 352-354,
Lipase 312, 314-317, 319, 320 366,368,369,371,372
Lipid bilayer 312, 314 Microinjection 156
Lipid phase separation 312, 314, 315, Microprojectile bombardment 156, 162-
324 164, 176-179, 183, 184, 187
Lipoxygenase 312, 316 Micropropagation 66, 70, 71, 75, 135,
Lisianthus (Eustoma grandiflorum) 168, 140, 141
176 Microsatellite 329, 330, 332, 333, 336
Living matter 355, 365-367
388
Microsomal membrane 313, 316
Mint secretory cell 277
Mitochondria 7, 19, 282, 317
Mitosis 3, 7, 93
Mobilizable plasmid 139
Molecular marker 16, 19, 86, 329, 334,
335, 337-340
Monocot167, 171,180,187,245,247
Monoecious species 2, 67, 73
Monohybrid 8, 9, 12
Monoterpene 296-298
Moondust 162
Moonshadow 162, 164
Moral Rights 247, 348
Morphological marker 68, 329
Movement protein 204, 205
Multiple allele 11, 22, 59
Multiple fruit 3
Mutagenesis 43, 44, 143, 147-151, 155
Mutagenic treatment 51, 108, 111, 113,
118, 120-125
Mutant9,20,35,36,43,58,59, 76, 77,
107-110, 112, 113, 116-118, 120-122,
124,125,147-149,204,208,209,220,
224,225,228-231,239-249,257,260,
262,276,280,281,285,287,318,
323,358,361,362
Mutant complementation 31, 35, 36
Mutant selection 149
Mutation 2, 3, 8, 18, 20, 21, 39, 42, 44,
51,55,58, 74, 76, 79,106-108,110,
112-117, 119-125, 134, 139, 141, 143,
147-149,151,208,220,222-224,227,
229,230,232,240-246,248,262,263,
280,284,285,329-331,333
Mutation breeding 105, 106, 108-117,
119-124, 139, 147, 148, 150
Mutuality principle 351, 352
MVA pathway 274, 276
MYB protein 261
MYC protein 261
Myrtaceae 187
NAFTA (North American Free Trade
Agreement) 349, 350, 352
Narcissus 1, 85, 94, 97-99, 248, 318
Native plant 129
Necrosis 167, 311
Nectary 283, 287
Neoxanthin 273,280,281,284
Neurosporene 278
New ornamental 129, 155, 156, 167, 263
Nicotiana 2, 53, 68, 71, 108, 187, 200,
248,259
No dominance 8, 9, 19
Non-climacteric fruit 318
Non-stigmatic pollination 155
Northern hybridization 32
Novartis II Decision 367, 369, 370
Novelty 50, 109, 110, 354, 357, 360, 363
NPH1 224-228
NSY 280,281
Nuclear translocation 222
Oligonucleotide 39, 40, 42, 208, 331
Open-pollination cultivar 53, 73
Orchid 1, 2, 5, 48, 53, 97, 99, 159, 178,
179,183,247,256,302
Organ identity 240, 242-245, 247, 248
Organogenesis 141, 143, 151, 168, 176,
181
Ornamental 2, 5, 7, 44, 47-51, 53, 54, 63,
71, 85, 86, 96, 105-112, 115-122, 125,
129, 1130, 141, 155-157, 174, 180,
183,187,188,197,199,203,205,
206,232,239,248,253,256,257,
264,265,306,307,329,334,335,
338-340
Ornithogalum 97, 131, 159, 179, 180
0. thyrisodies x 0. dubium 179
Oscillator 226-228
Osmophore 302
Osmoprotectant 209
Osmotin 199,201
Osteospermum 159, 180,336
0. ecklonis 180
Outbreeding 21, 53
Ovary 1-3, 5, 87-89,206, 318
Ovary culture 70, 88, 151
Ovule 1, 3-5, 69, 85, 88, 89, 97, 121, 149,
151,240,242,244
Oxygen function 280, 285, 286
Panicle 1

PAP204
Paris Convention for the Protection of
Industrial Property 348, 350-352, 355
Partial digestion 33
Patentability 349, 351-355, 360, 365-372,
374
Patent Rights 350, 352, 361
Pathogenesis-related 45, 186, 199
Pathotype 61, 62
PCR 40-42, 167, 176, 184,245, 329,
331-333, 340
PDS 163, 171, 176, 178, 179, 184,278,
280, 281, 283-285
PDS/ZDS inhibitor J852 284
Pelargonium 49, 53, 57, 61, 71, 74,76-
79,115,159,180-182,187,262- 265,
335,336
P. fragrans 181
P. graveolensi 181
P. hortorum 51, 59, 62, 76-78, 182
P. odoratissimus 181
P. quercifoliai 181
Pelargonium x domesticum 181
Pelargonium x hortorum 181
Pepper202,274,280,282,284-2 86
Pepper chromoplast 282
Perfect flower 2
Perianth 1, 5, 247, 248
Periclinal chimera 109, 115-118, 124
Perigynous 2
Peroxisome 299,317
Petal1, 2, 3, 69, 70, 114, 115, 162, 164,
180,181,186,239,224-248,255 ,257,
258,261,265,274,296,300-306 ,
311-322,361
Petal inrolling 311, 312, 315,318-320,
323
Petal senescence 311,318,320
Petunia 2, 5, 6, 49, 53, 54, 57, 58, 60, 65-
68,71,72,159,162,182,198,2 00,
203,239,242,245-247,256-260 ,262-
265,317,323,336,338,339
Petunia hybrida 159, 182, 239, 243, 257,
259-261
Phalaenopsis 53, 99, 179
Phenylpropanoid 296, 298-300
Phenylpropanoid pathway 253, 261
Phosphatase 34,211,312,314
Phosphatidic acid 312, 314
389
Phosphinothricin 160, 161, 167, 170,
180,208
Phospholipase 312-314
Phospholipid 312-316, 320
Photomorphogenesis 219, 233
Photoperiod 59, 66, 133, 220, 228, 230,
232,233
Photoperiodism 220, 229
Photoprotective carotenoid 281
Photoreceptor 220, 223-227, 230
PHOTOTROPIN 225
Phototropism 220, 224, 225
PHYA 165, 181,220-223,227,230,233
PHYB 165, 221-223, 226,227, 230
Phylogenetic tree 337
Phytoalexin 149, 199, 201, 300
Phytochrome 58, 220-230
Phytoene 56,278, 279, 281-284,286
Phytofluene 278
Phytol 274, 276
Pigment 13, 56,220,221,229,253,257,
259-261,263,273,274,277,278 ,280,
282-288, 298, 311
Pigmentation 44, 57, 120, 148, 253,261-
263,265,273,274,286,287
Pistil 1, 68, 87, 186, 303, 318
PISTILLATA 240
Plant Breeders' Rights 50, 349, 351, 356,
370
Plant form 58, 74, 114, 155, 163, 167,
176,180,182,183,219
Plant genetic system (PGS) 267
Plant resistance 197, 199,202,205,208,
212
Plant variety 349, 356, 357, 359, 360,
362-367, 369-373
Plant Variety Rights 349, 359
Plasmid 26, 29, 30,35-39, 157, 161-164,
166,168,169,176-178,180,367
Plasmid cloning vector 26, 29, 39, 37-39
Plastid lipid-associated protein 285
Plastid targeting 276
Plastoglobule 285
Plastoglobule-associated protein 281, 285
Pleiotropic effect 112, 125, 148, 209
Poinsettia (Euphorbia pulcherrima) 49,
160,182,336,338
Pollen 1, 3, 5, 8-11,19,21,63-65,67-71,
74, 79,86,87, 89,97, 121,122,149,
390
151,174,175,182,208,209,260,
295,303
Pollen tube 3, 86, 87, 89
Pollination 3-5, 8-11, 53, 54, 63-69, 71-
74,79,86-89,98,116,155,182,220,
229,295,311, 318
Pollination control 71
Pollination technique 87
Polygene 18, 58
Polyploid 4, 20, 54, 55, 57, 73, 74, 85,
89, 91, 94,97-99, 111, 112, 149-151,
166,334,339
Polyploidization 47, 55, 91, 94, 95, 97-
99, 139, 151
Polyploidy 52, 54, 55, 73, 85, 86, 98,
120, 122, 151
Post-fertilization 85, 88, 89, 99
Post-translational modification 43
Pot-plant species 47, 49
PPA (Plant Patent Act) 360-363, 365
Priority Rights 359
Process claim 371
Product claim 368, 370
Production value 48
Progeny testing 13, 63-65
Prolamellar body 282, 283
Prolycopene 281
Promoter 37, 42, 45, 157, 170, 171, 174,
175,178-180,186,187,198,203,209,
223,226,241,243,244,262,282-284,
287,306,321,323
Propagation 50, 52, 53, 59, 62, 63, 66,
73-75,77-79,97,106,108,111,112,
114, 117, 118, 121, 124, 125, 129,
130,135,140,141,143,334,357-359,
363,365,372,373
Propagation method 106, 121, 130
Protein-protein interaction domain (PAS)
223
Protoplast fusion 135
PR protein 199
PSY 278,281-285
PTOX 280,281,284
Public and morality 367, 374
Public domain 348
PVPA (Plant Variety Protection Act)
360, 363-366
Pyridoxal274
QTL 19, 285, 338, 339
Quantitative inheritance 17, 18
Quinone 276,284
Raceme 1
Radiation damage 123, 125
Radiation dose 123
Radiation treatment 122, 124
Random mating 63
RAPD86,96,97,329,331-336,338,339
Reactive oxygen species 199, 209,
Recalcitrant 139, 161, 183
Recessive 8-10, 12-14, 16, 19-22,51,57-
59,63,68,69, 71,120,149,248,339
Reciprocal translocation 21
Recombinant DNA 25, 26, 28, 38, 44,
368,370
Recombinant protein 42, 225
Recombined chromosome 3
Regional Convention and Agreements
348
Regular peloria 2
Regulatory gene 177,261-263,265,321
Remedy 365
Remnant seed 64
Repulsion 16, 17
Research exemption 359,
Restriction enzyme 26, 28, 32, 190, 330
Restriction map 28
Reverse transcriptase 35-37
RFLP 16, 86, 96, 329-334,336,338, 339
Rhododendron 5, 49, 53, 61, 110, 160,
183,336,338,339
Ribonuclease 198, 200
Ribosomeinactivating protein
(RIP)199
Rice 16, 44, 134, 136, 165, 170, 175,
180,187,197-202,204,206-208,210,
211,243,247,283,334,337
~-Ring 57,278,280,283,284
~-Ring hydroxylase 281
E-Ring hydroxylase 280, 281
Rosa hybrida 184
Rose 5, 48, 56, 60, 62, 64, 74, 76, 96,
105, 107, 110, 132, 136, 155, 160,

166,167,183-187,263,264,306,312,
314,334,335,337,339
S-adenosyl-L-methionine:benzoic acid
carboxyl methyltransferase 298, 301
S-adenosyl-L-methionine:salicylic acid
carboxyl methyltransferase 298, 301
S-adenosyl-L- methionine:(iso )eugenol
0-methyltransferase 300
Salt stress tolerance 209
Salt tolerance 211
Sandersonia 131, 155
SAR 199, 201-203
Satellite DNA 332
SCAR 333, 334
Scenedesmus obliquus 281
Scent gland 302
Scope of protection 351, 354,358,359
SDR 91-94
Secondary carotenoid 287
Secondary metabolite 253, 296, 298
Sectorial chimera 115
Seedling 5, 60, 65, 66, 69,74-77, 171,
172, 176
Seed-propagated crop 62, 79
Selective value 21
Self-incompatibility 6, 11, 44, 67, 68, 72,
86
Self-pollination 5, 7-9, 11, 69, 72, 73,
116
Senescence 44, 131, 157, 161, 163, 229,
263, 311-324
Senescence-induced gene 319, 321, 322
Senescence-induced leakiness 311
SEPALLATA (SEP) 245
Sexually reproduced plant 5, 6, 360, 363
Sexual polyploidization 91, 94, 95, 97-99
Sexual reproduction 1, 5, 335, 363
Shade avoidance 220,221,228,229
Shoot apex 113, 115, 242
Silver thiosulfate 131,318,322,323
Similarity 221,225,226,231,242,261,
262,300,301,302,337,362
Simple fruit 3
Sinapis alba 283
Slow growth 140, 146, 147
Solanaceae 166, 259
Somaclonal variation 119, 121, 139, 142-
144, 148, 151, 174, 184
391
Somatic embryogenesis 143, 144, 166,
174, 184, 185
Somatic mutant 77
Southern hybridization 32
Spike 1, 65
Spontaneous mutation 106, 107, 120,
121, 139, 143, 148
SSCP 330, 333, 334
Stability 42, 43, 51, 119, 121, 122, 125,
139-141,143,146-148,151,209,210,
287,355,357,360,364,367,370
Stable 32, 51, 63, 76, 116-118, 121, 122,
124, 141, 156, 157, 164, 165, 166,
168, 170-172, 177, 180, 181, 184, 186,
221,264,286,305,356,358,363
Stalk 1,
Stamen 1, 68, 244, 246, 247
State of the art 353
Sterol315, 316
Stigma 1, 3, 5, 87, 89, 182, 303
Stress resistance 60, 209
Structural flavonoid gene 261
Structural gene 25, 30, 35, 43, 261, 265,
286
Style 1-4, 68, 86-89, 318
Sui generis protection 349, 352, 366
Sulfometuron 208
Super crop 369
Synteny 340
Synthetic seed 144
Tangerine tomato mutant 281
Target sequence 27, 31, 32, 40, 331
TBA (EPO's Technical Board of
Appeals) 367-369, 371
Term of protection 351, 354, 362,364
TERMINAL FLOWER I (TFLl) 240-
242
Terminal oxidase 280, 281
Terpenoid 296
Tetraploid 20, 54, 55, 69, 70, 73, 74, 77,
78,94,95,98,99
Tetrasomic inheritance 20
Thiamine 27 4
Thidiazuron 144
Tobacco 59, 108, 143, 186, 197-208, 211,
243,247,262,265,283,284,287,350
a.-Tocopherol 277
392
Tomato 5, 16, 118, 161, 165, 174, 180,
197,198,200,202,204,207,208,
211,245,247,262,274,276-278,
280-287, 321, 322
Torenia 160, 186, 257-258, 260, 264
T. foumieri 160, 186
Transcription factor 209,211, 223,231,
240,242-244,261-263
Transformation 25, 26, 38, 44, 45, 105,
111, 118, 139, 140,143, 156-158,
161-187,208,209,239,244,247,306,
358
Transgenesis 44
Transgenic plant44, 139, 156, 157, 161-
177,179-187,201,204,206,207,209,
223,225,227,230,231,243,265,
282,306,323,366,369,370
Triacylglycerol 286, 317
Tripartite mating 39
Triploid block 44
Triploidy 54
TRIPS Agreement (trade-related aspects
of IPR) 349-352, 374
Trisomic 69
True-to-type 363
Trypsin inhibitor 206-208
Tubule 285
Tulipa 53, 85, 88, 89, 97, 99, 109, 260
T. gesneriana 44, 186, 247
Type II restriction endonuclease 27-29
UDPG-flavonoid-glucosyltransferase
(UFGT) 254
Umbel1, 78
Unfair competition 350, 357
Uniform 5, 6, 22, 51, 63, 73, 110, 114,
122,134,135,157,170,186,356,
357,363
Uniformity 51, 55, 63, 70, 73, 135,357,
360,364
Univalen 90-93
Unreduced 55, 91
UPA (Utility Patent Act 1952) 360, 361,
365,366
UPOV Convention 50, 125, 146, 348,
352,355-360,363,364,370,374
Variegation 20, 105, 114
Variety identification 335
Vase life 60, 131, 135, 155, 162, 167,
180,316,322,323
Vegetatively propagated crop 52, 74,
108, 111, 112, 115, 122, 123, 125, 146
Vegetative propagation 62, 97, 106, 111,
114,116,117,121,125,130,135,
140, 143
Vegetative reproduction 5, 13
Vernalization 133, 134,231,232,240,
241
Vertical resistance 61
Verticordia grandis 160, 187
Violaxanthin 273, 280, 281, 284
Virus transmission 112
Vitamin 274
Vitrification 141, 147
Volatile 273, 295-303, 305-307
Waiver 364
Wax flower 130, 134
Western hybridization 34
Whorl 1,239,240,244,246-248
WIPO (World Intellectual Property
Organization) Convention 348, 356
Written description 355, 357, 361
WTO (World Trade Organization)
Agreement 349, 351
Xanthamonas campestris 157, 182
Xanthophyll 283, 284, 286
Yield 10, 18, 22, 38, 43, 44, 51, 55, 63-
66, 74, 76, 78, 106, 130, 133, 135,
141,148,149,155,173,219,233,
274,276,278,280,283,285,307
Zantedeschia 89, 97, 155
ZDS 278, 280, 281, 283, 284
Zeaxanthin 273, 280, 284
Zeaxanthin epoxidase 281
Zygomorphic flower 2, 58, 107
Zygote3, 8, 19, 21, 88