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Advances in
Horticulture
Biotechnology
R E G E N E R A T I O N S Y S T E M S
Volume I: FRUIT CROPS, PLANTATION CROPS AND SPICES
H.P. Singh
V.A. Parthasarathy
K. Nirmal Babu
Westville Publishing House

New Delhi
H. P. Singh, V. A. Parthasarathy and K. Nirmal Babu (2011) Advances in Horticulture Biotechnology
— Regeneration Systems — Fruit Crops, Plantation Crops and Spices (Volume I), pp 484, Westville Publishing
House, New Delhi
© Publishers
First Edition 2011
ISBN 978-81-85873-65-7
All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted, by any
means, electronic, mechanical, photocopying, recording, or otherwise, without written permission from the
publisher.
Published by Mrinal Goel, Westville Publishing House

47, B-5, Paschim Vihar, New Delhi – 110 063, India
Tel: 011-25284742, Fax: 011-25267469
Email: westville_2002@yahoo.co.in
Printed at Salasar Imaging Systems, New Delhi

Foreword
Biotechnology
Indian agriculture stands out as
made a the
rapidfrontier
stride,area of research
converting theforcountry
crop improvement.
from food scarce The first
to
horticultural
sufficiency. But transgenic
challenges developed
for theinIndian
tomato in USA inin1980
agriculture 21st (Flavr
century Savr) has been
are much followed
greater than
by a large
before. Thenumber
growing of population
transgenicshas developed
to be fedinand potato, squash,
surplus has tocorn
be and recently
produced within declining
brinjal in
India. Recent advances in the fields of molecular genetics and recombinant
land, water and threat of climate change. The horticulture, which includes fruits, vegetables, DNA technology
has opened
spices, up new
flowers, andopportunities
medicinal and in agriculture,
aromatic plants,medicine, industrybeyond
has proved and environment protection.
doubt its potentiality
for gainful
The ability diversification.
to move genes across Initiatives
sexualtaken by Government
barriers and other stakeholders
has led to heightened interest in thehave impacted
conservation
the development
and sustainable and in terms of increased
equitable production,Since
use of biodiversity. productivity and availability
biodiversity of horticultural
is the feedstock for plant,
crops. The
animal emerging breeding
and microbial trend worldwide
enterprises,anditsalso in the country
conservation becomesis indicative of a paradigm
more important shift
for effective
in dietary
use. needs of the
The publication of people
genomewith maps rise
of in the income,
human which demand
and Arabidopsis for more
has opened up newhorticultural
vistas in
produce.
the area of Since the growing
genomic researchofand horticultural crops is
its application. rewarding
Today we have to the farmers
genome in terms
maps of returns
available for a
per unit
large area, the
number of sector
plants isand expected
animalsto including
contributeperennial
significantly forlike
crops fooddate
and palm,
nutritional security,
oil palm and
employment opportunity and poverty alleviation.
peach. Of course, we have to go miles before we develop maps for many of the tropical fruits
and vegetables.
The Indian Council of Agricultural Research is the premier agency which pioneered
systematic
There is research on agricultural
little doubt crops in the country.
that the biotechnology Horticulture
has opened researchopportunities
up uncommon in India received for
rd
very little attention till the 3 five year plan. To-day, the horticultural
enhancing the productivity, profitability, sustainability and stability of major cropping systems research in the country is
and has also created scope for developing crop varieties tolerant/resistant to biotic and abiotic8
being carried out at ten ICAR institutes (with 24 regional stations) and 5 Directorates and
Nationalthrough
stresses Research ancentres (on major
appropriate blendcrops).
of GMArea specific,
Mendelian andmulti-disciplinary
molecular breeding research is also
techniques.
being conducted fewer than 13 -All India Co-ordinated Research
These advances have also led to the possibility of undertaking anticipatory breeding to meet Projects at 215 centres located
at various research Institutes, and State Agricultural Universities. In addition, several net work
potential changes in temperature, precipitation and sea level as a result of global warming.
projects are now in operation. Research on horticulture is also being undertaken at several
Marker Assisted Selection has now become an integral part of breeding programmes. Many
multi-crop, multi-disciplinary Institutes. Departments of Horticulture in 34 Agricultural
novel and powerful markers such as SSRs and SNPs are available to the breeders for precise
Universities, three deemed to be Universities and one full fledged University of Horticulture
and rapid transfer of desired traits. The development of QTLs in perennial crops has become
and Forestry are also engaged in horticultural research. As a result, the country now has a
easier with the development of association mapping procedures. It is time that our scientists
sound research infrastructure in horticulture to meet the growing needs and expectations of the
take advantage of
fast developing these toolsindustry.
horticulture and apply them for
Among theimprovement
various areasofofthe crops native
research being to India.out
carried
world wide,the
While Biotechnology
benefits are clear,standsthere out asare
theconcerns
frontier area
aboutofthe research. India
short and is endowed
long term impact withofa
strong manpower in the area of Biotechnology. With the first
GMOs on the environment, biodiversity and human and animal health. There are also equityhorticultural transgenic developed
in tomato
and in USA
ownership in 1980
issues in (Flavr
relation Savr), the research has led
to biotechnological to the development
processes and products. of aThus,
large there
number is
of transgenics in potato, squash, corn and recently in brinjal in
need for transparent and truthful risk-benefit analysis in relation to GMOs, on a case-by-caseIndia. Resent advances in the
fields of
basis. molecular genetics
Biotechnology offersand recombinant
opportunities forDNA technology
converting has openedwealth
our biological up newinto opportunities
economic
wealth and new employment opportunities on an environmentally and socially move
in agriculture, medicine, and industry and environment protection. The ability to genes
sustainable
across
basis. sexual barriers has led to heightened interest in the conservation and sustainable and
(iv)
equitable usevast
With the of biodiversity, since biodiversity
available literature is the
scattered over feedstock
various for plant,
sources, animalfor
it is difficult andresearchers
microbial
breeding enterprises. Transgenic research should not be undertaken in crops/commodities
and students to remain abreast of all the biotechnology related developments in various where
our international trade may be affected Cultivation of GM crops should
horticultural crops. I compliment the efforts of Dr. H.P. Singh, Deputy Director General not be permitted in
designated “Agro-Biodiversity
(Horticulture), ICAR, New Delhi Sanctuaries”. Besides,
for compiling andwe should
editing theensure
seriesstrict safety
of the procedures
books entitled
so that the food
“Advances becomes safe Biotechnology”.
in Horticulture for consumption with no public outcry.
Commendable efforts have been made in this
treatise to areas
In the collate all the work
of genomics anddone on horticultural
bioinformatics, crops in of
the publication five thematic
genome mapareas, namely,
of human and
regeneration
Arabidopsis has systems,
opened molecular
up vistasmarkers, geneofcloning,
in the area genomictransgenics
research and andtoday
diagnostics.
we have The series
genome
will
mapsbeavailable
highly useful to allnumber
for a large plant biotechnologists in general
of plants and animals. and horticulturists
Genome maps of perennial in particular.
crops like
Date palm, oil palm and peach are already available. Of course, we have to go miles before we
develop maps for many of the tropical fruits and vegetables. Very good facilities for genomics
are available in India.
Marker Assisted Selection has become a part of many of the breeding programmes. Many
novel and powerful markers such as SSRs and SNPs are available to the breeders. The R.S.development
Paroda
of QTLs in perennial crops has become easier with the development of association mapping
Chairman
procedures. It is time that many of the institutes should develop mapping population for this
Trust for Advancement of Agricultural Science
purpose. It would not be long before we have the linkage and even genome map for most of the
crops native to India. In addition it also helps in generating employment New Delhi, Indiaand
opportunities
enhancing standards for global competitiveness. There is little doubt that the biotechnology has
opened up uncommon opportunities for enhancing the productivity, profitability, sustainability
and stability of major cropping systems. It has also created scope for developing crop varieties
tolerant/resistant to biotic and abiotic stresses through an appropriate blend of Mendelian and
molecular breeding techniques. It has led to the possibility of undertaking anticipatory breeding
to meet potential changes in temperature, precipitation and sea level as a result of global warming.
While the benefits are clear, there are concerns about the short and long term impact of GMOs on
the environment, biodiversity and human and animal health. There are also equity and ownership
issues in relation to biotechnological processes and products. Thus, there is need for transparent
and truthful risk-benefit analysis in relation to GMOs, on a case-by-case basis. Biotechnology
offers opportunities for converting our biological wealth into economic wealth and new
employment opportunities on an environmentally and socially sustainable basis.
With the vast available literature scattered over various sources, it is difficult for researchers
and students to know the status of science of biotechnology of various horticultural crops. The
attempt by Dr.H.P.Singh, Deputy Director General (Horticulture), ICAR, New Delhi is highly
a visionary approach. I am impressed by the volume of talent we have in India on biotechnology.
He has tried to collate all the work done on these crops in five thematic areas, namely,
regeneration systems, molecular markers, gene cloning, transgenics and diagnostics. The series
of volumes on these thematic areas will be highly useful to all plant biotechnologists in general
and horticulturists in particular.
Dr R.S. Paroda
Preface
Systematic research on fruits, vegetables and ornamental crops in India began in 1954
with the initiation of independent institutions and programmes. The research agenda is designed
relevant to national plans and priorities for the horticulture development. Today, 10 ICAR
institutes with 27 regional stations, 5 Directorates, 7 national research centres, 15 all India
coordinated research projects (AICRPS) with 223 research stations, 1 full fledged university
of horticulture, 25 state agricultural universities and 7 multi-disciplinary institutes of the ICAR
are engaged in horticulture research. In addition, a few R&D establishments of crop/commodity
boards and private sectors are providing research support to Indian horticulture. Besides, ICAR
operates over 330 ad-hoc research projects. Presently there are 29 revolving fund schemes and
many network projects both within ICAR and outside ICAR. It operates a massive seed project
encompassing all ICAR institutes and SAUs to meet the growing demand of quality seed
materials. Research system in horticulture is now geared to provide necessary technological
support to the expanding horticulture industry. The strength of horticulture biotechnology in
terms of man power and infrastructure is great in India. We have to channelize this for effective
improvement of horticulture crops and have to keep in mind, the tools of biotechnology can be
used across various disciplines and on various crops.
Production of quality planting materials is fast becoming an important input in disease
management programmes especially in production of disease/virus free plants. Many accredited
laboratories are being established to test the genetic fidelity and virus indexing of
micropropagated plants. Tissue culture raised plants also become an important source for
colonization and delivery of bio-control agents. In addition, efficient plant regeneration systems
are fast becoming important for in vitro manipulations and transgenic pathways for crop
improvement.
In the present series, volume 1 deals with available information on the micropropagation
and plant regeneration technologies developed for fruit crops, plantation crops and spices
while volume 2 deals with vegetables, ornamentals and tuber crops. These are comprehensively
covered with respect to individual crops by experienced scientists. Information on various
explants sources and media combinations are also presented in a concise form.
We are thankful to the contributors for their sincere effort to prepare comprehensive review
on the plant regeneration systems of different crops published in this volume. We gratefully
(vi)
acknowledge their contributions. We are also grateful to many others for their participation
and help in this publication. We hope this volume would be of use to students and researchers
in the field of biotechnology of fruit crops, vegetable crops, ornamentals, tuber crops, plantation
crops and spices.
New Delhi H. P. Singh

V. A. Parthasarathy
K. Nirmal Babu
Contents
Foreword ........................................................................................................................................ iii

Preface ............................................................................................................................................ v
Contributors ................................................................................................................................... xv
Acronyms ..................................................................................................................................... xvii
1. Biotechnology in Horticulture – Regeneration Systems
— Status, Progress and Future ................................................................................. 1
Introduction ................................................................................................................................ 1
Micropropagation and Commercialisation ................................................................................ 1
Production of Virus Free Plants ................................................................................................. 5
Plant Regeneration and Somatic Embryogenesis ....................................................................... 5
Somaclonal Variation and In vitro Selection ............................................................................. 6
In vitro Flowering, In vitro Pollination and Embryo Rescue ..................................................... 7
Development of Haploids .......................................................................................................... 7
Synthetic Seeds .......................................................................................................................... 7
Protoplast Culture and Development of Somatic Hybrids ........................................................ 7
Conservation of Germplasm through In vitro Conservation and Cryopreservation .................. 8
Long term Storage of Pollen .................................................................................................... 11
Production of Secondary Metabolites ...................................................................................... 12
Future Thrusts .......................................................................................................................... 12
FRUIT CROPS
2. Banana ........................................................................................................................... 15
Introduction .............................................................................................................................. 15
Micropropagation ..................................................................................................................... 16
Hardening ................................................................................................................................. 20
Production of Virus Free Planting Materials ........................................................................... 21
Safe Germplasm Movement — Technical Guidlines ............................................................... 26
Somaclonal Variation ............................................................................................................... 28
Macropropagation .................................................................................................................... 33
Somatic Embryogenesis ........................................................................................................... 34
Organogenesis .......................................................................................................................... 43
Embryo Culture and Embryo Rescue ....................................................................................... 44
(viii)
Anther Culture .......................................................................................................................... 46

Protoplast Culture .................................................................................................................... 50
Conservation of Musa Germplasm .......................................................................................... 52
Germplasm Exchange and Linkages ........................................................................................ 56
Future Perspectives .................................................................................................................. 56
3. Mango ............................................................................................................................. 73
Introduction .............................................................................................................................. 73
In vitro Propagation ................................................................................................................. 74
Somatic Embryogenesis ........................................................................................................... 75
Organogenesis .......................................................................................................................... 83
Embryo Rescue ........................................................................................................................ 83
Shoot Bud Culture .................................................................................................................... 84
Hardening ................................................................................................................................. 85
In vitro Germplasm Conservation ............................................................................................ 86
Future Perspectives .................................................................................................................. 86
4. Papaya ............................................................................................................................ 91
Introduction .............................................................................................................................. 91
Micropropagation ..................................................................................................................... 91
Plant Regeneration ................................................................................................................... 94
Protoplast Culture .................................................................................................................... 96
Anther Culture .......................................................................................................................... 97
Embryo Rescue ........................................................................................................................ 98
In vitro Conservation ............................................................................................................... 99
Future Perspectives ................................................................................................................ 100
5. Guava ...........................................................................................................................103
Introduction ............................................................................................................................ 103
Micropropagation ................................................................................................................... 104
Callus Culture ........................................................................................................................ 108
Somatic Embryogenesis ......................................................................................................... 111
Embryo Culture ...................................................................................................................... 113
Anther Culture ........................................................................................................................ 113
In vitro Conservation ............................................................................................................. 114
6. Citrus ............................................................................................................................121
Introduction ............................................................................................................................ 121
Plant Regeneration ................................................................................................................. 125
Rooting and Acclimatization ................................................................................................. 130
Protoplast Culture .................................................................................................................. 138
Production of Somatic Hybrids .............................................................................................. 140
Rootstock Improvement ......................................................................................................... 142
Scion Improvement ................................................................................................................ 145
Seedlessness ........................................................................................................................... 148
(ix)
In vitro Auto tetraploid production ........................................................................................ 148

Embryo Rescue ...................................................................................................................... 149
Haploid Production ................................................................................................................ 150
Rootstock Breeding ................................................................................................................ 152
Mutation Breeding and Mutant Selection .............................................................................. 156
Rootstock Breeding via Embryo Rescue ............................................................................... 157
Cryo Preservation ................................................................................................................... 159
7. Grape ............................................................................................................................173
Introduction ............................................................................................................................ 173
Pathogen Elimination and Production of Virus Free Planting Material ................................ 175
Hybrid Embryo Rescue .......................................................................................................... 177
Haploid and Dihaploid Production ........................................................................................ 178
8. Aonla ............................................................................................................................185
Introduction ............................................................................................................................ 185
9. Apple ............................................................................................................................191
Introduction ............................................................................................................................ 191
Organogenesis and Somatic Embryogenesis ......................................................................... 194
Embryo Rescue ...................................................................................................................... 197
10. Pear ...............................................................................................................................203
Introduction ............................................................................................................................ 203
Organogenesis ........................................................................................................................ 206
Synthetic Seeds ...................................................................................................................... 207
Haploid and Dihaploid Production ........................................................................................ 208
11. Peach ............................................................................................................................213
Introduction ............................................................................................................................ 213
Shoot Culture ......................................................................................................................... 214
Embryo Rescue ...................................................................................................................... 218
(x)
Somatic Embryogenesis and Organogenesis ......................................................................... 218

12. Plum .............................................................................................................................223
Introduction ............................................................................................................................ 223
Organogenesis and Regeneration ........................................................................................... 224
Embryo Rescue ...................................................................................................................... 227
13. Pomegranate ................................................................................................................233
Introduction ............................................................................................................................ 233
Organogenisis ......................................................................................................................... 234
Embryogenesis ....................................................................................................................... 237
Problems in Micropropagation .............................................................................................. 239
Anther Culture ........................................................................................................................ 240
In vitro Polyploidy ................................................................................................................. 240
Conservation .......................................................................................................................... 240
14. Annona .........................................................................................................................243
Introduction ............................................................................................................................ 243
Problems in Micropropagation .............................................................................................. 247
Organogenesis ........................................................................................................................ 248
Embryo Rescue ...................................................................................................................... 248
Haploids ................................................................................................................................. 248
Triploids ................................................................................................................................. 249
Mycorrhiza ............................................................................................................................. 249
15. Bael, Sapota and Wood Apple ...................................................................................253
Bael ...............................................................................................................................253
Organogenesis .......................................................................................................................... 25
Embryogenesis ....................................................................................................................... 257
Sapota ...........................................................................................................................257
Organogenesis ........................................................................................................................ 258
Embryogenesis ....................................................................................................................... 259
Wood Apple .................................................................................................................260
Organogenesis ........................................................................................................................ 260
Embryogenesis ....................................................................................................................... 262
16. Ber ................................................................................................................................265
Introduction ............................................................................................................................ 265
(xi)

Organogenesis ........................................................................................................................ 270
Embryogenesis ....................................................................................................................... 271
Embryo Culture and Embryo Rescue ..................................................................................... 272
Elimination of Viruses and other Microbes ........................................................................... 274
Polyploidy .............................................................................................................................. 275
Conservation .......................................................................................................................... 276
17. Datepalm ...................................................................................................................... 279
Introduction ............................................................................................................................ 279
Organogenesis ........................................................................................................................ 280
Embryogenesis ....................................................................................................................... 282
Problems Micropropagation .................................................................................................. 287
Production of Disease and Pest free Plantlets ....................................................................... 289
Germplasm ............................................................................................................................. 290
Embryo Culture ...................................................................................................................... 290
Haploids ................................................................................................................................. 290
PLANTATION CROPS
18. Coconut ........................................................................................................................ 299
Introduction ............................................................................................................................ 299
Production of Homozygous lines ........................................................................................... 302
In vitro Screening for Stress Tolerance .................................................................................. 302
Embryo Culture for Safe Movement of Germplasm .............................................................. 302
Cryopreservation .................................................................................................................... 306
19. Arecanut....................................................................................................................... 313
Introduction ............................................................................................................................ 313
Embryo Rescue ...................................................................................................................... 316
20. Oil Palm ....................................................................................................................... 319
Introduction ............................................................................................................................ 319
Oil Palm Tissue Culture ......................................................................................................... 320
Constraints in Micropopagation ............................................................................................. 323
Protoplast and Anther Culture ................................................................................................ 328
In vitro Conservation and Cryopreservation .......................................................................... 328
Genetic Transformation .......................................................................................................... 329
Somaclonal Variation ............................................................................................................. 331
21. Cashew ......................................................................................................................... 343
Introduction ............................................................................................................................ 343
(xii)
Micrografting ......................................................................................................................... 351

Somatic Embryogenesis and Organogenesis ......................................................................... 351
Embryo Rescue ...................................................................................................................... 352
22. Cocoa ............................................................................................................................357
Introduction ............................................................................................................................ 357
In vitro Multiplication ............................................................................................................ 358
Embryo Rescue ...................................................................................................................... 363
SPICES
23. Black Pepper ................................................................................................................369
Introduction ............................................................................................................................ 369
Plant Regeneration through Shoot Organogenesis ................................................................ 376
Plant Regeneration through Somatic Embryogenesis ............................................................ 382
Cell Suspension Cultures ....................................................................................................... 387
Anther Culture ........................................................................................................................ 389
24. Cardamom ...................................................................................................................395
Introduction ............................................................................................................................ 395
Plant Regeneration from Callus Cultures .............................................................................. 397
Characterization of Somaclones ............................................................................................ 398
Anther Culture ........................................................................................................................ 399
Synthetic Seeds ...................................................................................................................... 400
Conservation of Genetic Resources ....................................................................................... 400
25. Turmeric ......................................................................................................................405
Introduction ............................................................................................................................ 405
Micro Rhizomes ..................................................................................................................... 409
Plant Regeneration- Organogenesis and Somatic Embryogenesis ........................................ 411
Cell Suspension Culture ......................................................................................................... 414
Field Evaluation ..................................................................................................................... 415
Germplasm Exchange ............................................................................................................ 416
(xiii)

26. Ginger ...........................................................................................................................421
Introduction ............................................................................................................................ 421
Field evaluation ...................................................................................................................... 424
Microrhizomes ....................................................................................................................... 425
Plant Regeneration from Callus Cultures .............................................................................. 427
Somaclonal Variation ............................................................................................................. 429
Induction of Systemic Resistance .......................................................................................... 430
In vitro Polyploidy ................................................................................................................. 431
Anther Culture ........................................................................................................................ 431
Inflorescence Culture and In vitro Development of Fruit ...................................................... 432
Synthetic Seeds ...................................................................................................................... 435
Germplasm Conservation ....................................................................................................... 435
Production of Secondary Metabolites .................................................................................... 437
27. Tree Spices ...................................................................................................................443
Introduction ............................................................................................................................ 443
Plant Regeneration in Tree Spices ......................................................................................... 446
Synthetic Seeds ...................................................................................................................... 447
Production of Secondary Metabolites .................................................................................... 447
28. Seed Spices ...................................................................................................................451
Introduction ............................................................................................................................ 451
Application of Tissue Culture ................................................................................................ 451
Germplasm Enrichment through Tissue Culture .................................................................... 452
Somatic Embryogenesis and Synthetic Seed Technology: .................................................... 452
In vitro Conservation of Germplasm ..................................................................................... 453
In vitro Flowering ................................................................................................................... 453
In vitro Androgenesis and Double Haploids .......................................................................... 453
In vitro Mutagenesis .............................................................................................................. 457
In vitro Screening for Biotic and Abiotic Stresses ................................................................. 457
Production of Secondary Metabolites and Biotransformation .............................................. 458
Index .............................................................................................................................463
Contributors
Anitha Karun Maneesh Mishra

Central Plantation Crops Research Institute Central Institute for Sub-Tropical Horticulture
Kasaragod – 671 124 Rehmankhera P.O.
Kerala Lucknow – 227107
Uttar Pradesh
Anju Bajpai
Central Institute for Sub-Tropical Horticulture Malhotra S. K.
Rehmankhera P.O. Lucknow – 227 107 Indian Council of Agricultural Research
Uttar Pradesh Krishi Anusandhan Bhawan – II, Pusa Campus
New Delhi – 110 012
Hare Krishna
Central Institute of Temperate Horticulture Minoo D.
Mukteshwar – 263 138 Indian Institute of Spices Research
Uttarakhand Calicut – 673 012
Kerala
Jayanthi M.
Director of Oil Palm Research
Mir J. I.
Pedavegi – 534 450
Central Institute of Temperate Horticulture
Andhra Pradesh
Rengreth – 190 007
Srinagar
Leela Sahijram
Indian Institute of Horticultural Research Jammu & Kashmir
Hessarghatta Lake, Bangalore – 560 089
Karnataka More T. A.
Central Institute for Arid Horticulture
Madhu Kamle Beechwal
Central Institute for Sub-Tropical Horticulture Bikaner – 334006
Rehmankhera, P.O., Lucknow – 227107 Rajasthan
Uttar Pradesh
Nazeer Ahmed
Mandal P.K. Central Institute of Temperate Horticulture
Director of Oil Palm Research Rengreth – 190 007
Pedavegi – 534450 Srinagar
Andhra Pradesh Jammu & Kashmir
(xvi)
Neelam Shukla Senthil Kumar R.

Central Institute for Sub-Tropical Horticulture Indian Institute of Spices Research
Rehmankhera P.O. CRC Appangala
Lucknow – 227 107 Karnataka
Uttar Pradesh Kerala
Nirmal Babu K. Sheeja T. E.

Indian Institute of Spices Research Indian Institute of Spices Research
Calicut – 673 012 Calicut – 673 012
Kerala Kerala
Parthasarathy V. A. Singh Dhurendra

Indian Institute of Spices Research Central Institute for Arid Horticulture
Calicut – 673 012 Beechwal, Bikaner – 334 006
Kerala Rajasthan
Rajesh Pati Singh, H.P.

Central Institute for Sub-Tropical Horticulture Indian Council of Agricultural Research
Rehmankhera P.O., Lucknow – 227107 Krishi Anusandhan Bhawan – II, Pusa Campus
Uttar Pradesh New Delhi – 110012
Ramakrishnan Nair R. Sivalingam P.N.

Indian Institute of Spices Research Central Institute for Arid Horticulture
Calicut – 673 012 Beechwal, Bikaner – 334 006
Kerala Rajasthan
Ramesh Chandra Thimmappaiah

Central Institute for Sub-Tropical Horticulture Directorate of Cashew Research
Rehmankhera, P.O., Lucknow – 227 107 Puttur – 574 202, Dakshin Kannada
Uttar Pradesh Karnataka
Reshi T. A. Uma S.
Central Institute of Temperate Horticulture National Research Centre for Banana
Rengreth, Srinagar – 190 007 Tiruchirapalli – 620 102
Jammu & Kashmir Tamil Nadu
Saraswathi M. S. Verma M. K.
National Research Centre for Banana Central Institute of Temperate Horticulture
Tiruchirapalli – 620102 Rengreth – 190 007, Srinagar
Tamil Nadu Jammu & Kashmir
Selvarajan R. VijayaKumari N.
National Research Centre for Banana National Research Center for Citrus
Tiruchirapalli – 620 102 Shankar Nagar P.O, Nagpur – 440010
Tamil Nadu Maharashtra
Acronyms
2, 4-D : 2,4-dichlorophenoxyacetic acid

2iP : N-Isopentenyl amino purine
ABA : Abscissic Acid
AC : Acivated charcoal
ACC : Acetyl-coA-carboxylasa
AFLP : Amplification Fragment Length Polymorphism
AIPUB : Association for Improvement in Production and Utilisation of Banana
als : Acetolactate synthase (ALS)
AMF : Arbuscular mycorrhizal fungi
ANOVA : Analysis of varience
AP-PCR : Arbitarily primed PCR
BA : 6-benzyladenine
BAP : 6-benzyl aminopurine
bt : Bacillus thuringiensis
cat : Chloramphenicol acetyltransferase
chs : Chalone synthase
CF : Culture filtrate
CIRAD : Centre d cooperation Internationale en Resherche Agronomique pour le
diveloppement
CMS : Cytoplasmic male sterility
cp : Coat Protein
cry : Insecticidel Crystal Proteins (ICP)
CW : Coconut water
DAF : DNA amplification fingerprinting
DAS–ELISA : Double antibody sandwich– ELISA
DFMA : α- difluoromethyl argentine
DFMO : α- difluoromethyl ornithine
dhfr : Dihydropholate reductase (DHFR)
DMSO : Dimethylsulphoxide
ECS : Embryogenic cell suspension
EDTA : Ethelyenediamine tertra acetic acid
ELISA : Enzyme linked Immunosorbent assay
epsp : EPSP synthase (EPSPS)
FACS : Fluorescence activated cell sorter
FDA : Fluorescein diacetate
GA : Gibberlic acid
gent : Gentamycin acetyl transferase (GENT)
gus : α-glucuronidase (GUS)
(xviii)
Hepes : N-2-hydroxy ethane piperazine-N’-2-ethanesulphonic acid

hyg : Hygromycin phosphotransferase (HYG)
I50 : Inhibitor concentration resulting in 50% inhibition
IAA : Indole acetic acid
IBA : Indole 3- butyric acid
ICAR : Indian Council of Agricultural Research
IITA : International Institute of Tropical Agriculture
INIBAP : International Network for Improvement of Banana and Plantain
IPGRI : International Board for Plant Genetic Resources (Presently Bioversity International)
ISSR : Inernal Simple Sequence Repeats
K- Medium : Knudson orchid medium
Kin : Kinetin (6- furfuryl amine)
lac : α -galactosidase (LAC)
LS medium : Linsmair and Skoog medium
MES : 2-(N-morpholino) ethanesulphonic acid
MIC : Minimum concentration resulting in 100% inhibition
MS medium : Murashige and Skoog medium
NAA : Naphthalene acetic acid
NASH : Nucleic acid spot hybridisation
NBPGR : National Bureau of Plant Genetic Resources
nos : Nopaline synthase (NOS)
nptII : Neomycin phosphotransferase (NPT II)
NRCB : National Research Center for Banana
ocs : Octopine synthase (OCS)
PAGE : Polyacrylamide gel electro phoresis
P
CPA : P-
chlorophenoxy acetic acid
PCR : Polymerase chain reaction
PCV : Packed cell volume
PDA : Potato dextrose agar
PEG : Polyethylene glycol
PGR : Plant genetic resources
PLB : Protocorm like bodies
ppm : Parts per million
PPV : Plum pox virus
PVP : Polyvinylpyrolidone
QTL : Quantitative trait loci
RAPD : Random amplified polymorphic DNA
RH : Relative humidity
RT- PCR : Reverse transcriptase -PCR
SA : Salicylic acid
SCV : Settled cell volume
SDS : Sodium dodecyl sulphate
SH medium : Schenk and Hildebrandt medium
SSR : Simple (short) sequence repeat
TDZ : Thidiazuron
TERI : Tata Energy Rresearch Institute
TRIA : Triacontanol
TTC : 2,3,5-triphenyl tetrazolium chloride
V/V : volume/volume
V/W : volume/weight
WPM : Woody Plants Medium (Lloyd & Mc Cowan)
5
Guava
Ramesh Chandra, Madhu Kamle and Anju Bajpai
Introduction
Psidium guajava L. belongs to family Myrtaceae which comprises approximately 150
species of trees and shrubs, many of which have edible fruits. It is commonly called as guava,
“the poor man’s fruit” or “apple of tropics”. It is a popular tree fruit of the tropical and sub-
tropical climates of the world and is native to tropical America stretching from Mexico to Peru.
Guava is presently cultivated in most of the tropical and subtropical countries around the world
(Samson, 1986). It is a small tree with many branches and produces many small to medium
size fruits. The colour, size, flavour (tart to sweet) and characteristic musky odour vary between
the genotypes. Extensive genetic diversity exists and genotypes are adapted to a wide range of
soil and environmental conditions (Purseglove, 1968). Diversity exist in fruit size, shape, pulp
to seed ratio, seed characteristics, flavour, texture, colour of flesh, aroma, ascorbic acid content,
susceptibility to pest, diseases, frost, fruit cracking, growth, habit, flowering and fruit
characteristics and post-harvest characteristics (Batten, 1984). Guava is a diploid (2n = 22)
but natural and artificial triploids (2n = 33) and aneuploids exist. Triploids generally produce
seedless fruits (Jaiswal and Amin, 1992), but are shy bearers (Menzel, 1985). Seedling trees of
most guava cultivars vary in vigor and size. Natural cross pollination is common in guava
cultivars reaching up to 35% in some cases (Purseglove, 1968; Menzel, 1985) and responsible
for the variability observed in seedling trees.
India is one of the important countries as far as production and consumption of guava is
concerned, the total production of year 2004-05 being 16.86 lakh tons. Guava cultivation in
the country occupies 1,62,000 hectare land (NHB, Indian horticulture database 2005). The
fruit is an excellent source of vitamin-C (Rathore, 1976; Yadava, 1994), which is upto 2000
mg per 100gm of fruit (Campbell, 1984; Menzel, 1985; Martin et al., 1987) and abundant in
dietary fiber (5–7%), vitamin-A, pectin, phosphorus, calcium and potassium (Wilson, 1980
104 Advances in Horticulture Biotechnology (Vol-1)
and Yadava, 1994). Red fleshed fruit contains 3 mg of carotene/100 g of fruit. The predominant
non volatile organic compound of guava fruit includes citric, maleic, lactic, ascorbic and
galacturonic acid (Chan et al., 1971). The storage aroma of fruit is attributed to carbonyl
compounds (Mortan, 1987). Its leaves have been used to treat many ailments including cough
and pulmonary disease in Bolivia and Egypt (Batick, 1984). In Mexico guava leaves are
extensively used to stop diarrhoea (Lozoya et al., 1994) and for the alleviation of gastrointestinal
disorders, which is a common practice originally inherited from traditional Aztec medicine
(Lozoya et al., 2002).
Micropropagation
Guava was initially propagated through seeds but seedlings are variable in both plant and
fruit characteristics due to heterozygous nature of crop. Vegetatively propagated trees are
precocious in comparison to seedling trees, which start fruiting in just 2 to 3 years vis-à-vis
4–5 years for seedlings. It is propagated mainly by layering, inarching and budding. Several
clonal methods including rooting of succulents, greenwood, forkertor, patch budding, air layering
(marcottage or gooty) layering and inarching for propagating guava germplasm have been
discussed by Chandra (1965) and Samson (1986). Guava trees can be induced to set fruit and
produce good yield of marketable fruit during first season following clonal propagation
(Campbell., 1984). The rate of multiplication by these methods is slow and nature dependant.
Micropropagation method could assist in rapid and mass production of clonal stock of newly
released improved cultivars. Chandra (2001) has reviewed guava micropropagation in details.
Success of in vitro cultured plants through micropropagation of mature guava plants (Amin
and Jaiswal, 1987 and 1988) opens up the possibility of producing massive number of clonal
plants in the selected cultivars. Micropropagation is achieved by shoot tip explant from mature
trees (Jaiswal and Amin, 1987; Amin and Jaiswal, 1988; Loh and Rao, 1989; Papadatou et al.,
1990).
Micropropagation of guava assumes importance due to huge demand of its planting material
in many subtropical part of world. Critical steps for successful micropropagation of guava as
defined in literature are: explant collection, checking phenolic exudation, basal media
standardization and hormonal supplementation (Table 5.1)
Explant: Amin (1987) and Jaiswal and Amin (1987) have for the first time reported successful
plantlet formation from somatic tissue of mature guava plant. The best response of shoot
multiplication rate was reported on MS medium supplemented with 4.5 µM BA alone. However,
the survival and response of shoot-tip explants were not satisfactory. Rapid clonal multiplication
of guava through in vitro shoot proliferation from nodal explant of mature tree was reported
again by the same group (Amin, 1987; and Jaiswal and Amin 1987). The apical portion (5–7
cm) of shoots from new growth on mature branches and from basal sprouts of a 15 year old
guava plant was cultured. Siddiqui and Farooq (1997) used nodal explants of the glass-house
grown (2-3 month old) seedlings of guava plants for culture. They reported the use of only
0.4% HgCl2 treatment for 10 minutes to obtain the axenic cultures. They reported that multiple
shoot formation from nodal sections of young plants was obtained in MS medium supplemented
with 1 mg/l BAP alone after 2 weeks of incubation. Two explant sources were compared:
Guava 105
greenhouse grown plants (GHRP) and in vitro harvested axillary buds (IVDS). GHRP with
BAP at 2 mg l-1 gave 3.7 shoots per single node cutting with an average length of 0.7 cm.
Shoots 3.0 cm long was obtained with 0.5 mg/l BAP however, only 2.1 shoots per explant was
produced. For IVDS, the largest number of shoots (3.9 per explant) was obtained with BAP at
0.25 mg l-1, with an average shoot length of 1.6 cm. Generally lower concentrations of BAP
gave fewer but longer shoots. The highest number of roots and longest roots per shoot (5.4 and
2.0 cm, respectively) was obtained with 1 mg l-1 indole- 3-butyric acid (IBA) Ali et al. (2003).
Joshee et al. (2004) reported an efficient regeneration protocols for guava using germinating
embryos of cvs. Beaumont, Ka Hua Kula, and Lucknow-49 (L-49) cultivars as explants. Seeds
of these cultivars were germinated in the dark in MS basal medium supplemented with 2 mg/l
BAP. Both plant growth regulator and guava genotypes influenced shoot bud induction and
elongation. Shoot proliferation was the highest (10+) in cv. Beaumont embryo and nodal explants
when subjected to MS + 4 mg l-1 IAA. No phenolic exudation were observed in embryo explants
whereas the phenolic problem in case of explants from mature trees were solved by incubating
cultures in the dark at 18–20 oC for two to three weeks. It is concluded that the use of mature
embryo explants minimizes phenolic exudation problem in the medium and thus, improves
shoot proliferation.
Sterilization and Antioxidant Treatment: After sterilization, 1.0-1.5 cm nodal explants were
prepared aseptically and inoculated vertically on the media after a dip in solution of antioxidants
(75 mg l-1 citric acid and 50 mg l-1 ascorbic acid prepared in distilled water) (Jaiswal and Amin
1987). After 2-3 initial changes of medium, when phenolic exudation was checked, the explants
were oriented horizontally. This facilitated the rapid uptake of nutrients by the growing shoot-
tips. Prakash (1992) and Prakash and Tiwari (1993; and 1996) also reported in vitro clonal
propagation of guava. The explants (apical portion 5–10cm) were collected from 12 year old
guava plants of cv. Sardar. The shoots were collected, from new flush growth of the plant, in
distilled water containing 5ml l-1 teepol + 2 mg l-1 bavistin and streptomycin each + 75 mg l-1
ascorbic acid + 50 mg l-1 citric acid. The shoots were washed thoroughly under running tap
water and then kept under running tap water for 30–40 minutes, then washed with autoclaved
1% KCl followed with 1% NaOCl treatment for 5–7 minutes. The treated explants were washed
3–4 times with sterile distilled water. They reported that shoot-buds proliferated in MS medium
+ 3 mg l-1 BA + 0.4 mg l-1 IBA + 4 g l-1 PVP. Fitchet P.M. (1990) in his experiment uses shoot
tips of guava cv. Dimple, 10–25 mm in length which were sterilized in 0.2% HgCl2 for 10
minutes after which they were dipped in a solution of citric acid (75 mg l-1) and ascorbic acid
(50 mg l-1) followed by incubation either immediately or after 30 minutes. Untreated controls
were incubated immediately or left to dry for 30 minutes. The incidence of explant browning
was markedly reduced by dipping in the antioxidant solution and then leaving them to dry for
30 minutes; this treatment resulted in a higher survival rate. After 10 days on Murashige and
Tucker medium + 3% sucrose, the explants were divided into the shoot and 1 or 2 nodal
sections and these were incubated on the same medium with BA at 1 mg l-1. After proliferation
the individual plantlets were rooted in in vitro. A method for the quick establishment of aseptic
cultures in guava from mature field-grown stock plants for micropropagation through enhanced
axillary branching technique was reported by Meghawal et al. (2000). The maximum number
of aseptic explants with shoot proliferation was obtained by a combination of surface sterilizing
agents involving hydrogen peroxide (10%), silver nitrate (0.25%), and mercuric chloride (0.05%)
treatment of explants one by one for five, six and three minutes, respectively. The problem of
phenolic browning was also minimized to a great extent by agitation in antioxidant solution as
well as by proper drying of explant prior to inoculation. Meghawal et al. (2001) also developed
a simple method of partial etiolation of stock plants to minimize phenolic oxidation for increasing
the survival of culture during establishment phase was studied. Newly sprouted shoots from
15 years old stooling stumps of 2 guava genotypes, namely Allahabad Safeda and Aneuploid
No. 82, were used. The cultural establishment for micropropagation of Aneuploid No. 82
dwarfing rootstock was severely hampered by the browning phenomenon due to oxidation of
phenolic compounds. Partial etiolation of stock plants of Allahabad Safeda and Aneuploid No.
82 rootstock before explant excision resulted in early bud sprouting and significant increase in
explant survival. The lower degree of browning of culture medium was confirmed by recording
the lesser amount of total phenol exudation into the medium. Remirez et al. (2004) reported
that nodal segments collected from shoots of adult grafts on juvenile rootstocks from seedlings
grown under full sun and shade (40%), and from adult plants grown without and with shade
(80%) were cultivated in vitro. In addition, nodal segments from field grown plants were
cultivated in vitro under the following conditions: control; sixty days after dormex (hydrogenated
cyanamide 49%) application at 2% and 4%; sixty days after partial pruning; and 75 days of
80% shading. Sixty days after culture initiation, seedlings and adult plants under shade produced
no browning explants. The highest Percentage of Bud Break (PBB) (100%) was obtained at 20
days in explants of seedlings; at 40 days in explants of adult plants under shade. At sixty days,
adult and grafted plants without shade presented 40% and 35% browning explants, respectively,
and 85% and 80% bud break explants, respectively. At sixty days, the control treatment produced
65% bud break; the dormex treatment with the concentrations of 2% and 4%, 80% and 70%
PBB, respectively; the partial pruning, 65%. Dormex application at 2% increased PBB and
percentage of leaves formed to more than that of the control. in vitro cloning of Guava cv. Pant
Prabhat was obtained with treatment ethyl alcohol (70%) for 30 sec., mercuric chloride (0.1%)
for 5 minutes and sodium chloride (1%) for 8 min Mishra et al. (2007). Fortification of
500 mg l-1 citric acid, an oxidant to the MS media and initial incubation of cultures in complete
dark for 24 hours showed substantial reduction in phenolic browning of media and higher
survival of explants. Culturing for higher shoot proliferation in MS media containing 4.0 mg l-1
BA and 0.2 mg l-1 IBA was reported. Rooting was induced in half strength MS medium
supplemented with 0.4 mg l-1 IBA and 0.2 mg l-1 NAA.
Culture Media: The medium (MS + 4.5 µM BA) reported earlier (Amin, 1986., Jaiswal and
Amin, 1986) worked well with axillary shoot buds also. After proliferation, the explants were
sub cultured in wider culture vessels with more medium to meet increasing nutrients
requirements and space. 3–6 shoots per culture vessel were reported at 12 weeks of incubation.
Later Amin and Jaiswal (1988) reported a micropropagation method for guava (Psidium
guajava L.) ‘Chittidar’ using nodal explants of field-grown adult trees. Murashige and Skoog
(MS) revised medium containing BAP 1 mg l-1 was found to be the best when the axillary buds
grew out within 3-4 weeks. These shoots attained 3-5 cm in length and had 4–6 nodes after 4
weeks further culture in the same medium. Shoots obtained from the proliferation stage were
Guava 107
rooted on ½ strength MS medium containing IBA and NAA. Rooting rate was 70-90% in
shoots of the 5th and subsequent sub-cultures. The effect of sucrose, agar and pH on the growth
and proliferation of guava shoots was studied by Amin and Jaiswal (1989) with a view to
optimize the cultural conditions for consistent high production of shoots. MS medium with
1 mg l-1 BA and the sucrose containing media 30–45 g l-1 gave best results. In vitro growth and
proliferation of guava were also markedly affected by the concentration of agar and pH level
of the medium. The agar concentration more than 10 g l-1 yielded very poor result by producing
spindly, dry and less number of usable shoots. Khattak et al. (1990) reported that excised
shoots of Allahabad Safeda were culturedon a medium comprising MS salts + 0.7% agar + 3%
sucrose + 0.1mg l-1 BAP and cultured for 5 weeks. The apical portion (5–7 cm) of shoots from
new growth on mature branches and from basal sprouts of a 15-year-old guava plant was
cultured.
Rugini Olive medium (OM) supplemented with BAP 2 mg l-1was found efficient for
proliferating shoot cultures which were established from shoot tips excised from seedlings grown
in a growth chamber (Papadatou et al., 1990). Shoot explants were easily rooted in vitro using
OM with á-naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) at 0.5 and 1 mg l-1 for
both auxins respectively. Rooted shoots were readily established in peat-based compost (Amin
and Jaiswal, 1988). The early bud breaking was noticed on medium with lowest agar concentration
(5 g l-1). However, the brittle turgescent and water-soaked nature of culture on lower agar
concentration might be due to the changes in the matric potencial of media water (Debergh et al.,
1981). The regenerated shoots were multiplied on MS medium + 1mg l-1 BA + 0.2 mg l-1 IBA
and for shoot elongation 1 mg l-1 GA3 was added. Half strength of MS medium + 0.2 mg l-1 IBA
+ 0.2 mg l-1 NAA + 0.1% charcoal + 2% sucrose + 0.7% agar was used for rooting of regenerated
shoots. The regenerated plantlets were transferred to earthen pots filled with autoclaved FYM +
sand (1:1) and covered with glass beaker to maintain humidity. The humidity was reduced gradually.
Using this method they have produced hundreds of tissue cultured plantlets and successfully
transplanted in the field. Singh et al. (2002) reported the multiple shoots generated via direct
organogenesis on hypocotyl segments excised from in vitro germinated seedlings (45 day-old) of
Psidium guajava L. cv. Allahabad Safeda. Modified basal Murashige and Skoog (MMS) medium
supplemented with 6-benzylaminopurine (BAP), zeatin or thidiazuron with or without naphthalene
acetic acid (NAA) were tried. Thidiazuron (0.1 µM) along with 0.54 µM NAA gave the highest
response (44.6%) with the regeneration of 3.6 shoots per original explants. These shoots upon
subculture gave rise to about 5.0 shootlets per explant in shoot proliferation medium, i.e. MMS
supplemented with 2.22 ìM BAP + 2.32 µM kinetin. The regenerated microshoots elongated
using a quick dip of gibberellic acid (GA3 1.44 µM) prior to culture on MMS medium
supplemented with 0.88 µM BAP and adenine sulphate (54.29 µM) for 2 weeks. Rooting of
microshoots was achieved best on half strength MMS medium supplemented with 4.90 µM indole-
3-butyric acid along with 100 mg l-1 activated charcoal.
Perez et al. (2002) reported the multiplication of guava cv. Enana Roja Cubana (EEA
1840), starting from apexes and nodal segments of in vitro plants. The best results were obtained
when the apical segments were used as explants and cultured on MS medium supplemented
with 0.5 mg l-1 BAP [benzyl adenine] for 30 days, obtaining a multiplication coefficient of
4.03. Ali et al.(2003) reported an in vitro technique to clonally propagate guava. Different
concentrations of BAP (6-benzylaminopurine) and TDZ (thidiazuron) were used to regenerate
and micropropagate plants. Woody plant medium supplemented with 2 mg l-1 BAP [benzyl
adenine] was the best medium for culture initiation, while the same medium with lower rates
of BAP (1 mg l-1) produced the greatest number and length of shoots for aneuploid No. 82, an
important dwarfing rootstock for guava (Meghawal et al., 2003) . The effect of various basal
media (Murashige and Skoog’s medium or MS, half-strength MS, Woody Plant Medium, Lloyd
and McCown, and B5 medium) on the quality of shoots during proliferation was also significant.
The combined application of auxins (IBA and NAA) at 0.2 mg l-1 each resulted in a higher
frequency of rooting. Zamir et al. (2003) reported the in vitro mutagenesis in guava (Psidium
guajava L.) using axillary bud proliferation of shoot tips of guava cv. Safeda. Shoot tips were
irradiated with gamma rays at 15-90 Gy (intervals of 15 Gy) using 6 oCo gamma cell source
and cultured in Murashige and Skoog’s (MS) medium containing 3.0% sucrose,
6-benzylaminopurine [benzyl adenine] (BAP) and L-glutamine. Shoot proliferation was
observed after 7 weeks of culturing. Optimum shoot proliferation was recorded for the MS
medium supplemented with 1.0 mg l-1 BAP and 250 mg l-1 glutamine. Rooting of the cultured
shoots in half-strength MS medium supplemented with IAA and IBA. Sensitivity to radiation
was evaluated by determining the percentage shoot tip survival and shoot proliferation. The
LD50 (the rate at which 50% of the population died) was observed at 45 Gy. Rates of more
than 75 Gy were lethal to the explants. Raziuddin et al. (2004) reported the in vitro culture of
guava cv. Safeda. Mercuric chloride at 0.05% for 5 minutes, the combination of citric acid and
ascorbic acid (75: 50 mg l-1) as an antioxidant, 1 mg l-1 benzyl adenine and 500 mg l-1 L-
glutamine for shoot development, and 2.5 mg l-1 each of IAA and IBA for root development,
were the optimum concentrations for successful in vitro propagation of guava.
Mishra et al. (2007) reported a foolproof method of micropropagation through in vitro
shoot bud culture in guava cv. Allahabad safeda. Good shoot proliferation was reported on MS
medium fortified with 3 mg l-1 BAP and rooted on MS medium supplemented with 10 mg l-1 IBA.
Rooted shoots were acclimatized on autoclaved coconut husk fortified with 1/2 MS nutrient
solution + 1 mg l-1 Paclobutrazol (Fig 5.1).
Bajpai et al. (2007) developed regeneration system in Psidium spp. for wilt resistance
rootstock under in vitro conditions (Fig 5.2). The nodal buds excised from severely pruned
trees were used for initiation of sterile cultures and their multiplication. Role of sterilants and
antioxidants was also checked. Seasonal response and media constitution in different spp. was
clearly established for explant establishment and bud induction..
Callus Culture
Singh et al. (2007) reported the response of explant and media composition on callus
induction of guava seedlings cv. Pant Prabhat raised in vitro and ex vitro. Treatment of 0.1%
HgCl2 for 10 minutes on ex vitro grown seedlings was recorded maximum survival of explant
after 5 days of inoculation. Maximum callus induction in shortest time (33.43%) from in vitro
grown seedlings and ex vitro grown seedlings (28.42%) were observed in MS media containing
NAA (1mg l-1) + Kinetin (1mg l-1). Hypocotyl explants induced maximum callus in both types
Guava 109
A B
C D
E F
Fig. 5.1. Micropropagation of guava

A. Mother plant, B. in vitro shoot induction, C. Microshoot establishment,
D. in vitro rooting, E. Acclimatzation in cocopit and F. field establishment
Nodal explants (1.0- 1.5 cm)
-1
Surface sterilization 75 mg/l citric acid + 50 mgl ascorbic
acid pretreatment
Induction Media
(MS + BA 2mg l-1)
Frequent subculturing
Axillary
Bud Induction
Proliferation of shoots(MS +
BAP3 mg l-1 +GA 3 1 mg l-1)
Rooting of ind ividual

-1
shoots(MS +IBA 10mg l )
Acclimatized on autoclaved coconut

husk (1/2 MS nutrient solution + 1 mg l-1
Paclobutrazol)
Fig. 5.2. Scheme for in vitro regeneration of guava through nodal bud
Guava 111
of explant irrespective of hormonal combinations. Callus cultures were used under in vitro
selection scheme employed for screening of mesocarp derived tissue of P. guajava against the
culture filterate. A relatively short term callus culture was challenged with the toxin contained
in culture filterate. Preliminary results indicate that large scale callus mortality when treated
with 30 minutes. The use of selection pressure for involving resistance genotype is aimed at
increasing the mutation rate in comparison to somaclonal variation (Bajpai et al., 2007).
Somatic Embryogenesis
Somatic embryogenesis (SE) is defined as a process in which bipolar structures resembling
a zygotic embryo, develops from a non zygotic cell without vascular connections with the
original tissue. Somatic embryo are used for studying regulation of embryo development, but
embryogenesis is a multistep regeneration process starting with the formation of pro-
embryogenic masses followed by somatic embryo formation maturation, desiccation and plant
regeneration (Arnold et al., 2004). In vitro SE takes place in the absence of vascular connections
with the mother plant (Zimmerman, 1993) genotypes in shorter periods of time. As in somatic
embryogenesis there is no need to separate root induction, thus the plantlet can be multiplied
and acclimatized fast. The multiplication of true to type plants through somatic embryogenesis
will help in propagating elite and new varieties. Somatic embryogenesis (SE) is a new
advancement in vegetative propagation technology for plants, which has a major impact on
tree breeding and high value clonal forestry (Park, 2002 and Sutton, 2002). SE is a highly
efficient method for cloning genetically improved trees and offers the potential for storage and
testing of clones as well as production of unlimited numbers of plants. Thus, it is widely believed
that embryogenic cultures might eventually be used in commercial scale production of cloned
propagules of forest trees. However, considerably greater production rates and large scale
handling systems are required in order to use SE for mass production of somatic seedlings for
operational deployment (Sutton, 2002). Such feature makes it likely that cloned propagules
produced via somatic embryogenesis will have significantly lower costs per unit than those
produced using other micropagation system (Merkle, 1995). Somatic embryogenesis provides
an ideal experimental process for investigation of plant differentiation as well as the large
scale production of plants (Litz and Gray, 1992).
Chandra et al. (2004) for the first time have reported indirect embryogenesis in guava using
immature mesocarp explants. Using full strength MS media with 2, 4 –D (2 mg l-1), ascorbic acid
(100 mg l-1), L-glutamine (400 mg l-1) and sucrose (6%), a protocol for induction, maturation and
germination of somatic embryos via. callogenesis from mesocarp was developed (Figure 5.3).
The somatic embryos were retained in same medium where SE system could be perpetuated via
repetitive embryogenesis, which could be used in future for carrying out transgenic development
and in vitro selection against wilt resistance role of amino acids lglutamine and L proline was
evaluated for maturation and germination of somatic embryos in cv. Banarsi local. Here 0.87 µM
L proline and PEG 1.0% -2.0% influenced maturation of normaml somatic embryos resulting in
development of morphologically normal plants (Rai et al., 2009).
Somatic embryogenesis has been induced using 4cm long and 3.5 cm wide immature fruits
excised after 25–35 days after anthesis under the influence of 1 mg l-1 2, 4-D (Vilchez et al.,
2002). Kosky et al. (2005) reported the germination of somatic embryos of Psidium guajava
A B C
D E F
G H
Fig. 5.3. Somatic embryogenesis in guava.
A. Proembryo formation from callus; B. Globular shape embryo proliferation; C. Heart shaped embryo; D. Globular and
torpedo shape; E. Cotyledonary embryo development; F. Plantlet formation; G. Maturation of plants
H. In vitro acclimatization in cocopit.
cv. Cuban Red Dwarf EEA 18–40 in the RITA® system and on semisolid medium. Somatic
embryos were obtained from immature zygotic embryos which were cultured on the major
salts of MS medium at half strength, supplemented with 400 mg l-1 L-glutamine, 100 mg l-1
ascorbic acid, 60 g l-1 sucrose and 1 mg l-1 2, 4-dichlorophenoxyacetic acid (2, 4-D). Somatic
embryos at the heart and torpedo stages were transferred for germination into RITA® vessels
Guava 113
containing liquid half strength MS medium of the major salts supplemented with 0.25 mg l-1 6-
benzylaminopurine (6-BAP), 10 µg l-1 Biobras-6 (brassinosteroid analogue) and 20 g l-1 sucrose
or to semi-liquid medium of the same composition (solidified with 2.5 g l-1 Gellum Gum,
Spectrum®) in 250 ml glass vessels. The germination percentage, fresh weight and number of
somatic embryos with complete germination were determined. After 10 weeks of culture, the
highest germination percentage (91%) and fresh weight (1.22 g) were achieved in the temporary
immersion system, which was statistically superior to those obtained from semisolid culture
medium 81. 79% and 1.03 g respectively.
Rai et al. (2007) reported direct somatic embryogenesis in guava cv. Banarasi Local from
immature zygotic embryos. Best induction of somatic embryogenesis was achieved from 10
week old zygotic embryos containing 2, 4- D (4.52 µM) and sucrose 5%. Maximum number of
somatic embryos was produced when explants transferred to growth regulator free full strength
MS basal medium after 8 day treatment with 2, 4- D. Full strength MS basal medium containing
5% sucrose was most favorable for maturation of somatic embryos. Highest frequency of
conversion and normal plantlet production was recorded from elongated torpedo stages of
somatic embryos on half strength MS medium containing 3% sucrose. Over 90% of rooted
shoots survived acclimatization.
Embryo Culture
Embryo rescue and culture is a relatively simple in vitro technique and is useful when
embryos are slow to develop relative to the fruit, in cases of seed dormancy or where embryos
remain immature. Ramirez- Villalobos and Salazar Yamarte (1998) reported culturing of
immature embryos taken from adult field grown guava fruits on MS medium supplemented
with zeatin, ribozeatin, 2iP (2-isopentenyladenine), BA or Kinetin. Zeatin and ribozeatin were
each used at 0.1, 0.5 or 1 mg l-1 while, 2iP, BA and Kinetin were used at 1, 5 or 10 mg l-1. High
rates of callus formation were obtained in the ribozeatin, zeatin and 1 mg l-1 2iP. The highest
rate of somatic embryogenesis (60% after 25 days) has been obtained with 0.1 mg l-1 zeatin.
The 1mg l-1 2iP treatment generated structures that consisted of small, elongated, hyaline
appendices (84%of cultures after 50 days).
Anther Culture
Natural occurrence of haploids is rare and is confined to a few species (Kimber and Riley
1963). Haploids facilitate the detection of mutations which are usually recessive and difficult
to detect (Bajaj, 1983) and consequently have played an important role in the isolation of
resistant cell lines via in vitro selection. Babbar and Gupta (1986) for the first time reported the
anther culture system in guava and reported that anthers of Psidium guajava cultivated on
either Murashige and Skoog’s or Nitsch’s basal medium (BM) or the BM supplemented with
10"6 M benzylaminopurine (BAP) was observed to contain microspores undergoing androgenic
segmentations as well as a few multicellular microscopic embryoids. However, final
morphogenic response from such cultured anthers was the development of calli. These calli
had restricted growth accompanied by their early browning. Suspecting the browning to be
due to accumulation of polyphenols, the culture medium was fortified with polyvinyl pyrrolidone
(PVP). PVP increased sucrose concentration in the medium and cold pre-treatment of anthers
decreased the proportion of anthers turning brown as well as delayed the browning of calli, but
it was not possible to maintain the calli for differentiation. Cold pre-treatment significantly
increased the percentage of callusing anthers and also resulted in the early emergence of calli.
However, they could not develop the plantlet from anther culture of guava.
In vitro Conservation
The synthetic seed technology has been developed to use somatic embryos and/or other
micropropagules as seed analogues successfully in the field or greenhouse, and their mechanical
planting at a commercial level. The technology provides methods for preparation of seed
analogues called synthetic seeds or artificial seeds from the micro-propagules like somatic
embryos, axillary shoot buds, apical shoot tips, embryogenic calli as well as protocorm or
protocorm like bodies. For the last fifteen years, intensive research efforts have been made on
synthetic seed production in a number of plant species. Despite these researches, practical
implementation of the technology is yet to be fully realized due to limitations encountered with
the production, development, maturation and subsequent conversion of the micropropagules
into plantlets under in vitro or ex vitro conditions. The technology developed its achievements
and prospects as well as limitations resisting the application of the synthetic seed technology.
For commercial applications, somatic embryos must germinate rapidly and should be able to
develop into plants at least at rates and frequencies more or less similar if not superior to true
seeds. To achieve conversion of somatic embryos into plantlets and to overcome deleterious
effects of recurrent somatic embryogenesis as well as anomalous development of somatic
embryos on their conversion, it is necessary to provide optimum nutritive and environmental
conditions. The induction and control of somatic embryogenesis are dependent of the explant
source, the genotype of the mother plant, the type and level of growth regulators supplemented
to culture medium with 2, 4-D as the auxin normally employed for the induction of embryogenic
competence (Guerra et al., 1999). Once obtained, a somatic embryo can be stored as a synthetic
seed. The synthetic seed technology based on the encapsulation of somatic embryos in a hydrogel
capsule was proposed by Redenbaugh et al. (1986). The main advantages of this technology is
protection of the somatic embryos and ease in storage, conservation, transport and conversion
to plantlets (Onishi et al., 1994, Guerra et al., 1999).
Jaiswal et al. (2005) reported synthetic seed production system via somatic embryogenesis
using zygotic embryos as explant. Somatic embryogenesis was induced in ten week old zygotic
embryos cultured on MS basal medium containing 2 4-D (1 mg l-1) for 8 days and best
development of somatic embryo on full strength MS basal medium containing 3% sucrose and
devoid of 2 4-D. Full strength MS basal medium with 5% sucrose was recorded best for
maturation of somatic embryos. Cultures incubated at higher temperature (30 and 35 oC) shows
best maturation of somatic embryo earlier than 25 oC without affecting induction of somatic
embryogenesis. Maximum frequency of plantlet conversion was observed in ½ MS basal medium
supplemented with 3% sucrose. Somatic embryos of various morphological stages from different
weeks (8–14 weeks) old cultured were encapsulated. Eight weeks elongated and short torpedo
shape somatic embryos was observed best for encapsulation. Somatic embryo encapsulated in
Guava 115
2% sodium alginate (prepared in distilled water) and cultured on 3% sucrose supplemented

full strength MS basal medium shows optimum condition for germination of synthetic seeds.
Shoot tips encapsulated in calcium alginate beads fortified with full strength medium could be
converted into plantlets, thus facilitating storage and germplasm exchange (Rai et al., 2008).
Biswas et al. (2007) reported that efficient plant regeneration system suitable for genetic
transformation for cold hardiness enhancement in guava. Development of protocols for
morphogenesis and somatic embryogenesis was achieved using various tissue explants from
mature guava trees at Fort Valley State University. They developed ‘synseeds’ using somatic
embryos.
Future Perspectives
Guava is gaining popularity in India and is being cultivated in Bihar, Uttar Pradesh, Madhya
Pradesh, Karnataka, Gujrat and Andhra Pradesh. It is estimated that it is cultivated over an area
of 1.5 lakh ha in India with 1710.6 mt production. Although a large number of varieties of
guava have been selected, only few selections viz., Allahabad Safeda, Sardar, Pant Prabhat and
Lalit are being exploited commercially. A medium to tall, less seeded, colored variety with
good keeping quality and resistance to guava wilt disease is still a far cry. Wilt infected mother
plants of guava are playing a major role in spreading the disease beyond leaps and bounds. The
guava biotechnology industry presently has tremendous opportunities throughout the world
and efforts should be maintained to make perfect micropropagation techniques through shoot
bud culture and somatic embryogenesis. The rate of multiplication by conventional breeding
methods is slow and nature dependant while, micropropagation method could assist in rapid
and mass production of clonal stock of newly released improved cultivars.
Although a large number of reports have been published on plant regeneration through shoot
bud culture, none of the protocol is working for scaling up technology and for commercialization
of micropropagation technology; economics of the regenerated plants have to be worked out.
The multiplication of true to type plants through somatic embryogenesis will help in propagating
elite and new genotypes in shorter periods of time. As in somatic embryogenesis there is no need
for separate root induction, thus the plantlet can be multiplied and acclimatized fast. Shoot bud
culture has been fairly successful in guava but, the regeneration system via. somatic embryogenesis
and later synthetic seed production has an additional importance of further development into
somatic cell genetics, transgenic and in vitro selection against wilt. There is an urgent need to
produce disease free planting material in guava and to develop efficient protocol for transformation
system in guava so that suitable transgene could be transferred. There is a need to engineer genes
controlling ethylene biosynthesis and ethylene sensitivity in guava for better shelf life. Insertion
of genes encoding hydrolytic enzymes (which can degrade fungal cell wall) such as chitinase and
glucanase could also be helpful for controlling diseases in guava. Thus, biotechnology can help
in solving some of the long-standing problems of guava cultivation.
Efficient protocols need to be developed for androgenic haploid production system in
guava. Its applicability in haploid production, shortening of breeding cycle, rapid seed viability
test and propagation of rare plants are some of the areas to be exploited for harvesting the
potential of biotechnology.
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Printed at Salasar Imaging Systems, New Delhi

New Delhi H. P. Singh

Foreword ........................................................................................................................................ iii

Anther Culture .......................................................................................................................... 46

In vitro Auto tetraploid production ........................................................................................ 148

Somatic Embryogenesis and Organogenesis ......................................................................... 218

Micropropagation ................................................................................................................... 265

Micrografting ......................................................................................................................... 351

Future Perspectives ................................................................................................................ 416

Anitha Karun Maneesh Mishra

Neelam Shukla Senthil Kumar R.

Nirmal Babu K. Sheeja T. E.

Parthasarathy V. A. Singh Dhurendra

Rajesh Pati Singh, H.P.

Ramakrishnan Nair R. Sivalingam P.N.

Ramesh Chandra Thimmappaiah

2, 4-D : 2,4-dichlorophenoxyacetic acid

Hepes : N-2-hydroxy ethane piperazine-N’-2-ethanesulphonic acid

Fig. 5.1. Micropropagation of guava

Nodal explants (1.0- 1.5 cm)

Rooting of ind ividual

Acclimatized on autoclaved coconut

2% sodium alginate (prepared in distilled water) and cultured on 3% sucrose supplemented

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