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Plant Tissue Culture:An Introductory Text
Sant Saran BhojwaniPrem Kumar Dantu
Plant Tissue Culture:An Introductory Text
123
Sant Saran BhojwaniPrem Kumar DantuDepartment of BotanyDayalbagh Educational InstituteAgra, Uttar PradeshIndia
ISBN 978-81-322-1025-2 ISBN 978-81-322-1026-9 (eBook)DOI 10.1007/978-81-322-1026-9Springer New Delhi Heidelberg New York Dordrecht London
Library of Congress Control Number: 2012954643
� Springer India 2013This work is subject to copyright. All rights are reserved by the Publisher, whether the whole orpart of the material is concerned, specifically the rights of translation, reprinting, reuse ofillustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way,and transmission or information storage and retrieval, electronic adaptation, computer software,or by similar or dissimilar methodology now known or hereafter developed. Exempted from thislegal reservation are brief excerpts in connection with reviews or scholarly analysis or materialsupplied specifically for the purpose of being entered and executed on a computer system, forexclusive use by the purchaser of the work. Duplication of this publication or parts thereof ispermitted only under the provisions of the Copyright Law of the Publisher’s location, in itscurrent version, and permission for use must always be obtained from Springer. Permissions foruse may be obtained through RightsLink at the Copyright Clearance Center. Violations areliable to prosecution under the respective Copyright Law.The use of general descriptive names, registered names, trademarks, service marks, etc. in thispublication does not imply, even in the absence of a specific statement, that such names areexempt from the relevant protective laws and regulations and therefore free for general use.While the advice and information in this book are believed to be true and accurate at the date ofpublication, neither the authors nor the editors nor the publisher can accept any legalresponsibility for any errors or omissions that may be made. The publisher makes no warranty,express or implied, with respect to the material contained herein.
Printed on acid-free paper
Springer is part of Springer Science+Business Media (www.springer.com)
Dedicated to the most ReveredDr. M. B. Lal Sahab (1907–2002)D.Sc. (Lucknow), D.Sc. (Edinburgh),the visionary Founder Director of theDayalbagh Educational Institute, forthe inspiration and strength toundertake and complete the task ofwriting this book
Preface
Plant tissue culture (PTC) broadly refers to cultivation of plant cells,
tissues, organs, and plantlets on artificial medium under aseptic and
controlled environmental conditions. PTC is as much an art as a science.
It is the art of growing experimental plants, selecting a suitable plant
organ or tissue to initiate cultures, cleaning, sterilization and trimming it
to a suitable size, and planting it on a culture medium in right orien-
tation while maintaining complete asepsis. It also requires an experi-
enced and vigilant eye to select healthy and normal tissues for
subculture. PTC involves a scientific approach to systematically opti-
mize physical (nature of the substrate, pH, light, temperature and
humidity), chemical (composition of the culture medium, particularly
nutrients and growth regulators), biological (source, physiological status
and size of the explant), and environmental (gaseous environment inside
the culture vial) parameters to achieve the desired growth rate, cellular
metabolism, and differentiation.
The most important contribution made through PTC is the demon-
stration of the unique capacity of plant cells to regenerate full plants, via
organogenesis or embryogenesis, irrespective of their source (root, leaf,
stem, floral parts, pollen, endosperm) and ploidy level (haploid, diploid,
triploid). PTC is also the best technique to exploit the cellular totipo-
tency of plant cells for numerous practical applications, and offers
technologies for crop improvement (haploid and triploid production, in
vitro fertilization, hybrid embryo rescue, variant selection), clonal
propagation (Micropropagation), virus elimination (shoot tip culture),
germplasm conservation, production of industrial phytochemicals, and
regeneration of plants from genetically manipulated cells by recombi-
nant DNA technology (genetic engineering) or cell fusion (somatic
hybridization). PTC has been extensively employed for basic studies
related to plant physiology (photosynthesis, nutrition of plant cells, and
embryos), biochemistry, cellular metabolism, morphogenesis (organo-
genesis, embryogenesis), phytopathology (plant microbe interaction),
histology (cytodifferentiation), cytology (cell cycle), etc. Indeed the
discovery of first cytokinin is based on PTC studies.
Thus, PTC is an exciting area of basic and applied sciences with
considerable scope for further research. Considerable work is being
done to understand the physiology and genetics of embryogenesis and
vii
organogenesis using PTC systems, especially Arabidopsis and carrot,
which are likely to enhance the efficiency of in vitro regeneration pro-
tocols. Therefore, PTC forms a part of most of the courses on plant
sciences (Developmental Botany, Embryology, Physiology, Genetics,
Plant Breeding, Horticulture, Sylviculture, Phytopathology, etc.) and is
an essential component of Plant Biotechnology.
After the first book on ‘‘Plant Tissue Culture’’ by Prof. P. R. White in
1943, several volumes describing different aspects of PTC have been
published. Most of these are compilations of invited articles by different
experts or proceedings of conferences. More recently, a number of
books describing the methods and protocols for one or more techniques
of PTC have been published which should serve as useful laboratory
manuals. The impetus for writing this book was to make available an
up-to-date text covering all theoretical and practical aspects of PTC for
the students and early career researchers of plant sciences and agricul-
tural biotechnology. The book includes 19 chapters profusely illustrated
with half-tone pictures and self-explanatory diagrams. Most of the
chapters include relevant media compositions and protocols that should
be helpful in conducting laboratory exercises. For those who are inter-
ested in further details, Suggested Further Reading are given at the end
of each chapter. We hope that the readers will find it useful. Suggestions
for further improvement of the book are most welcome.
During the past two decades or so research in the area of plant
biotechnology has become a closed door activity because many
renowned scientists have moved from public research laboratories in
universities and institutions to the private industry. Consequently,
detailed information on many recent developments is not readily
available.
We would like to thank many scientists who provided illustrations
from their works and those who have helped us in completing this
mammoth task. The help of Mr. Jai Bhargava and Mr. Atul Haseja in
preparing some of the illustrations is gratefully acknowledged.
October 2012 Sant Saran Bhojwani
Prem Kumar Dantu
viii Preface
Contents
1 Historical Sketch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 Landmarks/Milestones . . . . . . . . . . . . . . . . . . . . . . . 8Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 10
2 General Requirements and Techniques . . . . . . . . . . . . . . . 112.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.2 Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2.1 Structure and Utilities . . . . . . . . . . . . . . . . 112.2.2 Washing Room . . . . . . . . . . . . . . . . . . . . . 122.2.3 Media Room . . . . . . . . . . . . . . . . . . . . . . 132.2.4 Glassware/Plasticware . . . . . . . . . . . . . . . . 142.2.5 Transfer Room . . . . . . . . . . . . . . . . . . . . . 142.2.6 Growth Room. . . . . . . . . . . . . . . . . . . . . . 152.2.7 Cold Storage . . . . . . . . . . . . . . . . . . . . . . 162.2.8 Greenhouse . . . . . . . . . . . . . . . . . . . . . . . 16
2.3 Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162.3.1 Glassware and Plasticware Washing . . . . . . 172.3.2 Sterilization . . . . . . . . . . . . . . . . . . . . . . . 17
2.4 Appendix I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222.5 Appendix II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 25
3 Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273.2 Media Constituents . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.2.1 Inorganic Nutrients . . . . . . . . . . . . . . . . . . 293.2.2 Organic Nutrients . . . . . . . . . . . . . . . . . . . 293.2.3 Plant Growth Regulators . . . . . . . . . . . . . . 313.2.4 Other Supplements . . . . . . . . . . . . . . . . . . 333.2.5 Undefined Supplements . . . . . . . . . . . . . . . 333.2.6 Gelling Agents . . . . . . . . . . . . . . . . . . . . . 34
3.3 pH of the Medium . . . . . . . . . . . . . . . . . . . . . . . . . 343.4 Media Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.4.1 Steps in the Preparation of CultureMedium . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.4.2 Use of Commercial Pre-Mixes . . . . . . . . . . 36Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 36
ix
4 Tissue and Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . 394.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394.2 Callus Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394.3 Suspension Cultures . . . . . . . . . . . . . . . . . . . . . . . . 40
4.3.1 Batch Cultures . . . . . . . . . . . . . . . . . . . . . 414.3.2 Continuous Cultures . . . . . . . . . . . . . . . . . 414.3.3 Medium for Suspension Cultures . . . . . . . . 434.3.4 Synchronous Cell Suspension Cultures . . . . 434.3.5 Determination of Growth in Suspension
Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . 434.3.6 Tests for Viability of Cultured Cells . . . . . . 44
4.4 Large Scale Cell Culture . . . . . . . . . . . . . . . . . . . . . 454.5 Single Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . 46
4.5.1 Isolation of Single Cells. . . . . . . . . . . . . . . 464.5.2 Culture of Single cells . . . . . . . . . . . . . . . . 464.5.3 Factors Affecting Single Cell Culture . . . . . 49
4.6 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . 494.7 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 50
5 Cytodifferentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515.2 Experimental Systems . . . . . . . . . . . . . . . . . . . . . . . 52
5.2.1 Tracheary Element DifferentiationIn Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . 52
5.2.2 Phloem Differentiation In Vitro . . . . . . . . . 535.3 Factors Affecting Vascular Tissue Differentiation . . . . 53
5.3.1 Growth Regulators . . . . . . . . . . . . . . . . . . 535.3.2 Other Factors . . . . . . . . . . . . . . . . . . . . . . 55
5.4 Cell Cycle and Tracheary Element Differentiation . . . 555.5 Changes Associated with Tracheary
Element Differentiation . . . . . . . . . . . . . . . . . . . . . . 565.6 Process of TE Differentiation . . . . . . . . . . . . . . . . . . 585.7 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . 595.8 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 60
6 Cellular Totipotency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636.2 Factors Affecting Shoot Bud Differentiation . . . . . . . 64
6.2.1 Culture Medium . . . . . . . . . . . . . . . . . . . . 646.2.2 Genotype . . . . . . . . . . . . . . . . . . . . . . . . . 676.2.3 Explant . . . . . . . . . . . . . . . . . . . . . . . . . . 676.2.4 Electrical and Ultrasound Stimulation
of Shoot Differentiation . . . . . . . . . . . . . . . 686.3 Thin Cell Layer Culture . . . . . . . . . . . . . . . . . . . . . 686.4 Totipotency of Crown Gall Tumor Cells . . . . . . . . . . 696.5 Ontogeny of Shoots . . . . . . . . . . . . . . . . . . . . . . . . 696.6 Induction of Organogenic Differentiation. . . . . . . . . . 70
x Contents
6.7 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . 73Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 74
7 Somatic Embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . 757.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 757.2 Factors Affecting Somatic Embryogenesis . . . . . . . . . 76
7.2.1 Explant . . . . . . . . . . . . . . . . . . . . . . . . . . 777.2.2 Genotype . . . . . . . . . . . . . . . . . . . . . . . . . 777.2.3 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . 787.2.4 Growth Regulators . . . . . . . . . . . . . . . . . . 787.2.5 Selective Subculture . . . . . . . . . . . . . . . . . 797.2.6 Electrical Stimulation . . . . . . . . . . . . . . . . 797.2.7 Other Factors . . . . . . . . . . . . . . . . . . . . . . 80
7.3 Induction and Development . . . . . . . . . . . . . . . . . . . 807.3.1 Induction . . . . . . . . . . . . . . . . . . . . . . . . . 817.3.2 Development . . . . . . . . . . . . . . . . . . . . . . 817.3.3 Single Cell Origin of Somatic Embryos . . . . 82
7.4 Synchronization of Somatic Embryo Development . . . 827.5 Physiological and Biochemical Aspects
of Somatic Embryogenesis . . . . . . . . . . . . . . . . . . . . 837.6 Molecular Markers and Somatic Embryogenesis . . . . . 847.7 Maturation and Conversion of Somatic Embryos . . . . 857.8 Somatic Embryos Versus Zygotic Embryo . . . . . . . . . 867.9 Large Scale Production of Somatic Embryos . . . . . . . 867.10 Synthetic Seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . 897.11 Practical Applications of Somatic Embryogenesis . . . . 907.12 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . 907.13 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 92
8 Androgenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 938.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 938.2 Androgenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
8.2.1 Techniques . . . . . . . . . . . . . . . . . . . . . . . . 938.3 Factors Effecting In Vitro Androgenesis . . . . . . . . . . 95
8.3.1 Genetic Potential. . . . . . . . . . . . . . . . . . . . 958.3.2 Physiological Status of the Donor Plants . . . 988.3.3 Stage of Pollen Development . . . . . . . . . . . 988.3.4 Pretreatments . . . . . . . . . . . . . . . . . . . . . . 988.3.5 Culture Medium . . . . . . . . . . . . . . . . . . . . 100
8.4 Origin of Androgenic Plants. . . . . . . . . . . . . . . . . . . 1008.4.1 Induction . . . . . . . . . . . . . . . . . . . . . . . . . 1018.4.2 Early Segmentation of Microspores . . . . . . . 1028.4.3 Regeneration of Plants . . . . . . . . . . . . . . . . 103
8.5 Diploidization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1048.6 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1058.7 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . 1068.8 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 110
Contents xi
9 Gynogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1139.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1139.2 Factors Affecting Gynogenesis . . . . . . . . . . . . . . . . . 113
9.2.1 Genotype . . . . . . . . . . . . . . . . . . . . . . . . . 1139.2.2 Explant . . . . . . . . . . . . . . . . . . . . . . . . . . 1149.2.3 Pre-Treatment . . . . . . . . . . . . . . . . . . . . . . 1159.2.4 Culture Medium . . . . . . . . . . . . . . . . . . . . 115
9.3 Origin of Gynogenic Plants . . . . . . . . . . . . . . . . . . . 1169.4 Endosperm Development . . . . . . . . . . . . . . . . . . . . . 1179.5 Abnormalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1179.6 Ploidy Level. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1179.7 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1179.8 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . 118Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 118
10 Triploid Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11910.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11910.2 Callusing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
10.2.1 Stage of Endosperm at Culture . . . . . . . . . . 11910.2.2 Culture Medium . . . . . . . . . . . . . . . . . . . . 121
10.3 Histology and Cytology . . . . . . . . . . . . . . . . . . . . . . 12110.4 Plant Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . 121
10.4.1 Culture Medium . . . . . . . . . . . . . . . . . . . . 12210.4.2 Cytology . . . . . . . . . . . . . . . . . . . . . . . . . 124
10.5 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12510.6 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . 12510.7 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 126
11 Zygotic Embryo Culture . . . . . . . . . . . . . . . . . . . . . . . . . . 12711.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12711.2 Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12711.3 Culture Requirements . . . . . . . . . . . . . . . . . . . . . . . 130
11.3.1 Mineral Nutrients . . . . . . . . . . . . . . . . . . . 13111.3.2 Amino Acids and Vitamins . . . . . . . . . . . . 13111.3.3 Carbohydrates. . . . . . . . . . . . . . . . . . . . . . 13111.3.4 Growth Regulators . . . . . . . . . . . . . . . . . . 13111.3.5 Natural Plant Extracts . . . . . . . . . . . . . . . . 132
11.4 Culture of Proembryos and Zygote . . . . . . . . . . . . . . 13211.5 Changing Growth Requirements of the Embryos . . . . 13311.6 Role of Suspensor in Embryo Development . . . . . . . . 13411.7 Precocious Germination . . . . . . . . . . . . . . . . . . . . . . 13511.8 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
11.8.1 Basic Studies . . . . . . . . . . . . . . . . . . . . . . 13511.8.2 Shortening of Breeding Cycle. . . . . . . . . . . 13711.8.3 Rapid Seed Viability . . . . . . . . . . . . . . . . . 13711.8.4 Propagation of Rare Plants . . . . . . . . . . . . . 13711.8.5 Haploid Production . . . . . . . . . . . . . . . . . . 13711.8.6 Transformation . . . . . . . . . . . . . . . . . . . . . 138
xii Contents
11.8.7 Production of Rare Hybrids . . . . . . . . . . . . 13811.9 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . 140Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 140
12 Somaclonal Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14112.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14112.2 Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14212.3 Methods to Assess Somaclonal Variation. . . . . . . . . . 14312.4 Origin of Somaclonal Variation . . . . . . . . . . . . . . . . 144
12.4.1 Pre-Existing Variability . . . . . . . . . . . . . . . 14412.4.2 In Vitro Induced Variations . . . . . . . . . . . . 145
12.5 Mechanisms Underlying Somaclonal Variation. . . . . . 14612.5.1 Changes in Chromosome Number
and Structure . . . . . . . . . . . . . . . . . . . . . . 14612.5.2 Gene Mutations . . . . . . . . . . . . . . . . . . . . 14712.5.3 Amplification of DNA. . . . . . . . . . . . . . . . 14712.5.4 Hypomethylation of DNA . . . . . . . . . . . . . 14712.5.5 Activation of Transposable Elements. . . . . . 148
12.6 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14812.6.1 Sugarcane . . . . . . . . . . . . . . . . . . . . . . . . 14812.6.2 Banana . . . . . . . . . . . . . . . . . . . . . . . . . . 14912.6.3 Geranium . . . . . . . . . . . . . . . . . . . . . . . . . 15012.6.4 Potato . . . . . . . . . . . . . . . . . . . . . . . . . . . 15012.6.5 Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15112.6.6 Mustard . . . . . . . . . . . . . . . . . . . . . . . . . . 15112.6.7 Tomato . . . . . . . . . . . . . . . . . . . . . . . . . . 15212.6.8 Finger Millet . . . . . . . . . . . . . . . . . . . . . . 152
12.7 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . 152Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 153
13 In Vitro Pollination and Fertilization . . . . . . . . . . . . . . . . 15513.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15513.2 In Vitro Pollination (IVP) . . . . . . . . . . . . . . . . . . . . 156
13.2.1 Terminology. . . . . . . . . . . . . . . . . . . . . . . 15613.2.2 Technique . . . . . . . . . . . . . . . . . . . . . . . . 15613.2.3 Preparation of Explant . . . . . . . . . . . . . . . . 15613.2.4 Factors Affecting Seed-Set
Following IVP . . . . . . . . . . . . . . . . . . . . . 15713.3 In Vitro Fertilization (IVF) . . . . . . . . . . . . . . . . . . . 158
13.3.1 Isolation of Egg, Central Celland Sperms . . . . . . . . . . . . . . . . . . . . . . . 160
13.3.2 Fusion of Gametes . . . . . . . . . . . . . . . . . . 16113.3.3 Culture of In Vitro Zygotes . . . . . . . . . . . . 162
13.4 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16813.4.1 Basic Studies on Fertilization
and Zygote Development . . . . . . . . . . . . . . 16813.4.2 Hybridization . . . . . . . . . . . . . . . . . . . . . . 16813.4.3 Transformation . . . . . . . . . . . . . . . . . . . . . 168
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13.5 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 171
14 Parasexual Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . 17314.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17314.2 Protoplast Isolation . . . . . . . . . . . . . . . . . . . . . . . . . 174
14.2.1 Factors Effecting Protoplast Isolation . . . . . 17514.2.2 Purification of Protoplasts . . . . . . . . . . . . . 17514.2.3 Viability of the Protoplasts. . . . . . . . . . . . . 176
14.3 Protoplast Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . 17614.3.1 PEG-Induced Fusion . . . . . . . . . . . . . . . . . 17614.3.2 Electrofusion . . . . . . . . . . . . . . . . . . . . . . 178
14.4 Protoplast Culture . . . . . . . . . . . . . . . . . . . . . . . . . . 18014.4.1 Culture Methods . . . . . . . . . . . . . . . . . . . . 18014.4.2 Cell Wall Formation . . . . . . . . . . . . . . . . . 18014.4.3 Cell Division and Callus Formation. . . . . . . 18014.4.4 Plant Regeneration . . . . . . . . . . . . . . . . . . 183
14.5 Selection of Somatic Hybrids . . . . . . . . . . . . . . . . . . 18414.6 Characterization of Somatic Hybrids . . . . . . . . . . . . . 18514.7 Consequences of Protoplast Fusion . . . . . . . . . . . . . . 18514.8 Symmetric Hybridization . . . . . . . . . . . . . . . . . . . . . 18614.9 Asymmetric Hybridization . . . . . . . . . . . . . . . . . . . . 18714.10 Cybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18914.11 Applications to Crop Improvement . . . . . . . . . . . . . . 19114.12 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . 19314.13 Landmarks in the History of Somatic
Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19314.14 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 198
15 Genetic Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19915.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19915.2 Gene Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
15.2.1 Agrobacterium Mediated Transformation . . . 20115.2.2 Direct Gene Transfer . . . . . . . . . . . . . . . . . 205
15.3 Selection and Identification of TransformedCells/Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20715.3.1 Selection . . . . . . . . . . . . . . . . . . . . . . . . . 20715.3.2 Analysis of Putative Transformants . . . . . . . 208
15.4 Regeneration of Transformed Plants . . . . . . . . . . . . . 20915.5 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
15.5.1 Herbicide Resistance . . . . . . . . . . . . . . . . . 20915.5.2 Insect Resistance. . . . . . . . . . . . . . . . . . . . 21115.5.3 Disease Resistance . . . . . . . . . . . . . . . . . . 21215.5.4 Virus Resistance . . . . . . . . . . . . . . . . . . . . 21315.5.5 Nutritive Quality of Food. . . . . . . . . . . . . . 21415.5.6 Abiotic Stress Tolerance . . . . . . . . . . . . . . 21515.5.7 Male Fertility Control . . . . . . . . . . . . . . . . 215
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15.5.8 Parthenocarpy . . . . . . . . . . . . . . . . . . . . . . 21615.5.9 Plants as Bioreactors . . . . . . . . . . . . . . . . . 21715.5.10 Biofuel . . . . . . . . . . . . . . . . . . . . . . . . . . 21815.5.11 RNA Interference (RNAi) Based
Improvement of Plant Products . . . . . . . . . . 21815.6 Biosafety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22215.7 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . 22315.8 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 225
16 Production of Virus-Free Plants . . . . . . . . . . . . . . . . . . . . 22716.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22716.2 In Vivo Thermotherapy . . . . . . . . . . . . . . . . . . . . . . 22816.3 In Vitro Therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . 229
16.3.1 Meristem-Tip Culture . . . . . . . . . . . . . . . . 22916.3.2 In Vitro Shoot-Tip Grafting . . . . . . . . . . . . 23416.3.3 Electrotherapy . . . . . . . . . . . . . . . . . . . . . 23516.3.4 Virus Elimination Through Other
In Vitro Methods . . . . . . . . . . . . . . . . . . . 23516.3.5 Practical Method of Virus Elimination. . . . . 236
16.4 Maintenance of Virus-Free Stocks . . . . . . . . . . . . . . 23616.5 Virus Indexing and Certification. . . . . . . . . . . . . . . . 236
16.5.1 Biological Indexing . . . . . . . . . . . . . . . . . . 23716.5.2 Molecular Assays . . . . . . . . . . . . . . . . . . . 238
16.6 Importance of Virus Elimination. . . . . . . . . . . . . . . . 23916.7 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . 24016.8 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 243
17 Micropropagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24517.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24517.2 Micropropagation of Orchids . . . . . . . . . . . . . . . . . . 24617.3 General Micropropagation Technique . . . . . . . . . . . . 249
17.3.1 Stage 0: Preparatory Stage . . . . . . . . . . . . . 24917.3.2 Stage 1: Initiation of Cultures. . . . . . . . . . . 25117.3.3 Stage 2: Multiplication . . . . . . . . . . . . . . . 25117.3.4 Stage 3: Shoot Elongation and Rooting . . . . 25517.3.5 Stage 4: Transplantation
and Acclimatization. . . . . . . . . . . . . . . . . . 25517.4 Factors Affecting Micropropagation . . . . . . . . . . . . . 258
17.4.1 Initiation of Cultures and ShootMultiplication . . . . . . . . . . . . . . . . . . . . . . 258
17.4.2 Rooting . . . . . . . . . . . . . . . . . . . . . . . . . . 25917.5 Problems Inherent with Micropropagation . . . . . . . . . 260
17.5.1 Hyperhydration . . . . . . . . . . . . . . . . . . . . . 26017.5.2 Contamination . . . . . . . . . . . . . . . . . . . . . 26117.5.3 Oxidative Browning . . . . . . . . . . . . . . . . . 26117.5.4 Recalcitrance of Some Plants . . . . . . . . . . . 26217.5.5 Off-Types . . . . . . . . . . . . . . . . . . . . . . . . 262
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17.5.6 High Cost . . . . . . . . . . . . . . . . . . . . . . . . 26317.6 Bioreactors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26417.7 Photoautotrophic Micropropagation. . . . . . . . . . . . . . 26617.8 The Indian Scenario of Micropropagation . . . . . . . . . 26717.9 Applications of Micropropagation . . . . . . . . . . . . . . . 26717.10 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . 26817.11 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 273
18 Production of Industrial Phytochemicals . . . . . . . . . . . . . . 27518.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27518.2 Strategies to Optimize Phytochemical
Production in Vitro . . . . . . . . . . . . . . . . . . . . . . . . . 27618.2.1 Culture Conditions . . . . . . . . . . . . . . . . . . 27618.2.2 Genetic Enhancement . . . . . . . . . . . . . . . . 27718.2.3 Elicitation . . . . . . . . . . . . . . . . . . . . . . . . 27818.2.4 Biotransformation . . . . . . . . . . . . . . . . . . . 27918.2.5 Immobilization of Cells . . . . . . . . . . . . . . . 28018.2.6 Permeabilization . . . . . . . . . . . . . . . . . . . . 280
18.3 Removal of Secreted Products . . . . . . . . . . . . . . . . . 28118.4 Hairy Root Cultures . . . . . . . . . . . . . . . . . . . . . . . . 28118.5 Bioreactors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28218.6 Commercialization . . . . . . . . . . . . . . . . . . . . . . . . . 28418.7 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . 285Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 285
19 Conservation of Phytodiversity . . . . . . . . . . . . . . . . . . . . . 28719.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28719.2 In Situ Conservation . . . . . . . . . . . . . . . . . . . . . . . . 28719.3 Ex Situ Conservation. . . . . . . . . . . . . . . . . . . . . . . . 28819.4 In Vitro Conservation . . . . . . . . . . . . . . . . . . . . . . . 288
19.4.1 Medium-Term Storage . . . . . . . . . . . . . . . . 28919.4.2 Long-Term Storage . . . . . . . . . . . . . . . . . . 292
19.5 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . 297Suggested Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . 298
About the Authors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Subject and Plant Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
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About the Book
Plant tissue culture (PTC) is basic to all plant biotechnologies and is anexciting area of basic and applied sciences with considerable scope forfurther research. PTC is also the best approach to demonstrate thetotipotency of plant cells, and to exploit it for numerous practicalapplications. It offers technologies for crop improvement (haploid andtriploid production, in vitro fertilization, hybrid embryo rescue, variantselection), clonal propagation (micropropagation), virus elimination (shoottip culture), germplasm conservation, production of industrial phytochemi-cals, and regeneration of plants from genetically manipulated cells byrecombinant DNA technology (genetic engineering) or cell fusion (somatichybridization and cybridization). Considerable work is being done tounderstand the physiology and genetics of in vitro embryogenesis andorganogenesis using model systems, especially Arabidopsis and carrot,which is likely to enhance the efficiency of in vitro regeneration protocols.All these aspects are covered extensively in this book.
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