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

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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

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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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