Plant Biotechnology, 4/e

The book ‘Plant Biotechnology’ is designed for the graduate students of Biotechnology, Botany, Life Sciences, and Agriculture who have a paper or part thereof in plant tissue culture and/or plant biotechnology. The book covers different aspects of the subject in 24 chapters devoted to plant tissue culture technology (10 chapters), transgenic technology  (7 chapters), gene editing, genomics (2 chapters), molecular markers (2 chapters), biosafety, and intellectual property rights. An attempt has been made to present comprehensive up to date information on the above aspects of plant biotechnology, and relevant technological details have been provided in a series of appendices given at the end of the book, The book has been written in simple language in a style I followed for teaching to my students. The concepts and techniques have been explained with the help of suitable diagrams and flowcharts, and every effort has been made to maintain readability and facilitate easy comprehension. It is expected to serve as the basic reading resource for the students preparing for their degrees and would be helpful in various competitive examinations.

1 Introduction to Plant Biotechnology............................................................................ 1 1.1 Introduction 1 1.2 Plant Breeding 1 1.3 Plant Biotechnology 3 1.4 Relevance of Plant Biotechnology to Sustainable Agriculture 5 1.5 Plant Biotechnology and Food Security 5 1.6 Nutraceuticals 6 1.7 Plant Biotechnology and Developing Countries 6 1.8 Plant Biotechnology in the Aid of Poor 7 1.9 Environmental Concerns 8 1.10 Biotechnology in India 8 1.11 Integration with Plant Breeding 9 1.12 Comparison Between Classical Plant Breeding and Genetic Engineering 12 1.13 Achievements 13 1.14 Some Disturbing Indications 13 1.15 Future Prospects 14 Questions 14 Suggested Further Readings 14 2. Plant Cell and Tissue Culture ..................................................................................... 16 2.1 Introduction 16 2.2 History of Tissue Culture Techniques 16 2.3 The General Technique of Tissue Culture 17 2.3.1 Laboratory Space 17 CONTENTS xii PLANT BIOTECHNOLOGY 2.3.2 Culture Room 18 2.3.3 Culture Vessels and Their Washing 18 2.3.4 Sterilisation 18 2.3.5 Surface Sterilisation 19 2.3.6 Nutrient Medium 22 2.4 Callus and Suspension Cultures 24 2.4.1 Callus Cultures 25 2.4.2 Suspension Cultures 25 2.4.3 Subculture 26 2.4.4 Estimation of Growth 27 2.4.5 Nuclear Cytology 27 2.5 Cloning 27 2.5.1 Isolation of Single Cells 27 2.5.2 Culture of Single Cells 28 2.5.3 Cell Viability Test 29 2.6 Shoot Regeneration 29 2.6.1 Ontogeny of Shoot Bud Development 30 2.6.2 Events during Shoot Regeneration 30 2.6.3 Factors Affecting Shoot Bud Regeneration 31 2.7 Somatic Embryogenesis 33 2.7.1 Developmental Pattern of SEs 33 2.7.2 Factors Affecting Somatic Embryogenesis 35 2.7.3 Molecular Aspects of Somatic Embryogenesis 37 2.8 Acquisition of Totipotency 39 2.9 Conclusions 41 Questions 41 Suggested Further Readings 42 3. Micropropagation ........................................................................................................ 43 3.1 Introduction 43 3.2 Meristem Culture 44 3.2.1 Explant 44 3.2.2 Culture Medium 44 3.2.3 Environment during Culture 45 3.3 Stages of Micropropagation 45 3.3.1 Selection and Preparation of Mother Plants (Stage 0) 45 3.3.2 Culture Initiation (Stage 1) 46 3.3.3 Multiplication (Stage 2) 48 3.3.4 Rooting of Shoots (Stage 3) 54 3.3.5 Transfer of Plantlets to Soil (Stage 4) 54 3.4 Choice of the Route for Micropropagation 58 3.5 Nutrient Media and Culture Environment 59 3.5.1 Medium Formulation 59 3.5.2 Growth Regulators 59 3.5.3 Physical Condition of Medium and Culture Environment 60 3.6 Applications of Micropropagation 60 3.7 Advantages of Micropropagation 60 CONTENTS xiii 3.8 Limitations of Micropropagation 61 3.8.1 High Cost of Propagules 61 3.8.2 Recalcitrance of Certain Species and Mature Trees 61 3.8.3 Browning of Medium 61 3.8.4 Hyperhydracity 62 3.8.5 Bacterial Contamination 62 3.8.6 Morphological and Somaclonal Variations 63 3.9 Micropropagation Industry in India 64 3.10 Conclusions 64 Questions 65 Suggested Further Readings 65 4. Virus-Free Plants ......................................................................................................... 67 4.1 Introduction 67 4.2 Shoot Meristem Culture 67 4.3 Thermotherapy 69 4.4 Thermotherapy Combined with Meristem Culture 70 4.5 Cryotherapy 71 4.6 Chemotherapy 71 4.7 In Vitro Shoot-Tip Grafting 72 4.8 Plantlet Regeneration from Callus Cultures 72 4.9 Virus-Indexing 72 4.10 Maintenance of Virus-Free Stocks 73 4.11 Applications and Limitations 73 4.12 Elimination of Pathogens Other Than Viruses 73 Questions 73 Suggested Further Readings 74 5. Germplasm Conservation............................................................................................ 75 5.1 Introduction 75 5.2 Cryopreservation 76 5.2.1 Choice of Material 77 5.2.2 Pre-culture 77 5.2.3 Cryoprotection 77 5.2.4 Freezing 78 5.2.5 Storage 78 5.2.6 Thawing 79 5.2.7 Reculture 79 5.3 Improved Methods of Cryopreservation 79 5.3.1 Vitrification 79 5.3.2 Droplet Method 80 5.3.3 Encapsulation-Dehydration 81 5.3.4 Encapsulation-Vitrification 82 5.3.5 Simplified Freezing Procedures 82 5.4 Slow-Growth Cultures 82 5.4.1 Low Temperature 83 5.4.2 Nutrient Restriction 83 xiv PLANT BIOTECHNOLOGY 5.4.3 Growth Retardants 83 5.4.4 High Osmoticum 83 5.4.5 Lower O2 Concentration 84 5.4.6 Culture Vessels 84 5.4.7 Restricted Illumination 84 5.4.8 Advantages of Slow-Growth Cultures 84 5.4.9 Limitations of Slow-Growth Cultures 84 5.5 DNA Clones 84 5.6 Effects on Genetic Constitution 84 5.6.1 Cryopreservation 85 5.6.2 Slow-Growth Cultures 85 5.7 Applications 85 5.8 Achievements 86 5.9 Advantages 87 5.10 Limitations 87 5.11 Conclusions 87 Questions 87 Suggested Further Readings 88 6. Biochemical Production from Cultured Plant Cells.................................................. 89 6.1 Introduction 89 6.2 Biochemicals from Cultured Plant Cells 91 6.3 Large-Scale Culture of Plant Cells 92 6.3.1 Agar Cultures 92 6.3.2 Suspension Cultures 92 6.3.3 Problems in Large-scale Culture of Plant Cells 92 6.4 Bioreactors for Plant Cells 93 6.4.1 Batch Bioreactors 93 6.4.2 Continuous Bioreactors 93 6.4.3 Multistage Bioreactors 94 6.4.4 Stirred Tank Bioreactor 94 6.4.5 Air-lift Reactor 95 6.4.6 Rotating Drum Bioreactors 95 6.4.7 Immobilised Cell Bioreactors 95 6.5 Improvement of Biochemical Production 96 6.5.1 Enhancing Biomass Yields 96 6.5.2 Improving Biochemical Content of Cells 97 6.6 Commercial Production of Shikonin 100 6.7 Biotransformation 100 6.8 Advantages 101 6.9 Limitations 101 6.10 Conclusions 101 Questions 102 Suggested Further Readings 102 CONTENTS xv 7. Haploid Production for Hybrid Sorting and Analytical Breeding...........................104 7.1 Introduction 104 7.2 Definitions 105 7.3 Androgenesis 106 7.3.1 Pathways of Early Development 107 7.3.2 Cytological and Biochemical Changes 107 7.3.3 Factors Affecting Androgenesis 108 7.3.4 Pollen Culture 111 7.4 Gynogenesis 112 7.5 Haploid Plants from Interspecific Crosses 112 7.6 Pollination with a Haploid Inducer Strain of the Same Species 113 7.7 Applications of Haploids 114 7.7.1 Rapid Isolation of Homozygous Lines 114 7.7.2 Gametoclonal Variation 117 7.7.3 Analytical Breeding 117 7.7.4 Production of all Male Populations 118 7.8 Achievements 118 7.9 Advantages 119 7.10 Limitations 119 7.11 Conclusions 119 Questions 120 Suggested Further Readings 120 8. Technology for Distant Hybridisation: I. Embryo Rescue and in Vitro Pollination ...............................................................121 8.1 Introduction 121 8.2 Embryo Culture 121 8.3 Preparation of Embryos for Culture 122 8.4 Culture Medium for Young Embryos 122 8.5 Culture Environment 124 8.6 Culture Strategies 125 8.7 Precocious Germination of Embryos 125 8.8 Applications of Embryo Culture 125 8.8.1 Embryo Rescue 126 8.8.2 Production of Haploid Plants 127 8.8.3 Propagation of Orchids 127 8.8.4 Shortening the Breeding Cycle 127 8.8.5 Overcoming Dormancy 128 8.8.6 Germination of Difficult to Germinate Seeds 128 8.8.7 Seed Testing 128 8.8.8 Basic Studies 128 8.9 In Vitro Pollination 128 8.9.1 Explant Preparation 129 8.9.2 Nutrition and Culture Conditions 129 8.9.3 Applications 130 xvi PLANT BIOTECHNOLOGY 8.10 In Vitro Fertilisation 131 Questions 132 Suggested Further Readings 132 9. Technology for Distant Hybridisation: II. Somatic Hybridisation ..........................133 9.1 Introduction 133 9.2 Protoplast Isolation 134 9.2.1 Enzyme Activities 134 9.2.2 Plant Tissues 135 9.2.3 Protoplast Purification 135 9.2.4 Protoplast Culture 136 9.3 Protoplast Fusion 136 9.3.1 High pH-High Calcium Treatment 136 9.3.2 Polyethylene Glycol Treatment 137 9.3.3 Electrofusion 138 9.4 Selection of Hybrid Cells 139 9.4.1 Visual Markers 140 9.4.2 Fluorescent Dyes 140 9.4.3 Complementation 141 9.4.4 Transgenic Selectable Markers 141 9.4.5 Culture of the Entire Fusion Mixture 141 9.5 Regeneration of Hybrid Plants 141 9.5.1 Symmetric Hybrids 144 9.5.2 Asymmetric Hybrids 144 9.5.3 Fate of Plasmagenes 144 9.5.4 Cybrids 145 9.6 Applications in Crop Improvement 146 9.7 Symmetric Somatic Hybrids 146 9.8 Asymmetric Hybrids 148 9.9 Cybrids and Cytoplasm Transfer 150 9.10 Genetic Transformation Using Protoplasts 151 9.11 Advantages of Somatic Hybridisation 151 9.12 Limitations of Somatic Hybridisation 151 9.13 Comparison Among Methods of Gene Transfer 152 9.14 Conclusions 152 Questions 153 Suggested Further Readings 153 10. Somaclonal Variation..................................................................................................155 10.1 Introduction 155 10.2 Definitions 155 10.3 Isolation of Somaclonal Variants 156 10.3.1 Screening 156 10.3.2 Cell Selection 157 10.4 Characterisation of the Variants 158 10.5 Molecular Basis of Somaclonal Variation 159 10.6 Origin of Somaclonal Variations 159 CONTENTS xvii 10.6.1 In Vivo Phenomenon Scaled up In Vitro 160 10.6.2 Novel Phenomena In Vitro 161 10.6.3 Factors Affecting Somaclonal Variation 161 10.7 Gametoclonal Variation 162 10.8 Somaclonal Variations and Induced Mutations 163 10.9 Size of Regenerant Population 163 10.10 Applications 164 10.11 Screening of Regenerants 164 10.12 Cell Selection 164 10.12.1 Application of Selection Pressure 165 10.12.2 Selection for Disease Resistant Variants 165 10.12.3 Stress Resistant and Other Mutants 166 10.13 Achievements 167 10.14 Advantages 168 10.15 Limitations 168 Questions 169 Suggested Further Readings 169 11. Recombinant DNA Technology..................................................................................171 11.1 Introduction 171 11.2 Restriction Endonucleases 171 11.2.1 Recognition Sequences 171 11.2.2 Cleavage Patterns 172 11.3 Modification of Cut Ends 173 11.4 Obtaining DNA Inserts for Cloning 174 11.4.1 cDNA Library 174 11.4.2 Genomic Library 174 11.4.3 Chemical Synthesis of Gene 175 11.4.4 Chemical Synthesis of Complete Gene 176 11.4.5 Gene Amplification Through Polymerase Chain Reaction 177 11.4.5.1 Procedure of PCR 177 11.4.5.2 Variations of PCR 179 11.4.5.3 Applications of PCR 179 11.4.5.4 PCR Versus Gene Cloning 180 11.4.5.5 Real-Time PCR 180 11.5 Isothermal Amplification of Nucleic Acids 183 11.5.1 Recombinase Polymerase Amplification 184 11.5.2 Nucleic Acid Sequence-Based Amplification 185 11.6 Vectors 186 11.7 E. coli Vectors 186 11.7.1 Plasmid Vectors 186 11.7.2 pBR322 186 11.7.3 pUCl8/19 187 11.8 Bacteriophage Vectors 188 11.8.1 l Phage Vectors 188 11.8.2 Phage M13 Vectors 189 11.9 Cosmid Vectors 189 xviii PLANT BIOTECHNOLOGY 11.10 Phasmid Vectors 190 11.11 Phagemid Vectors 190 11.12 Bacterial Artificial Chromosome (BAC) Vectors 191 11.13 Shuttle Vectors 191 11.14 Production of Recombinant DNA 192 11.14.1 Both Ends are Cohesive and Compatible 192 11.14.2 Both Ends Cohesive and Separately Matched 193 11.14.3 Both Ends Cohesive and Unmatched 194 11.14.4 Both Ends Blunt 194 11.14.5 One End Cohesive and Compatible, the Other End Blunt 194 11.15 Introduction of the Recombinant DNA into a Suitable Host 194 11.16 Selection of Recombinant Clones 195 11.16.1 Selection of Clones Containing Recombinant DNAs 195 11.16.2 Selection of Clone Containing a Specific DNA Insert 195 11.17 Gene Tagging 196 Questions 199 Suggested Further Readings 199 12. Transgenic Technology: I. Gene Constructs..............................................................200 12.1 Introduction 200 12.2 Applications of Transgenic Plants 200 12.3 Gene Constructs 201 12.4 Plant Promoters 202 12.4.1 Constitutive Promoters 202 12.4.2 Tissue-Specific Promoters 205 12.4.3 Inducible Promoters 205 12.4.4 Synthetic Promoters 209 12.4.5 Promoter Organisation 209 12.4.6 Some Useful Promoters/Expression Systems 211 12.5 Coding Region of the Desired Transgene 212 12.5.1 Gene Size 212 12.5.2 Codon Bias 212 12.5.3 Interfering Sequences 214 12.5.4 Context Sequence for Translation Initiation Codon 214 12.5.5 Untranslated Region of mRNA 214 12.5.6 Base Composition (G+C and A+T Contents) 215 12.5.7 Gene Construct Assembly 215 12.6 Reporter Genes 215 12.6.1 An Ideal Reporter Gene 215 12.6.2 Scorable Reporter Genes 215 12.6.3 Selectable Reporter Genes 216 12.6.4 Problems Posed by Antibiotic Resistance Reporter Genes 218 12.6.4.1 Excision of Antibiotic Resistance Genes 219 12.6.4.2 Use of Unlinked ‘Gene of Interest’ and Selectable Maker Gene 220 12.6.4.3 Non-antibiotic Resistance Selectable Reporter Genes 220 Questions 221 Suggested Further Readings 221 CONTENTS xix 13. Transgenic Technology: II. Vectors and Transformation Methods..........................223 13.1 Introduction 223 13.2 Plasmid Vectors 223 13.2.1 Molecular Biology of Agrobacterium Infection 223 13.2.2 Properties of Crowngall Cells 224 13.2.3 The Ti Plasmid 224 13.2.4 Organisation of T-DNA 225 13.2.5 Organisation of vir Region 226 13.2.6 Transfer of T-DNA 226 13.2.7 Integration of T-DNA into Plant Genome 229 13.2.8 Vectors Derived from pTi 229 13.2.9 Disarming 230 13.2.10 Co-integrate pTi Vectors 230 13.2.11 Binary Vector 232 13.2.12 Organisation of Plant Expression Vectors 235 13.2.13 Plant Artificial Chromosomes 236 13.3 Plant Virus Vectors 237 13.3.1 Cauliflower Mosaic Virus (CaMV) 237 13.3.2 Gemini Viruses 237 13.3.3 Tobacco Mosaic Virus (TMV) 238 13.3.4 Brome Mosaic Virus (BMV) 238 13.4 Transformation Techniques 238 13.4.1 Agrobacterium-Mediated Gene Transfer 238 13.4.1.1 Coculture with Tissue Explants 238 13.4.1.2 In Planta Transformation 239 13.4.1.3 Transformation of Soil-Grown Plants 240 13.4.2 Agroinfection 240 13.4.3 Direct Gene Transfers 240 13.4.3.1 Chemical Methods 240 13.4.3.2 Electroporation 241 13.4.3.3 Particle Gun Method 241 13.4.3.4 Microinjection 242 13.4.3.5 Fibre-Mediated DNA Delivery 242 13.5 Integration of Transgenes 244 13.6 Molecular Analysis of Putative Transgenics 244 13.6.1 PCR Analysis for Transgene Detection 244 13.6.2 Southern Analysis: Confirmation of Transgene Integration and Determination of the Number Integration Sites 245 13.6.3 Detection of Transgene Transcription 247 13.6.4 Assay for Transgene Protein Product 248 13.7 Phenotypic Analysis 248 13.7.1 Genetic Analysis of Transgenics 248 13.7.1 Transgene Inheritance 249 13.8 Genetic Transformation of Chloroplast 249 13.8.1 Vectors for Chloroplast Transformation 249 13.8.2 Selectable Markers for Chloroplast Transformation 250 13.8.3 Chloroplast Transformation Methods 250 xx PLANT BIOTECHNOLOGY 13.8.4 Development of Homoplastomic Lines 250 13.8.5 Some Successful Examples 251 13.8.6 Advantages of Chloroplast Transformation 251 13.8.7 Limitations of Chloroplast Transformation 252 13.9 Low Transgene Expression Levels 252 13.9.1 Premature Termination of Transcription 252 13.9.2 Splicing of cry Gene mRNA 252 13.9.3 mRNA Instability Elements 253 13.9.4 Biased Codon Usage 253 13.10 Suppression of Endogenous Genes 253 13.10.1 Antisense RNA Technology 253 13.10.2 Ribozyme Approach 254 13.10.3 Disruption of Endogenous Genes 255 13.10.4 Co-suppression of Genes 255 13.10.5 RNA-Mediated Interference (RNAi) 255 13.10.6 Gene Inactivation by Gene Editing 258 13.10.7 MicroRNA Manipulation 258 13.11 Increasing Transformation Efficiency 259 13.12 Transformation by A. rhizogenes 260 Questions 260 Suggested Further Readings 260 14. Transgenic Plants: I. Resistance to Abiotic and Biotic Stresses ...............................262 14.1 Introduction 262 14.2 Transgene Action 262 14.3 Development of Transgenic Crops 263 14.3.1 Discovery 264 14.3.2 Phase 1: Proof of Concept 264 14.3.3 Phase 2: Early Development 264 14.3.4 Phase 3: Advanced Development 265 14.3.5 Phase 4: Prelaunch Regulatory Submissions 265 14.3.6 Safety Assessment 265 14.4 First-, Second- and Third-Generation GM Crops 266 14.5 Herbicide Tolerance 266 14.5.1 Glyphosate Action 266 14.5.2 Strategies for Glyphosate Resistance 267 14.5.3 Genes for Tolerance to Other Herbicides 268 14.5.4 Achievements 268 14.5.5 Biosafety Concerns 269 14.6 Insect Resistance 270 14.6.1 The Crystal (Cry) Proteins 272 14.6.2 Toxic Action of Cry Proteins 274 14.6.3 Expression of Cry Genes in Plants 275 14.6.4 Other Genes for Insect Resistance 276 14.6.5 Limitations of Transgenic Insect Resistance 277 14.6.6 Achievements 278 14.6.7 Biosafety Concerns 278 CONTENTS xxi 14.7 Virus Resistance 279 14.7.1 Virus Coat Protein Gene 279 14.7.2 Other Viral Genes 280 14.7.3 cDNA of Satellite RNA 280 14.7.4 Antisense RNA Approach 280 14.7.5 RNA Interference (RNAi) Approach 280 14.7.6 Resistance Based on Artificial MicroRNAs (amiRNAs) 281 14.7.7 Achievements 281 14.7.8 Limitations 281 14.8 Resistance to Fungal and Bacterial Diseases 282 14.8.1 Immune Receptors 284 14.8.2 Modification of Decoy Proteins 286 14.8.3 Modification of Susceptibility Genes 286 14.8.4 Other Dominant Plant Resistance Genes 287 14.8.5 Other Approaches for Disease Resistance 287 14.8.6 Achievements 288 14.8.7 Limitations 288 14.9 Salinity and Drought Resistance 288 14.9.1 Osmoprotectants, Metabolites and Protective Genes 289 14.9.2 Transcription Factors 289 14.9.3 Protein Kinases 291 14.9.4 Salinity Stress (Ion Transporters) 291 14.9.5 Some Other Useful Genes 291 14.9.6 Achievements 292 14.9.7 Limitations and Biosafety Concerns 292 Questions 293 Suggested Further Readings 293 15. Transgenic Plants: II. Quality Modifications and Novel Features............................295 15.1 Introduction 295 15.2 Increasing Sugar Content of Sugarcane 295 15.3 Modification of Starch Quality 296 15.3.1 Starch Biosynthesis 296 15.3.2 Modifications of Starch 297 15.3.3 Starch Modification by Genetic Engineering 297 15.3.3.1 Starch Yield 297 15.3.3.2 Starch Quality 298 15.3.4 Achievements 299 15.3.5 Limitations 300 15.4 Modification of Oil Quality 300 15.4.1 Fatty Acid Biosynthesis 303 15.4.2 Modification of Oil Quality 304 15.4.3 Achievements 307 15.5 Modification of Seed Protein Quality 307 15.5.1 Types of Storage Proteins 308 15.5.2 Nutritional Quality of Proteins 308 15.5.3 Gene Addition (Expression of Heterologous Genes) 310 xxii PLANT BIOTECHNOLOGY 15.5.4 Modification of Endogenous Genes 310 15.5.5 Overexpression of Homologous Genes 311 15.5.6 Suppression of Endogenous Genes 311 15.5.7 Expression of Synthetic Seed Storage Protein Genes 311 15.5.8 Lysine Accumulation in Seeds 312 15.6 Nutraceuticals 312 15.6.1 Golden Rice 312 15.6.2 Purple Tomatoes and Other Nutraceuticals 314 15.7 Antinutritional Substances 314 15.8 Delayed Fruit Softening 314 15.9 Delayed Fruit Ripening 315 15.10 Delayed Senescence Carnation 315 15.11 Flower and Fruit Colour 316 15.12 Parthenocarpic Seedless Fruits 316 15.13 Other Quality Traits 317 15.14 Phytase, an Enzyme 317 15.15 Enhanced Biomass/Yield 318 15.16 Recombinant Hirudin 318 15.17 Male Sterility/Pollination Control 318 15.17.1 barnase-barstar System 318 15.17.2 Hormone-Inducible Male sterility Based on Bcp1 319 15.17.3 Other Male Sterility Systems 320 15.17.4 Achievements 322 15.18 Ornamental Plants: Modification of Flower and Foliage Colour and Plant Morphology 322 15.19 Accelerated Breeding 324 15.20 Safer Biofuel Crops 324 15.21 Phytoremediation 326 15.22 Targetting of Transgene Product into Chloroplast and Mitochondria 327 15.23 Problems in Gene Transfer 327 15.23.1 Transgene Silencing 327 15.23.1.1 Transcriptional Silencing 328 15.23.1.2 Post-Transcriptional Silencing 328 15.23.1.3 Amelioration of Gene Silencing 328 15.23.1.4 Problems Due to Gene Silencing 329 15.23.2 Associated Undesirable Features 329 15.23.3 Low Transformation Frequency 330 15.23.4 Random Integration 330 15.23.5 Contamination by Agrobacterium 330 15.23.6 Yield Penalty 330 15.24 Status of Transgenic Crops in India 330 Questions 332 Suggested Further Readings 333 16. Plant Molecular Farming ...........................................................................................335 16.1 Introduction 335 16.2 General Procedure of Plant Molecular Farming 335 16.3 Transgene Expression Systems 337 CONTENTS xxiii 16.3.1 Stable Nuclear Transformation 337 16.3.2 Stable Chloroplast Transformation 338 16.3.3 Hairy Root Cultures 339 16.3.4 Plant Cell Suspension Cultures 339 16.3.5 Transient Expression Systems 339 16.3.6 Agroinfiltration Using Integrative Expression Vectors 341 16.3.7 Transient Expression Based on Viral Vectors 341 16.3.8 Transient Expression Based on ‘Deconstructed’ Viral Vectors 342 16.4 Optimization of Recombinant Protein Accumulation 342 16.5 Plant Species Suitable for PMF 343 16.6 Production of Vaccines 346 16.7 Antibody Production 348 16.8 Production of Enzymes, Research Reagents and Other Non-Therapeutic Proteins 349 16.9 Therapeutic and Nutraceutical Protein Production 350 16.10 Production of Biopolymers 350 16.11 Biosafety Concerns and Regulatory Issues 351 16.11 Advantages of Plant Molecular Farming 351 16.12 Challenges in Plant Molecular Farming 352 16.12.1 Low Recombinant Protein Yields 352 16.12.2 Downstream Processing 352 16.12.3 Glycosylation of Recombinant Protein 352 16.13 Economic and Commercial Aspects of PMF 353 16.14 Future Perspectives 353 16.15 Conclusions 354 Questions 354 Suggested Further Readings 355 17. Adoption of GM Crops...............................................................................................356 17.1 Introduction 356 17.2 Potential Benefits from GM Crops 356 17.3 GM Crop Approvals 357 17.4 Some Recent Approvals 359 17.5 Commercial Cultivation of GM Crops 362 17.6 Adoption and Diffusion of GM Crops 364 17.7 Agronomic Impact of GM Crops 364 17.8 Sustainability of Agricultural Production Systems 365 17.9 Economic Benefits from GM Crops 366 17.10 Consumer Acceptance of GM Crops 367 17.11 Coexistence and Segregation Systems 367 17.12 Social Impact of GM Crops 368 17.13 Environmental and Ecological Impacts 368 17.14 GM Crop Adoption in India 368 17.15 Conclusions 369 Questions 370 Suggested Further Readings 370 xxiv PLANT BIOTECHNOLOGY 18. Gene Editing ...............................................................................................................371 18.1 Introduction 371 18.2 Gene Editing 371 18.3 Requirements for Useful Gene Editing in Plants 372 18.4 Site-Directed Nucleases 373 18.4.1 Meganucleases 373 18.4.2 Zinc-Finger Nucleases 374 18.4.3 Transcription-Activator Like Effector Nucleases 374 18.4.4 CRISPR-Cas9 SDN System 374 18.5 Nickases 375 18.6 Repair of Double-Strand Breaks 376 18.6.1 Non-Homologous End-Joining (NHEJ) 376 18.6.2 Homology-Directed Repair (HDR) 376 18.7 SDN Technology 376 18.7.1 SDN-1 Technology 377 18.7.2 SDN-2 Technology 377 18.7.3 SDN-3 Technology 378 18.8 Generalized Procedure for SDN Technology 378 18.9 Delivery of SDNs and Donor DNAs into Plant Cells 379 18.10 Off-Target Gene Editing 379 18.11 Oligonucleotide-Directed Mutagenesis 380 18.12 Base Editing in Plants 381 18.13 Mutation Detection 382 18.14 Applications of Gene Editing 383 18.15 Multiplexed Gene Editing 385 18.16 Isolation of Transgene-Free Mutant Lines 385 18.17 Programmable Transcription Factors 385 18.18 Recombineering 386 18.19 Regulation of Gene-Edited Crop Varieties 387 18.20 RNAi vs. Gene Editing 387 18.21 Gene Editing vs. Transgenic Technology 388 18.22 Merits of Gene Editing 388 18.23 Limitations of Gene Editing 388 18.24 Achievements 389 18.25 Future Perspectives 389 Questions 390 Suggested Further Readings 390 19. Genome Maps .............................................................................................................391 19.1 Introduction 391 19.2 Definitions 391 19.3 Genetic Maps 393 19.3.1 Linkage Maps 393 19.3.2 Cytogenetic Maps 393 19.3.2.1 Fluorescence In Situ Hybridisation (FISH) 393 19.3.2.2 Somatic Cell Hybridisation 394 19.3.2.3 Analysis of Small Changes in Polytene Chromosome Structure 394 CONTENTS xxv 19.3.3 Physical Maps 395 19.4 Genetic Markers 395 19.4.1 Morphological Markers 395 19.4.2 Protein-Based Markers (Biochemical Markers) 396 19.4.3 DNA Markers 396 19.5 The Human Genome Project 397 19.5.1 Mapping 398 19.5.2 Sequencing 398 19.5.3 Functional Analysis 398 19.6 Linkage Mapping of DNA Markers 398 19.6.1 Amplified Fragment Length Polymorphism (AFLP) 399 19.6.2 Sequence-Tagged Sites (STSs) 401 19.6.2.1 Polymorphic Sequence-Tagged Sites 401 19.6.2.2 Unique Sequence-Tagged Sites 402 19.6.2.3 Expressed Sequence Tags as STS Markers 402 19.6.3 Linkage Mapping of Molecular Markers 403 19.6.4 Map-Based Cloning 404 19.7 Physical Mapping of Genomes 405 19.7.1 Pulsed-Field Gel Electrophoresis (PFGE) 405 19.7.2 Automated Chromosome Sorting 406 19.7.3 Creation of Contigs 406 19.7.3.1 Chromosome Jumping 408 19.7.3.2 Creation of Contigs Using Unique STSs 409 19.7.3.3 Optical Mapping, Radiation Hybrid Mapping and Happy Mapping 409 19.8 Integration of Cytogenetic, Linkage and Physical Maps of a Species 411 19.9 Sequencing of Genomes 411 19.9.1 Clone-by-clone Sequencing 411 19.9.2 Shotgun Sequencing 412 19.9.3 Genome Sequence Compilation 413 19.9.4 Correction of Errors and Filling of Gaps in Genome Sequences 413 19.10 Genome Sequencing Projects 414 19.10.1 Benefits from Genome Sequencing Projects 415 19.11 Conclusions from Genome Maps/Sequences 416 19.12 Rice Genome Project 417 19.13 Conclusions from Plant Genome Sequences 417 19.14 Beyond the Genome Sequence 419 Questions 420 Suggested Further Readings 421 20. Genomics.....................................................................................................................422 20.1 Introduction 422 20.2 Definitions 422 20.3 Historical 423 20.4 Comparative Genomics 424 20.4.1 Non-orthologous Gene Replacement 424 20.4.2 Exon Shuffling 424 20.4.3 Horizontal Gene Transfers 424 xxvi PLANT BIOTECHNOLOGY 20.4.4 Genome Similarity 425 20.4.5 Conclusions from Comparative Genomics 425 20.4.6 Gene Order Comparisons 427 20.4.7 Phylogenetic Footprinting 427 20.4.8 Origin of New Genes 427 20.4.9 The Minimum Genome Size 428 20.4.10 Comparative Genomics of Mitochondria and Chloroplasts 429 20.4.11 Applications of Comparative Genomics 430 20.5 Gene Prediction and Counting 430 20.6 Genome Evolution 431 20.6.1 Genome Reduction in Disease-Causing Bacteria 431 20.6.2 Duplications in Origin and Evolution of Eukaryotic Genome 431 20.6.3 Gene Duplications Increase Genetic Diversity 432 20.7 Proteomics 432 20.7.1 Types of Proteomics 433 20.7.1.1 Structural Proteomics 433 20.7.1.2 Functional Proteomics 433 20.7.1.3 Expression Proteomics 433 20.7.2 Structural Analysis of Proteins 433 20.7.2.1 Sequence Alignment 433 20.7.2.2 Protein Structure 434 20.7.2.3 Functional Characterisation by Structural Analysis 434 20.7.3 Proteome Analysis 434 20.7.3.1 Two-Dimensional (2-D) Electrophoresis 434 20.7.3.2 Multidimensional Liquid Chromatography 435 20.7.3.3 Protein Microarrays 435 20.8 Functional Genomics 436 20.8.1 Expression Profiling 436 20.8.2 Transcriptome 436 20.8.3 Direct Sequence Sampling 437 20.8.4 DNA Arrays 438 20.8.5 Gene Function Determination 440 20.8.6 Protein Interactions 444 20.8.6.1 Comparative Genomics-Based Methods 444 20.8.6.2 Library-Based Methods 444 20.8.6.3 Phage Display 445 20.8.6.4 Yeast-Two Hybrid System 445 20.9 Metabolomics 446 20.10 Synthetic Genomes 447 20.11 Applications of Genomics 447 Questions 449 Suggested Further Readings 449 21. Molecular Markers: I. Markers and Marker-Trait Associations...............................450 21.1 Introduction 450 21.2 An Ideal Marker System 450 21.3 Molecular Marker Systems 451 CONTENTS xxvii 21.3.1 Restriction Fragment Length Polymorphism 451 21.3.2 Diversity Array Technology (DArT) 452 21.3.3 Random Amplified Polymorphic DNAs 452 21.3.4 Sequence Characterised Amplified Regions (SCARs) 453 21.3.5 Inter-Simple Sequence Repeats (ISSRs) 453 21.3.6 Simple Sequence Repeat (SSR) Markers 454 21.3.7 Cleaved Amplified Polymorphic Sequences (CAPSs) 455 21.3.8 Start Codon-Targeted (SCoT) Marker 455 21.3.9 Amplified Fragment Length Polymorphism (AFLP) 455 21.3.10 Single Nucleotide Polymophisms 456 21.4 Categories of Molecular Markers 456 21.5 Marker System Selection 457 21.6 Detection of Marker-Trait Associations 457 21.6.1 Association Mapping 458 21.6.2 Linkage Mapping 458 21.7 Mapping Populations 458 21.7.1 Recombinant Inbred Lines (RILs) 459 21.7.2 Doubled Haploids 460 21.7.3 Segregating Generations 461 21.7.4 Near-isogenic Lines (NILs) 461 21.8 Construction of Molecular Marker Maps 463 21.9 Mapping of Oligogenic Traits 465 21.10 Mapping of Quantitative Trait Loci (QTLS) 466 21.10.1 Basis for QTL Mapping 468 21.10.2 Methods for Detection of QTLs 468 21.10.3 Significance Threshold and Confidence Intervals for QTLs 470 21.10.4 Major and Minor QTLs 470 21.10.5 Factors Affecting QTL Detection 470 21.10.6 Some Examples of QTL Mapping 472 21.10.7 QTL Meta-Analysis 472 21.10.8 Nature and Function of QTLs 472 21.11 Developing Markers for Marker-Assisted Selection 473 21.12 Confirmation and Validation of Marker-Trait Associations 474 Questions 474 22. Molecular Markers: II. Applications of Molecular Markers.....................................475 22.1 Introduction 475 22.2 Genetic Diversity Analysis 475 22.3 Germplasm Characterisation and Conservation 476 22.4 Identification of Varieties and Hybrids 476 22.5 Selection of Parents for Crosses 477 22.6 Positional Cloning 477 22.7 Marker-Assisted Selection (MAS) 478 22.8 Marker-Assisted Back-Crossing 479 22.9 Gene Pyramiding 480 22.10 Bacterial Leaf Blight Resistant Version of Pusa Basmati 1 481 22.11 QTL Introgression 483 xxviii PLANT BIOTECHNOLOGY 22.12 Marker-Assisted Recurrent Selection 483 22.13 Genomic Selection 483 22.14 Novel Breeding Schemes 484 22.15 Advantages of Molecular Markers 484 22.16 Limitations of Molecular Markers 485 22.17 Achievements 485 22.18 Future Prospects 486 Questions 487 Suggested Further Readings 487 23. Biosafety Concerns and Regulation of GM Crops....................................................489 23.1 Introduction 489 23.2 The Conventional and Transgenic Varieties 490 23.3 Risk Assessment of GM Crops 491 23.3.1 Objective of risk assessment 491 23.3.2 Potential adverse effects of GM crops 492 23.3.3 Evaluation of Potential Harmful Effects 492 23.3.4 Procedure for Risk Assessment 492 23.3.5 Risk assessment dossier 493 23.4 Regulation of GM Varieties of Crops 493 23.5 Harmonisation of Biosafety Regulations 495 23.6 Socio-Economic Aspects of Regulatory Regimes 497 23.7 Biosafety Concerns Related to Transgenic Crops 497 23.8 Food Safety 498 23.8.1 The Concept of ‘Substantial Equivalence’ 498 23.8.2 Antibiotic Resistance Markers 500 23.8.3 Risk from other Transgenic DNA sequences 501 23.8.4 Allergenicity of the Transgene Product 501 23.8.5 Toxicity of Transgene Product 502 23.8.6 Contamination of Food by GM Crop Produce 502 23.8.7 Labeling of Transgenic Food 503 23.8.8 Completely Safe GM Food? 503 23.8.9 Organic Foods 504 23.9 Environmental Impact of GM Crops 504 23.9.1 Persistence of Volunteers 504 23.9.2 Effects on Non-target Species 505 23.9.3 Reduced Pesticide Use 505 23.9.4 Increased use of chemicals 506 23.9.5 Adverse Effects on Plant Biodiversity 506 23.9.6 Phytoremediation 507 23.9.7 Potential Environmental Benefits from GM Crops 507 23.10 Gene Transfers from Transgenic Plants 507 23.10.1 Vertical Transgene Transfers to Nontransgenic Varieties 508 23.10.2 Vertical Transgene Transfers to Related Species 508 23.10.3 Vertical Transgene Transfer-Containment Strategies 509 23.10.4 Terminator Seed Technology (The GURT Technologies) 512 23.10.5 Horizontal Transgene Transfers to Microorganisms 514 CONTENTS xxix 23.11 Relevant Agricultural Issues 514 23.11.1 Yield Drag 515 23.11.2 Transgene Instability and Gene Silencing 515 23.11.3 Variable Transgene Expression Across Environments 515 23.11.4 Resistance of Target Organisms 515 23.11.5 Generation of New Virus Strains 517 23.11.6 Genetic Vulnerability 517 23.12 Post-Release Monitoring of GM Crops 517 Questions 517 Suggested Further Readings 518 24. Intellectual Property Rights.......................................................................................520 24.1 Introduction 520 24.2 History of Intellectual Property Rights in India 520 24.3 Intellectual Property 521 24.4 Protection of Intellectual Property Rights 521 24.4.1 Trade Secret 521 24.4.2 Patent 521 24.4.2.1 Patent Requirements 521 24.4.2.2 Limits of a Patent 523 24.4.2.3 Procedure of Patenting 523 24.4.3 Copyright 523 24.4.4 Plant Variety Protection (PVP) 523 24.5 International Harmonization of Patent Laws 524 24.5.1 TRIPs 524 24.5.2 India and TRIPs 524 24.6 Protection of Biotechnological Inventions 525 24.6.1 Patenting of Genes and DNA Sequences 525 24.6.2 Gene Patents and Genetic Resources 526 24.6.3 Patenting of Life Forms 526 24.6.4 Should Life Forms Be Patented ? 527 24.6.5 IPR and Developing Countries 528 24.6.6 Broad Patents in Biotechnology 529 24.6.7 The Patent Imbroglio 529 24.7 Plant Breeder’s Rights (PBR) 530 24.7.1 Historical 530 24.7.2 A Comparison among UPOV Acts, PPVFR Act and Patents 531 24.7.3 Requirements for PBR 532 24.7.4 The Extent of Protection by PBR 532 24.7.5

Brahma Deo Singh, Ex-Emeritus Professor, School of Biotechnology, Banaras Hindu University, Varanasi, India obtained his Bachelor’s degree in agriculture from Allahabad Agricultural Institute, Allahabad, India and Master’s degree in Agricultural Botany from Government Agricultural College, Kanpur, India with first position in the university, and was awarded the University Gold Medal. He obtained his Ph.D. degree from University of Saskatchewan, Saskatoon, Canada. Prof. Singh has 40 years of teaching and research experience in genetics, plant breeding and plant biotechnology with research interests in genetics and breeding of pulse crops, plant tissue culture, biological nitrogen fixation, and molecular markers. He has published over 160 research papers in reputed journals and authored several books in genetics, plant breeding and biotechnology. He was awarded the First Prize of the Dr. Rajendra Prasad Puraskar in 1987 and 1990 by the Indian Council of Agricultural Research, New Delhi for the books Padap Prajanan and Anuvanshiki, respectively.