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EDITION
BiotechnologyFundamentals and Applications
S. S. Purohit
Ex-Head, P.G. Department ofBotany
Dungar College, Bikaner
*?
AGROBIOS (INDIA)
Contents
Chapter 1 1
BIOTECHNOLOGY:
AN OVERVIEW
Origin and Definitions 1
Definitions 2
Feeding the World 3
Multiple Faces of Biotechnology 5
The Power ofBiotechnology 7
Biotechnology: StrategiesforAgricultural Development 7
Genetic Engineering Techniques 8
Cloning Technology 8
Genetic Modification Technology 9
Hybrid Technologies 10
Gene Transfer 11
Isolation of Important Genes 11
Gene Transfer Technology 12
Molecular Markers 12
Marker-Assisted Selection: Fast Track
to New Crop Varieties 13
Molecular Shortcut 13
Marked Benefits 14
Marker-Assisted Selection 14
DNA Fingerprinting 15
Genetic Engineering of
Microorganisms Associated with
Plants 15
Genetically Modified (GM) Crops 15
Protein (or Enzyme) Engineering 16
Cell Culture Technology 16
Tissue Culture Techniques 17
Tissue Culture: Production ofTrue to
Type Plant 20
Tissue Culture Techniques to Increase
Genetic Variability 21
Protoplast Culture and
Micropropagation 21
Somatic Hybrids and Cybrids 21
Production ofVirus-Free Plants 22
Shoot Tip Micrografting 22
Micropropagation Techniques 22
Propagation by Axillary Shooting 22
Propagation by Direct or Indirect
Organogenesis 23
Improvement ofAxillary Branching 23
Somatic Embryogenesis Propagation 23
Synseeds 23
Somaclonal Variations 24
Haplodiploidisation 24
Gennplasm Conservation (Gene Bank) 24
Tissue Engineering Technology 25
DNA Chip Technology 25
Bioinformatics Technology 26
Microbial Biopesticides, Biofungicied,Herbicides and Agricultural Antibiotics 26
Use ofBiotechnology in Biocide
Production 27
Natural Protectionsfor Plants 28
Biosensor Technology 28
Biochips Technology 29
Biofdm Technology 29
Biosurfactents Technology 29
Monoclonal Antibody Technologies 29
New Approaches to Crop Production 30
Vie Genetic Engineering ofPlants 31
The Genetic Engineering of
Microorganisms Associated with
Plants 32
Genetic Engineeringfor CropProtection 33
New Approaches to Animal Agriculture 33
Animal Breeding 33
Pregnancy Tests 34
Growth Hormones 34
Booroola Gene 35
Muscle vs Fat 35
Fish Farming 35
Microorganisms Associated with Animals 36
Vaccines Against Animal Diseases 36
Vaccinesfrom Vaccinia Virus 37
Altering Intestinal Organisms 37
Bioprocessing Opportunities 38
Alternative Fuels 38
Alternative Feed and Food Sources 39
Other Products 39
Chapter 2 52
DNA, GENE, GENOME,PROTEIN SYNTHESIS AND
DNA CHIP TECHNOLOGY
The Physical Structure of DNA:
the Double Helix : 53
Native DNA 53
Sugar 54
Bases 54
Tint Phosphates 55
Types of DNA 56
Features ofA-DNA and B-DNA 57
Features ofZ-DNA :58
DNA in the Cell 58
Bends in DNA 59
Size, Organization, and Topology ofDNA 59
Supercoiled DNA 60
Genetic Polymorphism 60
DNA Replication 61
How Replication is Achieved? 62
Replication ofBacterial DNA 66
Ribonucleic Acid (RNA) 67
RNA can also Function as a Genetic-
Molecule 68
Gene 69
Prokaryotic Genes 72
Structure ofCyanobacterial Genes 73
Gene Arrangement in the CauliflowerMosaic Virus 73
Eukaryotic Genes 73
Organelle Genes 73
Gene that Codefor Proteins 74
Gene that Codefor tRNA and tRNA 75
Eukaryotic Gene Organization and
Expression 75
Prokaryotic Gene Organization and
Expression 76
Gene Isolation 78
Important Terms Related to Gene 7S
Genome 80
Genome ofBacteriophages 80
Genomes of Viruses ofEukaiyoticHosts .' 82
Retroviruses 82
Genomes ofMicroorganisms 83
Bacterial Genomes 84
Plasmids 84
Yeast (Saccharomyces cerevisiae) 89
Transposons 90
Genomes ofEukaryotes....: 9J
DNA Outside the Nucleus s 93
Genetic Code 94
Concepts ofDegeneracy 97
DNA Encodes Genetic Information 100
Molecular Cloning of DNA 101
Restriction Enzymes 101
DNA Fingerprinting 102
Mapping Genomes by Genetic
Techniques 102
Genetic and Physical Maps 103
vi
Markersfor Genetic Maps 104
Simple Sequence Length
Polymorphisms (SSLP) 105
Southern Hybridization 105
DNA Chip and its Technology 106
Structure 106
Chapter 3 109
APPLICATIONS OF
GENETIC ENGINEERING
Biological Molecular Markers Assisted
Breeding 109
Transgenic Plants 110
Engineered Resistance Against Herbicide 111
Modification ofthe Target 112
Resistance Against Glyphosate 112
Resistance Against Sulfonylurea and
Imidazolinone Herbicides 113
Resistance Against Phosphinothricin 113
Resistance Against Atrazine 113
Detoxification or Degradation ofHerbicide 113
Engineered Resistance Against Pest 114
Bacillus Tlutringiensis Endotoxins 114
Pseitdomonas Flourescens tliat
Secretes Bt Toxin 115
Protease Inhibitors 115
Genefor Other lnsecticidal SecondaryMetabolites 115
Engineered Resistance Against Viral
Infection 116
Cross Protection 116
Coat Protein Mediated Protection 117
Antisense RNA Mediated Protection 117
Satellite RNA Mediated Protection 117
Defective Interfering Mediated
Protection 117
Replicase Mediated Protection 117
Engineered Resistance Against FungalPathogen 118
Genetic Engineering and Plant Lipids 118
Genetic Engineering and Storage Proteins 119
Genetic Engineering for Nitrogen Fixation 120
Transfer ofNif-Genesfrom Klebsiella
Pneumoniae to E. Coli 120
Transfer ofNif-Genes of EukaiyoticOrganisms 120
Transfer ofNod-Genes to Increase
Host Range 120
Plants that Produce Plastic 120
Miscellaneous Applications 120
Chapter 4 122
ENZYMES IN GENETIC
ENGINEERING (NUCLEIC
ACID ENZYMOLOGY)
Storage of DNA and Handling of
Enzymes 123
Restriction Endonucleases 123
Types ofRestriction Enzymes 123
Naming ofRestsriction Endonucleases 124
Target Sites ofRestriction
Endonucleases 125
Restriction Endonucleases in Genetic
Engineering /29
Ligases 130
Activity ofLigases 130
Blunt-Ended Ligation 131
Source ofDNA Ligases 131
Applications ofDNA Ligases 133
Alkaline Phosphatase 133
Applications ofAlkaline Phosphatase 133
Polynucleotide Kinase 133
Applications ofPolynucleotide Kinase 134
Terminal Deoxynucleotidyl Transferase 134
SI Nuclease 135
DNA Polymerase I, Holoenzyme 135
DNA Polymerase I, Klenovv Fragment 136
T4 DNA Polymerase 136
Taq DNA Polymerase 137
Ribonuclease (RNAse H) 137
vii
Reverse Transcriptase 137
Poly (A) Polymerase 138
Deoxyribonuclease I 138
Chapter 5 139
GENE CLONING VECTORS
Plasmids 140
Replication ofPlasmids 140
Size ofPlasmids 140
Copy Number 141
Amplification ofthe Plasmid 141
Types ofPlasmids 141
Isolation ofPlasmid DNA 141
Criteria for Plasmid Cloning 142
Cloning Vectors Based on Bacterial
Plasmids 143
Plasmid Pbr322 144
Col El Plasmid DNA 146
Col E1 Amp Plasmid DNA 146
Pbr 325 Plasmid DNA 146
Pmb9 Plasmid DNA 147
Ptz Plasmids 147
Bacteriophage Vectors for E. Coli 147
Phage X as a Vector 148
Ml3 Bacteriophage 149
Genetic Organisation of Wild Type
Bacteriophage Ml3 150
Construction ofMl3 Based Vectors 151
M13P1 andM13Mp2 151
M13Mp7 151
M13-Plasmid Hybrid Vectors 151
Puc 118andPuc 119 152
The Pembl8 Cloning Vehicle 153
Cosmids 153
CosmidPhc79 753
CosmidPjb8 154
Cloning Using a Cosmid Vector 154
Vectors for Plant Cells 155
The Chromosome of Camv 156
Multiplication Cycle ofCamv 156
Camv as a Gene Vector 156
Shuttle Vectors 157
Yeast Episomal Plasmids (YEPss) 157
Yac Vectors 159
Expression Vectors 159
Gene Cartridges 162
Synthetic Regulator Sequences 163
Universal Translation Terminator 163
Portable Translation Initiation Sites
(PTIS) 163
Trpa Transcription Terminator. 163
Chapter 6 165
RECOMBINANTDNA
TECHNOLOGY
Isolation of Total Cellular DNA 165
Nucleic Acid Hybridization 166
Methods for Labelling Nucleic Acids 167
Methods of Labelling Nucleic Acid and
Probes 168
Nick Translation 168
Primer Extension Method 169
Methods Based on RNA Polymerases 169
End-Labelling of Nucleic Acids 171
Choice of Label 172
Radioactive Labels / 72
Non Radioactive Labels..... 172
Mapping Genes on SpecificChromosomes 173
Somatic Cell Hybrids '. 173
In Situ Hybridization '. / 73
Transposon Tagging 176
Genetic Linkage Mapping 176
Genomic Library 176
Colony Hybridization 178
Plaque Hybridization 178
Chromosome Walking 178
Chromosome Jumping.... 179
Cleaving DNA with Restriction Enzymes 179
Gel Eletrophoresis 180
viii
Blotting Techniques 181
Analysis ofDNA by Southern Blotting 181
Analysis ofRNAs by Northern Blot
Hybridizations 182
Analysis ofProteins by Western Blot
Techniques 183
Detection ofRflps 184
DNA Sequencing 185
Methodsfor Analyzing DNA Sequence 185
Sequencing by Chemical Degradation 185
Sequencing by Chain Termination 186
Automated DNA Sequence Analysis 189
Mechanical Shearing of DNA 190
Transcript Mapping Techniques 190
Primer Extension 191
SI Mapping 191
Synthesis of Cdna from Mrna*. 193
Preparation ofDouble-Stranded Cdna 194
Cdna Library 194
Searching for Gene Using Computers 195
Chapter 7 196
GENETIC ENGINEERING
TECHNIQUES
The Goal for Improved Crop Varieties 196
Transgenic Techniques 197
Steps for Developing New Crop Varieties 197
Bacteria: Nature's Genetic Engineers 200
Bacteria and Gene Transfers in Plants 200
How Bacteria are Used to ModifyPlants 200
Agrobacterium Mediated Gene Transfer 204
Plant-Transformation Vectors 205
Gene Cloning 205
Creating Recombinant DNA 206
DNALigase 206
T4 DNA Ligase 206
Linkers 207
Stop Linker 208
Adaptors 208
Double-Linkers 208
Terminal Transferase 209
Cloning in Bacteria and Eukaryotes 209
Cloning in Bacteria 209
Cloning in Eukaryotes 209
Amplification of DNAs by PolymeraseChain Reaction (PCR) 210
Applications ofPCR 211
Gene Transfer Technology 215
Sexual Method 216
Asexual Methods. 216
Biological Delivery System 218
Agrobacterium Tumefaciens: An
Important Vectorfor Genetic
Engineering 218
Disarmed Ti Vectors 222
Vectors 222
Gene Transfer Using Particle
Bombardment Technique 222
History 223
Applications ofBiolistic 223
Panicle Gun Design 224
Preparation ofDNA Coaled
Microprojectiles 224
Gene Gun 224
Modified Bombardment Devices 225
Factors Affecting Particle
Bombardment-Mediated
Transformation 226
Components ofthe Introduced Plasmid
DNA 228
Target Tissue 229
Tissue Treatment 231
The Fate ofthe Introduced DNA(s) 231
Gene Transfer by Microinjection
Technique 233
Applications ofMicroinjectionTechnique 234
Tlie Technique 235
Silicon Carbide Fiber-Vortex (Silicon
Whiskers) 236
Sonication 236
Elecuoporation ,237
ix
Poly-Cation Mediated DNA Uptake 238
Ca-DNA Co-Precipitation Method 238
Ultrasonication 239
Uv Laser Microbeam 239
Genetic Transformation in
Monocotyledonous Plants 240
Factors Involved in Agrobacterium-Mediated Transfer ofGenes to
Monocots 240
Direct Gene Transfer
Monocotyledonous Plants 240
Problems Involved in Gene Transfer 241
Expression System 241
Cellular Localization 241
Proteolysis, Protein Folding and
Prosthetic Group Acquistion 242
Precursor Availability 242
lnhibitoiy Environments 242
Side Reaction ofNew Compounds 242
Chapter 8 243
EXPRESSION OF INDUCED
GENES
Factors Affecting Gene Expression 243
Regulation ofGene Expression 243
Reporter Gene for Transient Expression 245
^-Glucuronidase Gene •. 245
Luciferase Gene 245
Chloramphenicol Acetyl TransferaseGene 245
Nopaline (Nos) Synthase Gene 246
Neomycin Phosphotransferase Gene
(Nptll) 246
Anthocyanins 246
Dihydrofolate Reductase Gene 246
Promoter Elements 246
Marker Gene 247
Inactivation of Foreign Gene Expression 247
Transgene Inactivation and
Methylation 248
Double Transfonnations and Gene
Silencing 248
Post-Transcriptional Gene Suppression 248
Chapter 9 249
GENE SILINCING AND
ANTISENSE TECHNOLOGY
Reasons for Gene Silencing 250
Types of Gene Silencing 251
Homology-Dependent Gene Silencing 251
Position Ejfects 251
Environmental Stress 251
Antisense Gene Silencing 252
Endogenous Genes Silencing by Co-
Suppression 252
Post-Transcriptional Gene Silencing
(PTGS) 253
Characteristics ofPTGS 253
Possible Mechanisms ofPost-
Transcriptional Gene Silencing 254
Elimination ofHomologous Transcripts 255
Ptgs ofEndogenous Genes and the
Presence ofSpecific Transgene Loci 255
Gene Silencing in Crop Plants 256
Tomato 256
Brassica 256
Rice 256
Development of Viral Resistance in
Plants 256
Effect ofTransgene Dose on Silencing 257
Silencing Depends on Similarity ofTranscribed Sequences 257
Class II Chilinase Host Genes are Not
Silenced by a Class I Transgenes 258
Silencing Occurs During Seedling
Development 258
Stability ofthe Silent State During
Vegetative Growth : 258
Genetic Factors Influencing Silencing 258
x
Silencing ofChitinase Gene Expression
is Likely to Be a Post Transcriptional
Event 259
Occurrence ofSilencing and Resetting 259
Antisense Technology 260
Formation ofAntisense mRNA 26J
Inhibition ofGene Expression byAntisense RNA 261
Plantsfrom Antisense Technology 261
Chapter 10 263
SCOPE, ACHIEVEMENTS AND
HAZARDS OF GENETICALLY
MODIFIED CROPS
Our Need 263
Fungal Resistance 265
Long Lived Fruits and Vegetable 266
Race-Specific Resistance Genes 267
Two-Component SystemsforNon¬
specific Resistance 267
Compounds Toxic to Fungi 268
Anti-Fungal Protein 268
Ribosome-lnactivating Proteins (RIPS) 269
Engineered Resistance Against Virus
Diseases 269
Coat Protein-Mediated Protection
(CPMP) 270
Defective Interfering RNA/DNA
Protection 271
Satellite RNA-Mediated Disease
Attenuation 271
Anti-Sense RNA/Ribozynie-MediatedProtection 271
Replicase and Protease-Mediated
Protection 271
Movement Protein-Mediated Protection 272
Resistance Tlirough Transgenic
Expression ofAntiviral Proteins 272
Herbicide Tolerance Genes 273
Herbicide Target Modification 273
Over Production ofTarget Enzyme 274
Detoxifying Enzymes 274
bisect Tolerance 274
Coat Protein Genesfor Virus
Protection 275
Light Regulated Genes 276
Antisense RNA 276
Transposable Elements 276
Seed Storage Proteins 276
Bioactive Peptides 276
Human Proteins 277
Enzymes 277
Antibodies 278
Vaccines 278
Industrial Products 278
Metal Resistance in Plants 279
Transgenic Plants with Higher Stress
Tolerance 279
Water Stress 279
Temperature Stress 279
Salt Stress 280
Strategyfor Stress Tolerant TransgenicPlants 280
Properties/Characters Requiredfor
Improved Stress Tolerance 280
Alteration of Oil Composition and
Content with Reference to Transgenic
Rapeseed 281
Recommendations and Priorities for
Future Research 282
Prioritiesfor Future Research 283
When Transgenes Wander, Should We
Worry? 2S4
Gene Flow in Crops 285
Plant Improvement are Not Absolutely
Safe 286
Why Bt Cotton Should Be Banned 286
Bt Cotton Creating Resistance to Bt? 287
xi
Chapter 11 288
ECO-SOCIAL IMPACT OF
GENETICALLY MODIFIED
CROPS /FOODS
Potential Hazards 288
Potential Ecological Impact of TransgenicVirus- Resistance in Plants 289
Areasfor the Assessment of the
Ecological Impact 290
Agricultural and Natural Habitats 290
Virus Location and Virus-Host Co-
Evolution 290
Virus Epidemiology 297
Fitness Impact 291
Plant Population Genetics 291
Assessment 292
RiskAssessment ofGene Flow
Associated with the Release of Virus
Resistant Transgenic Crop Plants 292
Adoption ofTransgenic Seeds: Profit or
Loss 292
Who Has Benefited? 293
Legal Rights in the New Biotechnology 294
What is a Patent? 294
Impact on Farmers and Consumers 295
Ethical and Practical Problems 295
Transgenic are Not Terminator Crops 296
The Hypothetical Risks 297
Government Alerted: Did Gm Foods
Reach India? 298
Transgenic Plants: Bioethical and Moral
Concerns 299
Bioethics 300
Ethical Concerns 300
Transgenic Plants: Risks, Benefits and
Impacts on Society and the
Environment 301
Transgenics and the Environment 302
Objections to Development and
Deployment ofTransgenic Crops. 303
Transgenic Trees 303
Transgenics and Human Health 304
Socio-Economic Impact and Benefits 305
Ownership ofGenes and the Need for
Patents 305
Biotechnological Development and Risks 306
Situation in the Developing Countries 306
Regulation ofAgricultural Products of
Biotechnology in Canada 308
Chapter 12 310
PLANT TISSUE CULTURE:
PRINCIPLES AND
METHODOLOGY
Importance ofTissue Culture Technique 310
Global Picture ofPlant Tissue Culture 311
History of Plant Tissue Culture
Researches 311
Tissue Culture Researches in India 314
Plant Tissue Culture: Principles 316
Callus Culture 316
Meristem Culture 317
Organ Culture 317
The Concept ofTotipotency of Cells 318
Contrasts Between Plant andAnimal
Cells ; 318
The Autonomous Organelles: Their
Behaviour During Growth Induction
and Morphogenesis 320
Sites and Modes ofAction ofGrowth
Regulating Substances 320
The Behaviour ofCarrot Cells in Vitro 320
Clonal Developmentfrom Animal and
Plant Cells 320
Plant Tissue Culture: Methods 321
Media Preparation: Area/Room 322
Culture Media, Washing Powder/
Liquid Detergent, Disinfectants 322
Aseptic Transfer ChamberArea 322
Environmentally Controlled Culture
Room 322
Analytical Room 323
xii
Acclimatization Room 323
Miscellaneous Items 324
Specifications ofLaboratoryEquipments 324
Aseptic Technique 325
Sterilization of Plant Tissues 325
Cleaning (Preparation ofGlasswares/Plastic Wares
(Autoclavable) 326
Sterilization 326
Surfactants 327
Control ofBacterial and FungalContaminants by Antibiotics in Plant
Tissue Culture 328
in Vitro Environment 329
Pretreatment to Explant Tissues Prior to
Culture 330
Procedure 330
Micropropagation Through
Organogenesis 332
Culture Media and Preparation 334
Media Components 335
Inorganic Salts 335
Plant Growth Regulators 336
Carbon Source 336
Gelling Agent 337
Amino Acids and Amides 337
Antibiotics 337
Natural Complexes 337
Additional Requirements 337
Sterilization ofMedia 342
Use and Storage ofCoconut Water 343
Related Procedures....:. 343
Ultraviolet Light 343
Preparation ofPhenol 343
Working with 32p Labelled Compounds 343
Silanization ofPlastic and Glassware 344
Preparation ofDialysis Tubing 344
Initiation of Embryogenesis in SuspensionCulture 347
Method. 348
Establishment of EmbryogenicSuspension Culture 348
Maturation ofEmbryos 357
Important Parameters for Consideration 351
Simplification ofthe Technique 351
Different Causes ofFailures (and
Remedies) 352
CHAPTER 13 353
HISTOLOGICAL AND
PHOTOGRAPHIC
TECHNIQUES FOR PLANTTISSUE CULTURE
Histological Techniques 353
General Considerationsfor ParaffinStudies 354
Immobilization ofSpecimensfor
Paraffin Sectioning 358
Dissection and Mounting ofSpecimens 360
Preparation of Specimens for ScanningElectron Microscopy 360
Photographic Methodsfor Plant Cell
and Tissue Culture 361
Type ofPhotographyfor Plant Cell and
Tissue Culture Research 361
Macropholography 361
Microphotography with
Slereomicroscopes 362
Focusing and Cropping the Image 363
Types ofthe Films 363
Composition 364
Chapter 14 365
PROTOPLAST TECHNOLOGY
Importance ofProtoplast Isolation and
Culture 365
Isolation of Protoplast from Various Plant
Parts 366
Enzymatic Method 367
Mechanical Method 367
Enzymic Isolation of Protoplasts: Method 368
xiii
Methods of Protoplasts Culture .....372
Laboratory Facilities 372
Enzyme Mixture and Osmotic Stabilizer
(Osmotica) 372
Purification of Isolated Protoplasts 374
Sedimentation and Washing 374
Flotation 374
Other Purification Methods 374
Protoplast Viability Testing 374
General Steps ofProtoplast Culture 375
Culture Media for Protoplast Culture 375
Protoplast Culture Mediafor Pc I
Group 375
Protoplast Culture Media Pc II Group 377
Prot oplast Culture Media Pc HI Group 377
Agar Embeded Culture 378
Microchambers 3 78
Hanging Drop Cultures (HDC)
Teclmiques 378
Multidrop Array (MDA) Techniques 378
Growth and Division of Protoplast 380
Protoplast Culture: Regeneration ofCell Wall 380
Development ofCallus/Wlwle Plant 380
Determination of Protoplast Plating
Efficiency 381
Technique for Isolation of Sub-Protoplasts 382
Handling of Regenerated Plantlets 382
Protoplast Fusion „382
General Steps ofProtoplast Fusion 383
Protoplast Culture: Regeneration of Plants 386
Organogenesis 387
Embryogenesis 387
Handling ofRegenerated Plantlets 387
Protoplast Culture: Uptake ofForeignMaterials 388
Incorporation ofForeign DNA 388
Incorporation ofNuclei 388
Incorporation ofChloroplasts 388
Incorporation ofCyanobacteria 389
Incorporation ofBacteria 389
Incorporation of Virus 389
Incorporation of Non-Biological Materials 390
Selection of Fusion Hybrids 390
Visual Selection 390
Fluorescent Labels 390
Fluorescence Activated Cells Sorting 390
Nutritional Selection 391
Drug Sensitivity and Resistance 391
Somatic Cell Hybridization or Cybrid or
Cytoplast 393
Protoplasts for Isolation of Cell
Components 393
Plasmalemma 393
Chloroplasts 394
Mitochondria 394
Vacuoles 394
Chapter 15 395
MICROPROPAGATION IN
PLANTS
Advantages ofMicropropagation 395
Other Advantages ofMicropropagationMethods 596
Advantages ofMicropropagation Over
Traditional Methods 398
Uses ofMicropropagation in
Horticultural Industry 398
Disadvantages ofMicropropagation 399
Commercial Uses of Micropropagation 400
Materials for Micropropagation 400
Plant Health, ,
403
Gennplasm Storage 403
Types of Micropropagation 404
Micropropagation Through Shoot Bud
Proliferation 404
Micropropagation ThroughAdventitious Buds 406
Meristern Tip Culture 408
in Vitro Tuberization '. 408
Somatic Embryogenesis 408
Methods of Micropropagation (General) 411
Which Material Should Be Used? 412
xiv
Stages ofMicwpropagation 412
Materials 412
Important Considerations and Precautions 416
Problems and Contamination in
Culture 416
Micropropagation in Forest Trees 418
Conifers 419
Commercial Aspects of Micropropagation 419
Large Potential Market 420
Futuristic Outlook of
Micropropagation 421
Chapter 16 422
SOMATIC EMBRYOGENESIS
Carrot as a Embryogenic System 423
Somatic and Zygotic Embryos 423
Totipotency and Role of Auxin 423
Proembryogenic Masses and Role ofAuxin 423
Acqitistion of Totipotency 424
Expression ofthe Programme in the
Absence ofAuxin 425
Methylated Cytosine Level and
Development 425
The Genetic Approach of Somatic
Embryogenesis 426
The Need. 426
The Process of Somatic Embryogenesis 426
Induction ofthe Embryogenic State 426
Recurrent Embryogenesis 427
Problems 429
Maintenance 429
Auxin vs Cytokininfor Induction of
Embryogenesis 429
How to Obtain Embryos from
Embryogenic Cultures? 429
Embryo Maturation and the Developmentof Germinability 430
Auxin Levels 430
Sucrose Level 430
Heat Shock Treatment 430
ABA Levels 430
Water Saturation 431
Genetic Control of Embryo Cloning 431
Genetic Variability : 431
Cytoplasmic Effects .;....431Applications of Somatic Embryogenesis 432
Mass Propagation 432
Scale-Up Potential 433
Use ofBioreactors 433
Protoplast Culture 433
Embryo Cloning and Gene Transfer 436
Somatic Embryogenesis in Trees 437
Chapter 17 440
SOMACLONALAND
GAMETOCLONAL VARIANT
SELECTION
Genetic Variations and CropImprovement 441
Why Variations Occur? 442
The Mechanism of Somaclonal Variation 442
Source Material and Culture Conditions 442
Detennination ofCell Number 442
Forms of Somaclonal Variation 443
Detection and Isolation of Somaclonal
Variants 443
Mutagenesis and Somaclonal Variation 444
Somatic Genetics of Nitrogen Metabolism 445
Methodfor Isolation ofDesired Variant
Cellsfor NaCl -Tolerantfrom Callus/
Suspension Cultures 445
Applications in Plant Breeding 446
Chapter 18 449
GYNOGENESIS AND CROP
IMPROVEMENT
Gynogenesis Induced by IrregularPollination in Situ 450
xv
Development of Useful Crop Varities 452
Gynogenesis Induced by in vitro Culture
of Unpollinated Female Reproductive.
Organs 452
Origin of Gynogenic Regenerants and
Methods of Chromosome Doubling 454
Conclusions 455
Chapter 19 456
SYNSEEDS OR SYNTHETIC
SEEDS
The Natural Seed 456
Development of the Concepts ofTissue
Culture and Artificial Seeds 456
Discovery of Synthetic Seeds 458
Uses and Limitations of Artificial Seeds 461
Potential Uses ofArtificial Seeds 463
Production of Synthetic Seeds 465
Encapsulation or Coating of SyntheticSeed 465
Gel Complexation via a DroppingProcedure 466
Automate Encapsulation Process 466
Mass Balance Concept 466
Steps of Commercial Artificial Seed
Production 467
Hydrogel Encapsulation ofArtificial
Seeds 468
Synthetic Seed and Forest Trees 469
Somatic Embryogenesis and Synseeds 470
Chapter 20 471
PLANT TISSUE CULTURE
SOME RELATED ASPECTS
Bio Village Concept 471
Qualifications Required to Join the
Training Course 472
Employmentfor Rural Youth 472
Efforts of Public Research Institutes 472
Production Criteria and Economics 473
Selection of CropsforMicropropagation 473
Selection ofLocation 474
Optimising Size of Production 474
Available Facilities and Production
Potential 475
Economic Analysis of the Project 475
Production Management 475
Order Processing 476
Planning for Production 476
Multirate 476
Passage 477
Operator Efficiency 477
Production Monitoring 477
Contamination: Economic Assessment 478
Calculation of Cost Price 478
Quality Control in Commercial Plant
Tissue Culture 478
Chapter 21 480
ANIMAL CELL AND TISSUE
CULTURE
Introduction 480
Advantages and Disadvantages of Tissue
Culture Methods 480
Advantages 481
Disadvantages 481
Primary and Established Cell Lines 481
Kinetics of Cell Growth.. 483
Established Cell Lines 483
Primary Cell Lines 483
Interaction Among Cells 484
Genetics of Cultured Cells 484
Metabolism 484
Animal Cell and Tissue Culture 485
Mechanical Technique 485
Biochemical Technique 485
Animal Tissue Culture Media 486
xvi
Culture Media Containing Naturally
Occurring Ingredients 486
Blood Plasma 486
Blood Serum 487
Serum Free Media 487
Tissue Extracts 488
Complex Natural Media 489
Chemically Defined Media 489
Chemically Defined Media 490
Other Tissue Culture Media 494
Laboratory Facilities for Animal Tissue
Culture 496
The Substrate on Which Cells Grow in
Laboratory 497
Treatment ofSubstrate Surfaces 497
Feeder Layers onSubstrate 498
The Gas Phasefor Tissue Culture 498
Culture Procedures 498
Preparation and Sterilization ofGlassware andApparatus 498
Preparation and Sterilization ofReagents andMedia 498
Preparation ofAnimal Material 500
Primary Culture, Cell Lines and Cloning 500
Disaggregation ofTissue and PrimaryCulture 500
Isolation ofTissue 501
Enzymatic Disaggregation 502
Disaggregation by Trypsin (forEmbryonic Tissue) or Trypsinization 503
Disaggregation by Collagenase (forEmbiyonic, Normal and MalignantTissues) 503
Mechanical Disaggregation 503
Separation of Viable and Nonviable
Cells : 503
Somatic Cell Fusion 503
Tissue Cultures 504
Primary Ex-Plantation Techniques 504
Slide or Coverslip Cultures 504
Flask Cultures 506
Test Tube Cultures 507
Animal Organ Culture 508
Culture ofEmbryonic Organs 508
Organ Cultures on Plasma Clots 508
Organ Cultures on Agar. 508
Whole Embryo Culture 509
Culture of Chick Embryo (Spratt, 1956) 509
Animal Tissue Cultures in Biomdical
Research-Genetics 509
Karyological Studies 509
Identification and Study ofHereditaryMetabolic Disorders 510
Somatic Cell Genetics 5JO
Tissue Cultures in Biomedical
Research-Virology and Host-Parasite
Relationships 510
Tissue Cultures in Biomedical
Research-Cancer 510
Chapter 22 511
IMMUNOLOGY, SEROLOGYAND VACCINES
Immunology 511
What is An Immune System? 512
Principles of Immunology 513
Immunoglobulins 513
Immunoglublin Classes and Subclasses 514
Diversity of Antibodies 518
Specificity ofAntibodies 520
Immunization 520
The Primary Antibody Responses 521
The Secondary Antibody Response 521
Catalytic Antibodies 521
Antigens 522
Proportion ofAntigens 522
Antigen-Antibody Binding 522
The Immune Response 523
Lymphocytes 524
Origin ofLymphocytes 524
B Cells or B Lymphocytes 524
Interaction ofTand B Lymphocytes 528
xvii
Accessory Cells (A Cells):
Macrophages 528
Immune System528
Immunological Tolerance 528
Serology529
Serological Tests 532
Biotechnology and Diagnoses of
Animal Diseases 533
Hybridomas Technology and Production
of Monoclonal Antibodies 534
Somatic Cell Fusion 534
Hybridoma Technology 534
Production ofMonoclonal Antibodies 535
Step I: Fusion and Culture of
Hybridomas 535
Step II: Cloning andPreservation of
Hybridomas 536
Step III: Production ofAntibody 536
Immunotoxins 538
Nucleic Acid Probe Technology 539
Disadvantages 539
Enzyme-Linked Immunosorbent Assays
(Elisa) 540
Brucellosis 540
Pseiidorabies 540
Bluetongue 540
Animal Vaccine Development and
Production 540
New Technologies 543
Vaccine Expression Systems 544
Expression ofViral Genes in
Eukaryotic Cells System 545
Use ofYeast as An Expression System 545
Control of Hepatitis-B Virus ThroughVaccines 545
Hepatitis-B: Plasma Derived Vaccine 546
Hepatitis-B: Genetically EngineeredVaccine 546
Microencapsulation in Medicine: a New
Approach 546
Polymeric Devices 546
Production of Human Insulin 547
Genetic Mapping for Human Health 548
Linkage Analysis is the Basis ofGenetic
Mapping 548
Chapter 23 550
ANIMAL HEALTH
BIOTECHNOLOGY
Wildlife Populations and Health 551
Immunity 552
Innate Immunity 553
Non Specific Defence Mechanisms 553
Phagocytosis 554
Animal Vaccine: Development and
Production 556
Advantages with Killed Vaccines 557
Disadvantages with Killed Vaccines 557
Advantages with Attenuated Vaccines 557
Disadvantages with Attenuated
Vaccines 558
Our Limitations 558
New Technologies 558
Vaccine Expression Systems 559
Expression of Viral Genes in
Eukaryotic Cells System 560
Use ofYeast as An Expression System 560
Control ofHepatitis-B Virus ThroughVaccines 561
Hepatitis-B: Plasma Derived Vaccine 561
Hepatitis-B: Genetically EngineeredVaccine 561
Microencapsulation in Medicine: a
New Approach 562
Production ofHuman Insulin 562
Types ofVaccines 563
Toxoids 564
Diphtheria Toxoid 564
Tetanus Toxoid 565
Diptheria, Tetanus and Pertusis
Vaccine (Absorbed) or D.P.T.
Vaccine 565
Interferon or Antiviral Substances 565
Nature oflnteiferon 565
xviii
Assay ofInterferon 565
Nature ofInterferon Genes 565
BiologicalActivity ofInterferon 566
Interference 566
Interference Within Inoculated Leaf 566
Synergism and Viruses 567
Chapter 24 569
ANTIMICROBIAL DRUGS AND
BIOTECHNOLOGY
Introduction 569
Chemolherapeutic Agents 569
Antibiotics 571
Antiviral Drugs 572
Antigungal 572
General Characteristics ofAntimicrobial
Drugs 573
Determination the Level of Antimicrobial
Activity 574
Minimum Inhibition Concentration
(MIC) 574
Minimum Bactericidal Concentration
(MBC) '. 575
How to Make a Concentration 575
Mechanisms of Action of Antimicrobial
Agents 575
Antimetabolitie Drugs 577
Factors Influencing the Effectiveness ofAntimicrobial Drugs 577
Other Antibacterial Drugs , 577
Sulfonamides or Sulfa Drugs 577
Quinolones 579
Antibiotics 579
Classification ofAntibiotics 579
Requirements ofAntibiotics 580
Mode ofAction ofAntibiotics 580
How Antibiotics Act? 580
Use ofAntimicrobial Drugs 581
Absorption, Metabolism and Extraction
ofAntibiotics 582
Route ofAdministration 582
Combinations ofAntibiotics 582
Doses ofAntibiotics 583
Demerits ofAntibiotic Therapy 583
Side Effects ofAntibiotics 583
General Principlesfor Use ofAntibiotics 5S3
Production ofAntiobiotics 584
Mode ofAdministration 584
Side Effects ofAntibiotics 584
Rational Use ofAntibiotics 585
Antibiotics and Plant Diseases 585
Antibiotics andAnimal Diseases 585
Antibiotics and Animal Feed 585
Antibiotics and Growth ofAnimals 586
Antibiotics and Food Preservation 586
Antibiotic Secretionfrom
Cyanobacteria 586
Antiviral Drugs 587
Hiv Drugs 567
Other Anti-Hiv Dnigs 588
Anticancerous Drugs 590
Chapter 25 591
APPLICATIONS OF ANIMAL
BIOTECHNOLOGY
Livestock Breeds and Their Productivityin India 591
Improving Livestock Production Under
Desert Conditions 592
Livestockfor Meat Production 592
Genetic Improvement Programme 593
Breeding Policy and its Implementation 593
Transgenic Animal Technology 594
Transfectgenic and SomatotransgenicAnimals and TheirApplications 596
Applications to Production and
Therapeutics 596
Genuine Transgenic Animals and Their
Applications 596
Enhanced and Tissue-Specific
Transgene Expression 597
xix
Production ofSheep TransgenicforGrowth Hormone Genes 598
Production ofTransgenic Cattle byPronuclear Injection 599
Methodsfor the Introduction ofRecombinant DNA Into Chicken
Embryos 601
Present States of Art 601
Gene Transfer by Microinjection 602
Other Methods 602
Use for Medical and Therapeutic Purposes 602
Genetic Engineering- Single Genes 604
Gene Mapping 604
Artificial Insemination and Germ Cell
Storage 605
Ectogensis 606
Amniocentesis 606
Significance ofAmniocentesis 607
Transplantation 607
Cloning Technique 607
Gene Therapy 608
Main Approaches ofGene Tiierapy 608
Genetic Counselling 611
in Vitro Fertilization and Embryo Transfer 613
Surrogate Motherhood .614
In Vitro Fertilization and Embryo
Transfer for Improved Breeding
Programmes 614
Success ofEmbryo Transplantation 615
Objectives and Applications ofEt 616
Superovulation 617
Physiological Basis of Superovulation 677
Factors Which Influence the
Superovulatory Response 617
Superovulation Using Pmsg 618
Freezing ofEmbryos 618
Frozen Embryos 619
Embryo Sexing 619
Success in Pre-Detennining the Sex 619
Micromanipulation ofEmbryos 620
Advantages ofCell Manipulation
Technique 620
Simple Method ofSplitting Embryos 621
Techniques of Nuclear Transplantation 621
Sources ofEggs and Embryos 621
Gene Injection (Introduction of ForeignCloned Genes) 622
Dense Cytoplasm and Gene Injection 622
Selective Animal Breeding 622
Intensity ofSelection and ReproductiveRate 622
Potential 623
Hazards of Artificial Breeding 623
Animals as Tennis Balls? 624
Down on the Farm 625
Time to Decide 625
Chapter 26 626
TRANSGENIC ANIMAL
TECHNOLOGY
The Concept of Transgene and
Transgenics 627
Integration of Microinjected SequencesInto the Embryo: a Case Study of Mice 628
Genotyping Transgenic Mice by PCR 630
Transgenic Outline 630
Rodent Cloning and Transgenesis 633
Expression ofForeign Genes in
Transgenic Mice 633
Specific Applications of TransgenicMice ...633
Transgenic Mice as Modelsfor Genetic
Engineering 635
Use of Transgenics in Animal Agricultureand Research Models 636
Transgenic Swine 636
Production of Sheep Transgenic for
Growth Hormone Genes 637
Insertion ofGenes 637
Expression ofTransgenes 638
Production ofTransgenic Cattle byPronuclear Injection 639
Collection ofEmbryos 639
Pronuclear Injection 639
xx
Preparation ofDNA 640
Culture and TransferofEmbryos 640
Collection and Analysis ofTissue 640
Methods for the Introduction of
Recombinant DNA Into Chicken
Embryos 641
Problems After Developing TransgenicAnimals 641
The Nature ofPatents 647
Animals as Tennis Balls? 642
Down on the Fann 642
Time to Decide 643
Problems After Developing TransgenicAnimals 643
The Nature ofPatents 643
Chapter 27 644
BIOTECHNOLOGY IN
FISHERIES
Genetic Requirements for Transgenic Fish
Production 648
Transgenic Constructs 648
Selection ofFish Species 649
Gene Transfer Technology in Fish 650
General StepsforDeveloping
Transgenic Fishes 651
Gene Transfer by Microinjection 651
Electroporation 656
Transfer ofTransgenes by Infectionwith Pantropic Retroviral Vectors 658
Fish Antifreeze Protein Gene Promoter
in the Production of GrowthHormone 658
Characterization of Transgenic Fish 665
Identification ofTransgenic Fish 665
Expression ofTransgenes 665
Patterns ofTransgene Integration ...667
Inheritance ofTransgenes 668
Application of Transgenic Fish in
Biotechnology 668
Biosynthetic Growth Hormone 668
Gene Transfer in Common Carp and
Channel Catfish 670
Future Prospectives 671
Future Tiirust Areas in Fish Gene
Transfer Technology 671
Other Methods 672
Chapter 28 673
BIOTECHNOLOGY AND
TRANSGENIC PLANTS
Advantages of Transgenic Plants 674
Other Uses 674
Medical and Veterinary Applications 675
Transgenic are Not Terminator Crops 675
Green Revolution 676
Genomics and Transgenic Plants 677
Gm Crops: Global Area Coverage 677
Genetic Engineering and TransgenicPlants 678
Uses of Transgenic Plants 679
Genetic Engineering and Plant Lipids 684
Techniques of Producing TransgenicPlants 686
Agrobacterium Mediated Gene
Transfer 688
Cloning in Bacteria and Eukaryotes 691
Gene Transfer Technology 692
Agrobacterium Tumefaciens: An
Important Vectorfor Genetic
Engineering 692
Artificial Delivery Systems 694
Expression of Induced Genes in
Transgenic Plants 694
Reporter Genefor Transient
Expression 695
Problems Involved in Gene Transfer 695
Scopes and Achievements 695
Our Need 695
Recommendations and Priorities for
Future Research 696
Priorities for Future Research 697
xxi
Alarming Issues Related to Transgenic
Plants 698
Adoption ofTransgenic Seeds: Profit or
Loss 698
Who Has Been Benefited? 699
Legal Rights in the New Biotechnology 700
Transgenic Trees 700
Transgenics and Human Health 701
Chapter 29 702
BIOTECHNOLOGY AND
FORESTRY
Selection of Trees for Indian Forestry 703
Forest Resources 704
Specific Recent Technologies Relevant to
Forestry 705
Clonal Propagation Including Tissue
Culture 705
Ciyopreservation and in vitro Storage 705
in Vitro Selection 705
Rapid Propagation of Some Forest Trees 707
Rapid Propagation ofEucalyptusTereticomis Sin : 707
Direct Somatic EmbryogenesisfromImmature Seeds ofRosewood
(Dalbergia latifolia Roxb.) 709
Micropropagution ofButeamonospenna (Lam) Taub 709
Molecular Genetic Markers 710
Genetically Modified Trees and Genetic
Transformation 711
Other Techniques 711
Microbial Breakdown of Cell Wall
Components 712
Applications of Biotechnologies to Forest
Soil Microbiology 712
Issues Surrounding the Use of Forest
Trees from Biotechnology 713
Issues Associated with Specific Types of
Traits for Trees 713
Herbicide Resistance 713
Reduced Flowering or Sterility 714
Insect Resistance 714
Wood Property Chemistry 714
Can Genetically Modified Trees Be Safely
Deployed in the Environment? 715
Equity of Access to Genetic Modification
Biotechnologies 716
Public Acceptance of GeneticallyModified Plants 716
Future Challenges 717
Chapter 30 718
BIOTECHNOLOGY OF
BIOFERTILIZERS
PRODUCTION
Biofertilisers/Microbial Inoculant 718
Types of Biofertilizers 719
Rhizobiuim Inoculant 779
Azotobacter Inoculant 725
Azospirillum Incoculant 726
Blue-Green Algae Inoculant 726
Azolla-Anabaena Symbiosis 727
Phosphate Mobilising Biojertilizers 727
Large Scale Inoculum Production 728
Future Thrusts 729
Nitrogen Transfer Between Nitrogen
Fixing and Non-Nitrogen Fixing
Mycorrhizal Plants 729
Symbiosis 729
Mycorrhizal Symbiosis 730
Nitrogen Transfer in MycorrhizalPlants 734
Nitrogen Nutrition in MycorrhizalPlants: 734
Leguminous Plants /Rhizobiaceae
Symbiosis 735
Non-Leguminous Plants / Frankiaceae
Symbiosis 736
Benefitsfrom Nitrogen Fixing Plants to
Non-Nitrogen Fixing Plants 737
xxii
Nitrogen Fixing Trees and Forest
Management 737
Mechanism of Nitrogen Fixation 738
Diazotrophic Microorganisms 740
Biological Nitrogen Fixation 741
Asymbiolic Nitrogen Fixation 741
Microorganisms 741
Mechanism ofAsymbiotic Nitrogen
Fixation 742
Requirements ofNitrogen Fixation 743
Symbiotic Nitrogen Fixation 743
Mechanism of Nitrogen Fixation in
Legumes 743
Theory ofVirtanen 744
Theory ofBurris and Wilson 744
Nitrogen Converters in the Soil 745
Cyanobacteria and Nitrogen Fixation 745
Nitrogen Fixing Fungi 746
Biochemical Aspects of Diazotrophy 746
Nitrogenase Producing E. Coli Cells 747
Genetics of Free-Living and SymbioticDiazotrophs 748
Organization ofNitrogen-FixationGenes 748
Nod-Genesfor Nodulation 749
Regulation of Nitrogen-Fixation Genes 749
Genetics of Symbiotic Diazotrophs 750
Transfer ofM/-Genes to Microorganisms 750
Transfer ofM/"-Genes and Development
of New Nitrogen-Fixing Plants 750
Nitrogen Fixation Research in India 752
Chapter 31 754
ENZYME AND
BIOTECHNOLOGY
Industrial Sustainability 754
Green Plastics 755
Biocatalysts 756
Properties ofEnzymes 759
Enzyme Classification 760
Enzymes as Chemo-Thermo Regulators 761
What Enzymes Do? 762
What is Enzyme Kinetics? 763
How Enzymes Work 763
Enzyme Activity 764
Multi-Substrate Enzymes 768
Enzyme Optima 769
Enzyme Inhibitors, Activators and
Inactivators of Enzymes 769
Reversible and Irreversible Inhibition 777
Enzyme Secretion by Plant Cells 771
The Mechanism of Protein Secretion 772
The Control ofSecretion 772
Sources of Enzymes 772
Microbial Enzymes 773
Mechanism of Enzyme Biosynthesis in
Microbial Cells 774
Induction 775
Repression 775
Catabolic Repression 775
Transcription Control During the
Growth Cycle 775
Genetics of Enzyme Formation 775
Isoenzymes 776
The Cause ofEnzyme Multiplicity 777
Primary or Genetic Isoenzymes 777
Isoenzymes Due to Multiple Genetic
Loci 777
Secondary or Post-Translational
Isoenzymes 778
Chapter 32 779
PRODUCTION OF ENZYMES
AND FERMENTATION
TECHNOLOGY
Solid Substrate Fermentation 779
Submerged Fermentation 780
Extraction ofEnzyme 783
Purification ofEnzyme 784
Production of Enzymes by Fermentation 786
xxiii
Oxidoreductases 786
Oxidases 787
Hydrolases 788
Penicillin Amidases (Penicillin Acylase)797
Transferases 797
Glycerol Kinase 792
Glucose Isomerase 792
Applications of Enzymes 793
Applications ofEnzymes in
Phaniiaceulical Industries 794
Applications in Therapeutics 795
Clinical Analysis 796
Chapter 33 799
ENZYME IMMOBILIZATION
Important Features EnzymeImmobilization 799
Disadvantages of Enzymes 800
Advantages ofImmobilized Enzymes 800
Properties ofImmobilized Enzymes 800
Types/Methods ofImmobilization 801
Choice ofImmobilization Method 805
Comparison and Evaluation ofImmobilization Techniques 806
Effects ofImmobilization on Kinetics
and Properties ofEnzyme 606
Immobilization Techniquesfor Cells 808
Covalent Binding 808
Selection of Suitable Strains and Mutants
of Microorganisms 809
Bacillus and Aspergillus as EnzymeSources 809
Nutrient MediaforEnzyme Production 809
Applications of Microbial Enzymes 810
Chapter 34 812
INDUSTRIAL FERMENTATION
TECHNOLOGY
The Components ofa Fermentor 813
Parts ofFermenturs 673
Peripheral Parts and Accessories 675
Alternative Vessel Designs 675
AdditionalAccessories and Peripherals 817
Types ofReactors 676
Different Types ofFermentorInstrumentation 627
Common Measurement and Control
Systems 623
Simple Continuous Culture 830
Fermentor Preparation and Use 637
Maintenance ofAseptic Conditions 836
Transfer of Oxygen 836
Chapter 35 839
ENZYME-BASED INDUSTRIAL
FERMENTATION
TECHNOLOGY
Induction ofFomentation Enzymes 539
Feedback Repression 840
Catabolic Repression 840
Hyperproduction by DeregulatedMutants - Gene Dosage 840
Production of Alcoholic Beverages 841
Wine 841
Beer 842
Whiskey 843
Miso : 843
Organic Acid Fermentation 843
Production of Vitamins 846
Vitamins: General Aspects 847
Vitamin B Complex 848
Vitamin B2 (Riboflavin) Production 850
Vitamin C (AscorbicAcid) 657
xxiv
Commercxial Production of Antibiotics 854
Screening ofAntibiotic Producers 855
^-Lactam Antibiotics ..855
Aminoglycoside Antibiotic 863
Tetracyclines 865
Chloramphenicol 866
Griseofitlvin 867
Macrolide Antibiotics 868
Rifamycins 869
Animal Feed Production and Medication
Types 870
Veterinary Therapeutics 870
Prophylactics 871
Growth Promoters 871
Animal Feed Types 872
Gibberellic Acid 872
Enzymes in Food Industry 875
Bitterness in Orange and Grape Fruit
Juices. 876
Chapter 36 977
APPLICATIONS OF ENZYMES
IN INDUSTRES
Applications in Pharmaceutical Industries 877
Synthesis ofAmino Acids 878
Synthesis ofNucleosides and
Nucleotides 878
Synthesis ofOther Compounds 878
Applications in Therapeutics 879
for Treatment ofGaucher's, Tay-Sach's, and Fabry's Diseases 879
Clin>il Analysis 880
Transferases 881
Ornithine Transcarbamylase 881
Creatine Kinase 881
Oxidoreductases or Dehydrogenases 881
Hydrolases.. 881
Chapter 37 883
BIOTECHNOLOGY
BIOMASS ENERGY
Renewable Energy 884
Composition of Biomass 885
Cellulose 885
Hemicellulose 886
Lignin 886
Types of Biomass 886
Terrestrial Biomass 886
Aquatic Biomass 886
Wastes as Renewable Source of Energy ....887
Types of Wastes 887
Composition of Wastes 888
Sources of Wastes 888
Industries 888
Agriculture 889
Methods for Energy Production 892
Pyrolysis of Wood 892
Gassification ofWood 893
Bariquetting S93
Liquefaction 893
The Biological Process (Byconversion) 893
Enzymatic Digestion 894
Anaerobic Digestion 894
Aerobic Digestion 895
Hydrolysis of Wood Elhanol 895
Etlianol Productionfor Energy 895
Conversion ofMethane Into the SyntheticGas 898
Factors Affecting Methane Formation 900
Tlie Experience ofIndia and China 901
Biogas Technology 901
Generation ofBio Gasfrom Cotton
Dust 902
Composition ofBiogas Slurry 904
Biogas Slurry for Agriculture 905
Energy and Fuel Using Microorganisms 909
Hydrogen as Energy Source 909
Production ofHydrogen 909
xxv
Petroplants 912
Hevea Rubber 912
Euphorbia 912
Guayuk and Russian Dandelion 913
Aak (Calotropis procera) 913
Jatropha Curcas 913
Algal Hydrocarbons 913
Chapter 38 915
BIOTECHNOLOGY
ENVIRONMENTAL
PROTECTION
Pollution - a Global Problem 915
How Does It Work? 916
Biotechnology and Pollution Control:
Some Aspects 916
Water Pollution 917
Microbiology ofLake 917
System ofSaprophytism in Stored
Water 918
Sewage 918
Biological Oxygen Demand 919
Bod Sensor .'919
Immobilised Microbial Cells and Waste
Water Treatment 920
Whole Cell Enzymes and
Biodegradation 921
Immobilised Cells and Enzymes for
Waste Water Treatment 921
Nitrate Reduction 921
Oxidation ofAmmonia in Waste Water 921
Biological Oxygen Demand (BOD) 922
Wheat Strach and Paper Mill Industry 922
Pulp Mill 922
Degradation ofPhenols 922
Methane Formation 922
Glucose Production 922
Lactose Hydrolysis 92J
Detoxificationfrom Cyanide 92J
Sewage Disposal and Treatment 923
Sewage Disposal 923
Physical Treatment 923
Biological Treatment 924
Septic Tanks 924
Trickling Filters 924
Oil Recovery 924
Oil Pollution 925
Air Pollution 926
Pesticides and Herbicides Pollution 926
Degradation of Herbicides, Insecticides
and Pesticides 926
Heavy Melals 926
Toxic Metal Accumulation 927
Microbes in Leaching of Metals 927
Recovery of Copper and Cobalt byBiopolymer Gels 927
Decay of Organic Matter 927
Plastic Industry 927
Chapter 39 929
BIOSENSORS, BIOCHIPS,
BIOFILMS AND
BIOSURFACTENTS
Biosensors 929
Analysis ofOrganic Compounds 930
Medical Sciences 930
Industrial Uses 930
Environmental Analysis (Protection) 930
Conventional Biosensor 931.
Microbial Biosensor 931
Development ofMicrobiosensor 932
Urea Sensor. 933
Alcohol Sensor 934
Biosensors Using Amorphous Silicon
Isfet : 935
Hypoxanthine and Inosine Sensor 935
Micro-Oxygen Electrode 936
Glucose and Carbon Dioxide Sensors
Using Micro-Oxygen Electrode 936
Integrated Multibiosensor 937
xxvi
Novel Biosensor Based on New
Transducers 937
Image Sensor. 937
Biochips 938
DNA Chip and its Technology 939
Structure 939
Biolllms 941
Biosurfactents 941
Chapter 40 942
BIOTECHNOLOGY AND
BIODIVERSITY
CONSERVATION
Biodiversity at Global Level 943
Biodiversity at Country Level 944
Animal Wealth 944
Species Diversity and Ecosystem Stability 945
Loss of Biodiversity: a Global Crisis 946
Listing of Threatened Biodiversity 947
ThreatenedAnimals 948
Causes for the Loss of Biodiversity 949
Proximate Causes 949
Biotechnology for Bioconservation of
Diversity 951
Biodiversity Conservation Methods 951
Conservation Methods 952
In-Situ Conservation 952
Ex-Situ Conservation 953
Biotechnology, Biodiversity and
Intellectual Property Rights (IPR) ..........954
Productivity and Diversity 954
Biodiversity- Means ofProduction or
Product 955
Politics ofPatents and Intellectual
Property Rights 955
Bio-Safety Protocol 957
Socio-Economic and Political Causes 957
Chapter 41 958
BIOTECHNOLOGY AND
ETHICS
Ethical and Practical Problems 959
Bioethics 960
Gene Therapy 961
Germ Line Gene Therapy Moratorium 967
Medical Privacy and Genetic
Discrimination 961
Xenotransplantation 963
Bio Statement of Principles 964
Transgenic Plants: Bioethical and Moral
Concerns 966
Ethical Concerns 966
Black Face of Biotechnology? 966
Chapter 42 968
BIOINFORMATICS
Sequencing DNA, RNA and Proteins 968
Terchniques for Determinatrion of
Proteins 969
Pattern Databasesfor Protein Families 970
Principles of Genome Annotation 970
Bioinformatics: Internet and Www 971
Database Searching and Available
Database 972
Annotated Sequence Database 973
Genome and Organism-SpecificDatabase 974
Miscellaneous Databases 974
Data Retrieval with Entrez and DbgetfLinkdb 975
Data Retrieval with Sequence
Similarity Searches (SRS) 975
Transcription Regulatory RegionsDatabase (TRRD) 976
Applications of Bioinformatics 986
Bioinfonnatics in Study of
Phylogenetics 987
xxvii
Bioinformatics Helps in Building
Phylogenetic Trees 987
Bioinformatics and Molecular
Phylogeny 988
Bioinformatics in Discovery ofDrugs 988
Pharmainformatics 989
Cheminformatics 989
Protein Interaction Informatics 997
Protein Interaction Databases 997
Chapter 43 992
NANOTECHNOLOGY
Basic Principles 993
Structural DNA Nanotechnology 993
Convergent Assembly 994
Important Characteristics 996
Brownian Assembly ,997
Molecular Manufacturing 998
Computers 999
Strong Materials 999
High Power-Density Motor-Generators 999
Destruction ofPathogens and Cancer
Cells : 999
Building More Molecular
Manufacturing Systems 999
Decisive Military Capabilities 7000
Molecular Mills 1001
Applications of Nanatechology 1002
Selected Bibliography 1004
Glossary 1025
Appendix: Some InterestingWorld Wide Web Sites 1048
xxviii
