Contents

Preface to the Third Edition

Preface to the First Edition

Abbreviations

List of color figures

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PART 1:PLANT TISSUE CUL TURE

1. INTRODUCTION

New technologiesOrigin of biotechnologyHistory

2. LABORATORYORGANIZATION

Washing facilityGenerallaboratory and media preparation areaTransfer areaCulturing facilities

Light unitsGreen housesLaboratory and personal safety

3. NUTRITIONMEDIUM

Units for solution preparationMedia composition

Inorganic nutrientsCarbon and energy sourceVitaminsGrowth regulatorsOrganic supplementsGelling agentPHProtocol: General methodology for medium preparation

4. STERILlZATIONTECHNIQUES

Preparation of sterile media, containers and small instrumentsSteam sterilization

Units of energy and pressure

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Dry sterilizationFilter sterilizationUltra violet sterilization

Maintenance of aseptic conditionsAlcohol sterilizationFlame sterilization

Sterilization of explantChemical sterilization

ProtocolsSterilization of seedsSterilization of buds, leaf, stem, roots, tubers, scales, etc.Sterilization of tissue for immature embryos, ovulesand flower buds for anther culture

5. TYPES OF CULTURE 33

CytodifferentiationVascular Differentiation

OrganogenicdifferentiationTypes of culture

Seed cultureEmbryo culture

Mature embryo cultureImmature embryo culture/embryo rescueApplications of embryo culture

Root cultureCallus cultureOrgan culture

Nucellus cultureApplication

Endosperm cultureApplication

Cell cultureProtoplast culture

ProtocolsProtocol for seed germination (Nicotiana)Protocol for embryo culture (Cereals-wheat, maize, barley rice, etc.)Protocol for embryo culture (Legumes-green gram, black gram,french bean, soybean, etc.)Protocol for callus induction (Nicotiana tabacum)Protocol for callus induction (Cereals-wheat, rice, maize, barley, etc.)

6. MICROPROPAGATION 49

Axillary bud proliferation approachMeristem and shoot tip cultureSud culture

Single node cultureAxillary bud method

Contents xiii

OrganogenesisOrganogenesis via callus formationDirect adventitious organ formation

EmbryogenesisAdvantages of micropropagationProblems associated with micropropagationProtocols

Meristem and node culture of potato (So/anum tuberosum)Proliferation ofaxillary buds (strawberry-Fragaria chiloensis)Organogenesis-adventitious shoot formationOrganogenesis vía callus formation (Nicotiana)Organogenesis via callus formation (Cereals-wheat, barley,maize, rice, etc.)Embryogenesis (carrot)

7. CELL SUSPENSION AND SECONDARY METABOLlTES 70

Types of suspension culturesBatch cultureContinuous culture

Open continuous cultureClosed continuous culture

Semi continuous cultureGrowth measurementsSynchronization of suspension culture cellsTechnique for single cell culture-Bergmann cell plating technique

ApplicationProduction of secondary metabolites

Morphological and chemical differentiationMedium composition for secondary product formationGrowth production patternsEnvironmental factorsSelection of celllines producing high amounts of a useful metaboliteProduct analysis

ApplicationProblems associated with secondary metabolite productionImmobilized cell systems

Polymers for immobilizationProduct release

BiotransformationSecondary metabolite production using genetically engineered plant cell culturesProtocols

Protocol for cell suspension culture (Nicotiana tabacum)Protocol for cell suspension culture (Cereals-wheat, rice,maize, barley, etc.)

xiv Introduction to Plant Biotechnology

8. IN VITRO PRODUCTION OF HAPLOIDS

Androgenic methodsAnther cultureMicrospore cultureThe various factors governing the success of androgenesis

GenotypePhysiological status of the donor plantsStage of polienPretreatment of anthersCulture media

Process of androgenesisThe ploidy level and chromosome doublingDiploidization

Significance and uses of haploidsProblemsGynogenic haploidsChromosome elimination technique for production of haploidsin cereals (Barley and wheat)Protocol

Protocol for anther culture of cereals (Rice, barley, wheat, etc.)9. PROTOPLAST ISOLATION AND FUSION

Protoplast isolationMechanical methodEnzymatic method

Physiological state of tissue and cell materialEnzymesOsmoticumProtoplast purificationProtoplast viability and densityCulture techniquesCulture medium

Protoplast developmentCell wall formationGrowth, division and plant regenerationSomatic hybridization

Protoplast fusionSpontaneous fusionInduced fusion methods

Treatment with sodium nitrateCalcium ions at high pHPolyethylene glycol methodElectrofusion

Mechanism of fusionIdentification and selection of hybrid cells

Chlorophyll deficiency complementation

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Auxotroph complementationComplementation of resistance markersUse of metabolic inhibitorsUse of visual characteristicsCompound selection system

Verification & characterization of somatic hybridsMorphologyIsoenzyme analysisChromosomal constitutionMolecular techniquesGenetic characterization

Chromosome number in somatic hybridsCybridsPotential of somatic hybridizationProblems and limitations of somatic hybridizationProtocol

Protocol for protoplast isolation and fu~ion10. SOMACLONAL VARIATION 130

NomenclatureSchemes for obtaining somaclonal variation

Without in vitro selectionWith in vitro selection

Factors influencing somaclonal variationFactors important during in vitro selection of somaclonal variants

Application of somaclonal variationBasis of somaclonal variationDisadvantagesGametoclonal variation

11. GERMPLASM STORAGE & CRYOPRESERVATION 146

CryopreservationRaising sterile tissue culturesAddition of cryoprotectants and pretreatment

Air Desiccation-FreezingPregrowth-DesiccationEncapsulation-DehydrationVitrificationAddition of cryoprotectants

FreezingStorageThawingDetermination of survival/viability

TIC method of stainingEvan's Blue staining

Plant growth and regeneration

xvi Inlroduclion lo Planl Biolechn,ology

Slowgrowth methodTemperatureLow oxygen pressureNutrient restrictionGrowth regulatorsOsmotics

AchievementsApplicationsLimitations

PART 11:GENETIC MATERIAL AND ITS ORGANIZATION

12. GENETIC MATERIAL

SugarsAmino acidsNucleotides

Structural formulae of nucleotidesNomenclature of nucleoside and nucleotide compounds

PolynucleotidesSignificance of differences between DNA and RNAShorthand notation for polynucleotide structure

Genetic materialDiscovery of DNADouble helix is a stable structureDNA replication

13. ORGANIZATIONOF DNA AND GENE EXPRESSIONDifferent forms of DNASupercoiling- Tertiary structure of DNA

Linking numberCruciform- Tertiary structure of DNAEukaryotic DNA organization into nucleosomesDNA contentDenaturation

DNA renaturationRenaturation rate and DNA sequence complexity-Cotcurves

Flow of genetic information: central dogmaOrganization of genes in DNA molecules

OperonMultigene family

Plant gene structure as discontinuous geneControl sequences

TATA boxAGGA boxOther regulatory elements

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Types of RNA moleculesMessenger RNA and processingRibosomal RNATransfer RNASmall nuclear RNA

TranscriptionNucleotide sequencesTranscription in prokaryotesTranscription in eukaryotes

Genetic code and translationGenetic codeTranslationPost-translational modifications

PART11I: RECOMBINANT DNATECHNOLOGY

14. BASICTECHNIQUES

Agarose gel electrophoresis

Pulsed field gel electrophoresisPolyacrylamide gel electrophoresis

Isoelectric focusing2-Dimensional gel electrophoresis

Nucleic acid blottingSouthern blot analysisNorthern blot analysis

Protein blottingWestern blot analysis

Dot blot techniqueAutoradiographyE. coli transformationProtocol

Agarose gel electrophoresis

15. GENE CLONING: Cutting and joining DNA MoleculesIntroductionEnzymes for cutting: Restriction endonucleases

Type 1, 11, 11I

Other restriction enzymesJoining DNA molecules

LigaseDNA modifying enzymes

KinaseAlkaline phosphataseTerminal transferaseDNA polymeraseS 1-Nuclease

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A-ExonucleaseExonuclease 111Bal31 nuclease

Linkersand adaptorsProtocol

Restrictive digestion of plasmid ONA16. GENE CLONING: Vectors

Cloning vectors-featuresBiology of E. coli K-12Plasmids

pBR322, pACYC184, pUC vectors, pUN121Yeast plasmid vectors

Yeast integrating plasmids, episomal plasmids, yeastreplicating plasmid, yeast centromeric plasmid, yeastlinear plasmid

Ti plasmidsCosmidsBacteriophagevectors

Biology of bacteriophage AA phage cloning vectors (Agt10,Agt11,EMBL3 and 4, Charon)M13 phage

PhagemidsYeast artificial chromosome (YAC)Bacterial artificial chromosome (BAC)P1 phage vectorsP1 derived artificial chromosome (PAC)TransposonShuttle vectorsExpression vectorsProtocols

Isolation of plasmid ONA: mini preparationIsolation of genomic ONA by SOS-Proteinase K method

17. GENE CLONING: cONA and Genomic Cloning and Analysis ofCloned ONASequences

Gene librariescONA cloning

Isolation of mRNASynthesis of first strand of cONASynthesis of second strand of ONACloning of cONAIntroduction to host cellsClone selection

Genomic cloningIsolation of ONA,Partial digestion

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Ligation ot tragment to a vectorPackaging)

Identitication and analysis ot cloned genesProbes

Approaches tor identitication ot genesColony and plaque hybridizationImmunological detectionSouthern blot analysis;

Detection ot nucleic acid sequencesRadioactive probe preparation and labeling

Nick translationRandomprimedPCR

Non-radioactivelabelingHorseradish peroxidase systemDIGBiotin-streptavidin

DNA sequencingSanger-CoulsonmethodMaxam-Gilbert methodHigh throughput DNA sequencing

18. POLYMERASE CHAIN REACTIONIntroductionProcedureComponents in a polymerase chain reactionInverse PCRReverse transcriptase mediated PCR (RT-PCR)

RACE: Rapid amplitication ot cDNA endsQuantitative RT-PCRAmplitication ot differentially expressed genes

Differential display reverse transcriptase PCRSuppression subtraction hybridization

Cloning ot PCR productsRestrictive site additionTIA cloningBlunt end ligationGenetic engineering by PCR

ApplicationsAdvantagesProblemsProtocol

PCR tor detection ot transgenes19. IN VITRO MUTAGENESIS

Site directed mutagenesisDeletion mutagenesis

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UnidirectionaldeletionsGap sealing mutagenesisLinker scanning mutagenesis

Cassette mutagenesisOligonucleotidedirected mutagenesis

Gappedduplex methoddUTP incorporation strategyPhosphorothioatemethod

Chemical mutagenesisPCR mediated in vitro mutagenesisRandom mutagenesis

InsertionalmutagenesisTransposon mediated insertional mutagenesisT-ONA mediated insertional mutagenesis

20. TRANSPOSON GENETIC ELEMENTS ANO GENE TAGGING 316

Transposable elements in bacteriaIS elementsComposite transposonsComplex transposons

Transposable elements in eukaryotesClassification

Class I elementsRetrovirusesLong terminal repeat retrotransposonsNon-LTR retrotransposons

Class 11elementsAc-Ds elementsEn/Spm transposable element family

Transposon gene taggingIsolation of transposable elementGene tagging: a) Non-targeted; b)TargetedCharacterization of transposon insertion mutation

Isolation of mutant geneIsolation of intact geneTransposon tagging in heterologous speciesSignificance (mutations, gene expression, vector development)

21. GENE ISOLATION 337

Chromosome mapsGenetic mapPhysical map

General strategies for cloning genes from plantsIsolation of genes coding for specific proteinsIsolation of genes which are tissue specific in functionCloning methods based on ONA insertionsSubtractive cloning

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Map based cloningIdentification of ONA markers linked to target gene andconstruction of genetic mapsConstruction of high resolution physical mapChromosomewalkingGene identification and testing by transformation

BLAST searchComparative sequence analysisGene expression analysisGenomic ONATransformationcONA transformationRNA interference (RNAi)

ChromosomejumpingChromosome landingapproach

22. MOLECULARMARKERS ANO MARKER-ASSISTEO SELECTION 356

MorphologicalmarkersBiochemical markersMolecularmarkersHistory of evolution of molecular markersNon-PCRbasedapproaches

RFLP (Procedure, construction of RFLP maps, uses of RFLPPCR based techniques

Random amplified polymorphic ONA(RAPO)ONAamplification fingerprinting (OAF)Arbitrarily primed polymerase chain reaction (AP-PCR),Amplified fragment length polymorphism (AFLP)Simple sequence repeats (SSR) or Microsatellites

Microsatellite directed PCR: unanchored primersMicrosatellite directed PCR: anchored primersRandom amplified microsatellite polymorphism (RAMP)Selective amplification of microsatellite polymorphic loci

Retortransposon-basedMarkersSequence-Specific Amplified Polymorphism (S-SAP):Inter-Retroransposon Amplified Polymorphism (IRAP)Retrotransposon-Microsatellite Amplified Polymorphism (REMAP)Retrotransposon-Based Insertional Polymorphism (RBIP)

Targeted PCR and sequencingSequence tagged sites

Sequence tagged microsatellites (STMs)Sequence characterized amplified regions (SCARS)Cleaved amplified polymorphic sequences (CAPS)Sequence related amplification polymorphism (SRAP)

Advantages of targeted PCR and sequencing approachesOisadvantages

Single Nucleotide Polymorphism (SNP)

xxii Introductlon to Plant Blotechnology

Molecular markers and plant diversityFingerprintingMarker assisted selection

Nearly isogenic line (NIL) strategyBulk segregant analysis (BSA)

ProtocolProtocol for RAPO analysis

23. GENE TRANSFER IN PLANTS

Transient and stable expressionMarker genes

ReportergenesOpine synthaseChloramphenicol acetyl transferaseBeta glucuronidase (GUS)Bacterialluciferase Lux F2Firefly luciferase LucGreen fluorescent proteinAnthocyanins

Selectable markersAntibiotic resistance markersAntimetabolite resistance markersHerbicide resistance markers

Chimeric gene vectorsGene transfer methods

Vector mediated gene transfer methodsAgrobacterium mediated ONAtransfer

Classification of AgrobacteriaTumor inducing principie and the Ti plasmidOrganization of Ti plasmidT-ONA transfer

Agrobacterium vectorsCointegrate vectorsBinary vectors

Transformation techniques using AgrobacteriumAgrobacterium mediated virus infection-Agroinfection

Viruses mediated gene transferCaulimovirusesGemini virusesRNA viruses

Vectorless or direct ONAtransferPhysical methods

ElectroporationParticle bombardment/microprojectile/biolisticsMacroinjectionMicroinjection

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Liposome mediated transformationSilicon carbide fiber mediated transformationUltrasound mediated DNAtransformationDNA transfer via pollen

Chemical methodsPEG mediated gene transferCalcium phosphate co-precipitationThe polycation DMSO techniqueDEAE dextran procedure

Status and expression of transferred genesGene silencing

Detection of intrusive DNACauses of gene silencing

DNA methylationHomology dependent gene silencingSuppression by antisense genesSilencing by RNA interferencePosition effectIncreased copy number

Strategies for avoiding gene silencingMethods for removal of marker genes

Co-transformationRemoval of marker genes by transposases

Transplastomics to overcome pollen gene transferModulation of marker gene expressionSelectable markers and safety

ProtocolsAgrobacterium mediated transformationGene transfer by biolistic method: transient expression

24. CHLOROPLASTAND MITOCHONDRIONDNA TRANSFORMATION 444

Chloroplast TransformationThe Plastid chromosomeSelectable markers

Dominant antibiotic resistance markers (Positive selection markers)Recessive selectable markers (Positive selection markers)Secondary positive selection markersNegative selection markers

Transformation methodsAgrobacterium mediated transformationParticle gun method

Gene replacementGene insertion

Other possible methods for introduction of DNA into plastidsPEG-mediated transformationGalistan expansion femtosyringe

xxiv Introductlon to Plant Blotechnology

DNA uptake by chloroplasts in vivoMicroinjection ot DNA into chloroplast in vivoDNA uptake by chloroplasts in vitro

Homoplastomic lines developmentAdvantagesApplicationsLimitations ot Chloroplast TranstormationMitochondrion transtormationTargetting ot Transgene Product into Chloroplasts and Mitochondria

25. TRANSGENICS IN CROP IMPROVEMENTResistance to biotic stresses

Insect resistanceResistance genes trom microorganisms

Bt toxin geneOther microorganism derived resistance genes

Resistance genes trom higher plantsProteinase inhibitors

Cowpea trypsin inhibitor gene (CpTI)a-Amylase inhibitor

LectinsResistance genes trom animals

Virus resistanceCoat protein mediated cross protectionNon-structural protein mediated resistanceAntisense and sense mediated resistanceSatellite RNA protectionDetective interfering RNAsPathogen targeted resistance

Mammalian oligoadenylate synthase geneRibozyme mediated resistance

Disease resistancePathogenesis related proteinsAnti-microbial proteinsEngineering toxin insensitivityPhytoalexinsManipulation ot disease resistance genes

Resistance to abiotic stressesHerbicide resistanceTransgenics tor quality

Transgenics tor improved storageLonger lite transgenic tlowersTransgenic tor tlower color and shapeTransgenics tor male sterilityTransgenics tor terminator seed

Mechanism in pure line seed productionTerminator technology tor use in hybrid seed production

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Transgenic plants as bioreactorsCarbohydratesLipidsProtein qualityEnzymesVitamin and mineralBiodegradableplasticProteins, peptides and vaccinesProduction of edible vaccine antigensAntibody Production in Plants (Plantibody)Production of Pharmaceutically Important Proteins in Plants

26. IMPACTOF RECOMBINANT DNA TECHNOLOGY

AgricultureStatus of transgenic cropsBenefits of transgenic cropsBiotech crops in IndiaInsect resistant plants through transgenic approach

AdvantagesLimitations/ApprehensionsStrategies for resistance management

Herbicidetolerant plants through transgenic approachArguments in favor of herbicide tolerance in crop plantsArguments against herbicide tolerant transgenics in crop plants

Plant based pharmaceuticalsProduction of biofuelsTransgenic crops in futureBiotechnology in Process IndustryBioremediationHealthcareapplicationsFuture

27. BIOSAFETYCONCERNS AND REGULATORY FRAMEWORK

Risk analysisRisk assessmentRisk managementRisk monitoringRisk communication

ContainmentBiological containmentPhysical containment

Biosafety levelsGood laboratory practicesGood laboratory practicesRisk Management in Field Trials

Physical strategies for confinementBiological strategies for confinementOther proceduresGeneral precautions

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National Biosafety Regulatory framework in IndiaRecombinant DNA Advisory CommitteeInstitutional Biosafety CommitteeReview Committee on Genetic ManipulationGenetic EngineeringApproval CommitteeState Biotechnology Coordination CommitteeDistrict Level Committee

Recombinant DNA safety GuidelinesRevised Guidelines for Research in Transgenic PlantsCross border movement of transgenic germplasm for research purposesBiosafety concerns

Risk to human healthRisk of toxicityRisk of allergiesAntibiotic resistanceEating foreign DNAUse of promoters of virus originChanges in nutritionallevel

Risk to environmentGene flow 01'dispersal from transgenics

Strategies to Prevent Gene Flow of Transgene EscapeResistance/tolerance of target organismsGeneration of new live viruses/super viruses

RecombinationTranscapsidation

Ecological issuesIncl'easedweedinessCreation of super-weedsLoss of biodiversity/reduction of cultivarsNon-target effectsPersistence of the transgene 01'transgene productGM crops affect the purity of other cropsIncreased use of chemicalsEffect on Rhizosphere and microfloraUnpredictable gene expression 01'transgene instability

Socio-economic and ethical considerationsInternational protocols and conventions on biosafety

Cartagena Protocol on BiosafetyWTO and other International Agreements

Regulatory frameworks in different countriesUSAEuropeanUnionCanadaAustraliaArgentinaAsian Region

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28. GENOMICS

IntroductionMapping of prokaryotic genomes

E. coli genomeMapping of eukaryotic genomes

Construction of linkage maps with molecular markersGene librariesScreening of libraries and constructing contigs

;:Se€juencing1New vectors BACs and PACs and the shot gun approach

Gene location in DNA sequencesSequence inspectionExperimental techniques

Hybridization testscDNA sequencing

Yeast (S. cerevisiae) genomeHumangenomeArabidopsisgenomeRicegenomeFunctionalgenomics

Computer analysisExperimental analysis

Gene inactivationGene overexpression

Phage displayYeast two hybrid system

Patterns of gene expressionGene expression assays by measuring levels of RNA transcriptsSAGEDNA chip technology

Oligonucleotide based chipscDNA based chipsHybridization and detection methodsDouble stranded DNA chipsCharacteristic features of DNA chips/microarraysApplications of DNA chips

ProteomicsMetabolomics

TerminologyTarget analysisMetabolite profilingMetabolomic approachesMetabolic fingerprinting

Analytical TechniquesMetabolomics is an important tool for functional genomics

Phenomics

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29. BIOINFORMATICS

Development of BioinformaticsRole of Internet in BioinformaticsOnlineBrowsingworking and downloadingSequences and NomenclatureFile FormatsFiles of Structural dataSubmission of SequencesDatabases

Primary sequence databasesSubsidiary (Secondary) sequence databasesComposite databases

Molecular Biology DatabasesBioinformatics Databases and analysis Services

GenBankEMBLdbESTMitoNCBI's Molecular Modeling Database (MMDB)The conserved Domain DatabasePDB: The Protein Data BankSWISS-PROT and TrEMBLBLASTRPS-BLASTVASTVAST Search

Specialized ResourcesPlant databasesUK Crop NetGenoplante

Bioinformatics Databases and Analysis ServicesBioinformatics tools for access to databases

Text based databases searchingSimilarity based database searching

Global vs. Local AlignmentAlignment algorithmsSearch parameters

Visualization toolsIntegrated Molecular Biology Database systemDatabase integration strategies

DataWarehousingLink Driven Federation

EntrezSRSLinkDB.

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Contents xxix"Semantic Integration

Detection of genesApplicationsChallenges

30. INTELLECTUAL PROPERTY RIGHTS 626

Protection of intellectual propertyWorld organizationsForms of protection

CopyrightTrademarkPatent

Patent applicationInternational patenting and patent cooperation treatyUses of technical information in patent documentsRevocation of patentPatenting of biological material

MicroorganismsPlant patentAnimal patentCloningBiological compounds

Patenting Procedure in IndiaGeographical indicationsTrade secretDesignsLayout design of integrated circuits

Plant breeders rightsUPOVFunctions of UPOV

Breeders exemptionFarmers privilege

Plant variety protection in IndiaFarmer's rightsAdvantages of PBRDisadvantages of PBR

Convention on Biological DiversitySome case studies on plant patents

Patenting of basmati rice in USARevocation of turmeric patentRevocation of neem patent

Appendix 654

Glossary 657Author index 676

Subject index 683