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Development of Strategies for the Constitutive and Tissue Specific Expression of Cry1A(c) Conferring Resistance to Lepidoptera Insect Pests in Transgenic Rice (Oryza Sativa L.)

Dissertation Title第2-3页
Sponsorship第3-11页
Certification第11-12页
Declaration第12-13页
Acknowledgement第13-15页
Abstract第15页
Part-I: Review第18-78页
    Chapter-1 Transgenic Crops: Current Challenges and Future Perspectives第18-39页
        1.1 Introduction第18-19页
        1.2 Mechanisms by Food Hazards may arise during the genetic engineering of crops第19-21页
            1.2.1 Inserted transgenes and their expression products第19页
            1.2.2 Secondary and pleiotropic effects of gene expression第19-21页
        1.3 Assessment of risks relative to traditional crop breeding第21-23页
            1.3.1 Inserted genes and their expression products第21-22页
            1.3.2 Secondary effects of gene expression第22-23页
        1.4 Hazards associated with some commercially important transgenes第23-32页
            1.4.1 Herbicide resistance transgenes第23-24页
            1.4.2 Insect resistance transgenes第24-26页
            1.4.3 Use of selectable marker genes第26-29页
            1.4.4 Grain quality alterations第29-30页
            1.4.5 Non target effects第30-32页
        References第32-39页
    Chapter-2 Approaches to Pest Control for Plant protection by Genetic Engineering第39-56页
        2.1 Need for approaches第39-40页
        2.2 Lectins第40-41页
        2.3 Arcelin第41页
        2.4 Amylase inhibitors第41-42页
        2.5 Ribosome- inactivating proteins (RIPs)第42页
        2.6 Cytokinin-mediated insect resistance第42-43页
        2.7 Lipid Oxidases- potent insecticidal proteins第43-44页
        2.8 Vegetative insecticidal protein from Bacillus thuringiensis,VIPs第44页
        2.9 Proteinase inhibitors第44-46页
        2.10 Other novel genes第46-47页
        Rreferences第47-56页
    Chapter-3 Bacillus thuringiensis and its application in agriculture第56-78页
        3.1 Historical background第56-57页
        3.2 Genetic manipulation of Bt第57页
        3.3 Mode of action第57-59页
        3.4 Diversification of genes encoding 8-endotoxins第59-60页
        3.5 Specificity and durability第60-61页
        3.6 Achieving adequate expression of Bt. in crop plants第61-63页
        3.7 Field trial testing of Bt. crops第63-67页
        3.8 The future of transgenic Bt. expressing plants第67-68页
        3.9 Other bacterial insecticidal proteins第68页
        References第68-78页
Part-II第78-161页
    Chapter-4 Assessment of role of various key factors involved in the culture system of Rice (Oryza sativa L.)第78-97页
        4.1 Introduction第78-79页
        4.2 Materials and methods第79-81页
            4.2.1 Plant materials and callus culture conditions第79-80页
            4.2.2 Callus induction第80页
            4.2.3 Plant regeneration第80-81页
        4.3 Results第81-91页
            4.3.1 Callus induction第81-88页
                4.3.1.1 Effect of growth regulators on embryogenic callus induction第82-85页
                4.3.1.2 Effect of sucrose concentrations on embryogenic callus induction第85-86页
                4.3.1.3 Comparison of carbon source on embryogenic callus induction第86页
                4.3.1.4 Impact of Casien Hydrolysate on callus Browning in XC-95第86-87页
                4.3.1.5 Effect of gelation types on embryogenic callus induction第87-88页
            4.3.2 Plant regeneration第88-91页
                4.3.2.1 Effect of phyta gel strength on shoot regeneration capacity第88-89页
                4.3.2.2 Effect of cytokinin concentration on shoot regeneration第89-90页
                4.3.2.3 Plantlet establishment第90-91页
        4.4 Discussion第91-94页
        References第94-97页
    Chapter 5 Development of Transgenic Rice Plants Expressing Resistance to Lepidoptera Insect Pests第97-145页
        5.1 Introduction第97-101页
        5.2 Materials and methods第101-112页
            5.2.1 Plant Materials and Tissue Culture Conditions第101页
            5.2.2 Transformation Vectors第101-102页
            5.2.3 Preparation of Competent Agrobacterium Cells第102-105页
            5.2.4 Transformation of plasmid DNA into Agrobacterium第105页
            5.2.5 Transformation Strategy; co-cultivation, selection and plant regeneration第105-106页
            5.2.6 Factor evaluation第106-107页
            5.2.7 PCR analysis第107-109页
                5.2.7.1 Mini-preparation of Genomic DNA第107-108页
                5.2.7.2 Thermal Reaction第108-109页
            5.2.8 RT (Reverse transcriptase)-PCR Analysis第109页
                5.2.8.1 RNA extraction第109页
                5.2.8.2 Reaction conditions第109页
            5.2.9 GUS Histochemical assay第109-111页
            5.2.10 Comparison of Morphological traits and Progeny Testing第111页
            5.2.11 Insect Bio-assay第111-112页
                5.2.11.1 Stripe stem borer(Chilo suppressalis)第111页
                5.2.11.2 . Rice Leaffolder {Cnaphalocrocis medinalis (Guenee)第111-112页
            5.2.12 Quantification of CrylAc Protein第112页
            5.2.13 Data analysis第112页
        5.3 Results第112-135页
            5.3.1 Plasmid DNA Transformation frequency studies in Agrobacterium第112-113页
            5.3.2 Factors affecting plant tissue transformation efficiency mediated by Agrobacterium第113-120页
                5.3.2.1 Effect of virulence induction medium (VIM)第113-115页
                5.3.2.2 Significance of Acetosyringone , a phenolic inducer compound第115-116页
                5.3.2.3 Impact of relationship between bacterial density and co-cultivation time第116-117页
                5.3.2.4 Effect of pH第117-118页
                5.3.2.5 Effect of carbon source type第118-119页
                5.3.2.6 Effect of Photoperiod conditions during co-cultivation第119-120页
            5.3.3 Comparison of different selection procedures on transformation efficiency第120页
            5.3.4 PCR and RT- PCR analyses for transgenes in the putative transgenic plants第120-121页
            5.3.5 Inheritance of the selective gene第121-122页
            5.3.6 Rl Agronomic performance analysis第122-123页
            5.3.7 Insect Bio-Assay第123-127页
                5.3.7.1 Strip stem borer (Chilo suppressalis)第123-125页
                5.3.7.2 Rice Leaffolder (Cnaphalocrocis medinalis -Guenee)第125-127页
            5.3.8 Quantification of CrylA(c) ICP levels 110 5.4 Discussion 111 5.4.1 Insect Resistance Evaluation第127-135页
        References第135-145页
    Chapter-6 Characterization of relative expression pattern of constitutive and tissue specific promoters in the transgenic Rice (Oryza sativa L.)第145-161页
        6.1 Introduction第145-146页
        6.2 Materials and methods第146-149页
            6.2.1 Plant materials第146页
            6.2.2 Transformation vectors第146-147页
            6.2.3 Transformation of plasmid DNA into Agrobacterium第147页
            6.2.4 Transformation Strategy; co-cultivation, selection and plant regeneration第147页
            6.2.5 PCR analysis第147-148页
            6.2.6 Substrates第148页
            6.2.7 Histochemical assay第148页
            6.2.8 Fluorometric assay第148-149页
                6.2.8.1 Lysis conditions第148-149页
                6.2.8.2 Reaction第149页
            6.2.9 Data analysis第149页
        6.3 Results第149-155页
            6.3.1 Production of transgenic rice lines第149-150页
            6.3.2 Molecular analysis of transgenic clones第150页
            6.3.3 Activity of Promoters in various vegetative tissues of transgenic lines第150-153页
                6.3.3.1 Strength of CaMV 35S promoter第150页
                6.3.3.2 Strength of tissue specific promoter第150-152页
                6.3.3.3 Overall Comparison of Promoters第152-153页
            6.3.4 Visualization of GUS activity in transgenic plants第153-155页
        6.4 Discussion第155-157页
        References第157-161页
Conclusion and perspectives第161页

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