| So far, the history of transgenic crops can be included in two parts. The first era deals with the development of such novel crops. As the time went on, various social, environmental and technical issues related to transgenic crops took their birth. The genetic engineering of crop plants is now moving towards the course of correction. Of the agronomic traits improved by using transgenic plants, genetic resistance to pests has attracted considerable attention. Now a days, a number of approaches to pest control via genetic engineering have been developed and genetically engineered crops expressed insecticidal characteristics. Use of Bacillus thuringiensis genes encoding 5 endotoxins with insecticidal characteristics is the major approach. In the Part-I of this dissertation, various strategies to combat issues faced by transgenic crops, approaches for pest control by genetic engineering and the application of Bacillus thuringiensis 5 endotoxins have been reviewed with significant details.In the recent years, the insect-resistance duration and the bio-safety of transgenic insect resistant crops has become a hot issue for publics, and many breeders are trying to seek efficient ways such as the use of different promoters, integration of more than one transgenes with various modes of actions etc. to cope with such problems. In this study, we established an efficient Agrobacterium-mediated transgenic system in rice {Oryza sativa L.). With the objective of prolonging the insect-resistance duration and achieving the spatially regulated expression of CrylA(c), two plant expression vectors i.e. PCAMB- CrylA(c)-CpTi harboring the two insecticidal genes each driven by CaMV 35S promoter, and PCAMB- CrylA(c)-RSuS-l containing single insecticidal gene CrylA(c) under the control of a vascular tissue specific (phloem) promoter RSuS-1. Another vector construct bearing CrylA(c) with CaMV35S promoter had also been used in the transformation experiments to have an efficiency comparison with the former two constructs. The major results in the Part-II of dissertation can be summarized as below; 1- An attempt was made to optimize conditions for an efficient embryogenic callus induction and plant regeneration system from mature seeds of japonica and indica rice {Oryza sativa L.) cultivars, Xiushui-11 and XC-95, respectively. Different factors such as plant growth regulators, casein hydrolysate, carbohydrate source and concentration,and various gelation conditions including gel type for callus induction and phyta gel concentration for shoot regeneration were studied in detail to have a clear idea about their role in the system. Overall, induction of embryogenic callus in case of japonica rice was the most efficient (78.0%) in the media supplemented with 2mg/L 2-4-D, 3% sucrose and 0.8% agar as gelling agent while in case of indica rice, 2mg/l of 2-4-D combined with 0.5mg/l of BAP and NAA each generated the best result ( 61.2%). Inclusion of 0.3% agarose (pure form) or phyta gel further enhanced the callus induction efficiency ( about 95% and 71.0%) in Xiusui-11 and XC-95, respectively. Higher concentration (300-500mg/L) of casein hydrolysate was found to significantly inhibit the browning of calli in XC-95. Role of cytokinin and phyta gel concentration was critical for shoot regeneration in both classes of rice. Increased strength of phyta gel was observed to enhance the shoot regeneration frequency. The optimal concentration of 2mg/L and 3mg/L for KT was necessary for higher differentiation frequency supplemented with 0.5% and 0.6% phyta gel for japonica and indica rice, respectively.2-During the plant transformation efficiency studies in the japonica (Xiushui-11) and indica rice cultivar (XC-95), the role of various factors responsible for virulence of Agrobacterium such as virulence induction medium (VIM), acetosyringone (AS), cell density and co-cultivation time, pH, carbon sources and light was assessed to establish a better transformation system for the rice cultivars used in the experiments. MS and AB media (as VIM) for japonica and indica rice, respectively, 100M AS, A6oo 0.4-0.6 for 10-15 minutes, pH 6.0 (VIM and COM), use of sucrose or glucose and dark conditions during co-cultivation period were found to be ideal for transformation process.3- A number of transgenic lines were developed harboring the CrylA(c) gene either alone or in combination with Cp 77, driven by CaMV35S or a tissue specific promoter, RSuS-l, mediated by Agrobacterium tumefaciens genetic transformation in japonica and indica rice. PCR analysis and histochemical assay revealed the successful transformation event in the tested transgenic lines. The transgenic construct strictly followed the Mendelian inheritance principle (3:1) in the subsequent generations as demonstrated by hygromycin assay result.4- Results of insect bio-assay showed comparable or slightly better performance of transgenic lines, R-l and R-5 containing CrylA(c) gene under RSuS-1 promoter than... |
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