| Pepper (Capshim annuum) is a kind of major vegetables in agriculture. During the recent three five-year-plans, China regarded breeding of pepper which can resist disease as the important project161. As the major harming pepper pest, Helicoverpa assulta, a kind of Papilio xuthus insect, whose larva feeds on flower, bud and fruit of pepper and can make lots of rot fruits to drop the yield. CryIA(c) gene, a kind of Bt genes expressing insecticidal crystal protein of Bacillus thuringiensis, codes 133kD crystal protein and just has a insecticidal scope of Papilio xuthus insect. So, the crylA(c) gene carried in the T-DNAs of binary vectors which contain nptll gene and gusA gene, were used to transform the explants of pepper via Agrobacterium transformation. Through stringent selection of the infected explants on the medium added with Kanamycin and following assays, the transgenic pepper plants were regenerated from the Kanamycin resistant explants. Selective breeding of pepper resistant to insects has active ecological significance and economical significance. The following is the results of experiments.1. Transform pKUC plasmid to two kinds of Agrobacterium tumefaciensThrough the way of direct transforming transform pKUC plasmid to EHA105 and LBA4404 Agrobacterium tumefaciens. The positive clones were selected by the medium added with Kanamycin. Enzyme cutting and electrophoresis confirmed that the pKUC plasmid was transformed to two kinds of Agrobacterium tumefaciens.2. Develop the in vitro culture system of high capacity and fast regeneration of explantsAfter compare, 12-16 days old seeds were confirmed to supply explants which have the strongest capacity of regeneration. Based on the MS medium, a series of medium were devised and selected by effect. The medium of inducingdifferentiation of explants is MS+ 6-BA5.0mg/L+IAA1.0mg/L; the medium of inducing elongation of sprouts is MS+ 6-BA3.0mg/L+ IAA1.0mg/L+GA31.0mg/L; the medium of inducing rootage of sprouts is 1/2MS+ IAA0.5mg/L. The inducing differentiation rate reached as high as 83.3%. The medium of inducing elongation of sprouts can effectively resolve the general problem of difficult to elongate the sprouts. The rootage rate reached 78%. The in vitro culture system of high capacity and fast regeneration of explants was successive developed in this experiment, which provide the base to transforming more genes of interest into pepper.3. Study on factors influencing Agrobacterium-mcdiated pepper transformationDuring transforming explants of pepper via Agrobacterium transformation, five factors, Agrobacterium type, genotype of pepper, explants, activation of vir gene culture medium and transformed methods, were set up to study the effect of Agrobacterium-mediatzd pepper transformation. Regarding the frequency of Km resistant callus cultureas standard , significant differences existed between Agrobacterium type and between genotype of pepper. When two days pre-cultured cotyledons were mixed with cell suspension of Agrobacterium in 5-7 minutes, the highest transformation frequency was obtained. The factors influencing Agrobacterium-mediaied pepper transformation were studied in systematism, in order to establish a universal high efficient and stability pepper gene transferring system.4. Assays on the transgenic pepperThrough stringent selection of the infected explants on the medium added with Kanamycin, eliminated some explants which without resistance to Kanamycin. Then the expression of GUS of the plants, regenerated from the Kanamycin resistant explants, were detected by histochemical method. Blue was found in organs of transformed plants. The results of molecular biological assays with PCR and Southern dot hybridization confirmed that the crylA(c) gene was integrated into genome of pepper. At last, the DNA sequence of special product of PCR wasanalyzed to confirm the integration status of gene to the genome. All assays proved that the transgenic pepper plants were gained in this experiment...
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