Cloning Of The Arabidopsis Npr1 Gene And Study The Genetic Transformation Of Citrullus Vulgaris

Experiments of obtaining more favorous watermelon were made with cultured Cotyledons of homozygous variety " HeShan" by combining plant tissue culture with Agrobacterium mediated transformation. In this research, we cloning of the Arabidopsis NPR1 gene, establishment of high efficiency regeneration system, determination of parameters involved in Agrobacterium mediated transformation ,Screening and characterization of transforming were systematically research .The results were summarized as follow: The NPRl gene was amplified from Arabidopsis thaliana genome DNA by DNA-PCR method. The DNA sequenced analysis showed that the sequence of amplified NPRl gene was the same as the published sequence. Therefore,the plant expression vector pCaMVNPR containing NPRl gene was constructed. The experiment will provide avenues for NPRl gene application in plant disease resistant genetic engineering. Success in establishment of high efficiency regeneration system was fundamental to the experiments. Many factors influenced regeneration frequency, such as explants varieties, explant age, and kinds of hormone. Cotyledons which aged 3~5days were the best explants. BA is a necessary factor to induce the differentiation of adventitious shoots, but the hormone concentration is unimportant. Add BA to the inducing medium, the explants can regenerate adventitious shoots. By this means, improve the regeneration and shorten the time of the regeneration. Adventitious will be observed in 8~12 days. The best induce inducing medium is MS+ BA1.0mg/ L to Cotyledons and to terminal bud, MS+ BA0.2mg/ L is the best. The ration of the regeneration is 96% with 100%. The Hygromycin was applied as a selectable marker at various concentrations. The evaluation of toxic damage indicated that lethal concentration of Hygromycin was>20mg/L, It caused the callus growth retardation and the lowest shoot regeneration frequency. The Agrobacterium-mediated transformation has been deep studied and worldwide for introducing foreign genes into different species, which is the best genetic transformation for the Dicotyledoneae. Effects the period of preculture, bacterial infection and co-cultivation on the transgenic efficiency were investigated. The results showed that the optimized period for preculture was one day, for bacterial infection was 10~15 minutes, and for co-cultivation three days. We used PCR to detect the transforming plantlets, four transformants were observed .the integration of target gene into watermelon genome primarily.