Study On Transformation Ofβ-1,3-Glucanases (BG2) And Chitinase (CH5B) Genes Into Watermelon

Watermelon[Citrullus lanatus(Thunb.) Mantsum.et Nakai] is an important horticultural crop and has been cultivated in the world for more than4000years. As a significant component of international fresh fruit trade, watermelon is preferred by most consumers because of their sweeter taste and aromatic odor.But pathogens have been identified and that cause million of metric tons of fruit are lost in fields to disease. Using traditional methods could not solve this problem effectively and many disadvantages still exist, such as environmental pollution, destruction of soil microorganisms. In addition, because of the narrow genetic background of watermelon, traditional breeding is difficult to improve resistent characters. With the development of biotechnology, genetic engineering technology offers a new way for improve new oriented varieties.In this study, a high frequency watermelon regeneration system had been developed using two inbred lines of watermelon, Citullus vulgaris W1-4and W1-12, it studied the effect factors on Agrobacterium tumefaciens-mediated transformation. Because of the transformation efficiency of Agrobacterium tumefaciens-mediated was too low; it failed to obtain transgenic plants.antifungal gene Chitinase and glucanase were successfully transferred into watermelon by ovary injection, respectively, The research studied the effect factors on regeneration system and the transformation by ovary injection and establishment of a highly efficient regeneration system and gemomic transformation for watermelon. The research provided some references for the application of ovary injection transformation technology in watermelon and provided a new germplasm resources of Anti-Fungal.The main results were summarized as follows:1) Establishment a highly efficient regeneration system for Citullus vulgaris W1-4and W1-12: the suitable medium for germination of watermelon was1/8MS medium with30g·L-1sucrose and solidified with7.0g·L-1agar; the best explants were from the seedings with their cotyledons in close contact and their color was just from yellow to pale green; the best explants were the cotyledonary nodes were excised nearly to be0.5cm×0.5cm long with the epicotyls; the medium of MS+6-BA1.5mg·L-1+IAA0.2mg·L-1and MS+6-BA1.0mg·L-1+IAA0.1mg·L-1were found the most suitable medium for bud organogenesis of W1-4and W1-12; The favorable shoot elongation medium for both inbred lines was MS+6-BA0.05mg·L-1and1/2MS+IAA0.1mg·L-1proved as an optimum medium for rooting of regenerated plants. 2) Construction of eukaryotic expression vector pB1-BG2and pBI-CH5B.3) The concentration of kanamycin was75mg·L-1for the bud in differentiate stage of W1-12.4) Agrobacterium tumefaciens-mediated transformation was found:pre-culturing for2d, then infecting for15min in the condition of OD600was0.5, co-culturing for3d with30mg·L-1AS in the medium, the highest transformation efficiency was2.2%,but we could not obtain transgenic plants.5) The ovary injection transformation technique used in watermelon was established:the optimal concentration of exogenous DNA was750-1000μg·mL-1; The highest transformation efficiency was at20h after self-pollination.6) The concentration of kanamycin screening in the fied was showed:The sensitivity of seedling to kanamycin was significantly different between W1-4and W1-12. In cotyledon stage, the optimal screening concentration of Wl-4and W1-12were75mg·L-1and50mg·L-1, respectively.7) Transgenic plants were examined by kanamycin screening and PCR amplification, W1-4and W1-12were gained7and16positive plants, respectively.