Study On Optimization Of Regeneration In Vitro Of Non-Heading Chinese Cabbage And Transformation Of Disease Resistence Gene

Non-heading Chinese cabbage (Brassica campestris ssp.chinensis Makino) is a verypopular leafy-vegetable in China, particularly in South of China. It plays a very importantrole in the year-round supply. However, downy mildew, virus disease and soft rot diseaseare so severe that the quality and yield are influnenced, we have to develop new varietiesusing genetic engineering method in addition to conventional methods.Brassica campestris was hard to regeneration in vitro, while gene engineer and otherhigh technology breeding techniques were based on the foundation of high frequency ofregeneration system. Studies on non-heading Chinese cabbage will offer important valuesin theoretics and practice.Based on four cultivars including Shulu, Suzhouqing, Liangbaiye and Aikangwuhao,effects of genotype, explant and hormone on shoot regeneration were evaluated to select theeasily regenerating variety, optimize their regeneration frequency. Proper concentration ofkanamycin, time of add kanamycin, different explant types, pre-cultivation andco-cultivation time were studied on the transformation system of non-heading Chinesecabbage using the Agrobacterium-mediated transformation method. The results are shownas follows:1. The regeneration frequency of Shulu was the highest among different cultivars. Theregeneration frequency of petiole with cotyledon was the highest among different explantstypes. Addition of TDZ (0.5 mg·L^-1) into the germination medium can make the seedlingsstronger which was beneficial to regeneration, leading to a regeneration rate of over 60%for all mediums. The regeneration rate of petiole with cotyledon was the highest in themedium MN+TDZ 0.5mg·L^-1+NAA 0.5mg·L^-1+AgNO₃ 7.5 mg·L^-1 and the ratereached 80%. The result indicated that TDZ played an obviously persistent role in inducingorganogenesis. 2. The optimal concentration of Kin was 15mg·L^-1 and we used the delayed-selectionmethod; hypocotyl had higher transformation efficiency than petiole with cotyledon; thepre-cultivation time and co-cultivation time were two days; after co-cultivation, thetransient expression of GUS test was positive and we got anti-kanamycin plants. ThePCR-test proved that the InBING gene have integrated into the plant genome ofnon-heading Chinese cabbage.