Establishment Of Agrobacterium-mediated Transformation System In Chinese Cabbage

Chinese cabbage(Brassica rapa L.ssp.Pekinensis)originating in China,is an important vegetable in China and Asia.The genetic transformation system will lay the foundation for molecular breeding and gene function verification of Chinese cabbage.In this study,the regeneration system of Chinese cabbage cultivar ’GT-24’ was optimized.On this basis,Agrobacterium-mediated transformation system was established and the transformed plants were obtained.The results are as follows:1.The effects of explants,sucrose,6-BA and AgNO3 on the adventitious bud differentiation were studied.The regeneration of Chinese cabbage ’GT-24’ in vitro was optimized.The results showed that:The optimum explants were cotyledon-petiole;the optimum bud induction medium was MS + 0.5 mg/L NAA + 5 mg/L 6-BA + 4 mg/L AgNO3 + 7 g/L agar + 30 g/L sucrose(PH=5.8),the differentiation rate of adventitious buds was 93.5%,which was 7.28%higher than that of the original regeneration system.2.Using the Agrobacterium tumefaciens-mediated method,the optimal culture system was established by screening the appropriate concentration of antibiotic hygromycin and antibacterial antibiotic terpene.the best pre-culture time.In the process of ’GT-24’genetic transformation,the OD600 of the Agrobacterium tumefaciens was 0.5,the infection time was 15 min,and the culture medium was MS + 0.5 Mg/L NAA + 5 mg/L 6-BA + 4 mg/L AgNO3 + 200 mg/L TMT + 25 mg/L Hyg + 7 g/L agar + 30 g/L sucrose(pH ＝5.8).Rooting medium of transgenic plants Add 1 mg/L NAA or 1 mg/L IB A.The recombinant plants were identified by PCR,RT-PCR and GUS histochemical staining.The results showed that the transgenic plants were transgenic.The average transformation rate was 2.49%.