Study On Agrobacterium-mediated Transformation System Of Brassica Campestris Ssp.chinensis Makino With Cold-resistant Gene

Several factors were evaluated for their effects on shoot organogenesls from explants of cotyledons of non-heading Chinese cabbage (Br4ice campestris ssp. Chinensis Makino) SuZhouQing, 98Hqiu-31, Duan-126 and AB line, and efficient shoot regeneration system of these four varieties was established respectively. The reporter gene GUS was transformed to non-heading Chinese cabbage by Agrobactem-mediated method to study the transformation condition. A cold-resistant gene THPI was transferred to non-heading Chinese cabbage.The following results were obtained: 1. Conditions and frequency of shoot differentiation of the four varieties in was apparently different. SuZhouQing had the highest shoot differentiation frequency 36.7% on MN media containing TDZ 2mgIL.. NM 0.5mg/L and AgNO3 7.5mg/L;the optimal differentiation medium of 98Hqiu-31 was the same as that of SuZhouQing with the shoot regeneration rate of 42.2%;and that of the AB line was 41.7% on MS+BAlOmg/L+NAA 0.3mg/L; the best condition of Duan-126 was MS-I-BA 5mg/L+NAA 0.1 mgIL+AgNO3 5mgIL, with the differentiation rate of 367%. 2. To varieties SuZhouQing.. Duan-126 AB line, AgNO3 had a significant effect on improving the regeneration rate of cotyledons. The best concentration varied with variety. 3. Seedling age influenced the regeneration rate apparently. 7-day old Seedlings had the highest regeneration rate, and when it was exceeded 9 days or shorted than 5 days, the differentiation rate decreased notably. 4. Antibiotic Kanamycin showed a inhibitive effect on shoot regeneration from cotyledons. The shoot regeneration was totally inhabited when Kanamycin was 5mgIL in media, and the calli formed were white. When Kanamycin was 1 Smg/L, the explants had no callus formed. 5. Reporter gene GUS was transferred into non-heading Chinese cabbage by Agrobactium-mediated method. After infection with A. tumefaciens, the explants were cultured on media containing Cb 500mg/L. Adventitious shoots were regenerated about 30 days later, then shoots were transferred to media with Gb 300mg/L and Km I Smg/L for selection, they were transferred to fresh selection media every 15 days. 7 non-heading Chinese cabbage plants were GUS positive and PCR positive, and they were further testified by Southern blot. 6. The cold-resistant gene Thp was transferred to non-heading Chinese cabbage. Only 2 plants with GUS positive were obtained by now.