青菜的高效再生和农杆菌介导B.t.及CpTI基因的转化

High Efficiency Regeneration and Agrobacterium-mediated Transformation of Brassica chinensis L. with B.t. Gene and CpTI Gene

分别对培养基中适宜的激素组成和AgNO3 添加浓度进行研究 ,建立了青菜下胚轴和子叶外植体的高效再生系统 ,青菜品种“矮抗青”的最高芽分化频率下胚轴可达 6 5 % ,子叶为 5 2 %左右。在此基础上 ,对影响转化频率的不同因素进行了探讨 ,共获 94株卡那霉素抗性植株。对转基因植株总DNA的Southernblot ting分析证明 ,B .t .基因和CpTI基因已整合到青菜植株的细胞核基因组中。经过抗虫性筛选试验 ,从转基因植株的T3 代筛选到 7个转B .t.基因的抗虫纯合株系和 5个转CpTI基因的抗虫纯合株系 。

We developed a high efficiency regeneration system of hypocotyl and cotyledons explants in Brassica chinensis L. based on the study of hormone composition and concentration of AgNO 3 in medium. The results indicated that when hypocotyls of 5 days were placed on the bud differentiation medium MSB+2ip 7.5 mg/L+IBA 0.1 mg/L+AgNO 3 7.5 mg/L(with 3% sucrose and 0.65% agar, pH 5.8), the percentage of explants which formed buds of hypocotyls was about 65% and that of cotyledons was about 52%(Table 2). Furthermore, some factors affecting the transformation frequency, i.e. the kinds and concentrations of antibiotics to inhibit the growth of Agrobacterium, preculture of hypocotyl explants, concentration of Agrobacterium and infection time, co-infection time, the time for supplementing the selecting kanamycin were also discussed. By means of Agrobacterium-mediated transformation, 94 kanamycin-resistant plantlets were obtained from hypocotyl of B. chinensis L. cv. Aikangqing. All of them survived after transplantation and grew well in pots. PCR and Southern blotting analysis of plant DNA confirmed that B.t. gene and CpTI gene were introduced into the B. chinensis L. (Chinese cabbage) genome(Figs.4,5 ). After strict examination of the resistance to insect of T 3 progenies of the transgenic plants, 7 transgenic plant pure-lines with B.t. gene and 5 transgenic plant pure-lines with CpTI gene desirable characters(the percentage of the resistance to insect about 60%-80%) have been established by selection and screening, and have passed the preliminary regional field tests of Chinese cabbage cultivars.