甘蓝下胚轴的高效再生和农杆菌介导B.t.基因转化甘蓝

HIGH EFFICIENCY REGENERATION AND Agrobacterium MEDIATED TRANSFORMATION OF HYPOCOTYL IN Brassica oleracea var.capitata WITH B.t.GENE

分别对取材时间和培养基组成进行研究，建立了甘蓝下胚轴外植体的高效再生系统，当选取6～7d苗龄的下胚轴，将其置于MSB＋BA2．5mg／L＋ZT1mg／L＋IBA0．1mg／L的芽分化培养基中，并于培养基中附加AgNO37．5mg／L时，最高的芽分化频率，“夏光甘蓝”母本“103”可达81．67％，父本“60天早椰菜”可达78．56％。在此基础上，我们对影响转化频率的不同因素，即：抑制农杆菌生长的抗生素种类及其浓度、预培养的有无及感染时的浓度、下胚轴外植体与农杆菌的感染时间和共培养时间、筛选时抗生素的加入时间进行了探讨。共获得100余株卡那霉素抗性植株，全部移栽成活，生长良好。对转基因植株总DNA进行Southernblotting分析，证明nptⅡ基因已整合到甘蓝植株细胞基因组中。

We have developed a high efficiency regeneration system of hypocotyl explants in Brassica oleracea var. capitata based on the study of the time of sampling and hormone composition in medium. The results indicated that when hypocotyls of 6~7 days wereplaced on the bud differentiation medium MSB+BA 2. 5 mg/L +ZT 1. 0 mg/L +IBA 0. 1mg/L(with 3 % sucrose and 0. 75 % agar,pH5. 8),supplemented with AgNO3 7. 5 mg/L,thehighest differentiation frequencies of 81. 67 % (Female parent '103')and 78. 56 % (male parent '60d Zaoyecai') could be obtained from hypocotyl explants, respectively. Furthermore,some factors affecting the transformation frequency,i. e. the kinds and concentrations of antibiotics for inhibiting the growth of Agrobacterium,preculture of hypocotyl explants,concentration of Agrobacterium and infection time,co-infection time,the time for supplementing theselecting antibiotic were also discussed. More than one hundred kanamycin-resistant plantshave been regenerated. All of them survived after transplantation and grew well in pots.Southern blotting analysis of plant DNA confirmed that nptⅡgenes have been introduced into the plant genome.