根癌农杆菌介导B.t.基因和CpTI基因对花椰菜的转化

用根癌农杆菌介导的方法 ,将B .t.基因和Cp TI基因分别导入花椰菜“杂交 75天”的父本和母本的无菌苗下胚轴切段细胞 ,都获得了转基因植株。经过预培养的下胚轴切段用根癌农杆菌 (LBA44 0 4/ pG BI4A2B ,含B .t .基因 ;LBA44 0 4/pBRLC ,含CpTI基因 )进行感染后 ,共培养 48h ,继续培养 30d后 ,将分化芽转移至筛选培养基上。 10d后 ,大多数分化芽的顶端变成紫色 ,2 0d后紫色芽逐渐变白死亡 ,而转化芽在选择培养基上长成小植株。小植株移至大田能正常生长、开花、结籽。PCR和Southernblot分析表明B .t和CpTI基因已整合在植物基因组中

Transgenic Plants Obtained from Agrobacterium-Mediated Transformation of Hypocotyl in Brassica oleracea var.botrytis with B.t.Gene and CpTI Gene

We have developed a high efficiency regeneration system of hypocotyl explants in Brassica oleracea var. botrytis based on the study of the time of sampling and hormone composition of medium. The results indicated that when hypocotyls of 5-6 d were placed on the bud differentiation medium containing MSB BA 3 mg/L ZT 1.5 mg/L GA 3 0.2 mg/L IBA 0.1 mg/L (with AgNO 3 7.5 mg/L, 3%sucrose and 0.65% agar, pH 5.8), differentiation frequencies of hypocotyl explants could be 61.8% (female parent "CA") and 58.4% (male parent "CB"). Some factors affecting the transformation frequency, i.e. the preculture of hypocotyl explants, concentration of Agrobacterium and infection, co-infection time, antibiotics kinds and concentrations for inhibiting the growth of Agrobacterium, the time of supplementation of the selecting antibiotic were also discussed (Tables 3,4). With the hypocotyl of aseptic seedling of Brassica oleracea var. botrytis "Zajiao 75 d" female parent "CA" and male "CB" as explant, through Agrobacterium-mediated transformation into the male and female parents, transgenic plants were regenerated. The precultured hypocotyl fragments were infected with Agrobacterium tumefaciens (LBA4404/pGBI4A2B containing B.t.gene; LBA4404/pBRLC containing CpTI gene). After being co-incubated for 48 h, 30 d after culture, and regenerated buds were transferred to selective medium 10 d later. The tops of most regenerated shoots turned purple, and 20 d later, the purple shoots turned white and then died, then the transgenic ones were into plantlets on the selective medium. 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 plant genome (Figs.4,5).