用in planta方法转化甘蓝型油菜

Genetic Transformation of Brassica napus with in planta Method

用inplanta方法进行遗传转化极大地促进了模式植物拟南芥的功能基因组研究 ,但在农作物中仍少有inplanta的成功报道。我们采用inplanta方法 ,用含有质粒pSG5 2 9、pHTT6 1 3和ApSCO的农杆菌菌株转化中油 82 1、湘油 1 3、宁RS 1、S2 5 0 1 1、华双 3号等甘蓝型油菜品种。在适宜的条件下 ,将根癌农杆菌菌液浸染甘蓝型油菜花序 1 0d至 1 5d ,每天浸染 1至 2次 ,共收获约 1 0 0 0 0 0粒受处理的种子。将种子播撒在含有 2 70mg·L- 1 卡那霉素的选择培养基中选择转化植株。卡那霉素抗性植株经PCR分析检测 ,结果在 5个供试品种中均获得了转基因植株 ,平均转化效率 0 1 %左右 ,各甘蓝型油菜品种间的转化效率无明显差异。这种方法方便、快速且无须经过组织培养阶段即可获得上百株转基因植株。

Genetic transformation with the in planta method has greatly promoted researches of functional genomics in model plant, Arabidopsis. However few of crops was reported being transformed with the method. Via in planta method, genetic transformations of different Brassica napus cultivars, i.e. Zhongyou 821, Xiangyou 13, Ning RS 1, S2501 1, Huashuang 3 were mediated by Agrobacterium containing plasmid pSG529, pHTT613 and ApSCO separatively. Under the suitable condition, the floral branches of Brassica napus were dipped in the suspension of Agrobacterium for 10 to 15 days. The 100 000 seeds obtained by in planta method were selected on selective medium supplemented with Kanamycin 270 mg·L -1 . The plants with Kanamycin resistance were analyzed by PCR. The result indicated that the alien genes were transferred into all the five cultivars, and the transformation frequency near by 0.1% was very close among the cultivars. Hundreds of transgenic plants were obtained by this simple, convenient, speedy and non tissued cultural method. The success of in planta method in B. napus showed that the foundation of the higher efficiency transformation method and the genomics study of Brassica napus had been laid.