Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants
Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants
复制标题
DOI：
10.1007/s00299-002-0560-y
复制
发表时间：
2003-02-01
影响因子：
6.2
通讯作者：
Stewart, CN
中科院分区：
文献类型：
--
作者：
Cardoza, V;Stewart, CN
一种生产转基因甘蓝型油菜的高效方案。通过对预处理时间和共培养时间这两个重要参数的优化，开发了西星植物。农杆菌介导的转化以下胚轴为外植体组织进行。绿色荧光蛋白（GFP）编码基因mGFP5-ER和eGFP的两种变体均在花椰菜花叶病毒35S启动子的组成表达下进行实验。将预处理时间优化为72 h，与农杆菌共培养时间优化为48 h，将转化效率从基线的4%提高到25%。mGFP5-ER的转化率为17%，eGFP的转化率为25%。用200 mg/l卡那霉素筛选转基因芽。在卡那霉素存在下，添加10 g/l蔗糖和0.5 mg/l吲哚丁酸的半强度Murashige和Skoog培养基上生根率为100%。
An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene - mGFP5-ER and eGFP - both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.