CaCl2诱导大豆花粉管通道农杆菌转基因研究

为了提高大豆花粉管通道法转基因效率,研究直接用农杆菌转化方法的可行性,以秋大豆品种上海本地青为受体材料,用花粉管通道法在三种CaCl2浓度水平下进行质粒pCAMBIA3301转化,又以GV3101、LBA4404、EHA105三种农杆茵菌株直接导入花粉管通道为处理进行了遗传转化.以质粒为外源基因平均结实率为36.83%,T0代种子平均成活率为60.89%,T1植株喷施除草剂后平均存活率为8.24%.以农杆菌直接转化平均结实率为15.92%.T0代种子平均成活率为20.40%,T1植株喷施除草剂后平均存活率为7.79%.对存活的植株提取DNA进行PCR检测,以质粒为外源基因的处理共获得20株转化苗,三种CaCl2浓度水平的转化效率分别为0.56%、1.64%、1.40%,有两种水平的转化效率约为传统转化效率的三倍.以农杆菌直接转化的处理共获得5株转化苗,转化效率分剐为0.17%、0.33%、0.29 %.

TRANSFOMING AGROBACTERIUM INTO SOYBEAN BY MEANS OF POLLEN TUBE PATHWAY INDUCED BY CACL2

In order to improve transgene efficiency of pollen tube pathway, and Study the possibility of direct transformation of Agrobacterium, pCAMBIA3301 at 3 concentration levels of CaCl2 was transformed into Shanghai Bendiqing Soybean receptors by means of pollen tube pathway, meanwhile, 3 different varieties of Agrobacterium GV3101,LBA4404,EHA105 were introduced into soybean as another treatment. When pCAMBIA3301 was served as foreign gene transformed by pollen tube pathway, an average seed--set percentage of 36.83% and an average seedling percentage of 60. 89% were achieved. An average seedling survival percentage was 8. 24~ when 0.2% Besta was conducted with T1 plants. When Agrobacterium GV3101, LBA4404, EHA105 were served as foreign gene transformed by pollen tube pathway, an average seed--set percentage of 15.92% and an average seedling percentage of 20.40% were achieved. An average seedling survival percentage was 7. 79% when 0.2% Besta was conducted with T1 plants. Leaf genome DNA from survived T1 plant of different treatments were analyzed by PCR, 20 positive plantswere obtained with pCAMBIA3301 as foreign gene, 3 concentration levels of CaCl2 gained 0.56%, 1.64%, 1.40?ficiency respectively, 2 of which were about 3 fold higher than that of traditional way. 5 positive plants were obtained Which 3 different varieties of Agrobacterium were directly introduced into soybean, gained 0. 17%, 0.33 %, 0.29 % efficiency respectively.