根癌农杆菌介导的高效大豆遗传转化体系的建立

利用根癌农杆菌对来自大豆成熟种子的胚尖进行遗传转化,研究了影响农杆菌介导大豆转化的各种因素,建立了一套优化的大豆遗传转化体系。研究结果表明:菌株KYRT1比EHA105和LBA4404具有更强的侵染能力;较酸的共培养基(pH5.4)、较低的培养温度(22℃)均有利于提高转化效率;恢复培养和分步抗性筛选方式有利于提高抗性组织的存活率和分化率。同时应用这种优化的遗传转化体系,获得了7个大豆品系的转基因植株,转化频率为4.29%-18%。经过PCR和Southern分析证明外源的双价抗虫基因cryIA(c)和pta已经整合到大豆的基因组中。

EFFICIENT AGROBACTERIUM-MEDIATED TRANSFORMATION OF SOYBEAN

To improve Agrobacterium tumefaciens mediated transformation of embryonic tips of soybean Glycine max (L.) Merr], the effect of several factors on transformation efficiency were examined by measuring transient expression levels of beta-glucuronidase and the number of resistant explants. The hypervirulent Agrobacterium tumefaciens strain KYRT1 was proved to be a better transformer than EHA105 and LBA4404. Improved transformation efficiencies were obtained when embryonic tips were incubated with an Agrobacterium suspension (A600=0.5) for 20 h. Optimized co-cultivation was performed in acidic medium (pH 5.4) at 22 degrees C in the dark for 5 days. Resting culture and step-by-step selection culture were beneficial to the survial of resistant explants. By combining the best treatments, transgenic soybeans of seven cultivars were obtained that simultaneously express the cryIA (c) and Pinellia ternata agglutinins (pta) genes. Most of the transgenic plants (about 70%) are fertile. The transformation frequency (the number of PCR-positive regenerated plants/the number of infected explants) x 100%] ranged from 4.29% to 18.0%. PCR and Southern analyses confirmed the stable integration of the binary insect resistance genes in the primary transgenic plants.