农杆菌介导甘蔗基因转化技术的优化

GFP Gene Transformation of Sugarcane Mediated by Agrobacterium tumefaciens

以甘蔗(Saccharum officinarum L.)品种ROC10为转化受体材料,以GFP基因为报告基因,通过对农杆菌介导遗传转化的一系列条件,包括农杆菌菌株及侵染菌液浓度、侵染时间、受体愈伤组织龄期、AS活化时间及使用浓度、共培养时间等进行优化,同时进行了便于vir基因活化和T-DNA转移的条件组合,如附加果糖和葡萄糖、酸性环境感染,低温共培养等,从而建立了一个可行、有效而又简便的农杆菌介导的甘蔗遗传转化体系。结果表明:农杆菌菌株EHA105优于LBA4404,其适宜侵染浓度D_(600)为1.3752;适宜受体愈伤组织龄期为25℃暗培养25d;适宜侵染时间为30min;适宜AS活化时间为2h,使用浓度为200μmol·L^(-1);适宜共培养时间为4d。获得2株有荧光信号的植株,对这2株植株进行斑点Southern杂交检测,均有阳性反应,证明GFP基因已整合到甘蔗基因组中并得到了有效表达,表明该转化体系确实是有效的。

Sugarcane (Saccharum officinarum) variety Roc10 was used as recipient material, and GFP gene as report gene. A series of genetic transformation conditions were optimized, including Agrobacterium tumefaciens strain and density for infection, infection time, callus age, activation time, density of AS and time of co -culturing. Meanwhile several optimized transformation conditions were combined to improve the activation of vir genes and T-DNA transfer, for instance, adding fructose and glucose, using acid infection condition and low temperature co -culture. Finally, a feasible, efficient and simple system of sugarcane genetic transformation mediated by Agrobacterium tumefaciens was established. The results were as follows: Agrobacterium tumefaciens strain EHA105 was better than LBA4404 and its best density for infection was D600=1.375 2; the best infection time was 30 minutes; callus cultured 25 days under 25μ was the best recipient material for transformation. To activate Agrobacterium tumefaciens, the best AS concentration used was 200μ mol·L-1, the best activation time was 2 hours, and the best co-cultivation time was 4 days. 2 transgenic sugarcane plants were obtained from co-culture of embryonic callus with Agrobacterium tumefaciens. The 2 plants showed fluorescent signal under fluorescent microscope, and also showed positive signals by dot southern blotting. The above results proved that GFP gene was successfully introduced into sugarcane and expressed effectively, which showed that the genetic transformation system of sugarcane mediated by A grobacterium tumefaciens wa effective.