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以预培养 4 d的小麦幼胚愈伤组织为受体 ,L B培养基上激活培养 3 d或 AB培养基上激活培养 4 d,并在WCC培养基中重悬的 C5 8c1农杆菌菌系为供体 ,将含有目的基因、标记基因的 p PTN2 4 9、p PTN 2 5 4、p PTN2 70、p SIS-GFP等重组 DNA质粒转入了小麦 ,经在含 10～ 5 0 mg/ L geneticin( G4 18)、5 0 mg/ L cefotaxime、5 0 m g/ L vancomycin、5 0 m g/ L ticillicin选择培养基上多次选择 ,移栽前利用 npt EL ISA检测 ,移栽后利用叶片离体退绿、PCR和 South-ern blot 4种方法综合检测 ,共获得了 2 9株转基因植株 ,转化频率平均为 0 .82 %。在所获得的转基因植株中 ,单拷贝整合植株占 65 .5 2 %。研究结果还表明 ,不同基因型的转化效率显著不同 ,除了 Bobwhite外 ,笔者也从扬麦 10号获得了转基因植株 ;EL ISA测定值的高低与外源 DNA整合的拷贝数有一定关系 ;初步建立了农杆菌介导稳定转化小麦的技术体系。
Four days pre-cultured wheat immature embryos callus as receptor, activated on LB medium for 3 days or AB medium activated for 4 days, and resuspended in WCC medium C5 8c1 Agrobacterium strains as The recombinant plasmids p PTN2 4 9, p PTN 2 5 4, p PTN2 70 and p SIS-GFP containing the target gene and the marker gene were transferred into wheat and transformed into wheat with 10 to 500 mg / L of geneticin (G4 18), 5 0 mg / L cefotaxime, 50 mg / L vancomycin, 50 mg / L ticillicin selection medium selection before transplanting using npt EL ISA detection, PCR, and South-ern blot. A total of 29 transgenic plants were obtained, with an average conversion frequency of 0.82%. Among the transgenic plants obtained, 65.52% were single-copy integrated plants. The results also showed that the transformation efficiency of different genotypes was significantly different. In addition to Bobwhite, the author also obtained transgenic plants from Yangmai 10. The level of EL ISA was related to the copy number of foreign DNA integration. Agrobacterium - mediated transformation of wheat stable technology system.