根癌农杆菌介导Cry1Ac基因转化‘雪青’梨获得转基因植株

 以‘雪青’梨叶片愈伤组织为外植体, 经根癌农杆菌介导将CaMV35S启动子调控下的Cry1Ac基因导入‘雪青’梨。698块愈伤组织和231个Kan^r芽与携带表达载体质粒pBX203的根癌农杆菌菌株LBA4404共培养3 d后, 转入含50 mg·L^-1 Kan的筛选培养基培养30 d, 15 d转接1次。结果表明,在MS + 5 mg·L ^-1 6-BA + 0.1 mg·L^-1 IAA筛选培养基中, Kan^r芽率为34.24% , 在1/2MS + 2 mg·L^-1 IBA+ 0.5 mg·L^-1 6-BA + 500 mg·L^-1AC筛选培养基中, 15.58% Kan^r芽生根。共获得再生植株32株, 经PCR和Southern-blot分析证明, 其中4株的基因组已成功导入和整合Cry1Ac基因。

Obtainment of Transgenic ''''Xueqing'''' Pear Plants with a Synthetic Cry1Ac Gene Mediated by Agrobacterium tumefaciens

By using calli from leaves of Xueqing pear ( Pyrus sp. ) as explants, the Cry1Ac gene regulated by CaMV35S promoter was introduced into Xueqing pear through Ag robacterium-mediated transformation.After co-culturing 698 calli and 231 Kan^r buds, respectively, with A. tum efaciens strain LBA4404 harboring aplasmid pBX203 for 3 days and transferred to the selective medium containing 50 mg·L^-1 kanamycin for 30days at a 15-day interval, 34.24% kanamycin2resistant (Kan^r ) buds ( adventitious shoots) regenerated on MS+ 5 mg·L^-1 6-BA + 0.1 mg·L^-1 IAA and 15.58% of Kan^r buds rooted on 1/2 MS + 2 mg·L^-1 IBA + 0.5mg·L^-1 6-BA + 500 mg·L^-1 AC. A total of 32 plants were obtained, but only 4 p lants were confirmed byPCR and Southern-blot analysis that the Cry1Ac gene was transferred and integrated into the genome ofXueqing pear.