摘要
用携带水稻蜡质基因反义 RNA片段的 p1 3 W4质粒的根癌农杆菌 EHA1 0 5 ,分别感染籼稻品种龙特甫 B、协青早B、珍汕 97B和 -3 2 B的幼胚愈伤组织 ,经共培养和在潮霉素 2 5～ 5 0 mg/L浓度下的二次筛选后 ,获得了对潮霉素表现抗性的愈伤组织 ,抗性愈伤组织的频率在 2 0 .1 %～ 2 7.1 %之间 ;抗性愈伤组织经分化培养 ,分化频率在 1 2 .0 %～ 2 1 .2 %。品种间绿菌分化情况存在较大差异 ,龙特甫 B的抗性愈伤组织分化成绿苗的频率为 8.8% ;珍汕 97B的抗性愈伤组织虽能分化 ,但是 ,无法获得绿苗。对转基因植株分别进行了 GUS活性测定和 PCR检测 ,结果显示 :获得的转基因植株 ,92 %的植株GUS检测呈阳性反应 ,并能正常结实 ,在 T1种子的胚乳中也能检测到 GUS染色的分离 ,证明外源基因确已导入这些植株中 ,而且 ,能正常遗传。在抗性愈伤组织的分化中 ,试验了潮霉素对分化的影响 ,结果显示 :不添加潮霉素 ,能明显地提高绿苗频率 ,但转基因植株的可靠性也随之下降。
Immature embryos of four indica rice varieties, Longtefu B ?Xieqingzao B?Ⅱ 32 B and Zhenshan 97 B, were used to induce calli, these calli were infected and transformed by A.tumefacien strain EHA105 harboring Ti plasmid p13W4 carrying waxy gene. After cocultivation and two times 25 50mg/L hygromycin resistant selections, 20.1% 27.1% resistant calli still survived, and the transgenic calli frequencies were no difference. These resistant calli were transferred to differentiation medium, and the differentiation frequencies of them were different, Longtefu B 21.2%, Xieqingzao B 12.0%,Ⅱ 32 B 15.1% and Zhenshan B 19.6%,respectively. But the green plant frequency was significantly different: 8.8% resistant calli of Longtefu B generated green plants, but no green plant was obtained in Zhenshan 97 B. All of plants were detected by GUS analysis and PCR amplification, the results showed that the anti Waxy gene had been integrated into these plants. The transgenic plants were normally fertile,and segregation of GUS active was observed among T0 seeds. The effect of hygromycin on calli was tested, no hygromycin differentiation medium can improve the plantlet frequency of resistant calli, but the reliability of transgenic plant decreased.
出处
《上海交通大学学报（农业科学版）》
2002年第1期7-12,共6页
Journal of Shanghai Jiaotong University(Agricultural Science)
基金
国家科技部专项 (ZJY-A-0 2 -0 2 )
上海市科委攻关项目 (0 13 912 0 19)
