论文部分内容阅读
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。
Agrobacterium tumefaciens strain EHA1 05 harboring the p1 3 W4 plasmid carrying the antisense RNA fragment of rice waxy gene was used to infect the immature embryos of indica variety Longtefu B, Xieqingzao B, Zhenshan 97B and -3 2 B respectively After the secondary screening of the tissue culture, co-culture and hygromycin 25 ~ 50 mg / L, the calli resistant to hygromycin were obtained. The frequency of resistant callus was 2 0 .1% ~ 2 7.1%; resistant callus by differentiation culture, the frequency of differentiation in 12.2% ~ 21.2%. There was a big difference in the differentiation of green bacteria among the cultivars. The resistance of Longtefu B to green seedlings was 8.8%. Although the resistant calli of Zhenshan 97B could differentiate, it could not get green seedlings . GUS activity assay and PCR assay were carried out on the transgenic plants respectively. The results showed that the transgenic plants obtained showed positive GUS test in 92% of the plants and normal sturdiness. GUS stained separation was also detected in the endosperm of T1 seeds , To prove that foreign genes have indeed been introduced into these plants, and, can be normal genetic. In the differentiation of resistant callus, the effect of hygromycin on differentiation was tested. The results showed that without adding hygromycin, the frequency of green plantlets could be significantly increased, but the reliability of transgenic plants also decreased.