利用基因枪法进行小麦遗传转化影响因素研究

Study on factors affecting microprojectile bombardment-mediated transformation of wheat(Triticum aestivum L.)

以生产上大面积推广的4个小麦品种为材料,对基因枪法转化小麦过程中的影响因素进行了研究。结果表明,在基因枪轰击时采用预培养10～15d的幼胚且在轰击前6h和轰击后18h经0 4mol L甘露醇处理,可明显提高植株再生率;轰击用金粉-DNA量以42-0 21μg 枪为宜;不同品种对筛选剂Bialaphos敏感性有差异,表现为豫麦18>豫麦34>豫麦70>温麦6号,最终确定适宜的筛选剂浓度为2～3mg L。利用上述的优化条件,通过愈伤组织培养、筛选和植株再生,4个品种共获得71株再生植株,其中豫麦18分化率最高达到34 7%,温麦6号分化率最低,未获得再生植株。对长势良好的39株再生苗进行PCR检测,全部呈阳性反应,表明上述优化条件不仅适用而且可行,具有一定的应用价值。

The acceptors were from 4 wheat cultivars including Yumai 18, Yumai 34, Wenmai 6 and Yumai 70. The factors affecting wheat microprojectile bombardment transfer were studied. The result showed that immature embryos pre-cultured for 10～15 days and treated with 0.4 mol/L mannitol could improve gene transformation frequency. The proper dosage of golden power-DNA was 42-0.21 μg per bombardment. The sensitivity to herbicide Bialaphos showed different between different cultivars among which Yumai 18,Yumai 34 were more sensitive than others cultivars. Based on optimizing gene transformation and plant regeneration condition, the 71 plants were achieved by calli culture, screening and plant regeneration among 4 cultivars. The frequency of plant regeneration from four genotypes used in this study was quite differenct. Yumai 18 was the best acceptor genotype among the 4 cultivars. It was 34.7% in Yumai 18 while Wenmai 6 didn't get regeneration plant. By PCR assay selecting wealthy 39 plants, 34 plants were positive individuals.