农杆菌介导小麦成熟胚愈伤组织的遗传转化研究

Genetic Transformation through Mature Seed-derived Callus of Common Wheat by Agrobacterium Tumefaciens

为建立和优化小麦成熟胚愈伤组织遗传转化体系,以筛选出的适合小麦成熟胚组织培养再生的普通小麦山农2618为材料,采用农杆菌介导法对其成熟胚愈伤组织进行转化,对影响转化效率的因素如侵染菌液的浓度、侵染时间、超声波处理、真空处理等进行了研究。结果表明,这些因素对小麦成熟胚遗传转化效率有很大影响。菌液侵染浓度OD600为0.8、侵染时间为60 min时转化效率最高。在此基础上辅以超声波处理、真空处理可明显提高转化效率,最高转化率可达8.1%。对转化愈伤组织和转化植株分别进行GUS染色和PCR检测,结果表明外源基因已整合到小麦基因组中。

Agrobacterium mediated transformation of common wheat through callus initiated from immature embryos has proved to be an effective way. But, obtaining a large number of immature embryos over a short period of time is a laborious work. To overcom this limitation, an alternative way of using callus initiated from mature seeds has to be established. Although there was also a problem of low transformation rate. In this essay the common wheat SN2616 was used,the regeneration of which has been established before, Cell density, inoculation time and treatments of sonication and vacuum infiltration on the transformation efficicency been assayed. Agrobacterium-mediated transformation assisted by a combination of 7.5 min sonication with 7.5 min vacuum infiltration resulted in the high est transformation frequency. The blue blots had improved the GUS expressed in the callus through histochemical GUS detection, And the existence and integration of BG： gene in transgenic plants were confirmed by PCR.