农杆菌介导海岛棉转化体系优化的研究

为建立高效规模化棉花转基因技术体系，解决棉花转化率受基因型限制、转化效率低以及转化苗畸形率高等问题。以棉花胚性愈伤组织作为受体，用农杆菌介导法将Bt基因导入海岛棉棉花品种‘新海16号’，在提高海岛棉遗传转化效率的同时，建立了相对高效地转基因再生植株成苗技术体系。结果表明：分3次依次继代于具头孢霉素浓度梯度的抗性愈伤增殖培养基，较3次均继代于含500 mg/L头孢霉素(Cef)的抗性愈伤增殖培养基MS₂，抗性愈伤增殖速度较快。且60天添加1次50 mg/L Kan时，转化苗成苗率达到最大值。降低NH₄⁺/NO₃^-水平对提高体胚发生率具有有效促进作用。选择继代90天后的愈伤组织，胚状体诱导率可达到最大值；继代180天以上的愈伤组织可以摒弃。经转化后所获的子叶畸形胚，具有较强再生能力，其产生的次生胚经再次培育出正常转基因植株这一途径可有效缩短转化周期。

Optimization of Agrobacterium-mediated Transformation System of Island Cotton

In order to establish an efficient and large-scale cotton transgenic technology system to solve the problems, such as the conversion of cotton genotypes restricted, low conversion efficiency and the conversion rate of seedlings deformities etc., by using the agrobacterium-mediated gene transformation method, the Bt gene was introduced into embryogenic callus of xinhai16 (Xinjiang Sea Island cotton). The study aims to improve Sea Island cotton genetic transformation efficiency and establish a relatively efficient regeneration of transgenic plants seedling technology system. The results showed that: the callus subculture on callus proliferation resistant medium containing cefotaxime, following three times, each subculture medium containing cephalosporins had a concentration gradient, or we could let callus subculture 3 times, each time subculture containing 500 mg/L cefotaxime resistant callus proliferation medium MS₂. Compared with the latter, the speed of resistant callus proliferation of the former is faster. What’s more, adding 50 mg/L Kan per 60 days, the seedling rate of transformed seedlings reached the maximum. Reducing the level of NH₄ /NO₃^- had an effective role in improving the rate of somatic embryogenesis. Selecting subculture callus after 90 days, embryo induction rate could reach the maximum value. After subculture time of 90 days, embryoid induction rate reached the maximum; more than 180 days of subculture time, the callus could be abandoned. After transforming, the cotyledon abnormal embryos were obtained with a strong ability to regenerate, its secondary embryos could produce normal transgenic plants again by cultivating and this approach could effectively shorten the conversion period.