农杆菌介导外源基因转化棉花上胚轴方法的研究

Study on the Transformation Method of Mediating Exogenous Gene into Cotton Epicotyl by Agrobacterium

[目的]介绍一种获得棉花转基因植株的新方法。[方法]以棉花感受态萌动的上胚轴作为外源基因转化的受体,用农杆菌介导法将携带有筛选标记NPTII（新霉素磷酸转移酶）基因及GFP（绿色荧光蛋白）报告基因导入棉花,研究了农杆菌介导法用于棉花感受态萌动上胚轴转化的预培养时间,浸菌时间,共培养时间及抗生素处理浓度等。[结果]在新疆棉花离体培养植株直接再生植株基础上,经3～5d预培养处理过的棉花上胚轴,在浸菌15 min共培养2 d后,经50 mg/L kan筛选,用1 000 mg/L头孢霉素抑菌,并用相对高含量细胞分裂素和低含量生长素的组合对其进行直接出芽培养,从而获得抗性小苗。[结论]用农杆菌转化处理棉花上胚轴是一种操作简单、重复性好、非基因依赖型的转化方法。

[Objective] The aim was to introduce a new method of obtaining transgenic cotton plants.[Method] With the competent germinating epicotyl of cotton as receptor in exogenous gene transformation,NPTII（neomycin phosphotransferase II） gene carrying selection marker and GFP（green fluorescent protein） reporter gene were mediated into cotton by agrobacterium-mediated transformation.The preculture time,bacterial-immersing time,co-culture time and antibiotics concn.,etc of using agrobacterium-mediated transformation in the transformation of competent germinating epicotyl of cotton were studied.[Result] Based on directly regenerated plant of in vitro plant of Xinjiang cotton,the cotton epicotyl treated by pre-culture for 3-5 d was immersed in bacteria for 15 min and co-cultured for 2 d.Cephalothin at 1 000 mg/L was used to inhibit bacteria after 50 mg/L kan selection.And the combination of cytokinin with relatively high content and auxin with low content was used in its directly budding culture to obtain resistant seedlings.[Conclusion] Treating cotton epicotyl by agrobacterium transformation was a simply operative and genotype independent transformation method with good reproducibility.