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以花椰菜赛雪的带柄子叶为外植体,以MS为基本培养基,GUS基因为报告基因,分析了遗传转化过程中的影响因子,如预培养时间、农杆菌菌液浓度、侵染时间、共培养时间、乙酰丁香酮浓度、延迟筛选时间等对外植体瞬间表达和稳定表达的影响。结果显示,以花椰菜的带柄子叶为外植体,预培养2d,农杆菌菌液为OD6000.3～0.4,侵染8min,共培养2d,乙酰丁香酮浓度为100μmol/L,延迟筛选7d,卡那霉素筛选压为5mg/L为最优的遗传转化方案,转化率最高可达35.7%。另外,GUS瞬间表达率和转化率并不存在绝对的相关性,但瞬间表达分析仍然可以作为外源基因进入受体细胞的指示。花椰菜农杆菌介导转化方案的优化研究为芸薹属蔬菜高效遗传转化提供了技术保障,有利于芸薹属蔬菜遗传育种与种质创新研究。
Using cauliflower with leaves and stems as explants, using MS as basic medium and GUS gene as reporter gene, the influencing factors of genetic transformation were analyzed, such as pre-culture time, Agrobacterium tumefaciens concentration, infection time , Co-culture time, acetosyringone concentration, delayed screening time on explant transient expression and stable expression. The results showed that with cauliflower cotyledons as explants, pre-cultured for 2 days, Agrobacterium tumefaciens OD6000.3 ~ 0.4, infection 8min, co-cultured 2d, acetosyringone concentration 100μmol / L, delayed screening 7d, Kanamycin screening pressure of 5mg / L is the best genetic transformation program, the conversion rate of up to 35.7%. In addition, there is no absolute correlation between GUS transient expression and conversion, but transient expression analysis can still be used as an indication of foreign gene entry into recipient cells. Optimization of Cauliflower-Agrobacterium-mediated transformation programs provided technical support for the efficient genetic transformation of Brassica vegetables, and was conducive to genetic breeding and germplasm innovation of Brassica vegetables.