国家重点基础研究发展计划(2009CB119000);“十一五”科技支撑计划(2007BAD57B04)
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。
An Agrobacteriummediated transformation system,using transient and stable transformation assays,was used to evaluate some factors influencing transformation in cauliflower.These included the precultivation time,the bacterial density,the inoculation duration time with Agrobacterium tumefaciens,the concentration of acetosyringone,the delay selection and the concentration of kanamycin in selection.Using cotyledons with 12mm petioles as explants,the best transformation parameters were:twoday precultivation,OD6000.30.4 of bacterial density,eightminute infection with Agrobacterium tumefaciens,twoday cocultivation,100μmol/L acetosyringone in the cocultivaiton medium and sevenday delay selection in kanamycin of 5mg/L.By optimizing the parameters on the procedure of Agrobacteriummediated transformation,a high transformation efficiency(35.7%)was demonstrated.The GUS transient expression could be an important indicator for cauliflower transformation.The development of an efficient Agrobacteriummediated transformation system opens up new opportunities for the functional characterization of genes and promotes the development of novel germplasm of Brassica vegetable genus.
于娅,刘莉莎,赵永钦,等.影响花椰菜农杆菌介导转化因素的研究[J].植物遗传资源学报,2010,11(3):320-325.
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