青花菜农杆菌遗传转化影响因素探究

Factors Affecting Agrobacterium Mediated Transformation of Broccoli(Brassica oleracea var.botrytis)

以青花菜"绿秀"为研究材料,以带柄子叶为外植体,采用根癌农杆菌LBA4404菌株进行农杆菌遗传转化,研究影响青花菜遗传转化的主要因素,结果表明:乙酰丁香酮浓度、OD600值、侵染时间等对瞬间表达都有一定的影响。因素中OD600值影响最大,AS浓度影响次之,再次是侵染时间。最适宜的试验条件为:乙酰丁香酮浓度为100μmol·L-1、OD600值为0.3、侵染时间为5 min。抗性植株经PCR检测、Southern杂交和GUS组织化学染色法,表明目的基因已整合到青花菜基因组中并得到了很好的表达。

An Agrobacterium mediated transformation system was used to evaluate some factors influencing transformation with the strain LBA4404 in broccoli varity " Lv Xiu"(using transient and stable transformation assays).These included the precultivation time,the bacterial density,the inoculation duration time with Agrobacterium tume faciens,the concentration of acetosyringone,the delay selection and the concentration of kanamycin in selection.Using cotyledons with 1mm petioles as explants,the best transformation parameters were:OD600 0.3 of bacterial density,five minute infection with Agrobacterium tume faciens,100μmol/L acetosyringone in the cocultivation medium.Stable integration of the transgenes in the plant genome was demonstrated by PCR and Southern hybrization analysis.Expression of the transgenes was confirmed by GUS staining.