农杆菌介导胡萝卜转化体系的诱导培养基优化

Inducing Medium Optimization for Agrobacterium-medieted Transformation System of Carrot(Daucus carota L.)

高频再生系统是外源基因成功转化的先决条件,但再生系统不等同于转化系统。为提高农杆菌共培养后胡萝卜的再生频率,以日本新黑田五寸参和林丰改良五寸参2个品种胡萝卜下胚轴为外植体,利用农杆菌介导法将人源白细胞介素-2基因(IL-2)导入其中,探讨了共培养后诱导培养基中激素浓度和筛选剂对再生的影响。结果显示:不同基因型胡萝卜的分化对激素浓度的反应不同,林丰改良五寸参在3种培养基上的胚性愈伤率差异不显著;日本新黑田五寸参胚性愈伤率和每个外植体分化的抗性株数随激素浓度的提高而呈上升趋势,2,4-D和6-BA浓度为1.0mg/L时胚性愈伤率和每个外植体分化的抗性株数最高,分别为87.50%和59株;日本新黑田五寸参以bar基因为标记基因、PPT作筛选剂时,分化较好。因此,遗传转化过程中,为提高共培养后胡萝卜胚状体的分化率,对共培养后的诱导培养基进行再优化是必要的。

Although high frequency of regeneration system determines the transfer of foreign gene, it is different from transformation system. In order to increasing the regenerating frequency of carrot by Agrobacteri- um transformation, the concentration of plant growth regulators and selective agents were studied on the re- generation of hypocotyls from 2 cultivars, which were transformed with A grobacterium tumefaciens containing recombinant human interleukin-2 gene （IL-2）. The results showed that the carrot of different genotypes responded differently with the plant growth regulators. There was no significant difference on the three kinds of callus inducing medium for cultivar Linfeng. In contrast, the rate of embryogenic callus and the number of resistant plants in cultivar Japanese black filed five inch was raised with increasing plant growth regulator concentration. The highest rate of embryogenic callus was 87.5% on the medium containing MS＋I.0 mg/L 2,4-D＋1.0 mg/L6-BA＋PPT, and the number of the resistant plants was 59. Therefore, it is essential to reoptimize the inducing medium with co-cultivation.