烟草转硝酸还原酶基因高效遗传转化研究

Optimization of the High Efficient Genetic Transformation for Tobacco NR Mediated by Agrobacterium

通过对农杆菌介导烟草遗传转化方法的优化,建立了快速高效烟草叶盘遗传转化体系。用OD600为0.5的农杆菌悬浮液浸染大小约1.0 cm×1.0 cm烟草叶盘5～10 min,再置于MS附加2.25 mg/L 6-BA和0.10 mg/LNAA的分化培养基上,在100 mg/L卡那霉素选择压下,21～28 d可获得抗性不定芽。经卡那霉素生根筛选和PCR扩增鉴定,其阳性转基因植株频率可达81.2%。

A rapid and high efficient transformation system has been established for Agrobacterium-mediated transformation of tobacco by optimizing the factors of transformation.The tobacco leaves were cut into 1.0 cm×1.0 cm discs and then were submerged in Agrobacterium suspensions（OD600=0.5） for 5～10 min,then were transferred to MS differentiation medium supplemented with 2.25 mg/L 6-BA and 0.10 mg/LNAA.The buds resistant to kanamycin were obtained in 21～28 days under 100 mg/L kanamycin selection.The positive rates of putative transgenic plants could reach 81.2% by using rooting selection of kanamycin and PCR identification.