Biotechnology Centre, Indian Agricultural Research Institute, 110012, New Delhi, India.
Plant Cell Rep. 1992 Jul;11(7):359-62. doi: 10.1007/BF00233366.
Immature stem segments of seven different genotypes of Brassica carinata produced shoots with variable frequencies when cultured in MS medium with BAP and picloram at 0.2 mg/l each. Line 171, which produced shoots with 100% efficiency from both cut ends of the expiant, was selected for testing the amenability of this regeneration protocol for genetic transformation. A non-oncogenic Agrobacterium tumefaciens containing plasmid PCV 730, a binary vector carrying resistance genes for kanamycin and hygromycin, was used. A cocultivation period of 4 d with a bacterial concentration of approximately 2.5×10 cells/ml, followed by a recovery period of 2 d, produced transformed shoots that could be selected and rooted in the presence of kanamycin at 15 mg/l. Transformation was confirmed by neomycin phospho-transferase assay and Southern blot analysis. Seed analysis of transformed plants indicated that kanamycin resistance was inherited in the progeny.
不同基因型的甘蓝型油菜未成熟茎段在含有 BAP 和 Picloram 的 MS 培养基中培养，每个浓度为 0.2mg/L 时，均可产生不定芽，但频率有所不同。171 号线在插条的两端都能以 100%的效率产生不定芽，因此被选中用于测试这种再生方案是否适合遗传转化。采用的根癌农杆菌是非致瘤型的，含有质粒 PCV 730，该质粒是一种携带卡那霉素和潮霉素抗性基因的二元载体。用浓度约为 2.5×10 个细胞/ml 的细菌进行 4 天的共培养，然后恢复 2 天，可产生转化的不定芽，在 15mg/L 的卡那霉素存在下可以进行选择和生根。通过新霉素磷酸转移酶检测和 Southern blot 分析确认了转化。对转化植株的种子分析表明，卡那霉素抗性在后代中是可遗传的。
