Shoot regeneration in stem expiants and its amenability to Agrobacterium tumefaciens mediated gene transfer in Brassica carinata
Shoot regeneration in stem expiants and its amenability to Agrobacterium tumefaciens mediated gene transfer in Brassica carinata
复制标题
甘蓝茎外芽再生及其对根癌农杆菌介导基因转移的适应性
作者：
S. B. Narasimhulu;P. Kirti;T. Mohapatra;S. Prakash;V. Chopra
7个不同基因型埃塞俄比亚芥的未成熟茎段在MS培养基中分别添加0.2mg/l的BAP和毒莠定时，产生不定芽的频率各不相同。选择171系，其从接种物的两个切割端以100%效率产生芽，用于测试该再生方案对遗传转化的适用性。使用含有质粒PCV 730的非致癌根癌农杆菌，所述质粒PCV 730是携带卡那霉素和潮霉素抗性基因的二元载体。共培养4天，细菌浓度约为2.5×10 - 15个细胞/ml，然后恢复期为2天，产生转化芽，可以选择和生根卡那霉素在15毫克/升的存在。通过新霉素磷酸转移酶测定和Southern印迹分析证实转化。转化植株的种子分析表明，卡那霉素抗性在后代中遗传。
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.