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利用农杆菌介导法用Bt cry1A(b)基因对印度栽培棉种进行非基因依赖型遗传转化和植株再生。将印度栽培棉品种Anjali(LRK 516)和LRA 5166与携带Bt cry1A(b)+nptII基因的根癌农杆菌LBA4404共培养,在含100μg·ml 1卡那霉素的筛选培养基上获得了可能的转基因直接再生苗。细菌浓度、共培养时间、感染组织的阶段和大小、标记筛选浓度、培养基成分和激素等都影响转化效果和效率。对程序进行了优化。经PCR、Southern杂交、ELISA等方法分别检测,证实了Bt cry基因的插入和表达。Southern分析表明转化植株存在3～5个基因拷贝,但CRY蛋白表达量非常低(为叶蛋白的0.003%～0.004%)且生化抗虫性很弱或基本不影响鳞翅目昆虫。尽管如此,该方案仍适用于其它CRY基因或重要经济型基因进行非基因依赖型遗传转化和再生,产生转基因棉花。
Non-gene-dependent genetic transformation and plant regeneration of Indian cotton cultivars using Bt cry1A (b) gene by Agrobacterium-mediated method. Indoculture of Indian cultivated cotton varieties Anjali (LRK 516) and LRA 5166 with Agrobacterium tumefaciens LBA4404 carrying the Bt cry1A (b) + nptII gene was made possible on a selection medium containing 100 μg · ml 1 of kanamycin The transgenic direct regeneration seedlings. Bacterial concentration, co-culture time, stage and size of infected tissue, marker screening concentration, medium composition and hormones all affect transformation efficiency and efficiency. The program has been optimized. By PCR, Southern blot, ELISA and other methods were detected, confirmed the Bt cry gene insertion and expression. Southern analysis showed that there were 3 to 5 copies of the gene in the transformed plants, but the expression level of CRY protein was very low (0.003% -0.004% of the leaf protein) and the biochemical pest resistance was weak or hardly affected Lepidoptera. Nevertheless, the program is still suitable for non-gene-dependent genetic transformation and regeneration of other CRY genes or important economic genes to produce transgenic cotton.