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试验以所建立的四倍体刺槐高频再生体系为基础,通过农杆菌介导法转化甜菜碱醛脱氢酶(BADH)基因,以GUS染色组织分析法为依据探讨了影响转化效率的各种因素,建立了优化转化体系,结果如下:20mg·L~(-1)乙酞丁香酮可显著提高转化效率;菌液浓度OD_(600)值0.3～0.7、预培养2d为宜;转化后暗培养对转化效率没有影响。并在以上研究基础上,成功地建立了高效、可重复的遗传转化体系,选择培养基上头孢霉素400mg·L~(-1)可有效地抑制农杆菌;卡那霉素50mg·L~(-1)时愈伤组织的白化死亡率达96.4％。经PCR检测,外源基因已成功的整合到植株的基因组DNA中,获得了15个转基因株系。
Based on the established high-frequency regeneration system of tetraploid R. pseudoacacia, the genes of betaine aldehyde dehydrogenase (BADH) were transformed by Agrobacterium tumefaciens, and the effects of transformation efficiency were discussed based on GUS staining. The results showed as follows: 20mg · L -1 acetosyringone could significantly improve the transformation efficiency; the concentration of OD_ (600) was 0.3 ~ 0.7 and pre-culture was suitable for 2 days; Culture has no effect on conversion efficiency. On the basis of the above studies, we successfully established an efficient and reproducible genetic transformation system. The selection of cefotaxime 400mg · L -1 could effectively inhibit Agrobacterium tumefaciens. The concentration of kanamycin 50mg · L ~ (-1) callus whitening death rate of 96.4%. After PCR analysis, the foreign gene has been successfully integrated into the plant genomic DNA, obtained 15 transgenic lines.