【摘要】 【目的】拟通过改进大豆外植体的组织培养、农杆菌侵染以及植株再生等关键性环节,构建快速、高效的大豆转基因技术体系,解决外源基因难以导入大豆的难题。【方法】成熟大豆种子在环境温度25～28℃、含有1mg·L-16-苄氨基嘌呤的MSB5培养基上催芽24h。保留大豆下胚轴、胚尖及1片子叶作为外植体,并在相同条件下预培养24h。将外植体转入2/3MSB5液体培养基(1mg·L-16-苄氨基嘌呤+100μmol·L-1乙酰丁香酮+携带有Ti质粒pBI121的根瘤农杆菌GV3101)中,在28℃黑暗环境下侵染21h。侵染后的外植体在28℃黑暗条件下共培养3d。外植体经无菌水处理后移栽到灭菌土中,并在适宜条件下培养。【结果】分子检测结果表明,本研究涉及的新型外植体的再生率高达90%以上(中品661为95%,垦农18为93%,绿75为90%),转化效率明显提高;而利用子叶节和胚尖进行培养获得的外植体的再生率仅50%和70%。此外,本研究涉及的大豆转化体系省去了外植体后期繁琐、耗时的组织培养步骤,再生周期明显缩短。【结论】与前人报道的大豆遗传转化体系技术相比,本研究所使用的方法更加快速、高效,可为农杆菌介导的大豆转基因、突变体构建等基因组学研究提供强有力的技术平台。更多还原

【Abstract】 【Objective】To develop a rapid and high efficient technique for soybean transformation, in this study, some key techniques for explants tissue culture, Agrobacterium tumeficiences infection and plants regeneration were studied.【 Method】Mature soybean seeds were germinated on MSB5-medium including 1 mg·L-1 N6-benzylaminopurine (BAP) at 25 - 28℃ for 24 h. The explants composed of hypocotyls, embryonic tips and one piece of cotyledons were pre-cultured at the same condition mentioned above for 24 h. Explants were then cultured on 2/3 MSB5-medium including 1 mg·L-1 N6-benzylaminopurine (BAP), 100 μmol·L-1 acetosyringone and Agrobacterium tumeficiences GV3101 carrying Ti plasmid pBI121 at 28℃ for 21 h. After co-cultivated in the dark at 28℃ for 3 d, transformants were sterilized with sterile water, and transferred to sterile soil directly and incubated with suitable conditions. 【Result】 The results of molecular test indicated that the regeneration ratio of new explants was more than 90% (95% for Zhongpin661, 93% for Kennong18 and 90% for Lv75, respectively), and the transformation ratio was increased significantly. But, at the same time, the regeneration ratios for cotyledonary nodes and embryonic tips were only 50% and 70%, respectively. Furthermore, as complicated and time-consuming tissue cultivation operations for transformants in the latter period were eliminated, the time for regeneration were reduced obviously. 【Conclusion】Compared with the soybean transformationtechniques reported before, the Agrobacterium tumeficiences-mediated genetic transformation system in this study was a more time-saving, efficient and powerful technique for soybean genomic studies on transformation, mutants construction and soon.更多还原