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以二穗短柄草3个品系BDR018,BD21,BD21-3的幼胚为外植体,研究了幼胚大小对胚性愈伤组织诱导和再生的影响。采用携带gus和bar基因双元表达载体(pDM805)的根癌农杆菌菌株AGL1对BDR018品系的幼胚愈伤组织进行了遗传转化。本实验探讨了影响幼胚愈伤组织诱导再生及遗传转化的几个因素。结果发现,幼胚大小介于0.5～1.0mm之间的愈伤组织诱导率最高。随愈伤组织年龄的增加,BDR018,BD21,BD21-3愈伤组织的再生率下降,白化率上升。愈伤组织年龄在5～8周范围内转化效率较高,平均转化效率为38.5%。真空处理5min和0.01%的Silwet L-77处理均可提高转化效率。对转化植株进行GUS基因化学组织检测和PCR鉴定,初步证明外源基因已整合再生植株基因组中。
The immature embryos of BDR018, BD21 and BD21-3 were used as explants to study the effects of immature embryo size on the induction and regeneration of embryogenic callus. The immature embryos callus of BDR018 strain was genetically transformed with Agrobacterium tumefaciens strain AGL1 carrying the binary expression vector of gus and bar genes (pDM805). This experiment explored several factors affecting immature embryo callus induction regeneration and genetic transformation. The results showed that callus induction rate of immature embryo size between 0.5 ~ 1.0mm was the highest. With the increase of callus age, the regeneration rate of BDR018, BD21, BD21-3 callus decreased, and the rate of albino rose. The callus’s transformation efficiency was high in the range of 5-8 weeks, and the average transformation efficiency was 38.5%. Vacuum treatment 5min and 0.01% Silwet L-77 treatment can improve the conversion efficiency. The GUS gene was detected by chemical organization and PCR identification of the transformed plants, which initially proved that the foreign gene has been integrated into the genome of the regenerated plant.