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以露地菊(Chrysanthemum morifolium)品种‘神韵’无菌苗叶片为外植体材料,研究植物生长调节剂配比对其愈伤组织诱导再生芽的影响,以建立离体再生体系。研究结果表明,露地菊‘神韵’在含NAA1.0mg·L-1+BA1.0mg·L-1的MS培养基上获得了最高的再生芽分化率。利用已经克隆的‘津田’芜菁BrDFR基因构建非抗生素筛选的表达载体。以露地菊‘神韵’的无菌苗叶片为受体,通过农杆菌介导进行BrDFR基因的遗传转化。以载体的PMI基因为筛选标记,通过甘露糖筛选获得了‘神韵’的遗传转化再生植株。经过PCR验证和Southern杂交检测证实BrDFR基因已经整合到‘神韵’基因组中。
The effect of plant growth regulators on callus induction of regenerated buds was studied using the leaves of ’Shenyun’ aseptic seedlings of Chrysanthemum morifolium as explants to establish an in vitro regeneration system. The results showed that the highest regeneration rate of shoot regeneration was obtained on MS medium with NAA1.0 mg · L-1 and BA1.0 mg · L-1. The non-antibiotic screened expression vector was constructed using the already cloned ’Tsuda’ turnip BrDFR gene. The germination of BrDFR gene was mediated by Agrobacterium tumefaciens mediated by aseptic seedling leaves of Dewdrop Chrysanthemum. The PMI gene of the vector was used as a screening marker, and the regenerated plant of ’Shenyun’ was obtained through mannose screening. After PCR validation and Southern hybridization test confirmed BrDFR gene has been integrated into ’Shen Yun’ genome.