乌拉尔甘草离体再生与遗传转化技术的研究

研究乌拉尔甘草离体再生受体系统和遗传转化技术方法,为甘草基因工程育种提供前期实验基础。以乌拉尔甘草茎段为外植体,试验筛选最适不定芽分化培养基和增殖壮苗培养基; 以乌拉尔甘草茎段为受体,利用农杆菌介导法对乌拉尔甘草进行GUS和GFP基因的遗传转化研究。结果表明,以乌拉尔甘草去腋芽茎段为外植体,其不定芽最适分化和增殖培养基配方为MS+0.1 mg/L TDZ+0.1 mg/L 6BA+0.1 mg/L IBA,不定芽分化率达38.8%,增殖倍数3.67,适宜的壮苗培养基为MS+0.5 mg/L NAA。与去腋芽茎段相比,带腋芽茎段的不定芽分化率达100%,能够在含抗生素的选择培养基MS+0.1 mg/L TDZ+0.1 mg/L 6BA+0.1 mg/L IBA+50 mg/L Kan+250 mg/L Car上存活,确定为最适转化受体;带腋芽茎段在分化培养基上预培养7 d后,经OD₆₀₀=0.6的菌液侵染10 min,共培养3 d,在选择培养基上培养20 d,其不定芽分化率为43.75%,经GFP荧光检测可得到78.88%的GFP基因瞬时表达率;随机选取5株再生转化植株,经PCR检测,均有GFP和GUS基因的目的条带,GFP基因表达较强烈,初步获得了5个株系的阳性转化植株。

In vitro research on receptor regeneration and genetic transformation in Glycyrrhiza uralensis

A study has been undertaken to investigate the system of receptor regeneration and genetic transfor-mation of Glycyrrhiza uralensis in vitro ,in order to provide a preliminary experimental basis for breeding G. uralensis via genetic engineering.Using stem segments as explants,tests were done to select the most appro-priate medium formula for adventitious bud differentiation,propagation and elongation.Furthermore,GUS and GFP genes were transferred into G.uralensis through Agrobacterium tumefaciens-mediated transformation methods.The results showed that the optimum formula for bud differentiation and proliferation was a Murash-ing and Skoog (MS)medium supplemented with 0.1 mg/L thidiaxuron,0.1 mg/L 6-benzyladenine and 0.1 mg/L indole-3butyric acid.Using this formula,the differentiation rate of adventitious buds was 38.8% and the rate of multiplication 3.67.The most suitable formula for bud elongation was a MS medium containing 0.5 mg/L α-naphthalene acetic acid.Compared to stems without axillary buds,the differentiation rate of adventi-tious buds reached 100%.Moreover,these buds can survive in antibiotic containers and so become the optimal transformation explants.Stems with axillary buds were pre-cultured for 7 days in the differentiation medium, then infected by an Agrobacterium tumefaciens solution (OD600 =0.6)for 10 min and co-cultured for 3 days, following which the explants were transferred to a selective medium based on the differentiation medium and supplemented with 50 mg/L kanamycin and 250 mg/L carbenicillin.After 20 days,the GFP transient expres-sion rate reached 78.88% and GFP expressed strongly by GFP detection.5 transformed plants were selected randomly for testing and the presence of GUS and GFP genes in the genome were confirmed by polymerase chain reaction,indicating that 5 transformed plantlets had been successfully produced.