草地早熟禾农杆菌介导法基因转化条件

A Study on the Conditions of Genetic Transformation into Poa pratensis L. by Agrobacterium Tumefaciens

以大青山草地早熟禾(PoapratensisL.cv.Daqingshan)和超级伊克利草地早熟禾(PoapratensisL.cv.Total eclipse)成熟种子诱导产生的胚性愈伤组织为材料,比较抗生素及筛选剂对愈伤组织生长和绿苗分化的影响。结果表明: 500mg/L的羧苄青霉素或头孢霉素对愈伤组织的生长与对照间差异不显著;羧苄青霉素可提高愈伤组织绿苗分化率, 而头孢霉素则有抑制作用;2个供试品种G-418的筛选浓度分别为50和70mg/L时,愈伤组织的褐化率达100%;在幼 苗分化期,G-418浓度为20mg/L时,2个供试品种的试管苗均为白化苗;在观察到愈伤组织周围的培养基上显现菌落 时,立即转移到筛选培养基上为宜。用农杆菌介导法获得了转苏云金芽孢杆菌杀虫晶体蛋白(B.t)基因植株。

Using the embryogenic calli induced from the mature seeds of Poa pratensis L. cv. Daqingshan and Total eclipse as recipient materials, authors of the paper studied the effect of carbenicillin, cefotaxime, and geneticin (G-418) on the callus growth and shoot regeneration. The result shows that the effect of 500 mg/L carbenicillin or cefotaxime to the growth of calli matches that of the control. Carbenicillin could increase the rate of shoot regeneration of calli, while cefotaxime checks it. The calli of the two species, “Daqingshan” and “Total eclipse”, screened separately with 50 mg/L and 70 mg/L of G-418 both turn brown. During the shoot regeneration period, 20 mg/L of G-418 turns all the shoots white. The time needed for culturing the embryogenic callus and agrobacterium tumefaciens together is determined by the appearance of colonies of the latter that could be detected with naked eyes. The callus should be transferred immediately to the screening medium. The recombinant plasmid carrying B.t gene and NPT Ⅱ gene are introduced into callus of “Total eclipse” by means of agrobacterium-mediated transformation. Transgenic plants confirmed by PCR analysis are thus obtained.