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利用种子和胚分别在两种培养基K3和K5诱导产生了早熟禾(Poa pratensis L.)一个品种Mado的胚性愈伤组织。K3培养基含有10.0μmol/L的二氯苯氧乙酸(2,4-D)、0.5μmol/L的苄氨基嘌呤(BAP)。K5培养基是K3另加0.5μmol/L的硫酸铜。光照条件为20～30μmol·m~(-2)·s~(-1)、16h光照、8h黑暗。温度保持在24℃。用携有bar基因和gus基因的pDM805质粒转化的农杆菌AGL1对胚性愈伤组织进行转化。共得到4个转基因株系。影响转基因效率的主要因素有愈伤组织的胚性、光照条件、共转化时间、抗生素浓度、选择压力。本研究建立了单子叶早熟禾农杆菌介导的转基因方案。
Induction of embryogenic callus of Mado, a cultivar of Poa pratensis L., was induced in both media, K3 and K5, respectively, using seeds and embryos. K3 medium contained 10.0 μmol / L dichlorophenoxyacetic acid (2,4-D) and 0.5 μmol / L benzylaminopurine (BAP). K5 medium is K3 plus 0.5 μmol / L copper sulfate. The light conditions were 20 ~ 30μmol · m ~ (-2) · s ~ (-1), 16h illumination and 8h darkness. The temperature is kept at 24 ° C. Agrobacterium AGL1 transformed with pDM805 plasmid carrying the bar gene and the gus gene is used to transform embryogenic callus. A total of four transgenic lines were obtained. The main factors affecting the efficiency of transgenic callus embryogenic, light conditions, co-transformation time, antibiotic concentrations, selection pressure. In this study, Agrobacterium tumefaciens-mediated transgenic program was established.