农杆菌介导法获得匍匐翦股颖转GO基因植株

Acquirement of GO Transgenic Plants by Agrobacterium tumefaciens in Creeping Bentgrass(Agrostis stolonifera L.)

以匍匐翦股颖2个品种PentA-4和L-93的成熟种子为外植体材料,附加2,4-D 4.0 mg/L和KT 0.05mg/L的稍作修改的MS培养基为愈伤组织诱导和继代培养基。琼脂条的用量为16 g/L时,50%以上的愈伤组织呈浅黄色或白色、干燥、颗粒状结构。颗粒状愈伤组织经过连续3次继代培养后,其绿苗分化率保持在60%以上。以颗粒状胚性愈伤组织为受体材料,用农杆菌介导法将含有葡萄糖氧化酶(GO)基因和新霉素磷酸转移酶基因(NPT II)的重组质粒导入匍匐翦股颖。愈伤组织经农杆菌LBA4404/pBGO感染和共培养后,在附加G-418 60mg/L的愈伤组织继代培养基上选择到具有卡那霉素抗性的愈伤组织。抗性愈伤组织分化的绿苗在附加G-418 30mg/L的绿苗分化培养基上进行筛选,获得了抗性再生植株。试管植株的叶片经淀粉-碘化钾显色反应,检测到转GO基因植株产生的过氧化氢。PCR分子检测结果证明,GO基因已整合到转化植株的基因组中。抗病性鉴定结果显示,转GO基因植株抗水稻纹枯病。

The mature seeds ofAgrostis stolonifera L. cv. Pent A-4 and L-93 were used as the explant materials to transform glucose oxidase （Go） gene and neomycin phosphotransferase （ NPTⅡ ）. The modified MS medium supplemented with 4. 0 mg/L 2,4-D and 0. 05 mg/L KT were used for callus induction and subculture. The yellowish or white dry pellet, obtained in the callus induction and subcuture medium with 16 g/L agar; showed the rate of shoot regeneration of over 60% after three subcultures through agrobacterium-mediated transformation, the recombinant plasmid carrying GO gene and NPTⅡ gene was introduced into Agrostis stolonifera L. with pellet embryogenic calli as recipient after infected with agrobacterium tumefaciens strain LBA4404/pBGO, calli with kanamycin resistance were screened out on the callus subculture medium supplemented wih 60 mg/L G-418, and regenerated plants with kanamycin resistance were acquired on the callus differentiation medium supplemented with 30 mg/L G-418. By color reaction with starch-KI, H2O2 was expressed in leaf of the transgenic plants. PCR analysis confimed that GO gene had been intergrated into the genome of the transgenic plants. Inoculation results indicated that GO transgenic plants were resistant to Rhizoctonia solani.