Perennial ryegrass (Lolium perenne), a graminaceous crop, is one of the most widely distributed grass in the temperate regions for forage and turf. However, its transformation mediated by Agrobacterium tumefaciens is largely recalcitrant and lagged. In this paper, an improved method for perennial ryegrass mediated by A. tumefaciens was developed. And the transgenic plants were successfully obtained. Embryogenic calli induced from mature embryos of a perennial ryegrass variety Tove were transformed by A. tumefaciens strain EHA105 containing the plasmid pCAMBIA2301. In this protocol, 0.1 mol/L mannitol or 5 mg/L silver nitrate (AgNO3) were added in preculture and coculture media. After selected with paromomycin and detected with PCR and GUS expression, we got 38 transgenic plants in all. The transgenic frequency was increased when 0.1 mol/L mannitol or 5 mg/L AgNO3 or 5 mg/L AgNO3 ＋0.1 mol/L mannitol was added in the pre-cultivation and co-cultivation media, which was 1.96, 1.59 and 2.95 times compared to control respectively. Moreover,the transgenic frequency was highest when the two materials were jointly used. The results indicated that mannitol and AgNO3 could improve the A. tumefaciens mediated transformation in perennial ryegrass evidently.%多年生黑麦草是优良的牧草和草坪草.农杆菌介导多年生黑麦草遗传转化非常困难.为了建立稳定的多年生黑麦草遗传转化体系,本研究以品种"多福"种子成熟胚为外植体诱导愈伤组织,携质粒pCAMBIA2301的根癌农杆菌EHA105介导,进行遗传转化.在预培养及共培养基中添加0.1 mol/L甘露醇或5 mg/L硝酸银,并分析了二者对转化的影响.经巴龙霉素筛选、PCR及GUS组织染色检测表明,共获得了转基因植株38株.添加O.1mol/L甘露醇或5 mg/L AgNO3或O.1 mol/L甘露醇+5 mg/L AgNO3,转化效率分别为对照的1.96,1.59和2.95倍.结果表明,在根癌农杆菌介导的多年生黑麦草愈伤组织遗传转化中,在预培养和共培养基中添加甘露醇或AgNO3能提高转化率,且当二者共同添加时可显著提高.
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