Study On Italian Ryegrass Transformation Of AVP1 Gene Mediated By Agrobacterium Tumefaciens

Italian Ryegrass(Lolium multiflorum Lam.) has great high nutritional value,and it is a good kind of forage plant.For a long time,biotechnology has been widely applied to improve its characteristics.The mature seeds of Italian Ryegrass were used as explants to establish high-frequency regeneration system, and the influences of varied factors on calls induction and growth were discussed.Based on that gene AVP1 was transformed into Lolium multiflorum Lam.by Agrobacterium-mediated.The transgenic Italian Ryegrass were gained.The main results were as following:(1)Establishment of high frequency regeneration system of Lolium multiflorum Lam..mature seeds of Italian Ryegrass were used for callus induction and differentiation.The results showed that①The effect of different ways of seeds treatment(with a cut or not)on callus induction was not significant.On this study,the mature seeds of Italian Ryegrass were used as explants.②The suitable medium for callus induction is a basical MS salt mixed with 5mg/L 2,4-D+300mg/L Hydrolysate.③The suitable medium for subculture is MS+5mg/L2,4-D+300mg/L Hydrolysate.④Callus with a higher regeneration are light yellow to green,compact fast-growing and green.⑤The suitable concentration of 6-BA in medium(MS) for plantlet regeneration is 0.2mg/L.(2)Establishment and optimization of Perennial ryegrass genetic transformation system by Agrobacterium-mediated method.The optimized genetic transformation procedure was:Callus were used as recipient material for transformation,the concentration of Agrobacterium tumefaciens suspension was OD600=0.5 added with AS of 150μmol/L,then the infected calli were incubated for 3 days.After that cultivated in the medium supplemented with 300mg/L G418 and 300mg/L Carb.(3)The transformation of AVP1 gene and the preliminary identification of transgenic plants,gene AVP1 was transformed into Lolium multiflorum Lam.Cultivar Barmultra by Agrobacterium-mediated, The transgenic Italian Ryegrass were gained.By PCR,RT-PCR analysis,the results indicated that AVP1 gene had already inserted into the genome in the plants and transcripted to mRNA.