Establishment Of Regeneration System Of Perennial Ryegrass And Agrobacterium Tumefaciens-mediated Transformation With AVP1

Perennial ryegrass(Lolium perenne L.)is one of the major cool-season turfgrass widely utilized both at home and abroad. For a long time, biotechnology has been widely applied to improve its characteristics. we choose three varieties for this reserch,which are Regal, Belle and Marathon. And we've established an efficient genetic transformation system. The results were as follows:(1)Establishment of high frequency regeneration system of perennial ryegrass .Different explants of Perennial ryegrass (Lolium perenne) were used for callus induction and differentiation on MS basic medium supplemented with different concentration of 2,4-D and 6-BA . The results showed that different explants have different induction and differentiation efficiency. Shoot tip and root tip were ineffective in callus induction. The callus induction and differentiation derived from mature seeds were superior to hypocotyl .When supplemented with 5-7mg/L of 2,4-D and 0.05-0.1 mg/L of 6-BA were propitious to callus induction from mature seeds and hypocotyl.And differentiation medium supplemented with 0.5-1.0 mg/L of 6-BA was the best.The mature seeds and hypocotyl were subcultured for 60 and 80 days,respectively,can get the highest differentiation efficiency .(2)Establishment and optimization of Perennial ryegrass genetic transformation system by Agrobacterium-mediated method.The optimized genetic transformation procedure was: Callus obtained after about 4 days cultured were used as recipient material for transformation , the concentration of Agrobacterium tumefaciens suspension was OD600=0.6 added with AS of 150μmol/L, treated with negative pressure, then the infected calli were incubated at 24°Сin the dark for 3 days.After that cultivated in the medium supplemented with 75mg/L Hygromyein and250mg/L Carb.(3)The transformation of AVP1 gene and the preliminary identification of transgenic plants.The resistant callus were transferred to the differentiation medium supplemented with 75mg/L Hygromyein and250mg/L Carb first, and then transferred to the regeneration medium for further root development and finally green plants.At last, performed PCR amplification,but no one positive sample was found.That means the AVP1 gene was not integrated to the genomic DNA of Perennial ryegrass.