Co-transformation Of Bar And P5CS Gene Using Bombardment On Perennial Ryegrass (Lolium Perenne L.)

Application of biotechnology in turfgrass breeding improved agronomic characteristic is a developing research. Perennial ryegrass (Lolium perenne L.) is a turfgrass of high quality. The conditions of establishing the high regeneration system of perennial ryegrass were studied, the genes of bar and P5CS were transformed into the calli using microprojectile bombardment resulted in the production of transgenic plants with improved tolerance to salt and drought stress, which offered a new germplasm to breed new perennial ryegrass with improving the salt and drought tolerance. The results were as follows: 1. Establishing the high regeneration system of perennial ryegrass with two different explants. By comparing test results, highly regenerative tissues of perennial ryegrass produced from mature embryo were induced and proliferated on MS medium containing 2,4-D 5mg/L and mannitol 0.2mol/L, and the best medium for the differentiation and germination of somatic embryos was 1/2MS with 2,4-D 1mg/L and KT 1mg/L, the rate of differentiation was 71%. Effecting factors of regenerative frequency were studied. The callus-induction medium and differentiation medium were the same for immature inflorescence of perennial ryegrass on MS supplemented with 2,4-D 3mg/L, 6-BA 0.5mg/L, and sucrose 3%, The rates of calli and shoots differentiation were 80%, and 81% respectively. The developmental stage of the inflorescences apparently wasn't an important factor for formation of the callus, which expanded a wide scope of useful explants.2. Establishing genetic transformation system of perennial ryegrass.(1) Sensitive test of calli to Glufosinate confirm that they were 1.2mg/L and 1.7mg/L.(2) Establishing genetic transformation system of perennial ryegrass from mature embryo.For bombardment, 4-week-old calli were used. After bombardment, the calli were allowed to recover 7 days before they were transferred to a selection medium with two chosen stages, which is 20 days respectively, and resistant calli were transferred to the regeneration medium containing Glufosinate. Shoots appeared within 4-5 weeks on this medium. Regenerated shoots were rooted on MS medium with Glufosinate and were transferred to soil after growing roots. PCR and PCR-Southern analysis indicated that 18 transgenic plants contained bar gene and the frequency of transformation was 0.72%. 5 of these 18 transgenic regenerate plants contained P5CS gene and the co-transformation frequency was 27.8%.(3) Establishing genetic transformation system of perennial ryegrass from immature inflorescence.For bombardment, 40-day-old calli were used. After bombardment, the calli were allowed to recover 7 days before they were transferred to a selection medium, and the calli were transferred to the regeneration medium containing Glufosinate. Shoots appeared within 3 months on this medium. PCR analysis indicated that transgenic plants only contained bar gene and the frequency of transformation was 2.7%.