| Perennial ryegrass (Lolium perenne L.) is a species of turfgrasses and foragegrasses with high quality. In the research area of turfgrasses, biotechnology has been widly applied to improve agronomic characteristics. In the present work, the prerequisites of establishing the high frequency regeneration system of perennial ryegrass were studied, bar gene was transferred into the embryonic calli by microprojectile bombardment and transgenic ryegrass plants were regenerated. This experiment offered a new protocol for improvement of ryegrass varieties.1. Establishment of high frequency regeneration system of perennial ryegrassBy comparing test results, highly regenerative embryonic calli of perennial ryegrass derived from mature embryos were induced and proliferated on MS medium containing 5mg/L 2,4-D and the induction rate of embryonic calli was 23.8%. The optimal medium for differentiation was MS medium containing 1mg/L 6-BA, the rate of differentiation was 33.3%.2. Establishment of genetic transformation system of perennial ryegrassSensitive test of calli to Bialaphos showed that the suitable concentration of Bialaphos in the medium was 2mg/L. Genetic transformation system of embryonic calli of perennial ryegrass was established by bombardment.Before bombardment, the vigorous calli were precultured on osmotic medium containing 0.25mol/L sorbitol and 0.25mol/L mannitol for 5～12h. In biolistic transformation, the embryonic calli were bombarded at the pressure 1350psi with 500μg gold particles per shot. After transformation, the calli were transferred onto selective medium, and were subcultured on selective medium for three times at 10 day intervals. The resistant calli were transferred onto the regeneration medium containing Bialaphos, and shoots appeared within 20 days on this medium. Regenerated shoots were rooted on MS medium and were transplanted to soil. PCR analysis indicated that 37 transgenic plants contained bar gene and the frequency of transformation was 1.54%. 22 of these 37 transgenic plants expressed gus gene. Basta resistant experiments showed that the transgenic plants of ryegrass can grow normally after painting with 100mg/L Basta solution. |
