Of Genetic Transformation System For Tall Fescue Establishment Of Salt-tolerant Plants Of D. Glomerata And Festuca Arundinacea In Vitro Screening

Tall fescue can be planted either for forage or for turf-type grass that spreads rapidly in China these years. Duckgrass is useful forage grass that prefers living under orchard trees and gets the name orchardgrass. The obvious defects such as less salt or heat tolerance ristrict the spreadness of tall fescue and orchardgrass. Traditional breeding is time-consuming and laborious, and has not resulted in salt-tolerant cultivars. Development of both cellular techniques and genetic engineering manipulation made it possible to decrease breeding time and create salt-tolerant cultivars of tall fescue or orchardgrass.In this research, multiple shoots were induced in vitro from sterilized seedling apices of tall fescue. MS medium supplemented with 7mg/L 2,4-D and 3mg/L 6-BA was the best inducing medium and that supplemented with 4mg/L 6-BA was the best subculturing medium for tall fescue .1-mm-long small shoot tips of tall fescue derived from the multiple shoot clumps subcultured for 5-7 days were infected with Agrobacterium LBA4404 harboring plasmid pCAMBIA1300-betA-als for 5 min with 0.5×10⁵pa negative pressure co-treatment. After co-cultured for 2 days on subculture medium, the shoot tips were transferred to medium supplemented with 150mg/L cefotaxime to inhibit the growth of Agrobacterium for 2 weeks. In the period of co-culture and inhibiting Agrobacterium, the shoots developed to 2-3cm high. After further 45 days subcultures on medium containing lvchuanglong (chlorsulfuron) at the concentration of 0.6mg/L, at a 15-day interval, the survival frequency of infected shoots was about 23.7%. Then, the survivals were transferred to medium without lvchuang to proliferate and larger shoots were transferrd onto rooting medium. Plantlets with vigorous roots were then transplanted into pots. Higher transformation efficiency was obtained at the following conditions: the bud tips were infected for 5 minutes under 0.5×10⁵pa in Agrobacterium suspension of OD₆₀₀ 0.8-1.0 and co-cultured for 2 days.DNA was extracted from leaves of transformed plants for PCR assay and PCR-Southern analysis. The primers were designed according to the coding sequences of betA gene. 20.9% of the lvchuanglong -resistant plants were PCR positive and gave positive signal at PCR-Southern blotting, indicating that the betA gene had been transferred into some of the transformed plants.Some of the transformed shoots were proliferated and then transferred to medium supplemented with 1.5% NaCl for 3 consecutive generations, they exhibited higher salt-tolerance compared with the controls.The routine transformation system of tall fescue had undesirable problems such as long-term in vitro cultures, genotype-dependence, high frequency of somaclonal variation of regenerants and low transformation frequency, limiting the development of tall fescue genetic engineering. This reseach established a short-term, less genotype-denpendence and efficient genetic transformation system for tall fescue.Multiple shoots of orchard grass and tall fescue were transformed onto medium supplemented with a range of concentrations (0.5%-2.5%) of NaCl for 3 consecutive generations' screening. Buds tolerated 1.5% NaCl were selected and salt-tolerant plantlets were regenerated. These buds or plantlets showed higher salt-tolerance compared with the controls.