Breeding Of Drought-and Salt-tolerant Lines By Bioengineering Techiniques In Tall Fescue (Festuca Arundinacea Schreb.)

Tall fescue (Festuca arundinacea Schreb.) is an open-pollinated, perennial, cool-season grass species widely used for forage and turf. In northern China, it is widely maintained on lawns, parks, public grounds and so on, for the characters of beautiful colour, high shoot density, cool tolerence and better shear-bear. The obvious defects such as less salt or heat tolerance ristrict the spreadness of tall fescue. Traditional breeding is time-consuming and laborious. Development of both cellular techniques and genetic engineering manipulation made it possible to decrease breeding time and create salt- and drought-tolerant cultivars of tall fescue.In this research, multiple shoots were induced in vitro from sterilized seedling apices of tall fescue. MS medium supplemented with 7 mg/L 2,4-D and 3 mg/L 6-BA was the best inducing medium and that supplemented with 4 mg/L 6-BA was the best subculturing medium for tall fescue multiple shoots.1-mm-long shoot tips of tall fescue derived from the multiple shoot clumps subcultured for 5-7 days were infected with Agrobacterium LBA4404 harboring plasmid pCAMBlA1300-PIS-als for 5 min with 0.5×10⁵pa negative pressure co-treatment. After co-culture and further 45 days subcultures on medium containing lvchuanglong (chlorsulfuron) at the concentration of 0.6 mg/L, at a 15-day interval, the survival frequency of infected shoots was about .23.7%. Then, the survivals were transferred to medium without lvhuanglong 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 PIS gene. 20.9% of the lvchuanglong -resistant plants were PCR positive and gave positive signal at PCR-Southern blotting, indicating that the PIS gene had been transferred into some of the transformed plants. The multiple bud clumps subcultured were a series of salt filtration and offspring breeding selection. Then salt-tolerant plantlets were regenerated and T₂ lines were obtained. The T₂ plants transferred into plastic pots containing sand were irrigated with brine of different concentration. Along with the increase of the salinity, the tillering ability of tall fescue was reduced, and the proline content was increased. Line 53 and 98 had higher proline content and tillering ability than control plants. The drought tolerance of these plants were also analyzed. Under drought stress conditions, line 53 and 98 had a lower percentage of ion leakage, better osmotic adjustment, slower reducing speed of chlorophyll and less lipid membrane peroxidation than control plants. It is obvious that line 53 and 98 had better salt and drought tolerance, revealing good potential in breeding salt- and drought-tolerant varieties.In addition, the transgenic tall fescue plants containing the E. coli. betA gene were also exposed to salinity and drought stress. During salinity stress, the salinity was increased gradually to the terminal concentration and maintained the terminal concentration for 20 days. During drought stress, the plants were wilted at 10 am and then were irrigated with temperate water to get right in the other moring. Among the six transgenic lines investigated, line 12 and 21 had a lower percentage of ion leakage, increased proline content, higher chlorophyll content and less lipid membrane peroxidation than wide-type plants. During drought stress, the content of glycinebetaine in transgenic plants was notable higher than wide-type plants. It is indicated that betA introduced into tall fescue improved the accumulation of glycinebetaine and consequently enhanced the salt and drought tolerance of these materials.