Optimization Of Regeneration System And Genetic Transformation Of Festuca Arundinacea

Tall fescue (Festuca arundinacea Schreb.), as a kind of elitecool-season turfgrass with characteristics of high tolerance to drought,barren and traffic, long green period and wide adaptability, has beenextensively applied in urban greening and sports field construction. Butsome cultivars of tall rescue are susceptible to fungal diseases, suchas Rhizoconia solani and Puccinia ssp. In this research, the regenerationsystems of several tall fescue cultivars had been established and aforeign lysozyme gene had been introduced into three tall fescue cultivarsby Agrobacturium tumefaciens-mediated transformation to breed thetransgenie lines resistant to fungal diseases.In this research, mature seeds of five varieties (Huntdogâ…¤,Crossfireâ…¡,Deminion, Dynasty and Xinxiu) were used as trial materialsand the main results were as follows:1 Establishment of regeneration systems of Festuca arundinacea(1) The calli induction and differentiation rates of 6 explants,including mature seed, seed without endosperm, seed without capsule,hypocotyl, young root and young leaf were comparied, which suggestedthat the seed without endosperm was the optimal explant to calli inductionwith a frequency of 76.5%, 27.8% over than that of the intact seeds. Thedifferentiation frequency of hypocotyl was up to 86.25%, a little higherthan the 81.32% of the seed without endosperm, but it was difficult toexcise the hypocotyls.(2) The effects of four basic medium, MS, MSM, CC and NB, on calliinduction, subculture and differentiation were investigated using the seed without endosperm as explants, and the results indicated that theMSM was the optimal basic medium for calli induction, and the calliinduction frequencies in medium MSM+9 mg/L 2, 4-D had reached 80%～92.5%,however the MS was the optimal basic medium for bud differentiation, thehighest frequency up to 89.5%.(3) A higher 2,4-D concentration was required for callus inductionof tall fescue, and 9 mg/L 2,4-D gave the highest induction frequency formost of cultivars. When supplement with 0.5～1 mg/L ABA in medium, thegermination and calli induction frequencies were strongly reduced, thecalli induction frequency even lower than 15%. A combination of 5 mg/L2,4-D +0.1 mg/L KT in subculture medium took the best effects on embryoniccalli induction, and a combination of 2.5 mg/L KT+ 2 mg/L 6-BA gave thehighest frequency of bud differentiation by 89.5%.In addition, the calli induction and bud regeneration frequencies hadextremly differences among different tall fescue cultivars, whichindicated it nessesary to select sutible cultivar for in vitro culture.2 genetic transformation of Festuca arundinacea via AgrobacturiumtumefaciensA foreign lysozyme gene resistant to fungal diseases had beenintroduced into three tall rescue cultivars via Agrobacteriumtumefaciens-mediated transformation. The embryonic calli havingsubcultured 20 days were infected with EHAI05 strain suspension ofOD₆₀₀≈0.4 for 20～30 min, and then co-cultured 2～3 days in dark condition.The infected calli were cultured in the selective medium with 30～40 mg/LG418 to regenerate the resistant seedlings. Finally, 72 resistant clonesderived from three cultivars were acquired, of which 83.3% seedlings wereidentified as transgenic individuals by PCR analysis.