The Study On Transformation Of DREB1A Gene Into Tall Fescue(Festuca Arundinacea Schreb.)

The influences of varied factors on calls induction and growth from explants of Tall Fescue (Festuca arundinacea Schreb.) were discussed, and plant regeneration system with high frequency was established. Embryonic callus of Tall Fescue were used as material for genetic transformation with the DREB1A gene by particle bombardment. The transgenic plants were obtained. At the same time, partial transgenic plants were treated with drought stress to check the actual results on drought resistance. Through the hard efforts, conclusions are showed as following.â… . In order to establish the tissue culture system of tall fescue (Festuca arundinacea Schreb.) for the transgenic study,callus induction and plantlet regeneration of four lines of tall fescue,Houndog 5,Crossfire II,Barlexus and Millennium, were studied in this experiment.The results indicated that: â‘ The best surface sterilization process for seeds is stirred in NaC10(active chlorin≥5%) one hour and then sterilized water for dehusking dissolving the skin of seeds in water without germs in seeds surface sterilization.The effect of different ways of seeds treatment (with a cut or not)on callus induction was not significant. â‘¡Millennium performed better than other lines of tall fescue in both callus induction and subculture. â‘¢The suitable medium for callus induction of Millennium is a basical MS salt mixed with 9mg/L 2,4-D+0mg/L 6-BA+300mg/L Hydrolysate. â‘£The suitable medium for subculture of Millennium is MS+9mg/L 2,4-D+0.05mg/L 6-BA+0.5mg/LKT+0.5mg/L CuSO₄. ⑤Callus with a higher regeneration are light yellow to green,compact fast-growing and green.â‘¥The suitable concentration of 6-BA and KT in medium (MS) for plantlet regeneration are 0.05mg/L and 0.2mg/L.â…¡. Hygromycin selection system for tall fescue transformants was standardized. Delayed selection was employed in biolistic gene deiver system. Two monthly callus subcultures on medium with 80mg/L hygromycin were followed by one monthly selection on plants regeneration medium containing 40mg/L hygromycin. Once emerged, shoot clusters were transferred to hygromycin-free rooting medium to develop into plants.â…¢. Biolistic gene deliver system for Tall Fescue was established. Plates containing the target tissue were placed 6 cm below the stopping mesh. Partice acceleration was done using Biorad PDS-1000/He device at 1100 psi under partial vacuum and each Petri plate was shot twice, using 500 μg gold particle (1μM) and lug DNA per bombardment. This optimized condition requires using embryonic callus of Tall Fescue. Embryonic callus were selected and arranged in the center of osmotic medium (callus induction medium containing 0.2M mannitol and 0.2M sorbitol) 4 h prior to transformation. After 16 h following bombardment, the callus were transferred to osmoticum-free medium. â…£. Transgenic Tall Fescue with foreign DREB1A gene were obtained. The transgenic plants were obtained. Through PCR and Southern blot analysis, 1 DREB1A-gene transgenic lines of cultivar 'Millennium' obtained. The total number of transgenic plants were 77.â…¤. Partial transgenic plants were treated with drought stress, and then the Relative leakage electrolytes, concentration of MDA, POD enzymes activity and concentration of chlorophylls are mensurated. The results showed that, transgenic plants have higher drought resistance than that of the control.