Incorporation Of Stress Tolerance-Related NHX1,CBF Genes Into Tall Fescue(Festuca Arundinacea Schreb.) By Particle Bombardment

Application of biotechnology in turfgrass breeding is being increasingly used in improving its agronomic characteristic. Tall fescue (Festuca arundinacea Schreb.) is a turfgrass with high quality. The conditions of establishing the high regeneration system of tall fescue were studied, and the genes of NHX1 and CBF were transformed into the calli using microprojectile bombardment. This study made a strong foundation for engineering tall fescue tolerant to salt, cold and drought stress. The results were as follows: 1. Study and establishing the high regeneration system of tall fescue.The regeneration system of the lower plumular axes from tall fescue young seedling has been established in this study. In order to establish a tissue culture system for tall Fescue, different ways of seeds treatment were done to investigate their effects on the germination and pollution rates of seeds, and the results showed that the optimum concentration of sodium hypochlorite was 50%, and the optimal time for treatment was 20 min. The lower plumular of tall fescue axes from two species sliced with two methods were used to induce callus, and the results showed callus induction rates sliced from one end increased 18.9% than that from two ends before plating. The lower plumular axes of tall fescue seedling were inoculated on the MS mediums with different 2,4-D concentration to induce calli, and the results indicated that 9 mg·L-1 2,4-D was the optimum concentration on calli induction, and the regenation rate of calli reached 80.7%. 2. The AtNHX1 gene from Arabidopsis was transferred into calli of tall fescue by microprojectile bombardment. Twenty-nine transgenic plants were obtained by PCR and Western-blotting analysis. In addition, the transgenic plants were more tolerant to 2 mg·L-1 phosphinothricin compared to control plants. 3. Arabidopsis CBF4 gene, which belongs to CBF family of transcription factors, is up-regulated by drought stress. Under the drive of RD29A inducible promoter, the plasmid (pGreen0229-RD29A-CBF4- DHA) containing a phosphinothricin resistance selectable marker was introduced into calli of tall fescue by microprojectile bombardment. Twenty-four transgenic plants were obtained by PCR analysis. 4. In our experiment, CBF3 was cloned into pGreen0229 vector under the drive of RD29A inducible and CaMV35S constitutive promoter respectively, and the plasmids (pGreen0229-RD29A-CBF3- DHA and pGreen0229-35S-CBF3- DHA) containing a phosphinothricin resistance selectable marker,were introduced into calli of tall fescue by microprojectile bombardment. Twenty-two transgenic plants were obtained. Both PCR and Western-blotting analysis showed that CBF3 gene was integrated into the genomes of transgenic plants.