| The influences of varied factors on callus induction and growth from different explants of Japanese lawngrass (Zoysia japonica Steud.) were discussed, and plant regeneration system with high frequency was established. Genetic transformation by biolistic bombardment and Agrobacterium infection was then studied and optimized. In order to breeding improved zoysiagrass varieties with better tolerance to drought, cold, salt and alkali through biotechnology, nearly four years' efforts were made on DREB1A transformation, conclusions showed as followings.I., In vitro plant regeneration system of Japanese lawngrass was established. Singles-factor and multiple factor orthogonal callus induction experiments were conducted on zoysiagrass seeds. The results showed that callus induction depended heavily on genotype, and 'Zenith' was easier for manipulation in vitro. MS medium containing 2,4-D 5mg/L, 6-BA or KT 0.1 mg/L, CuSO4 2.5 mg/L, gelling with phytagel was optimal for callus induction and growth. MS medium with 2-3 mg/L 2,4-D and 0.05 mg/L 6-BA was suitable for callus induction from hypocotyls of germination seedlings. Although the highest embryogenic callus frequency was obtained by culture shoot meristems on MS medium containing 2-4 mg/L 2,4-D, the manipulation was labor-consuming. Embryogenic callus should be collected deliberately and subcultured to fresh medium between 24-33 days' interval. The plant regeneration frequency depended on the callus quality, and half-strength MS medium containing KT 0.2 mg/L gave best results for somatic embryogenesis.II. Hygromycin selection system for zoysiagrass transformants was standardized. Delayed selection was employed in biolistic gene deliver system. Two monthly callus subcultures on medium with 50 mg/L hygromycin were followed by one monthly selection on plants regeneration medium containing 20 mg/L hygromycin. Once emerged, shoot clusters were transferred to hygromycin-free rooting medium to develop into plants. With respect to Agrobacterium-mediated transformation, the hygromycin concentration was as same as for the biolistic method, except for that 200mg/L cefotaxime was applied to inhibit the propagation of Agrobacterium.III. Biolistic gene deliver system for Japanese lawngrass was established. Plates containing the target tissue were placed 6 cm below the stopping mesh. Particle acceleration was done using Biorad PDS-1000/He device at 1100 psi under partial vacuum and each Petri plate was shot twice, using 500μ g gold particle(l μ M) and 1 μ g DNA per bombardment. This optimized condition required using four monthly embryogenic calli, which was cultured on subculture medium for about 25 days. Embryogenic calli 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 calli were transferred to osmoticum-free medium.IV. Agrobacterium-mediated zoysiagrass foreign gene transformation system was established. The embryogenic calli were precultured on fresh medium for 7 days, then were infected with Agrobacterium tumefaciens LBA4404 for 20 min with 5 min vacuum treatment. After 2 days of co-cultivation, the calli were washed thoroughly and transferred to medium containing hygromycin and cefotamixe for selection. 100 μ M acetosyringone was included in both infection and co-cultivation medium.V. Transgenic Japanese lawngrass with foreign DREBIA gene were obtained. For 'Zenith', 'Liaoning' and 'Qingdao' three cultivars, 16 individual hygromycin-resistant plants were recovered following biolistic bombardment. The putative transgenic plants frequency was 5.6%, 7.52% and 2.08% for 'Zenith', 'Liaoning' and 'Qingdao' respectively. Embryogenic calli of 'Zenith' were also proposed to Agrobacterium infection, and 2 individual hygromycin-resistant plants were obtained, giving a 0.80% transformation efficiency. The integration of DREBIA gene was confirmed in 11 individual plants through PCR-Southern or Southern blotting. |
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