Study On Regeneration System Optimization And Ethylmethylsulfone Mutation Of Cynodon Dactylon(L.)Pers Cv. Xinnong No.1

Cynodon dactylon was one of the warm season turfgrass that was widely applicated in China. At present,bermudagrass varieties applicated in the market mainly depended on foreign importion. Therefore, the exploitation of wild turfgrass resources and breeding new variety through traditional breeding methods combining with biotechnology was advocated. For bermudagrass biotechnology breed-ing, the progress was slow and the bottleneck lied in the difficulty of egeneration system establishment,the embryogenic callus rate was low, and it was difficult to differentiate. Moreover, there were no relevant reports of EMS mutagenesis bermudagrass until now. Therefore, this study investigated the effects of the hormones and additives on callus induction, embryonic callus obtaining and emb-ryogenic maintenance through using orthogonal design method and optimized the regeneration system of Cynodon dactylon(L.) Per cv. Xinnong No.1. On this basis, the vitro technology combining with EMS mutagenesis was explored, The main results were as follows:(1) For callus induction of Cynodon dactylon(L.) Per cv. Xinnong No.1, the influence sequence of three factors was 2,4-D >6-BA>Proline. MS salt supplemented with 2.0mg/ L 2,4-D,0. 01mg/ L 6-BA and 200mg/ L Proline was the optimized induction medium and callus induction rate was 69.5%; On the subculture of callus, the influence sequence of three factors on induction rate of embryonic callus, callus proliferation rate and browning rate were different. The influence sequence of the factors on induction rate of embryonic callus was 2,4-D>ABA>6-BA. The influence of the sequence factors on callus proliferation rate was 6-BA>ABA>2,4-D. The influence sequence of the factors on browning rate was 2,4-D >6-BA>ABA. The comprehensive results from three indexes showed that MS salt supplemented with 2.0mg/L2,4-D,0.2mg/L 6-BA and 2.0mg/L Proline was the optimized subculture medium. The embryogenic callus rate and callus proliferation rate was respectively 71.67% and 61.67% and the browning rate was low. the results of differentiation of bud showed that MS salt supplemented with 1.0mg/ L 6-BA was the optimized differentiation medium and Plantlet formation strength could reach 49.44%.(2) For embryogenic maintenance of Cynodon dactylon(L.) Per cv. Xinnong No.1, the influencesequence of 2,4-D,6-BA and CuSO4 on embryogenic callus proliferation rate, browning rate and plan-tle formation strength was different. The influence sequence of the factors on embryogenic callus proliferation rate was CuSO4>2,4-D>6-BA. The influence sequence of the factors on browning rate was 2,4-D>CuSO4>6-BA.The influence sequence of the factors on plantle formation strength was 2,4-D>CuSO4>6-BA. The medium-type experiment showed that the alternating medium was superior to the single medium on small plant formation strength. The comprehensive results from orthogonal test and Single-factor test showed that MS-N6 alternating salt supplemented with 1.0mg/ L 2,4-D, 2.0mg/L6-BA and 3.0mg/L CuSO4 was the optimized embryogenic maintenance medium. Embryogen-ic callus proliferation rate was 52.22% and plantlets formation was 53.33%. The browning rate at this point was lower.(3) EMS mutagenesis study of different concentrations and different treatment time on callus of Cynodon dactylon(L.) Per cv. Xinnong No.1 showed that the browning rate increased with the increasing of EMS concentration. Low concentration(0.2%, 0.4%) of EMS treatment had no significant influence on the callus death rate, while high concentrations of EMS treatment had significant effect. The callus death rate of 0.8%+2h, 1.0%+2h, 0.8%+4h, 0.6%+6h EMS treatment were close to the semi lethal dose. The same time relative differentiation rate decreased with the increase of EMS concentration. The relative differentiation rate of the 0.6%+2h、0.8%+2h、0.2%+4h and 0.4%+4h EMS treatment was close to 50%. The comprehensive results through callus death rate and relative differentiation rate in embryonic callus EMS treatment showed that the best dose was 0.8%+2h, and the death rate of embryogenic callus was 40.56% and the relative regeneration rate was 54.84%.