Tissue Culture In Vitro And Drought-Resistant Gene Transformation Of Ephedra Sincica

Ephedra Sinica is a traditional Chinese herbal medicine, and Ephedra Sinica's demand are very greatness. But the drought and salina soil confine Ephedra Sinica's yield. It is great significance to cultivate drought-resistance and salina-resistance Ephedra Sinica. Shoot in vitro callus inducement and regeneration effect of Ephedra in pH value, explant types, hormones, basic mediums and the ways that leaves touched medium were investigated. The best culture mode have been found from callus culture and suspend culture to regeneration culture. Drought-resistance gene lea3 gene was transformed into embryogenic embryos callus of Ephedra by particle bombardment. After being selected by Bialaphos, Bialaphos-resistant callus of Ephedra were filtrated and the transgene plants were obtained.The result callus inducement of Ephedra enucleate that different expend types have distinct result on callus inducement. The cotyledon and hypocotyl were better at callus inducement than radicel, and each inducement efficiency of cotyledon, hypocotyl and radicel is 65.2%, 57.4% and 1.2%. The basic mediums and hormones are also tremendous direct effect on callus inducement of Ephedra and the optimization condition of callus inducement is MS+2,4-D1.5mg/L+6-BA1.0mg/L+sugar30mg/L. In the research of Ephedra Sinica callus suspended culture, the ascendant medium was MS+2,4-D2.0mg/L+6-BA1.0mg/L +CH250mg/L+YE250mg/L+30g/L sugar+5g/L glucose. In the law of Ephedra Sinica callus growth, the ascendant medium was gained which was N6+2,4-D2.0mg/L+6-BA1.0mg/L+ 30g/Lsugar. The fleetly growth period was the 24^th-28^th after the callus was planted, and the POD and PPO activity of callus in every growth period approved tha the 24^th-28^th was the fleetly growth period. The same factors investigation of basic medium, hormones balance, and pH value et al. in callus regeneration the optimization condition of them was MS+NAA0.2mg/L+KT0.5mg/L+sugar30mg/L which pH was 5.8-6.0 and the callus regeneration efficiency was 15%.At building on callus culture system and callus regeneration system the plasmid pBY520 with drought-resistance gene lea3 gene was transformed into embryogenic embryos callus of Ephedra by particle bombardment. Bialaphos-resistant callus of Ephedra were obtained after 3 times Bialaphos selection and the efficiency of Bialaphos-resistant callus filtration was 12.83%. 7 Bialaphos-resistant were obtained and the differentiation was 12.5%. The ways of Plant DNA kit, SDS and CTAB were inspected in Ephedra Sinica DNA pick-up and theeffective and conveniency way was the way of CTAB. 5 positive controls with Iea3 gene were detected by PCR from 7 Bialaphos-resistant regeneration plants and the ultimately transgene efficiency was 1.1%.