| The high efficiency tissue culture system of Poa pratensis L. was established, through the study on the optimal condition of tissue culture and the impact factors of embryogenic regeneration . The BADH gene in Atriplex hortensis was transformed into Poa pratensis L. based on the system of optimized bombardment transfer technological parameters. The gene was proved to be integrated into the genome of Poa pratensis L. steadly, and the drought and salt resistant experiments showed that the drought and salt tolerance was improved evidently after drought and salt stress.1. A high efficiency tissue culture system of Poa pratensis L. regeneration, mainly for the seeds of the genotype "Nuglade" , was established successfully through the study on the influence factors of callus inducement, seedling differentiation, and rhizogenesis of 5 genotypes of Poa pratensis L. via the experiments of explants screening and different hormone concentrations. The relevant culture systems are:(1) Inducement culture medium ingredient : "MB5 + 2mg/L 2, 4-D + 0. 2mg/L 6-BA (or 0. 5mg/L KT) + 2mg/L ABA + 12mg/L gelrite + 60mg/L sucrose" .(2) Subculture media: "MB5 + 0. 5-lmg/L 2,4-D + 0. 2mg/L 6-BA (or 0. 5mg/L KT) + 2mg/L ABA + 12 mg/L gelrite + 60mg/L sucrose" .(3) The seedling differentiation culture media component: "MB5 + 3mg/L 6-BA (or 2mg/L KT) + 8mg/L gelrite + 30mg/L sucrose" .(4) The culture media for plant rhizogenesis: "1/2MS + 0. 8mg/L IAA" .(5) The callus should be moved to subculture medium in the light after being cultured for 3-4 weeks in 25±1°C in the dark. The temperature should be kept at 25 + TC during daytime, and 18 + TC during night. The light intensity should be kept at 1500-2000LUX for 16 hours per day.2. The experiments on screening the target callus of Poa pratensis L , high saturation solution treatment, subculture, reactivation afterbombardment, reveals that Nuglade is the best target genotype for high regeneration rate and high frequency transformation. The transformation rate could be improved if the differentiation culture media is pretreated with 0. 5M mannitol for 10-24 hours before bombardment. The transformation rate could also be enhanced if the subculture duration is controlled in three months to decrease the screening duration of resistant callus. The rate of transgenic plants could reach to above 79% when the bombarded callus is reactivated in basal media for 3 to 7 days and moved to the culture medium with 50-75mg/L G-418.3. The technique of bombard transformation was optimized through the experimental study on the pressure, times and distance of bombard, different purity and concentration of gold powder and DNA. It would be greatly helpful to turn directions of culture utensils and to upturn the receipt materials after each bombard. Good transformation result was gained when 11OOpsi pressure membrane was used, and bombard distance is 6cm. Bombard twice, and bombard with 250ug gold powders with 0. 5ug pBIN438 plastid DNA can make a best result. 34 individuals of BADH transgenic individuals were obtained with the bombard transformation method, among which 27 individuals were confirmed with PCR. The hybridization bands were present in most individuals in further hybridization validation, Which proved that BADH gene was transformed into the genome of Poa pratensis L.4. The experiments for drought and salt tolerance of the transgenic plants was conducted and the results were concluded as following:(1) The PCR analyses showed that 27 PCR positive individuals were found in all 33 BADH transgenic individuals, Further hybridization validation on the PCR positive individuals revealed the present of the hybridization bands in most individuals, which showed the BADH gene successful transformation of the genome of Poa pratensis L.(2) It was showed when treated in the culture medium with 25% PEG-6000 for 10 days, the transgenic plants could grow normally, whereas the check individuals turned yellow and withered, and the growth was retarded, When treated in the culture medium with 25% PEG-6000 for 20 days... |
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