The Establishment Of The Oat Genetic Transformation System And Elymus Establishment Of Regeneration System

This report describes the establishment of Elymus breviaristatus, it also contains the establishment of Oat's gnetic manipulation, ppIP gene which is from Pseudomonas pseudoalcaligenes is transmitted into Oat through Bacterium EHA 105 to breed new material of forage grass. The study of regeneration system of Elymus breviaristatus is essential to transmit ppIP gene into it.The regeneration system of Elymus breviaristatus is established. The reports show that the resource of explants and regulator combination are the key factors of influencing the induction of callus and regeneration. Three types of explants are compared and the highest callus ratio of which is embryo, even to 47.82％. The optimum medium is MS medium and the optimum regulator combination is 5mg/L 2,4—D+0.05mg/L KT+2mg/LABA. Meanwhile, the medium plus 1mg/L CH could obviously improve the quality and regenerating ability of callus. The culture time also influences regeneration. The regenerating ability of callus from embryo is highest to 48％. The optimum regeneration medium is 1/2MS+B5salt+NAA 0.5mg/L+KT 0.01 mg/L.The regeneration system of Oat861 is optimized: three types of explants are compared and the results is as the first one in which the callus induction ratio of callus from embryo is highest to 64％. The optimum medium is MS medium and the optimum regulator combination is 4mg/L 2,4—D. The regenerating ability of callus from embryo is highest to 54.3％. The optimum regeneration medium is 1/2MS+B5+6—BA1.0mg/L+KT0.1 mg/L.The screening process of Oat961 was established. The optimized screener is homomycin, and 45mg/L is the optimized concentration. The callus induced from matur embryo is determined to be the material for trangenetic manipulation, ppIP gene was transmitted into callus subcultured after 40 days through Agrobacterium and the callus been transgened were screened by homomycin. At last the transgenetic plants were regenerated. The optimized screening culture medium was determined: MS+2,4-D5.0+KT0.05+Hn45+Carb500. gus and mgfp gene of pCAMBIA1304were cut off by NcoI and BSTEII to avoid producing fusion protein with ppIP gene, infecting the activeness of ppIP protein. The cloned ppIP gene which is without signal peptide is connected to 35S promoter of pCANBIA1304 derectedly and the monocot expression vector pCAMBIA1304—ppIP is constructed. Then the AgrobacteriumEH105 was infected by constructed vector and the callus were infected by AgrobacteriumEH105. The callus infected by AgrobacteriumEH105 were cultured on co-culture medium. After 6 days of co-culturing they were moved to screening culture medium to screen resistant callus. The subculturing time, infecting concentration of Agrobacterium, time of co-culturing and temperature all influence the transformation of Agrobacterium.