Studies On Regeneration System And Transformation Of Green Fluorescent Protein(GFP) Gene By Agrobacterium In Chrysanthemum

Materials used in this study are cultivars of chrysanthemum, such as 'Ribenhuang','Yangguang','Guohuajifu'. The result showed that several factors(cultivars,plant growth regulators) affected callus,shoot and root inducement of chrysanthemum. A rapid and efficient regeneration system of chrysanthemum was established. The regeneration ability of cultivar 'Ribenhuang' was better than the other two cultivars. The best media for inducing callus,shoot and root were developed. At the same time, we found that shoots of cultivar 'Ribenhuang' regenerated directly from the leaf disc on the media MS+NAA(1mg/L)+6-BA(3mg/L). The shoot regeneration rate was 100%,and the propagation coefficient was 8.54. Roots were obtained when shoots were transferred onto the medium 1/2MS+NAA(0.2mg/L)+IBA(0.2mg/L)after 10 days,and the rooting percentage was 100%. Those were enough for genetic transformation. Genetic transformation was studied by using leaf discs by co-cultivation with agrobacterium. An optimal transformation system was obtained. The results showed that the genetic transformation rate was affected by several factors, such as the time of preculturation,OD600 of agrobacterium,the time of infecting and co-cultivation. We found that the genetic transformation rate was highest when without preculturaion ,OD600 for 0.6,infecting for 10 minutes and co-culturation for 4 days.At the same time ,the cap of filter paper(CFP) was used for improving transformation ratio. Leaf discs were transferred onto the media MS+ NAA(1mg/L)+6-BA(3mg/L)+Km(30mg/L)for regeneration resistant selction. Shoots from selection medium were cultured on medium MS +NAA(1mg/L) +6-BA(3mg/L) +Km(50mg/L) for selection again,and then on the media 1/2MS+NAA(0.2mg/L)+IBA(0.2mg/L)+Km(25mg/L)for rooting selectin. Integration of GFP gene into chrysanthemum was confirmed by using PCR amplification. The transgenetic seedlings werer obtained.