Establishment Of High Efficient Regeneration And Study On Genetic Transformation Of GAI Gene In Chrysanthemum

The leaves variety Ribenhuang of chrysanthemum (Dendranthema morifolium (Ramat.) Tzvel. ) was used as explants in this study. A rapid and efficient regeneration system of chrysanthemum was established.The result showed that the calli with light green colour were formed on the medium (MS + BA1.0 mg/L + NAA 0.5 mg/L+Sucrose30g/L+Agar5.5 g/L) after 2 week leaf discs were cultured and the callus induction rate was 97.5%.The shoots formed from the calli on the medium (MS + BA3.0 mg/L + NAA 1.0 mg/L+Sucrose30g/L+Agar5.5 g/L) after about 40 days and the shoot regeneration rate was 100% .The clustered shoots were cultured continued on the medium (MS+6BA4.0+NAA0.01+Sucrose30g/L+Agar5.5 g/L) for the shoots multipulication. Shoots were rooted on the medium (Half of MS + IBA 0.5 mg/L+Sucrose30g/L+Agar5.5 g/L) after culturing about fifteen days and the rooting percentage was 100%.The sensitivity concentration of Kan was tested for leaf discs of chrysanthemum. Using the GAI gene which control the stem of plant elongation as the target, Genetic transformation by Agrobacterium tumefaciens. and the affecter of preculture time, the infecting time and the co-cultivation time and AS was supplimented in the medium was studied. The results showed that the sensitivity cecentration of Kan was 20 mg/L for leaf discs of chrysanthemum. The genetic transformation rate was the highest when the explants were not precultured and infecting with Agrobacterium for 20 mim, co-cultivated with Agrobacterium for 2d, 100mg/LAS was supplimented in the medium and the infected leaf discs were cultured on the the medium whinout Kan for 3 days and than transfered to the medium whin Kan . The leaf discs were cultured on media (MS + BA1.0 mg/L + NAA0.5 mg/L+Cef 100mg/L+Km20mg/L about 20 days for callus induction.The callus were transferred onto media (MS + BA1.0 mg/L + NAA 0.5 mg/L+ Kan20mg/L+Cef 100mg/L)for resistant selction of regenerate shoots. The shoots from selection medium were cultured on the medium (MS + BA3.0 mg/L + NAA 1.0 mg/L+ Kan20mg/L)for selection again and then on the medium (1/2MS+NAA0.5mg/L+Kan5mg/L)for rooting selectin.PCR analysis of the transgenic plants demonstrated that five of seven were positive.