Study On Efficient Regeneration System And Agrobacterium-mdiated Transformation Of Tps1 Gene By Agrobacterium Into Chrysanthemum Lavandulifolium

Chrysanthemum grandiflorum is one of the most famous flower in China and cut flower in the world.Chrysanthemum lavandulifolium is used for this study.In order to enhancing the stress tolerance(drought,cold and salt) of Chrysanthemum lavandulifolium.TPS1 gene was introduced into chrysanthemum plants through Agrobacterium-mediated transformation.High efficient regeneration system of Chrysanthemum lavandulifolium with explants of leaf discs was established in this study.The optium media was obtained for shoots regeneration and rooting.Shoots regenernation rate was 98%of the total explants in the medium MS+6-BA(0.1 mg/L) +NAA(0.1 mg/L).Roots were 100%induced in the medium 1/2MS + NAA(0.1 mg/L). After the regeneration system was established,the selection pressure of kanamycin(Km) and phosphinothricin(PPT) for genetic transformation was investigated.When Km concentration was higher than 10 mg/L or PPT concentration was higher than 1 mg/L,the regeneration of leaf discs were completely inhibited.So the selection pressure of Km and PPT for genetic transformation was determined as 8mg/L and 0.8 mg/L.Agrobacterium tumefaciens-mediated transformation was used for genetic transformation of chrysanthemum leaf discs.Several experimental factors were evaluated in this study,we adopted preculture for 2 day,OD₆₀₀=0.6,infection for 10 min,co-culturation for 2 days.Then leaf discs were transferred onto the selection media MS+NAA(0.1 mg/L) +6-BA(0.1 mg/L) +Km(8 mg/L) +Cef(250 mg/L) for regeneration 15 days later,leaf discs were transferred onto the media MS + NAA(0.1 mg/L) + 6-BA(0.1 mg/L) + Km(8 mg/L) for further screening, shoots were differentiatied from leaf discs around 40 days later.Resistent shoots from selection medium were cultured on medium 1/2MS + NAA(0.1 mg/L) for rooting.PCR amplification showed that TPS1 gene had been integrated into 4 chrysanthemum plants.Preliminary data showed that transgenic plants displayed multiple phenotypic alterations:plants growed retardation,leaves become smaller,plants become thinner.