Improvement Of Regeneration Rate In Chrysanthemum Transformation Process And Transformation Of SVP Gene In ’Jinba’

In this study,by using the leaves of the ’Jinba’ aseptic seedlings as experimental material and agrobacterium-mediated leaf disc transformation,we repeated some of the regeneration and transformation systems which were reperted previously and found that there were still sme problems during the process of explant regeneration.Therefore experiments on explant types,seedling ages,explant inoculating methods and different concentrations of plant growth hormones were conducted to explore the effects of these four factors on leaf-induced callus and adventitious buds.Through previous experiments,four main factors were optimized and a high-frequency genetic transformation system of ’Jinba’ was established,which will lay the foundation for chrysanthemum variety improvement and provide a reference for future research on other genes transformation.The main findings are as follows:1.The best disinfection method for cut-flower chrysanthemum ’Jinba’ was determined.The optimal sterilization method for stem segments of cut-flower chrysanthemum ’Jinba’is soaking in 75% alcohol for 30 s,washing them once with sterile water,and then sterilizing with 20% sodium hypochlorite for 3min30 s,finally washing them twice with sterile water.2.The genetic transformation system of cut-flower chrysanthemum ’Jinba’ was established and optimized.Explant types,age,and plant growth regulators with different concentrations all have a certain effect on the genetic transformation system of cut-flower chrysanthemum ’Jinba’.With regard to the selection of explant age,when the sterile seedlings grew up,the regeneration ability of leaves gradually weakened with the increase of seedling age.25-day-old sterile seedlings were the best,with a 98% recovery rate and a 24.5% germination rate.In the choice of explant types,the callus induction ability of petioles and leaves were both above 95%.However,the callus differentiation ability of petioles was significantly higher than that of leaves.The petiole bud rate was twice that of the leaves,the former was 46.9% and the latter was 22.9%.In terms of plant growth regulators,the optimal callus and differentiation medium for cut-flower chrysanthemum ’Jinba’ was MS + 2mg / L6-BA + 0.5mg / LNAA,with a 98%recovery rate and a 30% germination rate.3.Some transgenic plants were obtained.A total of 34 transgenic resistant plants were obtained.After testing against the resistant plants by using the PCR method,10 SVP transgenic chrysanthemum ’Jinba’ plants were successfully obtained.