Agrobacterium Tumefaciens-Mediated Optimization Of Genetic Transformation System

Chrysanthemum morifolium is a perennial herbaceous flower of Compositae,because of its wide variety,rich in color and different forms of the world's favorite.In this study we obtained the sterile seedling first,using the top buds of the chrysanthemum cultivar 'C008';Secondly,The high frequency regeneration system was established by using the leaves of sterile seedlings of chrysanthemum cultivar 'C008' and 'A' as experimental materials;Then the factors affecting the genetic transformation were screened and analyzed,the genetic transformation system of two chrysanthemum cultivars were established,and the CBL gene was successfully transferred into the chrysanthemum cultivar 'C008' by Agrobacterium tumefaciens.This study laid a foundation for the research of new transgenic varieties and resistance.The main results are as follows:1.The acquisition of chrysanthemum cultivar 'C008' aseptic seedling:With 0.1%sodium chloride disinfection 5 min,the highest germination rate up to 92%.Chrysanthemum cultivar 'C008' stem cell proliferation medium is MS+0.3 mg·L-16-BA+0.3 mg·L-1NAA.2.Establishment of regeneration system of chrysanthemum cultivar 'C008' and 'A':The optimal medium for adventitious bud differentiation of chrysanthemum cultivar 'C008' and'A' is MS+3.0 mg·L-1 6-BA+0.1 mg·L-1 NAA and MS+1.0 mg·L-1 6-BA+0.3 mg·L-1NAA;The optimum medium for roots is 1/2MS+0.2 mg·L-1NAA and 1/2MS+0.3 mg·L-1NAA;The survival rate of transplanting seedlings is 84%and 80%;The optimal concentration of cepHalosporin is 50 mg·L-1 to inhibit the production of endopHytic fungus 'A'.3.Establishment of genetic transformation system of chrysanthemum cultivar 'C008'and 'A':The concentration of hygromycin of chrysanthemum cultivar 'C008' is 3.0 mg·L-1,the concentration of adventitious buds is 3.0 mg·L-1,and the inhibition concentration of Cef is 300 mg·L-1;The concentration of hygromycin of chrysanthemum cultivar 'A' is 1.0 mg·L-1,the concentration of adventitious buds is 3.0 mg·L-1,and the inhibition concentration of Cef is 300 mg·L-1;The genetic transformation system of chrysanthemum cultivar 'C008' is that Pre-culture for 2 d,the explants were infected for 8 min with diluted OD600=0.5-0.6 Agrobacterium culture liquild with 50 times,and then co-cultured for 2 d,delayed time for 2 d,and 41 strains of resistant plants were obtained,The PCR test showed that the positive plants were 5,which proved that the CBL gene had been transformed into the genome DNA of the Chrysanthemum cultivar 'C008',and the transformation rate was 12.2%.The genetic transformation system of chrysanthemum cultivar 'A' is that Pre-culture for 1 d,the explants were infected for 10 min with diluted OD600=0.5-0.6 Agrobacterium culture liquild with 50 times,and then co-cultured for 1 d,delayed time for 2 d,although resistant buds were produced,but did't get resistant plants.