Agrobacterium-mediated Genetic Transformation Of CmWRKY15-1 Gene To Chrysanthemum

Chrysanthemum morifolium is a perennial perennial flower belonging to the genus Chrysanthemum of Compositae.Chrysanthemum white rust,as a very serious worldwide disease,has been listed as a quarantine disease in many countries and regions.In this study,the effects of different concentrations of growth regulators on stem proliferation,callus induction and adventitious bud differentiation of resistant cultivar’C029’and’Ziyan’were studied,and a high-quality regeneration system was established.On this basis,an efficient genetic transformation system of’C029’and’Ziyan’was established.The overexpression lines of Cm WRKY15-1 gene from’Ziyan’were obtained,which provided theoretical basis for the control of chrysanthemum white rust and breeding of resistant varieties in the future.The main results are as follows1.The regeneration system of’C029’was established.The results showed that the best medium for stem proliferation of’C029’aseptic seedling was MS+1.0 mg·L^-16-BA+0.2mg·L^-1NAA;the best medium for callus formation of leaf disc was MS+1.0 mg·L^-16-BA+0.2 mg·L^-1NAA;the optimal medium for adventitious bud differentiation of leaf disc was MS+1.5 mg·L^-16-BA+0.5 mg·L^-1NAA.2.The regeneration system of’Ziyan’was established.The results showed that the best medium for stem proliferation of’Ziyan’aseptic seedling was MS+2.0 mg·L^-16-BA+0.3mg·L^-1NAA;the best medium for callus formation of leaf disc was MS+2.0 mg·L^-16-BA+0.2 mg·L^-1NAA;the optimal medium for adventitious bud differentiation of leaf disc was MS+2.0 mg·L^-16-BA+0.2 mg·L^-1NAA.3.The genetic transformation system of’C029’was established.The results showed that the critical concentration of kanamycin was 7.5 mg·L^-1;the optimal infection concentration OD₆₀₀was 0.6;the optimal infection time was 9 min;the best pre culture time was 2 d;the best co culture time was 2 d;the best delayed culture time was 2 d.4.The genetic transformation system of’Ziyan’was established.The results showed that the critical concentration of kanamycin was 20 mg·L^-1;the optimal infection concentration OD₆₀₀was 0.5;the optimal infection time was 7 min;the best pre culture time was 2 d;the best co culture time was 2 d;the best delayed culture time was 2 d.5.A total of 7 resistant lines were obtained in the overexpression system of’C029’.After verifying the integration and expression by PCR and qRT-PCR,we determined that there were 3 positive transgenic plants with a transformation effciency of 42.86%.6.A total of 9 resistant lines were obtained in the overexpression system of’Ziyan’.After verifying the integration and expression by PCR and qRT-PCR,we determined that there were 5 positive transgenic plants with a transformation effciency of 55.56%.