Establishment Of Receptor And Genetic Transformation System Of Chrysanthemum

Chrysanthemum morifolium is a major ornamental flowers in the northern autumn. It is very popular, with bright colors, dense flowers, long flowering period, low plants and extensive management features, widely used in urban greening. In this experiment, we researched on how to obtain aseptic seedings of Chrysanthemum morifolium ’China Red’、 ’Wanzhuang Shenfen’ ’C029’、’Sa’ and ’Sb’. To establish the regeneration system, we used the leaves of the aseptic seedings of 5 varieties of Chrysanthemum morifolium as explants and studied the effects of different concentrations of growth regulators on callus and adventitious buds. It laid the theoretical foundation for the breeding and improved varieties of Chrysanthemum morifolium. We established a ’China Red’ genetic transformation system and analyzed the influence of various factors on the Chrysanthemum morifolium genetic transformation, including Agrobacterium infection concentration and infection time, different treatments of the receptor and use of antibiotics. It could provide a reference for future research on Chrysanthemum morifolium gene transformation.The main results are as follows:(1)Regeneration system was established. The optimum sterilization time was 5min which sterilized explants with 1% sodium hypochlorite. The optimal proliferation mediums for stems were MS+0.3 mg·L-1 6-BA+0.3 mg·L-1 NAA; MS+0.3 mg·L-1 6-BA+0.5mg·L-1 NAA; MS+0.3 mg·L-1 6-BA+0.1 mg·L-1 NAA; MS+0.3 mg·L-1 6-BA+0.5 mg·L-1 NAA; MS+0.3 mg·L-1 6-BA+0.3 mg-L"1 NAA of’China Red’、’Wanzhuang Shenfen’、’C029’ ’Sa’ and ’Sb’. The highest rate of adventitious buds (89.6%) of ’China Red’ was obtained on MS+2.0 mg·L-1 6-BA+0.2 mg·L-1 NAA medium. Differentiation rates of ’Wanzhuang Shenfen’ ’C029’、’Sa’ and ’Sb’ were 60.4%、73.3%、29.2%、25.3%. The medium of 1/2MS+0.3 mg·L-1 NAA was the optimal for rooting of ’China Red’, which had rooting rate of 100%. ’China Red’ was easy to survive in hardening and transplanting, the survival rate up to 90.2%.(2)Genetic transformation system for ’China Red’ was established. The optimal critical concentration of kanamycin for leaf differentiation of ’China Red’ was 15 mg·L-1. The optimal critical concentration of kanamycin for rooting of ’China Red’ was 5 mg·L-1. The optimal concentration of Cefotaxime was 300 mg·L-1. Aseptic leaves of ’China Red’ was infected by Agrobacterium GV3101 containing the expression vectors pCAMBIA1301-CBL. The optimal conditions of genetic transformation:OD600=0.6、agrobacterium infection time for 7 min、 pre-culture 24 h、co-culture time for 48 h、delayed screen time for 2 d.