Elementary Studies On Regeneration Of Leaf Disks And Transformation Of Chrysanthemum 'Ribenhuang' And 'Jinba'

Chrysanthemum is a perennial herbage plant of the genus Chrysanthemum L. (Compositae), the history of chrysanthemum growing and breeding in China is over 1600 years. In this dissertation, we used chrysanthemum'Ribenhuang'and'Jinba'as the experimental materials, researched five aspects: secondary culture, construction of regeneration systems for chrysanthemum leaf disks, physiological and biochemical characteristics of vitrification, controlling of vitrification and construction of transgenic conditions.The main results of this studies are such as follows:1. The stem cut of'Ribenhuang'was the explant for successive transfer culture, the optimal medium was MS+ 6-BA 0.3 mg/L+ NAA 0.1 mg/L + sucrose 30 g/L+agar 7.5 g/L. The stem cut of'Jinba'was the explant to secondary culture, the optimal medium was MS+ 6-BA 1.5 mg/L+ NAA 0.5 mg/L + sucrose 30 g/L + agar 7.5 g/L.2.Cut the leaf of'Ribenhuang'into leaf disk (0.8cm×0.8cm), the leaf disk was the explant to establish the regeneration systems, the optimal medium was MS+ 6-BA 2 mg/L+ NAA 0.5 mg/L + sucrose 30 g/L + agar 7.5 g/L, regeneration frequency was 35%. Cut the leaf of'Jinba'into leaf disk (0.8cm×0.8cm), the leaf disk was the explant to establish the regeneration systems, the optimal medium was MS+ 6-BA 2mg/L+ NAA 0.1 mg/L + sucrose 30g/L + agar 7.5g/L, regeneration frequency was 61.7%.3. Compared the vitrification with the normal shoots on the physiological characteristics with'Ribenhuang'in tissue culture, the water content and soluble saccharides of the vitrification shoots were higher than the normal shoots, and the Chlorophyll a, Chlorophyll b, sucrose, soluble protein were lower than the normal shoots. The results indicated the vitrification's water metabolism was to seize up, protein synthesis was to be affected and the vitrification absorbed sucrose was different with the normal. 4. In this study, an orthogonal experiment was chosen to control the vitrification of'Ribenhuang', which contains four factors (agar concentration, surcose concentration, AC contents and seal material). The results indicated the agar concentration, surcose concentration and AC contents influenced the proliferation and reduced the vitrification rate significantly. Seal material with filtration pore reduced the vitrification rate significantly. The optimal medium for controlling the vitrification of'Ribenhuang'was MS+ 6-BA 0.3mg/L + NAA 0.1mg/L +agar 10g/L +sucrose 35g/L +AC 1g/L +seal material.5. Genetic transformation was studied on using leaf disks regeneration by co-cultivation with agrobacterium in time of preculture, co-culture time, selection pressure by Km and the minimal inhibitory concentrations. The results indicated that the optimal time of preculture was 24h, co-culture time was 2d, selection pressure was 30 mg/L, the minimal inhibitory concentrations was 400 mg/L.