Establishment Of In Vitro Rapid Micropropagation And Regeneration System Of Dahlia Pinnata

Dahlia pinnata, as an perennial flowering bulbs, belongs to the Compositae family Dahlia genus. It is a world-wide famous landscape greening material and is applied in flower borders, flowerbeds and pot flowers. The establishment of tissue culture rapid micropropagation and highly efficient regeneration system can lay foundations for the scale propagation of superior strains, germplasm conservation and the establishment of genetic transformation system, which is also significative for the germplasm innovation of Dahlia pinnata through the means of genetic engineering. In this study, different factors that effect the micropropagation and highly effective regeneration were studied, of which the establishment of leaf in vitro regeneration system was especially studied.The results were as follows:1, Normally rapid micropropagation①The explants comparison test was carried out using the stem tips and stems with axillary bud of Dahlia pinnata as material. The results showed that the stems with axillary bud were the better explants. The rate of survival was 75%. The method for disinfectant was 70% alcohol (washed for 30 s) and 0.1% HgCl2 or 5% NaClO(washed for 4 min)and then inoculate to the original medium which contained 2000mg/L AC. and finally established the sterile system.②The fitting strengthening medium was MS+6-BA 0.5 mg/L+NAA0.3 mg/+ 50 g/Lsugar+6.5 g/L agar +B950 mg/L.③The optimal multiplication medium was MS+6-BA 4 mg/L +NAA 0.1 mg/L. The multiplication coefficient could be reached to 6.8. The inferior medium was MS+KT 6.0 mg/L+ NAA 0.01 mg/L. The multiplication coefficient was 5.3. In which the sugar and agar was respectly 30 g/L and 6.5 g/L. The culture condition was 14/10 light period, 1 000～2 000 light intensity and 25℃±2℃.④The fitting rooting medium was 1/2MS+NAA0.1～0.3 mg/L+30.0 g/L sugar+6.5 g/L agar, the rooting rate was 92.0%, and the mean number of roots was 10.9.2, Highly effective regeneration The experiment was carried out using leaves, petioles, merithals of the test-tube plantlet as material which was orthogonal designed studing the combine and mixture ratio of the exogenous cytokinin(6-BA, KT, TDZ)and auxin(IBA, NAA). It also studied the physiological condition of leaf , the days for darking culture and so on. The results were as follows:①Contrast to other explants such as petioles and merithals,the leaves was the best explants for in vitro regeneration. The rate of adventitious shoots for leaf was significantly higher than petiole and merithal. The best physiological condition of leaf explant was 25 days of test-tube plantlets, enough expanding and 2 cm long.②This study achieved two ways of highly efficient regeneration, one way was through the path of callus tissue,the optimal medium was MS+6-BA 3 mg/L +IBA 0.2 mg/L,the Callus induction rate was 76.4%, regeneration rate was 88.0%, the number of adventitous sprouts per leaf was 7.5; the other was direct regeneration omitting callus induction, the optimal medium was MS+KT 7.0 mg/L+ NAA 0.05 mg/L, regeneration rate was 86.0%, the number of adventitous sprouts per leaf was 5.8.③Fifteen days dark pretreatment and then transferred to normal ray culture can significantly improve the callus induction rate to 89.0%. The regeneration rate of the highly effective regeneration system established by the in vitro leaf of test-tube plantlet of Dahlia pinnata was to keep at about 86.0% and the number of adventitous sprouts per leaf rised to 8.1.④The best ways on the medium was by the abaxial surface(leaf back)touching medium and the best cutting ways of leaves was Cutting the midrib vertically,the number of adventitous sprouts per leaf rised to 8.8.