Establishment Of Tissue Culture System Of Sinningia Speciosa And Cymbidium Grandiflorium And Pilot Study On CycD2 Gene Transformation To Cymbidium Grandiflorium

Sinningia Speciosa and Cymbidium grandiflorium are both popular ornamental flowers in recent years.The propagation of Sinningia Speciosa with seeds is limited since the pistils and stamens are degenerated. Cymbidium grandiflorium has a long growth period and a low reproduction rate by traditional breeding methods.The present protocol is trying to accelerate the propagation and elevate the reproduction quotiety of the two varieties by tissue culture ,which can solve the problem that supply falls short of demand and provide technical bases for their industrialization.Besides,Cymbidium grandiflorium has a long growth period and initiate to bloom in 3 years at least or even hard to bloom in some areas such as Canton,which highly degrade its commercial value. A brand-new method is required urgently to solve the problem. The present research utilize the Agrobacterium-mediated transformation and particle bombardment to transfer the cell cycle gene,CycD2, to 3 varieties of Cymbidium grandiflorium,LR515,LA516 and KY827,aiming to make it bloom earlier. The results were as flollows:1. Establishment of tissue culture system of Sinningia Speciosa : leaves are used as explants to induce callus in media of MS+BA1.0mg/L+NAA0.2mg/L . callus are proliferated in the same media and regenerated in media of MS+BA3.0mg/L+NAA0.2mg/L for plants and l/2MS+NAA0.2mg/L for roots.2. Elementary establishment of Cymbidium grandiflorium transformation system: shoots are used as explants to induce PLBs (protocorm-like bodies) in media of MS+6-BA1.0mg/L+NAA0.5mg/L and PLBs are regenerated in media of 1/2MS+ NAA0.2mg/L with La(NO3)3.XH2O and coconut farina as additive.The system has been improved through liquid shaking preparatory culture .3. LR515,LA516,KY827 are selected in the media respectively with 60, 60, 70mg/L Hygromycin as the highest selective concentration .The principle of "lower then higher " was adopted in use of antibiotics.4. Cymbidium grandiflorium has lower sensitivity to Agrobacterium, thus has lower transformed rate,but the residual bacterium is easy to be removed.5. Cymbidium grandiflorium cannot be exposed to intense osmosis environment in the particle bombardment.The short shot distance and 2 times bombardment can both debase the quotiety of resistant PLBs of LR515,but almost not affect KY827.6. The resistant seedling of Cymbidium grandiflorium to hygromycin are obtained.