Establishment Of High Efficient Regeneration System And Study On Genetic Transformation Of AFP Gene In Dendrobium

Orchidaceae is one of the largest families of flowering plants with more than 800 genera and over 25000 species that are commercially grown globally. Orchids are famous cut flowers and potted plants with high commercially values. Dendrobium are the largest genera of Orchidaceae and they are growed in tropical regions. Antifreeze proteins(AFP) gene will be introduced into Dendrobium by using of genetic engineering technigues in this study. The purpose is that it cultivate new material of Antifreeze Dendrobium and it maybe transfer new material of Antifreeze Dendrobium into the cold area.A cultivar of Dendrobium Madame Thong-In was used in this study. The induction of callus, shoot and root was affected by several factors including explant types, plant growth regulators etc. A rapid and efficient regeneration system was established. The bud was found to be the best explant in terms of regeneration ability in the tested five types of explants. The best media for inducing callus or shoot and root were developed. In the study. Buds with leaves removed would form calli on the medium MS+2,4-D(1.0mg/L)+ NAA(2.0mg/L)+KT(1.0mg/L)+coco-water(5.0%)+sucrose(3%)+agar(5.5g/L) after about 1 week, and the callus induction rate was 90%; The shoots of Dendrobium regenerated from the calli on the medium MS+BA(1.5mg/L)+NAA(0.2mg/L)+GA(1.0mg/L)+ coco-water(5.0%)+sucrose(3%)+agar(5.5g/L) after about 25 days, and the shoot regeneration rate was 82.0%; Roots were obtained when the shoots were transferred onto the medium 1/2 MS+IBA (0.5mg/L)+AC (0.5g/L)+coco-water (5.0%)+sucrose (3%)+agar (5.5g/L) after about 30 days, and the rooting percentage was 90%.The regeneration efficiency was high enough for genetic transformation of Dendrobium.After the establishing of high efficient regeneration system and confirming the concentration of selective agent Km for callus of Dendrobium, genetic transformation was studied with Agrobacterium tumefaciens and particle bombardment. An optimal transformation system was obtained. The results showed that the suitable Kan concentration was 150mg/L; (J)The genetic transformation rate was affected by several factors, such as the time of preculture, the time of co-cultivation, whether AS was supplimented in the medium in Agrobacterium mediated transformation. We found that...