Studies On Tissue Culture Andmolecular Genetic Diversity Of Wild Cymbidium In Qilnnling

Cymbidium plant has abundant resources in china, and some species were liked by Chinese, because of elegant fragrance, very high ornamental value and economic value. Wild orchid resources in Qinling were a wild Cymbidium resources distribution group in North-south division of china. With the development of orchid market, develop and cultivate of the wild orchids resources were particularly important for rich and save Cymbidium resources. While tissue culture, molecular markers and other biological technology also were widely applied to conservation, identification and breeding research the wild Cymbidium. In the paper, the tissue culture technology was used to study the various factors to perfect tissue culture system of wild Cymbidium. These factors could influence the success of tissue culture of wild Cymbidium. RAPD molecular markers technology was used to research and analysis genetic diversity of 95 materials from different natural forests in Qinling. The experimental results were as follows:1. The study on wild Cymbidium explants tissue cultureIn this experiment, wild Cymbidium goeringii and wild Cymbidium faberi were used as experimental material, peduncle,calyx,leaves and other plant material were used as the explants.Through these studies of the basic medium, growth regulator: 2.4 - D, 6 - BA, TDZ combination of factors and different explants. The experiment results showed the basic medium MS and 1/2MS could promoted the growth of wild Orchid explant, but the basic medium MS was more advantageous to growth of the wild Cymbidium faberi explant and 1/2MS basic medium was more advantageous to promotion growth of the of wild Cymbidium goeringii explant. NAA and 6-BA had good effect than 2.4 - D and TDZ on orchids explant training early. NAA and 6-BA were maintained a combination of concentration within a relatively stable ratio, and low growth regulator dosage that they could promoted the growth of wild Orchid explant. Leaf as explant was easy browning and peduncle more suitable than calyx as explant.2. Studies on browning of tissue culture of Wild Cymbidium in QinLingPolyvinyl Pyrrolidone (PVP), citric acid, activated carbon, and different time of dark cultivation were used to study the effect of the control on orchids browning. The results showed that treatment four was the best to control orchid browning and that was 1/2MS+AC 1 g/L+PVP 5 g/L+10 day's cultivation in the dark.3. Optimization and establishment of RAPD Reaction System on Wild CymbidiumIn this research, orthogonal design and Single factor design were used to screening and to establish optimal reaction system of RAPD that were used in Wild Cymbidium in QinLing. Through optimization experiments of reaction conditions and procedures, the reaction system RAPD of Wild Cymbidium in QinLing was established. The optimum concentration of five important components such as Mg²⁺, Taq DNA polymerase, dNTPs, primer, and template DNA in 25uL reaction system were 1.5².5umol/L, 2U, 0.16mmol/L, 0.6umol/L and 100ng.The amplified reaetion program was: 94℃,initial denaturation 3min; 94℃,1 min; 37℃,40s; 72℃90s; 40 times repeat; 72℃,7min.4. RAPD analysis of wild Cymbidium plant genetic diversity in QinLingIn order to perform the RAPD analysis, Genetic variation of 95 sample material of Cymbidium Wild Cymbidium goeringii and wild Cymbidium faberi in QinLing were investigated using the 16 screened arbitrary Primers. Through the Amplication, 145 discernible RAPD bands were scored, and polymorphic loci percentage to 100%. By clustering analysis showed that genetic diversity was rich between of Samples, genetic relationship with geographic distance, ecological environment and variety has great correlation, but was not entirely subjected to regional position.