Analysis Of Genetic Diversity For Dendrobium And Studies On The Extraction Technology Of Polysaccharide

The genus Dendrobium is the second biggest branch of Orchidaceae family. Among about 1500 Dendrobium species in the family Orchidaceae, mainly distributed in the tropical and subtropical Asia and North-Australia. Dendrobium substances have been used in traditional medicine for many centuries in Asian countries because of their therapeutic effects. Modern pharmacological studies have found that Dendrobium plants have various pharmacological effects, such as anti-cancer, anti-oxidant, immune modulation, and vasodilation effects. Most wild Dendrobium plants survive only in certain environments and are susceptible to the deterioration and fragmentation of natural ecosystems, which cause its high ecological diversity. Because of their wide and effective usage in traditional medicine and flower horticulture, Dendrobium plants have been subject to overexploitation for trades in both flower and medicinal herb markets in China, which lead to they are all endangered now. It is necessary to reinforce the collection and protection for Dendrobium genus.Molecular marker technique can reflect the genetic differences between species at the DNA level, which is an effective method to study the genetic diversity. ISSR molecular marker technique is simple, rapid, good stability, no species specificity, widely used in the study of genetic diversity. This study's research object is 30 Dendrobium species from the Fujian agriculture and forestry university in the Institute of Science, Technology and Economics of Tea Industry, using ISSR molecular marker technique to study the genetic diversity and phylogenetic relationship. Undoubtedly the study of the genetic diversity of Dendrobium species would be important not only for germplasm conservation but also for parental choice for breeding purposes.Dendrobium candidum Wall. ex Lindl. is one kind of Chinese traditional medicine in Dendrobium and has been in severe danger of extinction. The chemical studies of Dendrobium candidum had demonstrated that polysaccharides were the major active constituent. The extraction technology was optimized with the responsive surfaces methodology, choosing extraction temperature, liquid material ratio, extraction time and extraction number as factors. This study wills provide an important basis for the new extraction technology and intensive research of Dendrobium polysaccharides.The main results were as follows:(1) With the one-single and multi-level gradient testing method, to optimize system in five factors of template DNA, dNTP,10×PCR Buffer, primer, and Taq DNA polymerase. In the total volum of 20 ?L system which contained 3 ?L template DNA (10 ng·?L-1),1.5 ?L dNTP (2.5 mmol·L-1),3.0 ?L 10×PCR Buffer,3.5 ?L primer (4umol·L-1),0.2 ?LTaq DNA polymerase (5U·?L-1),8.8 ?LddH2O.(2) Screening 7 primers from the 20 ISSR random primers which can amplify clear bands. Genetic diversity of 30 Dendrobium germplasm by 7 polymorphic primers, which can amplify 30 polymorphic bands in 52 bands, the polymorphic rate was 57.7%, each primer amplified 7.4 polymorphic bands on average.(3) According to the results of ISSR-PCR amplified, similarity coefficients ranged from 0.60-0.93 by using Ntsys software. A dendrogram was constructed based on UPGMA cluster analysis and showed that the Dendrobium species could be majorly divided into two groups. The results showed that the genetic relationship was farther to the fifth sample and the rest of 29 Dendrobium, the first sample was close with the twenty-first sample, the third sample was close with the eighth sample, the tenth sample was close with the nineteenth sample, the twenty-sixth sample was close with the twenty-eighth.(4) On the basis of single-factors experiments, the Box-Behnken experimental design method were used to design the experiments and the responsive surfaces methodology analysis of 4 factors and 3 levels was adopted. The study established the quadratic regression equation for rate of polysaccharides. The equation is Y=14.03+1.05A+1.25B+0.12C+0.76D-0.29AB+0.34AC-0.098AD+0.21BC-0.20BD+ 0.29CD-0.94A2-0.76B2-0.64C2-0.22D2. The effects of the 4 factors in an order of decreasing magnitude:liquid material ratio(B)>extraction temperature(A)>extraction number(D)>extraction time(C). The optimal technical condition was extraction temperature 85?, liquid material ratio 1:37, extraction time 2.3h, extraction number 4 times. The extraction yield of polysaccharides was 14.98% in the optimal technical condition.