Study On Genetic Resources Evaluation, Tissue Culture And Molecular Identification Of Medicinal Plants

Bletilla striata, Bletilla ochracea, Bletilla formorsana, and Bletilla sinensis belong to Bletilla genus of Ochidaceae, distributing widely in most places in China. All of the four species are the raw material of Chinese medicine ’Baiji’. The dried pseudo bulb of Bletilla is usually used to stanch bleeding, subside a swelling, recover physical capacity and reduce phlegm, according to Compendium of Materia Medica. Modern pharmacological study also indicated that glucosan, phenols and many other components in Bletilla are of notable curative effect. However, due to overexploitation, Bletilla wild germplasm resources are at the risk of extinction. Therefore it is important to protect this endangered plant. Here, in this thesis, researches are carried out from three aspects:tissue culture of Bletilla striata, evaluation of Bletilla striata resources based on microsatellite marker and authentication of Bletilla striata using internal transcried sequence.Firstly, orthogonal test of L9 (34) was used in tissue culture experiment to find the best match of sucrose concentration, AC concentration, NAA concentration and organic substances. Results showed that 1% AC+0.5 mg/L NAA+25g/L sugar+ 10% banana juice was the best medium for plant rootage and strengthening. The order of effecting is AC concentation> organic substances> sugar concentation> NAA concentation.In this thesis, microsatellite markers are used to evaluate genetic diversity and genetic structure of B. striata, and lay the foundation of protecting the genetic resources of B. striata. Cross species amplifacation showed that the markers could also be used in the study of genetic diversity in the populations of B. formosana. Ten novel polymorphic microsatellite markers were selected from 59 markers to investigate the genetic diversity and population genetic structure of B. striata. Expected heterozygosity of three populations of B. striata from Anhui Province are HS (0.4958), HQ (0.6209) and SH (0.6961) respectively. Amova analysis showed that variance within populations were higher than variance among populations. Analysis for populations of B. formosana from Shanxi Province are XY (0.5932), ZY (0.6258), YS (0.6402), DF (0.7054), SY (0.6621) respectively, Amova analysis showed that variance within populations were higher than variance among populations.At present, market are loaded with fake B. striata medicines, which seriously break the order and damaged the the interests of customers. In this thesis, internal transcribed seqeunce (ITS) were used to identify B. striata and its adulterants such as Polygonatum cirrhifolium, Belamcanda chinensis, Pleione bulbocodioides, et al..rDNAITS sequences of Polygonatum cirrhifolium, Belamcanda chinensis, Pleione bulbocodioides were downloaded from GenBank. Relationship among B. striata, B. formosana, B. sinensis and other adulterants were established using UPGMA method. Results showed that commodities purchased from market belong to B. striata and B. striata were clearly identified from its adulterants. Molecular marker based on rDNA ITS can stably and accurately distinguish multi-origin medicinal materials in Chinese medicine.