Study On The Identification And Evaluation Of Leaf Bulb In White And

Bletilla of Orchidaceae is perennial herb, with ornamental and medicinal value. Bletilla is widely distributed in Asia, and the pseudobulb of Bletilla is a traditional Chinese herbal medicine, which have the very good effect on medicine,e.g. folding of the gas, permeability phlegm, hemostatic effect and so on. All of the four species are respectively Bletilla striata, Bletilla sinensis, Bletilla ochracea and Bletilla formorsana. The natural germination rate of Bletilla is low, and Bletilla striata of wild resources have been seriously damaged because of the habitat destruction and over excavation., so the protection of wild resources is necessary. Based on the discussion above, this study provided a theoretical basis for the utilization and protection of Bletilla striata.Firstly, This paper used single factor and multiple factors experiment to study pseudobulb induction. The experimental results show that:the MS culture medium was the most suitable culture medium for pseudobulb induction of Bletilla striata; 1 g/L activated carbon is the best concentration; the four factors (NAA,6-BA,PP333 and additives) on the induction rate of pseudobulb:additives>PP333>6-BA>NAA; on average grain diameter: 6-BA>PP333>additives>NAA. Finally confirmed the best culture medium was MS+NAA 1 mg/L+6-BA 0.8 mg/L+PP3330.5 mg/L+banana mud 10.0 g/L+activated carbon 1.0 g/L.Secondly, this paper used SRAP markers to establish and optimize SRAP-PCR reaction system.The22 samples of Bletillae were identified. The determined 20μL SRAP-PCR reaction system:(10×buffer) 2.0μL, MgCl2 1.5 mM, Taq polymerase 1.5 U, dNTP 0.4 mM, primers 0.4 M,20 ng template DNA. Twelve SRAP primer combinations generated a total of 189 clear amplification bands with 22 samples, Showing that Bletilla had high genetic diversity. Through the UPGMA clustering method, Bletilla striata, Bletilla ochracea and Bletilla formorsana are better separated.The four Unidentified samples are identified for Bletilla striata.Finally, The SRAP markers were applied to assess the level and pattern of genetic diversity in twelve populations of Bletilla striata. Eighteen SRAP primer combinations generated a total of 294 clear amplification bands, encompassing 240 (81.63%) polymorphic bands. A high level of genetic diversity was detected (PPB= 90.48%, H= 0.3494,1= 0.5096) at the species level. There was a moderate genetic differentiation (Gst = 0.2609) among populations. Three main clusters were detected by cluster analysis using the unweighted pair-group method with arithmetic average (UPGMA). Mantel test revealed that significant positive correlation was found between genetic distances and geographic distances (r= 0.6329; p<0.0001). Recommendations for conservation of the species resources are proposed.