Germplasm Identification And Tissue Culture And Propagation Of Tetrastigma Hemsleyanum, A Medicinal Plant

Tetrastigma hemsleyanum Diels & Gilg is an important endemic medicinal plant of China, which distributed widely in the middle and lower yangtze river and south region decades ago. However, the latest investigates showed that the wild populations of T. hemsleyanum were decreasing due to over utilization and habitat destruction. This study aims to resolve the problems about germplasm identification and rapid proliferation of T. hemsleyanum, and explore the cultivation condition simply. The main results are as follows:1. DNA barcode character analyses showed the numbers of variable sites which rbcL, matK, trnH-psbA and ITS had were 18,39,64 and 236 respectively. The mean interspecific distance/mean intraspecific distance of these four barcodes were 0.0063/0.0001,0.0049/0.0002,0.0184/0.0006 and 0.0745/0.0036 respectively. Species monophyly analyses showed ITS could identify 93.8% species, followed by trnH-psbA (66.7%), matK (61.1%), rbcL (22.2%). In NJ, UPGMA and ML analyses, the identification effect of multi-locus barcodes were better than singl-locus barcodes (except ITS). T. hemsleyanum could be identified by ITS and multi-locus barcodes (except matK+trnH-psbA). Obvious Barcoding gap wasn't found in four barcodes, but significant difference was tested between intra-and inter-specific distance. According to the results, we consider ITS is the most suitable barcode for identification T. hemsleyanum and other Tetrastigma species.2. The results of proliferation culture showed that BA worked better on shoot regeneration than KT and CPPU. Maximum proliferation multiple (7.27) was found in MS+BA4.0 with minimum shoots height (0.48 cm). Maximum shoots height (2.69) was found in MS+BA0.5+NAA0.1. MS+BA2.0,MS+BA2.0+NAA0.1,MS+BA4.0+ NAA0.1 have medium proliferation multiple (4.00-4.13) and shoots height (0.95-1.25cm). The results of rooting culture showed IBA was the most powerful one among four auxins tested in present study. All rooting rates achieved 100% and over 7.3 roots per shoot in three concentrations. According to the results, we consider MS+BA4.0 and MS+BA2.0+NAA0.1 as preferred proliferation medium and 1/2MS +IBA0.5 as the best rooting medium.3. Phenological observation showed T. hemsleyanum grew fastest in September and slowest in January, February, March and December. The results of cultivation test revealed the best planting spacing and shade rate were 0.1m×0.3m and 70%' respectively. Root yield couldn't be enhanced significantly in climbing growth model. Fertilizer was not necessary when planting in mountainous area.