Study On Tissue Culture And Molecular Identification Of Dendrobium Nobile

Dendrobium hancockii belongs to Dendrobium genus(Orchidaceae:Epidendroideae)and is a rare perennial herb.Its 'Huang Cao,medicinal materials was made of the stems of Dendrobium hancockii,and it is not only a valuable medicinal plant,but also a kind of ornamental plant.In recent years,D.hancockii has been excavated because of its high medicinal value and economic value,and its resources are greatly reduced with the restriction of environmental factors.In order to protect the rare traditional Chinese medicine,we did the following research.This research focuses on three aspects as follows:(1)The optimization of tissue culture system of D.hancockii and its application in bioreactor.In this study,we optimized the culture medium in seed germination,PLBs proliferation,and the culture metdium in rooting stage;In the process of seed germination,we found that,the optimal medium ingredients for seed germination of D.hancockii was:MS medium+0.5 mg/L NAA+1.5 mg/L 6-BA,meanwhile supplemented with sugar 30.0 g/L,and agar 7.2 g/L.In the process of PLBs proliferation,the optimal medium ingredients was:1/2MS medium+0.5 mg/L NAA+2.0 mg/L 6-BA,and supplemented with sugar 30.0 g/L,agar 7.2 g/L.In the process of seedling and rooting stage,the optimal medium ingredients for D.hancockii was 1/2MS medium+100.0 g/L potato extract+60.0 g/L banana extract+1.0 g/L peptone,meanwhile supplemented with sugar 30.0 g/L and agar 7.2 g/L.(2)Sequencing and analyzing of chloroplast genome of D.hancockii.The chloroplast genome of D.hancockii has a total length of 152,071 bp and is comprised of a large single copy region(LSC,85,004 bp),a small single copy region(SSC,14,485 bp),and two inverted repeat regions(IR,26,291 bp).The acquisition of the chloroplast genome sequence of D.hancockii has great significance in the development of the phylogenetic evolution,species identification of Dendrobium.(3)The identification of D.hancockii and its 'Huangcao' medicinal materials.In this study,we set up a rapid identification method based on RT ARMS-PCR technology.This technology based on real-time fluorescent quantitative PCR and combined with a mutant amplification block system technology,can identify D.hancockii and its 47 kinds of adulterate 'Huangcao' medicinal materials.We designed specific primers based on the high variability of chloroplast genome fragments,and three pairs of primers were filtered:BT2-BT4,CP4-CP5,LU5-LU6,which specificity to amplify the D.hancockii,but other 47 kinds of adulterants can't be amplify.Therefore,this method can identify the D.hancockii and its 'Huangcao' medicinal materials from the adulterants accurately and effectively.