The Tissue Culture Optimization Of Dendrobium Officinale And DNA Barcode Identification System Construction

As a valuable medicinal plants,Dendrobium,especially Dendrobium officinale has a huge market value and ornamental value.With harsh growing conditions under natural,low seed germination rate,Dendrobium officinale is facing with the current situation of wild resources which have been nearly exhausted.Therefore,a rapid propagation system is indispensable for sustainable development of Dendrobium officinale.What’s more,some Dendrobium are often mislabeled for a similar morphological features and adulterated by fake or other Dendrobium with low quality in the market.Based on this background,this paper carried out from the two aspects: the construction of rapid propagation system and rapid identification system on Dendrobium.The main results of this study were shown as follows:(1)The culture condition optimization of Dendrobium officinaleThis study conducted fast cultivation of Dendrobium officinale by using tissue culture technology,and optimized its growth condition from the three aspects: base medium,organic additives and hormones.The result was successful in shortening the Dendrobium officinale growth time from 8 months to 6 months,and showed that the optimum conditions for the cultivation of Dendrobium was B5+X+(20 ml/L coconut milk+80 g/L banana)+30 g/L sucrose+6.2 g/L agar,wherein X refers to hormone,rooting stage was 2 mg/L 1-Naphthylacetic acid + 0.5 mg/L 3-indolebutyric acid,and seedling stage was 0.5 mg/L 1-Naphthylacetic acid + 0.2 mg/L 3-indolebutyric acid.(2)Construction of DN A barcode identification system of DendrobiumThis experiment selected two kinds of commonly plant DNA barcoding gene : ribosomal DNA second internal transcribed spacer(internal transcribed spacer 2,ITS2)and chloroplast psbA-trnH spacer sequences to analysize 9 kinds of Dendrobium and its common fake——Bulbophyllum odoratissimum(a total of 55 samples).The analysis of the phylogenetic tree and interspecific and intraspecific differences showed that the ITS2 sequence had large difference between Bulbophyllum odoratissimum and Dendrobium,the maximum of intraspecific divergence was less than the minimum of interspecific divergence on Dendrobium,and the average interspecific divergence was 58 times that of the average intraspecific differences,thus indicated the feasibility of the ITS2 sequence used as DN A barcode for Dendrobium identification.While the psbA-trnH sequence can be used as an auxiliary sequence of ITS2 barcode sequence and not be used as a single barcode sequence for Dendrobium identification.(3)Construction of infrared spectroscopy identification system of DendrobiumExperiment scanned 9 kinds of Dendrobium and obtained characteristic spectra by using Fourier Transform Infrared Spectrometer technology(FTIR).By analyzing those characteristic and active ingredient of Dendrobium,results showed that the genetic relationship of 9 kinds of Dendrobium was generally consistent with DNA barcode analysis results.In addition,the Dendrobium officinale in four different places(Lishui,Wenzhou,Taizhou of Zhejiang province and Dali of Yunnan province)were analyzed by FTIR.We found that there were some differences in the peak height of the spectra of Dendrobium officinale from four different places,and the peak of Dendrobium officinale in Yunnan showed blue shift compared with the others.Therefore,FTIR can be used as an auxiliary means of DNA barcod e to establish a comprehensive identification system.