Establishment Of Plant Tissue Culture System For Anoectochilus Roxburghii:Development And Application Of Molecular Markers

Anoectochilus roxburghii(Wall.)Lindl,a kind of perennial precious traditional Chinese herbal medicines have wide medicinal value.In recent years,for the over-exploitation of human beings,Anoectochilus market is leading to tight situation,what is more,the Anoectochilus plants for sale are generally dry goods,it is difficult to recognize the specific form of the plant,and it is not easy to identify the authenticity.(1)Plant tissue culture system for Anoectochilus plantIn order to understand the impact of various tissue culture nutrients for Anoectochilus growth and proliferation and the optimization Anoectochilus tissue culture conditions and provide a theoretical basis for factory production Anoectochilus,we had Anoectochilus plants from Fujian and Taiwan for experimental subjects.We analyzed the impact of plant hormone 6-BA,NAA and IAA,the organic matter added mashed banana and mashed potatoes on Anoectochilus growing and proliferation.we found that,the influence of experimental factors was 6-BA>NAA>IBA>organic additive during the proliferation phase;and organic additives>IBA>NAA>6-BA during the growth phase,and they had fully illustrated that the nutrient media requirements of Anoectochilus plant in different growth stages varied,and the results provided guidance and reference for Anoectochilus tissue culture.In respect of the experimental study of the experimental point of view,it should choose a more stout stems Anoectochilus as explants for tissue culture propagation,the original bulbs and buds phase induction culture media formulations were:Taiwan Anoectochilus:6-BA 4mg/L + NAA 0.2mg/L + MS;Fujian Anoectochilus:6-BA 2mg/L + NAA O.lmg/L + MS;the culture medium formulation were:Taiwan Anoectochilus:6-BA 2mg/L + NAA 0.1mg/L + MS;Fujian Anoectochilus:6-BA 1mg/L + NAA 0.1mg/L + MS;and the rooting medium formulation stage were:NAA 0.1mg/L +1/2MS +60-80 g/L mashed banana.(2)ISSR-PCR analysisIn order to explore the genetic relationship among different species of A.Formosanus and complete the DNA finger-print of qualified species of A.Formosanus,and distinguish the affinis plants such as Goodyera matsumurana,Goodyera schlechtendaliana,Inter-simple Sequence Repeat(ISSR)technique was applied in this article to identify different species of A.Formosanus from Zhangzhou,Nanjing in Fujian Province,Guangdong Province,Guangxi Province,Taiwan Province and Vietnam and G.matsumurana from Fujian Province,and 9 pairs of UBC primers were selected.100 locus were totally amplified within 28 species,and 93 of them are polymorphic.There is high level of genetic diversity among different species.According to the analysis of the experiment,the 28 Anoectochilus samples can be clustered into 4 groups,however,the classification results of different approaches was not large in line,so we can probably infer that the genetic differences among Anoectochilus should be subject to environmental impact and growing environment as well as the subculture times.Moreover,9 new DNA fragments containing repeated sequences of AG,TC,GAA,CTT,GATA,CTAT compared in NCBI were determined specific in A.Formosanus,and 3 ISSR primers can distinguish species between A.Formosanus and G.Matsumurana were obtained.(2)cpSSR-PCR analysisIn order to take advantages of SSR advantages and plant chloroplast DNA,we had the Anoectochilus plants from Longyan,Zhangzhou and Taiwan for the studies.DNA sequencing of the Anoectochilus chloroplast were measured respectively,with the sizes of approximately 135kb,154kb and 215kb respectively,and they were similar to the general higher plant chloroplast DNA sequence size,in addition we measured the cpSSR message statistical analysis of Anoectochilus cpDNA sequence,the results showed that:Anoectochilus chloroplast DNA had any abundant dinucleotide and trinucleotide repeat sequences of SSR.Finally,we designed the 31 pairs cpSSR primers according to the sequencing results,with the help of PCR validation screening,we found that 25 pairs of the primers were able to amplify the target band in the Anoectochilus samples showing a generic feasibility primers,and there were 3 pairs of the primers were able to amplify stable target band in Anoectochilus plants and can not amplified the same target band in Goodyera plants indicating that these three pairs of primers were be suitable for the identification of Anoectochilus plants and Goodyera plants,and the results were expected to develop into molecule labeled primers for Anoectochilus quality monitoring.