A Fungal Symbiotic Germination Study On The Seeds Of The Endangered Orchid Medicinal Plant Gymnadenia Conopsea

Gymnadenia conopsea is a terrestrial,photosynthetic orchid that is widely but sporadicly distributed.It is not only a traditional medicine in China but also used as Tibetan medicine and Mongolian medicine,which has high medicinal value.However,G.conopsea species is more endangered because of it's low natural reproduction rate,undue artificial excavation and habitat environment destruction recently.For lack of endosperm,tiny orchid seeds need the aid of suitable fungi during germination.Based on the natural mycorrhizal symbiosis,exploring fungi that can effectively promote seeds germination of G.conopsea and applying them to the ecological restoration of wild populations is important for germplasm conservation of G.conopsea and resource regeneration.In this paper,we studied symbiotic seeds germination of G.conopsea,the main results are as follows:We studied mycorrhizal fungi community composition of G.conopsea between large geographic distance and found that members of the Tulasnellaceae were the most abundant fungal associates in Europe,while members of the Ceratobasidiaceae were the most abundant fungal associates of G.conopsea in China.However,none of the fungal OTUs of Sebacinaceae was shared between Europe and China.The data analysis showed that mycorrhizal communities of G.conopsea differed significantly among different regions and there is a general decrease in similarity with increasing geographical distance in mycorrhizal communities.Single peloton isolation and tissue isolation were used for isolating mycorrhizal fungi from roots of G.conopsea from Beijing and Tibet.Finally,4 strains belonging to Ceratobasidiaceae and 1 strain belonging to Tulasnellaceae were identified.By screening symbiotic seed germination fungi,we found that GS2 isolated from G.conopsea from Beijing could achieve seed germination of G.conopsea from Beijing and Jilin,and GZ1,GZ15 and GZ16 isolated from G.conopsea from Tibet could achieve seed germination of G.conopsea from Tibet.We found that G.conopsea requires specific fungi to promote protocorm formation and growth to a seedling by symbiotic seeds germination fungi specificity study.Environment conditions like medium and temperature and light were changed and studied.The results suggested that fungi is necessary for seed germination and the environmental conditions had insignificant influence on the symbiotic germination process,but light was an important factor in seedling establishment of G.conopsea.We observed morphological characteristics of the symbiotic germination seeds at the cellular and subcellular levels and explored molecular mechanism by transcriptome study.Two strains that had obvious germination effect were chosen as two treatment group for symbiotic germination.After 25 days,protocorms from two groups were collected for RNA extraction and transcriptome sequencing.Finally,27962 differentially expressed genes were obtained and analysis suggested that seed germination of G.conopsea was the result of the combined effect of molecular signal recognition and nutritional transport.To find out whether isolated fungi could still achieve seeds germination under natural environment,we carried out the study.For the first time,symbiotic seed germination of G.conopsea under natural environment succeed,which indicated that GS2 strains had good application prospect in artificial cultivation.Through the research of this paper,we knew differences of mycorrhizal fungi community composition of G.conopsea between large geographic distance,obtained fungal isolates that can promote symbiotic seed germinatin effectively and achieved seeds symbiotic germination under natural environment.The study results laid the foundation for artificial cultivation and ecological restoration of wild populations of G.conopsea.