The Preliminary Trial Of EGFP-labeled Transformation Of Mycorrhizal Fungus And Its Symbiotic Relationship With Bletilla Ochracea Schltr.(Orchidaceae)

The study on relationship between orchids and mycorrhizal fungihas always been a hot topic. In nature, germination of orchids' seed andgrowth of plant all depend on the symbiotic mycorrhizal fungi. Bletiliaochracea Schltr. is a terrestrial orchid and a kind of important Chineseherbal medicine in Guizhou Province. However, the wild resources oforchids are under the threat of existence due to being over-collected andtheir habitats almost being destructed nowadays.In this study, a symbiotic model between Bletilla ochracea(Orchidaceae) and the mycorrhizal fungi isolated from this orchid wasestablished. The growth-promoting endophytic fungi was screened andidentified, and the preliminary transformation of EGFP gene ofmycorrhizal fungus Sebacina sp. is trying to achieve. This study providesthe new ideas and references for the symbiotic relationships andinteractions between the Orchids and the mycorrhizal fungi, based onwhich can help us for understanding the role of mycorrhizal fungi in theprocess of artificial culture of wild orchids and provide the theoreticaldirection for this. The main results of the research are as follows:1. The2growth-promoting endophytic fungi of Epulorhiza sp. andSebacina sp. were screened and identified. Both fungi were isolated fromthe root of B. ochracea by tissue culture method. 2. The symbiotic relationship between B. ochracea and themycorrhizal fungi of Epulorhiza sp. and Sebacina sp. were rebuilt. Theresults showed that Epulorhiza sp. and Sebacina sp. had the significantpromotion of the germination with8.61%and18.43%higher than that ofthe control, respectively. The symbiotic mycorrhiza structure between2mycorrhizal fungi and B. ochrace was inspected under the microscope.3. The optimized condition for the production of protoplasts is: theyoung mycelia of Sebacina sp. was digested for1.5h at28?in thecombination of enzyme solution using1.2M KCl as the osmotic stabilizer.The protoplasts yield higher than5×107mL-1were obtained.4. Through preliminary PEG-mediated and Agrobacteriumtumefaciens mediated transformation of GFP gene to Sebacina sp.,hygromycin's dosage to Sebacina sp. was determined. This study hasprovided the genetic transformatiom for Sebacina sp. with a significantfoundation of theory and practice.