Optimization Of Fast Propagation System Of Bletilla Striata And Isolation-identification Of Mycorrhizal Fungi

The survival and reproduction of wild orchid plants depend on symbiotic fungi to a certain extent.By complex orchid mycorrhizal structure,symbiotic fungi helps orchid plants uptaking water and mineral nutrient when seed germination and seedling development as well as in the barren soil.Bletilla striata belongs to the genus Bletilla in Orchidaceae.Its pseudobulb contains plentiful Bletilla colloid which has the efficacy of hemostasis,tonifying lung and tissue regeneration.It is a precious Chinese medicinal herbs.The seeds of Bletilla striata are small,without endosperm,the embryo is very small with yellow-brown thin seed coat.The ability of its seed reproduction is weak.The growth cycle of seedlings is long.The transplant survival of the sterile tissue culture seedlings is low.The plant development is slow and the later growth is weak.The important reason is that the process of life is inseparable from the participation of endophytic fungi.The isolation and identification,species diversity and growth promoting effect of Bletilla striata endophytic fungi have become a hot topic in the upstream of Bletilla striata industry.Therefore,the purple flower Bletilla?Bletilla striata?in Xiushan County,Chongqing was as the research object.Its rapid propagation system was optimized.The endophytic fungi in its roots was isolated.The isolated strains were co-cultured with sterile seedlings to observe the effect of inoculation and to establish mycorrhizal symbiosis system.In the end,the mycorrhizal fungi which have a significant promoting effect on the growth of Bletilla seedlings were screened out.Their morphological and molecular identification was carried out.The research aims to provide a valuable theoretical basis for the rapid propogation and production of seedlings in Bletilla industry.The main results are as follows:?1?Optimization of the Bletilla seedling rapid propagation system.Under the same environmental conditions,aseptic Bletilla striata seeds were sown to 1/2MS,MS and KC plate culture medium.After a week,faster germination speed and higher germinating rate were observed on KC medium than those on 1/2MS and MS medium.Adding different concentrations of NAA and 6-BA in KC medium,the medium of KC+0.5mg/L6-BA+1.0mg/LNAA plate is better for germination of Bletilla striata seeds and easy to form protocorms.MS was chosen as the basic culture medium for strong seedling culture and the medium of MS+2.5mg/L6-BA+1.0mg/LNAA was better.Kinetin was to induce Bletilla rooting.These roots were stout.So,the better rooting medium was 1/2MS + 1.5mg/L KT + 0.5mg/L NAA.?2?Isolation and morphological characteristics of Bletilla endophytic fungi.Wild Bletilla striata roots were cultured in 4 different medium,and the induction rate in MMN medium was the highest,being 58.33%.28 strains of endophytic fungi isolated from Bletilla striata were divided into 7 groups according to colony morphology and microscopic characteristics of hyphae.These strains were co-cultured with bletilla plantlets.Two strains of beneficial endophytic fungi MF-1 and MF-2 were screened out.By morphological and rDNA-ITS sequence analysis,two strains were identified.The molecular sequence of MF-1 strain is more similar to the molecular sequence of Cladosporium sp.4₁8_M13F_E09₁9J than 99%.The molecular sequence of MF-2 strains and the molecular sequence of Cladosporium sp.Nankai₂0mbsf₄ are similar to 99%.Two strains identified all belonged to the genus Cladosporium,but belonged to different two species?strain?.After 2 months of inoculation rhizosphere of Bletilla seedlings with MF-1 and MF-2 seperately,root were observated under microscope.Result showed that MF-1 and MF-2 could invade the root tissue of Bletilla striata and colonize in cortical cells.Twe endophytic fungi strains could set a symbiotic mycorrhizal association with Bletilla roots.They belonged to orchid mycorrhizal fungi?OMF?.Mycelial colonization position was mainly concentrated in the middle part of the mature zone in root tips.The colonization of mycelium was not detected at the meristem zone and base part of mature zone in roots.?3?Effects of mycorrhizal fungi inoculation on growth and photosynthetic capacity of Bletilla plantlets.The endophytic fungi of MF-1 and MF-2 strains were inoculated at rhizosphyere of Bletilla plantlets respectively and co-cultured for 60 d.Compared with the non inoculation plantlets?CK?,the seedlings inoculated grew well with the dark green leaf and the developed roots which showed the growth promoting effect was remarkable.But the growth promoting effects of the two strains was not exactly the same.Bletilla striata seedlings inoculated with MF-1,plant growing,bulbous bulbis expanding and stem thickening are faster than inoculated MF-2.Bletilla striata seedlings inoculated with MF-2,the promoting growth for root was better than inoculation with MF-1 because the new root was stronger and the bud differentiated earlier from pseudo bulb.In leaves of Bletilla striata seedlings inoculated with MF-1 and MF-2,the relative content of chlorophyll increased by 43.67% and 27.05%,and the net photosynthetic rate increased by 56.65% and 30.29% respectively,the soluble sugar content increased by 82.01%,41.26%,the soluble protein content increased by 25.42%,48.20% respectively,the polysaccharide content in the protocorm was increased by 89.52% and 52.97% respectively.