| As an important plant resources, the Orchidaceae has high value in economic and scientific. However, wild orchid resource were decreasing and sufferring serious destruction because of the officinal and ornamental demand. Many orchids require endomycorrhizal fungi for their development and survival. Therefore, mycorrhization in tissue culture is a useful technique for the large quantity propagation of endangerous orchids. Mycorrhizal fungi application is essential for conservation of orchid species.To explore the mycorrhizal technique application to the conservation of endangerous Orchidaceae, fungi strains from natural roots of Doritis pulcherrima Lindl. were isolated and inoculated into the germfree orchid of D. pulcherrima. Through inoculation under tissue culture condition, some good strains that can promote the growth of orchids significantly were screened. Mycorrhization technique was used on D. pulcherrima under tissue culture and lichen culture condition. By using indicators such as fungi manage,mycorrhizae coloration,fungi re-isolation,growth of biomass, etc, it is confirmed that a symbioses culture system for fungi and orchids was established. The main results are summarized as the following:1) A system about asymbiotic germination and seedling propagation of D. pulcherrima was established. The best media for D. pulcherrima were selected as follows: The media hyponex 3 g/L + potatoes 30 g/L + peptone 0.5 g/L was the best for asymbiotic germination. MS + banana 70 g/L + peptone 1 g/L + active charcoal 1 g/L was the best for seedling propagation. MS + coconut milk 100 ml/L + BA 5 mg/L + NAA 0.5 mg/L was the best for adventitious bud propagation (propagation coefficient was 5). Lichen was the appropriate transplant substrate. The rate of young plant survive rate was 85﹪.2) An effective process to obtain orchid mycorrhizal fungi was established. The best separation condition were: using 75﹪alcohol to clean the root surface, then disinfected by 0.1﹪mercuric chloride for 8 min, taking the PDA which add environment factorial as a separate medium. 83 strains of fungi were isolated from the root of D. pulcherrima. The 73 strains isolated were classified to 17 genus of Ascomycetes and imperfect fungi, 10 genus need further identification.3) By the establishment of co-culture relationship of mycorrhizal fungi and D. pulcherrima, an effective way of mycorrhizal fungi selection was put forward. DE medium (Based on the experimental material improvement) was an effective symbiotic medium. By measuring the rate of fresh weight and dry weight of D. pulcherrima. Five strains that can promote the growth of orchid seedling significantly were selected.4) The technology of mycorrhization under tissue culture and potted culture condition was established. Through use of solid strain, seedlings of D. pulcherrima were mycorrhization in tissue culture. The fungal infection can be successfully done through mycorrhizal staining after 15 days of symbiosis culture.The survival rates of mycorrhizal seedlings were 100﹪(as the control of 63.33﹪). After 90 days of symbiosis culture, the fresh biomass of D. pulcherrima inoculated with fungi were higher than control. The fresh biomass of D. pulcherrima inoculated with F29 was 31.3﹪higher than control. Through use of liquid strain, seedlings of D. pulcherrima were mycorrhization in potted culture. After 90 d symbiosis culture , seedling survival and the fresh biomass of D. pulcherrima inoculated with fungi were higher than control. Seedling survival of D. pulcherrima inoculated with F29 was 35﹪higher than control. The fresh biomass of D. pulcherrima inoculated with F29 was 33.24﹪higher than control. Statistic showed that the difference between that of inoculation and control was extremely significant. All inoculated fungi were re-isolated from the root of mycorrhization seedling of D. pulcherrima.
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