Establishment Of In Vitro Ectomycorrhizal System Of Cathaya Argyrophylla And Abies Chensiensis

Cathaya argyrophylla and Abies chensiensis are species of Pinaceae native to China, and they are listed as first- and second-grade endangered plants by the State Environmental Protection Administration of China, respectively. Since the natural regeneration ability is getting lower, these two species are regarded as critically endangered conifers.On the basis of current ecological studies, my present study combined mycorrhizal methods and plant tissue culture techniques to induce in vitro ectomycorrhization using minimum seed sources, as well as to verify whether C. argyrophylla and A. chensiensis are ectomycorrhizal species. I also aimed to establish a culture system of in vitro ectomycorrhization, and to undertake primary researches of the functions of ectomycorrhizas. This study provided the useful information to unveil the reason why the survival of these species was endangered and to find out an approach for their regeneration. The results are described as follows:Establishment of in vitro culture system of C. argyrophylla and A. chensiensis using plant tissue culture techniques.The surface sterilization rate of C. argyrophylla seeds reached 100% with 70% ethanol and 30% H₂O₂ solution. The germination rate increased to be higher than 60% by removing 1/5 to 1/3 seed coat and endosperm.The surface sterilization rate of A. chensiensis seeds also reached 100% with each solution of 70% ethanol, 30% H₂O₂ and 0.2% HgCl₂. The germination rate was 30%.The modified MS medium (MMS) containing 1/4 MS macro-elements, 1/2 MS micro-elements, MS vitamins and MS Fe salt was shown to give optimal conditions for both species to grow as a result of comparing and screening various plant culture media such as WPM, SH and modified MS. The growth of C. argyrophylla and A. chensiensis seedlings was promoted on MMS medium containing 0.2 mg/l of BA and 0.02 mg/l of NAA.Establishment of in vitro mycorrhization system of C. argyrophylla and A. chensiensisCenococcum geophilum and RM medium was separately selected as a suitable isolate and an appropriate medium for ectomycorrhization. In vitro ectomycorrhizas formation between C. argyrophylla - C. geophilum and between A. chensiensis - C. geophilum were induced by using the modified plate dish method. Subsequently, the morphological and anatomical observation revealed that the synthesized ectomycorrhizas produced an obvious mantel in lateral roots and Hartig net structure between host cortex cells in both species. This is the first report that in vitro ectomycorrhizas were established in both species and that they were achieved at a laboratory condition.Improvement of the sterilization method of culture containers.Plastic culture containers, which are not suitable for sterilization by autoclaving, were successfully sterilized with the combination of ultraviolet-light irradiation, 70% ethanol and 0.1%NaOCl solution. This was essential for the induction system of in vitro ectomycorrhizal seedlings to be applied for practical use.Plant growth after in vitro seedlings were transplanted outDuring six months after in vitro ectomycorrhizal seedlings of C. argyrophylla were transplanted out, the growth of ectomycorrhizal and control seedlings are compared. It was shown that ectomycorrhizal fungi were distinctly effective on lateral root formation...