【作者】 邢树堂；

【导师】 李玉花； 傅禄敏；

【作者基本信息】 东北林业大学 ， 园林植物与观赏园艺， 2005， 硕士

【摘要】 银杉（Cathaya argyrophylla）和秦岭冷杉（Abies chensiensis）分别是我国特有的松科植物以及珍贵的一级和二级保护植物,由于其种群自然更新能力日趋低下,处于严重的濒危及渐危状态。本课题在现有的宏观生态学研究的基础上,以菌根学为主要研究方法并结合植物组织培养的技术,试图通过使用最小量种子资源来诱导该两种植物人工菌根的形成,验证银杉与秦岭冷杉为典型的外生菌根植物的理论:建立人工菌根苗的诱导技术,进行菌根功能的初步观察。从而为从根本上解开其濒危之谜,为寻找一条濒危松科植物种群复壮的途径提供有用的信息。本研究的具体结果如下: 1.运用植物组织培养技术建立了银杉及秦岭冷杉无菌幼苗的培养系 经70%酒精以及30%H₂O₂的表面消毒,银杉种子的无菌率可达100%;并且经过切割1/5-1/3的胚乳,其种子发芽率达到60%以上:经70%酒精、0.2%升汞溶液以及30%H₂O₂的多重表面消毒,秦岭冷杉种子的无菌率也可达100%,其发芽率为30%。 经对多种植物培养基（WPM、SH、改良MS）进行筛选和比较,确定了包含1/4 MS大量元素、1/2 MS微量元素、MS维生素及MS铁盐的改良MS（MMS）培养基为适合的基本培养基,在MMS培养基中添加0.2mg/L的6-BA以及0.02mg/L NAA,有利于银杉、秦岭冷杉幼苗的生长。 2.成功地建立了两种植物的人工菌根苗的诱导系 通过菌种以及菌根诱导培养基的筛选,确定了土生空团菌以及RM培养基的有效性;运用改良平板法技术,分别建立了银杉-土生空团菌以及秦岭冷杉-士生空团菌的人工菌根诱导体系;运用形态解剖学的研究技术,观察并证实了在两种植物的侧根中存在明显的菌套结构以及在侧根的皮层细胞间隙存在哈蒂氏网结构。首次在实验室水平上建立了该两种植物的人工菌根诱导系。 3.改良了培养容器的表面消毒法 通过紫外线照射与70%酒精或0.05%NaOCl的表面消毒的双重处理,使不能高压火菌的容器达到表面消毒效果,建立了银杉、秦岭冷杉人工菌根苗的实用化体系。 4.成功地移栽了银杉人工菌根苗 通过6个月的移栽试验,分别对银杉菌根苗以及非菌根苗进行了生长比较,其结果表明菌根菌对于植物侧根的形成有明显的促进作用。更多还原

【Abstract】 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更多还原