硬叶兰种子的迁地共生萌发及有效共生真菌的分离和鉴定

兰科植物的种子原地和迁地共生萌发技术是近年发展起来的开展兰科植物种子和共生真菌研究的有效方法。该研究对兰属(Cymbidium)附生植物硬叶兰(C. mannii)开展了种子的迁地共生萌发研究, 试图获得其种子萌发的有效真菌。利用硬叶兰成年植株根部周围的树皮、苔藓、枯枝落叶、腐殖质等作为培养基质, 进行种子的共生培养。在培养133天后, 成功地获得了处于不同阶段的已萌发种子、原球茎和幼苗, 并从原球茎中分离得到一种瘤菌根菌属(Epulorhiza)真菌。用所分离到的FCb4菌株和一种从兜唇石斛(Dendrobium aphyllum)分离到的胶膜菌属(Tulasnella) FDaI7菌株和硬叶兰种子在燕麦琼脂培养基上进行共生萌发, 设置不接菌作为对照处理, 以检验FCb4菌株对硬叶兰种子萌发的有效性。经过58天的培养, 不接菌的对照处理中种子没有萌发, 接种FCb4和FDaI7菌株的处理都有很高的种子萌发率, 两种接菌处理在不同光照条件下的种子萌发率均无显著性差异。但暗培养条件下, 种子萌发形成原球茎后, 表现出生长停滞的趋势, 仅有很少的原球茎继续生长达到幼苗阶段, 说明原球茎发育后期与幼苗发育阶段需要光照。在光照条件下, 接种FCb4菌株处理中达到幼苗阶段种子的比例为(25.67 ± 9.27)%, 显著高于接种FDaI7菌株处理的(3.04 ± 2.27)% (W = 56, p = 0.026, Mann-Whitney U-test), 表明此研究中分离到的瘤菌根菌属真菌能有效地促使硬叶兰种子萌发并生长发育到幼苗阶段。

Ex situ symbiotic seed germination, isolation and identification of effective symbiotic fungus in Cymbidium mannii (Orchidaceae)

Aims The in situ and ex situ seed baiting techniques, which have been developed in recent years, are effective methods to study the compatible mycorrhizal fungi of orchids. Our aim was to obtain the compatible mycorrhizal fungi of epiphytic Cymbidium mannii using the ex situ seed baiting technique. Methods Bark, moss, litter and humus around the roots of adult plants of C. mannii were collected as substrate to cultivate C. mannii seeds in the laboratory. The fungi were isolated from developed protocorms and identified by morphological and molecular characteristics. The effects of different mycorrhizal fungi and light on seed germination were examined by in vitro symbiotic germination. Important findings The isolated fungus was identified as a species of the genus Epulorhiza and was named as FCb4. After 58 days cultivation, seeds inoculated with FCb4 strain and FDaI7 strain (Tulasnella, isolated from Dendrobium aphyllum) had a high germination ratio, whereas seeds without the fungus failed to germinate. There was no significant difference between the germination ratios of seeds inoculated with FCb4 and FDaI7 strains, but FCb4 was significantly superior for seedling formation and development compared with FDaI7 in light. This indicated a lower degree of symbiotic fungal specificity on seed germination than the protocorm development stage in C. mannii. The fungus we obtained was effective for seed germination, and protocorm and seedling development for C. mannii. Germination and protocorm production were higher in the dark (0/24 h light/dark) than the light condition (12/12 h light/dark), whereas the subsequent protocorm development was better with light. These findings will aid in seedling production and reintroduction of C. mannii.