铁皮石斛种子的室内共生萌发

由于人为的采挖和原生境的破坏,使得铁皮石斛野生资源已濒临灭绝。因此,保护铁皮石斛野生资源及其生境,加快其野生资源的繁殖显得非常重要。以铁皮石斛种子(TTC染色显示种子生活力为77.65%)为材料,与分离自2种野生兰科植物根部的4株共生真菌 (C20来自铁皮石斛,L12,L24b 和 L28来自美花石斛)在燕麦培养基上进行共生萌发。经过18周的共生培养,4株真菌均不同程度地促进了铁皮石斛种子的萌发,其中菌株L24b (Epulorhiza)和L28 (Epulorhiza)显著提高了种子的萌发率,分别比对照高出26.51%和12.20%,但未形成幼苗,只是处于原生分生组织阶段(阶段3);菌株C20(Epulorhiza)和L12 (Alternaria) 虽没有显著提高种子的萌发率,但对原球茎的发育和幼苗的生长有明显的促进作用;而对照的种子仍然处于膨大转绿期,即萌发阶段(阶段2)。同时发现,TTC染色显示的铁皮石斛种子生活力要高于种子共生萌发的萌发率(除了菌株L24b)。研究结果表明:种子生活力染色检测的活力值只代表种子所具有的潜在的萌发能力,而不能代表实际的萌发率。在异地条件下,铁皮石斛与共生真菌间没有严格的专一性,可以与瘤菌根菌属、链格孢属真菌形成共生关系。菌株C20和L12能促进萌发后的种子进一步分化成幼苗。这两个菌株为铁皮石斛的人工优质栽培和野外种群的建立提供了可能。

Ex-situ symbiotic seed germination of Dendrobium catenatum

Dendrobium catenatum is a perennial, epiphytic orchid. It is not only an ornamental plant, but also an invaluable medicinal herb in China. It has the functions of benefiting the stomach and spleen, nourishing Yin and kidney, moistening the lungs, promoting the production of body fluid and improving immunity. Natural seed set is low and the wild resource of D. catenatum is scare. In recent decades, D. catenatum had been over-collected for its high profits, which led to drastic reduction of the wild populations of D. catenatum. In addition, D. catenatum's habitats were severely degraded. Together, these factors have driven D. catenatum to the verge of extinction. There is mature tissue culture technology of D. catenatum, but the survival rate of sterile plants in tissue culture is low, seedling growth rate is slow, and large scale production is difficult. Therefore, it's very important to protect the wild resources and habitats of D. catenatum. Effective ex-situ propagation of the species may help to boost the wild populations. It is well known that mycorrhizal fungi play an important role in the germination, growth and development of orchids, and understanding the relationship between orchids and mycorrhizae is important for the conservation of orchids. Our aim is to find the symbiotic fungi which can promote seed germination and production of symbiotic seedlings, and use them in the artificial cultivation and reintroduction. We used seeds (with seed viability of 77.65% based on TTC staining) harvested from wild plants of D. catenatum in Hunan province, and inoculated them with four symbiotic fungi that were isolated from the roots of D. catenatum (C20) and D. loddigessi (L12, L24b and L28) respectively in petri dishes with oat meal agar. Seed germination and protocorm development were observed using Dino-Litle Digital-Microscope and Olympus Microscope once a week. After 18-weeks cultivation, all four symbiotic fungi promoted germination of D. catenatum seeds, but to various degrees. Specifically, seeds co-cultured with strains L24b(Epulorhiza) and L28(Epulorhiza) had a significantly higher germination rates (88.41% and 74.10%, respectively) than that of the control(61.90%), but they were not able to form seedlings and were developed only to the stage of protomeristem(stage 3). Strains C20(Epulorhiza) and L12(Alternaria) did not significantly improve seed germination rate compared with control treatment. However, they promoted seeds to form symbiotic seedlings. During the same period of time, seeds in the control treatment were still in the stage of embryo enlargement with ruptured testa(=germination, stage 2). TTC staining showed that the seed viability is higher than the germination rate of symbiotic seed germination (excepted for strain L24b). Our study suggested that seed viability staining only represent the potential of germination instead of the real germination rate. The specificity between symbiotic fungi and D. catenatum is low in vitro because seeds of D. catenatum can form symbiosis between the fungi of Epulorhiza and Alternaria, and these two strains were from different orchids. Strains C20 and L12 may be used for high quality artificial cultivation as well as restoration of wild populations of D. catenatum.