Symbiotic Germination Technique of an Endangered Epiphytic Orchid Dendrobium sinominutiflorum: A Promising Conservation Application 濒危附生兰科植物勐海石斛 (Dendrobium sinominutiflorum) 的共生萌发技术:一种具有应用前景的保护策略

Abstract

Symbiotic fungi provide nutrients for orchid seed germination and seedling development, underpinning the long-term conservation and reintroduction. Dendrobium sinominutiflorum is a critically endangered orchid species, with limited natural populations and the role of symbiotic fungi in seed germination is still unclear. To ensure more effective conservation strategies for this endangered orchid species, this study developed a symbiotic seed germination and seedling development protocol. Using seed baiting technique, the in situ germinated protocorms were obtained. A compatible mycorrhizal fungus, Tulasnella sp. (strain MHSH), was isolated and identified by morphological and molecular analysis. Compared with asymbiotic seed germination on 1/2 Murashige and Skoog (1/2 MS) medium, symbiotic seed germination of D. sinominutiflorum by co-culturing with the MHSH strain on Oatmeal Agar (OMA) medium showed a higher protocorm growth rate and seedling formation rate. Transmission electron microscopy revealed that the infected cortical cells of symbiotic protocorms lacked distinct organelles and were colonized by fungal hyphae. The fungal hyphae penetrated, proliferated, and were subsequently digested within the protocorm cells, indicating that symbiotic protocorms exhibit a heterotrophic life and efficiently utilize fungal hyphae to support their growth and development. In contrast, asymbiotic protocorms contained abundant chloroplasts and amyloplasts, suggesting their capacity for autotrophic growth dependent on exogenous nutrients to sustain protocorm development. The symbiotic germination technique is a resource-saving and highly efficient propagation method, making it a promising ex situ conservation strategy. This study presents a technical protocol for screening seed germination-promoting fungi and establishing a symbiotic germination system, offering a feasible solution for the conservation of rare and endangered orchid species.