Long-term storage of mycorrhizal fungi and seed as a tool for the conservation of endangered Western Australian terrestrial orchids

The impact of seed drying, seed storage and development of testing procedures for seed viability assessment was undertaken for a selection of common taxa with congeners that are rare and endangered (Caladenia, Diuris, Pterostylis and Thelymitra). Freshly collected seed showed significantly lower levels of germination compared with seed that had been subjected to drying over silica gel for 24 h. Seed dried over silica gel for 24 h and plunged into liquid nitrogen exhibited a further increase in germination levels. Germination of seed stored at 4, 18 or 22 degreesC for 1 year was substantially higher than freshly collected seed (4 weeks after dehiscence), but germination was highest overall after storage of dried seed in liquid nitrogen (-196 degreesC). Mycorrhizal fungi that promote the germination and growth of plants were also successfully preserved in liquid nitrogen. The use of cryoprotectants on fungal isolates had no observable deleterious effects on fungal regeneration. Histochemical staining procedures (tetrazolium, fluorescein diacetate and Evans blue) substantially overestimated seed viability, relative to symbiotic seed germination, for most seed treatments indicating a need for re-evaluation of the effectiveness of staining procedures for testing viability. The implications of the long-term ex situ storage of orchid seed and fungal symbionts for the conservation of endangered orchids is discussed.