| Summary: | A greater understanding of the relationship between native orchids and their mycorrhizal symbionts is needed to ensure more effective orchid conservation strategies. A protocol for symbiotic seed germination and seedling development was developed for <i>E. geminiflorum.</i> Mature seeds were collected from a naturally occurring orchid population in Ecuador. Putative mycorrhizal fungi isolated from other native orchid species were used to screen their ability to facilitate germination and seedling development in vitro in either a 0/24 h or 12/12 h light/dark photoperiod at 20 °C. The mycorrhizal fungus <i>Tulasnella calospora</i> (UAMH 9824) isolated from <i>Spiranthes brevilabris</i> in Florida, USA, was also included in this study. Sterilization treatments using 0.3%, 0.5% sodium hypochlorite/ethanol or 2% calcium hypochlorite were tested for their effectiveness as sterilant and their subsequent effects on seed germination percentage. Effective surface seed sterilization was achieved with either 0.5% NaClO/ethanol or 2% calcium hypochlorite. However, significantly higher percentages of germinated embryos developed into protocorms when NaOCl solutions were used compared to the other treatments. Seed germination occurred in both photoperiods tested; however, delayed germination was observed under complete darkness. Seeds of <i>E. geminiflorum</i> germinated without fungal inoculation; however, co-culture with <i>Tulasnella</i> strains improved germination significantly. Seedling development was only observed when seeds were cultured in asymbiotic medium or co-cultured with <i>T. caloscopa</i> (UAMH 9824). Significantly longer seedlings were obtained when <i>T. calospora</i> was present in the culture compared with seedlings cultured in asymbiotic medium. The establishment of mycorrhizal associations was confirmed by the presence of pelotons in the roots of <i>E. geminiflorum</i> seedlings.
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