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BackgroundThe technique of symbiotic germination—using mycorrhizal fungi to propagate orchids from seed in vitro—has been used as one method to cultivate orchids in North America and abroad for > 30 years. A long-held assumption is that mycorrhizal fungi used for this purpose lose their effectiveness at germinating seeds over time with repeated subculturing.ResultsWe provide evidence for the lingering efficacy of one particular strain of Tulasnella calospora (266; UAMH 9824) to stimulate seed germination exemplified by the North American terrestrial orchid, Spiranthes cernua, as a case study. This fungus was originally acquired from roots from Spiranthes brevilabris in 1999 and sub-cultured during the two decades since. Seeds inoculated with the fungus in vitro developed to an advanced protocorm stage after 16 days, and leaf elongation was pronounced after 42 days. In a pilot study, seedlings co-cultured with Tulasnella calospora 266 were deflasked after 331 days and later transferred to soil under greenhouse conditions where they eventually initiated anthesis. During the course of two decades, seeds of 39 orchid species, cultivars and hybrids spanning 21 genera, germinated in vitro co-cultured with Tulasnella calospora 266. These orchids included temperate terrestrials and tropical epiphytes alike.ConclusionsThe sustained effectiveness of this fungus is noteworthy because it argues against the concept of mycorrhizal fungi losing their symbiotic capability through prolonged subculturing. This study serves as an example of why in situ habitat preservation is essential for the conservation of orchids as a source of potentially useful mycorrhizal fungi.

Background Seeds of temperate terrestrial (hardy) orchids are considered more difficult to germinate compared to their tropical epiphytic counterparts, presumably because they have higher levels of abscisic acid (ABA) in their seed coats which prevents seeds from germinating prematurely during winter dormancy. In nature, ABA is gradually broken down (stripped) by natural weathering, triggering germination. This process can be shortened artificially, however, by using chemical bleaching agents and cold-moist stratification with mixed results. In this study, we explored the use of fluridoneto break seed dormancy in a hardy orchid native to North America, Platanthera leucophaea (Nutt.) Lindl. This organic compound (IUPAC name: 1-methyl-3-phenyl-5-[3-(trifluoromethyl) phenyl] pyridin-4(1 H )-one) is a commercial herbicide that inhibits ABA biosynthesis. We added fluridone directly to agar media prior to seed sowing in vitro. Both symbiotic and asymbiotic germination techniques were applied that involved two different agar media, with and without added fluridone. Symbiotic germination was carried out using standard oatmeal agar inoculated with a mycorrhizal fungus ( Ceratobasidium ), whereas asymbiotic treatments utilized P723 agar medium. Results Seedling development within some of the replicate plates progressed to Stage 3 in all treatments, but development was marked in all asymbiotic plates containing fluridone leading to leaf elongation, 385 days after sowing. Conclusions As an herbicide, fluridone’s use as a media additive to propagate a rare photosynthetic orchid seems counterintuitive, but its use in vitro to stimulate seedling development has the potential to benefit conservation efforts for this and possibly other hardy orchid species.