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Orchids are among the most widely distributed plants but also the most threatened by biotic and abiotic factors. Their interactions with specific symbiotic fungi during germination in natural environments make their propagation challenging for conservation biologists. In this study, in the framework of the European conservation project LIFEorchids, the germination protocol for the endangered Mediterranean species Orchis patens was optimised, and the effect of controlled constant temperature in growth chambers with fluctuations of temperature occurring in natural environments was compared. Seed viability was also evaluated by using a double-staining technique. Minimum, maximum and average daily temperatures were recorded for six months, from July to December, and germination percentages, as well as developmental stages of seeds, were annotated, for both conditions, every month. Even if germination was obtained under both conditions, the effect of temperature fluctuations was evident, by increasing the germination percentage of seeds from about 10% under controlled conditions to 59%. The latter result was almost comparable with seed viability data, indicating a strong impact of temperature fluctuations in breaking seed dormancy. Our results are a confirmation that temperature oscillations play a more important role than the average temperature in seed germination and suggest avoiding a constant temperature to optimise germination protocols for European orchid species. more...