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The rare aquatic liverwort Riella helicophylla (Bory et Mont.) Mont., inhabitant of temporary shallow ponds around the Mediterranean basin, is considered threatened throughout its distribution range. In addition, little is known of its biology and ecology or of its role in such an important ecosystem where environmental conditions vary yearly in unpredictable ways. In these variable habitats, due to the seasonal fluctuation of water levels, there is no guarantee of yearly spore input into the spore bank. Spore germination rate and the effects of different culture media in an axenic culture establishment, as well as propagation procedures of R. helicophylla, were tested. New insights into the ecology and biology of R. helicophylla are given. Spore dormancy is documented, and the protocols for the in vitro culture establishment, propagation and acclimatization of this liverwort are developed. Dry storage at 20 ± 2 °C for about three months broke the dormancy of spores, which subsequently germinated in a high percentage (over 90%). A two phase (solid and liquid) culture media system was developed for the purpose of achieving fully developed gametophytes. The liquid phase contained electrolytes simulating brackish water.