A simple way to overcome the recalcitrance of the water fern Ceratopteris thalictroides (L.) Brongn. to cryopreservation

Ceratopteris thalictroides is a water fern very sensitive to both dehydration and low temperature. This study focuses on the cryopreservation of this species by encapsulation-dehydration technique, in particular on the effects of pre-culture step, alginate bead size and the physical conditions of culture on the cryopreservation efficiency. Encapsulated and non-pre-cultured gametophytes did not survive cooling with liquid nitrogen. When cryopreservation was preceded by a 2-week period of pre-culture, regrowth reached 42.1%. Reduction in the size of the alginate bead, and culture in total darkness resulted in improved gametophyte regrowth capacity (75.5% or 81.7%, respectively). The best results (91.3%) were obtained when all factors tested occurred simultaneously. The gametophytes recovered very quickly and sporophytes were formed within 4 weeks after rewarming. These simple improvements can be used, not only for the cryopreservation of gametophytes in cryptogams but also for some recalcitrant species of seed plants.