Cryopreservation of in vitro Grown Fern Gametophytes

Although the more prominent sporophyte and its spores are frequently the focus of study, the small, fragile thallus of the fern gametophyte has also been the subject of much research (Stokey, 1940; Miller, 1968; Dyer, 1979).Fern ametophytes are easily grown in culture, making them good candidates for physiological and developmental studies, but maintaining stock lines or lines from a number of species requires a consistent input of time and labor.Cryopreservation, or storage in liquid nitrogen (LN) at -196°C, has been usedto preserve a variety of living tissues of both vascular and nonvascular plants.Seeds, shoot tips, cell cultures, callus, protoplasts, pollen, and embryos of seed plants (Kartha and Engelmann, 1994;Stanwood, 1985; Pence, 1991), as well as fern spores and the gametophytes of bryophytes (Christianson, 1998; Pence, 1998; and Pence, submitted) have all been successfully maintained in LN.In this study, the possibility of using cryopreservation to preserve fern gametophytic tissue was explored.Two protocols, which have been used successfully with a variety of other tissues, were tested for preparing gametophyte tissue for cryopreservation: open drying of the tissues before LN exposure and the encapsulation dehydration procedure of Fabre and Dereuddre (1990). MATERIALS AND METHODS Gametophytecultures were initiated from spores of Davallia fejeensis Hook., Drynaria quercifolia (L.) John Sm., Cibotium glaucum (Sm.)Hook.& Arn., Adiantum trapeziforme L., Adiantum tenerum Swartz., and Polypodium aureum L. collected from fronds supplied by Mr. Jeff Kapella, Supervising Florist of the Krohn Conservatory (Cincinnati).For surface sterilization, spores were wrapped in small packages made by Pence, Valerie C .2000."Cryopreservation of in vitro Grown Fern