Exploration of cryo-methods to preserve tree and herbaceous fern gametophytes

Abstract

Fern gametophytes, derived from in vitro cultures, are a useful source of germplasm for ex situ conservation of endangered populations, scientific research using genetic stocks, mass production of ferns for plantings, and reproduction of species or hybrids that produce short-lived or infertile spores. Cryopreservation of gametophytic tissue is fairly established for mosses and liverworts, but is rare for ferns. The aim of this study was to develop methods for cryostorage of gametophytes from seven species of tree ferns originating from either the tropics or areas of mild winters and to compare these species' amenability to cryopreservation with two herbaceous species that originate in areas where winters are harsher. Efficacy of three cryoprotection methods - vitrification, encapsulation/vitrification, and encapsulation/dehydration - was compared in terms of overall survival, time to sexual maturity (recovery rate), and preparation time to acquire tissues tolerant of cryoexposure. The standard vitrification procedure using PVS2 and PVS3 was ineffective and damaged the naked fern prothalia even without cryoexposure. Encapsulated in alginate beads prothalia during chemical desiccation with vitrification solutions allowing about 50% survival of gametophytes. The encapsulation/dehydration technique consistently gave high survival and rapid recovery in cryoexposed gametophytes. Two-week-long agar preculture with 0.25 M sucrose and 10 μM ABA enhanced survival or recovery rate and greater than 80% survival was achieved for eight of the nine species used in the study. Cryopreservability of fern gametophytes may be related to adaptions for longevity and desiccation tolerance in their natural habitats and totipotency of cells in the meristem.