RESEARCH TO DEVELOP STRATEGIES AND TECHNOLOGIES FOR PRESERVING PLANT GENETIC DIVERSITY IN EX SITU GENEBANKS
Location: Plant Germplasm Preservation Research Unit
Title: Exploration of cryo-methods to preserve fern gametophytes
Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: January 1, 2010
Publication Date: January 1, 2011
Citation: Mikula, A., Makowski, D., Walters, C.T., Rybczynski, J.J. 2010. Exploration of cryo-methods to preserve fern gametophytes. pp. 173-192. In Fernandez, H, Kumar A, Revilla, MA (eds) Working with ferns: issues and applications. Springer, New York, NY.
Interpretive Summary: Problem: Very little is known about the biology of fern gametophytes, yet they appear to be ideal systems to study totipotency, organogeneis and mass regeneration of germplasm. Developing methods to cryopreserve this germplasm offers opportunity to explore the range of tissues that can be applied to cryopreservation techniques as well as to develop systems to study recovery mechanisms. Accomplishment: The paper describes survival following cryoprotection and cryoexposure of tree fern gametophytes. It demonstrates that preculture in ABA, encapsulation of prothalia in alginate beads, osmotic dehydration with concentrated sucrose and then air drying give excellent survival. Moreover, we observed a unique ability of the fern gametophyte to regenerate from a small number of cells without callus formation using cryoprotection procedures that were less effective. Impact: Cryopreservation capacity at NCGRP has now expanded beyond seed and vegetative material of angiosperms. We are now adept at preserving germplasm from very diverse plant materials and are developing model systems to study organogenesis during recovery of severe stress.
Fern gametophytes, derived from in vitro cultures, are a useful source of germplasm for scientific research using genetic stocks, mass production of individuals for plantings, ex situ conservation of endangered populations and reproduction of species or hybrids that produce short-lived or nonfunctional spores. Cryopreservation of gametophytic tissue is fairly routine 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 an herbaceous species, Phyllitis scolopendrium, that originates 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. Encapsulating prothalia in alginate beads protected gametophytes during chemical desiccation with vitrification solutions, allowing about 50% survival of gametophytes in Dicksonia fibrosa. The encapsulation/dehydration technique consistently gave high survival and rapid recovery in cryo-exposed gametophytes, though this technique was more complex and labor intensive than the other two. Preculture on agar for up to 2 weeks with 0.25 M sucrose and 10 µM ABA enhanced survival or recovery rate and greater than 80% survival was achieved for seven of the eight species used in the study. Cryopreservability of tree fern gametophytes may be related to adaptions for longevity and desiccation tolerance in their natural habitats and near totipotency of cells in the meristem.