Cryopreservation of Bryophytes: The Effects of Abscisic Acid and Encapsulation Dehydration

Abstract

Three species of liverwort and one species of moss were tested for their ability to tolerate desiccation and subsequent liquid nitrogen (LN) exposure. Riccia fluitans and Helicodontium cappelare were sensitive to desiccation and required either abscisic acid (ABA) or encapsulation in alginate beads with a 0.75 M sucrose pretreatment for 100% survival through drying and LN exposure. There was little effect ofABA on Plagiochila sp. although drying and LN exposure were successful with encapsulation. With Marchantia polymorpha optimal survival through drying and LN exposure required both ABA and encapsulation. These results suggest that encapsulation cryopreservation, in some cases in combination with ABA preculture, might be used as a method for long-term germplasm storage for desiccation sensitive bryophytes. With the decline in the area of natural plant habitats, ex situ preservation can be used as a supplemental tool for maintaining plant germplasm, offering insurance against the possibility of loss of organisms from the wild. While most ex situ preservation programs emphasize higher plants, there is a concurrent need to preserve the germplasm of nonseed plants, including bryophytes (Soderstrom et al. 1992). In the case of hepatics and mosses, there are a number of species which are considered endangered (Geissler et al. 1997; Tan et al. 1994a,b). The fact that biochemicals with antibiotic, neurotropic, insect antifeedant, and antitumor activity have been isolated from various species adds further weight to the argument for maintaining and exploring bryophyte germplasm (Asakawa et al. 1980; Fukuyama & Asakawa 1991; Joshi & Desai 1989; Spjut et al. 1992). Cryopreservation offers the potential for longterm maintenance of living tissues. Techniques for the preservation of cells, shoot tips, embryos, and other tissues in liquid nitrogen (LN) extend the concept of germplasm banking beyond the traditional banking of seeds and spores. Such ex situ preservation of rare plant germplasm can provide a resource for research as well as for reintroduction if a species is lost in the wild. In cases where tissues can survive desiccation, direct exposure to LN can be used successfully, for example, as with many seeds (Pence 1990; Stanwood 1985). If the tissues are sensitive to desiccation, other protective measures are needed, and a variety of techniques have been developed for cryopreserving plant tissues (Bajaj 1991). The following experiments were undertaken to examine the applicability of cryopreservation to the gametophytes of four in vitro grown bryophytes three liverwort species and one tropical moss species. Open drying and encapsulation dehydration were compared as protective measures, and the effect of preculture with abscisic acid (ABA) that has been implicated in conveying desiccation tolerance in other systems (Werner et al. 1991), was also ex-