Survival of Chlorophyllous and Nonchlorophyllous Fern Spores through Exposure to Liquid Nitrogen

Abstract

-Thirty-three species of ferns with nonchlorophyllous spores and five species with chlorophyllous spores were studied in regard to their ability to survive exposure to liquid nitrogen (LN). Air dried spores showed no inhibition of germination after LN exposure when planted on soil or growth medium. Spores of three species that were stored for 75 months either at 4?C, 20?C, or in LN showed no decrease in viability over that time, and spores of four other species were maintained successfully for 52 months in LN. Fresh chlorophyllous spores that were air dried, dried over silica gel, or prepared with the encapsulation dehydration procedure also showed good survival through desiccation and LN exposure. Spores of Osmunda regalis germinated well after 18 months of LN storage. These results indicate that both nonchlorophyllous and chlorophyllous spores are candidates for long-term germplasm storage at low temperatures, including storage in LN. Ex situ preservation of spores, particularly of rare species from threatened habitats, can be an important supplement to the maintenance of pteridophyte species in the wild. Many species of homosporous ferns produce nonchlorphyllous spores that can be stored for relatively long periods of time, although they can eventually lose viability in storage (Beri and Bir, 1993; Camloh, 1999). It has also been shown that hydrated spores of some of these species can remain viable in the soil spore bank for a number of years, and moist storage of spores ex situ has also been effective (Lindsay et al., 1992; Dyer, 1994). Other species of ferns and fern allies produce chlorophyllous spores, which generally have limited viability, even though they may survive desiccation (Lloyd and Klekowski, 1970). Low temperature storage has been shown to improve the longevity of dry seeds (Dickie et al., 1990), and lower temperatures improved the survival of spores of the tree fern, Cyathea delgadii during two years of storage (Simabukuro et al., 1998). Cryopreservation, or storage at -196?C in liquid nitrogen (LN), has the potential for maintaining viability in living tissues over long periods of time. Many seeds can survive direct exposure to LN (Stanwood, 1985; Pence, 1991), and spores of the endangered tree fern, Cyathea spinulosa have been successfully germinated after cryopreservation (Agrawal et al., 1993). In order to examine the possibility of extending cryopreservation to other species of ferns, spores of several nonendangered chlorophyllous and nonchlorophyllous species were tested for their ability to withstand LN exposure, and storage in LN was compared with other low temperature storage methods. Results from tests with these common species should provide direction for This content downloaded from 157.55.39.104 on Sun, 19 Jun 2016 05:51:11 UTC All use subject to http://about.jstor.org/terms AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 4 (2000) future work with rare or endangered germplasm, which is often in limited supply. MATERIALS AND METHODS Spores were obtained from a variety of sources, including from the American Fern Society Spore Exchange and from fronds collected in northwest Trinidad, at the Krohn Conservatory (Cincinnati) and at the Cincinnati Zoo and Botanical Garden. When fronds were collected, they were air dried for several days under ambient conditions in the laboratory in paper envelopes, with spores collected from the envelopes and by scraping the fronds. Samples of the spores were then transferred to 2 ml polypropylene cryovials and immersed directly into LN. After 1 hr, the vials were removed and placed on the benchtop to warm at ambient temperature for 20 min. LN exposed and control nonchlorophyllous spores of 33 species were tested for germination using one of the two following methods. 1) Spores of some species were sown on moist sterile soil (Metro Mix 250) in Magenta boxes or baby food jars with Magenta caps, and incubated at 26?C under CoolWhite fluorescent lights in a 16/8 hr light/dark cycle. 2) Spores of other species were germinated in vitro. Spores were wrapped in a small package made from Whatman No. 1 filter paper, and surface sterilized in a 1:20 dilution of commercial sodium hypochlorite for 5 minutes, followed by two rinses in sterile, ultra-pure water. The spores were then blotted onto medium consisting of half-strength Linsmaier and Skoog (1965) (LS) medium with 1.5% sucrose and 0.22% Phytagel (Sigma Chemical Co.), in 60 x 15 mm disposable petri dishes, approximately 15 ml/dish. For longer storage, nonchlorophyllous spores of three species were placed in multiple cryovials and stored at three temperatures: LN (-196?C), -20?C, and 4?C. The cryovials stored at -20?C and 4?C were placed inside 20 ml borosilicate scintillation vials with plastic screw caps, containing 2-2.3 g of silica gel. Spores from the three storage temperatures were sampled at 7, 13, 34, and 75 months by germinating on soil, as described above. Spores from four species collected in Trinidad were also cryostored in LN. Chlorophyllous spores of five species were also tested. These were obtained either from the Spore Exchange or from plants growing at the Cincinnati Zoo and Botanical Garden. In most cases these were air dried, as for the nonchlorophyllous spores. However, Osmunda regalis spores which were collected from plants grown at the Cincinnati Zoo and Botanical Garden were tested using two other procedures. One portion of the spores was dried in a desiccator with silica gel overnight, placed in a cryovial, and frozen and thawed as described above. The spores were then placed in a 100% humidity chamber for 3 hours, and surface sterilized and germinated in vitro as described above. A second portion of the spores was first surface sterilized and then encapsulated in alginate according to the procedure of Fabre and Dereuddre (1990). The spores were 120 This content downloaded from 157.55.39.104 on Sun, 19 Jun 2016 05:51:11 UTC All use subject to http://about.jstor.org/terms PENCE: SURVIVAL OF FERN SPORES blotted into a 3% solution of low viscosity alginic acid (Sigma Chemical Co.) and the spore suspension was added dropwise to a 100 mM solution of calcium chloride, where the drops gelled and formed alginate beads containing the spores. After 20 min the beads were transferred to a solution of LS medium containing .75M sucrose and incubated on a gyratoray shaker, 125 rpm, for 18 hours. They were then dried on filter paper under the air flow of a laminar flow hood for 4 hours, transferred to 2 ml polypropylene cryovials, immersed directly into LN, and maintained there for 1 hour. The beads with spores were warmed at room temperature for 20 minutes and then transferred to halfstrength LS medium for rehydration and growth. Some dried beads were also put into long-term LN storage, and a sample was removed after 18 months for growth and evaluation. Only a qualitative evaluation of spore germination was made, both on soil and in vitro, although an attempt was made to maintain approximately equal amounts of control and LN exposed spores. Positive spore germination was recorded if any germination was observed. With the nonchlorophyllous spores, no great differences in the rate of germination were observed between control and frozen spores of any species, as determined by gross observation. With chlorophyllous spores, a distinction was made between the germination of many spores and the germination of one or only a few spores, as indicated in Table 3.