Cryopreservation of in vitro grown fern gametophytes.

-TWO methods of protecting fern gametophyte tissues through exposure to liquid nitrogen (LN) were examined. In vitro grown gametophytic tissues from six fern species were exposed to LN after open drying or after encapsulation dehydration, with and without preculture on abscisic acid (ABA). Open drying itself decreased survival with little further effect from LN exposure, although survival was somewhat improved by preculture on ABA. In contrast, encapsulated tissues survived drying and LN exposure at rates comparable to controls (86-100%) irrespective of ABA preculture. Sucrose pretreatment of the encapsulated tissues was important for their subsequent survival through these procedures. Tissues prepared by encapsulation dehydration were successfully regrown after 3.5 years in LN storage. Thus, cryopreservation appears to be a technique which could be used for the stable preservation of in vitro cultures of fern gametophytes and for the long-term storage of rare or endangered germplasm of ferns. 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 gametophytes 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 used to 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 Gametophyte cultures were initiated from spores of Davallia fejeensis Hook. , Drynaria quercifolia (L.) John Sm. , Cibotium glaucum (Sm.) Hook. & Am. , 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 This content downloaded from 40.77. 167.24 on Mon, 19 Sep 2016 05:57:22 UTC All use subject to http://about.jstor. org/terms PENCE: CRYOPRESERVATION OF IN VITRO GROWN FERN GAMETOPHYTES folding pieces of Whatman No. 1 filter paper. The spores and packages were immersed in a 1:20 dilution of commercial sodium hypochlorite for 5 min, followed by two rinses in sterile distilled water. The packages were then opened, and the spores were blotted onto sterile germination medium, consisting of half-strength Linsmaier and Skoog (LS) (1965) salts and organics, with 1.5% sucrose and 0.22% Phytagel (Sigma Chemical Co.), in 60 x 15 mm disposable plastic petri dishes, approximately 15 ml/dish. The spores were incubated at 26? C under CoolWhite fluorescent lights in a 16/8 hr light/dark cycle. Once germination occurred and gametophytes were formed, the cultures were maintained by subculturing the tissue every 2-3 months onto fresh medium. In some experiments, gametophytes were precultured for one week on this same medium, with and without 10 ,LM abscisic acid (ABA), which was added to the medium after autoclaving. For open drying, tissues were cut into pieces, approximately 2-5 mm long, blotted onto sterile filter paper to remove excess moisture and placed in a sterile petri dish under the air flow of the laminar flow hood for 3 hrs. For encapsulation dehydration, the method of Fabre and Dereuddre (1990) was followed. Tissues were cut into small pieces, approximately 2-3 mm long, and transferred to a solution of 3% alginic acid in calcium-free MS medium plus 0.75 M sucrose. This solution, containing one or more pieces of gametophyte tissue, was then pipeted dropwise into a solution of 100 mM CaCl2, which caused the alginic acid to gel, encasing the tissue in an alginate bead. After 20 min, the beads were removed from the calcium solution and transferred to liquid MS medium containing 0.75 M sucrose, 25 ml in 125 ml flasks, and placed onto a gyratory shaker, 125 rpm, for 18 hr as a pretreatment. In one experiment, different concentrations of sucrose, ranging from 0-30% were tested in the pretreatment step. The pretreated beads were then blotted on sterile filter paper to remove excess moisture and placed on dry filter paper in sterile petri dishes under the air flow of the laminar flow hood to dry for 3-4 hours. Open dried tissues and dry encapsulated tissues were then placed into sterile 2 ml polypropylene cryovials and immersed directly into LN where they were left either for 1 hr or overnight (no difference was observed between these two LN exposure times). Tissues were thawed by placing the cryovials on the benchtop at ambient temperature for 20 min, after which the tissues or beads were removed and placed onto growth medium for rehydration and recovery. Survival was measured as the recovery of growth from each tissue piece for open dried tissue or the number of beads containing tissues resuming growth. As controls, some tissues were transferred to recovery medium after drying but without LN exposure. With the encapsulation dehydration procedure, tissues which had been pretreated for 18 hours in . 75 M sucrose but which had not been dried were cultured as an additional control. Fern gametophyte tissue from each species was also prepared by the encapsulation dehydration method for long-term cryostorage and banked in LN. After 3.5 years, samples of each were removed from storage and placed onto medium for rehydration and recovery growth. 17 This content downloaded from 40.77. 167.24 on Mon, 19 Sep 2016 05:57:22 UTC All use subject to http://about.jstor. org/terms AMERICAN FERN JOURNAL: VOLUME 90 NUMBER 1 (2000)