INDUCTION OF SPORE GERMINATION IN SCHIZAEA PUSILLA (SCHIZAEACEAE)

Requirements for spore germination in the rare and native New Jersey fern, Schizaea pusilla Pursh., were examined. Spores did not germinate in darkness and gibberellins (GA) did not induce germination in the dark. However, a dark pretreatment promoted germination in a subsequent light treatment and low temperatures during the dark pretreatment greatly enhanced germination in culture. Three wks of dark pretreatment were required for maximum germination. GA3 promoted germination in red light more effectively than GA4+7. Greater than ten days of continuous illumination was necessary for germination. Spores given red light reached half-maximum germination six days earlier than spores under white light. Red light promoted germination while blue light did not. Far-red light alone could stimulate germination and enhanced the promotive effect of red light; typical phytochrome photoreversibility was not observed. Blue light reduced the effect of red light. THE CURLY GRASS fern, Schizaeapusilla Pursh., is a rare bog-living leptosporangiate fern (Bierhorst, 1971). The anatomy and morphology of S. pusilla have been described (Britton and Taylor, 1901) but physiological processes involved in the development, growth and maintenance of this fern remain unstudied. A recent paper by von Aderkas and Raghavan (1985) repeated Britton and Taylor's (1901) morphological examination of spore germination and young gametophyte development in S. pusilla. Most fern species do not germinate in the dark (Miller, 1968). However, white light completely inhibits the germination of Botrychium dissectum spores (Whittier, 1973a, 1981) and even more extreme are Psilotum nudum spores which germinate only after six months in darkness (Whittier, 1973b). The spores of Anemia and Lygodium, thought to be close relatives of Schizaea, germinate in the dark, but only in the presence of gibberellic acid (GA3) (Naf, 1966; Weinberg and Voeller, 1969; Raghavan, 1976, 1977). The role of darkness and GA in spore germination is unstudied in Schizaea. Fern spore germination is promoted by red light in most fern species (Hartt, 1925; Mohr, 1963; Sugai, Takeno and Furuya, 1977; Furuya, Kadota and Uematsu-Kaneda, 1982; Huckaby, Kalantari and Miller, 1982; Sugai et al., 1984) and delayed by blue and far-red light (Sugai and Furuya, 1967, 1968; Sugai et al., 1977; Tomizawa, Sugai and Manabe, 1983). ' Received for publication 14 November 1985; revision accepted 23 April 1986. The support of DNA Plant Technology Corp., the Rutgers Research Council and Bureau of Biological Research, and Sigma Xi is gratefully acknowledged. Of course there are exceptions; for example, Onoclea sensibilis spores could be induced by far-red or blue light, but at greater fluence than red light (Towill and Ikuma, 1973). Various pigments play a major role in the germination of fern spores. These may include phytochrome and a blue light-absorbing pigment. To date, little is known about the photoregulation of spore germination in S. pusilla. The present study was designed to begin to fill these voids. MATERIALS AND METHODS-Spore collection and storage-Sporophylls of Schizaea pusilla Pursh. were collected from Webb's Mill bog, in the New Jersey pine barrens, in September 1983, 1984 and 1985. Sporangia dehisced in glass Petri dishes at room temperature (22 + 2 C) or at 4 ? 1 C. Some of the spores were immediately cultured (8-48 hr after dehiscence = "fresh spores") while the rest were stored dry in darkness at 4 C ("stored spores") for up to several months prior to culture. Light microscopy-To examine the early events (imbibition and cell division) of spore germination, dry and cultured spores were examined daily with a Nikon Optiphot fluorescence microscope and Nomarski optics. Spores were mounted in several drops of 1% (w/v) 4,6-diamidino-2-phenylindole and 1-5% (w/ v) dimethyl sulfoxide. Cell division was observed by counting the number of blue-fluorescing nuclei. The production of chlorophyll was monitored by counting the number of redfluorescing plastids. The spores were also stained with Sudan IV and I2KI to observe the presence of lipids and starch.