1 SPORE GERMINATION, GAMETOPHYTE MORPHOLOGY AND GAMETANGIA DEVELOPMENT IN A TROPICAL FERN, Cyclosorus striatus (Schum.) Ching (THELYPTERIDACEAE). Oloyede¹* F. A., Adu¹ B. and Akomolafe¹ F. G. ¹Department of Botany, Obafemi Awolowo University, Ile Ife, Osun State, Nigeria. * Corresponding author: E-mail: funsoji@oauife.edu.ng ABSTRACT The suitability of the fresh spores of Cyclosorus striatus (Schum.) Ching for propagation was studied to determine the ability of early germination of the spores, development of prothalli, gametangia and gametophytes on artificial medium. Fresh matured spores used for propagation were obtained from the matured sori of C. striatus fertile fronds collected from a stream at the Obafemi Awolowo University, Ile-Ife. The spores were cultured inside the Petri dishes labeled B, C, D, E containing modified Moore’s medium and Petri dish A containing sterilized distilled water as the control experiment. The cultures were incubated at 23 ± 2°C with cool fluorescent white light intensity, ca 1000 lux, 16 hr/d. The experiment was replicated twice. Spore germination was observed on day 10 while rapid rate of germination was observed on days 17, 24 and 31. The mean percentages of germination; 79%, 61%, 81%, and 65% were recorded in the Petri dishes B, C, D and E respectively, while no germination was observed in the control experiment (Petri dish A). Both antheridia and archegonia were uniserate while prothalli were slender, elongated and filamentous with cross walls but gametophytes were cordate shaped with rhizoids. This shows that the spores can be used propagation of the tropical fern. Keywords: Cyclosorus striatus, gametophyte, spores germination, prothallus. INTRODUCTION Cyclosorus striatus (Schum.) Ching is one of the living members of the Family Thelypteridaceae (Agnew, 1974) of the Order Filicales and Class Pteropsida (Sporne, 1975). In Nigeria, C. striatus is widely distributed in the Southwestern, humid ecological locations (Oloyede, 2008). The continuity and distribution of the species are enhanced by the massive spore production and 2 dispersal. Spore production is affected by several environmental factors, such as temperature, humidity and canopy cover (Odland, 1998; Greer and McCarthy, 2000; Arens, 2001). Spores generally show remarkable variations and are useful in systematic studies of ferns but in most cases, spore morphology is distinct at family, generic or species level (Taylor and Mickel, 1974). Germination of fern spores, growth and further development of the resulting gametophytes in artificial media is a well-studied area in pteridophytes and developmental biological researches (e.g Ganguly, et al., 2009; Hua, et al., 2009; Galan and Prada, 2010). Spores are widely used as starting materials for tissue culture technique in fern propagation (Hua, et al., 2009). Factors influencing sori formation in leptosporangiate ferns include: genetic factors, age of the plant and developmental stages of the frond (Opapeju, 1983); seasonal weather conditions and various environmental factors such as shade and exposure to sunlight (Ramsay, 1979; Oriola, 2007). Powell (1976) reported that in New Britain, a poultice of boiled fronds of C. striatus is used to treat sores and cold while the stems are used for lashings on houses in Papua New Guinea. Sori of C. striatus are arranged on the abaxial surface of the pinnule, each sorus containing few sporangia covered with indusia. Each sporangium contains a mass of small sized, light numerous spores. The objective of this work was to study the ability of the spores of C. striatus to germinate and produce prothalli, gametangia and gametophytes within few months using artificial growth medium. MATERIALS AND METHODS Mature fertile fronds of Cyclosorus striatus collected from a stream at Obafemi Awolowo University, Ile–Ife, were taken to the IFE Herbarium of the University for identification. The fronds were wrapped in paper envelopes and dried at room temperature for two weeks purposely for the sori to burst open and release the spores. The spores were stored inside 3 sterilized 15 ml centrifuge tubes at 4°C for two weeks. The Petri dishes were labeled A, B, C, D & E. The spores were surface sterilized in 0.1% mercuric chloride (w/v) solution for 5 to 8 minutes and rinsed three times with sterilized distilled water using sterilized filter paper. The spores were air dried inside micro flow chamber to avoid contamination. The Petri dishes containing agar were autoclaved at 121°C, 15 Ib/inch² pressure for 15 minutes before planting the spores to avoid contamination. The sterilized spores were sown in each of the Petri dishes containing modified Moore’s medium (Kato, 1969) solidified by 1% (w/v) agar maintained at 5.8 pH. 4 ml sterilized, deionized, distilled water was added to the Petri dish A, serving as the control experiment. 4 ml of modified Moore’s medium was added to each of the remaining four Petri dishes B, C, D and E containing the spores inside a micro flow chamber. The cultures were maintained at 23 ± 2°C and ca 1000 lux, light intensity16 hr/d in two replicates. Fresh nutrient was supplied every other day and spore germination was monitored with dissecting microscope inside the micro flow chamber. All the cultures were viewed after 10, 17, 21 and 31 days for spore germination and then daily for 16 weeks for prothalli, germetangia and gametophyte growth and development. The mean percentage germination of the spores was calculated for each of the Petri dishes after viewing and counting have been repeated ten times. RESULTS Germination of the spores started within ten days from sowing the spores in the Petri dishes B, C, D and E except Petri dish A, where there was no germination at all. The initial sign of germination was the changing of brownish colouration of the spores to green; spore wall broke creating a small opening (Fig. 1 B) for emergence of prothallial cells. Thereafter, there was rapid rate of germination of the spores in all the Petri dishes except A. The mean percentages of germination recorded for the Petri dishes B, C, D and E respectively from day ten through day 31 were 79%, 61%, 81% and 65% respectively. There was maximum germination of the spores as 4 almost all the spores germinated in all the Petri dishes by the end of the fourth week. Table 1 shows the mean percentage germination from each of the Petri dishes. The spores germinated to form slender, uniseriate, elongated, filaments with cells of e rhizoids developing first followed by the greenish protallial cells (Fig. 1 E (r)). This is Vittaria-type of spore germination. Rhizoids and hairs appeared after eight days; prothalli ten days; antheridia two weeks, archegonia three weeks while bisexual gametophytes appeared four weeks after sowing the spores. The first sets of gametophytes were hair-like, small sized and dark coloured. Long, slender, hair developed from the polar region of each spore. The hairs had elongated cells with slanting edges but the antheridia cells were smaller in size and more in number than the archegonia cells which were bigger and less in number (Fig. 1 F, G, H). The gametophytes were cordate in shape, possessed 12-14 rhizoids (Fig. 1 I r), and were bisexual for possessing both antheridia and archegonia cells together. This type of gametophyte growth and development is called Drynaria type. DISCUSSION C. striatus has mixed sori, hence maturity and dispersal of the spores take place at different stages of development. Thus the formation of sori is a gradual and progressive process in younger fronds. The maximum germination of the spores took place after 14 days because there were observable differences and increases in the mean percentage germination of the spores from day 10 through day 31(Table 1). The spore germination was of the Vittaria-type (Nayar and Kaur, 1968) while the gametophyte growth and development where rhizoids appeared first is Drynaria type. The Drynaria type of gametophyte development is a characteristic of Thelypteridaceae and Polypodiaceae (Nayar and Kaur, 1971). 5 6 Table 1: The mean percentage germination of the spores of Cyclosorus striatus. Mean Percentage germination (%) Petri dish Day 10 DAY 17 Day 24 A NGD NGD NGD B 50 80 C 30 D E Day 31 Mean NGD NGD 90 96 79 50 80 83 61 60 90 90 92 81 40 60 80 81 65 NGD: No germination observed 7 This mode of spore germination in which cells of rhizoids first developed is the most common way of spore germination in homosporous leptosporangiate ferns (Nayar and Kaur, 1971). This can be likened to Angiosperm (dicotyledon) seeds which first produce radicle during seed germination. The pattern of sexual (archegonia and antheridia) and hair development in C. striatus is similar to that of Pteris (Perez-Garcia and Fraille, 1986). Since the mean percentage spore germination was very high, this shows that C. striatus produces viable spores. This is likely helpful in the survival of this plant just as reported by Ganguly, et al. (2009) for Arthromeris himalayensis (Hook) as well as its distribution across many humid ecological areas. The result showed that the first prothalli cells started dividing to produce apical meristematic cells which continued to divide to form short but later long filaments with about 20 cells similar to the situation reported by Zhang, et al. (2008). The gametophytes are cordate and possessed 12-14 long, slender rhizoids (Fig. 1 I) being bisexual for possessing both the antheridia and archegonia together on each gametophyte. Our field observations revealed that croziers developed from rhizomes and unfold in C. striatus throughout the year producing massive, light, numerous spores in as much as enough moisture is available. Thus, there is a high reproductive capability in C. striatus. The high rate of germination of freshly harvested spores of C. striatus in Moore’s medium revealed the viability and the suitability to raise this plant from the spores. 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