Spore Germination and Gametophyte Growth of Polypodium Cambricum Fern

Quad. Bot. Amb. Appl., 18 (2007): 99-102.

Spore germination and gametophyte growth of Polypodium cambricum fern

S. MUCCIFORA, D. MARCHINI & L. M. BELLANI Department of Evolutionary Biology, Siena University, Via A. Moro 4, 53100 Siena, Italy

ABSTRACT. - Spore germination and gametophyte growth of Polypodium cambricum fern. - In controlled culture condi tions Polypodium cambricum L. spores germinate 12-14 days after sowing and reach maximum germination (99%) 40 days after sowing. The primary rhizoid and first protonemal cell arise from two unequal divisions of the original spore cell. Successive transverse divisions of the latter lead to the formation of an unbranched protonemal filament of 4-6 cells. The apical cell of the filament undergoes longitudinal division into two cells from which, through a series of divisions that pro duce spatula- and racket-like forms, a heart-shaped gametophyte arises. About 15 archegonia on the lower surface of the gametophytes just below the notched meristem and about 20 antheridia between the rhizoids characterize the sexually mature gametophyte. P cambricum gametophytes are readily available, easily cultured, and their fast growth and high germination make them potentially good material for studying the effects of contaminants on plant cells.

Key words: Polypodium cambricum L., spores, gametophyte, germination.

INTRODUCTION plates. Each plate was sown with 200 spores and three Unlike the sporophyte that is generally large and mor replicates were prepared. The plates were exposed to "day phologically complex, the fem gametophyte is small, mul light" tubes (15 W m-2), 12 h light/dark with a temperature ticellular, haploid and differentiated into rhizoids, photo regime of 20/23°C. Spore germination was followed under synthetic cells and reproductive structures and exhibits the a Leika Wild M 10 stereomicroscope and sexually mature same physiological metabolic phenomena as higher plants gametophytes were observed under a Leika DM MB light (DYER, 1979; BANKS, 1999; WADA, 2007). The gameto microscope. Spores were sputter-coated with gold and phyte is readily cultured in highly controlled environments. observed with a Philips XL 20 scanning electron micro The advantages of fem gametophytes as a model system scope at 10 kV. for studying plant biology have often been described (R.AGHAVAN, 1989; WADA & KADOTA, 1989; SUGAI, 1999). The present study is part of a research program con RESULTS AND DISCUSSION cerned with determining whether gametophytes could be Polypodium cambricum L. spores are monolete, good material for studying physiological and molecular anisopolar and bilaterally symmetrical, with an elliptic pro alterations induced by contaminants in plant cells. file and a major axis of about 80 Orn (Fig. la). Perine is Polypodium cambricum L. is a small fem found in Europe. absent and the exine shows in the aperture area roundish It is widespread in Italy, growing on cliffs, dry stone walls, tubercles which become smooth reliefs on the distal face tree bark and hot dry places of Tyrrhenian regions (Fig. la). (FERRARINI & al., 1986). Since no microscopic or submi Under controlled culture conditions, spores of P cam croscopic study of gametophytes of the species has ever bricum germinate 12-14 days after sowing. Spores were been carried out, the present research calls for detailed considered to have germinated when the rhizoid tip imme knowledge of all phases of gametophyte formation, from diately followed by the basal chlorocyte of the future pro spore germination to gametophyte maturity. toneme emerged from the ruptured spore wall. The spores showed 40% germination 15 days after sowing and reached maximum germination, about 99%, 40 days after sowing MATERIALS AND METHODS (Tab. 1), a particularly high percentage of spore germina Polypodium cambricum L. spores were gathered from tion and prothalli formation in modified Knop's medium. naturally growing sporophytes in Siena Botanical Garden. All gametophytes reached sexual maturity. Spores, sterilized in 1% sodium hypoclorite, were cultured The original spore cell underwent two unequal divi on Knop's medium modified according to VAUDOIS & sions, producing a newly formed gametophyte composed of TOURTE (1979), solidified with 1.5 % agar in sterilized Petri first rhizoid, first protonemal cell and a larger spore cell b

Fig. I. - a) A scanning electron micrograph of Polypodium cambricum L. spore; b-c). Unbranched protonemal filament. SC, spore coat; RZ, rhizoid; d-e-f) lnitial formation of the two-dimensional gametophyte (the arrow indicates the apical meristem); g-h) Initial fonnation of the heart-shaped gametophyte (the arrow indicates the developing notch); i) The mature cordate gametophyte; I) Portion of the lower surface of the mature gametophyte studded with archegonia (arrowheads) (the arrow indicates the notch); m) Light microscope photo graph of the lower surface of the mature gametophyte (the arrows indicate antheridia). 100 100 90 80 -1 70 c 0~ 0 60 ~ .Ec 50 ~ 40 ;;.,:;------! 30------· 20 ...----

10....------1

0 "!------~ 15 30 40 days after sowing

Values are the mean of three replicates. Vertical bars indicate standard deviation

Fig. 2 - Percentage germination of Polypodium cambricum L. spores 15 , 30 and 40 days after sowing.

within the spore coat (Fig. 1b ). The cell division pattern dur found similar to those of Dryopteris parasitica (REUTER, ing spore germination was similar to those reported in 1953) and A. trichomanes (MucCIFORA & GORI, 1995). Anemia phyllitidis (SCHRAUDOLF, 1981), Anemia mexicana About 15 archegonia were evident just below the (NESTER, 1985), Woodwardia radicans (CARAFA, 1990) and notched meristem on the lower surface of the gametophyte Asplenium trichomanes (MuccIFORA & GORI, 1995). The 90-100 day after sowing (Fig. 11). They were cylindrical and protonemal cell divided transversely and repeated equal bent towards the tip of the heart (Fig. 11). Two to three transverse divisions of the apical cell formed a protonemal weeks after archegonia formation, many spherical filament, consisting of 4-6 cells, that elongated along the antheridia appeared between the rhizoids (Fig. 1m). surface of the medium (Fig. 1c ). A further cell division per Limitation of archegonia and antheridia to the lower surface pendicular to the plane of the previous one gave rise to a two is a feature that this species shares with W radicans dimensional gametophyte (Fig. Id). The apical part of the (CARAFA, 1990), A. trichomanes (Mucc1FORA & GORI, latter produced an organized meristem of about 10 small 1995), Polystichum setiferum (MUCCIFORA & al., 1996) and cells (Fig. ld). The meristem always stands in the apical part Phyllitis scolopendrium (MucCIFORA & al., 2000). All P of the gametophyte. On the contrary, the meristem of most cambricum gametophytes observed were bisexual. previously described species develops laterally then shifts to The advantages of fem gametophytes as a model system apical position (PRAY, 1971; NESTER & SCHEDLBAUER, 1981; for studying plant biology are various. Multicellular, hap NESTER, 1985). Through a series of divisions, the meristem loid and morphologically simple, they differentiate into rhi cells formed a gametophyte that is first spatula-shaped and zoids, photosynthetic cells and reproductive structures, and then racket-shaped (Figs le - f). Subsequent divisions have the same physiological and metabolic processes as increased the size of the gametophyte. About 60 days after higher plants (DYER, 1979; BANKS, 1999; WADA, 2007). The sowing the meristem became indented (notch formation) use of gametophyte requires to kI10w the pattern of spore and the prothallus heart-shaped (Figs lg - h). Unicellular germination and development of gametophytes in highly unpigmented rhizoids were evident on the lower surface of controlled environment. P cambricum species for its readi the gametophyte near the tip of the "heart". About 80 days ly availability, the surprisingly high spore germination in after sowing, gametophytes reached final size of the heart vitro, and the quick development of gametophytes could be shape stage, terminating somatic development (Fig. Ii). The a good material for researches on the effects of contami developmental stages of P cambricum gametophyte were nants on plant cells. 101 REFERENCES lule. La cellula apicale de! protonema si divide lon gitudinalmente. Le due cellule figlie dividendosi BANKS J. A., 1999 - Gametophyte development in ferns. - formano in breve gametofiti a forma di spatola, poi Plant Mol. Biol. 50: 163-186. di racchetta ed infine un gametofito a forma di CARAFA A., 1990 - Gametophyte development of cuore. La comparsa sulla pagina inferiore de! Woodwardia radicans (L.) Sm.: effect ofpopulation gametofito di circa 15 archegoni in prossimita de! density and antheridiogen on sex expression. - setto de! cuore e numerosi anteridi tra i rizoidi Gior. Bot. Ital. 124: 571-580. denota la formazione di un gametofito sessualmen DYER A., 1979 - The experimental biology of ferns. - te maturo. La reperibilita deIla specie, I' elevata per Academic Press, London. centuale di germinazione delle spore i tempi relati FERRARINI E., CIAMPOLINI F., PICHI-SERMOLL! R ., vamente brevi di formazione di gametofiti sessual MARCHETTI D., 1986 - lconographia palynologica mente maturi in vitro, rende la felce P cambricum pteridophytorum Italiae. - Webbia 40: 1-202. un materiale di laboratorio idoneo per studiare gli Mucc1FORA S. & GORI. P., 1995 - Asplenium trichomanes effetti di differenti inquinanti sulle cellule vegetali. ssp. trichomanes gametophyte. A light and electron microscope study. - Caryologia 48: 265-274. M UCC IFORA S., BELLA I L.M., GORI P. , 1996 - Spermatogenesi in Polystichum setiferum (Forssk.) T Moore ex Woynar. - Gior. Bot. Ital. 130: 397. MuccIFORA S., Woo S., GORI P., 2000 - Ultrastructural fea tures of spermatocytes and spermatozoids in the fern Phyllitis scolpendrium (L.) Newm. subsp. scolopendrium. - Sex. Plant Repr. 12: 323-331. NESTER J. , 1985 - Spore germination and early gameto phyte development in Anemia mexicana Klotzsch. - Bot. Gaz. 146: 510-516. N ESTER J. & SCHEDLBA UER M., 1981 - Gametophyte devel opment in Anemia mexicana Klotzsch. - Bot. Gaz. 142: 242-250. PRAY T. , 1971 - The gametophyte of Anemia colimensis. - Am. J. Bot. 58: 323-328. RAGHAVA N V., 1989 - Developmental biology offern gametophytes. Cambridge University Press, Cambridge. REUTER R., 1953 - A contribution to the cell-physiologic analysis of growth and morphogenesis in fern pro thalli. - Protoplasma 42 : 1-29. SCHRAUDOLF H., 1981 - Cell division patterns during spore germination of Anemia phyllitidis L. Sw. - Beitr. Biol. Pflanzen 55: 285-288. SUGA I M ., 1999 - Developmental biology in fern gameto phytes - contributions by Japanese researchers. - Plant Morphol. 11: 32-41. VAUDOIS B. & TOURTE Y., 1979 - Spermatogenesis in a pteridophyte. I. First stages of the motile appara tus. - Cytobios 24: 143-156. WADA M ., 2007 - Th e fern as a model system to study pho tomorphogenesis. - J. Plant Res. 120: 3-16. WADA M. & KADOTA A., 1989 - Photomorphogenesis in lower green plant tissue. - Annu. Rev. Plant Physiol. Plant Mol. Biol. 40: 169-191.

RIASSUNTO - Germinazione delle spore e crescita dei gametofiti della felce Polypodium cambricum. - Le spore della felce Polypodium cambricum L. in condizioni di coltura controllate germinano 12-14 giomi dopo la semina e raggiungono ii massimo di germinazione (99%) 40 giomi dopo la semina. La cellula sporigena dividendosi produce un rizoide e la prima cellula de! protonema. Divisioni trasversa li successive di questa portano alla formazione di un filamento protonemale non ramificato di 4-6 eel- 102