Quad. Bot. Amb. Appl., 18 (2007): 99-102.
Spore germination and gametophyte growth of Polypodium cambricum fern
S.
MUCCIFORA,D.
MARCHINI& L.
M. BELLANIDepartment 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
Unlike the sporophyte that is generally large and mor- phologically complex, the fem gametophyte is small, mul- ticellular, haploid and differentiated into rhizoids, photo- synthetic cells and reproductive structures and exhibits the same physiological metabolic phenomena as higher plants (DYER, 1979; BANKS, 1999; WADA, 2007). The gameto- phyte is readily cultured in highly controlled environments.
The advantages of fem gametophytes as a model system 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- cerned with determining whether gametophytes could be good material for studying physiological and molecular alterations induced by contaminants in plant cells.
Polypodium cambricum L. is a small fem found in Europe.
It is widespread in Italy, growing on cliffs, dry stone walls, tree bark and hot dry places of Tyrrhenian regions (FERRARINI & al., 1986). Since no microscopic or submi- croscopic study of gametophytes of the species has ever been carried out, the present research calls for detailed knowledge of all phases of gametophyte formation, from spore germination to gametophyte maturity.
MATERIALS AND METHODS
Polypodium cambricum L. spores were gathered from naturally growing sporophytes in Siena Botanical Garden.
Spores, sterilized in 1 % sodium hypoclorite, were cultured on Knop's medium modified according to VAUDOIS &
TOURTE (1979), solidified with 1.5 % agar in sterilized Petri
plates. Each plate was sown with 200 spores and three replicates were prepared. The plates were exposed to "day- light" tubes (15 W m-2), 12 h light/dark with a temperature regime of 20/23°C. Spore germination was followed under a Leika Wild M 10 stereomicroscope and sexually mature gametophytes were observed under a Leika DM MB light microscope. Spores were sputter-coated with gold and observed with a Philips XL 20 scanning electron micro- scope at 10 kV.
RESULTS AND DISCUSSION
Polypodium cambricum L. spores are monolete, anisopolar and bilaterally symmetrical, with an elliptic pro- file and a major axis of about 80 Orn (Fig. la). Perine is absent and the exine shows in the aperture area roundish tubercles which become smooth reliefs on the distal face (Fig. la).
Under controlled culture conditions, spores of P cam- bricum germinate 12-14 days after sowing. Spores were considered to have germinated when the rhizoid tip imme- diately followed by the basal chlorocyte of the future pro- 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 (Tab. 1), a particularly high percentage of spore germina- tion and prothalli formation in modified Knop's medium.
All gametophytes reached sexual maturity.
The original spore cell underwent two unequal divi- sions, producing a newly formed gametophyte composed of first rhizoid, first protonemal cell and a larger spore cell
b
c
d
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
60
0 ~ c
.E 50
~ 40
;;.,:;- - - !3 0 - - - - - -·
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. 1 b ). The cell division pattern dur- ing spore germination was similar to those reported in Anemia phyllitidis (SCHRAUDOLF, 1981), Anemia mexicana (NESTER, 1985), Woodwardia radicans (CARAFA, 1990) and Asplenium trichomanes (MuccIFORA & GORI, 1995). The protonemal cell divided transversely and repeated equal transverse divisions of the apical cell formed a protonemal filament, consisting of 4-6 cells, that elongated along the surface of the medium (Fig. 1 c ). A further cell division per- pendicular to the plane of the previous one gave rise to a two dimensional gametophyte (Fig. Id). The apical part of the latter produced an organized meristem of about 10 small cells (Fig. ld). The meristem always stands in the apical part of the gametophyte. On the contrary, the meristem of most previously described species develops laterally then shifts to apical position (PRAY, 1971; NESTER & SCHEDLBAUER, 1981;
NESTER, 1985). Through a series of divisions, the meristem cells formed a gametophyte that is first spatula-shaped and then racket-shaped (Figs le - f). Subsequent divisions increased the size of the gametophyte. About 60 days after sowing the meristem became indented (notch formation) and the prothallus heart-shaped (Figs lg - h). Unicellular unpigmented rhizoids were evident on the lower surface of the gametophyte near the tip of the "heart". About 80 days after sowing, gametophytes reached final size of the heart- shape stage, terminating somatic development (Fig. Ii). The developmental stages of P cambricum gametophyte were
found similar to those of Dryopteris parasitica (REUTER, 1953) and A. trichomanes (MucCIFORA & GORI, 1995).
About 15 archegonia were evident just below the notched meristem on the lower surface of the gametophyte 90-100 day after sowing (Fig. 11). They were cylindrical and bent towards the tip of the heart (Fig. 11). Two to three weeks after archegonia formation, many spherical antheridia appeared between the rhizoids (Fig. 1 m).
Limitation of archegonia and antheridia to the lower surface is a feature that this species shares with W radicans (CARAFA, 1990), A. trichomanes (Mucc1FORA & GORI, 1995), Polystichum setiferum (MUCCIFORA & al., 1996) and Phyllitis scolopendrium (MucCIFORA & al., 2000). All P cambricum gametophytes observed were bisexual.
The advantages of fem gametophytes as a model system for studying plant biology are various. Multicellular, hap- loid and morphologically simple, they differentiate into rhi- zoids, photosynthetic cells and reproductive structures, and have the same physiological and metabolic processes as higher plants (DYER, 1979; BANKS, 1999; WADA, 2007). The use of gametophyte requires to kI10w the pattern of spore germination and development of gametophytes in highly controlled environment. P cambricum species for its readi- ly availability, the surprisingly high spore germination in vitro, and the quick development of gametophytes could be a good material for researches on the effects of contami- nants on plant cells.
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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
lule. La cellula apicale de! protonema si divide lon- gitudinalmente. Le due cellule figlie dividendosi formano in breve gametofiti a forma di spatola, poi di racchetta ed infine un gametofito a forma di cuore. La comparsa sulla pagina inferiore de!
gametofito di circa 15 archegoni in prossimita de!
setto de! cuore e numerosi anteridi tra i rizoidi denota la formazione di un gametofito sessualmen- te maturo. La reperibilita de Ila specie, I' elevata per- centuale di germinazione delle spore i tempi relati- vamente brevi di formazione di gametofiti sessual- mente maturi in vitro, rende la felce P cambricum un materiale di laboratorio idoneo per studiare gli effetti di differenti inquinanti sulle cellule vegetali.
