Fern spore germination in response to environmental factors

Abstract

Fern spore germination gives rise to the rhizoid and protonemal cell through asymmetric cell division, and then develops into a gametophyte. Spore germination is also a representative single-cell model for the investigation of nuclear polar movement, asymmetrical cell division, polarity establishment and rhizoid tip-growth. These processes are affected by various environmental factors, such as light, gravity, phytohormones, metal ions, and temperature. Here, we present a catalog of spore germination in response to different environmental factors. They are as follows: (1) Representative modes of light affecting spore germination from different fern species include red light-stimulated and far red light-inhibited spore germination, far red light-uninhibited spore germination, blue light-inhibited spore germination, and spore germination in the dark. The optimal light intensity and illumination time for spore germination are different among various fern species. Light response upon spore germination is initiated from the cell mitosis that regulated by phytochromes (PHYs) and cryptochromes (CRYs). AcPHY2, AcCRY3 and/or AcCRY4 are hypothesized to be involved in spore germination; (2) Gravity and calcium are crucial to early nuclear movement and polarity establishment of spores; (3) Gibberellin and antheridiogen can initiate and promote spore germination in many species, but abscisic acid, jasmonic acid, and ethylene pose only minor effects; (4) Spores can obtain the maximal germination rate in their favorable growth medium. Moreover, metal ions, pH, and spore density in the culture medium also affect spore germination; (5) Most fern spores germinate at 25°C, and an optimal CO₂ concentration is necessary for spore germination of certain fern plants. These provide valuable information for understanding fern spore germination in response to environmental factors.

Keywords

fern / spore / germination / environmental factors

Introduction

Ferns represent an ancient group of vascular plants, and the fern life cycle alternates between two distinct phases or generations: a sporophytic phase and a gametophytic phase. Fern spores generated from sporophytes can develop into gametophytes after germination. Gametophytes produce sperms and eggs to allow later fertilization and then develop into a new sporophytic generation. Following breaking of dormancy, the nucleus migration of fern spore occurs. Then rhizoids and protonemal cells are produced through the asymmetric cell division. The protonemal cell divides continuously and finally develops into a gametophyte. Obviously, spore germination is crucial to the alternation of fern generations. It is also a representative single-cell model for investigation of polarity establishment and growth processes, unlike the germination of plant pollen grains and seeds, which are two-/three-cellular and multicellular tip-growing systems, respectively ( Chatterjee et al., 2000; Dai et al., 2006). Investigation of fern spore germination will provide more information for the comparison of the germination mechanisms of fern spores, pollen grains, and seeds. Importantly, it is vital for understanding the mechanisms of single-cellular light response, cell nucleus migration, cytoskeletal dynamics and polar growth ( Dai et al., 2008). The spore germination of more than 200 fern species have been reported to be affected by various environmental factors including light, gravity, hormones (e.g., gibberellin (GA), antheridiogen (An), abscisic acid (ABA), jasmonic acid (JA), and ethylene), temperature, and nutrition. These fern species belong to 52 genera in 25 families, which represent diverse and somewhat disjointed features of spore germination in response to different exogenous and environmental factors (Fig. 1). Here, we present an extensive review as a catalog of existing data on environmental factors that affect fern spore germination.

The effect of light irradiation on spore germination

The quality, intensity, and duration of light are crucial to plant dormancy, germination, seedling development, reproduction, and shade avoidance ( Kami et al., 2010). Plants perceive diverse light signals using specialized information-transducing photoreceptors, such as phytochrome (PHY), cryptochrome (CRY), and phototropin (PHOT). Fern spore germination is also affected by such a diversity of light conditions.

Spore germination is regulated by PHY and CRY

Fern spores from species with photosynthetic gametophytes typically germinate in the light, while the species with subterranean, nonphotosynthetic, mycorrhizal gametophytes germinate in the dark (e.g., fern species from Psilotaceae, Ophioglossaceae, as well as species from Lycopodium and Tmesipteris) ( Whittier and Braggins, 1994; Whittier, 2006, 2008). It has been reported that the spore germination of two species, Ophioglossum crotalophoroides and Lycopodium clavatum, was induced in the dark and prevented by white light and red light, while initiated by far red light in a red/far red light reversible manner (Whittier, 2006, 2008). Similarly, spores of four species from Psilotum (P. nudum and P. complanatum) and Tmesipteris (T. lanceolata and T. sigmatifolia) also germinated in darkness (Whittier and Braggins, 1994).

Fern spore germination initiated from the cell mitosis is regulated by PHY and CRY ( Furuya et al., 1997). PHY is an approximately 120 kDa biliprotein encoded by a large multi-gene family. PHY switches its protein conformation between P_r (red light absorbing form with a maximum absorption peak at 666 nm) and P_fr (far red light absorbing form with a maximum absorption peak at 730 nm), thus to turn on (red light) or off (far red light) the first asymmetric mitosis of spores ( Raghavan, 1992). Experiments of partial (nuclear or cytoplasmic region) irradiation with a red/far red light microbeam on the darkness-imbibed spores from Adiantum capillus-veneris suggested that the PHY molecule was localized in the whole spores under darkness, but gradually migrated to the nuclear region under the irradiation with red light ( Tsuboi et al., 2012). Some PHY genes have been identified in Adiantum capillus-veneris, including two typical PHY genes (AcPHY1 and AcPHY2) ( Nozue et al., 1998a), a photoreceptor gene (AcPHY3) ( Nozue et al., 1998b), and a PHY-related sequence (AcPHY4) ( Nozue et al., 1997). The expression levels of AcPHY1 and AcPHY2 were similar in the spores under red light. The transient expression of the PHY and glucuronidase (GUS) fusion gene implied that it was AcPHY2, but not AcPHY1, that migrated into the nuclear region upon spore germination ( Tsuboi et al., 2012). In addition, AcPHY3 is a PHY-nonphototropic hypocotyl 1 (NPH1) chimera photoreceptor. Its N terminus is similar to PHY chromophore binding domain and PHOT, and the C terminus is a full-length blue light receptor domain ( Nozue et al., 1998b). As the photoreceptor of red/far red light and blue light, AcPHY3 can mediate red light-induced phototropic and polarotropic responses and chloroplast movement in Adiantum capillus-veneris protonemal cells ( Nozue et al., 1998b). However, the role of AcPHY3 during fern spore germination is still unknown.

The P_r-induced first mitosis of spores is inhibited by blue light under the regulation of cell nucleus-located CRY ( Furuya et al., 1997). CRYs function in the subsequent mitosis of protonemal cells which gives rise to protonema. In addition, the cell membrane-located PHY can inhibit the function of CRYs, resulting in the inhibition of protonema genesis. Five groups of CRY genes (AcCRY1-AcCRY5) have been cloned in Adiantum capillus-veneris ( Kanegae and Wada, 1998; Imaizumi et al., 2000; Suetsugu and Wada, 2003). All these AcCRYs contain an N-terminal photolyase-homologous domain and a C-terminal extension ( Kanegae and Wada, 1998). The C terminus of AcCRY2, AcCRY3, and AcCRY4 contains a STAESSSS-related amino acid motif, which is proposed to be important for the direct interaction between PHYs and CRYs by phosphorylation during spore germination ( Imaizumi et al., 2000). The expression of CRY-GUS fusion gene suggested that AcCRY1, AcCRY2, and AcCRY5 were localized in the cytoplasm, while AcCRY3 and AcCRY4 showed clear nuclear localization. AcCRY3 accumulated in the nucleus under darkness and red light, but not blue light, while the AcCRY4 tended to accumulate in the nucleus region under any treatment conditions ( Imaizumi et al., 2000). This implies that only AcCRY3 and/or AcCRY4 are involved in the inhibition of spore germination ( Imaizumi et al., 2000). Moreover, AcCRYs involved in the phototropism, apical swelling, and chloroplast orientational movement are proposed to be localized at the plasma membrane in protonema ( Hayami et al., 1992).

The effects of red light and far red light on fern spore germination

The spore germination of some fern species was induced by red light (Table 1), such as Onoclea sensibilis, Lygodium japonicum, Thelypteris kunthii, Pteris vittata, and Cheilanthes feei ( Nondorf et al., 2003). Longtime irradiation with red light was necessary for some fern spore germination. For example, 12 h illumination per day induced a high germination rate of Alsophila metteniana spores ( Du et al., 2009a). Similarly, Asplenium nidus spores initiated rhizoids under 12 h illumination of red light. Spores from some species germinate under red light irradiation for several days. For example, the maximum germination rate of Polypodium aureum spores was obtained after 5-day continuous red light illumination. However, for some fern spores, several minutes or seconds were sufficient to promote a high percentage of germination, such as spores of Cheilanthes farinosa and Onoclea sensibilis. On the contrary, intermittent irradiation with red light does not work effectively for some spore germination. Under a 24 h continuous irradiation of red light, Mohria caffrorum spores showed a 79.1% germination rate, while the germination rate was reduced to 15.4% with the intermittent red light irradiation (5 min per 1 h, 5 min per 4 h, and 5 min per 8 h) within a 24 h time course ( Reynolds and Raghavan, 1982). Similar regulation by red light has also been observed upon Thelypteris kunthii spore germination ( Huckaby and Raghavan, 1981).

In addition, short time far red light irradiation significantly inhibited spore germination of Adiantum capillus-veneris ( Furuya et al., 1997). However, far red light promoted the germination of spores from Mohria caffrorum and Onoclea sensibilis (Table 1). Intermittent illumination under far red light within 24 h was not conducive to Mohria caffrorum spore germination ( Reynolds and Raghavan, 1982). In addition, far red light also promoted initiation of Cheilanthes feei spore germination, but all the spores stopped germinating at the two-cell protonemal stage ( Nondorf et al., 2003). The reasons for lacking photoreversibility in those spores under far red light has been explained as follows: (1) spore germination was controlled by a low level of P_fr, which was produced by both red light and far red light; (2) once active P_fr was formed, it was rapidly used in the post-inductive phase, leading to the non-sensitity of spores to far red light; and (3) PHY was masked by some accessory pigments, which also promoted spore germination ( Reynolds and Raghavan, 1982). However, further study is necessary to prove these hypotheses.

There is a significant “switching effect” of red/far red light on spore germination of some fern species (Table 2). Spore germination (e.g., Adiantum capillus-veneris and Lycopodium clavatum) promoted by short time red light illumination can be reversed by the irradiation of far red light in a red/far red light reversible manner ( Furuya et al., 1997; Whittier, 2008). However, after 12 h irradiation with red light, the germination of Mohria caffrorum spores could not be reversed by 5 min or 1 h illumination of far red light ( Reynolds and Raghavan, 1982). In addition, a brief exposure of red light (5 min) could not repromote the germination of far red light-inhibited Thelypteris kunthii spores. On the contrary, if the spores were exposed to red light for 10 to12 h, or to a repeated 8 h red light plus 5 min far red light twice following an extra 8 h red light, the inhibition of far red light could be nullified ( Huckaby and Raghavan, 1981). This implies that enough time of red light illumination can nullify far red light-induced inhibition of spore germination.

Spores germinated under darkness are induced by far red light, but inhibited by red light. Spores from Psilotum (P. nudum and P. complanatum) and Tmesipteris (T. lanceolata and T. sigmatifolia) germinated only in the darkness ( Whittier and Braggins, 1994) (Table 1). Although Tmesipteris lanceolata spores could germinate in light, its gametophyte development never proceeded beyond the two-cell stage. Spores of Ophioglossum crotalophoroides and Lycopodium clavatum were able to complete germination under darkness and far red light, but were inhibited by red and white light ( Whittier, 2006, 2008). In addition, over 24 h far red light treatment followed by 1 to 2 min red light promoted almost complete Asplenium nidus spore germination and rhizoids initiation. Similarly, spores of fern Lygodium heterodoxum also germinated under far red light ( Pérez-García et al., 1994).

In addition, white light induced the spore germination of Alsophila metteniana, Cheilanthes feei, Lygodium heterodoxum, and Adiantum reniforme var. sinense, and the former two species also germinated well under yellow or green light conditions ( Pérez-García et al., 1994; Nondorf et al., 2003; Du et al., 2009a; Wu et al., 2010) (Table 1).

Blue light inhibition of spore germination

Blue light significantly inhibits fern spore germination of some species (Table 1). For example, blue light could not initiate the spore germination of Adiantum capillus-veneris and Polypodium aureum ( Furuya et al., 1997). For Alsophila metteniana spores ( Du et al., 2009a), blue light exposure only induced a 7.51% germination rate, which was lower than that under other light conditions. However, for certain species, blue light didn’t inhibit or only partially inhibit spore germination. An approximately 68% germination rate of Cheilanthes feei spores was obtained under blue light ( Nondorf et al., 2003). Additionally, the germination rate of Onoclea sensibilis spores under 5 min irradiation with blue light was similar to those under other light sources.

The mechanism of photoreversibility germination between blue light and red/far red light remains unclear (Table 2). Previous physiological studies have reported that blue light could not inhibit the red light-induced germination of spores from Cheilanthes farinosa, Polypodium aureum, and Mohria caffrorum ( Reynolds and Raghavan, 1982). For example, red light-promoted germination of Cheilanthes farinosa spores was reversed by a brief blue light, but the spores germinated again after a long time or high intensity of red light illumination. This is mainly due to the irradiation time of red light has reached the requirement for germination.

Light intensity and illumination time

The optimal light intensity for germination is different among various fern species. A relative low light intensity is more conducive to spore germination (Fig. 2). For example, when the light intensity increased from 20 μmol·m^-2·s^-1 to 50 μmol·m^-2·s^-1, the germination rate of Alsophila metteniana spores was increased from 55.25% to 80.45%, whereas the germination rate was decreased to 60.44% when the light intensity continued to increase from 60 μmol·m^-2·s^-1 to 100 μmol·m^-2·s^-1. Thus, 50 μmol·m^-2·s^-1 was the optimal light intensity for Alsophila metteniana spore germination ( Du et al., 2009a). Consistent with this, the germination rate of Cheilanthes feei spores also appeared to be increased under the light intensity from 50 μmol·m^-2·s^-1 to 100 μmol·m^-2·s^-1, and then declined with the light intensity increasing from 100 μmol·m^-2·s^-1 to 150 μmol·m^-2·s^-1, and the maximal germination rate occurred at light intensity of 100 μmol·m^-2·s^-1 ( Nondorf et al., 2003). Germination rates of four species from Tectaria (T. heracleifolia, T. incisa, T. mexicana, and T. transiens) were also induced with the increase of light intensity. The germination rates were over 65% under the irradiation with white light, while less than 40% under the leaf shade conditions ( Pérez-García et al., 2007). High light intensity inhibited the spore germination of Rumohra adiantiformis. The shortest germination time (8 days) was observed in a low light intensity (9% of nature light), whereas the germination time was extended to 23 days when the light intensity increased to 54% of natural light ( Brum and Randi, 2002). In some cases, low light intensity seemed more efficient for spore germination of some species in Dicksoniaceae, such as Dicksonia sellowiana, Cyathea delgadii, and Blechnum brasiliense ( Renner and Randi, 2004; Hiendlmeyer and Randi, 2007). Under 5%, 20%, 36%, and 50% of natural light irradiance, the spore germination rate of Dicksonia sellowiana was more than 85% over a 49-day incubation. Moreover, under 5% of natural light, the germination rate reached to 60% in a 21-day incubation ( Renner and Randi, 2004). Similarly, under 5% natural light, the spore germination rate of Cyathea delgadii and Blechnum brasiliense was 83.5% and 84%, respectively ( Hiendlmeyer and Randi, 2007). The highest spore germination rate of Polypodium lepidopteris was observed at 22% and 8% of natural light ( Viviani and Randi, 2008). Similarly, Gleichenella pectinata spores also showed the highest germination percentage at 5% to 20% of sunlight irradiance ( Santos et al., 2010).

The illumination time required for spore germination is diverse among different fern species (Fig. 2). Some fern spores need a long period of light irradiation for germination. For example, the spores of Asplenium nidus initiated rhizoids in 4 days under the irradiation with red light, and the maximal spore germination rate of Polypodium aureum was achieved in 5 days. In addition, after 4 days imbibition in darkness, the maximal spore germination rate of Thelypteris kunthii was detected under 7 days irradiation with white light or red light ( Huckaby and Raghavan, 1981). However, for spores from some fern species, such as Onoclea sensibilis, Dryopteris filix-mas, Dryopteris paleacea, and Polystichum minutum, short time red light irradiation (3 to 5 min) is enough for germination, In addition, treatment with red light for 24 h, Pteridium aquilinum spores required 3 to 5 days for germination, while the spores under red light for 12 h needed extra 3 to 4 days to germinate ( Niu et al., 2002). The differences in the requirement of illumination probably attribute to the specific properties of fern species, the spore longevity and inoculation density of spores.

The effects of gravity and Ca²⁺ on spore germination

Gravity acting as an important ecological factor is required for the morphogenesis and generative development of plant normal growth and plays key roles in determining the spatial orientation of higher plants ( Merkys et al., 1981). The polar development of Ceratopteris richardii spores is oriented by gravity. During the germination of Ceratopteris richardii spores, before the first mitosis took place, nuclear directional movement determined the asymmetry division of spore and the direction of rhizoid elongation. This period was called “polarity-determination window.” Roux and his collaborators ( Edwards and Roux, 1998; Chatterjee et al., 2000; Roux et al., 2003) have shown that gravity played a key role in nuclear migration. With the presence of gravity, the rhizoids of 94% spores grew in the direction of gravity. However, under the weightlessness condition, rhizoids exhibited random orientation and the cell nuclei also migrated to random positions ( Edwards and Roux, 1998). It has been shown that the influence of light on spore polarity was less than gravity ( Edwards and Roux, 1998). About 92% rhizoids grew downward (with respect to gravity) when spores were germinated in bidirectional light along the direction of gravity, or illuminated by uniform light from all directions. When the unilateral light was paralleled to the vector of gravity, 98% rhizoids grew downward. While the number of rhizoids growing downward was reduced to 81% with the light irradiation from the bottom of spores ( Edwards and Roux, 1998).

As an essential element for plants, calcium is required for building various cellular structures such as cell wall and membrane, and it also act as a counter-cation for inorganic and organic anions in the vacuole. Cytosolic Ca²⁺ concentration is also an obligate intracellular messenger coordinating responses to numerous developmental cues and environmental challenges ( White and Broadley, 2003). Calcium also plays key roles in the gravity response and polarity establishment during fern spore germination ( Chatterjee et al., 2000). In Ceratopteris richardii spores, calcium current moved along the opposite direction of gravity (from the bottom to the top of the spores) ( Chatterjee et al., 2000). Under nifedipine (a calcium-channel blocker) treatment, calcium transportation was disrupted, and the gravity-dependent spore polar development was also interrupted ( Chatterjee et al., 2000). A recent study has shown that gravity perception in single-celled spores of the Ceratopteris richardii was mediated by mechanosensitive calcium channels and membrane calcium pumps ( Salmi et al., 2011). In addition, two alternative models analyzing the role of calcium pumps and channels in the transcellular calcium current have been established. One hypothesis, which is favored, is that membrane calcium pumps and mechanosensitive calcium ion channels are uniformly distributed in the spore plasma membrane and are locally activated to generate the polar calcium current. The other hypothesis suggests that gravity must drive the redistribution of the calcium pumps and channels to the top and bottom of spores for the polar calcium current. Further work is required to identify the calcium channels and pumps involved in gravity-regulated polarity establishment of Ceratopteris richardii spores ( Salmi et al., 2011). In addition, it has been found that Ca²⁺ concentration had a significant effect on Dryopteris paleacea spore germination with the optimal Ca²⁺concentration of 0.1 mM ( Scheuerlein et al., 1989).

The effects of phytohormones on fern spore germination

GA, ABA, JA, and ethylene play regulatory roles in plant growth and development. GA stimulates plant elongation, promotes flowering, and induces seed germination and dormancy release. ABA plays a key role in plant stress response and is also known as the main regulator of seed dormancy maintenance. JA inhibits the germination of non-dormant seeds but stimulates the germination of dormant seeds. JA also inhibits root growth, prevents tuber formation, and involves in the development of flower organs and regulation of senescence. In addition, ethylene is a gaseous hormone that activates fruit maturation, stimulates germination, accelerates senescence, inhibits elongation, promotes horizontal growth, and initiates adventitious roots and programmed cell death. Importantly, phytohormones exhibit extensive cross-talk with each other and with environmental and developmental signaling pathways ( Wang and Irving, 2011). For fern spore germination, these hormones exhibited diverse regulation patterns.

GAs and An

The regulation mechanisms of GA on fern spore germination and seed germination are similar. The biosynthesis of endogenous GA in fern spores was suggested to be induced by red light via PHY system ( Kagawa and Sugai, 1991). It has been shown that both GA₃ (4 × 10^-7 M) and red light could induce the germination of Lygodium japonicum spores ( Kagawa and Sugai, 1991). For most fern species, GA promoted their spore germination (Fig. 3), including spores of Pteridium aquilinum ( Zhang and Niu, 1999), Athyrium multidentatum ( Guo et al., 2007), Nephrolepis auriculata ( Li et al., 1995). GA also functions in the spore maturation and dormancy breaking of Cyatheaceae fern species ( Jiang et al., 2002). Moreover, the duration of GA treatment has an impact on spore germination. For example, after 12 h and 24 h of GA treatment, the germination rate of Anemia phyllitidis spores was 30% and 70%, respectively. Interestingly, different GA molecules have diverse effects on spore germination. The spore germination rate of Schizaea pusilla was increased under 0.1 mM GA₃, but not 0.1 mM GA₄₊₇ ( Guiragossian and Koning, 1986). However, spores of some fern species are not sensitive to GA. For example, GA₃ treatment didn’t increase the spore germination rates of Pteridium aquilinum var. latiusculum ( Zhai et al., 2007) and Sphenomeris chinensis ( Ren et al., 2008).

An, secreted by mature fern gametophyte, is a small molecular and hydrophobic GA-like hormone. An can induce antheridium development ( Chiou and Farrar, 1997), which has been suggested to occur in over 50 fern species of six genera (i.e., Pteridium, Pteris, Anemia, Onoclea, Lygodium, and Ceratopteris) ( Chiou and Farrar, 1997). Various An purified from various ferns have been termed as Apt, Aps, Aan, Aon, Aly, Ace, and so on. Some of them were found to function in the initiation of spore germination in the dark ( Chiou and Farrar, 1997) (Fig. 3). Interestingly, some An secreted by certain fern gametophytes only act on the spores generated from the same fern species. For example, various An from Lepisorus thunbergianus, Phymatosorus scolopendria, Polypodium pellucidum, Campyloneurum angustifolium, Campyloneurum phyllitidis, Microgramma heterophylla, and Anemia phyllitidis only induced the spore germination of the corresponding species ( Chiou and Farrar, 1997). However, the An generated from Phlebodium aureum was able to promote spore germination of Onoclea sensibilis apart from its own spores in the dark. While Apt from Pteridium aquilinum did not have the species specificity and could induce many other fern spore germination in darkness ( Chiou and Farrar, 1997).

ABA and JA

ABA has little effect on fern spore germination. The spore germination rates of Mohria caffrorum and Matteuccia struthiopteris were almost unaffected under ABA treatment (Fig. 3). Similarly, JA had no influence on the germination and primary rhizoid initiation of Platycerium bifurcatum spores (Fig. 3), although it significantly promoted rhizoid elongation and early gametophyte development ( Camloh et al., 1996). This suggests that the pre-synthesis of mRNA, protein, and other storages in mature spores is adequate for spore germination, but the new mRNA synthesis stimulated by JA is necessary for the rhizoid elongation and early gametophyte development.

Ethylene

Ethylene can regulate the biosynthesis of RNA and protein, functioning in the plant growth and development (e.g. seed germination, plant mature and aging) ( Li and Han, 2000). However, high concentration of ethylene can inhibit fern spore germination by affecting DNA synthesis, nucleus movement, and cell division. Ethylene completely inhibited spore germination of Onoclea sensibilis spores in the dark (Fig. 3). 1 µM of ethylene led to the decrease of germination rate of Onoclea sensibilis spores to 40%. However, this inhibition could be partly removed by light illumination and CO₂ treatment. In addition, after absorbed into Ceratopteris richardii, (2-chloroethyl) phosphonic acid was degraded to produce ethylene, which promoted spore maturation, but reduced the germination rate of mature spores ( Warne and Hickok, 1987) (Fig. 3).

The effects of growth medium and spore density on fern spore germination

In comparison with solid medium, liquid medium was more suitable for the early development of gametophytes of Platycerium bifurcatum ( Camloh, 1993). The germination percentage was higher for Anemia phyllitidis spores in liquid medium ( Douglas, 1994). However, it was more suitable for Pteridium aquilinum spores to germinate on solid medium than in liquid medium, and the difference was probably caused by the species-specific or ecotype-specific response to liquid medium among different fern species, as well as the interaction between medium composition and spore age ( Sheffield et al., 2001). Medium composition is important for fern spore germination and gametophyte development (Table3). For spores from certain fern species, such as Schizaea dichotoma ( Cox et al., 2003), Pyrrosia lingua ( Du et al., 2009b), Osmunda japonica ( Yuan et al., 2002), and Alsophila podophylla ( Zhang et al., 2007), contained all the required nutrient for early growth and had high germination rates when germinated in the medium with relative low concentration of mineral nutrient (e.g., 1/8 Knop’s, 1/8 MS, 1/4 MS, or 1/10 MS). Similarly, 1/4 MS added with 15 g·L^-1 sucrose was suitable for Adiantum reniforme var. sinense spore germination and early gametophyte development ( Wu et al., 2010). Spore germination of Pteris cretica, Pteris wallichiana ( Xu et al., 2005; Zhang et al., 2008b), Pteris tripartita Sw ( Baskaran and Jeyachandran, 2012), and Dryopteris varia ( Ouyang et al. 2008) were more efficient in the 1/2 MS when compared to that in full strength MS. The optimal media for the germination of Adiantum raddianum spores was 1/2 MS plus 3.0 mg·L^-1 GA, 1.5 g·L^-1 carbon, 30 mg·L^-1 sucrose, and 6.5 g·L^-1 agar ( Tian et al., 2008).

Sucrose in the medium provides carbon source and osmolyte for spore germination. The size, coat, and reserve of fern spores among various species are different, which leads to diverse requirements for sucrose in medium. The spores of Pteris cretica ( Xu et al., 2005), Pyrrosia lingua, Dryopteris varia ( Ouyang et al., 2008; Du et al., 2009b), and Osmunda japonica ( Yuan et al., 2002) germinated normally in the medium with sucrose content less than 2%. Similarly, the highest germination rate (86%) of Colysis latiloba spores was observed at 1% sucrose medium ( Parajuli et al., 2013). However, the spore germination rates of some fern species (e.g., Pteridium aquilinum, Athyrium filix-femina, and Dryopteris expansa) cultured with 6.8% sucrose were higher than those with 1.7% sucrose in medium ( Sheffield et al., 2001). For Pteridium aquilinum var. latiusculum, high contents of sucrose, thiamine, pyridoxine, glycine, glutamate, kinetin, and extracts of Pteridium aquilinum leaves promoted spore germination and the growth of prothallus ( Bao et al., 2000). However, sulphites, oxybarbiturates, and lipophilic solvents inhibited spore germination of Lygodium japonicum, Pteridium aquilinum, Anemia phyllitidis, and Onoclea sensibilis ( Bannon et al., 1991; Sahi and Singh, 1994). In addition, adding a certain does of Eupatorium adenophorum extracts (5% to 50%) into the culture media would result in a decrease of spore germination of Macrothelypteris torresiana, Cibotium barometz, and Neocheiropteris palmatopedata ( Zhang et al., 2008a, 2008c, 2012).

Metal ions (e.g., Ca²⁺, Mn²⁺, and Mg²⁺) in the medium function as the signaling molecules or chelating ions of enzymes (e.g., phosphatase, ATP synthase, and nucleotidase) during fern spore germination, which is essential for cell polarity and enzyme activity. It has been proved that these ions were necessary for the spore germination of Onoclea sensibilis and Dryopteris paleacea ( Scheuerlein et al., 1989). However, Cd²⁺ and La³⁺ interfered with the activities of Ca²⁺ and other ions, leading to the inhibition of spore germination of Ceratopteris thalictroides ( Gupta et al., 1992) and Dryopteris paleacea ( Scheuerlein et al., 1989).

The pH of medium is also important for the germination of fern spores. The optimum pH value is varied from different fern species. Spores from Drynaria fortunei ( Chang et al., 2007) and Cheilanthes feei ( Nondorf et al., 2003) germinated on the medium with a relative wide range of pH from 3.7 to 9.7 and 4.5 to 8.5, respectively. However, some fern species only germinate in acidic or alkaline media. For example, the optimum pH for Alsophila metteniana spore germination was 4.7 ( Du et al., 2009a). Acidic pH also promoted a relative higher germination rates of Cyathea atrovirens (pH 4.0 to 6.5), Polypodium lepidopteris Kunze (pH 4.0 to 6.7), Ophioglossum palmatum L. (Ophioglossaceae) (pH 4.0), and Gleichenella pectinata (pH 4.5 to 5.0) spores ( Whittier and Moyroud, 1993; Viviani and Randi, 2008; Rechenmacher et al., 2010; Santos et al., 2010). Anemia mexicana and Anemia phyllitidis (Schizaeaceae) also germinated well at pH 5.0 to 6.5, but the germinability was very low in strong acidic conditions (pH≤4.5) ( Nester and Coolbaugh, 1986). However, Marsilea vestita spore germinated in alkaline condition (pH 7 to 8).

In addition, the spore density affects the utilization of nutrient in medium for spore germination and gametophyte growth ( Song et al., 2009) (Fig. 4). A high spore density would inhibit spore germination because of the competition for nutrient, physical impediment, biochemical inhibitors secreted by other spores, and a low density also causes a limitation of germination rate due to the individual biochemical basis of spores ( Ashcroft and Sheffield, 2000).This suggests that an optimum spore density is crucial for fern spore germination. For example, 3000 spores/cm² was the optimal inoculation density for Osmunda japonica spores germination ( Yuan et al., 2002). Ceratopteris thalictroides spores germinated completely with the inoculation density of 50 spores/mL medium ( Guo et al., 2010). The germination rate of Pteridium aquilinum spores was 43% to 52% with the intermediate density ranging from 187 to 2114 spores/mm², and the highest germination rate (52%) occurred at 360 spores/mm² ( Ashcroft and Sheffield, 2000).

The effects of temperature and CO₂ on fern spore germination

Temperature has a considerable influence throughout the plants development, such as plant morphology, flowering time, seed dormancy release, germination and vernalization. There is a considerable interaction between temperature and PHY-dependent light signaling pathways ( Franklin, 2009). For fern spore germination, temperature also plays an important role through a similar regulation mechanism. Temperature affects the cell membrane permeability and enzyme activity during spore germination. High temperature seemed to affect the activity of PHY thus to regulate fern spore germination ( Heschel et al., 2007). Some fern species can adapt to a wide range of temperature, and the optimum germination temperature is 25°C (Fig. 4), such as Microgramma lindbergii, Microgramma squamulosa, Polypodium pleopeltifolium, Polypodium latipes, Adiantopsis radiata ( Ranal, 1999), Argyrochosma formosa, Thelypteris glandulosa, Thelypteris serrata, Blechnum appendiculatum, and Dennstaedtia globulifera ( Juárez-Orozco et al., 2013). Similarly, spores of Marsilea vestita, Woodwardia radicans ( Quintanilla et al., 2000), Cheilanthes feei ( Nondorf et al., 2003), Rumohra adiantiformis ( Brum and Randi, 2002), Asplenium ruta-muraria ( Pangua et al., 1994), Polypodium lepidopteris ( Viviani and Randi 2008), Gleichenella pectinata ( Santos et al., 2010), Plagiogyria pectinata, Polystichum mickelii, and Hemionitis subcordata ( Juárez-Orozco et al., 2013), as well as species of Tectaria (T. heracleifolia, T. incisa, T. mexicana, and T. transiens) reached the highest germination rates at 25 °C. While spores of Culcita macrocarpa ( Quintanilla et al., 2000) and Microsorium pteropus ( Xue et al., 2008) cultured at 20°C had an approximately 85% germination rate (Fig. 4). In addition, for the spores of fern Sphenomeris chinensis ( Ren et al., 2008), a higher temperature (28°C) was better for germination (Fig. 4). Similarly, the spore germination rate of Alsophila metteniana was less than 46% when the temperature was lower than 15°C, and a higher germination rate (about 70%) was obtained under 20°C to 30°C ( Du et al., 2009a). The maximum spore germination rates of Onoclea sensibilis, Polypodium hirsutissimum, and Pteris denticulata were observed at about 30°C ( Ranal, 1999).

In addition, suitable concentration of CO₂ was necessary for Pyrrosia piloselloides spore germination (Fig. 4). Under 0.02% to 0.34% CO₂, spores had maximal germination rates ( Ong et al., 1998). However, high CO₂ concentration inhibited Onoclea sensibilis spore germination (Fig. 4). When the CO₂ concentration increased from 2% to 10%, the spore germination rate of Onoclea sensibilis reduced from 95% to 25%.

Conclusions

Fern spore germination is a fine-tuned cellular process regulated by various complicated genes/proteins interactive networks. Meanwhile, the complex germination processes are also sensitive to various environmental factors (e.g., light, gravity, Ca²⁺, hormones, and temperature). Most of the aforementioned environment-responsive mechanisms in spore germination are deduced from physiologic and ecological investigations. The unrelated nature of diverse methods/conditions used to generate these data makes it very difficult to draw a broad conclusion across the evolutionarily divergent clade of ferns. In this review, we just give a simple catalog of existing information, which would be a valuable contribution for future studies on ferns. To date, although the molecular and metabolic mechanisms during fern spore germination are still lacking, the update of ESTs and cDNA database for Ceratopteris, as well as the refreshed technologies for plant genome sequencing and transcriptomic/proteomic analysis, provide a well opportunity to investigate the fern spore germination in response to environment factors. Using these modern molecular, biologic and “Omic” approaches, further investigations of the Ca²⁺ signaling, phytohormone regulation, light/gravity response upon fern spore germination would be novel hot topics in the future.

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