Keywords

1 Introduction

Pteridophytes had been the dominant species of this planet about 280–230 million years ago (Sureshkumar and Ayyanar 2020), but at present they comprise of only 5% of the total vegetation of the world. The group includes over 12,000 species in the world, and India harbors 1100 species of ferns of which nearly 235 species are endemic (Chandra 2000; Dixit 2000); however, Fraser-Jenkins (2008) revisited the list and reported 47 species as endemic to India. Nearly 16% of global pteridophyte species are endangered and 22% are threatened (Brummitt et al. 2016). In India, 160 species are critically endangered, 82 are near threatened, and 113 are rare (Chandra et al. 2008). Fraser-Jenkins (2008) reported the high endemism is found in South India (27 sp.), followed by North Eastern India (7 sp.) and Western Himalayan ferns (2 sp.).

Pteridophytes constitute an important component of tropical and temperate biodiversity. They are ecologically important and have enormous potential as source of food, fodder, fiber, flavoring agents, aromatic oil, perfumes, dyes, and folk remedies. Besides their ornamental and medicinal values, aesthetic appeal, and exquisite foliage pattern which make them popular plants for landscaping, they have also been exploited as pollution indicator, insect repellents, antimicrobial agent, and phytoremediators (Shukla and Khare 2014). Ceratopteris richardii is recognized as an excellent model organism in developmental biology and physiological studies to identify and study mutants.

Epiphytic ferns grow in extreme environmental conditions which are very different from those of terrestrial species. Ferns found on bare bark are able to exploit microhabitats with low water availability; they drop their leaves under severe drought. Because of their habitat, the water availability is irregular; thus these plants are tending to endure drought stress. They develop different morphological, anatomical, and physiological adaptations necessary to conquer the epiphytic habit (Ranker and Haufler 2008). These include changes in nutrient and water uptake mechanisms (Watkins Jr et al. 2010), the evolution of desiccation tolerance in the gametophyte generation (Watkins Jr et al. 2007), alteration in sporophytic hydraulic and stomatal systems, and shifts in reproductive strategy (Watkins Jr et al. 2010; Pittermann et al. 2011). Epiphytic ferns show many water balance mechanisms like poikilohydry. Many species from the family Polypodiaceae like Pyrrosia longifolia shows a shift from C3 cycle to Crassulacean acid cycle for photosynthesis during transition from gametophyte to sporophyte generation (Martin et al. 2005). Successful epiphytic species would have required major modification in the gametophyte generation to include indeterminate growth, extreme stress tolerance, and an outcrossing breeding system.

In heterosporous taxa the gametophytes are endosporic, which is self-supportive in apt nutrition supply and ensures embryo development; however, in homosporous taxa the gametophytes are exosporic and dependent on the external environmental conditions. Pteridophytes display various other pathways of reproduction through apospory and apogamy (Ranker and Haufler 2008). In some ferns, the gametophytes also show vegetative propagation by means of dispersible gemmae. Gemma production has been commonly observed in epiphytic ferns. The fern Vittaria appalachiana is only known from its gametophytes (Chambers and Emery 2016). It multiplies by vegetative buds, or gemmae, and forms mats in dark, moist cavities and rock shelters in the Appalachian Mountains.

Ferns are long-lasting, stress tolerant (water, salt, heavy metals), fungal, and pest resistant and possess a number of useful phytochemicals. They play important role in restoration program. A large number of fern taxa in India have become threatened due to decreasing trend of their natural populations caused by changing land use pattern and overexploitation for commercial purposes. It will be pertinent to understand the diversity, distribution, ecology, reproductive biology, and present status in the context of threat of extinction and endangered situation and propose strategies for their conservation. In vitro culture of spores of ferns has been found beneficial for mass propagation, conservation of germplasm, and phytochemicals analysis. The program gives priority to the curation of plant collections and development of multiplication protocol, addressing reproductive constraints of populations. We studied the reproductive biology, mating system, and colonization potentiality in some fern species such as Acrostichum aureum L., Ceratopteris thalictroides (L.) Brongn., Tectaria polymorpha (Wall. ex Hook.) Copel., Phymatosorus scolopendria (Burm.f.) Pic.Serm., P. membranifolium (R.Br.) S.G. Lu, Christella dentata (Forssk.) Brownsey & Jermy, Macrothelypteris torresiana (Gaudich.) Ching, Platycerium bifurcatum (Cav.) C. Chr., Phlebodium aureum (L.) J. Sm., and Allantodia aspera (Blume) Ching and studied the effects of nitric oxide in the growth and development of gametophyte and gametangial production in Ceratopteris thalictroides.

2 Materials and Methods

Mature fertile fronds with well-developed sori of the abovementioned fern species were collected from Wayanad, Athirappilly, Pooyamkutty, and Calicut Kerala in the month of October 2016. Mature sporophylls were packaged in brown paper packets and stored in a desiccator at room temperature for the release of the spores. The Parker’s macro and Thompson’s micronutrient culture medium (P&T) was used for the culture of spores (Srivastava and Uniyal 2014). Periodically the spore germination percentage, gametophyte growth, differentiation, and gametangial ontogeny were observed under Olympus CX21 microscope and photographed using Olympus camera EP-1. Gametophytes in stock culture were observed at regular intervals and were isolated in separate petri plates containing P&T medium before initiation of gametangia. The ratios of gametophyte bearing male, female, bisexual, or neuter conditions were recorded. The gametophytes were transferred in other plates in two sets (Set 1, 25 petri plates with single gametophyte in each isolate culture; Set 2, 5 petri plates with 25 gametophytes in each composite culture). After the initiation of the gametangia in stock cultures, watering of all the isolate and composite populations was done from above with sterile distilled water twice a week to facilitate fertilization. Percentage of sporophytes was recorded in both the abovementioned sets (Srivastava et al. 2007). Genetic load and fern species are generally estimated by isolating gametophytes and counting the proportion that does not produced viable sporophytes in laboratory cultures (Klekowski Jr 1970). We calculated isolate potential and sib potential following Peck et al. (1990). Isolate potential (the frequency of successful intragametophytic events) is the number of sporophytes produced by isolated gametophytes divided by the total number of isolated gametophytes for that species. Sib potential (the frequency of successful intergametophytic events) is the number of sporophytes produced by paired gametophytes divided by the total number of gametophytes in the paired treatment for each species.

3 Results and Discussion

3.1 In Vitro Spore Culture

In vitro spore culture technique is a very important tool for mass propagation and reintroduction and to study the reproductive biology of fern species. Dyer (1979) discussed the spore storage techniques, spore germination, composition of the medium, and cultural conditions like temperature, light quality, and intensity that influence the gametophyte development. Spore sterilization is necessary before sowing (Wu et al. 2009). Sheffield et al. (2001) suggested that early developments of gametophyte occur better in solid media than liquid media. Chang et al. (2007) reported the light quality, pH, and concentration of sugar in the medium significantly influence the spore germination, early gametophyte development, and development of gametangia. Fern spores are relatively resistant to extreme conditions of the environment and remain viable in active metabolic state for quite long period of time. Viability of the spore varies from species to species. Generally, spore retains viability for 2–3 months, but some may have over a year like Onychium, Pityrogramma, etc. In some taxa which produce green spores like Osmunda, the spore remains viable for few days. Several cases of long viability are reported by Ballesteros (2010).

The spores are grown in artificial medium, the events of germination pattern and growth forms of gametophyte are recorded, and reproductive attributes are studied to establish their mating system. Regenerated sporophytes of rare taxa are hardened and reintroduced in their natural habitat that helps in their conservation in wild. In culture conditions, though spore persists in a viable condition for long, they require adequate moisture, optimal temperature and pH, oxygen, and light. Generally, spore on germination produces a primary rhizoid followed by the formation of an elongated uniseriate germ filament. In some more primitive taxa, in place of a germ filament, a mass or a plate of cells was formed.

For in vitro germination, the sori containing fronds can be sterilized using sodium hypochlorite and sown on Parker’s & Thompson’s culture media. Two different set of experiments comprising of composite gametophyte culture and isolate gametophytes culture can be established to investigate spore germination percentage, gametophyte development, sexual expression, mating system, reproductive barriers, genetic load, and colonization and regeneration potentiality.

Nayar and Kaur (1971) recognized three distinct types of the homosporous ferns, namely, Polar, Equatorial, and Amorphous, on the basis of patterns of spore germination. Polar germination is further classified into three types: Anemia, Osmunda, and Vittaria types. Equatorial germination is classified into six types: Cyathea, Christiopteris, Gleichenia, Hymenophyllum, Mecodium, and Trichomanes types, on the basis of the plane and sequence of cell divisions. Vittaria type is the most common type of germination pattern in ferns. Osmunda type is the simplest type of Polar germination exhibited in Osmundaceae. Anemia type is characteristic of Anemiaceae and Lygodiaceae. Among Equatorial germination is the Gleichenia type, the simplest type which occurs in the Gleicheniaceae, Dipteridaceae, Loxogrammaceae, and many of the Polypodiaceae. A variation of this is found in Christiopteris (Polypodiaceae). The Cyathea type of spore germination is observed in Cyatheaceae, Loxsomaceae, and Cheiropleuriaceae. Hymenophyllaceae shows a characteristic of tripolar growth. Trichomanes type occurs in Trichomanes; Mecodium type is found in the genus Mecodium.

In our study, Vittaria type and polar type of spore germination pattern were recorded in most of the species. Drynaria type gametophyte development pattern is observed in all epiphytic species presently studied. Much variation is observed among the terrestrial species. T. dentata and M. torresiana showed Drynaria type, T. polymorpha showed Aspidium type, and A. aspera showed Adiantum type. Aquatic species, C. thalictroides (Fig. 12.1a, b), and A. aureum showed Ceratopteris type. Following germination, the spore produced a rhizoid. In case of epiphytic species filament which appeared in 10–15 days after sowing, in aquatic species, it appeared much earlier (less than 10 days), while in terrestrial species, it delayed in T. polymorpha (25 days) and A. aspera (28 days). Some abnormally enlarged filaments and branched filaments were observed in the dense population as found in T. polymorpha and P. scolopendria.

Fig. 12.1
figure 1

Development stages of Ceratopteris thalictroides. (a) Spore, (b) filamentous prothallus, (c) two-dimensional prothallus, (d) spathulate prothallus, (e) male prothallus with antheridia, (f) asymmetric prothallus with apical notch, (g) bisexual cordate prothallus, (h) antheridium, (i) archegonium, (j) isolate population, (k) composite population, (l) sporophyte ready for hardening

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On comparing epiphytic, aquatic, and terrestrial fern growth pattern, we found that spores of aquatic and epiphytic ferns germinate early than those of terrestrial. Epiphytic species showed early germination. In Platycerium bifurcatum (Bhatia et al. 2018) and Phlebodium aureum, the spore germination began in 3–5 days, and in P. scolopendria and P. membranifolium, it took 7–10 days after spore sowing confirming that the spores of these two taxa require specific substratum and favorable conditions for germination just after released from the plants in nature. In general, the typical germination time was from 3 to 10 days. Among the terrestrial species, germination begun in 2–21 days. C. dentata germination begun in 2–3 days, in Macrothelypteris torresiana 5–7 days, and in Tectaria polymorpha 10 days, and Allantodia aspera took 21 days. In Allantodia aspera and T. polymorpha, spore took longer time in germination, indicating that viability of the spore germination in these two species is longer, and for prolonged period, therefore, population of these are comparatively healthier than other investigated species. Aquatic species took less time in spore germination. In Ceratopteris thalictroides, germination begun in 2–3 days, and Acrostichum aureum took 4–6 days to germinate (Bhatia et al. 2017). This indicates that plant take advantage of the prevailing moist conditions of the habitat and undergo rapid growth phase to complete the life cycle.

A comparative study on spore germination percentage in all species revealed that the germination percentage of epiphytic ferns is relatively low. The percentage of germination is highest in aquatic fern A. aureum (95%) (Bhatia et al. 2017) and lowest in epiphytic fern P. aureum (23.03%). However Allantodia aspera and M. torresiana being terrestrial fern showed germination as low as 24.59% and 26.92%, respectively, while P. bifurcatum being epiphytic fern showed as high as 85.39%.

Chang et al. (2007) reported the high germination (63.3%) occurred on Murashige and Skoog’s basal medium (half strength) supplemented with 2% sucrose at pH 7.7 under white light condition and red light, far red, blue, and white light caused maximum spore germination. Ranil et al. (2008) reported effective spore germination media, gametophyte morphology, and the successful raising of sporophytes from gametophytes in Cyathea walkerae Hook. Majumdar et al. (2010) obtained maximum germination at 0.023 μM IAA and maximum growth of gametophyte at 0.045 μM IAA, and maximum growth of sporophytes was observed in medium supplemented with 0.045 μM IAA+ 0.057 μM KN+ 0.023 μM IBA. Some protocols are optimized to obtain sporophytes in the laboratory in tree fern, Platycerium and Asplenium (Rybczyński and Mikuła 2011; Camloh and Ambrožič-Dolinšek 2010; Marszał-Jagacka and Kromer 2011).

3.2 Gametophyte: Growth Forms and Morphology

The fern gametophytes are haploid, simple in structure and independent of the sporophyte plant, but it has great potential to yield important and significant insights in many areas of plant development research. It can serve as a model system for experimental studies especially morphological studies and genetics (Mikuła et al. 2015). Its small size (1–1/2 cm) offers many advantages to culture in a petri dish, and all aspects of its growth and development can be observed and manipulated in a nondestructive way by using simple tools. Another advantage of using gametophyte is its relatively short maturation period which allows a rapid evaluation of treatment effects and execution of cumulative sequence experiments in a much lesser time as compared to the higher plants.

The fern gametophytes show certifiable criteria for taxonomic and phyletic studies. The morphology of the gametophyte, events of spore germination to the complete development of gametophyte, and gametangia formation are significant to complete and update revision of the species. The significance of the morphological diversity of species to the evolution and systematics of ferns cannot be properly assessed without further knowledge of morphological variation and reproductive strategies of ferns especially of epiphytic species. Fernandez and Revilla (2003) documented the nutritional, environmental, and other factors required for growth and development in different stages of the life cycle of ferns and highlighted some protocols for enhancing their morphogenic and reproductive capacity. De Brum and Randi (2002, 2006) reported the effect of irradiation, temperature, and cryopreservation on spore germination, gametophyte, and sporophyte development. Gametophyte shows a wide range of responses to different light intensities. Light can affect in the growth of the fern from spore germination to sporophyte development in terms of the length of photoperiod or its wavelength. Red light promotes spore germination while far red inhibits (Sou et al. 2015). In addition to far red, blue and UV lights also inhibit germination, and this effect can be neutralized by red light. Manipulation of the sucrose concentration in the medium stimulates the multiplication of the gametophyte, but its absence may induce the production of gemmae (Goller and Rybczynski 2007). Addition of sucrose is also reported to induce apogamy in many ferns (Atallah and Banks 2015), or it may inhibit spore germination as reported in Bolbitis costata (Majumdar et al. 2010) or promote it (Sheffield et al. 2001).

The prothallus of majority of ferns is one cell thick that permits to trace growth as a function of increase in surface area. It enables the investigator to make a rapid assay of relative development and to establish precise growth curves for an individual. Wada (2007) reported cell lineages of a prothallus and growth at cellular level. The sequence of effects of chemical treatment or surgical treatment can be observed throughout the entire living organism without killing it. It provides a means of studying morphogenesis at biochemical level. We recorded the events of gametangial initiation, fertilization, and sporophyte development in some epiphytic ferns. The morphological variations of the gametophyte and reproductive strategies of the epiphytic ferns are assessed in vitro, so that these characters can significantly be used in systematic studies of ferns.

A great variation in gametophytes has been exhibited by ferns like standard cordate-thalloid shaped, filamentous, ribbon shaped, strap shaped, and tuberous (Pinson et al. 2017). Cordate-thalloid gametophyte is most common in ferns, which has a distinct meristem in the apical notch and a well-defined midrib. Ribbon-like gametophytes are narrow, elongated, and laterally branched and lack midrib and well-organized meristem like in Loxogrammaceae, Vittariaceae, some Hymenophyllaceae, and some Polypodiaceae. Here the meristem is described as discontinuous marginal meristem (Imaichi 2008). Strap-like gametophyte are intermediates between cordate and ribbon like and much elongated, unbranched, and interrupted midrib and cordate apex with a definite apical meristem like in Grammitidaceae, some Lomariopsidaceae, Elaphoglossaceae, and Polypodiaceae. Tuberous and filamentous types are perennial and rare in ferns. Filamentous type have branched uniseriate filament with indefinite growth. These are found in Schizaea and in some Hymenophyllaceae. Tuberous type is subterranean, erect, and irregular in shape with uniseriate growing apex. Tuberous gametophytes are subterranean, cylindrical, or irregular in shape and very slow growing as found in Ophioglossaceae, Actinostachys, and Lophidium (Schizaeaceae), and Stromatopleris (Gleicheniaceae) possess tuberous gametophytes. Such diversity in the morphology of gametophytes enables them to grow indeterminately and branch to form clones of perennial gametophyte. Farrar (2003) documented the significance of gametophyte morphology in the classification of the ferns.

Nayar and Kaur (1971) described seven patterns of development in homosporous ferns, viz., Adiantum type, Aspidium type, Ceratopteris type, Drynaria type, Kaulinia type, Marattia type, and Osmunda type, on the basis of sequence of cell divisions during the stages of development, the region at which a meristem is established, and the resultant form of the adult thallus. In all, except the Marattia and Osmunda types, spore germination leads to a uniseriate, elongated, germ filament composed of barrel-shaped chlorophyllous cells and bearing one or more rhizoids at the basal end. Usually there is a sudden change which occurs in the plane of cell divisions, at a time when germ filament is two to ten0 cells long. However, it is often much delayed in some taxa, such as the Grammitidaceae, and results into more extensive uniseriate stage. Sometimes unfavorable conditions also delay this change. Some genera show no change in the plane of cell divisions, and the prothallus remains filamentous throughout their life as in Schizaea and Trichomanes.

In epiphytic species, two-dimensional prothallus was achieved in 15–20 days, in aquatic it occurred between 15 and 20 days, while in terrestrial species, it was formed in more than 25 days. Further divisions resulted in the development of spathulate thallus in 28–35 days. One or two terminal hairs on either side of the developing notch may be produced at the apex. The marginal cells in apical region of gametophytes usually became smaller and thick and transform into meristematic cells. Spathulate gametophyte developed in 21–35 days in epiphytic species with earliest emergence in P. bifurcatum (Bhatia et al. 2018) and P. scolopendria in 25 days, P. aureum in 28 days, and M. nigrescens in 35 days.

In terrestrial species, spathulate stage was achieved in more than 30 days. M. torresiana showed spathulate stage in 20 days, C. dentata in 30 days, and A. aspera and T. polymorpha in 40 and 42 days, respectively. Many unicellular hairs developed on the margins and a few superficial hairs at the spathulate stage in most of the species of our study. In aquatic species, spathulate growth was very fast developed much early on the 15th day in C. thalictroides, and A. aureum showed spathulate stage (Fig. 12.1c–e) in 20 days.

In epiphytic species cordate gametophyte was achieved much early in 35–42 days. The earliest development was observed in P. bifurcatum (35 days) and delayed in M. nigrescens showed last (42 days). However, terrestrial ferns showed wide range of days in development of cordate thallus in C. dentata and M. torresiana in 50 days and T. polymorpha and A. aspera in 60 days. In C. thalictroides, cordate thallus (Fig. 12.1f, g) developed in 28 days, followed by A. aureum in 40 days. In C. thalictroides, A. aureum, and A. aspera, an asymmetric lopsided thalloid shape gametophyte was observed (Bhatia et al. 2017). P. bifurcatum (Bhatia et al. 2018), P. scolopendria, P. membranifolium, and P. aureum showed persistent gametophyte which grew for more than 2 years. In the terrestrial species, C. dentata and M. torresiana gametophyte remained for more than a year. However, they developed profuse clone forming branching in older gametophytes. Perhaps the long-lived gametophyte growth strategy adopted by ferns in the epiphytic habitat applies more generally, and the terrestrial ferns develop the competitive advantage over mosses in the terrestrial habitat.

De Brum and Randi (2002, 2006) and Chang et al. (2007) reported the effects of pH, light quality, presence of different sugars, and different concentrations of sucrose in the medium on spore germination, early gametophyte development, and change in the reproductive phase. Spore storage at low temperature has become a profitable tool for ex situ fern conservation (Li et al. 2010; Quintanilla et al. 2002).

3.3 Gametangial Sequence and Reproductive Biology

The main functions of gametophyte are sex determination and gamete production and develop a system for mating. Verma (2003) mentioned three types of mating system in homosporous ferns, each expressing different level of inbreeding. These are intragametophytic mating or intragametophytic selfing (mating between gametes developed from same gametophyte), intergametophytic selfing (mating between gametes from different gametophyte developed from same parent sporophyte), and intergametophytic crossing (mating between gametes derived from different sporophytes). The intragametophytic selfing results in a totally homozygous sporophyte. Development of both male and female gametes simultaneously and the gametophyte genotype which must be free from any genetic lethal defect are the two prerequisites for the success of this system. Intergametophytic crossing is equivalent to outbreeding in heterosporous ferns. The survival of the completely homozygous sporophytes may be hampered by deleterious recessive alleles because lethal recessive gene in homozygous condition causes death and removes the individual from the progeny during natural selection. Lethal or deleterious recessive alleles can be eliminated by natural selection in homozygous sporophytes over a long period of time, and alternatively “frequent occurrence of intragametophytic selfing will eliminate recessive lethal effect rapidly” (Sessa et al. 2016). Wubs et al. (2010) reported that these deleterious recessive genes usually expressed as abortive embryos which appear as swollen tissue within the archegonium or small and abnormal sporophytic tissue or abnormalities in the production of the first few fronds or roots.

Haufler (2002) explained the correlation between homozygosity and polyploidy through a model. Wood et al. (2009) reported that homosporous ferns have higher number chromosomes as compared to the heterosporous fern. There is an excellent correlation between the utilization of polyploidy and the evolution of taxa in the homosporous pteridophyta. Polyploidy provided the means to main inter-locus heterozygosity and release of variability via occasional homoeologous chromosome pairing and recombination (Haufler 2014). Gametangial pattern in the present study showed that all the species studied were protandrous. Antheridia appeared in the terrestrial species with earliest in 30 days in C. dentata and in 35 days in M. torresiana. However, it took more time in A. aspera (42 days) and T. polymorpha (55 days). Antheridia appeared much early in the aquatic species with earliest in C. thalictroides (Fig. 12.1h) in 15 days and in A. aureum in 35 days. In epiphytic species such as P. bifurcatum, the antheridia appeared in 35 days (Table 12.1) followed by 40–50 days in P. scolopendria, P. aureum (Bhatia et al. 2017), and P. membranifolium. In the aquatic species, C. thalictroides archegonia appeared as early as in 20 days (Fig. 12.1i and Table 12.2) and in A. aureum in 40 days. Among the terrestrial species, C. dentata and M. torresiana, it was formed in 50 days, and A. aspera and T. polymorpha developed archegonia in 63 days and 65 days, respectively. In case of epiphytic species, archegonia developed in moderate time. In P. bifurcatum (Table 12.1) and P. aureum, it developed in 49 days and in P. scolopendria and P. membranifolium in 60–65 days.

Table 12.1 Chronological changes in the sex ratio of a composite culture in Platycerium bifurcatum (Cav.) C. Chr
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Table 12.2 Chronological changes in the sex ratio of a composite culture in Ceratopteris thalictroides (L.) Brongn
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We observed formation of dimorphic gametophytes in the composite population. Male gametophytes were small, spathulate, and ameristic, whereas bisexual gametophytes were large, cordate, and meristic. In C. thalictroides, such dimorphism (Fig. 12.1f, g) was prominent and well established under the influence of antheridiogen. Later, these early formed male prothalli may become bisexual as found in A. aureum, C. dentata, M. torresiana, and A. aspera. Bisexual gametophyte in P. aureum (Bhatia et al. 2017) and P. bifurcatum appeared in 70 days (Table 12.1), and in P. membranifolium and P. scolopendria, it appeared in 80 days. Among the terrestrial species, earliest bisexual gametophyte developed in M. torresiana in 50 days, in C. dentata and T. polymorph in 61–65 days, and in A. aspera in 84 days. In aquatic species, bisexual stage appears as early as in 15 days in C. thalictroides (Fig. 12.1g and Table 12.2) and in A. aureum in 42 days.

There was a sexual gap of 15–20 days in the expression of antheridia followed by the formation of archegonia in P. membranifolium, P. scolopendria, and P. bifurcatum (Bhatia et al. 2018). Therefore the mating between the gametangia originating from an individual gametophyte did not occur, which suggest that the gametophytes escaped intragametophytic selfing for sporophyte production. However, in case of P. membranifolium, there was no sporophyte in isolated population; thus the possibility of intragametophytic selfing is very rare. This indicates that though both the sexes developed in the same thallus, the lap in their emergence did not provide enough opportunity for their mating or they may be incompatible. However, there were numbers of antheridiate as well as archegoniate gametophytes in composite culture, which have imparted the gametangial fusion through intergametophytic selfing by the fusion of gametes which arise on two or more gametophytes of common parental origin. Thus intergametophytic mating was shown by P. scolopendria, P. membranifolium, and P. bifurcatum (Table 12.3).

Table 12.3 Breeding behavior of different populations in Platycerium bifurcatum (Cav.) C. Chr
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In aquatic species C. thalictroides and A. aureum and terrestrial species C. dentata, M. torresiana, T. polymorpha, and A. aspera, the sexual gap between antheridia and archegonia was less than 15 days (Table 12.4). In epiphyte P. aureum, antheridia appeared with a gap of 5 days after the expression of archegonia. It is suggested that both the sexes occur on an individual gametophyte for a very short gap; hence these genera exhibited possibilities for gametic union between gametes developed either on the same gametophyte or two different gametophytes of common parental origin. The gametophytes remained bisexual with a slight gap of a day. Therefore, they performed reasonable intragametophytic selfing as well as intergametophytic selfing between the gametangia arisen from the common parent origin. As both the gametangia expressed with a short gap of period, therefore, they did not impart cent percent selfing and must have resulted in the reasonable rate of genetic load.

Table 12.4 Breeding behavior of different populations in Ceratopteris thalictroides (L.) Brongn
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Wubs et al. (2010) studied species to colonization over longer distances and found a mixed mating system in Asplenium scolopendrium with outcrossing when possible and occasional selfing when needed. The resulting sporophyte, which is completely homozygous, shed large amounts of spores over time. Each year this creates a bed of gametophytes in the vicinity of the mother plant. Any unrelated spore, which arrives, is then selectively favored to reproduce and contribute its genes to the new population. Thus, while selfing facilitates initial colonization success, inbreeding depression promotes genetically diverse populations through outcrossing. The results provide further evidence against the overly simple dichotomous distinction of fern species as either selfing or outcrossing. Page (2002) discussed some important ecological strategies of ferns and allied plants and their underlying selection pressures, based on an extensive survey of tropical and temperate species. Khare (2003) described Dryopteris cochleata as a good colonizer in disturbed areas due to the presence of intragametophytic selfing, intergametophytic selfing, as well as intergametophytic crossing. Lott et al. (2003) suggested that the mating system of ferns shows enough plasticity to colonize a diversity of habitat. Outcrossing is density dependent; thus intragametophytic selfing would likely be the primary mode of reproduction in the pioneer population. As the density increases, opportunities for outcrossing would be greater as number of spores and resulting gametophytes increases. The presence of potential antheridial system supports this hypothesis, since they increase gametophyte density, particularly of males by inducing dark germination of spores. Khare et al. (2005) concluded that the species has less genetic diversity and less genetic load; thus it is a potential colonizer. Srivastava and Uniyal (2014) reported that Polystichum lentum is a good colonizer because sufficient numbers of sporophytes were developed by selfing but a few plants were found in the natural habitat of its occurrence.

Sex in an organism may be determined genetically or environmentally or by the interaction of the two. In organisms that exhibit environmental sex determination, individuals in poor quality habitat, low nutrient availability, high population density, or who are slow growing would expect to develop into males (DeSotot et al. 2008; Quintanilla et al. 2007). In many ferns antheridiogen, a pheromone secreted by bisexual gametophyte can control the sex expression in undifferentiated spores present in the in the surrounding. Though it is generally species specific, some of them show a broad range of plants to act upon (Jimenez et al. 2008). In many homosporous ferns including C. richardii, ACE has been shown experimentally to effect the gender decision of undifferentiated spores. The absence of antheridiogen ACE directs the spores toward hermaphrodite development and contains antheridia, archegonia, and a notch meristem (Tanurdzic and Banks 2004). The lack of ACE is thought to lead to expression of both female and male genes. In the presence of ACE, spores tend toward male development through a process called induction, which is thought to be a consequence of the signal transduction system initiated by ACE that leads to the suppression of female genes (Strain et al. 2001). This signal transduction system is reactive to ACE within a narrow window of development, approximately 3–6 days from sowing, and ACE exposure outside of this window has no effect (Ganger and Sturey 2012). Not all spores grown in the presence of antheridiogen develop into males, or its absence leads to total bisexual gametophytes (Atallah and Banks 2015).

Relative position of antheridia and archegonia on the bisexual or unisexual prothalli in a population directly affects the breeding system of the species. This can vary among species, with the presence or absence of antheridiogen system and with density of the gametophyte population. Antheridiogen may act in two possible ways. It may act directly through signal transduction by suppressing female genes or alternatively may slow down the growth and development coupled with limited resources responsible for induction. ACE was reported to have a negative effect on growth of male gametophyte in a dosage-dependent manner in Woodwardia radicans (Quintanilla et al. 2007), Onoclea sensibilis, and C. richardii. ACE effected the growth of bisexual gametophyte as well, but the effect is not dosage dependent, and gametophytes remain receptive to ACE only prior to the decision to become bisexual (Ganger and Sturey 2012).

3.4 Sporophyte Production

Among the epiphytic species investigated in the present study, P. bifurcatum, P. scolopendria, and P. membranifolium showed intergametophytic mating system, whereas P. aureum showed mixed mating type. All terrestrial species studied, viz., C. dentata, M. torresiana, A. aspera, and T. polymorpha, exhibited mixed type of mating system showing both intra- and intergametophytic mating type. However, intergametophytic mating was predominant as expressed by high percentage of sporophyte production in composite culture than that of isolate culture. Aquatic species C. thalictroides and A. aureum exhibited mixed type of mating system. Majority of the ferns studied have potential of both intragametophytic (gametophytic selfing) and intergametophytic (sporophytic selfing) and exhibited mixed mating type.

In the present study, sporophytes were produced in earliest composite culture, in aquatic species C. thalictroides in 40 days (Fig. 12.1k and Table 12.4) followed by terrestrial M. torresiana in 65 days, A. aureum in 77 days, C. dentata in 85 days, T. polymorpha in 90 days, and A. aspera in 120 days. In epiphytes sporophyte emerged little late. In P. aureum first sporophyte appeared in 80 days in composite population and 88 days in isolate population. In P. bifurcatum sporophyte emerged in 120 days in composite and 145 days in isolated population (Table 12.3). In P. scolopendria sporophyte developed in 150 days in composite and 95 days in isolated cultures. In P. membranifolium sporophyte developed in 170 days in composite cultures and no sporophyte in isolated culture.

The sporophyte production in composite culture in epiphytic species revealed that P. bifurcatum showed minimum sporophyte production percentage (20%) (Table 12.3), followed by P. scolopendria (50.66%), P. membranifolium (54.66%), and P. aureum (80%). Among the terrestrial species, the percentage of sporophyte production in composite culture was minimum in A. aspera (29.33%) followed by T. polymorpha (36%) and C. dentata (44%) and maximum in M. torresiana (96%). Aquatic species shows high percentage of sporophyte production (A. aureum 68% and C. thalictroides 73.33%) (Fig. 12.1l and Table 12.4).

The species M. torresiana (96%), A. aureum (68%), and C. thalictroides (73.33%) with high sporophyte production showed mixed type of mating, i.e., both the intra- and intergametophytic selfing mode were favored in these species. Minimum percentage of sporophytes developed in isolated culture of A. aureum (25%), followed by C. dentata (32%), A. aspera (36%), T. polymorpha (56%), C. thalictroides (Fig. 12.1j) (60%), and M. torresiana (84%).

Majority of the ferns have potential of both intragametophytic (gametophytic selfing) and intergametophytic (sporophytic selfing) and exhibited mixed mating type. Among the epiphytic species, Phlebodium aureum, P. scolopendria, and P. membranifolium showed intergametophytic mating system, whereas Platycerium bifurcatum showed mixed mating type. All the terrestrial species studied, Thelypteris dentata, Macrothelypteris torresiana, A. aspera, and Tectaria polymorpha, exhibited mixed type of mating system. Aquatic species Ceratopteris thalictroides and Acrostichum aureum exhibited mixed type of mating system. Less genetic load seems to be found in M. torresiana and C. thalictroides. Mixed mating type provides flexibility in ferns and allows them to exercise both evolutionary and ecologically strategies. They use intragametophytic mating to establish new populations through long-distance wind dispersal of single, minute spores and simultaneously maintaining significant genetic variation through intergametophytic mating.

Makowski et al. (2016) advocated the application of the in vitro tissue culture and cryopreservation methods for propagation and conservation of the fern Osmunda regalis. They reported increased number and early development of sporophyte by integrating two techniques. Ravi (2016) induced polyembryony in Pteris tripartita in half strength MS basal media with 3 mg/L of BA and 30% sucrose and found similarity of these sporophytes to the normally developed sporophytes. Fernandez and Revilla (2003) reported that low concentration of ammonia in the media (Knops, Knudson, or 1/4 MS) was most effective for gametophytic growth of Osmunda regalis. Gemmae formation was also reported for the first time in this species with sucrose and in darkness.

3.5 Effects of Nitric Oxide (NO) in the Gametophyte Development and Gametangial Production in Ceratopteris Thalictroides L

The immense effects NO on plant cell functioning and plant development and response to changes in the environmental conditions have been studied. Nitric oxide (NO) and its role in physiology especially in regulating blood pressure and relieving heart conditions were first discovered in animal cell. The enzyme responsible for NO generation in animal organisms is nitric oxide synthase (NOS) with the participation of O2and NADPH.

It is has also been found to be involved into the diverse biological activities in plants. Nitric oxide is a crucial signaling molecule with diverse physiological functions in plants. It plays an important role in plant growth and development, starting from germination to flowering, ripening of fruit and senescence of organs, respiratory metabolism, as well as plant response to abiotic and biotic stressors. NO play important role in the disruption of seed dormancy and seed germination (Arc et al. 2013) and root nodules (Boscari et al. 2013). Baudouin and Hancock (2014) discussed NO-dependent protein modification during germination of seeds. NO has a key role in the control of stomatal apertures (Gayatri et al. 2013).

In plants, nitric oxide can be produced by any of the four routes: (1) by nitric oxide synthase, (2) by plasma membrane-bound nitrate reductase, (3) by mitochondrial electron transport chain, or (4) by non-enzymatic reactions. SNP treatment (100 μM) is also reported to inhibit hypocotyl growth in potato, lettuce, and Arabidopsis (Beligni and Lamattina 2000). Nitric oxide has been found to effect photosynthesis, photorespiration, and photosynthetic electron transport chain directly.

Growth and development of gametophyte under the influence of sodium nitroprusside (SNP – a NO donor) were recorded in Ceratopteris thalictroides. In general germination of spores was found to be increased by SNP treatment at higher concentration (50 μM and 100 μM) while inhibited at lower concentrations (5 μM and 25 μM). The percentage of germination observed in first week was minimum at 5 μM NO (11.35 ± 18.19), and with the increase in NO concentration, the percentage germination increased. The percentage germination at 5 μM and 25 μM was less than that of controls. At 50 μM (91.14 ± 20.09) and 100 μM (94.72 ± 6.95), the percentage germination in first week was much higher than that of the control. The promotion was found to be higher at higher concentrations of the NO (50 μM and100 μM) (Table 12.5).

Table 12.5 Average number of prothallial cells and average number of rhizoids produced in different treatments in 2 weeks
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SNP supplementation in the growth medium inhibited the vegetative growth of the prothallus. The growth was found to be retarded by NO treatment. The growth of the prothallus was recorded in terms of number of prothallial cells and rhizoidal cells. The growth was found to be retarded with the change in concentration of NO as compared to the control. Lower concentrations (5 μM & 25 μM) were found to be more effective in inhibiting the growth. In the second week, the number of prothallial cells observed was very less in all treatments than that of the control (197.7 ± 26.80). The number of rhizoidal cells in control (9.7 ± 1.95) was much higher than that found in all NO treatments. With increase in NO concentration, the number of prothallial cells as well as rhizoids increased, indicating that growth inhibition is less effective at higher concentration of SNP (50 and 100 μM). With increase in NO concentration, the number of prothallial cells increased indicating that growth inhibition is less effective at higher concentration of SNP (NO). However, 50 μM SNP has no inhibitory effect on the number of prothallial cells; rather it was found to promote the number of rhizoids. The number of rhizoid (15.5 ± 6.36) was much higher than that of control (10.6 ± 2.17) (Table 12.5).

Two-dimensional stages reached in 15 days with all the treatments of NO, which was achieved in 7 days in control. However, there was a quantitative increase in the percentage of two-dimensional stage with the increase in NO concentration, but the number of two-dimensional prothalli decreases. Spathulate stage reached in 4 weeks in all the treatments. In bisexual prothalli, usually the basal cell was not divided and remained short and broad. In the fifth week, the thallus developed a notch in the apices and resulted into cordate type in all the treatments. The number of prothallial cells and rhizoids increased with the increase in concentration of SNP, but it was less than that of control. But 50 μM showed exceptionally higher values for the number of prothallial cells and rhizoids; however, it was less than that of control.

At higher NO concentrations (50 μM and 100 μM), growth of the prothalli was showing exceptional results. In some gametophytes the cells of the lower portion were highly elongated. Gametophytes were much asymmetrical and dissected. Some gametophytes were showing abnormal growth as the apical notch was lacking in them. Their middle portion grew continuously extending the two sides like drooping branch. In general, all NO treatments resulted in the browning and degeneration of cells after fifth week. At lower concentration (5 μM NO), it occurred after 5 weeks, but with the increase in concentration, it was found to be delayed. At 25 μM NO and 50 μM NO, it occurred in 6 weeks (Table 12.5), while in 100 μM NO, it took 7 weeks. The primary gametophyte was also found degenerating at its lower portion, and new secondary gametophyte was also developed.

In many homosporous fern species, the sexual generation is featured by sexual dimorphism, where male, female, and bisexual (hermaphroditic) prothalli are formed. Such sexual phenotypes are induced by antheridiogen, which is secreted by the young gametophytes especially at the cordate stage (Tanaka et al. 2014; Atallah and Banks 2015). Antheridiogen is released into the external environment, which induces the development of antheridia in the nearby asexual gametophytes (Strain et al. 2001). In our experiment, Ceratopteris thalictroides showed male prothalli, female prothalli, as well as bisexual prothalli.

Antheridia formation was promoted at higher concentrations of NO (50 μM and 100 μM). At these higher concentrations, majority of the prothallus, antheridia developed in large numbers in 2 weeks which took 3 weeks in control. Both the numbers of male prothalli and the numbers of antheridia per prothallus were increased with the concentration of NO. Although under controlled conditions (without NO), the formation of male and female prothalli was recorded; however, no female prothalli were formed under all the treatments of NO. Later a few bisexual prothalli were found at late spathulate or early quadrate stage. In general, development of bisexual stage was delayed by NO treatment. Bisexual prothallus appeared in 28 days in all concentration (5 μM and 25 μM and 100 μM) while concomitant with control at concentration at 50 μM (21 days). The quantitative increase was found in number of bisexual prothalli with the increase in concentration of NO. In 7 weeks (49 days), sporophyte was developed in cultures supplemented with 5 NO only, while in all other treatments, no sporophyte was observed in 7 weeks. In control setup, first sporophyte was produced after 40 days of spore sowing.

Nitric oxide shows a dose-dependent effect on the growth and development. Low micro-molar concentrations produced an increase in the rate of leaf expansion, whereas it does not show any promoting effect at higher concentrations by using NO donor sodium nitroprusside (SNP) as reported in wheat and pea seedlings (Tian and Lei 2006). Low micro-molar concentrations of SNP are found to induce root growth in maize. Treatment with SNP led to the inhibition of primary root length and led to a higher number of lateral roots of tomato seedlings (Correa-Aragunde et al. 2006) and adventitious root formation in cucumber (Pagnussat et al. 2002). SNP treatment enhances the rate of photosynthesis, chlorophyll content, transpiration rate, and stomatal conductance in cucumber seedlings (Fan et al. 2007). SNP has been found to decrease the level of enzymes that regulate photosynthesis in wheat. It is possible that ABA function as an antagonist of antheridiogen is also involved in the formation of antheridia (Romanenko et al. 2020).

4 Conclusions

The data on the reproductive biology of the species is an essential aspect which would help in propagation and multiplication of the threatened taxa. Information on spore germination studies is prerequisite for large-scale multiplication. Species showing low regeneration capacities are sorted and screened to find out the problems associated with their regeneration. The study is one step forward toward multiplying species with the help of biotechnology, and efforts will be directed toward their reintroduction to the natural habitats. From conservation point of view, it is important to identify the specific sites, besides the existing locations where reintroduction of the endangered/threatened species can be made through ecological niche modeling.

The data presented here are an important step toward a more thorough understanding of the capacity for intragametophytic mating in homosporous ferns and can serve as a guide for researchers wishing to know about the mating system(s) potentially in use by taxa of interest.

The fern gametophytes are haploid, simple in structure, and independent of the sporophyte plant, but it has great potential to yield important and significant insights in many areas of plant development research. It can serve as a model system for experimental studies especially morphological studies and genetics. Its small size (1–1/2 cm) offers many advantages to culture in a petri dish, and all aspects of its growth and development can be observed and manipulated in a nondestructive way using simple tools. Another advantage of using gametophyte is its relatively short maturation period which allows a rapid evaluation of treatment effects and execution of cumulative sequence experiments in a much lesser time as compared to the higher plants. The prothallus of majority of ferns is one cell thick that permits to trace growth as a function of increase in surface area. It enables the investigator to make a rapid assay of relative development and to establish precise growth curves for an individual and help in tracing cell lineages of prothalli and development at cellular level. The sequence of effects of chemical treatment or surgical treatment can be observed throughout the entire living organism without killing it. It provides a means of studying morphogenesis at biochemical level.

Nitric oxide plays a key role in improving early growth of plants. It can be can be used as a potential stress ameliorant in salt-affected soils. However, further studies are suggested to explore the NO-mediated biochemical mechanisms responsible for various aspects like growth and metabolism, sex expression, and different stress tolerance. It would be interesting to investigate the interaction between NO and plant hormones particularly IAA actively involved in growth activities like spore germination, gametophyte form, sex expression, and sporophyte development of plant species.