Sporophyte Formation Research Articles

Effects of nutrient concentration, germanium dioxide and seeding density on sporophyte production in the kelp Ecklonia radiata

Abstract Overcoming low frequencies of transition from the gametophyte to the sporophyte life history phase is vital to enable efficient hatchery production of the kelp Ecklonia radiata (Phaeophyceae) for aquaculture, restoration and conservation. Therefore, we investigated the effects of nutrient strength (full to 1/20 strength Provasoli enriched seawater (PES)), germanium dioxide strength (full and half strength) and application period (no application, 9-16, 9-30, 16-23 and 16-30 days post seeding), and seeding density (2, 4, 12 and 18 spores mm-2) on sporophyte production in E. radiata in three laboratory experiments. Nutrient strength and seeding density significantly affected sporophyte transition frequency, sporophyte formation frequency, and sporophyte length, with all three metrics consistently decreasing with decreasing nutrient strength and increasing seeding density. Sporophyte transition frequencies were more than four times higher in the full-strength nutrient treatment (58.2 %) compared to the 1/8 and 1/20 strength treatments (12.5 and 3.0 %, respectively), and 18% higher in the 2 spores mm-2 seeding density treatment (100.0 %) compared to the 18 spores mm-2 treatment (84.3 %). Germanium dioxide application period but not strength significantly impacted sporophyte transition frequency and sporophyte formation frequency. Transition frequencies were more than two-thirds lower when GeO2 was applied from day 9 onwards (<25 %) compared to day 16 onwards and no application (>75 %). Based on these results, we recommended using full strength PES, half strength GeO2 from day 16 onwards only if diatom contamination is an issue and seeding at a density of ≤10 spores mm-2 to maximise sporophyte production.

Gametophytes of Five Species of Rajasthan Ferns under Cultural Conditions

Gametophytes of seven species of ferns raised from spores on agar medium, are described. These are either cordate (Cheilanthes, Hypodematium, Cyclosorus and Tectaria) or lobed, thalloid (Actiniopteris, Asplenium and' Adiantum). All are homosporous ferns but in some of the taxa distinction is observed between antheridia and archegonia bearing prothalli (Cheilanthes, Hypodematium and Tectaria). Variations in morphology, sex expression and sporophyte formation are discussed.

In Vitro Multiplication and Ontogeny of Ferns Tectaria zeylanica (Houtt.) (Dryoptridaceae) from South India under Current Climate Conditions

The oak leaf fern, Tectaria zeylanica (Houtt.) Sledge, is an endangered fern in India, native to the Southeast Asian region. For the ex-situ conservation of this endangered fern, spores were collected and cultured aseptically on full-strength KC Medium with sucrose at pH 5.8. The spores are monolete with a prominent winged perispore. The exine ruptures at the laesura, and three or four germ filaments grow out, following a Vittaria-type germination pattern. The prothallial development was of the Aspidium type. Adult gametophytes are cordate and much broader than their length. Antheridia develop between 120 and 140 days. They are characteristically 3-celled, with a basal cell, a median cell, and an opercular cell. The female sex organs, archegonia, develop between 150 and 170 days. They are superficial and arranged on the lower side of the prothallus near the midrib and the meristematic region. Fertilization and subsequent development of the sporophytes were observed after four months. The highest percentage of sporophyte formation was observed on KC medium fortified with Kin 0.5 mg/l. Sporophytes had uniseriate, unicellular, and multicellular hairs, similar to those found on adult prothalli. The highest percentage of root formation was observed in the medium with IBA + NAA (1.0 mg/l). The in vitro raised plants showed a 54% establishment during hardening in the field. The present study has established a reproducible protocol for the ex-situ conservation of this endangered oak leaf fern.

Unicellular and multicellular developmental variations in algal zygotes produce sporophytes.

The emergence of sporophytes, that is, diploid multicellular bodies in plants, facilitated plant diversification and the evolution of complexity. Although sporophytes may have evolved in an ancestral alga exhibiting a haplontic life cycle with a unicellular diploid and multicellular haploid (gametophyte) phase, the mechanism by which this novelty originated remains largely unknown. Ulotrichalean marine green algae (Ulvophyceae) are one of the few extant groups with haplontic-like life cycles. In this study, we show that zygotes of the ulotrichalean alga Monostroma angicava, which usually develop into unicellular cysts, exhibit a developmental variation producing multicellular reproductive sporophytes. Multicellular development likely occurred stochastically in individual zygotes, but its ratio responded plastically to growth conditions. Sporophytes showed identical morphological development to gametophytes, which should reflect the expression of the same genetic programme directing multicellular development. Considering that sporophytes were evolutionarily derived in Ulotrichales, this implies that sporophytes emerged by co-opting the gametophyte developmental programme to the diploid phase. This study suggests a possible mechanism of sporophyte formation in haplontic life cycles, contributing to the understanding of the evolutionary transition from unicellular to multicellular diploid body plans in green plants.

Protocol: an improved method for inducing sporophyte generation in the model moss Physcomitrium patens under nitrogen starvation

BackgroundLand plants exhibit a haplodiplontic life cycle, whereby multicellular bodies develop in both the haploid and diploid generations. The early-diverging land plants, known as bryophytes, have a haploid-dominant life cycle, in which a short-lived multicellular body in the diploid generation, known as the sporophyte, develops on the maternal haploid gametophyte tissues. The moss Physcomitrium (Physcomitrella) patens has become one of the most powerful model systems in evolutionary plant developmental studies. To induce diploid sporophytes of P. patens, several protocols are implemented. One of the conventional approaches is to grow approximately one-month-old gametophores for another month on Jiffy-7 pellets made from the peat moss that is difficult to fully sterilize. A more efficient method to obtain all tissues throughout the life cycle should accelerate studies of P. patens.ResultsHere, we investigated the effect of nitrogen conditions on the growth and development of P. patens. We provide an improved protocol for the sporophyte induction of P. patens using a BCD-based solid culture medium without Jiffy-7 pellets, based on the finding that the formation of gametangia and subsequent sporophytes is promoted by nitrogen-free growth conditions. The protocol consists of two steps; first, culture the protonemata and gametophores on nitrogen-rich medium under continuous light at 25 °C, and then transfer the gametophores onto nitrogen-free medium under short-day and at 15 °C for sporophyte induction. The protocol enables to shorten the induction period and reduce the culture space.ConclusionsOur more efficient and shortened protocol for inducing the formation of sporophytes will contribute to future studies into the fertilization or the diploid sporophyte generation of P. patens.

Optimising the zoospore release, germination, development of gametophytes and formation of sporophytes of Ecklonia radiata

The kelp Ecklonia radiata has become a target for controlled cultivation. However, to date there are no standardised protocols for the hatchery stage of this species that result in high rates of germination, gametophyte development and transition to sporophytes. Therefore, the objective of this study was to quantify the effect of photoperiod, light intensity, temperature, nutrient media and use of GeO2 on the key hatchery processes of germination, gametophyte development and transition to sporophytes in controlled laboratory experiments. Germination of E. radiata was high (≥ 85%) throughout the study, regardless of treatments. Temperature had a major effect on the length of gametophytes, which increased with increasing temperature. The formation of sporophytes was favoured when individuals were maintained under 17 °C continuously, while reduced by approximately 30% when using F/2 compared to PES nutrient media. Overall, the recommended conditions for the hatchery stage of E. radiata are to maintain cultures under a 12 h L:12 h D photoperiod at 17 °C as this resulted in higher germination rates, good gametophyte development and higher transition to sporophytes compared to other treatments. Moreover, the use of GeO2 has to be limited to no more than 2 days as extended use has detrimental effects on the development of sporophytes. Finally, storage of sorus-bearing fronds of sporophytes up to 4 days after the collection from the field generally increased the number of released zoospores and is a simple mechanism to increase the fertility of brood stock.

Microscopic Stages of North Atlantic Laminaria digitata (Phaeophyceae) Exhibit Trait-Dependent Thermal Adaptation Along Latitudes

Kelp forests in the North Atlantic are at risk of decline at their warm temperature distribution margins due to anthropogenic temperature rise and more frequent marine heat waves. To investigate the thermal adaptation of the cold-temperate kelpLaminaria digitata, we sampled six populations, from the Arctic to Brittany (Spitsbergen, Tromsø, Bodø [all Norway], Helgoland [Germany], Roscoff and Quiberon [both France]), across the species’ entire distribution range, spanning 31.5° latitude and 12-13°C difference in mean summer sea surface temperature. We used pooled vegetative gametophytes derived from several sporophytes to approximate the genetic diversity of each location. Gametophytes were exposed to (sub-) lethal high (20-25°C) and (sub-) optimal low (0-15°C) temperature gradients in two full-factorial, common-garden experiments, subjecting subsets of populations from different origins to the same conditions. We assessed survival of gametophytes, their ability to develop microscopic sporophytes, and subsequent growth. We hypothesized that the thermal performance of gametophytes and microscopic sporophytes corresponds to their local long-term thermal history. Integrated gametophyte survival revealed a uniform upper survival temperature (UST) of 24°C among five tested populations (Tromsø to Quiberon). In contrast, following two weeks of thermal priming of gametophytes at 20-22°C, sporophyte formation at 15°C was significantly higher in southern populations (Quiberon and Roscoff) compared to the high-latitude population of Tromsø. Between 0-15°C, survival of the Arctic population (Spitsbergen) was negatively correlated with increasing temperatures, while the southern-most population (Quiberon) showed the opposite. Thus, responses of survival at low, and sporophyte formation at high temperatures, support the concept of local adaption. On the other hand, sporophyte formation between 0-15°C peaked at 6-9°C in the Quiberon and at 9-12°C in the Spitsbergen population. Sporophyte growth rates (GR) both in length and width were similar for Spitsbergen, Tromsø and Quiberon; all had maximum GRs at 12-15°C and low GRs at 0-6°C. Therefore, responses of sporophyte formation and growth at low temperatures do not reflect ecotypic adaptation. We conclude thatL. digitatapopulations display trait-dependent adaptation, partly corresponding to their local temperature histories and partly manifesting uniform or unpredictable responses. This suggests differential selection pressures on the ontogenetic development of kelps such asL. digitata.

Apomictic fern fathers: an experimental approach to the reproductive characteristics of sexual, apomict, and hybrid fern gametophytes.

Apomixis and hybridization are two essential and complementary factors in the evolution of plants, including ferns. Hybridization combines characteristics from different species, while apomixis conserves features within a lineage. When combined, these two processes result in apo-sex hybrids. The conditions leading to the formation of these hybrids are poorly understood in ferns. We cultivated spores from 66 fern samples (43 apomicts, 7 apo-sex hybrids, and 16 sexuals), and measured their development in vitro over 16 weeks. We evaluated germination, lateral meristem formation rates, sexual expression, and production of sporophytes and then compared ontogenetic patterns among the three groups. The three examined groups formed antheridia (male gametangia) but differed in overall gametophyte development. Sexual species created archegonia (female, 86% of viable samples), but no sporophytes. Apomicts rarely created nonfunctional archegonia (8%) but usually produced apogamous sporophytes (75%). Surprisingly, apomictic and sexual species showed similar development speed. The sexually reproducing parents of viable studied hybrids formed about twice as many meristic gametophytes as the apomictic parents (39% vs. 20%, respectively). We present the most thorough comparison of gametangial development of sexual and apomictic ferns, to date. Despite expectations, apomictic reproduction might not lead to earlier sporophyte formation. Apomicts produce functional sperm and thus can contribute this type of gamete to their hybrids. The development patterns found in the parents of hybrids indicate a possible increase of hybridization rates by antheridiogens. The apo-sex hybrids always inherit the apomictic reproductive strategy and are thus capable of self-perpetuation.

Topoisomerase 1α is required for synchronous spermatogenesis in Physcomitrium patens.

DNA topoisomerase 1 (TOP1) plays general roles in DNA replication and transcription by regulating DNA topology in land plants and metazoans. TOP1 is also involved in specific developmental events; however, whether TOP1 plays a conserved developmental role among multicellular organisms is unknown. Here, we investigated the developmental roles of TOP1 in the moss Physcomitrium (Physcomitrella) patens with gene targeting, microscopy, 3D image segmentation and crossing experiments. We discovered that the disruption of TOP1α, but not its paralogue TOP1β, leads to a defect in fertilisation and subsequent sporophyte formation in P. patens. In the top1α mutant, the egg cell was functional for fertilisation, while sperm cells were fewer and infertile with disordered structures. We observed that the nuclei volume of wild-type sperm cells synchronously decreases during antheridium development, indicating chromatin condensation towards the compact sperm head. By contrast, the top1α mutant exhibited attenuated cell divisions and asynchronous and defective contraction of the nuclei of sperm cells throughout spermatogenesis. These results indicate that TOP1α is involved in cell division and chromatin condensation during spermatogenesis in P. patens. Our results suggest that the regulation of DNA topology by TOP1 plays a key role in spermatogenesis in both land plants and metazoans.

Effects of seawater temperature and seasonal irradiance on growth, reproduction, and survival of the endemic Antarctic brown alga Desmarestia menziesii (Phaeophyceae)

Endemic Antarctic macroalgae are especially adapted to live in extreme Antarctic conditions. Their potential biogeographic distribution niche is primarily controlled by the photoperiodic regime and seawater temperatures, since these parameters regulate growth, reproduction, and survival during the entire life cycle. Here we analyzed the upper survival temperature (UST) of juvenile sporophytes and the temperature range for sporophyte formation from gametophytes of Desmarestia menziesii, one of the dominant endemic Antarctic brown algal species. This process is a missing link to better evaluate the full biogeographical niche of this species. Two laboratory experiments were conducted. First, growth and maximum quantum yield of juvenile sporophytes were analyzed under a temperature gradient (0, 5, 10, 12, 13, 14, 15, and 16 °C) in a 16:8 h light:dark (LD) regime (Antarctic spring condition) for 2 weeks. Second, the formation of sporophytes from gametophytes (as a proxy of gametophyte reproduction) was evaluated during a 7 weeks period under a temperature gradient (0, 4, 8, 12, and 16 °C), and two different photoperiods: 6:18 h LD regime simulating winter conditions and a light regime simulating the Antarctic shift from winter to spring by gradually increasing the light period from 7.5:16.5 h LD (late winter) to 18.5:5.5 h LD (late spring). Sporophytes of D. menziesii were able to grow and survive up to 14 °C for 2 weeks without visible signs of morphological damage. Thus, this species shows the highest UST of all endemic Antarctic Desmarestiales species. In turn, gametophyte reproduction solely took place at 0 °C but not at 4–8 °C. The number of emerging sporophytes was six times higher under the light regime simulating the transition from winter to spring than under constant short day winter conditions. There was a negative relationship between the number of sporophytes formed and the gametophyte density at the beginning of the experiment, which provides evidence that gametophyte density exerts some control upon reproduction in D. menziesii. Results strongly indicate that although sporophytes and gametophytes may survive in warmer temperatures, the northernmost distribution limit of D. menziesii in South Georgia Islands is set by the low temperature requirements for gametophyte reproduction. Hence, global warming could have an impact on the distribution of this and other Antarctic species, by influencing their growth and reproduction.

Enhancing In Vitro Production of the Tree Fern Cyathea delgadii and Modifying Secondary Metabolite Profiles by LED Lighting.

The tree ferns are an important component of tropical forests. In view of this, the enhancement of in vitro production of these plants is needed. Thus, the effect of different light-emitting diodes (LEDs) as well as control fluorescent lamps (Fl) and a 3-week-long period of darkness at the beginning of in vitro culture on micropropagation of the tree fern Cyathea delgadii Sternb. was analysed. Moreover, the photosynthetic pigment content and secondary metabolite profiles were estimated. The period of darkness contributed to a high production of somatic embryo-derived sporophytes and a low production of gametophytes. The formation of new sporophytes was stimulated by RBY (35% red, 15% blue, and 50% yellow) and B (100% blue) lights when the stipe explants or whole young sporophytes were used in the culture, respectively. The elongation of the roots and leaves was stimulated by RBfR light (35% red, 15% blue, and 50% far red), while root production increased under RBY light. The RB (70% red and 30% blue) and B lights stimulated the accumulation of chlorophyll better than Fl light. The most abundant metabolite found in the plant extracts was trans-5-O-caffeoylquinic acid (1.013 µg/mg of dry weight). The extract obtained from plants growing in a greenhouse had the best antioxidant activity.

Culture types and period impact gametophyte morphogenesis and sporophyte formation of eastern bracken

BackgroundLiquid suspension culture efficiently proliferates plant cells and can be applied to ferns because it rapidly increases the fresh weight of gametophytes. This study assessed gametophyte proliferation and sporophyte production of Pteridium aquilinum var. latiusculum using a suspension culture method.ResultsThe growth curve linear phase of gametophyte cells was confirmed between 9 and 18 days of culture, and the subculture cycle was determined to be 2 weeks. A double-strength MS medium (fresh weight, 18.0 g) containing 2% sucrose and NH4+:NO3− (120 mM, 40:80) was found to be the optimal liquid medium. Gametophytes obtained after suspension culture for 18 days did not normally form sporophytes in an ex vitro soil environment. However, this issue was resolved after changing the culture type or extending the culture period to 6 weeks. A short suspension culture period increased the fresh weight of fragmented and homogenized gametophytes but yielded numerous relatively immature gametophytes (globular forms of branching gametophytes, BG). Furthermore, differences in gametophyte morphogenesis and development were indicated by changes in endogenous phytohormone content. BG with immature development exhibited high accumulation of zeatin, jasmonic acid, and salicylic acid, and relatively low levels of abscisic acid and indole-3-acetic acid. The immature development of gametophytes directly affected sporophyte formation.ConclusionsThis study maximized the advantages of liquid suspension culture using eastern bracken gametophytes and provides data to resolve any associated issues, thus facilitating efficient bracken production.

Assessment of the independent and combined effects of copper and polycyclic aromatic hydrocarbons on gametogenesis and sporophyte development of the kelp Lessonia spicata (Phaeophyceae, Ochrophyta)

Coastal shores near the industrial park of Quintero Bay in central Chile exhibit increasing concentrations of copper (Cu) and polycyclic aromatic hydrocarbons (PAHs), well above international standards. This raises concern about their combined toxic effects on early development stages of kelps. Accordingly, we aimed to assess more accurately the independent and combined effects of Cu and PAHs on gametogenesis and sporophyte development in the kelp Lessonia spicata from central Chile by in vitro cultivation. Independent Cu and PAH trials were performed using increasing nominal concentrations of Cu and PAHs in the ranges 0.8–200 µg L−1 and 0.05–100 µg L−1, respectively. Cu and PAH median effective concentrations (EC50) on gametogenesis and early sporophyte formation were calculated using DRC in the R environment. Then, combined EC50 Cu + PAH trials were conducted to determine their effects on gametogenesis and sporophyte formation. Cu EC50 values on gametogenesis and sporophyte formation were up to three orders of magnitude lower than EC50 reported previously on spore germination in kelps. The gametogenesis (EC50 = 1.39 µg L−1) was more sensitive to Cu than sporophyte formation (EC50 = 11 µg L−1). Inversely, sporophyte formation (EC50 = 0.04 µg L−1) was more sensitive to PAHs (EC50 = 0.11 µg L−1). Considering the entire exposure period, the combined EC50 Cu + PAH exposure was the most harmful and rapid for L. spicata microscopic stages, especially the synergistic effect on early sporophytes. This highlights the need to acquire an integrated knowledge of the seasonal variation of pollutants and their combination on highly intervened coasts.

The Inheritable Characteristics of Monoecy and Parthenogenesis Provide A Means for Establishing A Doubled Haploid Population in the Economically Important Brown Alga Undaria pinnatifida (Laminariales, Alariaceae).

Monoecy and parthenogenesis exist in certain male and female gametophytes of the brown alga Undaria pinnatifida. The inheritance of these traits is not known. In this study, we made a cross between a male and a female gametophyte clone which could exhibit monoecy and parthenogenesis phenotypes, respectively, and obtained their next-generation gametophyte offspring. We found that under conditions suitable for gametogenesis, all of the male offspring (n=100) exhibited monoecy phenotype and all of the female offspring (n=100) only formed oogonia and underwent parthenogenesis, suggesting that monoecy and parthenogenesis phenotypes are inheritable. Then, we established a doubled haploid (DH) population through monoecious selfing and parthenogenesis, and evaluated the young sporophyte growth and the maximum quantum yield (Fv /Fm ) of 10 "male" and 10 "female" DH lines. On day 60, the average length of the "male" DH lines was significantly larger than that of the "female" DH lines, while their average Fv /Fm values were not significantly different. Monoecious selfing seemed superior to parthenogenesis as the sporophyte formation efficiency, and the young sporophyte growth was better in the former than in the latter. We also crossed two monoecious gametophytes with another male gametophyte, and a parentage analysis showed success of obtaining hybrid sporophytes, indicating that the female gametes released by the monoecious gametophyte can actually be fertilized by sperm. The approach of establishing a DH population proposed here will be useful in genetic breeding and quantitative trait loci mapping in U. pinnatifida and may be applicable to other kelp species.

SEC6 exocyst subunit contributes to multiple steps of growth and development of Physcomitrella (Physcomitrium patens).

Spatially directed cell division and expansion is important for plant growth and morphogenesis and relies on cooperation between the cytoskeleton and the secretory pathway. The phylogenetically conserved octameric complex exocyst mediates exocytotic vesicle tethering at the plasma membrane. Unlike other exocyst subunits of land plants, the core exocyst subunit SEC6 exists as a single paralog in Physcomitrium patens and Arabidopsis thaliana genomes. Arabidopsis SEC6 (AtSEC6) loss-of-function (LOF) mutation causes male gametophytic lethality. Our attempts to inactivate the P.patens SEC6 gene, PpSEC6, using targeted gene replacement produced two independent partial LOF ('weak allele') mutants via perturbation of the PpSEC6 gene locus. These mutants exhibited the same pleiotropic developmental defects: protonema with dominant chloronema stage; diminished caulonemal filament elongation rate; and failure in post-initiation gametophore development. Mutant gametophore buds, mostly initiated from chloronema cells, exhibited disordered cell file organization and cross-wall perforations, resulting in arrested development at the eight- to 10-cell stage. Complementation of both sec6 moss mutant lines by both PpSEC6 and AtSEC6 cDNA rescued gametophore development, including sexual organ differentiation. However, regular sporophyte formation and viable spore production were recovered only by the expression of PpSEC6, whereas the AtSEC6 complementants were only rarely fertile, indicating moss-specific SEC6 functions.

Synthetic seed production using eastern bracken (Pteridium aquilinum var. latiusculum) gametophyte: Effect of gametophyte density and short-term storage conditions

This study proposed the production of gametophyte-derived synthetic seeds and a practical plug seedling method for eastern bracken. Synthetic seeds were produced by mixing fragmented gametophyte and alginate matrix. The gametophyte regenerated regardless of the syringe tip size and the injection molding type employed, to successfully form the sporophyte. The synthetic seeds were successfully stored short-term under cold storage (4 °C). The storage period influenced the percentage of sporophyte formation, which was 70.8% or more until day 7, but decreased to 56.9% or less after day 14 of storage. However, the storage period of 7 days is sufficient for short-distance transport. The development of gametophyte-derived synthetic seeds improves the logistics of transport and handling ferns, and it enables establishment of plug seedling cultivation.

Drynaria bonii spore culture: optimization of culture conditions and analysis of gametophyte and sporophyte development

Drynaria bonii H. Christ is a medicinal plant that has traditionally been used for the treatment of chronic diseases and tuberculosis. However, D. bonii has not yet been cultivated or propagated. In this study, we optimized the conditions for the spore culture of D. bonii to produce sporophytes. First, we evaluated the effects of various factors, such as temperature, light intensity, potting material, and pH, on spore germination and early gametophyte development. Under optimal culture conditions, 95–99% of the spores germinated within 2 weeks of culture. Next, based on these results, we established a culture system for gametophyte development and sporophyte production. Mature gametophytes first appeared 5 months post-germination (mpg), and at 12 mpg, the rate of production of heart-shaped gametophytes reached 56.6%. Sporophytes at the early first-leaf stage first appeared at 6 mpg, and at 12 mpg, the rate of formation of sporophytes reached 15.4%. Interestingly, 6.3% of all gametophytes produced multiple sporophytes. Additionally, 42–62% of the gametophytes also produced multiple secondary gametophytes, indicating a high potential of D. bonii gametophytes to generate new gametophytes and, subsequently, sporophytes. When transferred to new trays or pots, sporophytes grew well and showed 100% survival. Overall, we conclude that this spore culture system can be successfully used for the propagation of D. bonii sporophytes.

In vitro Phytohormonal Regulation of Fern Gametophytes Growth and Development

Interest in ferns as objects of biotechnological research is determined by the characteristics of their development and reproduction, which is important for understanding the evolutionary pathways of seed plants. The previous published data on the phytohormones involvement in the regulation of fern gametophytes ontogenesis during in vitro cultivation are analyzed and summarized. The possibility of fern reproduction through spores application as explants using in vitro methods is considered. The effects of exogenous treatment with cytokinins, auxins, gibberellins, ethylene, jasmonic acid, and brassinosteroids on spore germination, gametophyte morphology and development are analyzed. The roles of gibberellins and antheridiogen in the formation of fern gametophytes’ sexual dimorphism are discussed. Data on disturbances of the sexual process in ferns and the phytohormones’ involvement in the formation of apogamic sporophytes are summarized.

Practical methodology for gametophyte proliferation and sporophyte production in green penny fern (Lemmaphyllum microphyllum C. Presl) using mechanical fragmentation

Propagation of gametophytes and sporophytes using mechanical fragmentation has been considered a suitable method for mass production of ferns. This study aimed to develop a practical propagation method for Lemmaphyllum microphyllum C. Presl, which is a fern of significant ornamental and medicinal value. Gametophytes were obtained through in vitro spore germination and used for propagation experiments. The gametophyte was mechanically fragmented using a scalpel into small fragments, which were then used to investigate gametophyte proliferation. In addition, the gametophyte was fragmented using a blender and then used to study sporophyte formation. Optimal proliferation conditions of the gametophyte were determined using Murashige and Skoog (MS) basal medium (double-, full-, half-, quarter-strength), Knop medium, and medium components (sucrose, nitrogen sources, activated charcoal), at various concentrations. The fresh weight of the gametophyte was 14-fold higher than that of gametophytes (300 mg) used as culture material, when cultured on double-strength MS. Moreover, 1 g of the gametophyte fragmented in 25 mL of distilled water formed more than 430 sporophytes in a soil mixture in an area of 7.5 cm2. The sporophytes were successfully cultivated in the greenhouse after acclimation. A large-scale production method for L. microphyllum that can be easily implemented in a fern production farm is outlined.

Differential gene expression profiling of one- and two-dimensional apogamous gametophytes of the fern Dryopteris affinis ssp. affinis

Apomixis was originally defined as the replacement of sexual reproduction by an asexual process that does not involve fertilization but, in angiosperms, it is often used in the more restricted sense of asexual reproduction through seeds. In ferns, apomixis combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells of the prothallium (apogamy). The genes that control the onset of apogamy in ferns are largely unknown. In this study, we describe the gametophyte transcriptome of the apogamous fern Dryopteris affinis ssp. affinis using an RNA-Seq approach to compare the gene expression profiles of one- and two-dimensional gametophytes, the latter containing apogamic centers. After collapsing highly similar de novo transcripts, we obtained 166,191 unigenes, of which 30% could be annotated using public databases. Multiple quality metrics indicate a good quality of the de novo transcriptome with a low level of fragmentation. Our data show a total of 10,679 genes (6% of all genes) to be differentially expressed between gametophytes of filamentous (one-dimensional) and prothallial (two-dimensional) architecture. 6,110 genes were up-regulated in two-dimensional relative to one-dimensional gametophytes, some of which are implicated in the regulation of meristem growth, auxin signaling, reproduction, and sucrose metabolism. 4,570 genes were down-regulated in two-dimensional versus one-dimensional gametophytes, which are enriched in stimulus and defense genes, as well as genes involved in epigenetic gene regulation and ubiquitin degradation. Our results provide insights into free-living gametophyte development, focusing on the filamentous-to-prothallus growth transition, and provide a useful resource for further investigations of asexual reproduction.