Rhizoid Formation Research Articles

ВОЗДЕЙСТВИЕ КОБАЛЬТА И МЕДИ НА РОСТ ПРОРОСТКОВ БУРОЙ ВОДОРОСЛИ FUCUS DISTICHUS SUBSP. EVANESCENS (FUCALES, PHAEOPHYCEAE)

The data of cobalt and copper effect on the development of Kamchatka brown alga Fucus distichus subsp. evanescens is presented in the paper. The observations were carried out under laboratory conditions with temperature of 10°C, natural light and a photoperiod from July, 6 to August, 6. The cultivation media contained metals at nominal concentrations of 5, 10, 20, and 50 µg/l. It was found that Co addition to the medium mostly stimulated germlings growth. The average growth rate varied from 15.6 to 23.5 µm/day during entire experiment period but did not exceed 16.1 µm/day in the control group. On the contrary, a significant decrease of growth in groups grown in excessive copper media was revealed. By the end of the experiment the rate of germlings growth in media with copper of 10, 20, and 50 µg/l did not exceed 6 µm/day; in the media with minimum metal content was 11.2 µm/day. Also the abnormal formation of germlings rhizoids, the presence of extra- and intracellular copper deposits and underdevelopment of hyaline hairs were observed in all cultures with excessive copper content. The obtained results can be used to predict the responses of Fucus littoral communities to cobalt and copper environment pollution.

The single Marchantia polymorpha FERONIA homolog reveals an ancestral role in regulating cellular expansion and integrity.

Plant cells are surrounded by a cell wall, a rigid structure that is not only important for cell and organ shape, but is also crucial for intercellular communication and interactions with the environment. In the flowering plant Arabidopsis thaliana, the 17 members of the Catharanthus roseus RLK1-like (CrRLK1L) receptor kinase family are involved in a multitude of physiological and developmental processes, making it difficult to assess their primary or ancestral function. To reduce genetic complexity, we characterized the single CrRLK1L gene of Marchantia polymorpha, MpFERONIA (MpFER). Plants with reduced MpFER levels show defects in vegetative development, i.e. rhizoid formation and cell expansion, and have reduced male fertility. In contrast, cell integrity and morphogenesis of the gametophyte are severely affected in Mpfer null mutants and MpFER overexpression lines. Thus, we conclude that the CrRLK1L gene family originated from a single gene with an ancestral function in cell expansion and the maintenance of cellular integrity. During land plant evolution, this ancestral gene diversified to fulfill a multitude of specialized physiological and developmental roles in the formation of both gametophytic and sporophytic structures essential to the life cycle of flowering plants.

Cell tip growth underlies injury response of marine macroalgae.

Regeneration is a widely observed phenomenon by which the integrity of an organism is recovered after damage. To date, studies on the molecular and cellular mechanisms of regeneration have been limited to a handful of model multicellular organisms. Here, the regeneration ability of marine macroalgae (Rhodophyta, Phaeophyceae, Chlorophyta) was systematically surveyed after thallus severing. Live cell imaging on severed thalli uncovered the cellular response to the damage. Three types of responses–budding, rhizoid formation, and/or sporulation–were observed in 25 species among 66 examined, proving the high potential of regeneration of macroalgae. The cellular and nuclear dynamics were monitored during cell repair or rhizoid formation of four phylogenetically diverged species, and the tip growth of the cells near the damaged site was observed as a common response. Nuclear translocation followed tip growth, enabling overall distribution of multinuclei or central positioning of the mononucleus. In contrast, the control of cell cycle events, such as nuclear division and septation, varied in these species. These observations showed that marine macroalgae utilise a variety of regeneration pathways, with some common features. This study also provides a novel methodology of live cell imaging in macroalgae.

Asymbiotic Germination and Seedling Development of Terrestrial Orchid Bletilla striata Using in vitro and ex vitro Cultures

Procedures for asymbiotic germination and seedling development under in vitro and ex vitro conditions were investigated for Bletilla striata. Five different asymbiotic germination media (½ P6668 ¬Phytamax Orchid Maintenance Media, ½ P6668 ¬Phytamax Orchid Maintenance Media with coconut water, P723 PhytoTechnology Orchid Seed Sowing Media, P723 PhytoTechnology Orchid Seed Sowing Media with coconut water, and ½ MS media) for in vitro culture and sphagnum moss for ex vitro culture were examined for their effectiveness for 8 weeks on seed germination and seedling development of B. striata. Germination occurred in all media, however, the best germination rate was obtained in P723 medium with coconut water while the lowest frequency was obtained in sphagnum moss. Moreover, the highest leaf parameters of the seedlings of B. striata developed in ½ MS medium when the seedlings in sphagnum moss showed the lowest results. Furthermore, all germinated protocorms showed rhizoid formation in all media.

Changes in Cell Wall Structure During Rhizoid Formation of Silvetia babingtonii (Fucales, Phaeophyceae) Zygotes.

We examined the ultrastructure of the cell wall and immunolocalization of alginates using specific antibodies against M-rich alginates and MG blocks during rhizoid formation in fucoid zygotes, Silvetia babingtonii. The thallus region of 24-h-old zygotes had a cell wall made of three layers with different fiber distribution. In the 12-h-old zygotes, three layers in the thallus were observed before rhizoid formation, namely the inner, middle, and outer layers. During rhizoid elongation, only the inner layer was apparent close to the rhizoid tip area. Immunoelectron microscopy detected M-rich blocks of alginate on the inner half of the cell wall, irrespective of the number of layers in the thallus and rhizoid regions. The MG blocks were seen to cover a slightly wider area than M-rich alginate blocks. It was suggested that parts of M in mannuronan would be rapidly converted to G, and MG-blocks are generated. Transcriptome analysis was performed using 3 -, 10 -, and 24-h-old zygotes after fertilization to examine the relationship between gene expression and alginate synthesis over time. The expression of two mannuronan C5-epimerase homologs that convert mannuronic acid into guluronic acid in alginates was upregulated or downregulated over the course of the examination.

Distinct Functions of Ethylene and ACC in the Basal Land Plant Marchantia polymorpha

Ethylene is a gaseous phytohormone involved in various physiological processes, including fruit ripening, senescence, root hair development and stress responses. Recent genomics studies have suggested that most homologous genes of ethylene biosynthesis and signaling are conserved from algae to angiosperms, whereas the function and biosynthesis of ethylene remain unknown in basal plants. Here, we examined the physiological effects of ethylene, an ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) and an inhibitor of ethylene perception, silver thiosulfate (STS), in a basal land plant, Marchantia polymorpha. M. polymorpha plants biosynthesized ethylene, and treatment with high concentrations of ACC slightly promoted ethylene production. ACC remarkably suppressed the growth of thalli (vegetative organs) and rhizoids (root-hair-like cells), whereas exogenous ethylene slightly promoted thallus growth. STS suppressed thallus growth and induced ectopic rhizoid formation on the dorsal surface of thalli. Thus, ACC and ethylene have different effects on the vegetative growth of M. polymorpha. We generated single and double mutants of ACC synthase-like (ACSL) genes, MpACSL1 and MpACSL2. The mutants did not show obvious defects in thallus growth, ACC content and ethylene production, indicating that MpACSL genes are not essential for the vegetative growth and biosynthesis of ACC and ethylene. Gene expression analysis suggested the involvement of MpACSL1 and MpACSL2 in stress responses. Collectively, our results imply ethylene-independent function of ACC and the absence of ACC-mediated ethylene biosynthesis in M. polymorpha.

In vitro spore germination and phytoremediation of Hg and Pb using gametophytes of Pityrogramma calomelanos

Few pteridophytes have proven the capacity to accumulate and remediate heavy metals from contaminated soils. Pityrogramma calomelanos, a non-seasonal fast-growing, a cosmopolitan fern, is a good indicator of environmental conditions, was used in the present study. The life cycle of ferns alternates with haploid gametophyte and diploid sporophyte. The present study was undertaken to access the effect of mercury, in form of mercury (II) chloride [(HgCl2)] and lead as lead nitrate [Pb(NO3)2] in developmental studies using in vitro spore germination. Periodic recording of the germination, protonemal growth, rhizoid formation and differentiation of sex organs in different concentrations of heavy metals were conducted for a period of 6 weeks. It was found that the percentage of spore germination and the number of protonemal cells reduced significantly causing developmental defects in the presence of HgCl2 as compared to Pb(NO3)2 (p < 0.0001). A significant decrease in the number of archegonial count and chlorophyll content was observed in different concentrations of the heavy metals tested. Gametophytes of P. calomelanos recorded lead uptake of 646.51 ± 0.93 mg/kg in treatments of 25 ppm of lead and high mercury accumulation up to 1,885 ± 1.98 mg/kg at 10 ppm of mercury, indicating successful uptake of heavy metals. Novelty statement: Pityrogramma calomelanos is gaining interest amongst pteridologists upon proving its exclusive capacity of phytoremediation. It is superior in comparison to the most popular, patented fern Pteris vittata. Our study demonstrates the effective use of the promising fern in its simplest, nonvascular form of gametophytes as a phytoremediation agent in controlled conditions.

Cytokinin signaling coordinates development of diverse organs in Marchantia polymorpha

ABSTRACTCytokinins play an essential role in plant growth and development. A recent study showed that the cytokinin signaling pathway was conserved in the liverwort Marchantia polymorpha, and that it controlled gemma cup and rhizoid formation during thallus development. Here we show that the type-B response regulator, MpRRB, is mainly localized in the nucleus. Moreover, observations of thalli revealed that the distribution of air pores and the shape of the thallus margin are impaired in cytokinin-deficient lines and those defective in cytokinin signaling. This suggests that cytokinins regulate cell division and/or differentiation of precursor cells derived from the apical cell, thereby coordinating development of various organs produced on the thallus.

Cytokinin Signaling Is Essential for Organ Formation in Marchantia polymorpha.

Cytokinins are known to regulate various physiological events in plants. Cytokinin signaling is mediated by the phosphorelay system, one of the most ancient mechanisms controlling hormonal pathways in plants. The liverwort Marchantia polymorpha possesses all components necessary for cytokinin signaling; however, whether they respond to cytokinins and how the signaling is fine-tuned remain largely unknown. Here, we report cytokinin function in Marchantia development and organ formation. Our measurement of cytokinin species revealed that cis-zeatin is the most abundant cytokinin in Marchantia. We reduced the endogenous cytokinin level by overexpressing the gene for cytokinin oxidase, MpCKX, which inactivates cytokinins, and generated overexpression and knockout lines for type-A (MpRRA) and type-B (MpRRB) response regulators to manipulate the signaling. The overexpression lines of MpCKX and MpRRA, and the knockout lines of MpRRB, shared phenotypes such as inhibition of gemma cup formation, enhanced rhizoid formation and hyponastic thallus growth. Conversely, the knockout lines of MpRRA produced more gemma cups and exhibited epinastic thallus growth. MpRRA expression was elevated by cytokinin treatment and reduced by knocking out MpRRB, suggesting that MpRRA is upregulated by the MpRRB-mediated cytokinin signaling, which is antagonized by MpRRA. Our findings indicate that when plants moved onto land they already deployed the negative feedback loop of cytokinin signaling, which has an indispensable role in organogenesis.

Arabinogalactan Proteins and the Extracellular Matrix of Charophytes: A Sticky Business.

Charophytes represent the group of green algae whose ancestors invaded land and ultimately gave rise to land plants 450 million years ago. While Zygnematophyceae are believed to be the direct sister lineage to embryophytes, different members of this group (Penium, Spirogyra, Zygnema) and the advanced thallus forming Coleochaete as well as the sarcinoid basal streptophyte Chlorokybus were investigated concerning their vegetative extracellular matrix (ECM) properties. Many taxa exhibit adhesion phenomena that are critical for affixing to a substrate or keeping cells together in a thallus, however, there is a great variety in possible reactions to e.g., wounding. In this study an analysis of adhesion mechanisms revealed that arabinogalactan proteins (AGPs) are most likely key adhesion molecules. Through use of monoclonal antibodies (JIM13) or the Yariv reagent, AGPs were located in cell surface sheaths and cell walls that were parts of the adhesion focal zones on substrates including wound induced rhizoid formation. JIM5, detecting highly methyl-esterfied homoglacturonan and JIM8, an antibody detecting AGP glycan and LM6 detecting arabinans were also tested and a colocalization was found in several examples (e.g., Zygnema) suggesting an interplay between these components. AGPs have been described in this study to perform both, cell to cell adhesion in algae forming thalli and cell to surface adhesion in the filamentous forms. These findings enable a broader evolutionary understanding of the function of AGPs in charophyte green algae.

Auxin Function in the Brown Alga Dictyota dichotoma.

Auxin controls body plan patterning in land plants and has been proposed to play a similar role in the development of brown algae (Phaeophyta) despite their distant evolutionary relationship with land plants. The mechanism of auxin action in brown algae remains controversial because of contradicting conclusions derived from pharmacological studies on Fucus In this study, we used Dictyota dichotoma as a model system to show that auxin plays a role during the apical-basal patterning of the embryo of brown algae. Indole-3-acetic acid was detectable in D. dichotoma germlings and mature tissue. Although two-celled D. dichotoma zygotes normally develop a rhizoid from one pole and a thallus meristem from the other, addition of exogenous auxins to one-celled embryos affected polarization, and both poles of the spheroidal embryo developed into rhizoids instead. The effect was strongest at lower pH and when variable extrinsic informational cues were applied. 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid, an inhibitor of the ABC-B/multidrug resistance/P-glycoprotein subfamily of transporters in land plants, affected rhizoid formation by increasing rhizoid branching and inducing ectopic rhizoids. An in silico survey of auxin genes suggested that a diverse range of biosynthesis genes and transport genes, such as PIN-LIKES, and the ATP-binding cassette subfamily (ABC-B/multidrug resistance/P-glycoprotein) transporters from land plants have homologs in D. dichotoma and Ectocarpus siliculosus Together with reports on auxin function in basal lineages of green algae, these results suggest that auxin function predates the divergence between the green and brown lineage and the transition toward land plants.

Interactive effects of temperature and light on reattachment success in the brown alga Fucus radicans

Abstract Fucus radicans is an endemic habitat-forming brown macroalga in the Baltic Sea that commonly complements its sexual reproduction with asexual reproduction. Asexual reproduction in F. radicans takes place through formation of adventitious branches (hereafter fragments), but the exact mechanisms behind it remain unknown. We assessed experimentally the importance of two environmental factors determining the re-attachment success of F. radicans fragments. By combining different light conditions (daylength and irradiance; high or low light) and water temperature (+14°C and +4°C), we mimicked ambient light and temperature conditions of winter, spring/autumn and summer for F. radicans. Fragments were able to re-attach in all tested conditions. Temperature and light had an interactive impact on re-attachment: the combination of high temperature and high light level resulted in the highest re-attachment success, while light level had no effects on re-attachment success in cooler water temperature and the re-attachment success in high temperature under low light levels was very low. The results suggest that rhizoid formation, and thus re-attachment success, may depend on the net primary production (metabolic balance) of the fragment. However, whether the re-attachment and asexual reproduction success simply depends on photosynthetic capacity warrants further mechanistic studies. Understanding the mechanisms of asexual reproduction in F. radicans is important in order to assess the dispersal capacity of this foundation species.

Tetraspore germination of two vulnerable marine algae,Gelidium canarienseandG. arbusculum(Rhodophyta, Gelidiales)

AbstractGelidium canarienseandGelidium arbusculumcoexist in the upper sublittoral zone in the Canary Islands and are the only Gelidiales registered as vulnerable species. Spore germination and the formation of rhizoids are vital steps for the successful growth of new plants. We investigated the initial germination stages of tetraspores and the growth of the primary rhizoid in these two vulnerable species. Both Gelidiales exhibitedGelidium-type germination. However, liberated tetraspores ofG. canarienseandG. arbusculumgerminated earlier than those of other species of the Gelidiales. The growth curves of primary rhizoids were similar in both species.

Développement D’un Stock De Semences (Seedstocks) De L’algue Rouge Gelidium Corneum (Gelidiaceae, Rhodophyta)

Gelidium corneum is a species of red algae notable for its commercial important as an agarophyte in Morocco. Several regions from the Moroccan Atlantic show that this alga is an endangered species due to the excessive tearing. Hence, the repopulation of these areas is necessary. The in vitro culture of the species was carried out in three media: enriched seawater medium (PES medium (Provasoli Enriched Seawater, Provasoli 1968)), medium with seawater (SW) and medium with artificial seawater, with the addition of polyamines (putrescine (put), spermidine (spd), and spermine (spr)) as a growth regulator in the three media. The results obtained are very significant, especially in PES medium with a growth rate of 95%. Rhizoid formation and attachment of explants have been noted, especially in PES + Put medium.

The phaeophyte Hizikia fusiformis extracts suppress rhizoid and blade formation in seaweeds

Background: Plants, including marine algae, produce allelochemicals that influence the growth, survival, and reproduction of other organisms.&#x0D; Questions: To identify natural algicidal or antifouling allelochemicals, we screened 18 common seaweed extracts for suppression of rhizoid and blade production in a convenient Porphyra suborbiculata monospore assay.&#x0D; Species study and data description: Addition of extract from the most potent phaeophyte, Hizikia fusiformis , suppressed rhizoid formation, rhizoid number, rhizoid length, blade formation, and blade length.&#x0D; Study site and dates: Seaweed thalli for methanol extraction were collected on the coast of Korea from October 2012 to July 2015.&#x0D; Methods: Extracts were tested using the P. suborbiculata monospore assay system.&#x0D; Results: The 50 % suppression doses were 15 µ g ml-1 for rhizoid formation, 2.4 µ g ml-1 for rhizoid number, 13 µ g ml-1 for rhizoid length, 6 µ g ml-1 for blade formation, and 11 µ g ml-1 for blade length. The H. fusiformis extract also suppressed rhizoid and blade production in leafy green ( Ulva pertusa ) and brown ( Undaria pinnatifida and Ecklonia cava ) seaweed spores, as well as suppressing diatom settlement.&#x0D; Conclusions: The allelochemicals that suppressed or eliminated competing seaweed species may be efficacious for new seaweed control technologies, including the development of antifouling or algicidal agents based on natural products.

Molecular phylogeny and detailed morphological analysis of two freshwater Rhizoclonium strains from contrasting spring types in Egypt and Italy

Two Rhizoclonium strains thriving in contrasting spring types (slightly hot spring/ambient, shaded pool spring) and biogeographic areas (El-Farafra Oasis in the Western Desert of Egypt/River-Po Plain in Northern Italy) were studied in depth based on field and cultured material, bright-field and fluorescence microscopy, and phylogenetic molecular analyses (SSU and LSU rRNA gene sequences). This polyphasic approach revealed that the two Rh. strains clearly belonged to the Rhizoclonium clade but differed in some of their key diagnostic features. The Egyptian Rhizoclonium strain, isolated from the spring-fed (Ain El-Balad) agricultural ditch in El-Farafra Oasis, was described with the working name ‘Rhizoclonium sp. 10.6 μm from a desert, slightly-hot spring’ based on its smaller cell diameter, ecological and phylogenetic molecular traits compared to allied morphospecies. Moreover, it was highlighted that the L/D ratio can be more than 3.0 within this group. The Italian Rhizoclonium strain, collected from the Fontanile Valle Re-shaded, pool ambient spring (Emilia-Romagna Region), was virtually identical to other strains identified as Rh. hieroglyphicum from China and Japan, due to its highly supported congruence in morphological characteristics and phylogenetic position. This study provided the first partial LSU and SSU rRNA gene sequences for European Rh. hieroglyphicum based on available literature. Phenotypic plasticity of rhizoid formation was also observed in both Rhizoclonium strains studied using culturing approaching techniques. Our investigation also confirms that a lot of work with a variety of approaches is still needed to assess the ecological preferences, morphological plasticity, and phylogenetics of freshwater Rhizoclonium taxa worldwide.

Whole-Plant Regeneration via Epidermal Cells in the Axis of the Early Devonian Rhynie Chert PlantRhynia gwynne-vaughaniiKidston et Lang

Premise of research. The Early Devonian Rhynie chert is an invaluable source for studying the morphology, ecology, and life cycles of early land plants, but information regarding morphological development in the Rhynie chert plants is sorely lacking.Methodology. Serial petrographic thin sectioning of the Rhynie chert reveals asexual propagules developing from the epidermal cells in the axes of the plant Rhynia gwynne-vaughanii. Three distinct developmental stages recording the formation of stomata, rhizoids, apical growth, and vascular tissue were observed. Similar propagules are also found developing from hemispherical projections, also on the axes of Rhynia.Pivotal results. The developmental pattern resembles that seen in somatic embryogenesis, a form of asexual reproduction that occurs naturally in extant plants as well as in in vitro, producing nutritionally independent plantlets from somatic plant cells.Conclusions. Our new observations on these propagules significantly contribute to a better underst...

녹조류 청각 추출물에 의한 해조류 가근 및 유엽형성 촉진

Living organisms can maintain or extend their territories by producing allelochemicals that influence the growth, survival, and reproduction of other organisms. To identify natural biostimulants of positive allelochemicals, we screened 18 common seaweed extracts for enhancement of rhizoid and blade production in a convenient Porphyra suborbiculata monospore assay. By addition of methanolic extract from the most potent green seaweed, Codium fragile, 100% and 50% enhancement doses reflecting the amount of C. fragile extract required to enhance rhizoid formation (in terms of number of spores with rhizoids per total spores tested) were approximately 100 and 50 μg/ml, respectively, in the P. suborbiculata monospore culture. The C. fragile extract quickly enhanced rhizoid formation, rhizoid numbers per rhizoid-holding spore, rhizoid length, blade formation (in number of spores with blade per total spores tested), and blade length from most monospores at early culture days. The extract enhanced rhizoid formation after 2 days of culture significantly, rhizoid numbers per rhizoid-holding spore after 3 days, rhizoid length after 3 days, blade formation after 2 days, and blade length after 1 day, respectively, from most monospores. The allelochemicals that enhanced favorite seaweed species may be efficacious for new seaweed management technologies, including the development of biostimulant agents based on natural products.

Evaluation of salinity effects on the release, adhesion, and germination of the tetraspores of Gelidium floridanum (Rhodophyta, Florideophyceae)

The release, adhesion, and germination of tetraspores determine the successful development of the red seaweed Gelidium floridanum. The aim of this study was to investigate the effects of salinity on the initial development of G. floridanum tetraspores. To accomplish this, tetraspores were released and allowed to germinate under salinity of 24, 28, 32, 36, and 40 psu for a period of 6 to 24 h. Samples were processed for light, confocal, and transmission electron microscopy to analyze the ultrastructural characteristics, as well as analyses of the cell viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)) and quantification of photosynthetic pigments. At 32 psu, tetraspores exhibited normal characteristics with germ tube formation after 6 h and rhizoid formation after 24 h. Under hyposalinity of 24 psu, tetraspores did not form a germ tube after 6 h, and no development at all occurred after 24 h. Thus, the development of tetraspores was inhibited, resulting in nonviable cells or disorganized cytoplasmic contents. Tetraspores cultivated in hypersaline conditions (40 psu) showed a delay in development. After only 6 h of culture, samples in both hyposaline and hypersaline conditions showed differences in photosynthetic pigments, and such pigments tend to stabilize after 24 h. Based on these results, we can conclude that this species has only limited tolerance to salinity in the range of 28 to 36 psu and that salinity above or below these values is sufficient to change the pattern of development of G. floridanum tetraspores.

Morphology and ploidy level determination of Pteris vittata callus during induction and regeneration.

BackgroundMorphological and ploidy changes of the arsenic hyperaccumulator, Chinese brake fern (Pteris vittata) callus tissue are described here to provide insight into fern life cycle biology and for possible biotechnology applications. Pteris vittata callus was studied using transmission and scanning electron microscopy, and flow cytometry.ResultsCallus induction occurred both in light and dark culture conditions from prothallus tissues, whereas rhizoid formation occurred only in dark culture conditions. Callus tissues contained two types of cells: one actively dividing and the other containing a single large vacuole undergoing exocytosis. Sporophytes regenerated from callus asynchronously form clusters of cells in a manner apparently analogous to direct organogenesis. Extracellular matrices were observed in actively-growing callus and at the base of regenerating sporophytes. Callus tissue nuclei were found to be primarily diploid at induction and throughout maintenance of cultures indicating that callus cell fate is determined at induction, which closely follows apogamous sporophyte development. Presence of a dense extracellular matrix in conjunction with sporophyte development suggests a link between the suspensor-like activity of the embryonic foot during normal fern embryo development and the suspected functions of extracellular matrices in angiosperms.ConclusionsFurther investigation could lead to a better understanding of genes involved in P. vittata embryo development and apogamous sporophyte development. The methodology could be useful for in vitro propagation of rare and valuable fern germplasm.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-014-0096-6) contains supplementary material, which is available to authorized users.