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.

     

Efficient induction of apospory and apogamy in vitro in silver fern (Pityrogramma calomelanos L.)

Silver fern (Pityrogramma calomelanos L.) is a terrestrial or lithophytic herbaceous fern used for ornamental and medicinal purposes. In its farina it produces the cytotoxic and anticancer compound dihydrochalcone. In vitro induction of apospory and apogamy, and direct field establishment of aposporous gametophytes and subsequent sporophyte development has been accomplished. Half-strength Murashige and Skoog (MS) medium with 3.33 μM N⁶-benzyladenine (BA) and 2.32 μM kinetin (Kn) showed earlier development and produced higher numbers of aposporous gametophytes than half-strength MS basal medium. Crozier explants developed higher numbers (mean value 29.2) of gametophytes, but were slower than frond explants (mean value 23.2). The gametophytes originated from the epidermal hairs progressed from uniseriate filamentous to cordate through bi-, tri- and multiseriate and spatulate stage with the development of antheridia. Reduction in the nutrient and sucrose concentrations in the media favoured apogamy. Sucrose-free 1/10 strength MS medium and agar plates developed a mean of 30.4 and 29.9 sporophytes, respectively in the light. The greenhouse-established gametophytes developed sporophytes. The established sporophytes ex vitro showed 95% survival rate. Apogamous sporophytes and the source plant showed the same chromosome numbers (2n=116). The established protocol accomplishes apogamy and apospory in silver fern, and the aposporous gametophytes can be used for genetic transformation and development of transgenic silver fern.     

Propagation by partial tissue culture of Austral Bracken (Pteridium esculentum) for revegetation

Austral Bracken (Pteridium esculentum) is a native fern common in many Australian ecosystems and is needed in large numbers for revegetation projects. The main limiting factor for the propagation of locally sourced material is spore availability. A mass propagation system was developed by combining tissue culture and nursery‐based systems. Spores collected over the summer months from wild populations were germinated in vitro on ½ MS medium containing 0.15% w/v activated charcoal. Gametophytes were rapidly multiplied on the same medium. In vitro sporophyte development was unreliable although sometimes prolific. However, gametophytes transferred to a pine bark potting medium with added coir, on a capillary bed in a fog house, produced sporophytes reliably. Across different seasons and populations, 75–100% of the gametophyte explants developed sporophytes within about 9 weeks. Three hundred propagated ferns planted into two field sites within their provenance origins had a survival of 92 and 95% respectively, 3 or 4 months after planting. This report delivers a ready‐to‐use and reliable protocol for the mass propagation of bracken fern of local origin to the revegetation industry.     

Production of aposporous gametophytes and calli from Pteris vittata L. pinnae strips cultured in vitro

Murashige and Skoog's modified medium in 1% Difco Bacto-agar supplemented with sugar alcohols (sorbsitol, mannitol), growth regulators (1-naphthalenacetic acid, 2,4-dichlorophenoxyacetic acid, benzyladenine, kinetin) and sugars (fructose, glucose, sucrose) induced aposporous gametophytes from pinnae of Pteris vittata cultured in vitro at lower concentrations of all the mentioned components. Aposporous gametophytes and vegetative calli were produced at higher concentrations. The calli regenerated sporophytes when cultured on MS medium without growth regulators. The gametophytes grew vegetatively on MS medium but produced sporophytes when transferred into 0.1 strength MS medium. This is the first report of simultaneous production of calli and gametophytes from fern explants.     

In vitro polyploidization of mature ostrich fern sporophytes through rejuvenation

An in vitro method is described for producing ostrich fern (Matteuccia struthiopteris (L.) Todaro) polyploids from mature sporophytes as a possible means of plant improvement in this economically important fern species. The procedure is based on rejuvenating adult sporophytes (2n) to enable the aposporous production of diploid (2n) gametophytes, and then mating the gametophytes to produce tetraploid (4n) sporophytes. The adult sporophytes were rejuvenated by culturing excised shoot tips for a minimum of three months in a liquid medium (Murashige and Skoog salts) under conditions of extreme carbohydrate deprivation (0.01% sucrose). Apospory was induced by culturing leaves excised from the rejuvenated shoots for two months on a semi-solid medium lacking sucrose, resulting in the production of diploid gametophytes. The gametophytes were transferred to fresh medium and grown to sexual maturity for one or two months, then floated on the surface of a liquid medium containing 0.01% sucrose for up to two months to promote opening of the sex organs. Subsequent self-fertilization resulted in the successful production of tetraploid sporophytes in 11 of the 14 diploid clones in which polyploidization was attempted. Tetraploids (4n=156) were confirmed by cytological examination. This method permits polyploidization of mature, fully characterized plants.     

Gametophyte contribution to sporophyte growth on the basis of carbon gain in the fern <Emphasis Type="Italic">Thelypteris palustris</Emphasis>: effect of gametophyte organic-matter production on sporophytes

At an early stage of growth gametophytes support the sporophytes of ferns. Young sporophytes become independent of gametophytes when the first leaves develop. Although large fern gametophytes produce multiple archegonia simultaneously, only one sporophyte is typically established on one gametophyte. The number of sporophytes is believed to be controlled in two possible directions, from gametophyte to sporophyte or from preceding sporophyte to another sporophyte. To investigate the effects of gametophytes on their sporophytes, we studied the relationship between organic matter production by gametophytes and the growth of young sporophytes of Thelypteris palustris. We cut gametophytes in half (CGs) to reduce the gametophytes’ production of matter. There was no significant difference between the growth of sporophytes on intact gametophytes (IGs) and that on CGs. According to our estimates, based on the rate of organic matter production, the large gametophyte was able to produce two or more sporophytes. The resources required for CGs to make similar-sized sporophytes was twice that for IGs. In polyembryony each of the multiple sporophytes was similar in size to the single sporophytes. Resource limitation does not seem to explain why fern gametophytes establish single sporophytes.     

Influence of tissue culture conditions on apogamy in Dryopteris affinis sp. affinis

The naturally-occurring apogamy of some ferns can be modified by culture conditions and growth regulators. Gametophytes of the apogamic fern Dryopteris affinis sp. affinis L., were cultured on Murashige and Skoog (MS) basal medium. Changes in concentration of MS medium components, sucrose, agar and different pH values were tested. The addition of benzyladenine (4.43 M) and naphthalene acetic acid (0.53 M) enhanced sporophyte proliferation on the gametophytes. After one month in culture, the gametophytes formed callus with a high morphogenic capacity. Culture of calli on medium without growth regulators yielded about 10,000 sporophytes per 1 g fresh weight of callus. This pattern of differentiation slowed with time to a point where only gametophyte regeneration was observed.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - F.W. fresh weight - MS Murashige & Skoog medium - NAA 1-naphthalene acetic acid - SE standard error     

The effect of exogenous and endogenous phytohormones on the in vitro development of gametophyte and sporophyte in <Emphasis Type="Italic">Asplenium nidus</Emphasis> L.

The fern Asplenium nidus L. is in great demand as an ornamental plant. The aim of this work was to investigate the influence of phytohormones in promoting a gametophytic and sporophytic growth in homogenized sporophytes tissue. Exogenous application of 0.5 and 5 μM N ⁶-benzyladenine, 0.05 and 0.5 μM indole-3-acetic acid (IAA), and 0.3 and 3 μM gibberellic acid (GA₃) favoured sporophyte regeneration, whereas gametophyte regeneration took place when plant material was cultured in a hormone-free liquid MS medium. The endogenous contents of the auxin IAA, the cytokinins trans-zeatin, trans-zeatin riboside, dihydrozeatin, dihydrozeatin riboside, isopentenyladenine and isopentenyladenosine, and the gibberellins GA₁, GA₃, GA₄, GA₇, GA₉ and GA₂₀ in growing gametophytes and sporophytes were evaluated. Similar levels of the auxin and cytokinins and qualitative differences in the gibberellins were found between both generations.     

APOGAMY AND SOMATIC RESTITUTION IN THE FERN CERATOPTERIS

A mutant stock of the fern Ceratopteris has been derived from an inbreeding study following an interspecific hybridization between two diploid species. The mutant is characterized by gametophytes that produce non-functional spermatozoids and are incapable of selfing. Sporophytes develop apogamously from the mutant gametophytes and, although they are initially haploid and sterile, portions of the fronds later become doubled somatically and behave like tissues of sexually derived homozygous sporophytes. The mutant segregates from sporophytes in a 1:1 ratio when crosses are made with wild type gametophytes. Certain aspects of the behavior are similar to those seen in some naturally occurring apomictic ferns.     

Antheridiogen,dark spore germination,and outcrossing mechanisms in Bommeria (Adiantaceae)

Isozymic analyses of the patterns of genetic variability in sporophyte populations have demonstrated that most fern species have outcrossing breeding systems. However, because fertilization takes place during the ephemeral, diminutive gametophyte generation, direct observation of breeding systems in nature has not been possible. Recent discoveries of soil-bound spore banks suggested that genetic diversity could be stored beneath the surface and subsequently released by appropriate chemical cues. Previous studies demonstrated that Bommeria sporophytes are the product of outcrossing, that their gametophytes carry high levels of genetic load, and that they produce and respond to antheridiogen. Research reported here demonstrated that Bommeria spores can survive long-term storage but will not germinate in the dark. Antheridiogen, however, will release spores from this light requirement and stimulate germination. Higher concentrations of antheridiogen result in higher germination rates. Gametophytes grown in the dark on antheridiogen-enriched agar form antheridia and release actively swimming sperms. Thus, spores housed beneath the soil surface could remain dormant until stimulated to germinate by antheridiogen secreted by surface-dwelling, archegoniate gametophytes. Sperm released from these subterranean gametophytes could fertilize eggs on the surface. Because spores housed in the soil are likely to be genetically different than those at the surface, heterozygous sporophytes would be more likely to result. Discovering that Bommeria species contain all of the prerequisites for this proposed outcrossing mechanism provides an explanation for the maintenance of genetic diversity in some fern populations.     

C3 photosynthesis in the gametophyte of the epiphytic CAM fern Pyrrosia longifolia (Polypodiaceae)

Sporophytes of some epiphytic species in the fern genus Pyrrosia exhibit Crassulacean acid metabolism (CAM), generally considered to be a derived physiological response to xeric habitats. Because these species alternate between independent sporophytic and gametophytic generations yet only the sporophyte has been characterized physiologically, experiments were conducted to determine the photosynthetic pathways present in mature sporophytes, immature sporophytes, and gametophytes of Pyrrosia longifolia. Diurnal CO₂ exchange and malic acid fluctuations demonstrated that although the mature sporophytes exhibited CAM, only C₃ photosynthesis occurred in the gametophytes and young sporophytes. Consideration of the above results and those from previous studies, as well as the life cycle of ferns, indicates that the induction of CAM probably occurs at a certain developmental stage of the sporophyte and/or following exposure to stress. Elucidation of the precise mechanisms underlying this C₃-CAM transition awaits further research.     

EFFECTS OF BENLATE ON FERN GAMETOPHYTE DEVELOPMENT

The widely used fungicide, benlate, was tested for its effect(s) on gametophyte development in the fern Ceratopteris thalictroides (L.) Brongn. The active ingredient of benlate, benomyl, represents 50% of the fungicide by weight. Seven concentrations of benomyl were tested on C. thalictroides gametophytes: 0, 0.001, 0.01, 0.1, 1.0, 10.0 and 100 mg/l. Five developmental stages were observed for possible effects of benomyl. These were 1) germination, and the initiation of 2) antheridia, 3) notch meristems, 4) archegonia, and 5) sporophytes. Overall inhibition was greatest at 100 mg/l benomyl. At 10 mg/l and 100 mg/l, sporophyte initiation was completely blocked. This was probably the consequence of two characteristics found only on gametophytes growing on medium containing 10 mg/l and 100 mg/l benomyl. These characteristics were lack of sperm motility and production of callus growths in the areas proximal to the notch meristems, just proximal to the younger archegonia. Besides blocking the completion of sexual reproduction, the highest concentrations tested also produced smaller (cell number) and chlorotic gametophytes (especially at 100 mg/l). The bigametophyte population (made up of hermaphroditic and male gametophytes) was changed from 51% hermaphrodites (at 0 mg/l benomyl) to 26% hermaphrodites at 100 mg/l. This would, since only hermaphrodites possess archegonia, also decrease the potential for the production of sporophytes.     

Apospory in leaf culture of staghorn fern (Platycerium bifurcatum)

The induction, origin, morphology, and ploidy of aposporous gametophytes produced on juvenile leaves of the fern Platycerium bifurcatum (Cav.) C. Chr. were studied. Leaf explants were grown on modified Murashige and Skoog medium with 0%, 0.01%, 0.1%, 1%, or 2% sucrose. A low sucrose concentration (0.01%) and wounding of the adaxial side of the leaf significantly increased the induction of aposporous gametophytes (90% of leaves produced gametophytes). Regeneration began as a proliferation of mainly epidermal cells on both sides of the leaf; subsequent development was similar to that shown by gametophytes originating from spores. Flow cytometric analysis of sporophytes and aposporous gametophytes revealed that both forms had the same ploidy level. On the basis of these findings, we propose a set of conditions which regularly and reproducibly induces apospory on most of the leaf explants of the fern P. bifurcatum.     

POPULATIONAL AND GENETIC STUDIES OF A HOMOSPOROUS FERN—OSMUNDA REGALIS

The mating system and the genetic system of the homosporous fern Osmunda regalis were investigated. Seven populations from western Massachusetts were sampled. All the sporophytes investigated were found to be heterozygous for zygotic lethals. Morphological studies of the gametophytes indicated an intergametophytic mating system when the gametophytes were spatially and chronologically situated to exchange male gametes. Genetic studies evidenced a genetic system based upon duplicate loci.     

Tree Ferns in the Interior and at the Edge of a Mexican Cloud Forest Remnant: Spore Germination and Sporophyte Survival and Establishment1

The three most common tree fern species in a Mexican montane cloud forest fragment (Alsophila firma, Lophosoria quadripinnata and Sphaeropteris horrida) were selected for laboratory and transplant experiments. The objectives were: (1) to determine the percentage of spore germination and gametophytes producing sporophytes, and (2) to compare early establishment of sporophytes at the edge and in the interior of the forest. Percent spore germination varied between 16 and 86 percent, and the number of gametophytes that produced sporophytes was high (>50%). Survival and growth of sporophytes differed between species and habitats. Survival was greater at the edge than in the forest interior for Lophosoria, but it was similar for Alsophila and Sphaeropteris. Number and length of fronds were higher at the forest edge for individuals of Alsophila and Lophosoria, but not for Sphaeropteris. RGR was higher at the forest edge than in the forest interior for sporophytes of the three species. This study suggests that the forest edge is an appropriate habitat for establishment of Alsophila and Lophosoria, but Sphaeropteris is apparently a forest interior species.     

Enhancement of Apospory in Liquid Culture of Matteuccia struthioptens

Apospory in the fern Matteuccia struthiopteris, which is reportedhere for the first time, was induced more readily in liquidculture than on agar medium. It occurred more frequently fromdetached meristems than from juvenile leaves. Gametophytic outgrowthsbearing rhizoids, but lacking sex organs were found occasionallyon roots. Aposporous gametophytes isolated from liquid culturewere induced to form sporophytes either sexually or apogamously.Both types of sporophyte also behaved aposporously in liquidculture. Matteuccia, fern, apospory, apogamy, liquid culture, detached meristem     

Changes in the Level of the Protein Nitrogen during Growth of the Gametophyte and the Initiation of the Sporophyte of Dryopteris borreri Newm.

Estimations of the amounts of organic nitrogen in spores anddeveloping gametophytes of Dryopteris borreri, a fern reproducingapogamously and lacking archegonia, have shown that the nitrogenin the gametophytes increases exponentially. The logarithmicrate of increase of the nitrogen remains unchanged during theinitiation and emergence of the sporophyte. Gametophytes aboutto produce sporophytes yielded only filamentous growths whentransferred to a medium containing 8-azaguanine, and the increasein their content of organic nitrogen was very small. The resultsare discussed in relation to similar estimations made by otherworkers upon the gametophytes of Dryopteris erythosora(Eat.)Schott, a fern reproducing sexually. The interpretation placedupon the results obtained for D. erytkrosora is questioned,and a new interpretation of these results, together with thosefor D. borreri, is related to the difference in morphology ofthe two generations in the life cycle of the Polypodiaceousferns.     

Natural soil spore banks — can they be used to retrieve lost ferns?

Some fern species form spore banks — reservoirs in the soil of viable spores which remain dormant while buried but germinate in light if brought to the surface. The recently discovered characteristics of these spore banks are described. Enough is now known to suggest that they might have a role in the conservation of endangered fern species as alternatives toex situ collections of sporophytes, gametophytes and spores, the relative merits of which are also considered. Mature sporophytes of several British species have now been raised from natural spore banks in soil samples; if this proves to be possible also for endangered species, some interesting options become available. The possibilities are discussed of augmenting surviving populations and increasing their genetic diversity, even perhaps of retrieving lost populations, by reintroduction of spore bank-derived plants or by stimulating regeneration from spore banksin situ. Botanic gardens are well placed to provide the further research, the regular monitoring of endangered populations, and the taxonomic and horticultural support required to realise these possiblities.     

Contribution of fern gametophytes to the growth of produced sporophytes on the basis of carbon gain

Sporophytes appeared on most gametophytes of Thelypteris palustris (Salisb.) Schott that reached a certain size, which is interpreted to be a critical size of gametophytes for the production of sporophytes. After sporophytes were produced, attached gametophytes ceased dry weight growth, but the gametophytes which did not produce sporophytes grew successively. It was hypothesized that matter produced by gametophytes was being supplied to young sporophytes. Photosynthesis and respiration of gametophytes with attached sporophytes were not significantly different from that of gametophytes without sporophytes. Photosynthetic activity of gametophytes dropped from 0.18 to 0.03 mol CO₂ g^–1 s^–1 during the growth period. The higher photosynthetic rates of gametophytes in the early growth stage were important for reaching the critical size for sporophyte production in a short time. Sporophytes in the one leaf stage averaged 0.14 mol CO₂ g^–1 s^–1 of photosynthetic activity. The results show that sporophytes that had expanded the first leaf grow by their own photosynthetic production. Gametophytes allocated the photosynthate for sporophytes and it was an important aid before the one-leaf stage. The supportive role of gametophytes ended at that stage.     

Micropropagation and phase change in Blechnum spicant and Pteris ensiformis

Rhizomes of juvenile sporophytes of Blechnum spicant L. and Pteris ensiformis L. cultured on MS medium with N⁶-benzyladenine alone (0.44–4.4 M) or in combination with naphthaleneacetic acid (0.053–0.53 M), respectively, gave rise to several proliferation centres located at the epidermal and inner parenchyma of this organ after one month in culture. Subculture of these rhizomes for one month in growth regulator-free medium allowed organization of internal proliferation centres, and regeneration of a large number of sporophytes. Subculture in the above-mentioned proliferation medium, induced phase change and development of aposporous gametophytes from surface-localized proliferation centres. Addition of naphthaleneacetic acid to the culture medium promoted proliferation of green globular bodies (GGB) in B. spicant and both rhizogenesis and callogenesis processes in P. ensiformis. GGB derived from rhizomes of B. spicant produced numerous sporophytes and from the point of view of micropropagation it is a good system to regenerate sporophytes in this species.Abbreviations BA-6 benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - MS Murashige and Skoog (1962)