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.

     

Biological and nutritional aspects involved in fern multiplication

Gametophytes of several species of ferns were mechanically triturated and the resulting homogenates cultured in vitro for propagation purposes. Differences in the time period from spore culture to sporophyte development were perceivable between species. For those species with a fast life cycle and high sporophyte production such as Woodwardia virginica and Dryopteris affinis sp. affinis, homogenization of gametophytes can be considered to be excellent method for propagation, yielding hundreds of sporophytes in a short period of time. Sporophyte formation was inhibited in O. regalis by the succesive application of homogenization to gametophytes regenerated by this technique. The effect of the culture medium composition on fern production was also studied in O. regalis and P. ensiformis gametophytes. In these species, sporophyte formation increased when the gametophytes were cultured in a medium containing water+0.7% agar. Addition of sucrose inhibited gametophyte development and induced their necrosis. The 1/2 dilution of Murashige and Skoog basal medium, without sucrose, favoured leaf expansion in P. ensiformis sporophytes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Gametogenesis and chromosome number in Postelsia palmaeformis (Laminariales, Phaeophyceae)

Gametophytes of the ‘sea palm’, the kelp Postelsia palmaeformis Ruprecht, produced gametes whether or not chelated iron was supplied in the culture medium, in contrast to the inhibition of gametogenesis seen with the absence of iron in many other kelps. As gametogenesis proceeded, every cell of the gametophytes was converted into a gamete so that the gametophytes did not continue to grow vegetatively. The portion of the life history from spore release through germination, gametophyte growth, gametogenesis, fertilization and growth of the young sporophyte was completed in 9–10 days under laboratory conditions. Chromosome counts showed that sporophytes had a diploid number of 26–34 chromosomes while sporangia and gametophytes had a haploid number of 14–17 chromosomes, indicating a typical haplodiplophasic life history as seen in other Laminariales.     

IAA-induced apogamy in Platycerium coronarium (Koenig) Desv. gametophytes cultured in vitro

Apogamous sporophytes were produced on Platycerium coronarium gametophytes cultured in the presence of indole-3-acetic acid (IAA). The percentage of apogamy as well as the total number of apogamous sporophytes produced per gametophyte clump were highest in the presence of 40 M IAA. When ethylene was allowed to accumulate in the culture vessel in the presence of an optimum level of IAA, the percentage and total number of apogamous sporophyte production decreased significantly. Using light microscope and confocal laser scanning microscope we have shown that nuclear size can be used as a quick parameter to estimate the ploidy level of P. coronarium.Abbreviations CLSM confocal laser scanning microscope - IAA Indole-3-acetic acid - MS Murashige and Skoog     

Generational differences in response to desiccation stress in the desert moss Tortula inermis

BACKGROUND AND AIMS: Active growth in post-embryonic sporophytes of desert mosses is restricted to the cooler, wetter months. However, most desert mosses have perennial gametophytes. It is hypothesized that these life history patterns are due, in part, to a reduced desiccation tolerance for sporophytes relative to gametophytes. METHODS: Gametophytes with attached post-embryonic sporophytes of Tortula inermis (early seta elongation phenophase) were exposed to two levels of desiccation stress, one rapid-dry cycle and two rapid-dry cycles, then moistened and allowed to recover, resume development, and/or regenerate for 35 d in a growth chamber. KEY RESULTS: Gametophytes tolerated the desiccation treatments well, with 93 % survival through regenerated shoot buds and/or protonemata. At the high stress treatment, a significantly higher frequency of burned leaves and browned shoots occurred. Sporophytes were far more sensitive to desiccation stress, with only 23 % surviving after the low desiccation stress treatment, and 3 % surviving after the high desiccation stress treatment. While the timing of protonemal production and sporophytic phenophases was relatively unaffected by desiccation stress, shoots exposed to one rapid-dry cycle produced shoots more rapidly than shoots exposed to two rapid-dry cycles. CONCLUSIONS: It is concluded that sporophytes of Tortula inermis are more sensitive to rapid drying than are maternal gametophytes, and that sporophyte abortion in response to desiccation results from either reduced desiccation tolerance of sporophytes relative to gametophytes, or from a termination of the sporophyte on the part of the gametophyte in response to stress.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of <Emphasis Type="Italic">Drynaria fortunei</Emphasis>, a fern species source of Chinese medicine “Gu-Sui-Bu”

This study reports spore germination, early gametophyte development and change in the reproductive phase of Drynaria fortunei, a medicinal fern, in response to changes in pH and light spectra. Germination of D. fortunei spores occurred on a wide range of pH from 3.7 to 9.7. The highest germination (63.3%) occurred on ½ strength Murashige and Skoog basal medium supplemented with 2% sucrose at pH 7.7 under white light condition. Among the different light spectra tested, red, far-red, blue, and white light resulted in 71.3, 42.3, 52.7, and 71.0% spore germination, respectively. There were no morphological differences among gametophytes grown under white and blue light. Elongated or filamentous but multiseriate gametophytes developed under red light, whereas under far-red light gametophytes grew as uniseriate filaments consisting of mostly elongated cells. Different light spectra influenced development of antheridia and archegonia in the gametophytes. Gametophytes gave rise to new gametophytes and developed antheridia and archegonia after they were transferred to culture flasks. After these gametophytes were transferred to plastic tray cells with potting mix of tree fern trunk fiber mix (TFTF mix) and peatmoss the highest number of sporophytes was found. Sporophytes grown in pots developed rhizomes.     

RADIOACTIVE TRACER STUDY OF SPOROPHYTE NUTRITION IN HEPATICS

Sporophytes of Fossombronia foveolata, Lophocolea heterophylla, Pellia epiphylla, Ptilidium pulcherrimum, and Riella affinis were surgically isolated from host gametophyte tissues and treated with ¹⁴CO₂. Sporophytes of all five species are capable of fixing CO₂ in the light. Sporophyte/gametophyte ratios for ¹⁴CO₂ fixation/mg fresh weight range between 0.12 and 0.39. Corresponding ratios for chlorophyll content are 1.07 to 3.30. Of the total ¹⁴CO₂ fixed by excised Lophocolea sporophytes, 40% can be attributed to the photosynthetic activity of haploid spores. Enveloping gametophytic tissues (calyptra and pseudoperianth) inhibit photosynthesis of attached sporophytes by as much as 50%. For sustained growth, sporophytes rely on organic nutrients supplied by the gametophyte: radioactivity of Lophocolea sporophytes increases significantly after application of ¹⁴C-glucose to host gametophytes. Surgically isolated sporophytes develop slowly in mineral culture, without significant increase in dry weight. The assumption that hepatic sporophytes are at least partly autonomous with respect to organic nutrition (an assumption that figures prominently in speculation on the evolutionary origin of the sporophyte) is confirmed.     

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.     

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.     

GROWTH INHIBITION IN GAMETOPHYTES AND OAT COLEOPTILES BY THELYPTERIN A AND B RELEASED FROM ROOTS OF THE FERN THELYPTERIS NORMALIS

Gametophytes, when grown in the immediate vicinity of a Thelypteris normalis sporophyte—in soil or in sterile culture on agar—showed a reduced number of cells and an altered gross morphology. This is attributed to the action of the thelypterins, which are inhibitors released from T. normalis sporophytes. Growth inhibition of the gametophytes was greatest when thelypterins were added during early stages of gametophyte development. Removal of thelypterin A permitted resumption of growth. Thelypterin A noncompetitively inhibited auxin-enhanced elongation of A vena coleoptiles. The growth of T. normalis root segments was not affected by thelypterins. The growth of young sporophytes of T. normalis was inhibited by mature sporophytes.     

Photosynthetic Rates of Sporophytes and Gametophytes of the Fern, Todea barbara

The photosynthetic rates of intact sporophytes or gametophytes of the fern Todea barbara grown in sterile culture were measured using an infrared gas analyzer. Sporophytes consisted of single whole plants with roots and leaves grown in tubes of agar. Gametophytes were grown as several plants covering the surface of the agar. Sporophytes had photosynthetic rates at light saturation of 8.50 microliters CO₂ per hour per milligram dry weight and 1,300 microliters CO₂ per hour per milligram chlorophyll, whereas rates for gametophytes were lower, 2.36 microliters CO₂ per hour per milligram dry weight and 236 microliters CO₂ per hour per milligram chlorophyll.     

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.     

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.     

INTERGENERIC HYBRIDIZATION AMONG FIVE GENERA OF THE FAMILY LESSONIACEAE (PHAEOPHYCEAE) AND EVIDENCE FOR POLYPLOIDY IN A FERTILE PELAGOPHYCUS×MACROCYSTIS HYBRID

Hybridization was attempted by combining gametophytes between intergeneric pairs among the following taxa in the Lessoniaceae: Macrocystis pyrifera ( L.) C. Agardh , M. integrifolia Bory, M. angustifolia Bory , Pelagophycus porra ( Leman) Setch ., Nereocystis luetkeana ( Mert.) Post & Rupr ., Dictyoneurum californicum Rupr ., and Dictyoneuropsis reticulata ( Saud.) Smith. Hybrid sporophytes were produced in some combinations involving Macrocystis × Pelagophycus and Macrocystis × Dictyoneurum, and in all combinations of Dictyoneuropsis × Dictyoneurum. This is the first report of intergeneric hybrids involving Dictyoneurum. Gametophytes of P. porra had 16–24 chromosomes. Gametophytes from a fertile Macrocystis-Pelagophycus hybrid were crossed with Macrocystis and Pelagophycus gametophytes. Hybrid male gametophytes and Pelagophycus female gametophytes produced sporophyte progeny, but hybrid males with Macrocystis females did not. A single hybrid female gametophyte did not produce gametophytes in combination with hybrid males , Pelagophycus males or Macrocystis males. The hybrid gametophytes had approximately 30 chromosomes. It is hypothesized that the hybrid is an alloploid, containing a complete set of Macrocystis and Pelagophycus chromosomes, which may have allowed meiosis and sporogenesis to proceed normally in the hybrid sporophyte found in the sea. Thus, reproductive isolating mechanisms appear to operate at both pre- and postzygotic stages, and both can be overcome in intergeneric hybrids .     

NORTHERN AND SOUTHERN HEMISPHERE HYBRIDS OF MACROCYSTIS (PHAEOPHYCEAE)1

All combinations of individuals of Macrocystis pyrifera (L.) C. Agardh, M. integrifolia Bory, and M. angustifolia Bory hybridized. Gametophyte isolates obtained from 18 individuals were used, including M. pyrifera and M. integrifolia from the extremes of their Northern Hemisphere ranges along the Pacific Coast of North America and M. pyrifera and M. angustifolia from Tasmania, Australia. All combinations of gametophytes produced sporophytes of normal morphology, with the exception of crosses involving three gametophyte isolates. One female (M. integrifolia) and two male (M. pyrifera and M. angustifolia)gametophyte isolates were unable to produce normal sporophytes in combination with gametophytes of the opposite sex. Some cultures of female gametophytes produced abnormally shaped parthenogenetic sporophytes. Gametophytes and sporangia of M. pyrifera had n= 16 chromosomes. The M. integrifolia female gametophyte that was unable to produce normal sporophytes had n = ca. 32 chromosomes. These results show that these species of Macrocystis have not become reproductively isolated. Although these species may be considered conspecific according to the biological species concept, we recommend that they continue to be recognized as separate species based on morphological differences.     

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.     

扇蕨配子体发育与濒危机制的探讨

采用改良Knop’s固体培养基、原生境腐殖质土和红壤分别培养扇蕨（Neocheiropteris palmatopedate）孢子,光学显微镜及解剖镜下观察记录其孢子萌发及配子体发育过程,比较了3种培养方法对其配子体发育和有性繁殖的影响,并在此基础上探讨了扇蕨的濒危原因。结果表明：成熟孢子黄褐色,赤道面观为豆形,极面观椭圆形,单裂缝。孢子萌发类型为书带蕨型,原叶体发育为槲蕨型。成熟原叶体呈心脏形。毛状体在原叶体阶段出现。有性生殖周期长及配子体发育成幼孢子体的百分率低是扇蕨在配子体世代的主要濒危原因。此外,红壤固有的理化性质导致扇蕨配子体发育极其缓慢、精子器和颈卵器发生的时间间隔过长使其不能受精产生孢子体。原生植被遭受破坏引起的林下腐殖质土消失、红壤裸露,加剧了扇蕨的濒危。     

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     

Mycothallic/mycorrhizal symbiosis of chlorophyllous gametophytes and sporophytes of a fern, Pellaea viridis (Forsk.) Prantl (Pellaeaceae, Pteridales)

Gametophytes of Pellaea viridis that appeared spontaneously on the surface of substratum originating from an ultramafic area were found to form mycothallic symbiosis with arbuscular mycorrhizal fungi (AMF) under laboratory conditions. In gametophytes and sporophytes grown with Glomus tenue, abundant arbuscule formation was observed at both stages. In gametophytes, the fungus was found in the region where the rhizoids are initiated. If G. intraradices was added to the soil, the gametophytes were colonised mostly by G. tenue, and roots of sporophytes were colonised by G. intraradices. The presence of AM fungi in both gametophytes and sporophytes of P. viridis resulted in the development of larger leaf area and root length of the sporophyte. The analysis of gametophytes from the Botanical Garden in Krakow (Poland) showed that cordate gametophytes of Pteridales, namely Pellaea viridis (Pellaeaceae), Adiantum raddianum and A. formosum (Adiantaceae), were also mycothallic.     

GENETIC LOAD IN OSMUNDA REGALIS POPULATIONS

Osmunda regalis sporophytes form haploid spores which develop into functionally hermaphroditic gametophytes. The self-fertilization of such gametophytes results in zygotes which are completely homozygous. Spore samples collected from sporophytes in natural populations were used to establish gametophyte cultures. The majority of these gametophytes were unable to form viable embryos when only self-fertilization was possible. Controlled selfing and crossing experiments revealed that the inability of these homozygous embryos to develop normally is attributable to the presence of recessive lethals. To account for this genetic load, an hypothesis is proposed integrating the morphology and ecology of the gametophyte generation with the polyploid genetic system of the sporophyte generation.