Effects of CO2 on growth and photosynthesis of Pyrrosia piloselloides (L.) Price gametophytes

The effects of CO2 concentration on spore germination, growth, and net photosynthetic rate (PN) of gametophytes of a tropical epiphytic fern, Pyrrosia piloselloides, were investigated over a 100-d period. Increasing CO2 concentration stimulated spore germination and enhanced gametophytic growth. The appearance of sexual organs and formation of sporophytes were accelerated with higher CO2 during growth. Radiant energy saturated PN and dark respiration rate also increased with increasing CO2 concentrations during growth.     

Developmental morphology of strap-shaped gametophytes of Colysis decurrens: a new look at meristem development and function in fern gametophytes

Background and Aims

The gametophytes of most homosporous ferns are cordate–thalloid in shape. Some are strap- or ribbon-shaped and have been assumed to have evolved from terrestrial cordate shapes as an adaptation to epiphytic habitats. The aim of the present study was to clarify the morphological evolution of the strap-shaped gametophyte of microsoroids (Polypodiaceae) by precise analysis of their development.

Methods

Spores of Colysis decurrens collected in Kagoshima, Japan, were cultured and observed microscopically. Epi-illuminated micrographs of growing gametophytes were captured every 24 h, allowing analysis of the cell lineage of meristems. Light microscopy of resin-sections and scanning electron microscopy were also used.

Key Results

Contrary to previous assumptions that strap-shaped Colysis gametophytes have no organized meristem, three different types of meristems are formed during development: (1) apical-cell based – responsible for early growth; (2) marginal – further growth, including gametophyte branching; and (3) multicellular – formation of cushions with archegonia. The cushion is two or three layers thick and intermittent. The apical-cell and multicellular meristems are similar to those of cordate gametophytes of other ferns, but the marginal meristem is unique to the strap-shaped gametophyte of this fern.

Conclusions

The strap-shaped gametophytes of C. decurrens may have evolved from ancestors with a cordate shape by insertion of the marginal meristem phase between the first apical-cell-based meristem and subsequent multicellular meristem phases. Repeated retrieval of the marginal meristem at the multicellular meristem phase would result in indefinite prolongation of gametophyte growth, an ecological adaptation to epiphytic habitats.     

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.     

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.     

A methodology for large-scale Athyrium sheareri gametophyte proliferation and sporophyte production using tissue culture

Tissue culture methods using gametophytes are considered the easiest ways to mass-produce fern sporophytes. The aim of this study was to develop a practical propagation method for the ornamental fern, Athyrium sheareri. The gametophytes obtained from in vitro spore germination were used as experimental materials. We used the chopping method to investigate the culturing conditions for proliferating gametophytes and the blending method for evaluating the mass production of sporophytes in mixed soil. Gametophyte proliferation was determined via Knop medium, various concentrations of Murashige and Skoog (MS) basal medium (1, 1/2, 1/4), and media components (sucrose, nitrogen source, and activated charcoal). The fresh weight of the gametophytes increased by more than 24-fold in 1/2 MS medium. In addition, 1 g of gametophyte could produce a maximum of 255.3 sporophytes in a mixed soil of 7.5 cm² area. Treating gametophytes with exogenous plant growth regulators promoted the formation and growth of sporophytes. The cultivated young sporophytes were acclimated and successfully grown in greenhouses. We developed a mass production protocol for A. sheareri sporophytes suitable for field application, which is expected to have commercial value.

     

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.     

Tree fern trunks facilitate seedling regeneration in a productive lowland temperate rain forest

Seedling regeneration on forest floors is often impaired by competition with established plants. In some lowland temperate rain forests, tree fern trunks provide safe sites on which tree species establish, and grow large enough to take root in the ground and persist. Here we explore the competitive and facilitative effects of two tree fern species, Cyathea smithii and Dicksonia squarrosa, on the epiphytic regeneration of tree species in nutrient-rich alluvial forests in New Zealand. The difficulties that seedlings have in establishing on vertical tree fern trunks were indicated by the following observations. First, seedling abundance was greatest on the oldest sections of tree fern trunks, near the base, suggesting that trunks gradually recruited more and more seedlings over time, but many sections of trunk were devoid of seedlings, indicating the difficulty of establishment on a vertical surface. Second, most seedlings were from small-seeded species, presumably because smaller seeds can easily lodge on tree fern trunks. Deer browsing damage was observed on 73% of epiphytic seedlings growing within 2 m of the ground, whereas few seedlings above that height were browsed. This suggests that tree ferns provide refugia from introduced deer, and may slow the decline in population size of deer-preferred species. We reasoned that tree ferns would compete with epiphytic seedlings for light, because below the tree fern canopy photosynthetically active radiation (PAR) was about 1% of above-canopy PAR. Frond removal almost tripled %PAR on the forest floor, leading to a significant increase in the height growth rate (HGR) of seedlings planted on the forest floor, but having no effects on the HGRs of epiphytic seedlings. Our study shows evidence of direct facilitative interactions by tree ferns during seedling establishment in plant communities associated with nutrient-rich soils.     

Gametophyte ecology and demography of epiphytic and terrestrial tropical ferns

Factors that influence the distribution of ferns are poorly understood and likely reflect the ecology of both the sporophyte and the gametophyte generation. Little study has been done on the ecology of the gametophyte generation, especially in regard to tropical species. The goal of this study was to examine demography and the influence of light and disturbance on the distribution of the gametophytes of several tropical epiphytic, hemiepiphytic, and terrestrial fern species. Through a series of observational and experimental studies, we found that increased terrestrial gametophyte density and richness were related to both increased light and disturbance. By contrast, increased light had no influence, and increased disturbance negatively affected epiphytic density. Over a 25-mo demographic study, epiphytic and hemiepiphytic species had significantly greater longevities and lower recruitment rates than terrestrial species. Such unique strategies may have evolved in response to different disturbance regimens between the two habitats. Terrestrial species encounter and are adapted to more frequent disturbance and have invested in rapid gametophyte growth and recruitment. Epiphytic species may be more influenced by bryophyte competition, and in habitats of relatively low disturbance, they have invested in greater size and longevities. In such systems, gametophytes are able to survive for years waiting for favorable recruitment conditions.     

Ethylene is a modulator of gibberellic acid-induced antheridiogenesis in <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> gametophytes

In fern (Anemia phyllitidis) gametophytes cellulose in the walls of the antheridial zone cells which was organized in clusters and spots was transformed via dispersed form to fibrillar arrangement (layered in oblique and perpendicular array in relation to the transverse direction of cell expansion) during antheridiogenesis induced by gibberellic acid (GA₃) and/or enhanced by 1-aminocyclopropane-1-carboxylic acid (ACC). In the ACC-treated gametophytes, where antheridia were not induced, the cellulose was arranged in the same manner. Aminooxyacetic acid (AOA), which inhibits antheridiogenesis and development of fern gametophytes, produced in the cell walls both random and longitudinal type of organization of cellulose microfibrils, however, in the GA₃/AOA-treated plants the oblique type was also observed. The total numbers of cells with perpendicular and/or oblique type of cellulose microfibrils in the GA₃-, GA₃/ACC-and GA₃/AOA-treated gametophytes corresponded to the average number of antheridia formed. Moreover, it was found that the extracts from the gametophytes treated with GA₃ or with the mixture of GA₃ and ACC contained significantly less soluble sugars but more α-amylase-and endoglucanase-released sugars than the extracts from the gametophytes of the other series. Thin layer chromatography of the samples from the cell wall extracts hydrolyzed by endoglucanase contained xylose and cellobiose which suggested that these sugars built the xyloglucans, hemicellulose polymers responsible for tethering of walls of fern gametophyte cells like in higher plants.     

Identifying fern gametophytes using DNA sequences

Identification of fern gametophytes is generally hampered by low morphological complexity. Here we explore an alternative: DNA‐based identification. We obtained a plastid rbcL sequence from a sterile gametophyte of unknown origin (cultivated for more than 30 years) and employed blast to determine its affinities. Using this approach, we identified the gametophyte as Osmunda regalis. To evaluate the robustness of this determination, and the usefulness of rbcL in differentiating among species, we conducted a phylogenetic analysis of osmundaceous fern sequences. Based on our results, it is evident that DNA‐based identification has considerable potential in exploring the ecology of fern gametophytes.     

蕨类植物配子体发育与生理生态研究进展

蕨类植物配子体为单倍体,结构简单,独立于孢子体生活,在研究其对环境的响应以及揭示其机理上都具有独特的优势。该研究从我国和国际两个分支出发,梳理了近年来全球范围内相关的文献资料,透视了蕨类配子体的发育和生理生态前沿科学和研究动态。在发育部分以研究进展为主要内容,国内研究以传统植物蕨类植物的配子体形态和发育的观察为主,而国外学者更关注于新技术和新方法在传统学科中的运用,如X光透射技术和流式细胞术。生理生态部分分为光合与呼吸作用、土壤逆境的响应、气候变化的响应以及对化感物质的响应4个板块。在光合作用的研究中,发现蕨类配子体会在光强的变化下产生自我保护机制,碳水化合物和脂质是配子体能量代谢中的重要指标。在土壤逆境的响应研究中,对砷有超富集作用的蜈蚣草配子体和耐高盐的铁角蕨配子体是配子体研究中较为突出的材料。荷兰地区广泛存在的耳蕨属蕨类,哥斯达黎加热带雨林的20种蕨类植物及水生蕨类槐叶萍,成为了证明配子体成活率和温度之间重要关系的实验例证。在化感作用研究中,主要通过紫茎泽兰根、茎和叶水提液对扇蕨等4种蕨类配子体的作用,证明了入侵植物对于蕨类植物配子体生长发育具有危害作用。在美国佛罗里达的一类爬树蕨也发现了同样的入侵植物现象。此外,还对几个新兴技术在配子体研究的前景进行了展望,并对已有技术进行了描述。该研究以多个角度介绍了国内外配子体的研究进展,希望有助于促进我国学者对该领域的深入研究。     

Growth and development of fern gametophytes in an airlift fermenter

Spores of the fernsPteridium aquilinum andAnemia phyllitidis were grown in an airlift fermenter and subsequent growth and development of gametophytes was monitored. Both species produced greater biomass than that generated in any other solid- or liquid-based culture system tested.Pteridium generated more tissue thanAnemia in every system. The morphology of airlift-grown gametophytes was similar to that of soil-grown plants; fewer gametophytes with perturbed development were observed in airlift cultures than in the other liquid-based systems. No attempt was made to optimise airlift conditions for the species and tissue employed, so it is concluded that airlift cultivation is a promising system for the bulk production of fern gametophytic tissue.     

Phenology and wintering capacity of sporophytes and gametophytes of ferns native to northern Japan

Summary To investigate life history adaptations to cold climates, the leaf development, sporulation period, growing stage of gametophytes, and the frost and drought resistance of sporophytes and gametophytes of 67 fern species native to Kokkaido were studied. Most ferns common in Hokkaido are summer-green with leaves developing during late May to June and decaying during October. Most of the ferns in Hokkaido sporulate during August to early September. Spores dispersed from June to September germinate before winter begins, forming vegetative prothallia. Gametophytes mature only in the following summer. Thus in Hokkaido the gametophytes as well as perennial sporophytes are exposed to severe winter conditions. In order to correlate the life cycles of temperate ferns with winter cold stress, frost resistance of gametophytes, rhizomes, and leaves of sporophytes were determined. Maximal frost resistance of rhizomes reflects the stress conditions of their habitats: rhizomes of forest understory ferns are damaged at-5°to -17.5°C, epiphytic ferns and ferns of habitats exposed to severe frost sustained temperatures of -20° to-40°C. The leaves of winter-green and evergreen ferns resist frost ranging from -25° to -40°C. The leaves of summer-green ferns are killed by late frost below -5°C. With some exceptions, gametophytes of ferns growing on the forest floor resist frost to -40°C and are much hardier than sporophytes. These results suggest the possible restrictive effects of cold climate on the life span of leaves as well as on the sporulation period. If winter cold is one of the decisive factors for seasonality expression and habitat distribution of ferns, the sensitive generation must be the sporophyte rather than the gametophyte. The hardier gametophyte is therefore able to colonize habitats in which the sporophyte is excluded by frost if mechanisms of vegetative propagation are evolved.Contribution No. 2451 from The Institute of Low Temperature Science     

Negative phototropism in young gametophytes of the fern Schizaea pusilla

Schizaea pusilla is a rare fern that occurs in acidic bogs and is one of the few fern species that maintains a filamentous gametophyte throughout its development. To expand our knowledge of the physiology of this fern, phototropic responses were examined in young gametophytes. In contrast to germ filaments of other fern species, apical protonemata of young gametophytes are negatively phototropic in continuous white, red and blue light at all fluence rates tested. The expression of phototropic curvature is not limited by time since apical protonemata are also negatively phototropic when they are given brief exposures of light and then placed in the dark. In other lower plant groups such as mosses and some algae, the direction of phototropic curvature can change depending on light quality and intensity, but in young gametophytes of Schizaea, negative phototropic curvature was observed in all conditions studied. Blue light is the most effective in promoting the negative phototropic response in Schizaea.     

Inhibition of two-dimensional growth and suppression of ribonucleic acid and protein synthesis in the gametophytes of the fern, Asplenium nidus, by chloramphenicol, puromycin and actinomycin D

Chloramphenicol and puromycin at appropriate concentrations inhibited the induction of two-dimensional growth in the gametophytes of the fern Asplenium nidus without drastically inhibiting germination and continued filamentous growth. Similar responses to actinomycin D were reported earlier. Radioautographic techniques were employed to study the pattern of ribonucleic acid and protein synthesis in gametophytes which were treated with chloramphenicol, puromycin and actinomycin D. Uptake of H³-uridine into ribonucleic acid was strongly inhibited by all three antibiotics. Chloramphenicol and puromycin were not as effective as actinomycin D in inhibiting H³-leucine incorporation. The results are discussed in relation to the quality of light and antibiotics on two-dimensional growth in the gametophytes.     

Rapid in vitro propagation of Matteuccia struthiopteris (L.) Todaro – an edible fern

A rapid and efficient in vitro plant regeneration method was developed for Matteuccia struthiopteris (L.) Todaro (Ostrich fern). Side shoots, originating in meristems of sectioned rhizomes, were used as explant material. A very high rate of meristem multiplication was achieved by culturing the explants in half-strength MS liquid medium supplemented with 2.0 mg/l N-(4-Pyridyl)-N′-phenylurea (4-PU) and 0.5 mg/l thidiazuron (TDZ). Multiplication of the shoot primordia was faster in suspension culture than on solid medium. Rhizogenesis and growth of regenerants were best achieved on hormone-free one-quarter-strength MS solid medium amended with 0.4% agar and 1.0% activated charcoal. Regenerated plantlets continued to grow after transfer to soil in a phytotron. Received: 19 March 1998 / Revision received: 17 July 1998 / Accepted: 3 August 1998     

RNA and protein metabolism in the particulate fractions of the gametophytes of bracken fern during growth in red and blue light

Summary The metabolism of RNA and protein in the gametophytes of bracken fern (Pteridium aquilinum) is affected by the quality of light in which they are grown. When sporelings were grown as two-dimensional gametophytes in blue light, particulate fractions separated from the sporelings exhibited greater incorporation of uridine-³H and leucine-³H into RNA and protein, respectively, than those from sporelings grown as one-dimensional protonema in red light. After various periods of exposure of gametophytes to red or blue light in the presence of uridine-³H, the nuclei-rich fraction showed the highest specific activity in RNA, and irrespective of incubation time, blue light was more effective than red light. The possibility that enhanced synthesis of RNA in the nucleus in response to blue light is significantly related to the morphological growth pattern of the gametophytes, is discussed.     

ACTION OF PURINE AND PYRIMIDINE ANALOGS ON THE GROWTH AND DIFFERENTIATION OF THE GAMETOPHYTES OF THE FERN ASPLENIUM NIDUS

The purine analogs, 8-azaadenine, 8-azaguanine, 8-azaxanthine and 8-azahypoxanthine, and the pyrimidine analogs, 2-thiocytosine, 5-fluorouracil, 2-thiouracil and 6-azauracil, inhibited the induction of 2-dimensional growth in the gametophytes of the fern Asplenium nidus L. In contrast, thymine analogs such as 5-fluorodeoxyuridine, 2-thiothymine, 6-azathymine and 5-bromouracil caused non-specific growth inhibitions without suppressing 2-dimensional growth. Subinhibitory concentrations of 8-azaxanthine, 8-azahypoxanthine, and 2-thiouracil promoted both 1-dimensional and 2-dimensional phases of growth of the gametophytes. Inhibitory effects of the analogs were observed on treatment of the spores or of gametophytes of different ages. Gametophytes growing in the analogs for different periods of time recovered from inhibition on transfer to the basal medium.