Squash Techniques in the Cytological Investigation of Mosses

Abstract

Photosynthetic activity of attached sporophytes is very low (a few per cent or less) compared with that of associated gametophyte structures (perianth, bracts and uppermost leaves) in Cephalozia bicuspidata and Lophocolea heterophylla, or with even small areas of thallus in Pellia epiphylla. Photosynthetic uptake of ¹⁴CO₂ by developing sporophytes of P. epiphylla, C. bicuspidata and L. heterophylla is at most a few per cent of the ¹⁴C translocated subsequently from the gametophyte, and could be negligible. In L. heterophylla, the perianth, bracts and uppermost leaves appear to play only a limited role in nutrition of the sporophyte, the leafy shoots making a major contribution. In C. bicuspidata the perianth and leaves of the short archegonial shoot may provide a substantial part of the nutrition of the sporophyte. There is some indication in all three species that translocation from the gametophyte is most active when the sporophyte reaches full size but is still green, declining in the final stages of maturation of the capsule.     

Orthophosphate Absorption by the Sporophyte of Funaria Hygrometrica During Maturation

The absorption of phosphate by the sporophyte of Funaria hygrometrica,during its maturation, has been studied using ³²P. More radioactivitywas found in the capsule when the absorption occured throughthe seta alone than when it look place through the leaves ofthe gametophyte. Each stage of capsule development studied isdescribed from transverse sections and electron micrographs.Phosphate is accumulated very actively by the capsule in theyoungest stage when the spores are being formed. The exchangesbetween the capsular tissues and the spores are low.     

Distribution of Potassium, Calcium and Magnesium in the Gametophyte and Sporophyte Generations of Funaria hygrometrica Hedw

Analyses have been made of the K, Ca and Mg in separated sporophytes,spores and gametophytes of the moss Funaria hygrometrica duringsporophyte development. Degeneration of the gametophyte wasaccompanied by loss of K and a gain in Ca while the K contentof the developing sporophyte increased more rapidly than thatof Ca. The presence of an air–gap in the expanding capsulesignificantly influenced the observed cellular location of ions.Relative to the sporophyte the spores were shown to have a higherK and a lower Ca concentration. The behaviour of Mg was intermediatebetween K and Ca throughout. Funaria hygrometrica, potassium, calcium, magnesium, cation distribution, gametophyte, sporophyte     

Structure and Function of Transfer Cells in the Sporophyte Haustorium of Funaria hygrometrica Hedw: III. TRANSLOCATION OF ASSIMILATE INTO THE ATTACHED SPOROPHYTE AND ALONG THE SETA OF ATTACHED AND EXCISED SPOROPHYTES

Translocation of products of photosynthesis from gametophyteto sporophyte was examined in the moss Funaria hygrometricaHedw., as an adjunct to companion studies on the ultrastructureof the sporophyte haustorium and its capacity for absorptionof sugars in vitro. Labelled products derived from gametophyticphotosynthesis are transported to the sporophyte at an approximatelylinear rate for up to 12 h after a pulse treatment with ¹⁴CO₂.Large sporophytes receive label at a greater rate than smallerones. Transport is inhibited under conditions of water stress,and by lack of light, though darkening the sporophyte alonehas no effect. Movement of label from the haustorium along theseta occurs at a velocity of 1–3 mm h^–1, and issimilar to the onward movement of label derived from [³H]glucosesupplied to the haustorium in vitro.     

Assessment of photosynthetic performance in the two life history stages of Alaria crassifolia (Laminariales,Phaeophyceae)

Understanding of the physiological responses of kelp to environmental parameters is crucial, especially in the context of environmental change that may have contributed to the decline of kelp forests all over the world. The current study presents the photosynthetic characteristics of the macroscopic sporophyte and microscopic gametophyte stages of the brown alga Alaria crassifolia from Hokkaido, Japan, as determined by examining their photosynthetic responses over a range of temperature and irradiance using dissolved oxygen and chlorophyll fluorescence measurements. Net photosynthetic rates of the sporophyte were consistently higher than those of gametophyte across temperature gradients and irradiance levels. Photosynthesis–irradiance curves at 8°C, 16°C, and 20°C revealed similar initial slopes (α = 0.4–0.9) on the two life history stages, but higher compensation (E _c = 4–7 μmol photons m^?2 s^?1) and saturation irradiances (E _k = 53–103 μmol photons m^?2 s^?1) for the sporophyte than for the gametophyte (E _c = 0–7 μmol photons m^?2 s^?1; E _k = 7–10 μmol photons m^?2 s^?1). Both stages exhibited chronic photoinhibition, as shown by the failure of recovery in their maximum quantum yields (F _v/F _m) following high irradiance stress, with greater possibility of photodamage at low temperature. Gametophytes were less sensitive to low temperatures than sporophytes, given their relatively stable F _v/F _m response. Nevertheless, temperature optima for photosynthesis of both stages coincide with each other at 20–23°C, which correspond to the growth and maturation periods of A. crassifolia in Japan. This species is also likely to suffer from thermal inhibition as both GP rates and F _v/F _m decreased above 24°C.     

<Emphasis Type="Italic">Macrocystis</Emphasis> mariculture in Chile: growth performance of heterosis genotype constructs under field conditions

Recent progress in Macrocystis mariculture is based on clonal stock cultures of gametophyte parents. Batches of up to 10⁵ genetically identical sporophyte seedlings can be produced at any time in the laboratory and explanted in the field for production of biomass. Sexual crosses of selected Macrocystis pyrifera gametophyte parents of different geographic origin along the coast of Chile showed heterosis and produced sporophyte batches with superior growth performance. Starting from zygotes, after 10 weeks in the laboratory and 5 months in the sea, our best hybrid genotypes grew up to 11 kg fresh weight per frond, which corresponds to 66 kg m⁻¹ of line in a commercial mariculture installation. In contrast, average yields of 14.4 and 22 kg m⁻¹ are reported in the literature for traditional methods. Additional experiments, including inter-specific crosses M. pyrifera × M. integrifolia and their performance in different climate zones of Chile, confirm that heterosis is a powerful tool for crop improvement in Macrocystis. It opens the possibility to construct tailor-made heterosis genotypes with maximum productivity and/or other desired properties for any given locality.     

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.

     

ROLE OF SETTLEMENT DENSITY ON GAMETOPHYTE GROWTH AND REPRODUCTION IN THE KELPS PTERYGOPHORA CALIFORNICA AND MACROCYSTIS PYRIFERA (PHAEOPHYCEAE)1

Laboratory studies were used to examine how variation in the density of spore settlement influences gametophyte growth, reproduction, and subsequent sporophyte production in the kelps Pterygophora californica Ruprecht and Macrocystis pyrifera (L.) C. Ag. In still (non-aerated) cultures, egg maturation in both species was delayed when spores were seeded at densities 300 · mm^?2. Although the density at which this inhibition was first observed was similar for both species, the age at which their eggs matured was not. P. californica females reached sexual maturity an average of 4 days (or ～ 30%) sooner than did M, pyrifera. As observed previously in field experiments, per capita sporophyte production was negatively density dependent for both species when seeded at spore densities of 10 · mm^?2. Total sporophyte production (i.e. number · cm^?2) for both species, however, was greatest at intermediate densities of spore settlement (～ 50 spores · mm^?2). In contrast, total sporophyte production by P. californica steadily increased with increasing spore density in aerated cultures; highest sporophyte density was observed on slides seeded at a density of 1000 spores · mm^?2. Preliminary experiments with P. californica involving manipulation of aeration and nutrients indicate that inhibition of gametophyte growth and reproduction at higher densities of spore settlement in non-aerated cultures was probably caused by nutrient limitation.     

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.     

The development of the placenta in the anthocerote Phaeoceros laevis (L.) Prosk

The development of the placenta in the anthocerote Phaeoceros laevis (L.) Prosk. was studied by transmission electron microscopy. By the time the sporophyte emerges from the involucre, a conspicuous placental region is formed by the intrusive growth of sporophyte foot haustorial cells into the adjacent gametophyte vaginula tissue. The separation of gametophyte cells by haustorial cells and their incorporation into the placenta are preceded by the loosening and swelling of their walls and the formation of a periplasmic space. This process causes the disruption of the plasmodesmata, and may eventually result in the complete isolation and consequent degeneration of the cells. Crystals are commonly observed in the vacuoles of gametophyte placental cells. Crystals become more abundant during cytoplasmic degeneration, and are released in the placental lacunae that result from the complete dissolution of gametophyte cells. During the subsequent phase of capsule elongation, the gametophyte placental cells that retain the symplastic connection with the adjoining gametophyte parenchyma develop a wall labyrinth typical of transfer cells. Obliteration of the wall labyrinth by deposition of lightly staining wall material is observed later in sporophyte development, in concomitance with capsule dehiscence. Crystals are negative to the periodic acid/thiocarbohydrazide/silver proteinate test for carbohydrates whilst they are completely digested by pepsin or protease, denoting protein composition.Abbreviation PATAg periodic acid/thiocarbohydrazide/silver proteinate     

LIFE HISTORY AND SYSTEMATIC POSITION OF AKKESIPHYCUS LUBRICUS (PHAEOPHYCEAE)1

The life history in culture of Akkesiphycus lubricus Yamada et Tanaka, an alga which has been placed in the Coilodesmaceae or the Punclariaceae, Dictysiphonales, was studied. In culture the species alternates between a microscopic filamentous gametophyte and a macroscopic polystichous sporophyte, a pattern common to the Dictyasiphonales and Laminariales. However, it has a unique anisogamous dioecious gametophyte. Fusions between mac-ro-gametes and micro-gametes were not observed, Macro-gametes or zygotes germinated, mostly developing into sporophytes that formed unilocular sporangia and the rest developed into reduced gametophytic flaments again. The gametophyte matures in 50C short-day conditions, corresponding to winter in Hokkaido. The sporophyte develops normally and matures only in low-temperature conditions irrespective of daylength. In regard to iits systematic position, Akkesiphycus lubricus is considered to have a closer relationship with the Laminariales than with the Dietyosiphonales in the following characters; lack of pyrenoids; early stages of parenchyma formation in the sporophyte; direct development of sporophytes from gametes or zygotes without forming a besal system zoospore becomes almost empty after germination by the migration of cell contents into a germ lube; formation of macro-gametangia by direct conversions of mother cells of mother cells of fertile branches; and micro-gametangia formed in clusters showing closeresemblance to the antheridia of Pseudochorda nagii (Tokida) Inagaki.     

A hundred-year-old question: is the moss calyptra covered by a cuticle? A case study of Funaria hygrometrica

Background and Aims

The maternal gametophytic calyptra is critical for moss sporophyte development and ultimately sporogenesis. The calyptra has been predicted to protect the sporophyte apex, including the undifferentiated sporogenous region and seta meristem, from desiccation. We investigate the hypothesis that this waterproofing ability is due to a waxy cuticle. The idea that moss calyptrae are covered by a cuticle has been present in the literature for over a century, but, until now, neither the presence nor the absence of a cuticle has been documented for any calyptra.

Methods

The epidermis of the calyptra, leafy gametophyte and sporophyte sporangia of the moss Funaria hygrometrica were examined using scanning and transmission electron microscopy. Thicknesses of individual cuticle layers were quantified and compared statistically. The immunochemistry antibody (LM19) specific for pectins was used to locate cell wall material within the cuticle.

Key Results

A multi-layered cuticle is present on the calyptra of F. hygrometrica, including layers analogous to the cuticular layer, cell wall projections, electron-lucent and electron-dense cuticle proper observed in vascular plants. The calyptra rostrum has a cuticle that is significantly thicker than the other tissues examined and differs by specialized thickenings of the cuticular layer (cuticular pegs) at the regions of the anticlinal cell walls. This is the first documentation of cuticular pegs in a moss.

Conclusions

The calyptra and its associated cuticle represent a unique form of maternal care in embryophytes. This organ has the potential to play a critical role in preventing desiccation of immature sporophytes and thereby may have been essential for the evolution of the moss sporophyte.     

Calymperastrum,a new genus of Pottiaceae

Abstract

Annual growth rate and spore and capsule production was studied in 50 colonies of the epixylic hepatic, Ptilidium pulcherrimum in northern Sweden. Radial growth rate was independent of colony size but varied from year to year (mean value 5.2 mm year^?1, range 3.5–6.3 mm year^?1). Size at first sporophyte production was estimated to be 68 cm² and the age of first reproduction was estimated to be about 9 years. It was estimated that antheridia were formed the third year. No correlation between growth rates and reproduction was observed. Capsule density as well as capsule and spore production increased significantly with increased colony size. Differences between years in spore and capsule production (up to six-fold) were observed. The mean number of spores per capsule was 27 400 but significant differences between colonies (range from 18 000 to 44 000) were observed.     

Substratum preference,spore output and temporal variation in sporophyte production of the epixylic moss Buxbaumia viridis

Abstract

Occupancy and sporophyte numbers of Buxbaumia viridis on patches of decaying wood were investigated during a 4-year period (1996-99). Temporal variation in sporophyte number was investigated in relation to precipitation. Spore number per sporophyte was counted and spore number per substratum patch area and forest area were calculated. To predict the occupancy of B. viridis on decaying logs and stumps in a forest, the patch size (suitable wood area) was the most important variable. Decomposition stage was included in the model as a quality factor, expressing the higher and more stable humidity in late wood-decay stages. The suitable wood area of each patch was determined first and foremost by the stage of decomposition and diameter of the decaying wood. Only 16% of all wood patches considered suitable for production of sporophytes were occupied. The probability of a patch being occupied increased linearly with patch size to approximately 7 dm²; above this size the probability of being colonized was close to one. Both the number of sporophytes and the number of occupied patches were correlated with precipitation amounts during the summer months, with a reduction of occupied patches of 73% in the dry year 1999, compared with mean values for 1996-98. Spore number per sporophyte was correlated with length and width of the capsule, and varied between 1.4 and 9.0 million, with a mean value of 6.0 million. It is suggested that the rarity of B. viridis is caused by a low probability of patches being occupied because of their short longevity and small size, together with the facts that the species is dioicous, short-lived, sensitive to desiccation of the substratum, and has a gametophyte that is so minute it cannot compete with larger bryophytes.     

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.     

MORPHOLOGY AND ANATOMY OF NEW SPECIES OF SCHIZAEA AND ACTINOSTACHYS

The gametophyte, old embryo, and sporophyte of Schizaea pseudodichotoma sp. nov., sporophyte and female parental gametophyte of S. diversispora hybr. nov. (S. pseudodicholoma X probably S. dichotoma), sporophyte of S. rhacoindusiata sp. nov., and gametophyte, old embryo, and sporophyte of Actinostachys macrofunda sp. nov. are described. The taxonomy of Schizaea is discussed and the system of Diels is strongly supported. The two sectional names used by Diels, Euschizaea Hook, and Lophidium Rich, are replaced by Pectinatae Prantl and Schizaea respectively. The prime morphological significance of Schizaea pseudodicholoma lies in its leafless embryo and its simple leaf differing from other species in its section, and that of Actinostachys macrofunda lies in its reduction to nearly complete heterotrophic existence and its frequent multiple annulus. Fungal hyphae have been traced from Schizaea and Actinostachys through the substratum and into root nodules of Casuarina and into roots of two other angiosperms.     

A method to establish an “immortalized F2” sporophyte population in the economic brown alga Undaria pinnatifida (Laminariales: Alariaceae)

Studies of quantitative trait loci based on genetic linkage maps require the establishment of a mapping population. Permanent mapping populations are more ideal than temporary ones because they can be used repeatedly. However, there has been no reported permanent sporophyte population of economically important kelp species. Based on the characteristics of the kelp life cycle, we proposed a method to establish, and then constructed experimentally, an “immortalized F₂” (IF₂) population of Undaria pinnatifida. Doubled-haploid “female” and “male” sporophytes were obtained through the parthenogenesis of a female gametophyte clone and the selfing of a “monoicous” gametophyte clone (originally male), respectively, and they were used as the parents. The F₁ hybrid line was generated by crossing the female and male gametophytes derived from the respective female and male parents. Full-sibling F₂ gametophyte clones, consisting of 260 females and 260 males, were established from an F₁ hybrid sporophyte. Thirty-five females and 35 males were randomly selected and paired to give rise to an IF₂ population containing 35 crossing lines. A parentage analysis using 10 microsatellite markers confirmed the accuracy of the IF₂ population and indicated the feasibility of this method. This proposed method may be adapted for use in other kelp species, and thus, it will be useful for genetic studies of kelp.     

Alternation of generations – unravelling the underlying molecular mechanism of a 165‐year‐old botanical observation

Characteristically, land plants exhibit a life cycle with an ‘alternation of generations’ and thus alternate between a haploid gametophyte and a diploid sporophyte. At meiosis and fertilisation the transitions between these two ontogenies take place in distinct single stem cells. The evolutionary invention of an embryo, and thus an upright multicellular sporophyte, in the ancestor of land plants formed the basis for the evolution of increasingly complex plant morphologies shaping Earth's ecosystems. Recent research employing the moss Physcomitrella patens revealed the homeotic gene BELL1 as a master regulator of the gametophyte‐to‐sporophyte transition. Here, we discuss these findings in the context of classical botanical observations.     

A proposed reclassification of Bryum,Anomobryum and Brachymenium (Musci,Bryaceae)

Abstract

The traditional generic classification in the Bryoideae (Bryaceae) is based primarily on details of the sporophyte, particularly peristome reduction and capsule inclination. Recent research, however, has suggested that closely related species in Anomobryum can have very distinct capsules, varying from complete peristomes and inclined capsules to reduced peristomes and erect capsules. The suggestion is made here that sporophyte features, especially those related to peristome features, may obscure phylogenetic relationships. A revised classification is proposed, based on analyses of 100 species of Bryum, Anomobryum, and Brachymenium and sixteen sporophytic and gametophytic characters. The revised classification groups species with similar gametophores but is supported by at least some sporophytic characters. Analysis of variance and principal components analysis indicate that both the traditional and revised classification provide effective summaries of the variation within the 100 species examined. The new classification consists of three informal groups of species which can be recognized at generic rank. One comprises Bryum species, including those with reduced peristomes and inclined capsules. A second comprises all species of Anomobryum, as well as species of Bryum and Brachymenium with similar gametophores. The third group consists of the rosulate species of Bryum and Brachymenium. Phylogenetically, the second group may be more closely related to Pohlia than to the species in the first and third groups. The third group appears to be most closely related to Rhodobryum and Plagiomnium. Suggested names are provided for groups one and three, while the name Bryum is retained for group two. No nomenclatural changes are made, however, until more research is done.