SEXUAL DEVELOPMENT OF ONOCLEA SENSIBILIS ON AGAR AND SOIL MEDIA WITHOUT THE ADDITION OF ANTHERIDIOGEN

Onoclea sensibilis gametophytes grown on agar, unsterilized soil or ashed soil without the addition of antheridiogen developed antheridia after the notch meristem was formed. There was a linear increase in maleness through time on all three media. Antheridial production on agar was delayed nearly 3 wk. Agar had a significantly higher proportion of females than ashed soil or unsterilized soil. It was concluded that agar is an inappropriate medium for experiments on the mechanism of sexual development in Onoclea gametophytes.     

Control of heat-shock response of young gametophytes of the sensitive fern is at the translational level

Young gametophytes of the sensitive fern, Onoclea sensibilis,respond to heat-shock by synthesizing in excess certain proteinsthat are made at normal growth temperature. Enhanced proteinsynthesis occurred during a 2 h heat-shock at a range of temperaturesbetween 38 °C and 50 °C. Although a temperature of 50°C proved lethal, a 5 min pulse at 50 °C resulted inenhanced synthesis of heat-shock proteins which continued forseveral hours at 25 °C. After heat-shock at 50 °C for10 or 15 min, the gametophytes temporarily lost their capacityfor protein synthesis but normal protein synthesis was resumedwithin 24 h of heat-shock. A heat-shock at 38 °C precedingone at 50 °C did not have any protecting effect on the gametophytes.In vitro translation of poly(A)+ RNA isolated from heat-shockedgametophytes yielded several proteins including heat-shock proteins.The results suggest that, rather than activating genes encodingnew messages for the synthesis of stress proteins, heat-shockof gametophytes of O. sensibilis triggers a controlling systemwhich enhances the translation of certain messages that aresynthesized at normal growth temperature. Key words: Onoclea sensibilis, heat-shock response, protein synthesis, sensitive fern, in vitro translation     

Effects of Population Density on Sex Expression in Onoclea sensibilis L. on Agar and Ashed Soil

Population density affected the sex expression of agar-growngametophytes of Onoclea sensibilis L. The time of onset of sexualitywas advanced, the proportion of females was increased, and thegrowth rate of individuals was greater at lower densities. Populationdensity had no effect on the sex expression of Onoclea grownon ashed soil, and there was no difference in growth rate ofindividuals grown on ashed soil at different densities. Covariateanalysis, using thallus width as a measure of growth rate, indicatedthat the effect of density on sex expression was mostly associatedwith growth rate. The differing effects of population densityon agar and ashed soil demonstrate that substrate influencessex expression in Onoclea. This influence is most dramatic insingle-gametophyte cultures, where agar cultures produced 97per cent females and ashed soil cultures 100 per cent males. Onoclea sensibilis L., sensitive fern, fern gametophytes, sexuality, population density     

Regulation of Protein Synthesis during Photomorphogenesis of Gametophytes of the Fern Onoclea sensibilis

Gametophytes of the fern Onoclea sensibilis grow as filaments in the dark and in red light and become planar in blue light. Pulse-labeling 4-day-old gametophytes with [³⁵S]methionine at different times after transfer to dark, red, and blue light environments revealed higher rates of amino acid uptake and protein synthesis in blue light than in red light or in the dark. Characterization of the extant and newly synthesized soluble proteins by one- and two-dimensional gel electrophoresis showed that the patterns of protein accumulation and synthesis in gametophytes exposed to short periods of red or blue light were qualitatively indistinguishable from those of gametophytes maintained in the dark. However, some striking increases and decreases in the levels of certain polypeptides were noted and these changes were accentuated during continued growth of gametophytes in the different environments. The results show that photomorphogenesis of gametophytes of O. sensibilis is associated with quantitative rather than qualitative changes in the population of mRNAs available for translation.     

Dark Growth and the Associated Phenomenon of Age-dependent Photoresponses in Fern Gametophytes

The cross-sectional area of the growing tip of a fern filamentundergoes an age-dependent decline over the period of darknessthat precedes an experimental light treatment. This dark-mediateddecline accounts for the appearance of age-dependent responsesto light treatments. Moverover, monochromatic irradiation experimentsshow that the cross-sectional areas of filaments do not manifestsignificant age-dependent variation in response to light treatmentsof sufficient energy. Because it is the cross-sectional arearather than the elongation rate that exhibits the direct responseto light in the control of filament growth, it follows thatthere is no evidence for age-dependent changes in photosensitivityof the filaments. Regression analyses establish that pre-germinationlight treatments do not affect subsequent patterns of dark growth;instead, stress calculations suggest that the tapering of thecross-sectional area may be referable to a state of stress-straindisequilibrium within the filament tip. These and other characteristicsof age-dependent growth responses of fern filaments are summarizedin a general working model. fern gametophytes, age-dependent photoresponses, stress analysis, Onoclea sensibilis     

REGIONS OF CELL WALL EXPANSION IN THE PROTONEMA OF A FERN

Protonemata of Onoclea sensibilis were grown for 7 days in darkness and were then transferred into light on new media, either liquid or agar-solidified, which contained 0.15% colchicine. The growth of individual plants was observed on solid media in microchambers. Unequivocal evidence was obtained that cell wall expansion and an increase in cell diameter occurred in regions well behind the apex of the protonema. This finding is related to an hypothesis which proposes light-induced changes in microtubule orientation and cell wall structure as an explanation for certain changes in cell form in fern gametophytes.     

The Effects of Centrifugation on Asymmetric Cell Division and Differentiation of Fern Spores

We have investigated the effects of centrifugation on sporepolarity, asymmetric cell division, and rhizoid differentiationin the sensitive fern Onoclea sensibilis L. Centrifugation at10000 g for 30 min produces a random orientation of spores withstratification of the cell contents. After centrifugation atmost early stages of development, the nucleus retains its normalpattern of migration from the centre of the ellipsoidal sporeto the proximal face and then to an end of the spore, withoutregard to the orientation of stratification. This indicatesthat the polarity of the spore is stable to centrifugation.As long as the nucleus migrates to an end of the spore and asymmetriccell division occurs, the small cell differentiates into a rhizoid.The arrangement of large cytoplasmic organelles appears to haveno influence on nuclear migration, asymmetric cell division,or rhizoid differentiation. The only period during developmentwhen centrifugation blocks asymmetric cell division is immediatelypreceding and during mitosis and cytokinesis. Spores centrifugedat this stage do not complete nuclear migration, and symmetriccell division results, with neither cell differentiating intoa rhizoid. The source of the stable polarity of the spore isdiscussed. cell polarity, rhizoid differentiation, centrifugation, Onoclea sensibilis L., sensitive fern, fern spores     

Fern antheridiogen: Cancellation of a light-dependent block to antheridium formation

In the light (200 ft-c), prothalli of Onoclea sensibilis failed to form antheridia at any stage of development unless they were exposed to the antheridium-inducing hormone of Pteridium aquilinum (abbreviation: A_Pt). If prothalli pregrown for 7 days in the light were transferred to near-darkness (ca. 0.8 ft-c), plus sucrose, then antheridium formation set in spontaneously ca. 15–19 days after transfer.Prothalli exposed to near-darkness, plus sucrose, plus A_Pt, began to form antheridia within 4 days. This lag period was shortened to 3 and 2 days if, after transfer to near-darkness, the prothalli were exposed to A_Pt with a delay of 5 and 12 days, respectively. These, and other results, led to the conclusion that a block to antheridium formation gradually decayed upon exposure to near-darkness and was reestablished in the light within 24–36 hr. The sucrose requirement for antheridium formation in near-darkness became operative after the block to antheridium formation had decayed. Antheridiogen was not detected during spontaneous antheridium formation in near-darkness.The results suggest that A_Pt and near-darkness interchangeably cancel a light-dependent block to antheridium formation, A_Pt acting more promptly than near-darkness.The observations indicate that this block must remain canceled while initials emerge and undergo differentiation.     

<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.     

GROWTH REGULATION BY ETHYLENE IN FERN GAMETOPHYTES. II. INHIBITION OF CELL DIVISION

Ethylene, a natural product of sensitive fern (Onoclea sensibilis L.) gametophytes, has been demonstrated to inhibit cell division in light-grown prothallia. When plants were grown on Knop's solution plus 1 % sucrose under 300 ft-c or more of white light, all ethylene concentrations from 1–1000 μl/liter reduced the rate of increase of cell number by about one-half. The over-all rate of increase of cell number was regulated by various environmental and chemical factors, but regardless of the rate established in control cultures, ethylene treatment of 1–1000 μl/liter produced a relative 50 % depression of cell number. Ethylene was specific for inhibition of cell division and was not a general inhibitor of growth. The ethylene inhibition did not result from a reduction of photosynthesis or energy supply. Further demonstration of ethylene as the active gaseous component resulted when cultures were grown in small enclosed containers with an ethylene absorbent, mercuric perchlorate, and consequently the cell number of gametophytes was restored to the level of unenclosed controls.     

Electrical Changes in the Apical Cell of the Fern Gametophyte during Irradiation with Photomorphogenetically Active Light

Electrophysiological procedures were used to evaluate cellular responses of fern (Onoclea sensibilis L.) gametophytes to photomorphogenetically active light. Red, far red, and blue light caused rapid changes in the membrane potential of the apical cell of the gametophyte filament; other cells in the filament were not similarly responsive. Measurements made with one electrode in the apical cell revealed that the membrane potential depolarized in red light and repolarized in far red light. Irradiation with blue light caused a hyperpolarization, the rapidity of which was dependent on a red light pretreatment. More refined measurements with one electrode in the tip of the apical cell and another in the base of the cell showed that both red and blue light treatments cause the apical cell to behave as a dipole. Because of the profound, long-term morphological changes that follow light irradiation in this organism, it was hoped to use it to elucidate the role that electrical parameters play in determining subsequent developmental events.     

Interaction between Antheridogens and Fatty Acids in Fern Spore Germination

Certain fatty acids in the C₅ to C₁₈ range, at concentrations as low as 10⁻⁵ M, were found to inhibit the germinations of spores of the sensitive fern, Onoclea sensibilis L. The addition of gametophytic culture flltrates of the bracken fern, Pteridium aquilinum (L.) Kuhn, containing antheridogen A, was found to overcome this inhibition and allow the spores to germinate and the gametophytes to develop in a normal fashion. Some fatty acids were found to increase the antheridium-inducing potency of antheridogen A as much as 10-fold. An effect similar to this may promote the diecious reproduction of ferns.     

Antheridia and Archegonia of the Apogamous Fern Pteris cretica

The external appearance of gametophytes and gametangia of Pteriscretica closely resembled those of sexually reproducing ferns.Antheridia were fully functional but archegonia were not. Despitethe formation of egg, ventral and neck canal cells, the flask-shapedarchegonia failed to open and Pteris spermatozoids failed toexhibit a chemotactic response to them. Cryo SEM revealed thatthe topmost cells of mature archegonia had collapsed, and thisis thought to account for the loss of archegonial function. Pteris cretica, fern, apogamy, antheridia, archegonia, cryo SEM     

Antheridiogen production and response in Polypodiaceae species

Antheridiogen chemicals secreted by living fern gametophytes have been shown to influence production of male gametangia and thus mating systems in a large number of terrestrial fern species. Antheridiogens have not previously been thought to be prevalent in the Polypodiaceae, a large family composed mostly of tropical epiphytes. This study presents bioassay methods more sensitive than previously used to detect antheridiogen and demonstrates that antheridiogens are also operative in the Polypodiaceae and in epiphytic species. Seven species in six genera (Campyloneurum angustifolium, C. phyllitidis, Lepisorus thunbergianus, Microgramma heterophylla, Phlebodium aureum, Phymatosorus scolopendria, and Polypodium pellucidum) were tested for the presence of an antheridiogen system. All species tested except P. aureum were induced to produce antheridia precociously by their own antheridiogen and by that of Pteridium aquilinum (APt). Phlebodium aureum responded to APt and promoted antheridium formation in Onoclea sensibilis but did not respond to its own antheridiogen. Spores of all species except P. aureum were induced to germinate in darkness by antheridiogen of the same species and by APt and to form antheridia in the dark, further enhancing the possibility of intergametophytic mating.     

Gene Activity during Germination of Spores of the Fern, Onoclea sensibilis: RNA and Protein Synthesis and the Role of Stored mRNA

Pattern of ³H-uridine incorporation into RNA of spores of Onocleasensibilis imbibed in complete darkness (non-germinating conditions)and induced to germinate in red light was followed by oligo-dTcellulose chromatography, gel electrophoresis coupled with fluorographyand autoradiography. In dark-imbibed spores, RNA synthesis wasinitiated about 24 h after sowing, with most of the label accumulatingin the high mol. wt. poly(A)^–RNA fraction. There was noincorporation of the label into poly(A) ⁺ RNA until 48 h aftersowing. In contrast, photo-induced spores began to synthesizeall fractions of RNA within 12 h after sowing and by 24 h, incorporationof ³H-uridine into RNA of irradiated spores was nearly 70-foldhigher than that into dark-imbibed spores. Protein synthesis,as monitored by ³H-arginine incorporation into the acid-insolublefraction and by autoradiography, was initiated in spores within1–2 h after sowing under both conditions. Autoradiographicexperiments also showed that the onset of protein synthesisin the cytoplasm of the germinating spore is independent ofthe transport of newly synthesized nuclear RNA. One-dimensionalsodium dodecyl sulphate-polyacrylamide gel electrophoresis of³⁵S-methionine-labelled proteins revealed a good correspondencebetween proteins synthesized in a cell-free translation systemdirected by poly(A) ⁺RNA of dormant spores and those synthesizedin vivo by dark-imbibed and photo-induced spores. These resultsindicate that stored mRNAs of O. sensibilis spores are functionallycompetent and provide templates for the synthesis of proteinsduring dark-imbibition and germination. Key words: Onoclea sensibilis, fern spore germination, gene expression, protein synthesis, sensitive fern, stored mRNA     

THE EFFECT OF AUXIN AND GUANINE ON CELL EXPANSION AND CELL DIVISION IN THE GAMETOPHYTE OF THE FERN,ONOCLEA SENSIBILIS

Miller , J. H. (Yale U., New Haven, Conn.) The effect of auxin and guanine on cell expansion and cell division in the gametophyte of the fern, Onoclea sensibilis. Amer. Jour. Bot. 48(9): 816–819. Illus. 1961.—Auxin and guanine promote cell expansion in 0. sensibilis gametophytes. The optimum concentration of auxin for total expansion is 10^−-5 M, but the optimum for elongation is 10^−-6 M. Above this concentration the cells expanded isodiametrically. Guanine is active at higher concentrations than auxin. Increasing concentrations of auxin progressively inhibit red light-induced cell division, while guanine has no effect on cell division. Neither kinetin nor adenine promotes cell expansion or cell division.     

THE RELATIONSHIP BETWEEN THE PROMOTION OF ELONGATION OF FERN PROTONEMATA BY LIGHT AND GROWTH SUBSTANCES

Germinating spores of the fern Onoclea sensibilis L. were grown in darkness, so that they developed as filaments (protonemata). Brief daily exposure of the filaments to red, far-red or blue light increased the rate of filament elongation. Filament elongation was also promoted by indoleacetic acid. When filament elongation was promoted with both indoleacetic acid and exposure to light, the growth promotions caused by red and far-red light were additive to auxin-induced growth. Blue light promoted elongation only at sub-optimal concentrations of auxin. Elongation induced by guanine was additive to red- and far-red-induced elongation. Gibberellic acid had no effect on elongation under any condition. Blue-light-induced elongation resembled auxin-induced elongation in its requirement for exogenous sucrose and sensitivity to inhibition by parachlorophenoxyisobutyric acid. Red and far-red light were active regardless of the presence or absence of sucrose and promoted elongation at a concentration of parachlorophenoxyisobutyric acid which completely inhibited blue-light-induced elongation.     

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.     

SPERMATOGENESIS IN A HOMOSPOROUS FERN,ONOCLEA SENSIBILIS

A detailed study of spermatogenesis in a homosporous fern, Onoclea sensibilis L., is presented from the formation of the first spermatogenous cell to the release of the sperm. Two different walls are deposited around the developing spermatids at specific developmental stages as opposed to one wall reported for other species. Most ultrastructural changes that occur in Onoclea during spermatid differentiation resemble those described in previous studies on other fern species, with the following exceptions: 1) A previously undescribed structure appears during midspermatid stage. This dense layer of amorphous material with a row of evenly spaced light areas occurs between the anterior portion of the mitochondrion associated with the multilayered structure and the anterior plasmalemma of the spermatid. 2) An early stage in blepharoplast formation resembles that which occurs in the heterosporous fern Marsilea, in contrast to that which has been reported in Platyzoma, the only other homosporous fern studied at this stage. 3) The osmiophilic crest does not form as early as reported in other ferns. 4) The cap cell of Onoclea is removed intact, rather than collapsing or forming a pore during sperm release. Observations are reported on the number of sperm per antheridium, the time course of spermatogenous cell mitosis, and of differentiation of spermatids into sperm. In Onoclea, an antheridium may contain either 32 or 64 sperm. Regardless of the final number of sperm, each has approximately the same volume.