Reproductive biology of the Pteridophyta. II. Theoretical considerations

Because of the homothallic nature of many pteridophytes, two categories of mating are possible: intragametophytic selling (the origin of both gametes from a single gametophyte) and inter-gametophytic mating (the origin of each gamete from a different gametophyte).Various morphological and genetical criteria (placement of the gametangia on the thallus, their sequence of ontogeny, the capacity for simple polyembryony and genetic self-incompatibility) can be used to indicate the relative probability of intragametophytic selfing or intergametophytic mating. Only the former has genetic significance (i.e.complete homozygosity); if the latter is evidenced, then detailed studies of population variability are required to ascertain the breeding system. Three types of reproductive systems involve the gametophyte generation: intragametophytic selfing, intergametophytic mating and apogamy. Apogamy generally offers the shortest gametophyte generation and the least evolutionary potential, intergametophytic mating systems generally have the longest gametophyte generation and the greatest evolutionary potential, and intragametophytic mating systems are intermediate in both respectS. It is envisioned that the interaction between gametophyte ecology and evolutionary potential is important in the evolution of a taxon's reproductive system.     

球子蕨孢子的无菌繁殖

以球子蕨成熟孢子为外植体,研究了不同激素及浓度对其孢子萌发、愈伤组织诱导、丛生芽分化及生根的影响。结果表明:孢子萌发最适培养基为1/2MS+2%蔗糖,20d后萌发率达55.7%;诱导愈伤组织的最适培养基为MS+0.5mg·L-1KT+0.5mg·L-12,4-D,诱导率达36%,愈伤组织为绿色颗粒状;颗粒状愈伤组织在不添加激素的MS培养基中即可生长出大量丛生芽,转化率可达49.3%;低浓度(0.2mg·L-1)的IAA可有效促进幼孢子体苗生根。     

球子蕨孢子的无菌繁殖

以球子蕨成熟孢子为外植体,研究了不同激素及浓度对其孢子萌发、愈伤组织诱导、丛生芽分化及生根的影响。结果表明：孢子萌发最适培养基为1／2MS＋2％蔗糖,20d后萌发率达55．7％;诱导愈伤组织的最适培养基为MS＋0．5mg·L-1 KT＋0．5mg·L～2,4-D,诱导率达36％,愈伤组织为绿色颗粒状;颗粒状愈伤组织在不添加激素的MS培养基中即可生长出大量丛生芽,转化率可达49．3％;低浓度（0．2mg·L-1）的IAA可有效促进幼孢子体苗生根。     

Carbon Dioxide and Ethylene Control of Spore Germination in Onoclea sensibilis L

Regulation of spore germination in the fern Onoclea sensibilis L. was investigated by applying CO₂ alone and in combination with ethylene. Sterile spores were sown aseptically on Knops solution in loosely capped culture tubes, enclosed individually in 2-liter chambers, and grown under continuous white light. When maintained in enclosed containers with the ethylene-absorbent mercuric perchlorate and with atmospheres enriched up to 2% CO₂ (v/v), spores germinated without any inhibition. Higher levels of applied CO₂ were progressively inhibitory. Inhibition by CO₂ was reversible. When CO₂ was permitted to escape and spores were exposed subsequently to ambient laboratory air, recovery from inhibition occurred within 48 hours. Also, inhibition by CO₂ was specific, since the same degree of inhibition resulted regardless of whether spores were treated with exogenous CO₂ for 48, 72, or 96 hours. The effect on germination of 1 μl/l added ethylene depended upon the amount of applied CO₂. When containers of KOH were enclosed and ambient CO₂ was absorbed, inhibition of germination by 1 μl/l exogenous ethylene was 90%. When CO₂ was applied in concentrations from 0.25 to 1.0% (v/v), CO₂ increasingly antagonized the inhibitory action of 1 μl/l added ethylene. Thus, photoinduced germination of spores was regulated by competitively interacting levels of CO₂ and ethylene.     

The role of calcium ions in phytochrome-mediated germination of spores of Onoclea sensibilis L.

Phytochrome is confirmed to be the photoreceptor pigment in the germination response of Onoclea sensibilis L. by demonstrating red-far-red (R-FR) photoreversibility. External Ca²⁺ is required for this response with a threshold at a submicromolar concentration. Ethylene glycol-bis(-amino-ethyl ether)-N,N,N,N-tetraacetic acid, La³⁺ and Co²⁺ reversibly inhibit germination. Lanthanum only inhibits germination when applied before or during irradiation, indicating that the external Ca²⁺ requirement is transient, although in the absence of Ca²⁺ the R-stimulated system remains maximally poised to accept the ion for over 4 h after irradiation. The ability to respond to Ca²⁺ 4.1 h after R-irradiation is not reversed by FR-irradiation, indicating that Ca²⁺ transport has been uncoupled from phytochrome. Barium and Sr²⁺, but not Mg²⁺ can substitute for Ca²⁺. Artificially increasing the concentration of intracellular free Ca²⁺ with the ionophore A 23187 stimulates germination in the dark. The Ca²⁺-calmodulin antagonists, trifluoperizine and chlorpromazine, reversibly inhibit germination. Calcium is required in phytochrome-mediated fern spore germination; it may be acting as a second messenger.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FR far-red light - R fed light     

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     

Reproductive biology of the Pteridophyta IV. An experimental study of mating systems in Ceratopteris thalictroides (L.) Brongán

Gametophytes of the fern Ceratopteris thalictroides originating from four different geographic areas were studied morphologically and genetically. Intraspecific variability was found among the collections for gametophyte morphology and the frequency of lethal sporophytic genotypes. The differences in gametophyte morphology were shown to result in a different mating system for each collection.     

Initial events in the tip-swelling response of the filamentous gametophyte of Onoclea sensibilis L. to blue light

Several inhibitors were applied to filamentous gametophytes of the fern Onoclea sensibilis in the attempt to characterize how electrical phenomena might be involved in the tip-swelling response to blue light (BL). The membrane potential of the apical cell in the typical fern filament rests near-120 mV in darkness, but irradiation with blue light causes the membrane to hyperpolarize at a steady rate of 2.6 mV min^-1 until it reaches a new stable value between-130 and-135 mV. In darkness, 10^-4M salicylhydroxamic acid (SHAM), an inhibitor of BL-mediated absorbance changes in putative plasma-membrane fractions from maize coleoptiles, has no observable effects on the membrane potential or on filamentous growth. A SHAM pretreatment before BL irradiation causes approx. 70% inhibition of the membrane hyperpolarization as well as a comparable reduction in the growth response; however, SHAM has no effect in experiments where its application follows the onset of the electrical response. Exposing the filaments to 10^-5M Na₃VO₄, an inhibitor of the plasma-membrane ATPase, depresses the membrane potential in darkness. Depending on the timing of application, Na₃VO₄ prevents the initiation of or blocks further increases in the BL-mediated hyperpolarization. Application of Na₃VO₄ causes an immediate cessation of growth in both darkness and BL. These findings implicate the involvement of a plasmalemma-bound flavin-cytochrome complex and ATP-driven proton pump in the initial events of this growth response to blue light.     

Expression of two cloned mRNA sequences during development and germination of spores of the sensitive fern,Onoclea sensibilis L.

A library of complementary DNA (cDNA) clones has been prepared from poly(A)⁺RNA of spores of the sensitive fern, Onoclea sensibilis L. By differential hybridization with labeled probes made to poly(A)⁺ RNA of spores, gametophytes and leaves, two spore-specific clones (pOSS68 and pOSS194) were selected and characterized. Northern blot analysis showed that RNA sequences homologous to the two cDNA clones first appear in the post-meiotic spore and increase in abundance during spore maturity. Both RNA sequences decay during photoinduced germination of the spores and do not reappear in the gametophytes. In spores imbibed in the dark under conditions which do not favor germination, no significant decrease in pOSS194-mRNA abundance is noted. In contrast, the decrease in pOSS68 mRNA in dark-imbibed spores parallels that observed in photoinduced spores. The predicted amino-acid sequence of pOSS194 has a striking similarity to the early light-inducible proteins expressed during the greening of etiolated pea and barley seedlings, whereas that of pOSS68 shows some homology to proteins encoded by late-embryogenesis-abundant mRNAs of angiosperm embryos.Abbreviations bp base pairs - cDNA complementary DNA - ds double-stranded - ELIP early light-inducible proteins - LEA late embryogenesis abundant - nt nucleotide - ss single stranded This work was partially supported by a NASA grant (NAGW-901) and by an allocation from the Research Challenge Investigators' Fund of the Ohio State University to V.R. Thanks are due to Mr. Clayton L. Rugh for sequencing our clones and to Dr. Paul A. Fuerst for help in the computer search of sequence alignments.     

Experimental studies on mating systems and genetic load in Onoclea sensibilis L. (Aspleniaceae: Athyrioideae)

Sporophytes from natural populations of Onoclea sensibilis L. from eastern North America and Japan were investigated to determine their mating system and the frequency of recessive deleterious genes (genetic load). Morphological and physiological criteria in the gametophyte generation indicate that this species has a high probability of intergametophytic mating in nature. All sporophytes exhibited some degree of heterozygosity for genetic load, expressed either as zygotic lethals or leaky lethals. Effects of antheridogen on gametophyte sex ratios and polyembryony are described.     

Reproductive Biology of the Pteridophyta

Because of the homothallic nature of many pteridophytes, two categories of mating are possible: intragametophytic selling (the origin of both gametes from a single gametophyte) and inter-gametophytic mating (the origin of each gamete from a different gametophyte).Various morphological and genetical criteria (placement of the gametangia on the thallus, their sequence of ontogeny, the capacity for simple polyembryony and genetic self-incompatibility) can be used to indicate the relative probability of intragametophytic selfing or intergametophytic mating. Only the former has genetic significance (i.e.complete homozygosity); if the latter is evidenced, then detailed studies of population variability are required to ascertain the breeding system.
Three types of reproductive systems involve the gametophyte generation: intragametophytic selfing, intergametophytic mating and apogamy. Apogamy generally offers the shortest gametophyte generation and the least evolutionary potential, intergametophytic mating systems generally have the longest gametophyte generation and the greatest evolutionary potential, and intragametophytic mating systems are intermediate in both respectS. It is envisioned that the interaction between gametophyte ecology and evolutionary potential is important in the evolution of a taxon's reproductive system.     

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.     

Red Light Stimulates an Increase in Intracellular Calcium in the Spores of Onoclea sensibilis

Red light (R) stimulates an increase in the total concentration of intracellular calcium in the spores of Onoclea sensibilis L. as determined by atomic absorption spectroscopy. Subsequent exposure to far-red light inhibits the R-induced increase in intracellular calcium. The majority of the increase occurs 5 minutes after the onset of irradiation. The calcium antagonist, La³⁺, inhibits both germination and the R-induced increase in intracellular calcium. The R-induced increase in calcium is sufficient to account for an increase in the concentration of intracellular calcium ions from 0.1 micromolar to 1 to 10 micromolar. Large detectable changes in other elements tested are not required for germination.     

Effects of caffeine on germination and differentiation in spores of the fern, Onoclea sensibilis

During spore germination in the fern, Onoclea sensibilis L., the nucleus moves from a central position to one end, and an asymmetrical cell division partitions the spore into two cells of greatly unequal size. The smaller cell differentiates directly into a rhizoid, whereas the larger cell and its derivatives give rise to the prothallus. In the presence of 5 mM caffeine, the nuclei of most of the spores undergo mitotic replication, whereas cell wall formation is blocked. Multinucleate single cells are produced, which are capable of growth, but no rhizoid differentiation occurs. In some cases a partial cell wall is produced, but the nucleus moves through the discontinuity back to the center of the spore, and the enucleate, incompletely partitioned small “cell” fails to differentiate into a rhizoid. In less than 1% of the spores a broad protuberance, whose wall is yellow-brown, is formed in a multinucleate single cell. The color, staining reaction to ruthenium red, and ultrastructural appearance of the protuberance resemble that of the rhizoid wall. It appears that infrequently in the caffeine-treated spores, a feature which is characteristic of rhizoids is expressed, in the absence of asymmetric cell division, in a cell which otherwise is unable to produce a rhizoid. The results are interpreted to mean that the spore has a highly localized, persistent differentiated region. For rhizoid differentiation to occur, a nucleus must be confined in that region – a confinement which normally is accomplished by the geometrically asymmetric first cell division of germination.     

Reversal by Light of Ethylene-induced Inhibition of Spore Germination in the Sensitive Fern Onoclea sensibilis: An Action Spectrum

Spores of the fern Onoclea sensibilis L. normally germinate to produce two cells of unequal size. The larger cell divides to produce the familiar heart-shaped prothallus. The smaller cell elongates and differentiates into the rhizoid but normally does not divide again. Onoclea spores germinate in complete darkness. Dark germination can be completely inhibited by ethylene gas (10 microliters per liter is saturating). This inhibition can be reversed by light. Broad band colored light studies were designed to determine which area of the spectrum was most effective in overcoming ethylene inhibition. White light treatment resulted in 17% germination. Blue light treatment resulted in 1% germination. Red light treatment resulted in 15% germination. Red light, therefore, was most effective and accounted for most of the effects of white light. A detailed action spectrum was constructed using narrow band interference filters in the wavelength range from 350 to 764 nanometers. The action spectrum has only one major peak at 711 nanometers.     

How to model a complex trait. 1. General considerations and suggestions

Usually, when complex traits are at issue, not only are the loci of the responsible genes a priori unknown; the same also holds for the mode of inheritance of the trait, and sometimes even for the phenotype definition. The term mode of inheritance relates to both the genetic mechanism, i.e., the number of loci implicated in the etiology of the disease, and the genotype-phenotype relation, which describes the influence of these loci on the trait. Having an idea of the genetic model can crucially facilitate the mapping process. This holds especially in the context of linkage analysis, where an appropriate parametric model or a suitable nonparametric allele sharing statistic may accordingly be selected. Here, we review the difficulties with parametric and nonparametric linkage analysis when applied to multifactorial diseases. We address the question why it is necessary to adequately model a genetically complex trait in a linkage study, and elucidate the steps to do so. Furthermore, we discuss the value of including unaffected individuals into the analysis, as well as of looking at larger pedigrees, both with parametric and nonparametric methods. Our considerations and suggestions aim at guiding researchers to genotyping individuals at a trait locus as accurately as possible.     

Cell expansion in the filamentous gametophyte of the fernOnoclea sensibilis L.

Filamentous gametophytes of the fernO. sensibilis were exposed to paired combinations of light of different qualities, hormones and cations in the attempt to elucidate the underlying processes that regulate cell expansion. Simultaneous treatments with high-pH buffers or the auxin antagonistp-chlorophenoxyisobutyric acid abolished blue-light-mediated expansion but did not influence growth in red light. In contrast, the red-light response was preferentially altered by the ethylene absorbant KMnO₄ or the Ca²⁺ chelator ethyleneglycol-bis(-aminoethyl ether) N,N-tetraacetic acid. The Ca²⁺ ionophore A23187 caused a significant reduction in cell expansion under both blue and red irradiation. A marked promotion of expansion was mediated by high concentrations of indole-3-acetic acid, but this effect was dependent on the presence of low-pH buffers. The ethylene-generating agent 2-chloroethylphosphonic acid decreased the magnitudes of both photoresponses; this inhibition was further enhanced by high Ca²⁺ concentrations. These findings and those with other plants are interpreted in terms of two independent control mechanisms for cell expansion: 1) a blue light photoreceptor-auxin-hydrogen ion system, and 2) a phytochrome-ethylene-calcium ion system.Abbreviations APW-X artificial pond water (the associated number designates pH) - CEPA 2-chloroethylphosphonic acid - EGTA ethyleneglycol-bis(-aminoethyl ether)N,N-tetraacetic acid - IAA indole-3-acetic acid - PCIB p-chlorophenoxyisobutyric acid