THE INITIATION OF SPOROPHYTES BY OBLIGATE APOGAMY IN CHEILANTHES CASTANEA

The initiation of apogamous sporophytes in Cheilanthes castanea was recorded by daily photography of individual gametophytes. Whereas an ordinary embryo arises from a zygote, apogamous embryos of C. castanea originate from one to three initial cells which occur just behind the apical region of the prothallus. The initial (or initials) produce cells with small chloroplasts behind the sinus of the gametophyte. The appearance of cells with smaller chloroplasts than those normally found in gametophytes is the first indication that apogamy is occurring. The cells with small plastids produce a group of densely-cytoplasmic meristematic cells. The size of the meristematic mass increases until shoot and root apices of the apogamous embryo are organized.     

THE INFLUENCE OF GROWTH SUBSTANCES ON THE INDUCTION OF APOGAMY IN PTERIDIUM GAMETOPHYTES

Experiments employing sequences of three media demonstrated the effect of growth substances on the induction of apogamy. The most effective sequence was 4% sucrose 1–14 days, 4% sucrose plus growth substance 15–28 days, and 0.1% sucrose 29–56 days. In this sequence concentrations of NAA, IAA, and GA promoted apogamous shoot formation. A higher NAA concentration than optimal for shoot formation stimulated apogamous root formation in all medium sequences. Kinetin was without effect or inhibitory to apogamy. Combination of kinetin/GA or GA/NAA concentrations did not increase the apogamous response. One combination of the kinetin/NAA concentrations had a synergistic effect on the apogamous shoot formation. Additions of GA to the synergistic kinetin/NAA combination had an antagonistic effect on the apogamous response.     

The role of ethylene in the induction of apogamous buds in Pteridium gametophytes

Summary The level of induced apogamy in gametophytic colonies of Pteridium aquilinum (L.) Kuhn is altered by varying the number of colonies per culture vessel or by including a constant number of colonies in culture vessels of different volumes. In either case, placing vials of mercuric perchlorate, an absorber of ethylene, within the closed culture vessels reduced the apogamous response to a very low level. Production of ethylene by the gametophytes was demonstrated by gas chromatography. Ethylene supplied in a continuous-flow system promoted the apogamous response above that of an air control.     

Exogenous and endogenous growth regulators on apogamy in Dryopteris affinis (Lowe) Fraser-Jenkins sp. affinis

This work showed for the first time the relationship between the effect of exogenous auxins and gibberellins on apogamy in Dryopteris affinis (Lowe) Fraser-Jenkins sp. affinis and its endogenous contents during early apogamic events. The addition of NAA (0.53 and 5.37 μM) or GA₃ 2.8 μM to an MS solid medium significantly increased apogamous sporophyte formation. BA induced brown callus that regenerated sporophytes in a hormone-free medium. The endogenous contents of GA₁, GA₃, GA₄, GA₇, GA₉ and IAA were determined by GC–MS in gametophytes cultured on MS solid medium, before and during early stages of apogamous embryo development. The accumulation of both GA₉ and IAA before embryo development was evident as high levels of GA₄ in the earliest analysed stage of embryo development and high levels of GA₃ in elongating shoots were found. The role of gibberellins on apogamy was also supported by data showing a decrease in the percentage of gametophytes developing embryos because of the addition of flurprimidol to the culture medium.     

金粉蕨配子体发育及其无配子生殖的研究

采用光学显微镜和半薄切片技术对金粉蕨(Onychium siliculosum(Desv.)C.Chr.)的配子体发育及其无配子生殖进行了研究。金粉蕨孢子黄褐色,具三裂缝,孢子萌发时先产生假根,然后长出原叶体细胞,萌发类型为书带蕨型。原叶体细胞经丝状体、片状体发育为原叶体,发育类型为水蕨型。金粉蕨配子体分枝发达,成熟配子体呈丛状。多次培养表明金粉蕨只能产生精子器,不能产生颈卵器,为专性无配子生殖。半薄切片观察表明配子体生长点下方的细胞经分裂产生一群小型细胞,由它们发育为无配子生殖胚。小型细胞先分化出无性胚芽,再分化出胚根。胚芽与胚根,胚与配子体之间由分化出的导管连接。     

INTERPOPULATIONAL VARIATION IN THE GAMETOPHYTES OF PELLAEA ANDROMEDAEFOLIA

The development and mature morphology of the gametophytes from both sexual and apogamous populations of the fern Pellaea andromedaefolia were investigated. While most sexual examples were indistinguishable, some differences were noted. An insular collection was distinctive in its variability and irregularity of form. Although the latter was a representative of var. pubescens, other collections of the variety could not be distinguished from var. andromedaefolia on the basis of gametophytic characteristics. The apogamous gametophytes were decidedly more variable in development and often very different from sexual thalli. The mature asexual thalli tended to be more irregular in form and usually sharply divergent from the typical cordate type characteristic of the sexual populations. Each of the five apogamous samples was unique with respect to gametophyte development. The differences among the gametophytes of the various populations do not correlate with the sporophytic characteristics which differentiate the two varieties of the species.     

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     

Regulation of a Sub-sexual Life Cycle in a Moss: Evidence for the Occurrence of a Factor for Apogamy in Physcomitrium

As demonstrated in our earlier studies, the differentiationof apogamous sporophytes on the secondary protonema of the mossPhyscomitrium pyriforme Brid. requires an exogenous supply ofsucrose in the medium. In the present work, similar differentiationwas observed in the leaf cells of aged gametophytes. The experimentsindicate an accumulation in the leaves of a sporophytic factorwhich initiates a de novo differentiation of sporophytes fromleaf cells without the intervention of sexual reproduction.In the absence of sucrose, the factor for apogamy was not present.Highlight intensity (5000–6000 lx) also inhibited itsproduction. There was no evidence that its presence interferedwith or inhibited production of gameto-phores. Growth regulatorssuch as IAA and kinetin altered only the effectiveness of thissporophytic factor, demonstrating that it was endogenous. Sporogenesisin the apogamous sporophytes took place without orthodox meiosis. Results obtained by using different exogenous environments forthe in vitro differentiation of callus into gametophytes orsporophytes are also reported. These support our contentionthat there is an accumulation of a sporophytic factor in thegametophytic callus cells, which is diluted during the processof differentiation. The morpho-regulatory influence of lightin the differentiation of apical cells with three cutting facesfrom unorganized callus is also considered.     

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.     

THE APOGAMOUS LIFE CYCLE OF TRICHOMANES PINNATUM—A CONFIRMATION OF KLEKOWSKI'S PREDICTIONS ON HOMOEOLOGOUS PAIRING

The gametophyte of Trichomanes pinnatum is filamentous save for its club-shaped archegoniophores. Gametangial structure is consistent with that of related species. The apogamous embryo originates from the archegonial jacket and contiguous tissue such that there is no external indication of apogamy. The life cycle follows the Döpp-Manton scheme. Specimens were studied which were homozygous for a paracentric inversion and these showed chromosomal bridges and associated fragments at meiosis-I, thus confirming cytologically the occurrence of homoeologous pairing.     

Induction of apogamy inEquisetum arvense

InEquisetum arvense, apogamous sporophytes were produced on medium containing 5×10^?6–5×10^?8 g/ml kinetin. NAA, IAA, GA₃, glucose and saccharose were ineffective for the induction of apogamy. On medium containing 5×10^?7–5×10^?8 g/ml kinetin, the gametophytes passed into sporophytic structures directly. On medium containing 5×10^?6 g/ml kinetin, some gametophytes passed into sporophytic structures directly, and others became a callus-like cell mass from which an apogamoun shoot arose. The results of the morphological observations on them were reported and compared with the sexually produced sporophyes. The apogamous sporophytes induced by 5×10^?7 g/ml kinetin were haploid in their nuclear phase and some of those induced by 5×10^?6 g/ml kinetin had a tendency to become diploid.     

The effect of light quality on the induction of apogamy in prothalli of Pteridium aquilinum

Summary Pteridium gametophytes cultured on a medium containing 4% sucrose under various qualities of light exhibit different developmental responses. Under far-red illumination (690–730 nm) apogamy is produced without enhanced gametophytic growth, thus allowing a distinct separation of the specific induction of apogamy from a general enhancement of gametophytic growth.     

37 Production, survival and morphology of kelp (protista) "sporophytes" arising from selfings, apogamy and hybridizations

The production and survival of sporophyte morphologies that arose from selfings, parthenogenesis, male apogamy and reciprocal hybridizations of Costaria costata (C.A. Agardh) Saunders, Laminaria saccharina (L) Lamouroux, Alaria marginata Postels and Ruprecht, Eisenia arborea Areschoug, Lessoniopsis littoralis (Tilden) Reinke, Macrocystis integrifolia Bory, and Nereocystis leutkeana (Mertens) Postels and Ruprecht was tracked for 215 hybridization trials for 30 weeks. Each trial consisted of selfings for each species, solo cultures of males and females for each species and reciprocal crossings. At the end of 30 weeks, "sporophyte" survival in the 215 cases was 13% for selfings, 10% for parthenogenesis, 6% for apogamy, and 16% for the reciprocal hybridizations. The morphologies of sporophytes resulting from selfings were typical of the selfed species. Parthenosporophytes and reciprocal hybrids usually had the distinctive morphology of the female parent. Male, apogamously derived "sporophytes" often had reduced morphological features. For example, A. marginata male apogamous sporophytes often lacked a midrib. The sporophyte morphologies that arose in the reciprocal crossings are considered to be putative hybrids. Molecular evidence is required to establish their genetic origins.     

INDUCTION OF APOGAMY IN MEGAGAMETOPHYTES OF ZAMIA INTEGRIFOLIA

Ovules of Zamia integrifolia Ait. were dissected and megagametophytes and embryos excised and cultured. Induction of apogamous, haploid roots and leaves by auxin and kinetin, in combination with glutamine, asparagine and alanine, was observed. The organs resembled their normal diploid counterparts. 2,4-D at 1 ppm was more effective than 1 ppm IAA, but less effective than 5 ppm IAA. The highest percentage of apogamy was 59% after 5 months of culture. In several cultures embryoids were formed both by diploid excised embryos and by haploid megagametophtyes.     

Ultrastructural and cytochemical aspects of induced apogamy following abscisic acid pre-treatment of secondary moss protonema

Abscisic acid (ABA) treatment of secondary protonema of Physcomitrium pyriforme Brid in the presence of sucrose does not prevent cell division but results in shorter cells with vesicular cytoplasm and an accumulation of lipid. When transferred to sucrose medium without ABA and with low irradiance isodiametric intercalary cells are cut off which give rise to apogamous sporophytes either directly or after the formation of a small amount of callus. The organization of the cells leading up to the apogamous sporophyte is described. The cells initiating the sporophyte develop dense cytoplasm and the walls become labyrinthine and callosed, but they do not form any recognizable placenta. It is proposed that labyrinthine walls are a consequence of a perturbation of cell wall metabolism as growth changes from gametophytic to sporophytic. The use of the term transfer cell for this kind of cell is questioned and the need for a causal approach to the investigation of labyrinthine walls is stressed.     

In vitro studies on apogamy, apospory and controlled differentiation of rhizome segments of the fern, Ampelopteris prolifera (Retz.) Copel.

Apogamy was induced in the fern Ampelopteris prolifera by culturing the gametophytes on mineral nutrients supplemented with various concentrations of sucrose. Higher concentrations (5–8%) of sucrose were detrimental to prothallial growth, while in lower concentrations (2–3 %) apogamy was delayed. Gametophytic callus was induced from the germinating spores by culturing them on 2,4-D rich (3–5 mg/1) media. The differentiation of this gametophytic callus was conditioned by sucrose and auxin concentrations of the medium. In the presence of sucrose, calli responded like prothalli, while in the presence of 2,4-D, differentiation was delayed or completely inhibited. Apospory was induced on the sexual cotyledonary and juvenile sporeling leaveS. Leaves with petiole, excised from aseptically raised plants from excised cultured buds, also exhibited apospory, while no success was achieved with the excised leaves of the parent plantS. Rhizome segments of various length were cultured on media containing different concentrations of sucrose. The differentiation of rhizome segments into gametophytes or sporophytes was conditioned by the length of the rhizome segments and the sucrose concentration of the medium. The possible significance of all the results is discussed.     

THE GENETIC STRUCTURE OF SPOROPHYTIC AND GAMETOPHYTIC POPULATIONS OF THE MOSS,FUNARIA HYGROMETRICA HEDW

Patterns of phenotypic and genotypic variability in two populations of the moss, Funaria hygrometrica, were investigated using measurements of gametophytic and sporophytic morphology, sporophytic reproductive output, spore germination, gametophytic growth rates and tolerances of copper, cadmium, and low nutrient conditions, and electrophoretically detectable enzyme variation. The two populations differed in all traits measured, but complete monomorphism within populations at 14 enzyme loci suggested that each represented a single clone. Variability in gametophytic growth rates and responses to different experimental media, however, occurred among haploid sib families (families of meiotic progeny derived from the same sporophyte) and among sibs within families within both populations, suggesting high levels of genetic variability. Low mean reproductive output and a high level of variability among sporophytes in a mine site population probably reflected heavy metal toxicity. Based on this study, in combination with previous work on F. hygrometrica (Shaw, 19906), somatic mutation and/or nongenetic effects appear to contribute significantly to phenotypic variability in natural populations.     

Further Embryological Studies on the in Vitro Apogamy in Oryza saliva L.

In the cultured young ovaries of rice, the processes of megagametophyte develop- ment could be switched to the formation of various abnormally organized embryo sacs and then to the initiation of synergid apogamy. The main pathway leading to apogamy was found to go via a linearly oriented 4-nucleate embryo sac to the formation of a li- nearly oriented egg apparatus, from which it was usually the chalazal synergid giving rise to an apogamous proembryo, and the micropylar synergid degenerated. The proembryo thus produced was located at the base of a vacuolated egg cell (Plate Ⅰ, 1–7). The second pathway went through a nonlinearly oriented 4-nucleate embryo sac to the formation of an egg apparatus in which the two synergids were located at one side of the egg and oriented longitudinally. In this case it was often the chalazal synergid that could be triggered to apogamy, resulting in a hook-shaped proembryo embracing the egg cel1 from one side (Plate Ⅰ, 8–11). When ovaries with nearly matured embryo sac were cultured, in a few cases where apogamy was induced, the proembryos observed were all situated at one side of the egg and were hook-shaped (Plate Ⅰ, 12). All these pathways are summarized in a diagram (Fig. 23). Some interesting changes were observed in the synergid and the egg cell of the cultured ovaries by PAS reaction and mercuric bromphenol blue staining. The egg cells, in contrast to in vivo condition, often contained abundant starch grains,. The synergids and synergid proembryos were rich of cytoplasmic protein (Plate Ⅱ, 13, 14). We supposed that the egg may supply some nutrients as well as stimulants to the developing synergid in the course of apogamy. The distribution of starch and protein in apogamous embryoids during subsequent development was also described in this paper (Plate, 15–22).     

SEASONAL LIGHT AND TEMPERATURE INTERACTION EFFECTS ON DEVELOPMENT OF LAMINARIA SACCHARINA (PHAEOPHYTA) GAMETOPHYTES AND JUVENILE SPOROPHYTES1

In previous studies, Laminaria saccharina L. (Lamour.) sporophytes were found to exhibit two major peaks of sporogenesis and an annual life cycle in Long Island Sound, New York. Young sporophytes were observed shortly after the sporogenesis peaks in early autumn and spring, but most of the mature sporophytes decayed during summer. A new study was conducted to determine if the spring sporogenesis activity contributed to the recruitment observed in autumn through oversummering of gametophytic and juvenile sporophytic stages, as previously suggested. Reproduction and growth in gametophytes and growth in juvenile sporophytes were studied under crossed gradients of light and temperature. Periodic outplantings of substrata seeded with gametophytic and sporophytic stages to the field were conducted to assess actual survival. The optimum temperature and light conditions for gametophyte development, growth and reproduction varied with the time of year meiospores were obtained. Most of this variation was attributable to temperature. A seasonal adaptation to temperature in most developmental stages was observed. Higher temperatures resulted in greater numbers of male gametophytes. Gametophytic stages could develop at all times, suggesting that oversummering in this stage was possible. Juvenile sporophytes had a narrower optimum temperature range and again photon fluence rate contributed little to observed variances. Out planting of sporophytic stages at various times during the year indicated only sporophytes prepared from autumn and winter could survive summer conditions. The thalli of these plants grew rapidly in spring and eroded back to the meristematic region in summer. Most of these plants then quickly became reproductive, resulting in another autumn sporogenesis peak. Gametophytic and sporophytic outplantings prepared from spring meiospores did not survive the summer. Thus, the recruitment observed in autumn can only be the result of the previous autumn's sporogenesis activity. The sporogenous activities of spring and early summer appear to be unimportant, despite the fact that all reproductive indices are superior at those times.