Proceedings of the XVII International Congress on Sexual Plant Reproduction July 9-13, 2002, Lublin, Poland

Embryogenesis is a critical stage of the sporophytic life cycle, during which the basic body plan of the plant is established. Although zygotic embryogenesis is induced by fusion of the sperm and egg nuclei, plant cells can initiate embryo development without fertilization. For example, cultured somatic and male gametic cells can be induced to undergo somatic and microspore embryogenesis, respectively. Embryogenesis in vitro represents a powerful tool to manipulate plant development. After characterizing in situ embryo development in flax, we followed the cytological, morphological and some biochemical features of zygotic embryo development in embryo cultures. We also induced direct and indirect somatic and gametic embryo formation in flax. There is a strong indication that somatic and gametic embryogenesis is a stress response and that it is a way the plant cell realizes its survival strategy under completely changed and unusual conditions.

Key words: Linum usitatissimum L., zygotic embryogenesis, somatic embryogenesis, gametic embryogenesis, embryo-like structure.

Sexual reproduction in angiosperms is an interactive process involving the sporophyte, gametophytes, embryo and endosperm as well as the environment, aimed at achieving pollination, fertilization and dispersal. This interaction occurs via an interface with nutrients and signals outside the cell and even outside the plant. Sexual reproduction has a history. In water, algae have different types of sex organs and gametes, and in some cases the female gamete stays on the plant. The zygote uses water movement and gravity for dispersal. Some algae have alternation of generations in the life cycle, and only the gametophyte functions in sexual reproduction. On land, ferns and mosses inherited alternation of generations, with oogamy and zygote development on the gametophyte, with wind dispersal of the meiospore. In angiosperms, heterospory and the retention of the megaspore, megagametophyte and embryo on the sporophyte lead to a seed with gravity and biotic dispersal. The history of sexual reproduction is based on sex determination, due to cross-fertilization and recombination. Sex differentiation is manifested in the increasing complexity of interaction in the nutrient supply, the retention of the gametophyte or even the embryo, and the type of vector of dispersal. Regulation of sexual reproduction in angiosperms is governed mainly by the sporophyte, with the expression of new genes for biotic pollination and seed dispersal. In the heterotrophic gametophyte some gene expression is suppressed. The development of sexual reproduction is due to the communication between the organism and a dynamic environment.

Key words: Sex differentiation, dispersal, interaction, regulation, environment

The theory of critical periods in plant ontogenesis has been elaborated from studies of integral morphogenetic processes on different levels. The periodization of the development of various reproductive structures (anther, microspore, pollen grain, ovule, megagametophyte, egg cell, zygote and embryo) has been worked out from data on morphogenesis using systemic and complex morphophysiological approaches. Critical phases, stages and periods have been revealed, for example the stage of autonomy in different flowering plants, by means of culture in vitro. The concepts of "critical period" and "critical mass" in relation to embryonal structure periodization are discussed here. Also addressed are the question of allometry and the significance of morphogenetic fields and rhythms of cell division for revealing critical periods and the management of ontogenesis. Examination of the genesis and structure of anthers and ovules in various flowering plant species has permitted us to discover general regularities in their development and the occurrence of three common critical periods: premeiotic, meiotic and postmeiotic. Embryo development in angiosperms is characterized by two common phases (proembryonal/blastomerization and embryonal/organogenesis) and five critical periods (zygote and proembryo, globular, heart- shaped, torpedo-shaped, and mature embryo). The combination of common and specific critical periods and stages determines the taxon-specific morphogenesis of reproductive structures and contributes to the plasticity and tolerance of the reproductive systems of different species of flowering plants, and of ontogenesis as a whole.

Key words: Critical period, critical mass, morphogenetic fields, reproductive structures, switching over development program.

Stigma receptivity of Platanthera chlorantha was examined in a laboratory experiment. Flowers were hand-pollinated at bud stage and at different intervals between the beginning and the 14th day of anthesis. Afterwards, pollen tube growth was examined by fluorescence microscopy. Stigma receptivity was also tested for the presence of peroxidase using the Peroxtesmo Ko test. The efficiency of hand-pollination in flowers of varying ages was confirmed by capsule formation. Pollen tubes started to germinate 6 h after pollination. The stigma was overgrown with pollen tubes 24 h after pollination. The stigma became receptive at the bud stage. Receptivity lasted 15 days on average. Pollen did not germinate on stigmas with a dry surface, in flowers with a dehiscent perianth. Pollination did not affect stigma receptivity. Pollen tubes germinated from pollinaria deposited on the stigma additionally 6 days after the first pollination. Fluorescence microscope observations of pollen tube germination produced results corresponding to those obtained with the Pertexmo Ko test.

Key words: Stigma receptivity, pollen tube, pollination, Platanthera chlorantha, Orchidaceae.

The structure of nectaries in the flowers of Myosotis sylvatica Hoffm. was studied by light microscopy, scanning electron microscopy and transmission electron microscopy. A nectariferous gland of irregular disc shape surrounds the bottom of the four-lobed ovary. From the upper side, both the nectary and the ovary are protected by ligulate folds of a widening corolla tube with epidermis outgrowths. Nectar is secreted by modified stomata situated only on the top and abaxial nectary surface adjoining the corolla tube. Stomata are irregularly distributed, forming aggregates of 2-3 each. On the longitudinal sections of the receptacle, the nectariferous tissue is distinguished by small cells, dense protoplast content, and intercellular spaces of different sizes. Branches of phloem bundles are observed at the base of the nectariferous tissue.

Key words: Myosotis sylvatica Hoffm., Boraginaceae, nectaries, corolla outgrowths, morphology, anatomy.

Variation in the level of self-incompatibility (SI) was quantified in 14 Polish lines of cabbage (Brassica oleracea L. var. capitata) pollinated in various temperature/humidity conditions. Two methods were used to measure the expression of incompatibility: counts of seed set and counts of pollen tubes penetrating the styles 48 h after self-pollination. The level of self-incompatibility varied distinctly depending on the cultivar (from 0 to 65% seed-containing siliques). The genetic background of the material determined the influence of environmental factors on SI. Lower temperature (12 C) and higher humidity (90%) positively affected bud pollination, possibly due to a less stressful protocol. The observed great genotypic variation suggests it may be difficult to find a universal method suitable for breaking the self-incompatibility barrier for the species. The method may have to be tailored to the particular genotype.

Key words: Bud pollination, cabbage, humidity, seed production, self-incompatibility, temperature.

This work is the first characterization at cellular and subcellular level of the main cellular events occurring in the first stages of microspore embryogenesis. Microspore embryogenesis was induced in two varieties of Citrus clementina (Nules and Monreal). The results showed that one of the most responsive stages for embryogenesis was the vacuolate microspore. Microscopic analysis revealed specific features of the young proembryos still surrounded by the exine: large nuclei, clear areas in the cytoplasm, starch accumulation, and an increase in the thickness of the wall under the exine. Immunogold labelling with JIM 5 antibody showed a high amount of non-esterified pectins in the surrounding cell wall. After exine rupture, different cell types were detected in late proembryos. As embryogenesis proceeded, the normal pattern of development was observed, including heart-shape, torpedo and cotyledonar embryos.

Key words: Citrus, haploids, microspore embryogenesis, ultrastructure, cytochemistry.

The paper reports a comparative study of storage protein synthesis and enzyme activity during zygotic and somatic embryogenesis of silver fir. The SDS-PAGE profiles of storage proteins in zygotic and somatic embryos were similar but not identical. Six storage protein fractions were detected in zygotic embryos, as compared with eleven fractions in somatic embryos. The principal storage protein of zygotic embryos was represented by the 43 kDa fraction, and in somatic embryos by the 53 kDa fraction. Peroxidase activity was lower in the precotyledonary and cotyledonary stages of somatic embryos than in the corresponding developmental stages of zygotic embryos. However, following desiccation, the mature somatic embryos possessed three times higher peroxidase activity than the mature zygotic embryos. The reverse was true of the specific activity of esterase, which was higher in zygotic embryos than in somatic embryos in all stages of development.

Key words: Abies alba Mill., silver fir, embryogenesis, storage proteins, enzymes.

Cuticle visualized by auramine O fluorescence on developing Arabidopsis thaliana embryos was investigated. Localization of the cuticle was studied on embryos of the zygotic wild Ler ecotype and nine lines of embryonic mutant: CS 2330, CS 3009, CS 3016, CS 3023, CS3025, CS 6330, CS 6340, CS 6343 and CS 6346. In Arabidopsis Ler ecotype embryogenesis, a fluorescing cuticle layer appears on the globular embryo and persists during successive stages of development. Such a layer does not occur on the suspensor. In a similar way, fluorescing cuticle envelops the entire globular and older embryo of embryonic mutants, although the embryos of different mutant lines reach different developmental stages.

Key words: Embryo, embryonic mutants, cuticle, fluorescence, Arabidopsis.

Reproduction of Echium vulgare L. was examined in situ at two contaminated sites (Silesia, Poland): a zinc spoil heap in Katowice-Wełnowiec, and the surroundings of the Żelazny Most copper post-flotation waste reservoir. Plants from uncontaminated sites (near Rymanów) were studied as the control material. We compared the reproductive capacity of plants in the stress conditions to that of plants from uncontaminated sites. Degenerative processes and abnormalities found in the reproductive organs of plants from polluted sites, more intensified in the population from Żelazny Most, resulted in lowered fertility of plants. In germination tests on standard soil and from polluted sites, seeds from the control plants had the lowest germination on soil from Żelazny Most, and seeds from Żelazny Most had significantly delayed germination and had higher germination on standard soil than on polluted soil from their site; on the other hand, seeds from Katowice-Wełnowiec had higher germination on waste heap soil than on standard soil. The results suggest that among the populations from polluted sites, the one from Katowice-Wełnowiec is more advanced in selection for tolerance.

Key words: Echium vulgare L., heavy metals, pollution, embryological disturbances, germination.

Pollen grains of Pinus wallichiana, P. mugo, P. ponderosa and Ephedra distachya germinated at various intensities on in vitro cultured placentas of 32 angiosperm species. Pollen of the same gymnosperms did not form tubes on stigmas of pistils cultured under the same conditions as the placentas. Pollen of several angiosperms germinated on semi in vitro cultured opened ovules of Larix decidua and nucelli of Taxus baccata. Angiosperm pollen did not germinate in vivo in the pollination drop secreted by ovules of T. baccata. This report shows that (1) gymnosperm pollen produces fully formed tubes on ovules of angiosperms but do not germinate on their stigma, and (2) pollen representing angiosperms is able to germinate and form tubes on ovules of gymnosperms.

Key words: Semi in vitro pollination, gymnosperms, angiosperms.

This study uses cytochemical tests, electron spectroscopic imaging and electron energy loss spectroscopy techniques to identify and localize the reserves inside the generative cell of Hermodactylus tuberosus pollen. Cytochemical probes applied to sections observed by light and transmission electron microscopy indicated that the generative cell contains large osmiophilic bodies probably made of phytic acid rich in P and Ca. The significance of the rich granulations in generative cells of Hermodactylus pollen is discussed in relation to floral biology and environmental conditions. In comparison, the vegetative cytoplasm contains (a) lipid droplets formed by unsaturated lipids and related to vacuoles, (b) lipid bodies with larger dimensions, irregular in shape and very rich in Ca, (c) bodies stained in polysaccharide tests as well as lipid probes tentatively identified as glycolipid granulations, and (d) small granules very rich in P and Ca interpreted as phytin granules.

Key words: Hermodactylus, pollen, reserves, cytochemistry, ESI, EELS.

This paper describes the distribution of Ole e 3 allergen and its transcripts in the developing anther and in mature olive pollen. Northern blot and RT-PCR analyses over the course of pollen development show that Ole e 3 transcripts accumulate exclusively in mature pollen. This gene therefore corresponds to a "late gene." The sequences amplified by RT-PCR display high identity with those already reported for Ole e 3, including two Ca2+-binding motifs. Immunoblot analysis of the allergen shows that Ole e 3 accumulates during the same stage as the corresponding transcripts, suggesting a transcriptional regulation mechanism for the expression of the gene. Finally, the use of transmission electron microscopy techniques has shown that (a) the allergen is located mainly in the vicinity of membrane systems and in the aperture regions of the mature pollen grain, and (b) Ole e 3 transcripts are detectable after using in situ RT-PCR. These data are significant clues to the biological roles of the protein in olive pollen biology. Such putative functions are discussed.

Key words: Allergen, expression, localization, Olea europaea L., Ole e 3, olive.

In order to study microtubule organization during pollen germination and pollen tube growth in Olea europaea, we applied immunofluorescence microscopy using mouse monoclonal antibody against ?-tubulin as primary antibody and FITC-conjugated goat antimouse IgG as secondary antibody. DAPI enabled observation of the vegetative nucleus entering the emerging pollen tube before the generative cell. The latter then overtakes the vegetative nucleus once both are inside the pollen tube. The generative cell remains ahead of the vegetative nucleus until it is finally divided into two gametes. This cell division occurs when the generative cell is close to the tip of the pollen tube. Possible connections between microtubules and nuclear migration in the pollen tube are discussed.

Key words: Pollen tube, microtubules, movement, generative cell, vegetative cell nucleus.

Conserved 14-3-3 proteins have been shown to play regulatory roles in eukaryotic cells, including cell cycle control and differentiation. We were interested in the possible involvement of 14-3-3 proteins in the embryogenic process of barley (Hordeum vulgare L.). Barley microspore-derived embryo development was used as a model system. Immunolocalization of three barley 14-3-3 isoforms, 14-3-3A, 14-3-3B and 14-3-3C, was carried out using isoform-specific antibodies. In immature microspore-derived embryos, 14-3-3C was specifically expressed underneath the L1 layer of the shoot apical meristem and in the scutellum. Comparative studies showed that 14-3-3C was also expressed underneath the L1 layer of the shoot apical meristem and in the scutellum of immature zygotic embryos. We further demonstrated that 14-3-3C expression is restricted to L2 layer-derived cells of in vitro shoot meristematic cultures. Our results reveal that 14-3-3C isoform tissue-specific expression is closely related to defined events during differentiation processes in embryogenesis and in vitro meristematic cultures.

Key words: Barley, 14-3-3, embryogenesis, L2 layer.

Zygotes fertilized in planta developed into fertile plants in vitro. Microspore cultures and ovaries derived from the same species were tested as nurse cells. With both types of feeder system about 20% of the isolated zygotes were able to regenerate into plants. The morphology, cytological properties and development rhythm of the zygotes resembled those of the in vivo course of zygotic evelopment, except that the first division appeared symmetrical in contrast to the asymmetrical division observable in planta. The results indicate that ovaries may have the same nurse effect as microspores on zygotes cultured in vitro. Using ovaries as a nurse system is much less time-consuming than using isolated microspore cultures.

Key words: Embryo regeneration, growth factors, nurse cells, isolated zygotes.

A four-year study examined the degree of self-pollination and self-fertility in the following cultivars of Ribes nigrum L.: Ben Alder, Ben Lomond, Ben Nevis, Ben Tirran, Ceres, Ojebyn, Titania and Triton. The percentage of flowers setting fruit largely depended on the cultivar and on the pollination treatment. The most fruits (over 60%) were obtained in free-pollination conditions; 44-64% fruit set occurred when flowers were pollinated with their own pollen, and ~20% after self-pollination. In cultivars Ojebyn, Ben Nevis, Ben Lomond, Ben Alder, Ben Tirran and Triton, which are largely self-fertile, there was no significant difference in fruit set between own pollen and free pollination. Cultivars Ceres and Titania had the lowest degree of self-fertility (~44%). Flowers of Ben Lomond were most capable of self-pollination (~45%). All cultivars examined require pollinating insects during flowering for satisfactory yield.

Key words: Black currant, Ribes nigrum, self-pollination, self-fertilization.

This study investigated cross-reactivity between allergens of Cupressaceae and Fagaceae pollen. Human IgE raised against Cupressus arizonica pollen allergen was used to demonstrate the presence of related allergens on ultrathin sections of Fagaceae pollen (Quercus ilex, Castanea sativa, Fagus sylvatica). Tissue localization of the cross-reactive allergen was investigated by immunogold electron microscopy. TEM observations showed that IgE raised against C. arizonica allergen recognizes epitopes on Fagaceae pollen. The cross-reactive allergens can be found on the wall and in the cytoplasm.

Key words: Pollen allergen, immunogold labelling, cross-reactivity.

To study mitochondria and plastid involvement in plant development, particularly in sexual reproduction, we made use of the Arabidospis thaliana T-DNA insertion collection developed in Versailles. Mutants affected in the nuclear genes that encode proteins predicted to be targeted to organelles were identified using two complementary strategies. In the first (forward genetics), mutants chosen for their sterile or gametophytic lethal phenotype were screened for T-DNA insertion in genes encoding mitochondrial or plastid proteins after systematic sequencing of the Flanking Sequence Tag (FST). The second (reverse genetics) enabled us to identify other mutants using the following tools: the systematic A. thaliana proteome analysis, bioinformatics software to predict the sub-cellular localisation of putative proteins, and the FST sequencing program FlagDB. Preliminary results for the first set of 82 putative mutants are presented and discussed.

Key words: Reproduction, sterility, mitochondria, plastids, protein targeting, T-DNA insertion mutants.

Screening of male gametophytic mutants from the Versailles collection of T-DNA transformants allowed us to isolate and characterize two novel genes: KINKY POLLEN (KIP) and POKY POLLEN TUBE (POK), which are required for correct tip growth in Arabidopsis thaliana. As KIP and POK are expressed in all plant tissues, though to a higher level in pollen and roots, their roles may not be restricted to tip growth only, but might extend to more general elongation mechanisms. Both genes are duplicated in the Arabidopsis genome. Specific roles for each duplicate, indicated by mutant phenotypes, will be discussed. Moreover, KIP and POK proteins have putative orthologs in all eukaryotes investigated, suggesting that they may be crucial proteins required for correct polar growth in all eukaryotic species.

Key words: Pollen tube, tip growth, duplication, Arabidopsis thaliana.

Pollen of Lagerstroemia indica was collected from polluted (SO2, NOx, CO, HC, airborne particulate matter) and less polluted areas of Tehran city, Iran. Some pollen from less polluted areas was exposed to polluted air for 10 and 20 days. To determine the effect of air pollution on proteins, pollen extracts were analyzed by the Bradford method and SDS-PAGE. Study of pollen structure by light and scanning electron microscopy showed that air pollution increased the number of shrunken and fragile pollen. Particle agglomeration and cellular material release were increased in polluted pollen. Total protein content and the staining intensity of protein bands by SDS-PAGE were decreased in polluted pollen.

Key words: Lagerstroemia indica, air pollution, pollen structure, protein release.

In Galanthus nivalis during the progamic phase, both the embryo sac and somatic cells of the ovule change their ultrastructure and physiology, as observed by light, fluorescence, and electron microscopy. Fresh ovules from buds, opening flowers, and from cross-pollinated flowers were stained in toto to detect pectins, acidic polysaccharides, proteins, lipids, callose, free calcium ions and membrane-bound calcium. These substances were found only in the micropylar part of fertile ovules. All stainings were negative in sterile ovules. In EM, the somatic cells in the micropylar part of the ovule were observed to develop secretion activity. Their exudates passed to the intercellular spaces, mainly to the micropylar canal. The amount of the exudate increased after pollination. Free or loosely bound calcium ions were present in extracellular regions of the micropylar part of fertile ovules. The substances detected in the micropylar exudate of fertile ovules are suggested to support and direct pollen tube growth to the embryo sac.

Key words: Galanthus nivalis, ovule, micropyle, exudates, pectins, calcium.

The actin cytoskeleton of endosperm and of the mature endosperm chalazal haustorium cell of Rhinanthus serotinus was examined by immunohistochemistry and epifluorescence microscopy. A prominent actin cytoskeleton composed of numerous cross-linked filaments is present at the distal pole of the chalazal haustorium cell. Thick, longitudinally oriented bundles of microfilaments localize in transvacuolar cytoplasmic strands. A meshwork of delicate actin filaments surrounds the large polytene nuclei; some of the filaments radiate from the nuclear envelopes. Abundant and clearly visible actin filaments also occur at the proximal pole of the haustorium cell. A network of microfilaments in cortical cytoplasm and F-actin arrays associated with nuclei are found in endosperm proper cells.

Key words: Rhinanthus serotinus, cytoskeleton, F-actin, immunolabelling, rhodamine-phalloidin, endosperm, chalazal haustorium.

This work presents data on the genesis of ovule and seed structures in Dioscorea nipponica Makino and examines morphogenetic correlations in their development.

Key words: Dioscorea, reproductive biology, embryology, seed.

Comparison of maize mutants pam1, mac1 and normal plants (variety Belaya noch') showed that the formation of female reproductive organs is very similar between them. One afunicular ovule develops in an ovary; it is ortho-campylotropous and bitegmic, with a hypostase. The postament, podium and nucellar cap are differentiated in the nucellus. The integuments, epidermal in origin, arise from a common initial zone, but their differentiation is separated in time. In the subepidermal layer of the apical part of the primordium, the complex of initial cells is distinguished. Some of them differentiate as archesporial cells. In normal plants and pam1, only one of them transforms into the megasporocyte, while up to 6-8 do in mac1. Polygonum-type embryo sacs are formed. In pam1 the majority of megasporocytes do not undergo meiosis, while the surrounding ovular tissues continue their development. Analysis of the distribution of polysaccharides and proteins revealed similarities in their spatial and temporal coordination. The differences are quantitative, character, and probably conditioned by the number of megaspores and embryo sacs. 

Key words: Zea mays, ovule morphogenesis, polysaccharides, proteins, mac1, pam1.

A study of Gentiana cruciata L. (Gentianaceae), Gymnadenia conopsea (L.) R.Br. (Orchidaceae) and Luzula pedemontana Boiss. et Reut. (Juncaceae) showed differences in the number and characteristics of critical stages in ovule and seed development. The shared critical stages explain the general direction of the formation of reproductive structures and surrounding tissues. The taxon-specific critical stages may have different implications in a given species: they may (1) verify that the ovule belongs to a specific type, (2) indicate their lability in different taxa with the same ovule type, or (3) coincide in species with various ovule types.

Key words: Ovule, seed, development, histochemistry, critical stages.

The comparative morphogenetic study of reproductive organs of chrysanthemums is conducted using morphological analysis. Morphological changes during the development of a generative shoot and organogenesis from first through ninth phases were described. A flat apex becomes spherical in form. An inflorescence apical meristem initiates involucral bract. An initiation of floret primordia is followed an acropetal sequence. Floral meristem produces corolla, then 5 staminata primordia and two-lobed pistillata primordia. The ray floret and the disc floret both form 5-lobed corolla, but 2 lobes of the corolla of the ray floret stop their growth. Staminata primordia of the ray floret produce sterile staminodia or do not continue their development. At phase IX during flowering, 4 stages are identified. A dehiscence of introrse anthers and anthesis occur in a closed disc floret. There are protoandria and different arrangements of mature anthers and mature stigmata in disc florets.

Key words: Chrysanthemum, Chrysanthemum x hortorum hort., Dendranthema grandiflora hort., morphogenesis of generative shoot, organogenesis.