Western white pine (Pinus monticola Dougl.) reproduction: I. Gametophyte development

Male and female gametophyte development are described from light and transmission electron microscope preparations of ovules from first and second year Pinus monticola Dougl. seed cones. In the first year of development, pollen tubes penetrate about one-third the distance through the nucellus. The generative cell and tube nucleus move into the pollen tube. The megagametophyte undergoes early free nuclear division. First-year seed cones and pollen tubes become dormant in mid-July. In the second year, seed cones and pollen tubes resume development in April and the pollen tubes grow to the megagametophyte by mid-June. Early in June the generative cell undergoes mitosis, forming two equal-size sperm nuclei that remain within the generative cell cytoplasm. The generative cell has many extensions and abundant mitochondria and plastids. The megagametophyte resumes free nuclear division, then cell wall formation begins in early July. Cell wall formation and megagametophyte development follow the pattern found in other Pinaceae. Three to five archegonial initials form. The primary neck cell divides, forming one tier of neck cells. Jacket cells differentiate around each central cell. The central cell enlarges and becomes vacuolate; then vacuoles decrease in size and the cell divides, forming a small ventral canal cell and a large egg. Plastids in the central cell engulf large amounts of cytoplasm and enlarge. This process continues in the egg, and the peripheral cytoplasm of the egg becomes filled with transformed plastids. Mitochondria migrate around the nucleus, forming a perinuclear zone. The wide area of egg cytoplasm between these two zones has few organelles. A modified terminology for cells involved in microgametophyte development is recommended. Received: 9 December 1999 / Revision accepted: 30 April 2000     

Embryology ofHeliotropium scabrum andH. strigosum (Boraginaceae)

The embryology ofHeliotropium scabrum andH. strigosum has been studied. The development of the anthers follows the dicotyledonous type, the tapetal cells become binucleate. The pollen grains are shed at the two-celled stage. Megaspore tetrads are linear and the development of the megagametophyte corresponds with the Polygonum type. The endosperm is cellular. The embryo development follows the Onagrad Type, i.e. the Capsella variation inH. scabrum and the Nicotiana variation of the Solanad Type inH. strigosum. The pericarp is differentiated into a one-layered epicarp with bulbous-based, unicellular hair, a 5–6-layered chlorenchymatous mesocarp and a 6–7-layered endocarp. The seed coat consists only of the thickened portions of the epidermis.     

YAO is a nucleolar WD40-repeat protein critical for embryogenesis and gametogenesis in Arabidopsis

Background  

In flowering plants, gametogenesis generates multicellular male and female gametophytes. In the model system Arabidopsis, the male gametophyte or pollen grain contains two sperm cells and a vegetative cell. The female gametophyte or embryo sac contains seven cells, namely one egg, two synergids, one central cell and three antipodal cells. Double fertilization of the central cell and egg produces respectively a triploid endosperm and a diploid zygote that develops further into an embryo. The genetic control of the early embryo patterning, especially the initiation of the first zygotic division and the positioning of the cell plate, is largely unknown.     

RELATIONSHIP BETWEEN MALE AND FEMALE GAMETOPHYTE DEVELOPMENT IN RYE

The development of male gametophyte and female gametophyte within a floret of rye (Secale cereale L.) was examined. Generally, meiosis in microsporocytes and in megasporocytes occurs simultaneously in most florets, but the period from zygotene to tetrad meiosis in the megasporocyte progresses more slowly than that in the microsporocyte. When the female gametophyte has one nucleus and no vacuoles, the male gametophyte has a single, eccentric nucleus. By the time the female gametophyte develops to the vacuolated one-, two-, four-, and eight-nucleate stages and to the growth and differentiation of the egg apparatus stage, the male gametophyte reaches the two-celled pollen stage. As the female gametophyte matures, the male gametophyte also reaches maturity. The duration of male gametophyte development from microspore mother cell and the duration of female gametophyte development from megaspore mother cell are the same in most florets. The relationship between sexual development of cross-pollinated rye is similar to that of self-pollinated wheat (Triticum aestivum L.). It seems that the relationship is not related to the breeding system.     

Production and viability of unreduced gametes in triploid interspecific blueberry hybrids

Summary Three triploid (2n=3x=36) blueberry hybrids were obtained by hand-pollinating approximately 7,000 flowers of tetraploid highbush blueberry cultivars (based on Vaccinium corymbosum L.) with pollen from the diploid species V. elliottii Chapm. Meiotic analysis of these triploids revealed trivalents, bivalents and univalents in all metaphase I cells, with lagging chromosomes evident at anaphase I. Pollen of the three triploids was mostly aborted and did not stain with acetocarmine. However, the three triploids did produce from 0.9%–1.3% giant pollen grains that stained with acetocarmine and were present as monads, dyads or triads, rather than the normal tetrads. Pollination of 10,853 flowers of hexaploid V. ashei Reade cultivars with pollen from the triploids produced 266 berries, which averaged fewer than two fully-developed seeds per berry. One triploid clone showed partial female fertility when crossed to hexaploids, self-pollinated, or intercrossed with other triploids. Ploidy levels of the resulting hybrids were determined.Florida Agricultural Experiment Station Journal Series No. 8672     

Quantitative,three-dimensional analysis of alfalfa egg cells in two genotypes: Implications for biparental plastid inheritance

In alfalfa (Medicago sativa L.), plastids are inherited biparentally. Patterns of plastid transmission vary according to the genotypes involved, but there is a strong bias in favor of male plastid transmission. Previous cytological studies on the male gametophyte of this species have not provided an adequate explanation for the differences in plastid transmission frequencies among genotypes. In the present study, we compared egg cells from genotypes classified as strong or weak plastid transmitters to determine whether plastid transmission strength is correlated with egg cell structure before fertilization. We found that plastids in the mature egg cells of the strong female (genotype 6–4) are significantly larger than in mature eggs of the weak female (genotype CUF-B), and that significantly more plastids are positioned in the apical portion of the mature egg cell of genotype 6–4 than in CUF-B. Immature eggs in the two genotypes show the same pattern as mature eggs with regard to plastid number and polarization. Since only the apical portion of the egg cell/zygote gives rise to the functional embryo, these results indicate that the potential input of female plastids, in terms of plastid size and number, may be an important factor in determining the inheritance patterns of these organelles in alfalfa.Support for this work by the United States Department of Agriculture under grant 88-37234-3876, the National Science Foundation under grant DCB-9103658, the Organized Research Fund of Northern Arizona University, and the Arizona Agricultural Experiment Station is gratefully acknowledged. We are indebted to Dr. Craig Caldwell, Northern Arizona University Computer Visualization Laboratory, for his expert help with the computer graphics.     

A role for LORELEI,a putative glycosylphosphatidylinositol‐anchored protein,in Arabidopsis thaliana double fertilization and early seed development

In plants, double fertilization requires successful sperm cell delivery into the female gametophyte followed by migration, recognition and fusion of the two sperm cells with two female gametes. We isolated a null allele (lre‐5) of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)‐anchored protein implicated in reception of the pollen tube by the female gametophyte. Although most lre‐5 female gametophytes do not allow pollen tube reception, in those that do, early seed development is delayed. A fraction of lre‐5/lre‐5 seeds underwent abortion due to defect(s) in the female gametophyte. The aborted seeds contained endosperm but no zygote/embryo, reminiscent of autonomous endosperm development in the pollen tube reception mutants scylla and sirene. However, unpollinated lre‐5/lre‐5 ovules did not initiate autonomous endosperm development and endosperm development in aborted seeds began after central cell fertilization. Thus, the egg cell probably remained unfertilized in aborted lre‐5/lre‐5 seeds. The lre‐5/lre‐5 ovules that remain undeveloped due to defective pollen tube reception did not induce synergid degeneration and repulsion of supernumerary pollen tubes. In ovules, LORELEI is expressed during pollen tube reception, double fertilization and early seed development. Null mutants of LORELEI‐like‐GPI‐anchored protein 1 (LLG1), the closest relative of LORELEI among three Arabidopsis LLG genes, are fully fertile and did not enhance reproductive defects in lre‐5/lre‐5 pistils, suggesting that LLG1 function is not redundant with that of LORELEI in the female gametophyte. Our results show that, besides pollen tube reception, LORELEI also functions during double fertilization and early seed development.     

Pollenfertility and seed formation in theOrnithogalum umbellatum/angustifolium complex (Liliaceae/Scilloideae)

The pollen fertility and seed formation of six species of theOrnithogalum umbellatum/angustifolium complex and of seven related species were studied. Four types of pollen grains could be recognized. The pollen fertility varied greatly in this complex and is not related to the ploidy level. The seed formation ofO. umbellatum showed an adaptation to a subcontinental-Mediterranean climate, that ofO. angustifolium to an Atlantic climate. In both cases raindrops seem to be important for pollination, in view of the absence of insect pollinators. After open pollination 113 seedlings were obtained in four species. Their chromosome numbers were determined. Nearly all the cultivated seedlings were aneuploid, which points to a positive selection of euploids in nature, because aneuploid individuals are rare in the wild.Biosystematic Studies on theOrnithogalum umbellatum/angustifolium Complex III.—Previous parts of this series are Part I: Taxonomy. Proceeding Kon. Ned. Acad. Wet. series C,85 (4), 563–574 (1982) andvan Raamsdonk (1984).     

Reproductive biology of henequen (Agave fourcroydes) and its wild ancestor Agave Angustifolia (Agavaceae). i. Gametophyte development

The pathways of micro- and megagametophyte development in Agave fourcroydes (henequén) and A. angustifolia were studied. We used histology and light microscopy to observe anther ontogeny and ovary differentiation in relation to flower bud size. Both species have the same sexual reproductive strategies and gametophyte development that may be divided into three phases: (1) premeiotic, which includes the establishment of the megaspore mother cell and the pollen mother cell; (2) meiotic, the formation of mature microspores and functional megaspores; (3) postmeiotic, which encompasses the development of mature pollen grains and the formation of the embryo sac. A successive type microsporogenesis was found in both species with formation of T-shaped tetrads and binuclear pollen grains. In vitro germination tests revealed very low pollen fertility. The female gametophyte is formed from two micropylar megaspore cells after the first meiotic division (bisporic type). Male and female gametogenesis occur asynchronously with microsporogenesis finishing before macrosporogenesis. The results so far show that the formation of male and female gametophytes in henequén is affected at different stages and that these alterations might be responsible for the low fertility shown by this species.     

Apomixis in plants: structural and functional aspects of diplospory inPoa nemoralis andP. palustris

Summary Data on structural and functional aspects of mitotic diplospory and later stages of apomictic seed formation are reported forPoa palustris andP. nemoralis. In this study, the plant material of two Russian populations ofP. nemoralis andP. palustris were used for transmission electron microscope observations. Seed formation was investigated by phase contrast microscopy in two populations ofP. nemoralis collected in The Netherlands. The processes of transformation of the megasporocytes to the megaspore mother cells of diplosporous embryo sacs, and thereafter to one- and two-nucleate diplosporous embryo sacs (Antennaria type) were characterized by an increase of cell size, structural and functional reorganization of the nucleus, nucleolus, and cytoplasm, and cell isolation as a result of thickening of the cell wall. These were accompanied by an increase in the cell metabolic activity inferred from visual evidence of the activation of nucleus, nucleolus, endoplasmic reticulum, dictyosomes, mitochondria, and from the appearance of a dense population of ribosomes and polysomes. The diplosporous embryo sac of the Antennaria type was characteristic for bothP. nemoralis andP. palustris. No signs of the presence of synaptonemal complexes were observed during the process of diplosporous-embryo-sac megaspore mother cell differentiation and division. About 90–95% of the diploid egg cells of diplosporous embryo sacs were able to produce apomictic embryos. These embryos developed before anthesis. However, many of them degenerated at the globular stage because of lack of endosperm. The ultrastructural events occurring during the process of diplospory of apomictic species, and meiosis and megagametogenesis of sexually reproduced plants are discussed.Abbreviations DMC megaspore mother cell of diplosporous embryo sac - TEM transmission electron microscopy - ER endoplasmic reticulum     

Arabidopsis hapless mutations define essential gametophytic functions

In flowering plants, the egg develops within a haploid embryo sac (female gametophyte) that is encased within the pistil. The haploid pollen grain (male gametophyte) extends a pollen tube that carries two sperm cells within its cytoplasm to the embryo sac. This feat requires rapid, precisely guided, and highly polarized growth through, between, and on the surface of the cells of the stigma, style, and ovary. Pollen tube migration depends on a series of long-range signals from diploid female cells as well as a short-range attractant emitted by the embryo sac that guides the final stage of tube growth. We developed a genetic screen in Arabidopsis thaliana that tags mutant pollen with a cell-autonomous marker carried on an insertion element. We found 32 haploid-disrupting (hapless) mutations that define genes required for pollen grain development, pollen tube growth in the stigma and style, or pollen tube growth and guidance in the ovary. We also identified genomic DNA flanking the insertion element for eleven hap mutants and showed that hap1 disrupts AtMago, a gene whose ortholog is important for Drosophila cell polarity.     

An efficient protocol for plant regeneration from protoplasts of the moss <Emphasis Type="Italic">Atrichum undulatum P. Beauv in vitro</Emphasis>

A system for plant regeneration from protoplasts of the moss, Atrichum undulatum (Hedw.) P. Beauv. in vitro, is first reported. Viable protoplasts were isolated at about 9 × 10⁵ protoplasts g⁻¹ fresh weight from 10 to 18 days protonemata. For regeneration of protoplasts, viable protoplasts were cultured in liquid–solid medium containing surface liquid medium MS (0.4 M mannitol) and subnatant solid medium Benecke (0.3 M mannitol) at 20 °C under a 16-h photoperiod white light after 12 h preculture in darkness at 20 °C. The great majority of protoplasts follow a regenerative sequence: formation of asymmetric cells in 2–3 days; division of the asymmetric cells to 2–3 cells in 4–5 days, and further develop to produce a new chloronemal filament in 15 days. Juvenile gametophyte can be visible in 20 days. The plating ratio of cell cluster regenerated from protoplasts reaches up to 45%. Transient expression experiments indicate the electroporation uptake of DNA is possible.     

Diploidization in megagametophyte-derived cultures of the gymnosperm Larix decidua

Tested haploid embryogenic lines (n=12) of Larix dedicua Mill, initiated from megagametophyte tissue were maintained on half-strength LM medium without growth regulators. The cultures were analyzed for ploidy level after 1–9 years. All lines tested were found to have doubled (2n=24) their chromosome number at the end of the experiment, though there were a few lines that still gave occasional haploid counts. Flow cytometric data of embryogenic tissue confirmed these results. Protoplasts were stained in ethidium bromide, and cultured human leucocytes and chicken erythrocytes were used as internal standards. Haploid megagametophytes from immature seeds of L. decidua and known diploid culture lines of a related hybrid (L. x eurolepis) were also analyzed by flow cytometry. Haploid reference material had 12.3–13.6 pg DNA per cell, whereas formerly haploid callus lines had an average of 25.0 pg DNA per cell. The one exception was a known, genetically unstable line of L. decidua (34.8 pg DNA per cell). The diploid cell line of L. x eurolepis had 27.6 pg DNA per cell. The results show that spontaneous diploidization of megagametophyte lines is relatively rapid and that both haploid and dihaploid lines are embryogenic in larch.     

Syncyte formation in the microsporangium of <Emphasis Type="Italic">Chrysanthemum</Emphasis> (Asteraceae): a pathway to infraspecific polyploidy

Polyploidy, which is thought to have played an important role in plant evolution and speciation, is prevalent in Chrysanthemum (x = 9). In fact, polyploid series are known in C. zawadskii (2x, 4x, 6x, 8x, and 10x) and C. indicum (2x, 4x, and 6x), but the mechanism by which polyploidization occurs is unknown. Here we show that in diploid individuals of both C. zawadskii and C. indicum, the fusion between two adjacent pollen mother cells (PMCs) occurs at a frequency of 1.1–1.3% early in the first meiotic division. While possessing the chromosomes of both PMCs, the fused cell or syncyte undertakes subsequent meiotic division processes as a single large PMC, producing four 2n pollen grains that are able to germinate. Despite their low frequency, syncyte formation may have played a major role in the production of infraspecific polyploids in Chrysanthemum.     

Unreduced gametes in diploid Medicago and their importance in alfalfa breeding

Summary In the genus Medicago, it is known that 2n gametes have been important in the evolution and breeding of cultivated alfalfa, which is a natural polysomic polyploid (2n=4x=32), however little is known on the frequency of male and female 2n gametes in diploid relatives of alfalfa. To obtain data on the frequency of 2n gametes, more than 12,000 2x–4x and 4x–2x crosses were made in 1982 at Madison (USA). Diploid parents in crosses were from four populations of M. coerulea, two of M. falcata and one diploid population of cultivated M. sativa which was derived by haploidy. The tetraploid seed parent in the crosses was a male-sterile M. sativa clone and vigorous tetraploid M. sativa plants were used as pollen parents. Each of 274 diploid plants was utilized both as male and as female. Of the 548 cross combinations, 266 crosses produced variable quantities of seeds which were sown in 1983 in a greenhouse at Perugia (Italy); the plants were subsequently space transplanted in the field in 1984. The identification of ploidy level of these genotypes was made on the basis of morphological characters, plant fertility, pollen stainability and chromosome counts.Of the 515 plants analyzed, the majority behaved as normal tetraploids indicating that many diploid plants produced 2n gametes. Diplogynous and diplandrous gamete production was not correlated with each other, which indicated a different genetic control of 2n sporogenesis in the 2 sexes. Only 4 F₁ triploid plants confirmed the presence of a very effective triploid block in alfalfa. In consequence, bilateral sexual polyploidization is a more likely alternative for the origin of tetraploid alfalfa than triploid bridges. The present study showed that it is possible to efficiently identify genotypes able to produce high frequencies of 2n gametes within natural populations of diploids Medicago that are useful in alfalfa breeding.Part of this study was conducted at the Agronomy Department, University of Wisconsin, Madison, Wis, USA, while one of us (F. Veronesi) was in receipt of financial assistance provided by the National Research Council of Italy; part was conducted at Centro di Studio per il Miglioramento Genetico delie Piante Foraggere, C.N.R., Perugia, Italy. The paper was presented at the Eucarpia Fodder Crops Section Meeting, Svalöv, Sweden, 16–19th September 1985     

Embryological studies of Oxalis debilis Kunth

The study of pollen and female gametophyte development of Oxalis debilis was carried out to elucidate the reasons for the absence of seed production in this species. The formation of an incomplete callosic wall separating the microspores of the tetrad was observed; therefore, cytoplasm connections were present between the microspores. Microspores of different sizes and with different grades of vacuolation were observed in the mature anther. Only few microspores divide mitotically and form the generative and vegetative cell. The cytoplasm of the vegetative cell may accumulate different substances in reserve. Pollen grains are 8–12?colpate, and the morphology and electron density of the exine varies. Because flowers in full anthesis have all ovules at the megasporocyte stage with the nucellar epidermis intact, fecundation could not occur. The production of only a few viable pollen grains and the absence of successful megagametophyte development prevent fertilization and account for the absence of fruits and seeds formation.     

Patterns of gametophyte dominance ofIridaea splendens(Rhodophyta) in Vancouver Harbour,Vancouver, British Columbia,Canada

This paper presents results of field and laboratory work on one component of population structure ofIridaea splendens (Setchellet Gardner) Papenfuss (Gigartinaceae, Rhodophyta), the seasonal change in abundance of the gametophyte life-history stage. Investigations of this genus by others (using a variety of sampling and identification techniques) have shown a seasonal gametophyte dominance, sporophyte dominance, or some combination of these two. Gametophyte stages ofIridaea splendens produce kappa carrageenan and tetrasporophyte stages lambda carrageenan; a chemical colorimetric test using resorcinol can be used to identify these stages regardless of the presence of reproductive structures. In this paper we report on the proportion of gametophytes determined both by this chemical test and by the presence of reproductive structures, and on one possible determinant of the seasonal change in dominance. Analysis of field samples using the resorcinol test showed that from June–August the gametophyte stage predominates and from December–February the tetrasporaphyte stage is most common, both in reproductive and non-reproductive thalli (the latter tested as three size classes). Examination of reproductive structures gave similar results. One factor suggested in the literatures as a possible determinant of the haploid/diploid ratio is apomeiosis (in the formation of the tetraspores); our results show this to be unimportant in the Vancouver Harbor populations.Presented at the XIIIth International Seaweed Symposium, University of British Columbia, Vancouver, Canada, August 1989.     

Experimental induction of triploid plants ofPhysalis through anther culture

Summary Anther culture ofPhysalis minima L. (2 n=48) was successful and embryos and plantlets could be obtained 5–6 weeks after culture. MS medium containing CM was essential for pollen division. 16.7% of the cultured anthers produced plantlets. All the pollen plantlets were triploid (3 n=72). Regeneration of multiple shoot buds was obtained from cultured leaf and stem expiants of pollen plantlets.     

Studies in the embryology ofHeliantheae (Compositae)

The embryology ofLagascea mollis, Zinnia angustifolia andGalinsoga parviflora has been studied. The anther archesporium is hypodermal and consists of a single row of 6–8 cells, there are two layers below the epidermis of the anther and a periplasmodial tapetum. Ripe pollen grains are tricolpate and 3-celled. The ovary contains a single ovule, but in a few cases ofGalinsoga parviflora two ovules have been found. The female archesporium is unicellular, but sometimes more than one archesporial cell occurs inLagascea mollis andZinnia angustifolia. The embryo sac development is of the Polygonum type, the synergids are hooked, antipodal cells show great variation. The endosperm development is cellular inGalinsoga parviflora and peripheral layer persists in the mature seed. The embryo development conforms to the Senecio variation of the Asterad type. The pericarp structure and cmbryological features support the disputed systematic position ofLagascea withinHeliantheae. Part of a Ph.D. thesis, accepted at Andhra University.