Structural Modifications of the Female Gametophyte Induced by Temperature in Nicotiana tabacum

Effect of environmental conditions on formation of Nicotiana tabacum L. megagametophyte was studied. It was established, that unfavorable temperatures can specifically modify the structure of embryo sacs (ES). At low temperature (9/5 °C), ES with a reduced number of cells or with egg-like synergide(s) can be formed; at high variable (40/25 °C) or constant (37 °C) temperatures, ES with excessive numbers of cells or with synergide-like egg cells arise. Total frequencies of the changed ES patterns varied from 8 up to 35 % per plant and depended on the plant genotype and conditions of exposure. This revised version was published online in July 2006 with corrections to the Cover Date.     

The megagametophyte in Anarthria (Anarthriaceae,Poales) and its implications for the phylogeny of the Poales

The development and structure of the megagametophyte of Anarthria (Anarthriaceae), Aphclia, and Centrolepis (Centrolepidaceae) are described. Anarthriaceae has tenuinucellate ovules and the Polygonum type of megagametophyte development, both characters typical of the Poales. However, it lacks the anticlinally elongated nucellar epidermis and numerous large starch bodies observed in the megagametophyte of Centrolepidaceae, both characters also present in Restionaceae. This relatively generalized megagametophyte structure is consistent with data from the chloroplast genome, which suggest that Anarthriaceae are not as closely related to Restionaceae as previously assumed. New data from the megagametophyte are analyzed cladistically together with other available information on the poalean families. The results show that there are two possible positions for Anarthriaceae: either as sister to Poaceae. Joinvilleaceae, Restionaceae, Ecdeiocoleaceae, and Restionaceae, or as sister to only the latter three families. The new data also allow a critical reevaluation of the phylogenetic position of Centrolepidaceae, which is either basal to the poalean clade (based on microgametophyte data), or embedded in the Restionaceae (based on anther structure and megagametophyte data).     

The development of onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.) Embryo sacs <Emphasis Type="Italic">in vitro</Emphasis> and gynogenesis induction in relation to flower size

Summary The development of embryo sacs (ES) in vitro and induction of gynogenesis were studied in onion flower bud culture. Explants were divided into three groups according to their size at inoculation: (a) small flower buds (2.3–3.0 mm in diameter); (b) medium flower buds (3.1–3.7 mm); and (c) large flower buds (3.8–4.4 mm). For histological study, excised ovaries were fixed at inoculation and then at 3-d intervals until day 12, and after 2 and 3 wk of culture. Some explants were cultured until embryo emergence, i.e., 3–5 mo. In total, 2592 ovules were examined histologically. At inoculation, 83% of ovules in small flower buds contained a megaspore mother cell; in 17% of ovules, two-nucleate ES occurred. In medium flower buds two-nucleate, four-nucleate, and mature ES were present at frequencies of 15%, 46%, and 40%, respectively. In large flower buds, only mature ES occurred. In vitro conditions did not disturb meiosis and megagametophyte development in non-degenerated ovules. Regardless of the developmental stage at inoculation, only mature ES occurred on day 12. Gynogenic embryos were found after 2 wk of culture, indicating that embryos developed in mature ES exclusively. Embryos were detected in 5.4% of histological studied ovules; however, the number of embryos after 3–5 mo. was higher (12.4%). The parthenogenetic origin of the embryos is discussed. In addition, ES containing endosperm only (6.5%) and both endosperm and embryo (0.4%) were observed.     

Cytological basis for a tetraspory in Cupressus sempervirens L. megagametogenesis and its implications in genetic studies

 The processes of megasporogenesis and early megagametogenesis were cytologically investigated in Cupressus sempervirens L. in order to elucidate, at the cellular level, the origin of the megagametophyte. After pollination, sporogenous tissue developed in the chalazal region of the nucellus, but only one megaspore mother cell differentiated and divided meiotically without cell-wall formation. This led to the development of a cell with four nuclei which directly functioned as a megaspore. The C. sempervirens megagametophyte is thus tetrasporic, in contrast to the majority of conifers where the megagametophyte is monosporic. The consequenses of this observation are discussed from a genetics point of view. Received: 15 August 1997 / Accepted: 19 September 1997     

THE MEGAGAMETOPHYTE OF HYDRASPERMA TENUIS LONG FROM THE UPPER DEVONIAN OF IRELAND

Hydrasperma tenuis ovules containing cellular megagametophytes have been recovered from Upper Devonian (stage) strata near Ballyheigue, County Kerry, Ireland. The megagametophytes are elliptical in transverse section and obovate in longitudinal section. The vegetative cells are organized into three, distinct, concentric zones and exhibit the radial orientation characteristic of centripetal development. Up to three archegonia may be present at the apical end of the megagametophyte. They are elliptic to terete in cross-section and elliptic in longitudinal section. Archegonia are thought to be sunken with each possessing a canal-like connection to the surface of the megagametophyte. Archegonial jacket cells surround the egg cavity. A low, broad tent-pole occurs at the top of the megagametophyte. This report represents the oldest, structurally-preserved gymnosperm megagametophyte.     

Megagametophyte abnormalities of near-isogenic female partial-sterile soybean mutants (Glycine max; Leguminosae)

Megagametogenesis of soybean, Glycine max (L.) Merr., cultivars Clark and Clark k2, and F1 hybrid of Clark (female parent) crossed with Clark k2 (male parent) were studied using stereo light microscopy and confocal scanning laser microscopy. Reproductive development in Clark and Clark k2 plants was compared to F1 hybrid plants. In mature pods, 6.4% of the ovules of Clark, 8.1% of the ovules of Clark k2, and 41.4% of the ovules of F1 hybrid plants were aborted. This female partial sterility was due to incomplete megagametophyte development: undeveloped polar nuclei—or developed but not in a position for fertilization; increased megagametophyte wall thickness; abnormal shape and/or premature degeneration of synergids and intact synergids throughout the life of the ovule; egg cell not well-developed or absent; and megagametophyte remaining uninucleate. Each of these abnormalities contributed to either lack of double fertilization or early megagametophyte abortion. Electronic Publication     

A COMPARATIVE STUDY OF OVULE AND MEGAGAMETOPHYTE DEVELOPMENT IN FIELD-GROWN AND GREENHOUSE-GROWN PLANTS OF GLYCINE MAX AND PHASEOLUS AUREUS (PAPILIONACEAE)

A study of ovule and megagametophyte development in field- and greenhouse-grown plants of Glycine max (L.) Merrill and Phaseolus aureus Roxb. reveals several consistent features for both species. These features include: a multiple archesporium, enlargement of a primary sporogenous cell directly into a megasporocyte, production of unequal dyad cells, a functional chalazal megaspore, Polygonum-type development, and a hypostase. A filiform apparatus was not observed in either species. Several marked differences in development also occur. Phaseolus usually produces one sporogenous cell per ovule; Glycine produces 2–3 sporogenous cells per ovule. Meiosis II is synchronous in Phaseolus but nonsynchronous in Glycine. Linear tetrads are produced in Phaseolus, whereas linear and T-shaped tetrads are found in Glycine. Starch grains accumulate in the mature megagametophyte of Glycine but are absent at that stage in Phaseolus. The usefulness of the modified clearing fluid, benzyl benzoate-4½, for the study of ovule and megagametophyte development in Glycine max and Phaseolus aureus is here demonstrated. In addition, the study indicates for both species that megagametophyte development in plants grown under field conditions is markedly similar to development in plants grown in the more uniform conditions of the greenhouse. Accordingly, these findings suggest generally that embryological data collected from plants grown under greenhouse conditions will reflect those from plants found in nature.     

Einfluß von Textur,Gehalt an organischer Substanz,Dichte und Luftgchalt des Bodens auf die Vermehrung von Heterodera schachtii Schmidt, 1871 an Zuckerrüben

Defense reactions of embryo and megagametophyte (endosperm) of European black pine (Pinus nigra Arn.) were studied by in vitro technique. As a tester, basidiomycete Phaeolus schweinitzii (Fr.) Pat. was used. Both, defense reaction of embryo and very strong defense reaction of megagametophyte were found. Some substances which may be involved in defense reactions are discussed.     

Intergeneric pollen – megagametophyte relationships of conifers in vitro

 Germinating pollen from larch (Larix occidentalis), Sitka spruce (Picea sitchensis) and white pine (Pinus monticola) were co-cultured with megagametophytes dissected from cones of other genera (Pseudotsuga menziesii, Larix×eurolepis and Pinus monticola). Pollen was presented to megagametophytes possessing archegonia which were either alive, degenerating or dead. In addition, pollen was presented to fertilized megagametophytes and to megagametophytes that had been cut in half. Megagametophyte penetration by pollen tubes and male gamete release into archegonia were verified by serial sections of glycomethacrylate-embedded specimens. Pollen tubes penetrated through any part of the apex of the megagametophyte. Division of the body cell into the two gametes was regularly observed. Delivery of gametes was confirmed between spruce and larch. Pollen tubes also penetrated fertilized megagametophytes, dead or degenerating archegonia as well as wounded and/or cut surfaces. This demonstrates the inability of the male gametophyte to optimize its mating efforts, since it is unable to differentiate between healthy and unhealthy archegonia. The megagametophyte cells are unable to optimize male selection. They may produce secretions of a generally attractive nature, as pollen is attracted to the apex of the megagametophyte, but archegonia themselves do not produce pollen-specific signals of either a promotive or inhibitory nature. These results open new avenues for the development of novel breeding strategies where natural breeding barriers may be bypassed. Received: 19 March 1998 / Accepted: 29 April 1998     

Characteristics of ovary, ovule and mature megagametophyte in Rhododendron L. (Ericaceae) and their taxonomic significance

PALSER, B. F., PHILIPSON, W. R. & PHILIPSON, M. N., 1991. Characteristics of ovary, ovule and mature megagametophyte in Rhododendron L. (Ericaceae) and their taxonomic significance. The ovary, ovule and megagametophyte at the time the latter is mature are described for 177 species representing all subgenera, sections and most subsections recognized in Rhododendron. All three, but particularly the ovary, vary considerably. The ovary is compared among species as to size; shape of apex–tapered vs. depressed; relative dimensions; indumentum–five basic hair types; wall structure including crystal distribution and frequency, stomata, cuticular ornamentation; locule number, shape and size relative to radius; wall thickness relative to radius; placenta size, shape, depth of cleft, level of cleft junction, presence or not of a decurrent placental stalk ridge; number of ovules, their arrangement and orientation on placenta; and presence and distribution of internal stomata and hairs. The nectary which girdles the base of the ovary also varies in size, shape, indumentum, stomata and some internal features. The ovule is anatropous, unitegmic and tenuinucellate with the lateral and micropylar nucellus disappearing completely and the megagametophyte elongating into the micropyle in all species. Also common to almost all are an endothelium, hypostase, differentiated epidermis–most often tanniniferous, and starch in integument around egg apparatus and micropyle. There are differences in size, shape, proportion occupied by gametophyte and micropyle, thickness of integument, degree of differentiation of some features, amount and area of starch and occurrence of chalazal and micropylar tails or of incipient micropylar and/or chalazal appendages. The megagametophyte, which develops according to the Polygonum pattern, has two distinct portions, the chalazal bounded by the endothelium with small antipodal cells at its end and the usually broader micropylar part within the micropyle containing a rather large egg apparatus with distinctive synergids; starch is present in the central cell and the polar nuclei are most often fused. Differences occur in proportions of the parts to one another, amount of starch, etc. Sixty-three characters were entered onto a computer, clustered by two different techniques and dendrograms constructed. Personal analysis and both dendrograms show section Vireya to be characterized by a syndrome of distinctive features and clearly separated from the rest of the genus. Subgenus Hymenanthes also has its particular syndrome but is somewhat less distinct from the remainder of the genus. Even less distinct but still grouped together are species of section Choniasirum and of section Sciadorhodion. Many species of section Rhododendron and of subgenus Tsutsusi tend to cluster together but section Rhododendron and most sections of the azalea complex are more generalized and moderate in the ovary, ovule and megagametophyte characters and do not separate sharply from one another.     

Spatio-Temporal Variations in Starch Accumulation During Germination and Post-Germinative Growth of Zygotic and Somatic Embryos of Pinus pinaster

During germination and post-germinative growth of Pinus pinaster Ait. seeds, triglycerides are hydrolysed and concurrently the embryo accumulates starch. In this study, the spatio-temporal variation of starch accumulation was described in zygotic embryos associated (ZE⁺) or not (ZE⁻) to their megagametophyte and in somatic embryos (SE). In germinating ZE⁺, starch was accumulated in the growing tissues, following closely the spatio-temporal pattern of triglycerides depletion. In contrast, in ZE⁻ and SE, starch was only found in cortical cells close to the culture medium. In germinating ZE⁺, the spatio-temporal variations of starch accumulation can be thus interpreted as the result of the changing contact between the megagametophyte and the growing tissues and also of the existing interactions between triglyceride hydrolysis and the allocation of sucrose exported from the megagametophyte. This revised version was published online in July 2006 with corrections to the Cover Date.     

Regulation of loblolly pine (Pinus taeda L.) arginase in developing seedling tissue during germination and post-germinative growth

After seed germination, hydrolysis of storage proteins provides a nitrogen source for the developing seedling. In conifers the majority of these reserves are located in the living haploid megagametophyte tissue. In the developing loblolly pine (Pinus taeda L.) seedling an influx of free amino acids from the megagametophyte accompanies germination and early seedling growth. The major component of this amino acid pool is arginine, which is transported rapidly and efficiently to the seedling without prior conversion. This arginine accounts for nearly half of the total nitrogen entering the cotyledons and is likely a defining factor in early seedling nitrogen metabolism. In the seedling, the enzyme arginase is responsible for liberating nitrogen, in the form of ornithine and urea, from free arginine supplied by the megagametophyte. In this report we investigate how the seedling uses arginase to cope with the large arginine influx. As part of this work we have cloned an arginase cDNA from a loblolly pine expression library. Analysis of enzyme activity data, accumulation of arginase protein and mRNA abundance indicates that increased arginase activity after seed germination is due to de novo synthesis of the enzyme. Our results suggest that arginase is primarily regulated at the RNA level during loblolly pine seed germination and post-germinative growth.     

Occurrence of unique three-celled megagametophyte and single fertilization in an aquatic angiosperm-Dalzellia zeylanica (Podostemaceae-Tristichoideae)

Angiosperms are characterized by the occurrence of double fertilization. However, Podostemaceae is considered an exception with the presence of only single fertilization (syngamy) though two male gametes are formed conventionally. To determine the cause for the failure of double fertilization in Dalzellia zeylanica (Gardn.) Wight, we closely tracked the movement of the male gametes from the time of pollen tube initiation to the time of entry into the megagametophyte to affect fertilization. We report for the first time, the presence of a novel type of three-nucleate/three-celled mature megagametophyte consisting of two synergids and an egg cell in D. zeylanica. Therefore, of the two male gametes formed in this plant, one fuses with the egg cell resulting in syngamy, whereas the other male gamete eventually degenerates due to the absence of its partner i.e. single polar nucleus of the central cell that degenerates prior to the entry of the pollen tube into the synergid. The present work not only highlights the highly reduced nature of megagametophyte but also the occurrence of single fertilization resulting in sperm selection in D. zeylanica.     

Analyses to determine the role of the megagametophyte and other seed tissues in dormancy maintenance of yellow cedar (Chamaecyparis nootkatensis) seeds; morphological, cellular and physiological changes following moist chilling and during germination

Yellow cedar (Chamaecyparis nootkatensis) seeds exhibit prolonged dormancy following their dispersal from the parent plant. Embryos excised fully from their enclosing seed tissues exhibit 100% germination, indicating that the seed tissues enclosing the embryo (the testa, remnants of the nucellus and the megagametophyte) play an inhibitory role and prevent radicle emergence. As part of an assessment of the role of seed tissues in the dormancy mechanism of yellow cedar seeds, light microscopy was used to examine changes within the major structures of the seed following a 90 d war (26C)/cold (4C) moist treatment ('stratification') and during germination. In the micropylar tip of the seed, the nucellus forms a hard nucellar cap covering the radicle. The nucellar cap is composed primarily of degenerated cells; histological staining with ruthenium red revealed a predominance of pectins. There were no obvious cellular or morphological differences (detected by light microscopy) between mature seeds subjected to a 3 d soak and seeds subjected to a 3 d soak and the 90 d dormancy-breaking treatment. However, just prior to germination there was an outward projection of the nucellar cap through the micropyle, which appeared to be caused by the extension of highly folded proteinaceous strands lying immediately in front of the radicle. When the testa was removed, the embryo enclosed within the intact megagametophyte was incapable of germination. If, however, the megagametophyte surrounding the embryo was slit or the embryo surrounded by an intact megagametophyte was subjected to a 3d rinse in water, some germination occurred, perhaps as a result of an enhanced release of inhibitors from the megagametophyte. After stratification, dormancy of yellow cedar seeds is broken; concurrent with dormancy breakage, there was a mechanical weakening of the megagametophyte. The embryo also underwent changes that included an increase in turgor and a reduced sensitivity to highly negative osmotic potential. It is concluded that coat-imposed dormancy of yellow cedar seeds is enforced by mechanical restraint of the megagametophyte as well as a leachable chemical inhibitor (most probably ABA).     

DEVELOPMENT OF THE OVULE AND MEGAGAMETOPHYTE IN SAXIFRAGA HIERACIFOLIA

Wiggins , Ira L. (Stanford U., Stanford, Calif.) Development of the ovule and megagametophyte in Saxifraga hieracifolia. Amer. Jour. Bot. 46(10): 692–697. Illus. 1059.—Buds of Saxifraga hieracifolia collected in the vicinity of Point Barrow, Alaska, fixed, sectioned, and stained by standard methods, revealed that the archesporial cell in the ovule of this species is hypodermal and gives rise to the megaspore mother cell and a small number of parietal cells. Occasionally 2 megaspore mother cells occur within an ovule. Meiosis in the megaspore mother cell produces a linear tetrad of megaspores, the chalazal one of which normally gives rise to a monosporic, Polygonum-type megagametophyte. The polar nuclei fuse near the chalazal end of the megagametophyte and the antipodal cells disintegrate prior to fertilization. A distinct filiform apparatus and a marked lateral “spur” develop on each synergid. Vacuolation in the egg cell and in the synergids follows the usual pattern. Only a single integument surrounds the nucellus.     

APOMIXIS AND SEXUALITY IN THREE SPECIES OF AMELANCHIER,SHADBUSH (ROSACEAE,MALOIDEAE)

We used Nomarski differential interference contrast microscopy of cleared, whole ovules to examine megasporogenesis and megagametogenesis in tetraploid (N = 34) individuals of three species of Amelanchier in Maine. Amelanchier canadensis and A. stolonifera conform to the general pattern of apomixis in the Maloideae by being aposporous and by frequently forming more than one megagametophyte per megasporangium. These species are also pseudogamous; both self and foreign pollen elicit fruit set. Amelanchier bartramiana follows a sexual pattern by producing a triad of megaspores and almost always only one megagametophyte per megasporangium. This boreal shrub, strikingly distinct morphologically and in its habitat preference from other North American species of the genus, is primitive in its sexuality and self-incompatibility relative to other species we have studied.     

Development of cycad ovules and seeds. 1. Implication of the ER in primary cellularisation of the megagametophyte in <Emphasis Type="Italic">Encephalartos natalensis</Emphasis> Dyer and Verdoorn

Very little is known about the pre- and post-shedding megagametophyte development and utilisation of accumulated reserves, respectively, in cycads (Zamiaceae). In the present study on developing ovules of the recalcitrant-seeded species, Encephalartos natalensis, cells of the megagametophyte were found to become progressively packed with starch and protein as the two main storage reserves, a limited number of discrete lipid bodies, and occasional mitochondria all of which appeared to be embedded in a homogeneous matrix. ER-derived vesicles (and not Golgi-derived vesicles) appeared to be the principal contributors of the primary cell wall components, pectin and xylan, during megagametophyte cellularisation. This was confirmed by the use of enzyme-gold localisation. High-pressure freezing (HPF) and freeze substitution (FS) of samples the following season showed that while the apparently featureless cytomatrix of the megaspore was an artefact of conventional fixation, there was still an insignificant occurrence of Golgi bodies during primary wall formation. When enzyme-gold localisation was employed on the HPF-FS material, label for pectin and xylan was found only in the regions of ER and vesicles and not in any of the few Golgi bodies or their associated vesicles. Immunocytochemistry revealed that pectin and xylan were restricted to the ER and not to any vesicles or to the occasional Golgi body that was found. This suggests that the ER exclusively, is involved in the deposition of these primary cell wall components during the cellularisation of the E. natalensis megagametophyte. While cellularisation took place over approximately 1–2 weeks, subsequent development of the megagametophyte cells involved the accumulation of storage reserves, this phase lasting approximately 8 months—after which the seeds were shed whether pollination/fertilisation had recently occurred, or not.     

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     

Nuclear and cell fusion cause polyploidy in the megagametophyte of common cypress, Cupressus sempervirens L.

In common cypress, Cupressus sempervirens L., the megagametophyte persists in mature seeds as a polyploid endosperm containing cells with even and odd series of DNA contents: 1C, 2C, 3C, 4C, 5C etc., where C is the amount of DNA in the haploid genome. In this study, cytometrical, histological and cytochemical investigations were performed in order to determine the behavior of megagametophyte nuclei during the reproductive cycle. Unexpected nuclear alterations due to a continuous process of nuclear fusion were observed in the megagametophyte, leading to polyploidization and consequently to intense food-reserve synthesis. During the free nuclear stage, the megagametophyte exhibited only sporadic nuclear fusion and limited food-reserve production. When cellularization took place, multinucleated compartments were observed in which nuclei fused, producing odd and even series of DNA contents as proved by flow-cytometric analysis. This polyploidization process considerably increased after fertilization and during embryo development, and was accompanied by increased food-reserve synthesis. During these later stages, fusion mainly involved nuclei of contiguous cells and was preceded by the disintegration of their adjacent walls. Mitoses with incomplete phragmoplast differentiation were also observed to yield polyploid nuclei. Finally, in mature seeds the endosperm still exhibited multinucleate cells and fusion nuclei, and contained high amounts of storage products. The results are interpreted as an alteration of DNA contents in the megagametophyte cells in relation to specific metabolic activity during seed development. Received: 2 September 1998 / Accepted: 31 December 1998