EMBRYO SAC LACKING ANTIPODAL CELLS IN ARABIDOPSIS THALIANA (BRASSICACEAE)

Ultrastructure of the embryo sac lacking antipodals in prefertilization stages in Arabidopsis thaliana has been examined 2 hr before and 5 hr after manual cross pollination. The cytoplasm of both synergids before fertilization is rich in ribosomes, mitochondria, and rough endoplasmic reticulum, and also contains several microbodies and spherosomes. The filiform apparatus includes electron-dense material and a fibrous part. Many cortical microtubules appear in the filiform apparatus area. One of the two synergids degenerates before fertilization. The synergids, the egg cell, and central cell have a rich cytoskeleton of microtubules; only the synergids appear to contain microfilaments. At the chalazal end, the antipodals are initially present but degenerate by the time of pollination in most embryo sacs in the starchless line studied. The embryo sac is completely surrounded by a wall containing an electron-dense layer, separating it from the nucellus, including the chalazal end. When the antipodals have degenerated, the electron-dense layer disappears at the chalazal end only, and the wall between the central cell and the nucellus is homogeneous. Between the central cell and nucellar cells no plasmodesmata are found. The membranes of both antipodal cells at the chalazal end of the embryo sac appear sinuous, like those of transfer cells. The central cell has plastids preferentially distributed around the nucleus, but the other organelles are randomly distributed. The central cell in the embryo sac and the adjacent chalazal nucellar cells show a transfer-cell function in the embryo sac after the antipodals degenerate.     

Some aspects of organization and histochemistry of the embryo sac ofScilla sibirica sato

Summary The present investigation deals with some of the organizational and histochemical aspects of the embryo sac ofScilla sibirica. Both the synergids and egg cell are invested by PAS-positive complete walls. The filiform apparatus comprises an elaborate system of fibrillar projections, showing extensive ramifications. The micropylar region of the embryo sac wall from where the filiform apparatus originates is composed of three distinct layers. On a histochemical basis it may be surmised that, unlike the egg cell, the synergids are metabolically very active. Two kinds of wall ingrowths (i) massive and highly branched very much akin to the filiform apparatus, and (ii) small tuberculate wall projections, are unique to the antipodal cells of S.sibirica. Small tuberculate projections have also been observed along the wall of the central cell adjacent to the nutrient-rich nucellar cells. The antipodals and the central cell show the presence of starch grains and abundant total proteins. All the cell types in the embryo sac ofS. sibirica are structurally so organized as to meet the requirements of its nutrition during pre- and postfertilization development. The presence of abundant PAS-positive granular substance in the cells of nucellar epidermis probably establishes a gradient which assists in the pollen tube growth.     

粉叶小檗大孢子发生和雌配子体形成

采用石蜡切片方法对粉叶小檗（Berberis pruinosa Franch.)的大孢子发生和雌配子体形成过程进行了研究。主要结果如下：雌蕊1枚,子房单心皮,边缘胎座,2枚胚珠倒生,具双珠被,厚珠心,珠孔由内外两层珠被共同形成,呈“Z”字形;单孢原,位于珠心表皮下;直线形大孢子四分体,合点端的1个大孢子发育为功能大孢子,胚囊发育类型为蓼型;成熟胚囊中,2个极核在受精前融合为次生核;3个反足细胞不发达,较早退化;"品"字形卵器,其中助细胞发达且具丝状器。     

宁夏枸杞大孢子发生和雌配子体发育的细胞学研究

采用半薄切片技术和组织化学染色法对宁夏枸杞大孢子发生和雌配子体发育过程中的细胞结构变化及营养物质积累特征进行了观察。结果表明,(1)宁夏枸杞为中轴胎座,多室子房,倒生胚珠,单珠被,薄珠心类型。(2)位于珠心表皮下的孢原细胞可直接发育为大孢子母细胞,减数分裂后形成直线型大孢子四分体,合点端第一个大孢子发育为功能大孢子,胚囊发育类型为蓼型,具有珠被绒毡层。(3)初形成的胚囊外周组织中没有营养物质积累,成熟胚囊时期出现了大量的淀粉粒且呈珠孔端明显多于合点端的极性分布特征。(4)助细胞的珠孔端具有明显的丝状器结构,呈PAS正反应表现出多糖性质,成熟胚囊具有承珠盘结构。     

Megasporogenesis, female gametophyte development and embryonic development of Ambrosia L. in China

The megasporogenesis, female gametophyte development and embryonic development of Ambrosia artemisiifolia L. and Ambrosia trifida L. of genus Ambrosia L. in China were studied using conventional paraffin section technology and optical microscopy. The results show that both A. artemisiifolia L. and A. trifida L. have a bilobed pistil stigma, two carpels, one chamber, basal placenta, unitegmic, tenuinucellate, anatropous ovule, and well-developed integumentary tapetum. Megaspore mother cells are directly developed from archesporial cells originated from the nucellar cells under the nucellar epidermis and further undergo meiosis to form linear tetrads. The megaspore at the chalazal end develops into a functional megaspore and the other three megaspores are degraded. The development of embryo sac is monosporic type. After three consecutive mitosis, mononucleate embryo sac becomes a mature embryo sac with two synergids and one egg cell at the micropylar end, a central cell at the center and three antipodal cells at the chalazal end. Most antipodal cells are mononucleate or binucleate, only few are trinucleate. The embryonic development process contains four stages: globular embryo, heart-stage embryo, torpedo-stage embryo and mature embryo. The development of endosperm is cellular type.     

Ultrastructure of endopolyploid antipodals inAconitum vulparia Rchb.

Summary The ultrastructure of antipodals ofAconitum vulparia Rchb. was studied at two stages of development: at the earlier stage the endosperm has several nuclei, at the later one the endosperm is multinucleate. Over the investigated period the antipodal size enlarges distinctly. The wall ingrowths increase in size and number. Finally, they occur throughout the antipodal walls except for a small area in the extreme chalazal part, sunk deep into the nucellar podium. There are no plasmodesmata in the antipodal cell walls. The cytoplasm is dense and rich in ribosomes; it shows weak vacuolation. The rough endoplasmic reticulum is well developed. At the later stage dilated cisternae of endoplasmic reticulum are formed. Mitochondria, plastids and active dictyosomes are abundant. At the later stage some giant mitochondria are present; their matrix contains a large clear area with fine fibrils and an aggregation of fibrillar material. At this stage of development plastids have two types of inclusions: electron-transparent vacuoles and aggregations of electron-dense granules. The giant endopolyploid nuclei are considerably larger than those at the mature embryo sac stage; they are lobed on all sides.During the studied periodA. vulparia antipodals seem to be at their most active state.     

A CYTOCHEMICAL STUDY OF EMBRYO SAC DEVELOPMENT IN STELLARIA MEDIA

Megasporogenesis and embryo sac development in Stellaria media were investigated using cytochemical methods for the demonstration of nucleic acids, proteins, and polysaccharides. RNA concentrations were high in the archesporial cells, low in the megaspore mother cell, and increased again to high concentrations with the formation of the megaspore and 2-, 4-, and early 8-nucleate embryo sac. RNA levels were also high in the egg and primary endosperm nucleus but low in the synergid and antipodal cells. Nucleolar size and vacuolation were indicative of RNA synthetic activity. Protein concentrations were parallel in concentration and distribution to those observed for RNA. Polysaccharides were conspicuously absent from all stages except the synergids and nucellar cells. Feulgen-stained DNA was demonstrable in the antipodal cells, megaspore mother cell, and megaspore cell, but was not visible in the 2-, 4-, or early 8-nucleate embryo sac. Feulgen staining was also absent from the egg and primary endosperm nucleus but was visible in the synergids and antipodals. Histones were difficult to visualize anywhere except in the egg cytoplasm and the nuclei of the antipodals.     

Iatercellular Migration of Protoplasm and Its Relation to the Development of Embryo Sac in Nucellus of Wheat

During the development of the ovule before pollination, deterioration of successive layers of nucellar tissue, beginning from the nnermost, constantly takes place and consistently forms a zone of disorganization surrounding the periphery of the enlarging embryo sac. Nucellar tissue deteriorates much more profusely near the antipodal end of the sac. "Nuclear extrusion" taken as an indication of intercellular movement of the protoplasm which has undergone partial disassembly, can be seen among the nucellar tissues and between the nucellus and the embryo sac. The intruding nuclear fragments, some of which assume the form of nucleolus, can be found in the antipodal cells. The results interpreted according to our previous hypothesis, are as follows. The nucellar cell by means of intercellular movement of its own protoplasm in the state of partial disassembly, furnishes the embryo sac with composite units of various polymers and organelles. Consequently, the antipodal cells proliferate and flourish The interrelationship between nucellus and embryo sac has been discussed from the viewpoint of supply and utilization of nourishment, which is necessary for the rapid development of the embryo sac.     

Antipodal cells persist through fertilization in the female gametophyte of Arabidopsis

Key message

Extended antipodal life-span.

Abstract

The female gametophyte of most flowering plants forms four cell types after cellularization, namely synergid cell, egg cell, central cell and antipodal cell. Of these, only the antipodal cells have no established functions, and it has been proposed that in many plants including Arabidopsis, the antipodal cells undergo programmed cell death during embryo sac maturation and prior to fertilization. Here, we examined the expression of female gametophyte-specific fluorescent reporters in mature embryo sacs of Arabidopsis, and in developing seeds shortly after fertilization. We observed expression of the fluorescence from the reporter genes in the three antipodal cells in the mature stage embryo sac, and continuing through the early syncytial endosperm stages. These observations suggest that rather than undergoing programmed cell death and degenerating at the mature stage of female gametophyte as previously supposed, the antipodal cells in Arabidopsis persist beyond fertilization, even when the other cell types are no longer present. The results support the concept that the Arabidopsis female gametophyte at maturity should be considered to be composed of seven cells and four cell types, rather than the previously prevailing view of four cells and three cell types.     

Optimization of conditions for in situ hybridization and determination of the relative number of ribosomes in the cells of the mature embryo sac of maize

Summary We have initiated experiments to understand the molecular regulation of embryo sac development in flowering plants by a study of ribosome synthesis and accumulation. Because of the very small size of the embryo sac and the large volume of ovule tissue it is embedded in, in situ hybridization with nucleic acid probes is presently the only practical method for such molecular measurements on individual cells of the embryo sac. Methods of tissue preparation, sectioning and screening of ovules for embryo sac containing sections, in situ hybridization using a ribosomal mRNA probe, and staining were optimized. Relative densities of silver grains for individual cells of the mature maize (W22) embryo sac were determined from in situ hybridizations. The silver grain counts are directly related to the numbers of ribosomes. Volumes of individual cells were determined by confocal microscope image analysis, and this permitted the calculation of the relative total numbers of ribosomes in individual cells of the embryo sac and nucellus. The central cell has a volume 260 times that of a nucellar cell at the micropylar end of the ovule, 15 times that of the egg cell, 30 times that of a synergid, and 130 times the volume of an antipodal cell. The mature maize embryo sac has 20 or more antipodal cells. The central cell has approximately 200 times the number of ribosomes as are present in a nucellar cell, about 7 times as many ribosomes as are in the egg cell, 14 times as many ribosomes as in each synergid, and about 80 times the ribosome content of individual antipodal cells. The data are discussed with respect to the utilization of the ribosomes following fertilization in the early embryo and endosperm.     

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.     

Transport of Disassembled Protoplasm from Degenerated Nucellus into Embryo Sac and Its Role in Feeding the Proliferating Antipodals in Wheat

The wheat embryo sac is pear-shaped and deeply imbedded in fleshy nucellus of uneven thickness, which, in mm, is enclosed partially by two layers of integument and is in intimate connection with the procambium around the chalazal region (Text fig. 1 ). This connection seems to be the main inlet passage of nutrient in the ovule. Accordingly the nutrient has to pass from cell to cell and to be incorporated in the nucellus before it is fed to the enlarging embryo sac. Though as a whole the nucellus is transitory in existence, establishment of new peripheral layers and decline of inner layers occur at the same time. While cells in the outer layers multiply by mitosis; cells in the intermediate layers begin to exhibit “nuclear extrusion” (an indication of transcellular movement of protoplasm) which becomes more frequent in inner layers (Text Fig. 2); and cells in the innermost layer, embracing the embryo sac, actively undergo disintegration, showing walls in rupture and cellular contents in disassembly and in retreat (Fig. 7,8). A distinguished feature of high activity of ATPase located in extruding nucleus has been observed in chalazal region (Fig. 4) and in degenerating nucellag cells (Fig. 5). The embryo sac is delimited from the nucellus by an incomplete envelop at the mycopylar end, and the envelop is reinforced by successive deposition of wall debris of the diminished nucellar layers (Fig. 9); whereas at the chalazal end the envelop is lacking and the anfipodals can communicate directly with the disintegrating layer of the thickened portion of nucellus. The antipodals grow steadily as the embryo approaches maturity and the number of cells can be increased 7–8 times(from 3 to 20 or more). Conceivably, proliferation of the antipodals is sustained at the expense of the disintegration of nucellar tissue. The present investigation has confirmed our previous statement that transport of disassembled protoplasm is involved in the feeding of antipodals by nucellus. Some electron micrographs are chosen to reveal details of this particular process. Some findings of special interest are listed below: 1. Cells that make up the nutritional pathway at the chalazal end are small, closely packed, and rich in mitochondria. Its wall is thickened irregularly by heavy deposition of el ectron-translucent material and is interspersed with prominent bundles of plasmodesmata (Fig. 2). It seems likely that the inlet passage is predominantly symplastic in nature. 2. Wi thdrawal of cell contents from the nucellar tissue at the early stage is carried out by efflux of nuclear substance (karyoplasm) through enlarged openings on the nuclear envelop, and by exokaryosis of vesicles packed with ribosome-like granules (Fig. 6). These vesicles can then be trapped in the ER cavity. Breakdown of the endomembrane system follows next. A multitude of small vacuoles, vesicles (coated or not) impregnated with sap, fibrils and granules respectively, deformed mitocondria, chromatic aggregates, etc. can be found in suspension within the deteriorating cell (Fig. 7, 10). In addition, degradation of polysaccharides can also take place. Withdrawal of the cell content from shrinking nucellar layer and its flow into the antipodal section is through the ruptured wall where the cellulose skeleton is turned loose and fluffy at the opening. The protoplasmic fragments in transport are those structures of definite submicroscopic constitution, resulted from disassembly and disinteg ration of the protoplasm and from reassorment of protoplasmic constituents. 3. The antipo dal cells are separated from each other by partial walls riddled with cytoplasmic canals. The naked portion of the ceU can be in direct access to the invading fragments, which can be utilized and incorporated by the antipodal cell and participate in the building of new cell possibly by reorgnization (Fig. 12a) which may be a special mode of cell proliferation in antipodals. Amitosis of antipodal nucleus also has been observed (Text Fig. 3). Discussion is made in regards to the physiological significance of the nutritional supply in form of protoplasmic fragment and of the self-propelling mobility of the fragment. Alth ough the antipodals still proliferate to some extent even after fertilization, they meet the same fate as its predecessor, the nucellus, and soon vanish during the establishment of en dosperm. In food transport, the interconversion of polymer-monomer of saccarides, etc. is fre quently involved. In the present case of material transport, interconversion at higher levels plays a dominant role as shown in the assembly and disassembly of protoplasmic constituents and in the orgnization and disorgnization of ephemeral tissue.     

水稻胚囊壁的形成与发育观察

通过透射电镜对水稻(Oryza sativa L.)功能大孢子形成开始至胚囊成熟期间胚囊壁的形成与发育进行观察,结果表明:水稻胚囊壁是在原有功能大孢子壁的基础上,通过与其周围退化珠心细胞留下的壁相叠合,使壁加厚。功能大孢子近合点端壁存在胞间连丝,其中个别胞间连丝可保留到八核胚囊。胚囊壁上内突最早于四核胚囊近珠孔端发生。八核胚囊形成后,内突的发育在胚囊不同的细胞中表现不同,其中以中央细胞最具特点,表现为先在中央细胞与珠心相接的近珠孔端和近合点端两个区域的胚囊壁上形成,以后近珠孔端胚囊壁上的内突大量增加,而近合点端的却增加不明显,中部胚囊壁上的内突出现的时间相对较晚。到胚囊成熟时,近珠孔端胚囊壁上内突的分布密度最大,中部次之,近合点端的最小,三个区域上内突的形态各异。反足细胞与珠心相接的胚囊壁上内突的形成时间较早,但以后的发育却相对缓慢,数量增加不明显。2个助细胞交界处胚囊壁上的丝状器在胚囊未明显膨大时已形成。卵细胞除在与助细胞交界处的壁外,其它部位不形成明显的内突结构。     

Ovule,Female and Male Gametophyte and Fertilization of Kingdonia uniflora Balfour F. et W. W. Smith

The structure of ovule, female and male gametophyte, double fertilization and the distrubution of starch grains during the fertilization have been studied. The main results are as follows: ( 1 ) Ovule The ovule is anatropous, unitegmic and tenuinucellate. The nucetlus appears cylindric, since megaspores and embryo sac development, its internal cells of nucellus become disorganized, so that only a single layer of epidermal cells remains toward the side of the micropyle, On the other hand, the integument is not as long as nucellus, as a result micropyle is not formed. And no vascular bundle is found in the integument. (2) Female gametophyte The mature embryo sac is slender and is composed of an egg cell, two synergids, a central cell and three antipodal cells. The egg cell is situated slightly away from the tip of embryo sac. Some of them contain starch grains. Synergids occupy the tip of embryo sac. Its wall at micropylar region appears irregular in thickenes and irregular in ingrowths to form the filiform apparatus. The centrateell is very large, and strongly vacuolated Two polar nuclei come to contact closely with each other, but not fuse, or to fuse into a large secondary nucleus before fertilization. The polar nuclei or the secondary nucleus are usually situated at the middle-lower position of the central cell or nearer to the chalazal end above the antipodal cell. It is different from egg cell, no starch grains are found here. In most embryo sacs three antipodal cells are found. They are not as large as those in other plants of Ranunculaceae. But six antipodal cells or the antipodal cell with two nuclei may rarely be found. Like synergid, the wall of them appears not only irregularly thickened, but clearly with irregular ingrowths. In a few antipodal cells the starch garins are usually found near the nucleus. By the end of fertilization, antipodal cells become disintegrated. (3) Male gametophyte Most pollen grains are two-celled when shedding, and rich in starch grains. A few of them contain single nucleus or three-celled. (4) The double fertilization The fertilization of Kingdonia unifiora Balfour f. et W, W. Smith is wholly similar to some plants of Ranunculaceae studied. First, the pollen tube penetrates a degenerating synergid. And the pollen tube discharges its contents with two sperm nuclei into the degenerating synergid cell. One of the two sperms fuses with the nucleus of the egg, and the other fuses with two polar nuclei or the secondary nucleus of the central cell. If one sperm nucleus at first fuses with one of the polar nuclei, and then the fertilized polar nuclei again fuses with other polar nucleus. Secondly, the fertilization of the polar nuclei or the secondary nuclei completes earlier than that of the egg. The primary endosperm nucleus begins to divide earlier than the zygote. It seems that one of the sperm nuclei come to contact with egg nucleus, the other has already fused with polar nuclei or the secondary nucleus. The zygote with a single nucleolus appears until the endosperm with 16–20 cell. Thirdly, before and after fertilization there are one to some small nucleoli in egg nucleus and polar nuclei or secondary nucleus. However they increase in quantity from the beginning of the fusion of male nucleis. These nucleoli quite differ from male nucleoli by their small size, and most of them disappear at the end of fertilization. It may be concluded that the small nucleoli increase in quantity is related to the fusion of male and female nuclei. In the duration of fertilization, in ovule starch distribution is in the basal region of integument. But in embryo sac, onlysome egg cells, or zygotes contain starch grains, a part of which was brought in by pollen tube. Sometimes the starch grains are found in some synergids and antipodal cells. No starch grains are found in the central cell.     

Inorganic salts modify embryo sac development in sexual and aposporous Cenchrus ciliaris

Summary Sexual and aposporously apomictic plants of buffelgrass (Cenchrus ciliaris L.) form megaspore tetrads. In sexual plants the chalazal megaspore develops into a single Polygonum type embryo sac. In aposporous plants the megaspores degenerate, and one or more un-reduced nucellar cells form Panicum type embryo sacs. Apospory is conditioned by gene A; the dominant allele of gene B is epistatic to A and preserves sexual reproduction. We recently observed that heavy application of (NH₄)₂SO₄ to the soil induced multiple embryo sacs in a sexual line. Therefore we tested the effect of salt stress on embryo sac formation in sexual and aposporous genotypes. One molar solutions of CaCl₂, NaCl, (NH₄)₂SO₄, NH₄Cl, NaNO₃, or Na₂SO₄ were applied to the soil of greenhouse plants every day or two starting at the archespore stage. Some of the pistils in salt-treated plants of sexual genotypes AaBb, aaBb, and aabb showed features not seen in untreated controls: (1) multiple Polygonum type embryo sacs in 1%–7% of pistils depending upon the salt; (2) embryo sacs without antipodals (0%–7%); (3) embryo sacs protruding through the micropyle (1%–16%). Some pistils of salt-treated obligately aposporous lines, but not controls, developed Polygonum type embryo sacs (4%–13%) and protruding embryo sacs (0%–6%). There was no ion specificity for induction of abnormal features. We postulate that salt stress suppresses the developmental priority of nucellar embryo sacs over megaspores in aposporous lines and of the chalazal megaspore over other megaspores in all lines. This may permit megaspores of aposporous plants to form reduced Polygonum type gametophytes, and permit more than one megaspore to form reduced embryo sacs in all lines. Protrusion of sacs and failure of antipodal formation in reduced embryo sacs may be the consequence of uncoordinated expansion of the embryo sacs and surrounding tissue.Joint contribution of the Department of Biology, The Pennsylvania State University, and USDA-ARS, U.S. Regional Pasture Research Laboratory. Names of products are included for the benefit of the reader and do not imply endorsement or preferential treatment by USDA     

The Embryology of Nyssa sinensis Oliv. (Nyssaceae)

The flower develops in March and blossoms in early May in Nanjing. The cytokinesis of microsporocytes is simultaneous and most tetrads are tetrahedral. The tapetum is secretory and the nuclei become polyploid at last. The style is solid and most ovaries are unilocular, rarely bilocular. The ovule is pendulous, anatropous and unitegmic. The nucellus is pseudocrassinucellate. An obturator formed by transmitting tissue covers the micropyle. The raphe vascular strand extends into the integument when it reaches the chalaza and on a whole keeps a “U” shape. The endothelium cell is uninucleate. In most cases no nucellar cap is formed. No hypostase is found below the embryo sac. The archesporium is one-celled. The embryo sac development conforms to the Polygonum or Allium types. The degeneration of the megaspores in the linear tetrad usually occurs from the chalazal toward the micropylar end. Two synergids persist during fertilization. Three antipodal cells are uninucleate and ephemeral. Two polar nuclei fuse at the time of fertilization. The fertilization type accords with porogamy. The syngamy is premitotic. The development of endosperm is cellular. The initial four successive divisions of the primary endosperm cell are transverse-verticaltransverse-transverse subsequently, giving rise to sixteen cells of the early endosperm. The mature embryo is straight and nearly as long as the endospermous seed. The cotyledons are more or less cordate at base. The seedoat is thin and composed of 5-11 layers of compressed cells. Neither embryo nor endosperm contain the alkaloid camptothecine. The major similarities of Nyssa sinensis to the American nyssas in embryology, which may be a counted as the generic features, are the polyploid tapetum cells, the unitegmic ovule with U-shaped vascular strand, the direct enlargement of the archesporial cell to produce the megasporocyte, the pseudocrassinucellus, the usual absence of the nucellar cap, the Polygonum or Allium type of the embryo sac development, the first degeneration of the metachalazal megaspore, the ephemeral antipodal cells, a single nucleolus in the nucleus ofthe primary endosperm cell, the more or less cordate base of the cotyledons.     

Ultrastructural characterization of apospory in Panicum maximum

The nucellar ultrastructure of apomictic Panicum maximum was analyzed during the meiocytic stage and during aposporous embryo sac formation. At pachytene the megameiocyte shows a random cell organelle distribution and sometimes only an incomplete micropylar callose wall. The chalazal nucellar cells are meristematic until the tetrad stage. They can turn into initial cells of aposporous embryo sacs. The aposporous initials can be recognized by their increased cell size, large nucleus, and the presence of many vesicles. The cell wall is thin with few plasmodesmata. If only a sexual embryo sac is formed, the nucellar cells retain their meristematic character. The aposporous initial cell is somewhat comparable to a vacuolated functional megaspore. It shows large vacuoles around the central nucleus and is surrounded by a thick cell wall without plasmodesmata. In the mature aposporous embryo sac the structure of the cells of the egg apparatus is similar to each other. In the chalazal part of the egg apparatus the cell walls are thin and do not hamper the transfer of sperm cells. Structural and functional aspects of nucellar cell differentiation and aposporous and sexual embryo sac development are discussed.     

Fertilization and Histochemical Investigation on Pulsatilla chinensis (Bung) Regel

Fertilization and variation of protein and starch grains in Pulsatilla chinensis (Bung) Regel have been studied at light microscopic level with histochemical test. Based upon the observations, the main conclusions are summarized as follows: The mature pollen grains are two-celled in which the generative cell shows the stronger protein staining than the vegetative cell. And vegetative cells are full of starch garins. When the pollen tube enters into the embryo sac, one synergid is destroyed, or in a few cases synergids are intact. Occasionally two synergids are disorganized as pollen tube penetrates. However, most of the remaining syuergids break down during fertilization, only in a few cases it remains till early stage of embryo development. The contents discharged by the pollen tube consist of two sperms, which stain intensely blue with protein dyes, a great amount of protein and starch grains. Mature female gametophyte (embryo sac) consists of an egg apparatus, central cell, which has a huge secondary nucleus, and antipodal apparatus which retain in course of fertilization. A few of embryo sac contain two sets of egg apparatus, a central cell with two huge secondary nuclei and two sets of antipodal apparatus. In some nucleoli of the central cell the comb-like structure pattern may be detected clearly. There are 1–2 small nucleoli in some egg cells and central cells. All the cells in embryo sac show protein positive reaction. According to the different shades of the color in cells, its may be arranged in the following order: antipodal cells, synergids, central cell and egg cell. Only a few small starch grains are present near nuclei of central cell and egg cell before fertilization, but no starch grains remain in most of the central cell, the synergids and antipodal cells. The fertilization is of the premitotic type. The fusion of the sexual nuclei progresses in the following order: 1, sperms approach and lie on the egg nucleus and secondary nucleus; 2, sperm chromatin sinks themselves into female nucleus, and male nucleolus emerges with the sperm chromosome; and 3, male nucleoli fuse with the nucleoli of egg nucleus and central cell nucleus, and finally forming the zygote and the primary endosperm cells respectively. Nevertheless, as it is well known, the fertilization completes in central cell obviously earlier than that in egg cell. Though it has been explained in cereals and cotton, in Pulsatilla chinensis the main reason is that nucleolar fusion of the male and female nucleoli in egg nucleus is slower than that in secondary nucleus. And the dormancy of the primary endosperm nucleus is shorter than that of the zygote. In the process of fertilization, histochemical changes are considerably obvious in the following three parts: 1, from the begining of fusion of male and female nuclei to form zygote and primary endosperm cell, Protein staining around female nucleus appears to increase gradually; 2, no starch grains are detected in embryo sac. Though only starch grains are carried in by pollen tube, they are completely exhausted during this period; and 3, near completion of fertilization starch grains appear again in zygote, however, not yet in primary endosperm nucleus till its dividing for the first time. The present study reveals that antipodal cells and synergids seem to play a significant role in nutrition of the embryo sac during the fertilization.