HISTOCHEMISTRY AND ULTRASTRUCTURE OF RICE (ORYZA SATIVA) ZYGOTIC EMBRYOGENESIS

Rice embryo development was examined, histochemically and ultrastructurally, from the time of fertilization to embryo maturity. At the time of fertilization, the megagametophyte consists of an antipodal mass of 10–15 cells, parietally positioned along the placental side of the central cell, and, at the micropylar end, two partly fused polar nuclei and the egg apparatus. Hydrolysis of adjacent nucellar tissue suggests the secretion of hydrolytic enzymes by the antipodal mass. The antipodal cells stain intensely for RNA and protein, indicating that they are metabolically active. The egg, supported by two overarching synergids, occupies a small, wall ingrowth-lined pocket of the central cell that quickly fills with cellular endosperm after fertilization. The endosperm cells, initially supplied with nutrients from wall ingrowth-derived vesicles, are digested and utilized by the embryo as a nutritive source. The developing embryo is also supplied with assimilates via the nucellus at the base of the embryo until about 8 days after fertilization. After 8 days, the embryo is no longer connected to the nucellus, and the nucellar cells at the base of the embryo are crushed. The zygote is not structurally polarized and contains a central nucleus, amyloplasts, lipid bodies, dictyosomes and extensive dilated ER. The first division of the zygote is transverse and unequal and occurs about 4 hours after fertilization. Embryo development is rapid, and within 24 hr, the embryo consists of 5–8 cells. Organ development begins with scutellum emergence in the 3-day-old embryo. The shoot apex organizes and the coleoptile develops from scutellum tissue at 4 days postfertilization, the epiblast emerges at 5 days, and the vascular bundle and root apex differentiate by 6 days after fertilization. Starch begins to accumulate in the basal cells of the 3-day-old embryo and deposition proceeds acropetally over the next 9–10 days. Lipid accumulation begins in the basal scutellum in the 6-day-old embryo and also proceeds acropetally. Storage protein synthesis is first detected in 6-day-old embryos and accumulation again proceeds acropetally, reaching the apex of the scutellum of the 25-day-old embryo. The ultrastructure of the 24-hr-old embryo is distinctive. The cells are characterized by numerous vesicles, heterochromatin and extensive nuclear evaginations.     

ORCHID EMBRYOLOGY: MEGAGAMETOPHYTE OF EPIDENDRUM SCUTELLA FOLLOWING FERTILIZATION

The megagametophyte of Epidendrum scutella, an orchid, was examined with the electron microscope after the entrance and discharge of the pollen tube. The pollen tube enters the embryo sac by growing through the filiform apparatus of a synergid and discharges through a terminal pore into the degenerating cytoplasm of the synergid. The synergid nucleus appears pushed to one side by the discharge of the pollen tube. What is believed to be the remains of the vegetative nucleus has been found in the degenerate synergid, but no trace of the sperm cytoplasm has been seen. The zygote is approximately the same size as the egg. The ribosomes become grouped into polysomes. Both the egg and the zygote apparently completely lack dictyosomes. The polar nuclei partially fuse before fertilization, but fusion of the sperm nucleus with the polar nuclei does not occur and no endosperm is produced. Polysome formation occurs in the central cell and large amounts of tubular, smooth ER are seen. The antipodals remain following fertilization, undergoing ultrastructural changes similar to the central cell.     

星星草受精作用及其胚与胚乳早期发育的观察

利用常规石蜡切片法对星星草[Puccinellia tenuiflora(Griseb．)Scribn．et Merr．]受精过程及胚与胚乳的早期发育进行了观察,主要结论如下：(1)开花后2h,花粉管破坏1个助细胞,释放2个精子,精子呈逗点状。(2)开花后2～3h,2个精子分别移向卵细胞与极核。(3)开花后3～5h,精核分别贴附于卵细胞与极核的核膜上。(4)开花后5～10h,精核与卵核融合,雄性核仁出现,合子形成。(5)开花后5～6h,精核与极核融合,并出现雄性核仁,形成初生胚乳核,精核与极核的融合比与卵核融合要快。(6)开花后20h左右,合子分裂。(7)开花后8h,初生胚乳核。     

羊草受精作用及其胚与胚乳早期发育的观察

利用常规石蜡制片方法研究了羊草受精过程及胚与胚乳的早期发育,其主要结果为：(1)授粉后1h,花粉管破坏1助细胞,释放2精子。精子为眼眉状,难以区分其细胞质鞘;(2)授粉后1～2h,2个精子分别移向卵细胞与极核;(3)授粉后2～3h,精核分别贴附于卵细胞与极核核膜上;(4)授粉后3～10h,精核与卵核融合,并出现雄性核仁,形成合子;(5)授粉后3～4h,精核与极核融合,并出现雄性核仁,形成初生胚乳核,精核与极核的融合比与卵核融合快;(6)传粉后20h,合子分裂,合子的休眠期为10h左右;(7)传粉4h,初生胚乳核分裂,初生胚乳核没有休眠期;(8)羊草双受精作用属于有丝分裂前配子融合类型;(9)胚胎发育属于紫菀型,胚乳发育属于核型胚乳。     

Observations on Fertilization in Sugar Beet

The whole process of double fertilization in sugar beet has been observed, the main results are as follows: About 2 hours after pollination, the pollen grains germinate, the sperms in the pollen tube are long-oval. 15 hours after pollination, the pollen tube destroys a synergid and releases two sperms on one side or at the chalazal end of the egg cell. The sperms are spherical each having a cytoplasmic sheath. 17 hours after pollination, one sperm enters the egg cell, and the sperm nucleus fuses with the egg nucleus rapidly. 21 hours after pollination, the zygote is formed. In the meantime, the primary endosperm nucleus has divided into two free endosperm nuclei. 25 hours after pollination, the zygote begins to divide, forming a two-celled proembryo. The dormancy stage of the zygote is about 4 hours. In the meantime the endosperm is at the stage of four free nuclei. 17 hours after pollination, the sperm nucleus comes into contact and fuses with the secondary nucleus. The sperm nucleus fuses with the secondary nucleus, faster than the sperm with the egg. he first division of the primary endosperm nucleus is earlier than that of the zygote, it takes place about 20 hours after pollination, the dormancy stage of the primary endosperm is about 2 hours. The endosperm is free nuclear. The fertilization of sugar beet belongs to premitotic type of syngamy. From the stage of zygote to the two-celled proembryo, it can be seen that addition- al sperms enter the embryo sac, but polyspermy has not been observed yet.     

竹节参雌配子体发育的研究

本文报道了竹节参(Panax japonicus C.A.Mey)雌配子体(胚囊)的发育过程。竹节参大孢子母细胞减数分裂产生线形排列的大孢子四分体。胚囊发育属蓼型,由合点端大孢子发育而成。游离核胚囊时期,胚囊珠孔端的细胞器种类和数量都较胚囊合点端多;胚囊合点端相邻的珠被细胞中有含淀粉粒的小质体,与胚囊珠孔端相邻的退化中的非功能大孢子中则有含淀粉粒的大质体和大类脂体。成熟胚囊中,反足细胞较早退化;极核融合成次生核;卵细胞高度液泡化,细胞器数量较少;助细胞则有丰富的细胞器和发达的丝状器。PAS反应表明,受精前的成熟胚囊中积累淀粉粒。次生核受精后,很快分裂产生胚乳游离核,到几十至数百个核时形成胚乳细胞。卵细胞受精后则要经过较长的休眠期。     

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     

向日葵胚囊的超微结构和雌性生殖单位问题

本文对向日葵胚囊中卵细胞、助细胞与中央细胞开花前和传粉后的超微结构变化进行了研究。着重报道了不同发育时期这三种细胞之间特定区域的界壁的消长动态。在此基础上结合现有文献资料探讨了由三者共同组成“雌性生殖单位”以适应受精作用的问题。     

ULTRACYTOCHEMICAL LOCALIZATION OF PLASMA MEMBRANE-ASSOCIATED PHOSPHATASE ACTIVITY IN DEVELOPING TOBACCO SEEDS

Plasma membrane-associated phosphatase activity was found in integumentary cells of developing tobacco ovules from the megaspore tetrad stage to seed maturity. Enzyme activity is greatest in the innermost layers of the integument from the mature megagametophyte stage on. The egg, zygote, and synergids almost totally lack plasma membrane-associated reaction product, while the antipodals show some activity at their chalazal ends. The endosperm has much plasma membrane-associated phosphatase activity in most of its cells during development, but it is primarily the outermost plasma membranes of the surface cells of the embryo that have associated reaction product. It is concluded that the plasma membrane-associated phosphatase activity is related to active transport of assimilates and that the integument is the most important site of active transport in the young ovule. After fertilization, in addition to the innermost layers of the integument, the endosperm and the outermost cells of the embryo become involved in active transport, which continues to seed maturity.     

水稻胚囊超微结构的研究

水稻(Oryza sativa L.)胚囊成熟时,卵细胞的合点端无细胞壁,核居细胞中部,细胞器集中在核周围,液泡分散于细胞周边区域。助细胞珠孔端有丝状器,合点端无壁,核位于细胞中部贴壁处,细胞器主要分布在珠孔端,液泡主要分布在合点端。开花前不久,一个助细胞退化。中央细胞为大液泡所占,两个极核靠近卵器而部分融合,细胞器集中在极核周围和靠近卵器处,与珠心相接的胚囊壁上有发达的内突。反足细胞多个形成群体,其增殖主要依靠无丝分裂与壁的自由生长,反足细胞含丰富活跃的细胞器,与珠心相接的壁上有发达的内突。开花后6小时双受精已完成,合子和两个助细胞合点端均形成完整壁。合子中开始形成多聚核糖体、液泡减小。退化助细胞含花粉管释放的物质,其合点端迴抱合子。极核已分裂成数个胚乳游离核,中央细胞中细胞器呈活化状态。反足细胞仍在继续增殖。讨论了卵细胞的极性、助细胞的退化、卵器与中央细胞间界壁的变化、反足细胞的分裂特点等问题。     

Microtubules in early development of the megagametophyte of <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Food storage tissue in the seeds of gymnosperms is female gametophyte (megagametophyte) that develops before fertilization, whereas, in seeds of angiosperms, food is stored as endosperm initiated by double fertilization. The megagametophyte is haploid, and endosperm is usually triploid, at least initially. Despite differences in origin, ploidy level, and developmental trigger, the early events of female gametophyte development in ginkgo are very similar to nuclear endosperm development in the seeds of angiosperms. In both, development begins as a single cell that undergoes multiple mitoses without cytokinesis, to produce a large syncytium. This study provided evidence that microtubule involvement in organization of the syncytium into nuclear cytoplasmic domains (NCDs) via nuclear-based radial microtubule systems is a critical developmental feature in the ginkgo megagametophyte, as it is in endosperm. Once the initial anticlinal walls have been deposited at the boundaries of NCDs, cellularization proceeds by the process of alveolation. Continued unidirectional growth of the alveolar walls is an outstanding example of polar cytokinesis. Ginkgo megagametophyte development appears to occur uniformly throughout the entire chamber, whereas nuclear type endosperm usually exhibits distinct developmental domains. These observations suggest that there is a fundamental pathway for the development and cellularization of syncytia in seed development.     

Gamete membrane dynamics during double fertilization in Arabidopsis

In the double fertilization of angiosperms, one sperm cell fertilizes an egg cell to produce a zygote, whereas the other sperm cell fertilizes a central cell to give rise to an endosperm. There is little information on gamete membrane dynamics during double fertilization even though the cell surface structure is critical for male and female gamete interactions. In a recent study, we analyzed gamete membrane behavior during double fertilization by live-cell imaging with Arabidopsis gamete membrane marker lines. We observed that the sperm membrane signals occasionally remained at the boundary of the female gametes after gamete fusion. In addition, sperm membrane signals entering the fertilized female gametes were detected. These findings suggested that plasma membrane fusion between male and female gametes occurred with the sperm internal membrane components entering the female gametes, and this was followed by plasmogamy.     

FINE STRUCTURE AND COMPOSITION OF THE EGG APPARATUS BEFORE AND AFTER FERTILIZATION IN QUERCUS GAMBELII: THE FUNCTIONAL OVULE

A study of the egg apparatus of Quercus gambelii was made at both the light and the electron microscope levels. This investigation was concerned primarily with the changes that occur in these cells before and after the process of fertilization and what role, if any, is played by the synergids in this phenomenon. The synergids before fertilization are, on the basis of ultrastructure, healthy, intact, functional cells. They have numerous mitochondria, dictyosomes, endoplasmic reticulum, ribosomes, and a typical nucleus. A prominent filiform apparatus is present, but the cell wall only extends a short distance around the micropylar end of the cells. Just before fertilization, one of the synergids degenerates. This is the synergid that receives the pollen tube and its discharge, including both male gametes. Dictyosomes increase in number and activity in the other synergid (persistent synergid) after fertilization. Eventually a complete cell wall forms around both of the synergids. No plasmodesmata are present in these walls. The egg has numerous mitochondria, dictyosomes, endoplasmic reticulum, and ribosomes, both free in the cytoplasm and attached to the endoplasmic reticulum. Lipid bodies are characteristic of this cell. A cell wall is present only around the micropylar end of the egg. After fertilization, little change occurs in the zygote. The number and activity of the dictyosomes increase, apparently in correlation with cell wall formation. The number of lipid bodies increases. The zygote is approximately the same size as the egg. Plastids are scarce, and starch grains are typically absent from all cells of the egg apparatus. It is suggested that the synergids function in the secretion of chemotropic substances that guide the growth of the pollen tube. Comparisons are made between the egg apparatus of Quercus gambelii and that of the other plants studied thus far.     

Cytological Studies of the Double Fertilization in Rice

Detailed studies on the process of double fertilization in rice were conducted in the present work. The results are summarized as follows: 1. In the embryosac 30 minutes after anthesis, the pollen tube has already reached the micropyle in every specimen. In some cases, it has even entered further into the embryosac and discharged its contents, including the two male gametes. 2. 1½ hours after anthesis, the male gamete enters into the egg cell. As soon as it comes in contact with the egg nucleus, it increases in size. 2 hours after anthesis, the male nucleus is found inside the female one. A male nucleolus is now clearly discernible. 3. The male nucleolus is gradually growing until it reaches the size of the female one, and then the fusion of the two takes place. The fusion is generally completed and the zygote is formed 7 hours after anthesis. 4. The first mitotic division of the zygote occurs 9 hours after anthesis. 5. The fusion of the male gamete and the polar nucleus proceeds in a similar way as that of the male and female gametes, but it takes a much shorter time usually being completed within 3 hours after anthesis. 6. The male gamete enters into one of the polar nuclei and reveals its nucleolus which increases rapidly in size and then unites with that of the polar nucleus. As soon as the union is completed, the nuclear membrane between the closely contacted parts of the two polar nuclei disappears and the primary endosperm nucleus is formed. 7. The first mitotic division of the primary endosperm nucleus begins right after its formation. 8. With the fusion of the male and female gametes and the development of the zygote, the mitochondria in the cytoplasm surrounding the nucleus increase in size and number. However, in the central cytoplasm about the polar nuclei they show no notice- able change during the fertilization process. 9. Based on the facts that in the embryosac a secondary pollen tube is often seen in every stage of the fertilization process and that additional nucleoli are also observed sometimes in the egg nucleus, the authors believe that polyspermy most probably exists in rice plants, and that this may be one of the causes of polyploid plants often found in rice field as reported by several authors.     

The Ultrastructural Study on the Early Embryonic Development of Radish

The ultrastructure of the mature embryo sac, the early stages of the embryo and endosperm development of common radish, Raphanur sativus was examined. The embryo sac consists of 7 cells with antipodal ceils disappeared when it matures. The egg cell is highly polarized. The wall surrounded the chalazal end of the egg cell is incomplete, showing a discontinuous structure of an electron dense material deposited intermittently in the space between the two plasma membranes of the egg cell and central cell. The synergid has filiform apparatus, rich in organelles and well developed ER. The two polar nuclei of the central cell are located near the egg apparatus because of the big vacuole, and the finger-like protrutions from the cell wall, as that in synergid, are found. The first division of the zygote occurs 4–5 days after pollination and the development of the embryo follows the Onagrad type, and the structure of the embryo cell is quite simple for containing small quantity of ER, plastids and other organelles. The primary endosperm nucleus deviates 2 days earlier than zygote. The endosperm is of nuclear-endosperm containing chloroplasts, well developed ER, and plentiful of mitochondria and golgi bodies and the nodule-like aggregation in both. the chalazal and micropylar ends of the embryo sac during the early development appeared, and cell wall starting at the micropylar end by freely-growing forms about 16 days after pollination.     

THE ULTRASTRUCTURE AND HISTOCHEMISTRY OF THE SYNERGIDS OF COTTON

The composition and ultrastructure of the synergids of cotton were studied. The cells were found to be surrounded by a partial wall composed of cellulose, hemicellulose, and pectins. The structure of the wall was observed to consist of an unusual fibrillar arrangement. The filiform apparatus was demonstrated to be an extension of the wall at the micropylar end of the cell. Large amounts of ER surround the filiform apparatus. Also associated with the latter are large numbers of plastids and mitochondria. The nucleus is large and contains a single, large nucleolus and, frequently, 1 or more micronucleoli. The nuclear membrane contains membrane-bound vesicles but has few extensions into the cytoplasm. The ER is oriented parallel to the long axis of the cell and decreases in concentration from the micropylar to the chalazal end of the cell. Dictyosomes are common throughout the cell but are more numerous in the midportion where they are closely associated with the ER. The chalazal end of the cell is occupied by vacuoles rich in an inorganic compound which leaves a considerable residue of ash. Spherosome-like bodies are common throughout the cell. Both the plastids and mitochondria show evidence of division. Ribosomes are numerous and are both free and associated with the ER, nucleus, plastids, and mitochondria. The function of the synergids is proposed to be the absorption, storage, and transport of compounds from the nucellus. On the basis of this function, it is suggested that the synergids act by providing material to the egg and the developing embryo and endosperm and that they are involved in the growth of the pollen tube into the embryo sac.     

Transmission of male cytoplasm during fertilization in Nicotiana tabacum

Serially sectioned embryo sacs of Nicotiana tabacum were examined during fertilization events using transmission electron microscopy. After pollen tube discharge, the outer membrane of the sperm pair is removed, the two sperm cells are deposited in the degenerate synergid and the sperm cells migrate to the chalazal edge of the synergid where gametic fusion occurs. During fertilization, the male cytoplasm, including heritable organelles, is transmitted into the female reproductive cells as shown by: (1) the cytoplasmic confluence of one sperm and the central cell during cellular fusion, (2) the occurrence of sperm mitochondria (distinguished by ultrastructural differences) in the zygote cytoplasm and adjacent to the sperm nucleus, (3) the presence of darkly stained aggregates which are found exclusively in mature sperm cells within the cytoplasm of both female cells soon after cell fusion, and (4) the absence of any large enucleated cytoplasmic bodies containing recognizable organelles outside the zygote or endosperm cells. The infrequent occurrence of plastids in the sperm and the transmission of sperm cytoplasm into the egg during double fertilization provide the cytological basis for occasional biparental plastid inheritance as reported previously in tobacco. Although sperm mitochondria are transmitted into the egg/zygote, their inheritance has not been detected genetically. In one abnormal embryo sac, a pair of sperm cells was released into the cytoplasm of the presumptive zygote. Although pollen tube discharge usually removes the inner pollen-tube plasma membrane containing the two sperm cells, this did not occur in this case. When sperm cells are deposited in a degenerating synergid or outside of a cell, this outer membrane is removed, as it apparently is for fertilization.     

天竺葵雌性生殖单位的超微结构

应用透射电镜研究了临近受精时天竺葵(Pelargonium hortorum Bailey)胚囊中的卵细胞、助细胞和中央细胞的结构。证明了卵细胞与助细胞以及助细胞与助细胞之间从合点端至珠孔端有很大的面积以质膜分界,仅珠孔端少部分以壁分隔。卵细胞与中央细胞之间同样缺乏细胞壁。在卵细胞的合点端,两质膜不同程度地分离形成宽窄相间的间隙。在间隙的絮状基质中存在小泡,这些小泡的产生似与卵和中央细胞中周质内质网的活动有关。推测小泡为多糖性质,可能为合子新壁的建造提供物质。卵细胞质中含巨大线粒体,质体和内质网也较丰富。基于超微结构的特征,可认为卵细胞具高度的生理合成活动的潜能。中央细胞极核位于珠孔端与卵器细胞毗邻,有利于在双受精作用中同时发生精细胞与卵细胞和精细胞与中央细胞核的融合。中央细胞的侧壁在珠孔端形成内突,具传递细胞的特点,表明这是雌配子体向孢子体摄取营养的重要部位。助细胞的细胞质含丰富的细胞器,这与多数植物中的相似,但具几个明显的特征,即核中存在微核仁,内质网形成圆球体或脂体,线粒体富集在丝状器的附近。传粉后花粉管进入胚囊之前,两个助细胞中一个退化。