A Contribution to Pollination and Fertilization of Amentotaxus argotaenta

The present investigation was conducted during 1980–1982, and mater- ials collected from Jin-Fo shan (Golden Buddha Mountain), at a height of 1400-1600 m, Sichuan province, China. Pollination of Amentotaxus argotaenia began to proceed last week of May, and came into bloom the first week of June. The male strobiles were almost entirely wilting at June 12–15. Thus, florescence of Amentotaxus spread over a period of 3 weeks. While the pollen grains approaching to maturity, most of the microspores divide to form a larger tube cell and a smaller antheridial initial. In this case the mature pollen grains of Amentotaxus consist of two cells. Then pollen grains are attracted down into the pollen chamber in the apex of the nucellus after pollination. The pollen chamber of Amentotaxus in longitudinal section looks like a flask in shape and is very much similar to that of Ginkgo biloba. As pollen grains at pollen chamber begin to germinate, the antheridial initials divide again to give rise to a spermatogenous cell and a sterile cell. At first, the spermatogenous cell is of a size only 11–13 μ in diameter. When the pollen tube reaches the middle part of the nucellus, the spermatogeneous cell is of a size about 30 μ. In the middle of July, pollen tube approaches the top of the female gametophyte. In this time, the spermatogenous cell has already been mature enough and is of 58–85 μ in diameter. The nuclei of spermatogenous ceils, 30–36 μ in size, are usually lying in the lateral side of the cytoplasm at its micropylar end. From the middle to the end of July, spermatogenous cells divide to form two unequal sperms, one of which is larger than the other and is the functional one. The large sperm is almost round in shape and about 56 μ in diameter. The small sperm is elliptic in shape, non-functional, and about 33 μ in diameter. The nuclei of the large and small sperms are about 40 μ and 26 μ, respectively. In some cases there are lateral pollen tube and sperms in the ovules of Amentotaxus, or the pollen tube even grows toward the lower part of female gametophyte in the chalazal end and there are well developed sperms in such a case. In the middle of July, nucleus of the central cell divides to form a ventral canal nucleus and an egg nucleus. The former then breaks down quickly and the latter continues to develope and moves toward the central part of the egg cell gradually. It is interesting to note that there are a number of nucleolus-like grains in the cytoplasm of the egg cell in Amentotaxus. The large nueleolus-like grains contain a larger central vacuole with several smaller vacuoles surrounding it. These grains show a positive reaction and blue colour by PAS and aniline blue black or coomassie brilliant blue, respectively. The above facts show that the nucleolus-like grains contain not only po- lysaccharides, but also protein. Similar grains may also found in the developing pollen tube. This is a unique feature in Amentotaxus and even in Gymnosperms. Otherwise, there are often two groups of the dense cytoplasm under the egg nucleus in Amentotaxus. Fertilization of Amentotaxus took place around July 20–29 (1980–1982). Interval between pollination and fertilization was about two months. After male nucleus fuses entirely with the female nucleus, the zygote begins to divide by mitosis. During fertilization, in addition that the large sperm enters the egg cell and fuses with the egg nucleus, the small sperm, tube nucleus, and sterile cell are often delivered into the egg cell. But they are disintegrated gradual]y and eventually. It is worthy to note that the nucleolus-like grains and the starches in pollen tube are also released into the egg cell. Then enlargement, fusion, and budding in the nucleolus-like grains may be found within the cytoplasm of the egg cell after fertilization. The history of investigating Amentotaxus found in 1883 has been lasting a long period of 100 years. But researches in sex production has never been studied before. The present work has shown that fertilization in Araentotaxus is very much similar to that in Taxus, Pseudotaxus, and Torreya. In other words, they all belong to the same type, that is, mitosis of zygote taking place after fusion of the two sexual nuclei. This condition constitutes one of the features of Taxaceae. But fertilization in Cephalotaxaceae is different from that of Taxaceae in having mitosis taking place before fusion of the two sexual nuclei. Pollination of Amentotaxus is similar to that of Cephalotaxus with dual-cell pollen grains at shedding stage. On the other hand, interval between pollination and fertilization in Austrotaxus lasts for 13.5 months, and this is the longest one in Taxaceae, and it is similar to that of Cephalotaxus proceeding for 14 months. To sum up, from the point of view of pollination, fertilization, and embryogenesis, Amentotaxus could be considered a primitive type in Taxaceae. Perhaps an order of systematic position of the genera belonging to Taxaceae can be arranged thus: Amentotaxus, Austrotaxus, Taxus, Pseudotaxus, and Torreya. And Cephalotaxaceae may be related to Taxaceae by way of Amentotaxus.     

Studies in Fertilization of Fokienia

Material of Fokienia hodginsii was collected in 1964 from Fengyangshan (alt. 1000–1400 M) in Lungchuan county, Chekiang province. This paper deals with the fertilization in Fokienia. It includs the structure of male and female gametes as wed1 as the process of fusion of their nuclei and cytoplasm respectively. The division of the spermatogenous cell of Fokienia occurred by the end of June (1964) and two sperms similar in shape and size were formed when pollen tube reached the top of archegonia. Two equalsperms look like two hemispherical bodies conjoined togather. The sperm possesses cell wall and is about 65 μ in diameter. Its nucleus is rather large and about 45–50 μ in diameter. There is a nucleolus in the nucleus. Outside the nucleus the dense cytoplasm forms the deep colored zone, some 10 μ in thickness. This zone is separated from the nucleus by a narrow perinuclear zone, and from the plasmalemma by a marginal zone. The perinuclear zone is about 2 μ thick, and the mariginal zone is from 3 to 4 μ thick. Both zones have transparent cytoplasm. When the archegonium is formed, the central cell has a small nucleus which is located below the neck ceils. At the middle of June (1964), the central cell divides to form the ventral nucleus and the egg nucleus. The egg nucleus sites primarily at the upper part of archegoninm and has only one nucleolus. Then the egg nucleus increases gradually in sim and moves to the central part of the archegoninm. In mature archegonium there are usually 4–5, rarely 6–7 nucleoli in the egg nucleus, each of them is about 15 μ in diameter. The egg cell in Fokienia hodginsii is about 500 in length. The female nucleus is larger than the male one. After egg cell matures, its cytoplasm increases gradually, while the central vacuole decreases gradually and almost disappears completely after fertilization. It is interesting to note that there are 1–2 dense cytoplasm masses at the upper or lower part of egg nucleus. The shape of the mass is similar to that of the egg nucleus but no membrane is formed. These cytoplasm masses are about 50–70 μ in diameter in some cases. The fertilization of Fokienia took place at the end of June when the growing tip of pollen tube had reached the top of the archegoninm. Then the neck cells become disorganized and degenerated. It is possible that all the cytoplasmic contents of pollen tubes are released into the archegoninm. Before fertilization, the cytoplasm around the sperms and sterile cell and tube nucleus are in front of these two sperms. Then the sperms separate from each other and come down into the cytoplasm of the egg. When the mede nucleus contacts with the egg nucleus, both become flattened along their contact surface. Then the nuclear membranes of both sperm and egg nuclei become ultimately disintegrated. Thus the fusion process is complete. However, it is nia, though the opposite is the case in an exceptional example. When the sperm nucleus passes into the cytoplasm of egg cell, its cytopasm is released inside the archegonium along with it. During the course of fusion of the male and female nuclei, tile fertilized nucleus is surrounded by both female and male cytoplasm. Thus the male cytoplasm along with the peripheral cytoplasm of the egg cell invests the two nuclei lying in contact and forms a dense neocytoplasm. When the zygote divides, the neoeytoplasm is full of the starch grains and a dense cytoplasm sheath is formed. After fertilization, the fused nucleus moves toward the base of the egg cell. It seems that the movement of the fused nucleus is not a simple mechanical movement but turned over repeatedly toward the base of the arehegonium. Sometimes the position of the sperm and egg nuclei makes a turn of 180. At the same time the track of the fertilized egg nucleus with vacuoles in the archegonium may be traced. After zygote moves into basal part of the archegonium, first intranuclear mitosis occurs. The nuclear envelop of zygote disappears gradually at the telophase of the first mitosis. Then division of the free nuclei of proembryo follows. From fertilization to the stage of proembryo formation, the second sperm may sometimes enter into the cytoplasm of the egg cell. Mitosis of the second sperm nucleus may take place in the upper part of the archegonium. In addition, there are often several supernmnerary nuclei (as many as 7–8 in number) in the same egg cell. These nuclei are also surrounded by dense cytoplasm. They may persist for some time and be recognizable at somewhat later stages of the proembryo or even after the elongated suspensors are formed. In some cases, there are some cell groups above the upper tier of proembryo. These cell groups are also surrounded by dense cytoplasm. Either the supernumerary nuclei or cells are surrounded by the dense cytoplasm. Probably they are derived from the mitosis or amitosis of the second sperm. Investigations on submicroscopic structures of sperm and egg in relation to the fertilization of Cupressaceae have been carried out extensively during the last decade. The fate of male cytoplasm has been debated for a long time and this problem attracted attention again in the nineteen seventies. At last the concept of neocytoplasm has been established soundly based upon the information from observation of electron microphotographs. The neocytoplasm is also visible under the light microscope though the components are not recognizable. The sperms of Fokienia are similar to those of Cupressus funebris, Juniperus communis, Sabina virginiana, Tetraclinis articulata, Chamaecyparis pisifera as well as the genus Thujopsis and others. Two sperms are all effective in fertilization and this is the common phenomenon of the family Gupressaceae.     

Development of Gametophytes in Fokienia Cupressaceae

The structure of the ovule and the development of the gametophytes in Fokienia, an endemic genus of Cupressaceae are described in some detail. Two wings, one small and one large, are developed along the micropylar end of the ovule and two resin canals are present in each of them. The material collected in the middle of April was already pollinated and the pollen began to germinate on the nucellus. The sterile cell, tube cell and spermatogenous cell have been formed in the tube in the first collection of April 17. At the end of June the division of the spermatogenous cell results in two sperms of Similar size and shape and the division plane is usually parallel to long axis of the pollen tube. Both sperms are effective in fertilization. 4096 free nuclei (actual counting, 3733—4224 ones) are produced through 12 times of repeated divisions of the functional megaspore, then cell walls appear among the free nuclei and cellular female gametophyte is formed. The number of archegonia varies from 6 to 16, mostly 9–12. The archegonial complex is enclosed by 2–3 layers of jacket cells. The neck cells are usually 4 in number, arranged in 1–2 layers. The central cell divides and results in the formation of one ventral canal nucleus and one egg nucleus. Fertilization takes place in the middle of the archegonium. The development of the gametophytes of Fokienia is more or less similar to that of Sabina.     

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.     

CYTOLOGICAL BASIS FOR CYTOPLASMIC INHERITANCE IN PSEUDOTSUGA MENZIESII. II. FERTILIZATION AND PROEMBRYO DEVELOPMENT

Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) ovules were used to study male gamete formation, insemination of the egg, and free nuclear and cellular proembryo development. Two male nuclei form as the pollen tube either reaches the megaspore wall or as it enters the archegonial chamber. No cell wall separates them. They are contained within the body-cell cytoplasm. A narrow extension of the pollen tube separates the neck cells and penetrates the ventral canal cell. The pollen tube then releases its contents into the egg cytoplasm. The two male gametes and a cluster of paternal organelles (plastids and mitochondria) migrate within the remains of the body-cell cytoplasm toward the egg nucleus. Microtubules are associated with this complex. The leading male gamete fuses with the egg nucleus. The zygote nucleus undergoes free nuclear division, but the cluster of paternal organelles remains discrete. Free nuclei, paternal and maternal nucleoplasm, maternal perinuclear cytoplasm, and the cluster of paternal organelles migrate en masse to the chalazal end of the archegonium. There, paternal and maternal organelles intermingle to form the neocytoplasm, the nuclei divide, and a 12-cell proembryo is formed. The importance of male nuclei or cells, the perinuclear zone, and large inclusions in cytoplasmic inheritance are discussed in the Pinaceae and in other conifer families. This completes a two-part study to determine the fate of paternal and maternal plastids and mitochondria during gamete formation, fertilization, and proembryo development in Douglas fir.     

红松配子体的发育过程

红松是我国主要用材树种,材质优良。我国主要分布在长白山和小兴安岭,随着森林工业的发展,大面积的原始森林迅速减少,人工更新日趋重要。在生产中采用种子育苗,因此对红松种子的发育规律应全面掌握,以便为种子园建立、良种选育采取有效措施提供理论依据。根据国内外有关报导,红松雌雄配子的形成、受精作用已有一定的研究^[14、15],但这些研究只侧重在小孢子母细胞的减数分裂和受精作用。故对雌雄配子体的发育过程仍需深入研究,尤其是授粉后的雄配子体发育和精子进入颈卵器的过程,均未见详细报导。     

Fertilization in Pinus Bungeana

Pinus bungeana is a species endemic to China and as yet its embryology has not been reported. The present paper deals with its process of fertilization in some details. 1. The development of the male gamete and the structure of the archegonium. The spermatogenous cell has already divided into two uniqual male gametes in the middle of May (in 1978, at Peking), about ten days before fertilization. Both sperms are spheroidal to ellipsoidal. The larger sperm is about 94 × 65 μm and the smaller one, about 72 × 58 μm in size. As the pollen tube approaches the archegonium the two sperms move toward the apex of the tube together with the remaining contents. Generally the larger sperm precedes the smaller one. The cytoplasmic contents also contain a sterile cell, 3—43×2—29 μm in size and a tube nuleus, 15—30 μm in diamter, besides the sperms. A mass of starch grains of more or less similar to sperm in size is also included in the contents of the pollen tube. Generally 3—4, even up to 7–8 pollen grains germinate normally within an ovule. Therefore, many sperms (up to 14—16) may be present on the same nucellus. The archegonium is elongato-ellipsoidal, about 870 ×500 μm in size. Arehegonia are single, 2—(3—5) in number, with 2 neck cells and a layer of jacket cells. The central cell divided in the middle of May and gave rise to the ventral canal cell and the egg. As the archegonium matures the cytoplasm becomes radiate fibrillae around the egg nucleus. The egg nucleus is large, 150—226 μm in diameter. One large nucleolus, 22—25 μm in diameter and sometimes up to 50; small nueleoli are present within the nucleus. 2. Fertilization Pollination takes place in the first week of May and fertilization will be effected from the end of May to the first week of June of next year. The interval between pollinatin and fertilization in P. bungeana is about thirteen months and the lapse of time is almost similar to most of the Pinus so far recorded. When the pollen tube contacts the archegonium through the neck cells all its contents are discharged into the egg cell. Usually the larger sperm fuses with the egg nucleus and the rest of the contents stays in the upper part of the egg cell. It is interesting to note that the nonfunctional second sperm also moves toward the egg nucleus and often divides by mitosis; and this phenomenon is not reported elsewhere. At the earlier stage of the fusion between male and female nuclei the male nucleoplasm is dense and finely granular while the female nucleoplasm is thin and coarsely granular, hence the boundary between them is very clear. The nuclear membranes of both nuclei persist for a long time. After the male nucleus sinks into the female nucleus completely, both nuclei begin to divide and enter into the prophase and then the metaphase simultaneously. By this time the paternal and maternal chromosome sets with their spindles still remain at certain distance from each other. Then the paternal chromosomes with their spindle move gradually toward the maternal ones. At first a multipolar common spindle appears as the maternal and paternal spindles with their chromosomes merge together. Finally a regular bipolar spindle is formed and both the maternal and paternal chromosomes become arranged on the equatorial plate. In the meantime, the process of fusion is complete and the zygote is at the stage of metaphase. At the moment the spindle looks greater in width than in length, being about 80×65—70 μm in size. 3. Supernumerary nuclei and sperms. The ventral canal cell degenerates soon after its formation. While the supernumerary sperms divide usually after their entrance into the egg cell. Therefore, the supernumerary nuclei probably derive directly from the smaller sperms or indirectly from mitoses of the larger ones Generally the nucleoplasm of the supernumerary nuclei is rather thin while the nucleoplasm of the undivided sperms is rather dense. This shows that the former is in the state of degeneration. The supernumerary nuclei of P. bungeana are as many as 7, their usual size being 43—58×32—43 μm. In the upper part of some egg cells there are still secondary smaller sperms about the size of 36 × 29 μm, Their volume is just about half of the usual smaller sperm. Probably they are derived from the division of the smaller sperms.     

She's the boss: signaling in pollen tube reception

In angiosperms, the sperm cells are carried within the pollen tubes (male gametophytes) to the female gametophyte so that double fertilization can occur. The female gametophyte exerts control over the male, with specialized cells known as synergids guiding the pollen tubes and controlling their behavior when they enter the female gametophyte so that the sperm cells can be delivered to the egg and central cell. Upon pollen tube arrival at the ovule, signal transduction cascades mediated by receptor-like kinases are initiated in both the synergid and the tip of the pollen tube, leading to synergid cell death and pollen tube rupture. In this review, we discuss the role of these receptors and of newly discovered members of the pollen tube reception pathway.     

Western white pine (Pinus monticola Dougl.) reproduction: II. Fertilisation and cytoplasmic inheritance

Fertilisation and proembryo development are described from transmission electron micrographs emphasising the origin and fate of the maternal and paternal mitochondria and plastids. During central cell and egg development mitochondria migrate toward the nuclei, forming a perinuclear zone consisting predominantly of maternal mitochondria and polysomes. At the same time, maternal plastids transformed and at fertilisation are excluded from the neocytoplasm. The pollen tube releases two sperm nuclei into the egg with cytoplasm from the generative cell and the tube cell. The leading sperm nucleus fuses with the egg nucleus and a small number of paternal mitochondria and plastids are taken into the perinuclear zone. The second sperm nucleus degenerates. As the zygote nucleus undergoes mitosis followed by free nuclear division and nuclear migration to the chalazal end of the archegonium, maternal and paternal organelles intermingle within the neocytoplasm. The result is paternal inheritance of plastids and biparental, but predominantly maternal, inheritance of mitochondria. This pattern is consistent within the Pinaceae but differs from some other conifer families. Received: 9 December 1999 / Revision accepted: 30 April 2000     

Double fertilization in Gnetum gnemon (Gnetaceae): its bearing on the evolution of sexual reproduction within the Gnetales and the anthophyte clade

The fertilization process in Gnetum is critical to our understanding of the evolution of sexual reproduction within the Gnetales, a monophyletic group of nonfiowering seed plants that are the closest living relatives to flowering plants. Although much is known about the fertilization process in Ephedra, which is basal within the Gnetales, little is known about sexual reproduction in the derived sister groups Gnetum and Welwitschia. Ovules of Gnetum gnemon were collected at various stages after hand pollination and processed for light, fluorescence, and electron microscopy. Approximately 5 d after pollination, pollen tubes reach sexually mature female gametophytes, which are coenocytic. At that time, a binucleate sperm cell is found within each pollen tube. Within 7 d of pollination, double fertilization events occur when each of two sperm nuclei released from a pollen tube fuses with a separate, undifferentiated female nucleus within the free nuclear female gametophyte, which lacks differentiated egg cells. The products of double fertilization are two viable zygotes; endosperm is not formed. The lack of differentiated egg cells in Gnetum gnemon is unparalleled among land plants and the documentation of a regularly occurring process of double fertilization is congruent with the hypothesis that a rudimentary process of double fertilization evolved in a common ancestor of angiosperms and Gnetales.     

Invvestigations on Early Embryogenesis of Actinidia chinensis Planch var. chinensis

This paper deals with early embryogenesis of Actinidia chinensis var. chinensis. 1. Ovary superior consists of 34—45 carpels. Each carpel contains 11–45 ovules. The ovule is uni-integument and tenuinucellar. The ovule is anatropous. The archesporium is formed by a single cell, and directly develops into megaspore mother cell. Sometimes the archesporium consists of 2–3 cells, but only one of them develops into megaspore mother cell and the others are degenerated. 2. The mature pollen grain is two-celled and the embryo sac belongs to olygonum type. In most embryo sacs two polar nuclei are fused before fertilization. One of the synergids was destroyed as the pollen tube penetrated into embryo sac the other one disappeared after fertilization. In most cases the antipodal cells became degenerated in fertilization process, only some remained until the first division of primary endosperm nucleus. 3. In Beijing area the double fertilization of Actinidia chinensis occurred 30–72 hours after pollination. In the fertilization one sperm fused with egg nucleus and the other sperm fused with the secondary nucleus as usual. The fusion of the secondary nucleus with sperm was in advance of the fusion of the egg nudeus. 4. The endosperm is cellular type.     

Paternal cytoplasmic transmission in Chinese pine (Pinus tabulaeformis)

Summary. In this paper, the stages of normal sexual reproduction between pollen tube penetration of the archegonium and early embryo formation in Pinus tabulaeformis are described, emphasizing the transmission of parental cytoplasm, especially the DNA-containing organelles – plastids and mitochondria. The pollen tube growing in the nucellus contained an irregular tube nucleus followed by a pair of sperm cells. The tube cytoplasm contained abundant organelles, including starch-containing plastids and mitochondria. The two sperm cells differed in their volume of cytoplasm. The leading sperm, with more cytoplasm, contained abundant plastids and mitochondria, while the trailing one, with a thin layer of cytoplasm, had very few organelles. The mature egg cell contained a great number of mitochondria, whereas it lacked normal plastids. At fertilization, the pollen tube penetrated into the egg cell at the micropylar end and released all of its contents, including the two sperms. One of the sperm nuclei fused with the egg nucleus, whereas the other one was retained by the receptive vacuole. Very few plastids and mitochondria of male origin were observed around the fusing sperm and egg nuclei, while the retained sperm nucleus was surrounded by a large amount of male cytoplasm. The discharged tube cytoplasm occupied a large micropylar area in the egg cell. In the free nuclear proembryo, organelles of maternal and paternal origins intermingled in the neocytoplasm around the free nuclei. Most of the mitochondria had the same features as those of the egg cell, but some appeared to be from sperm cells and tube cytoplasm. Plastids were obviously of male origin, with an appearance similar to those of the sperm or tube cells. After cellularization of the proembryo, maternal mitochondria became more abundant than the paternal ones and the plastids enlarged and began to accumulate starch. The results reveal the cytological mechanism for paternal inheritance of plastids and biparental inheritance of mitochondria in Chinese pine. Correspondence and reprints: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing 100094, People’s Republic of China.     

水稻双受精过程的细胞形态学及时间进程的观察

应用常规石蜡切片和荧光显微镜观察水稻（Oryza sativa）受精过程中雌雄性细胞融合时的形态特征及时间进程,确定合子期,为花粉管通道转基因技术的实施提供理论依据。结果表明：授粉后,花粉随即萌发,花粉管进入羽毛状柱头分支结构的细胞间隙,继续生长于花柱至子房顶部的引导组织的细胞间隙中,而后进入子房,在子房壁与外珠被之间的缝隙中向珠孔方向生长,花粉与花粉管均具有明显的绿色荧光。花粉管经珠孔及珠心表皮细胞间隙进入一个助细胞,释放精子。精子释放前,两极核移向卵细胞的合点端：两精子释放于卵细胞与中央细胞的间隙后,先后脱去细胞质,然后分别移向卵核和极核,移向卵核的精核快于移向极核的精核：精核与两极核在向反足细胞团方向移动的过程中完成雌雄核融合。大量图片显示了雌雄性核融合的详细过程以及多精受精现象。水稻受精过程经历的时间表如下：授粉后,花粉在柱头萌发：花粉萌发至花粉管进入珠孔大约需要0．5小时：授粉后0．54,时左右,花粉管进入一个助细胞,释放精子：授粉后0．5—2．5小时,精卵融合形成合子：授粉后约10．0小时,合子第1次分裂,合子期为授粉后2．5-10．04,时：授粉后1．0-3．04,时,精核与两极核融合：授粉后约5．0小时,初生胚乳核分裂。’     

水稻双受精过程的细胞形态学及时间进程的观察

应用常规石蜡切片和荧光显微镜观察水稻(Oryz a sativa)受精过程中雌雄性细胞融合时的形态特征及时间进程, 确定合子期, 为花粉管通道转基因技术的实施提供理论依据。结果表明: 授粉后, 花粉随即萌发, 花粉管进入羽毛状柱头分支结构的细胞间隙, 继续生长于花柱至子房顶部的引导组织的细胞间隙中, 而后进入子房, 在子房壁与外珠被之间的缝隙中向珠孔方向生长, 花粉与花粉管均具有明显的绿色荧光。花粉管经珠孔及珠心表皮细胞间隙进入一个助细胞, 释放精子。精子释放前, 两极核移向卵细胞的合点端; 两精子释放于卵细胞与中央细胞的间隙后, 先后脱去细胞质, 然后分别移向卵核和极核, 移向卵核的精核快于移向极核的精核; 精核与两极核在向反足细胞团方向移动的过程中完成雌雄核融合。大量图片显示了雌雄性核融合的详细过程以及多精受精现象。水稻受精过程经历的时间表如下: 授粉后, 花粉在柱头萌发; 花粉萌发至花粉管进入珠孔大约需要0.5小时; 授粉后0.5小时左右, 花粉管进入一个助细胞, 释放精子; 授粉后0.5-2.5小时, 精卵融合形成合子; 授粉后约10.0小时, 合子第1次分裂, 合子期为授粉后2.5-10.0小时; 授粉后1.0-3.0小时, 精核与两极核融合; 授粉后约5.0小时, 初生胚乳核分裂。     

黑节草双受精过程及胚胎发育的研究

黑节草从传粉到受精约需１３０ｄ,精子在花粉管中形成,胚囊发育属蓼型胚囊,因反足细胞较早退化,故受精前胚囊多只由卵器和中央细胞组成。精卵核融合时,精核染色质进入卵核后凝集成颗粒状,并在原位与卵核的染色质融合,雌、雄性核仁一直维持至合子的第一次分裂期前。双受精作用正常,属于有丝分裂前配子融合类型,初生胚乳核发生２－３次分裂后逐渐退化消失,胚的发育局限于球形胚阶段。     

云南松雌雄配子体的发育

云南松(Pinus yunnanensis Fr.)雄配子体于10月在小孢子叶腹面产生二个小孢子囊,内有许多进行分裂的造孢组织细胞。第二年一月下旬至二月初小孢子母细胞进行减数分裂。在分裂期间,细胞内所贮存的淀粉粒的分布发生变化。二月初四分体小孢子形成,绒毡层细胞解体。二日中旬单核花粉粒形成,外壁扩展形成二个异极对称的气囊。三月花粉在四细胞时期散发。 雌配子体于二月上旬在珠心皮下分化出孢原细胞。二月下旬大孢子母细胞进入减数分裂期。三月初直列四分体大孢子形成,珠孔端三个退化,合点端一个功能大孢子进入有丝分裂期,形成约32个游离核的配子体。次年三月初雌配子体形成,四月初中央细胞核分裂,四月底颈卵器成熟,卵核周围产生辐射状原生质纤丝。五月初受精开始。云南松雌雄配子体的发育与亚热带分布的P.roburghii相似。     

侧金盏花双受精进程研究

应用荧光显微镜和常规石蜡切片观察侧金盏花(Adonis amurensis)花粉管生长和受精作用的全过程。结果表明,侧金盏花为湿型柱头,授粉后1–2小时,花粉粒与柱头识别;授粉后2–4小时,花粉粒萌发;授粉后4–6小时,花粉管进入柱头。侧金盏花的受精模式为珠孔受精,授粉后10小时,精子被释放;授粉后30小时,精核与卵核融合;授粉后7天合子形成;授粉后15天合子进入分裂期,合子休眠期为8天。2个极核在受精前不融合,授粉后14–16小时,精核与1个极核融合;授粉后20–22小时,受精极核与另1个极核融合形成初生胚乳核。双受精作用属于有丝分裂前配子融合型。通过实验确定了侧金盏花受精过程的雌雄性细胞融合形态变化与相应经历的时间及其合子休眠期。研究结果丰富了侧金盏花胚胎学资料,对其今后的育种及转基因研究具有重要意义。     

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.     

Development of ovulate cones from initiation of reproductive buds to fertilization in Cephalotaxus wilsoniana Hay

Cephalotaxus wilsoniana Hay, is endemic to Taiwan. This study was performed morphologically and anatomically to investigate reproduction in this species for the purpose of conservation. The duration from reproductive bud formation to fertilization in C. wilsoniana lasts about one year and five months. Buds are initiated in late January and differentiate into one vegetative bud and 3 female cones in late February. A female cone is constructed with 4 pairs of decussate opposite bracts. A small ridge-like secondary axis sits on the axil of each bract. Two ovules are borne on both sides of each secondary axis. A lysogenous pollen chamber begins to be formed from the degenerative tissues on the top end of the nucellus in early March. In late March the megasporogenous tissue is differentiated in the core center of the nucellus, and the micropyle closes gradually after pollination. By late July, pollen tubes have developed in the pollen chamber, and the megaspore mother cell appears. Then the functional megaspore becomes active in mid-October. The 8 free nucleate macrogametophyte appears in late December. From January to late March of the following year, the elliptical cyst-like female gametophyte keeps growing through continuous divisions of its free nuclei. The cyst layer of protoplast thickens in early April. In mid-April, cell walls begin to form among free nuclei. The archegonia are initiated in late April. Pollen tubes extend their tips to the macrogametophyte in early May, and each tube with 2 spermatozoids reaches a mature archegonium with an egg needed to perform fertilization in late May. Generally, only 1-(3) ovules in each cone can become mature.     

FERTILIZATION IN BARLEY

The mature embryo sac of barley consists of an egg, two synergids, a central cell, and up to 100 antipodal cells. At shedding the male gametophyte is 3-celled, consisting of a vegetative cell with a large amount of starch and two sperms having PAS+ boundaries. Before pollination the nucleus and cytoplasm of each synergid appear normal. After pollination the nucleus and cytoplasm of one synergid undergo degeneration. The pollen tube grows along the surface of the integument of the ovule, passes through the micropyle, and enters the degenerate synergid through the filiform apparatus. The pollen tube discharges the vegetative nucleus, two cellular sperms, and a variable amount of starch into the degenerate synergid. Soon after deposition the sperms migrate by an unknown mechanism to the chalazal end of the degenerate synergid. Sperm nuclei then enter the cytoplasm of the egg and central cell, ultimately resulting in the formation of the zygote and primary endosperm nucleus, respectively. Sperm boundaries do not enter egg or central cell, but it was not possible to determine the fate of other sperm components. Degenerate vegetative and synergid nuclei remain in the synergid after fertilization, constituting what are considered to be X-bodies in barley. The second synergid degenerates during early embryogeny.