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.     

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.     

Studies on Fertilization in Glycine max

This work studies the whole process of fertilization in Glycine max. The results are summarized as follows: 1. The type of ripened pollen grain of soybean was two-cell type. The generative cell was divided mitotically into two spermatids within the pollen tube. 2. In the 6th hour after self-pollination, the pollen tube entered into the embryo sac and released two sperms. Before the fusion of the male and female nuclei, the cytoplasmic sheath of the spermatids falled off. The cytoplasm of the male gamete did not fuse with that of the egg cell. 3. In the 6th hour after self-pollination, one spermatid nucleus come in contact with the egg nucleus and the other with the secondary nucleus, The contact of the sperimatid nucleus with the egg nucleus was a little earlier than that with the secondary nucleus. 4. The spermatid nucleus entered into the egg nucleus; the chromatic granules of the spermatid despiralized. After the complete fusion of the spermatid nucleus with the egg nucleus, the egg nucleus was darkly stained and the chromonemata increased, afterward the male nucleus appeared. 5. In the 28th hour after self-pollination, the zygote begun the first mitotic division. It took about 20 hours to fuse the male and female gametes, and to form the zygote untile before first mitotic division. It means that the zygote dormancy stage of soybean was about twenty hours. 6. In the 7th hour after self-pollination, the spermatid nucleus fused with the secondary nucleus. The process was similar to that of the spermatid nucleus with the egg nucleus. The chromatic granules gradually despiralized within the secondary nucleus, and the male nucleoli appeared. The velocity of the fusion of the spermatid nucleus with the secondary nucleus was faster than that of the other spermatid nucleus with the egg nucleus. 7. In the 10th hour after self-pollination, the secondary nucleus begun the first mitotic division. 8. The fertilization of soybean was the premitotic type.     

短柄五加开花后雌蕊的发育状态与受精作用的研究

短柄五加（ＥｌｅｕｔｈｅｒｏｃｏｃｕｓｂｒａｃｈｙｐｕｓＨａｒｍｓ．）开花当天,花药散粉,而雌配子体需经４～５ｄ才发育成熟。证实短柄五加为雄蕊先熟植物。开花第５天,成熟胚囊的比率为５７６９％,其余为退化和不育胚囊。开花第６天,胚囊开始受精。开花第１０天,受精胚囊占胚囊总数的５３５７％。柱头的可授期自开花后第４～５天开始,自花粉萌发至雌雄性核融合大约有２～３ｄ的间隔期。短柄五加受精过程与一般被子植物相同,其受精作用属于有丝分裂前配子融合类型。观察并统计了合子中雌性核仁的数目、存在状态,指出短柄五加合子中从雄性核仁出现到与雌雄性核仁融合为一个大核仁需经历３ｄ左右;如果以胚乳游离核数目为对照,大部分合子中雌雄性核仁的融合发生在３２～１２８个胚乳游离核时期。大多数合子是以雌雄性核仁融合为一个大核仁后进入合子分裂期;少数合子的雌雄性核仁不经融合也进入合子分裂期。观察到多精入胚囊、多精入卵以及成熟胚囊退化的现象。讨论了被子植物受精过程中有关受精终结的标志等问题。     

The Fusion of Male and Female Nuclei in Fertilization of Higher Plants

Studies on the fusion of male and female nuclei in fertilization of Helianthus an- nuus L., Triticum aestivan L., Gossypium hisutum L., Hosta caerulea Tratt., and Pinus tabulaeformis Carr. were made in the present work. The results are summarized as follows: 1. The essential process of the fusion of male and female nuclei during syngamy in four species of angiosperms studied may' be generalized as follows: (1) the male nucleus made contact with the female one, (2) followed by the fusion of nuclear membranes between the male and female nuclei. (3) then the despiralization of male spireme happened and male nucleolus made its appearance inside of the fertilized egg nucleus (4) the male chromatin dispersed and make its appearance indistinguishable from that of the female chromatin, (5) the male and female nucleoli fused together to form a larger nucleolus as a sign of completion of the fusion of the two nuclei. In the first mitotic division of the zygote there was only one common mitotic spindle. 2. The essential process of the fusion of egg and sperm nuclei during syngamy in a gymnosperm-Pinus tabulaeformis could also be outlined as follows: (1) the sperm nucleus made contact with the egg nucleue, (2) the fusion of nuclear membranes happened between the male and female nuclei, (3) the male and female ehromatins condensed to form two separate groups of chromatin threads together with the very apparent apperance of the male and female nucleoli at this stage, (4) the male and female chromosomes grouped respectively in their own spindles while both nucleoli disappeared, (5) then the two spindles fused together and all the chromosomes arranged to form a common equatorial plate, (6) finally two daugter nuclei resulted from the mitotic division. 3. Based on the facts that there were two different patterns of the fusion of male and female nuclei in fertilization discribed, all of these accounts are in general accord with the condition usually described that there are two types of fertilization, the pre- mitotic and postmitotie syngamy in higher plants. The type of angiosperm fertilization and the mechanism of promoting the zygote to divide after fertilization are discussed, and the nuclear fusion in sexual reproduction has been compared with that of somatic cell hybridization.     

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.     

The Development of Gynoecium After Anthesis and Fertilization in Eleutherococcus brachypus (Araliaceae)

Eleutherococcus brachypus Harms. is a protandrous plant because the female gametophyte delays its maturation until the fifth day after anthesis and pollen shelling. On the fifth day after anthesis, about 57.69% of the embryo sacs matured and the rest degenerated or failed to develop. Fertilization began in the embryo sac on the fifth day. On the tenth day fertilization took place in 53.37 % of the total of embryo sacs. The stigma became receptible after 3 to 4 days of anthesis. It took 2 to 3 days from the germination of pollen grains on stigma to the fusion of male and female nuclei. The process of fertilization in E. brachypus is not different from most other angiosperms. It belonged to the type of premitotic syngamy. The observations and statistical analysis were made on the number feature of male and female nucleoli in the zygote. The result indicated that it took three days or so from the appearance of male nucleolus in the zygote to its fusion with the female nucleotus. Refering to the number of free nuclei of the endosperm, the fusion of male and female nucleoli in most of the zygotes occurred in the stage of 32 to 128 nuclei of the endosperm. Most zygotes con-tained a big nucleolus resulting from the fusion of male and female nucleolus and proceeding to mitosis. A few without fusion could also proceed to the mitotic stage. Features of multiple sperms entering the embryo sac or entering the egg cell and the degeneration of mature embryo sacs were observed as well. The sign of the termination of fertilization in angiosperms was discussed.     

The Development of Gynoecium after Anthesis and Fertilization in Eleutherococcus senticosus (Araliaceae)

The development status of gynoecia in Eleutherococcus senticosus flowers is different from that in ordinary plants. Female gametophytes of E. senticosus have not become mature until the 6th day after anthesis. On the 6th day, 82.25% of embryo sacs in female plants, and 67.25％ of those in hermaphroditic plants become mature, while the rest are sterile, immature or degenerated with no fertilized embryo sacs observed. At the same time, all embryo sacs degenerated and flowers withered in male plants. On the 7th day, a few embryo sacs in female and hermaphroditic plants start being fertilized. Accompanying the differentiation of embryo sacs, styles of female and hermaphroditic flowers start to expand and their nectaries become mature gradually. After the 4th or 6th day of anthesis, stigmatic papillae become conspicious and stigmata become white and open. In the meantime, the stigmata become receptive and the nectaries get active or reactive. By the 9th or 10th day, 40～65 ％ of embryo sacs in female plants and 25～41% of those in hermaphroditic plants have been fertilized. The whole process of fertilization in E. senticosus was observed. About 2 or 3 days after pollination, the two sperm nuclei start to fuse with the egg and the secondary nucleus. The fertilization of E. senticosus belongs to the premitotic type of syngamy. The essential process of the fusion of male and female nuclei during syngamy may be generalized as follows: (1) the contacting of male nucleus with the female one; (2) the fusion of nuclear membranes between the male and female nuclei; (3) the despiralization of male spireme and the appearance of male nucleolus inside the fertilized female nucleus; (4) the dispersion of male chromatin and the mergence with the female chromatin, which is the sign of completion of the fusion of the two nuclei. In addition, degeneration types of mature embryo sacs were observed. And typical polyspermy and a series of cases in which extra sperms enter the em-bryo sac are recorded.     

糜子双受精过程的细胞学观察

糜子(Panicum miliaceum L.)受精的全过程在开花后3小时内完成。开花后20分钟,花粉管到达珠孔,30分钟进入胚囊并释放精子;雌、雄性核融合发生在开花后30分钟至3小时。精核与卵核和极核融合的过程基本相同,但总是先完成与极核的融合。开花后2小时,初生胚乳核形成,随后立即分裂。开花后3小时,合子形成,此时胚乳含两个游离核。开花后8—10小时,合子进入分裂期。合子的休眠期约5—7小时。受精作用属于有丝分裂前配子融合的类型。     

Studies on Fertilization and Embryogenesis of Red Vinespinach (Basella rubra L.)

This paper presents detailed report on the process of fertilization and the develop- ment of embryo and endosperm of Basella rubra L. The results obtained are summarized as follows: About 4–6 hours after anthesis a great deal pollen grains germinated on the stig- ma; 6–11 hours, the pollen tube passed through the style; 11–16 hours, the pollen tube reached the ovary cavity. About 16–18 hours, one sperm nucleus entered into the egg and the other one entered into the secondary nucleus. In most cases, after 16–24 hours the double fertilization had completed. After 2–8 days, two-celled proembryo was first shown. Finally, the proembryo gave rise to multicellular globular embryo proper. The development of the embryo of Basella rubra L. conforms to the Asterad type. Whether the Asterad type had a high frequency needs further to be studied. Although the fertilization of the sperm nucleus with the secondary nucleus began later, the fusion of two sexual nuclei and the development of the endosperm proceeded often quickly. After 20 hours, the free nuclei period began, and after 2–4 days the free nuclei of endosperm were rapidly formed. The endosperm of Basella rubra L. is a nuclear type. And at the time When the seed had ripened the endosperm tissues were all absorbed by the developing embryo. The author observed that there are different distributions of the vegetative nucleus and two sperms in the pollen tube, and that two sperms entered a egg or secondary nucleus. The changes of the starch accumulated and distributed et al. in the floral organs are also studied and discussed.     

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.     

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.     

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。     

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。     

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。     

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.     

番茄受精作用及其间隔期的研究

利用常规石蜡切片法研究了番茄受精作用的全过程,具体研究结果为:(1)授粉后2 h,花粉粒在柱头上萌发;约2~4 h,花粉管长入柱头,且末端膨大;约8 h后,生殖细胞进入分裂期;并于约两小时后,分裂为两个精细胞。(2)约14 h,花粉管进入子房腔;约18~24 h,花粉管进入胚囊,破坏一个助细胞,并在其珠孔端释放两个精子;随后被释放的精子移到卵细胞与次生核附近。(3)授粉后约30 h精核进入卵细胞;约34 h,精核与卵核融合,并在卵核内出现分散的雄性染色质,进而出现雄性核仁;44~50 h,雌、雄性核仁融合,形成合子;合子的休眠期为10 h左右。60 h之后,合子分裂形成二细胞原胚。(4)约26 h,另一个精子的精核与次生核核膜相贴伏,随后与之融合;约30~34 h,次生核内出现分散的雄性染色质,随之出现雄性核仁;约38~42 h,雌、雄性核仁融合,形成初生胚乳核。约44 h后,初生胚乳核进行有丝分裂,形成两个胚乳细胞。番茄胚乳发育属于细胞型。初生胚乳核无休眠期。(5)精子与次生核的融合比与卵核的融合快。(6)番茄的受精作用属于有丝分裂前配子融合类型。     

The kinetics of cytological events during double fertilization in Zea mays L.

By using a clearing method, the process of double fertilization in Zea mays L. (line A 188) was analysed and the precise sequence of events was determined. The period from pollen tube arrival to gamete fusion was relatively short, possibly less than 1 h. The karyogamy was of premitotic type, and the time from the contact of male and female nuclei to the fusion of male and female nucleoli was about 5 h in the egg cell and 3 h in the central cell. In the central cell, the sperm nucleus fused with either one of the polar nuclei or the secondary nucleus, the latter being observed for the first time in maize. The zygote was in the resting period for 13–16 h before division commenced, changing the cell polarity during karyogamy and the resting period. The primary endosperm nucleus divided immediately after karyogamy was completed in the central cell. The embryo sacs with two-celled proembryos contained four to eight endosperm nuclei. The timetable of fertilization events could be a standard for further studies on in vitro fertilization at the cytological and molecular levels.     

Mitosis and Amitosis of Generative Cell Nuclei in Artificially Germinated Pollen Tubes in Zephyranthes candida(Lindl.)Herb.

This paper deals with the comportmem of the vegetative nucleus and its spatial association with the generative cell and sperm cells in the artificially germinated pollen tubes of Zephyranthes candida (Lindl.) Herb. before and after generative cell mitosis with the use of DNA-specific fluochrome 4′,6-diamidino-2-phenylindole (DAPI). The induction of amitosis and abnormal mitosis of generative cell nuclei by cold-pretreatment of the pollen prior to germination was studied in particular. In normal case, the generative cell, after appressing to the vegetative nucleus for certain time, underwent mitosis to form two sperms, while the vegetative nucleus became markedly elongated, diffused, and exhibited blurring of its fluorescence. After division, a pair of sperms remained shortly in close connexion with the vegetative nucleus. Then the vegetative nucleus returned to its original state. In the pollen tubes germinated from cold-pretreated pollen, amitosis of some generative cell nuclei were frequently observed. Amitosis took place via either equal or unequal division with a mode of constriction. During amitosis, the dynamic change of vegetative nucleus and its intimate association with generative cell afore described did not occur. Sperm nuclei produced from amitosis could farther undergo amitisis resulting in micronnclei. Factors affecting the amitosic rate of generative cells, such as pollen developmental stage, temperature and duration of cold-pretreatment, were studied. Besides amitosis, cold-pretreatment also induced some abnormal mitotic behavior leading to the formation of micronuclei. Based on our observations and previously reported facts in other plant materials, it is inferred that the vegetative nucleus plays an important role in normal mitosis of generative cell and development of sperms.