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     

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.     

Cytological Mechanism of Cytoplasmic Inheritance in Pinus tabulaeformis: II. Transmission of Male and Female Organelles During Fertilization and Proembryo Development

In an earlier report the ultrastructure and nucleoid organelles of male gamete in Pinus tabulaeformis Carr. have been described. Presently, the ultrastructure of the cytoplasm of the egg cell and pollen tube—immediately before fertilization and during cytoplasmic transmission of male gametophyte—has been described for the same species. The fate of parental plastids and mitochondria in the proembryo has also been followed. The mature egg cell contains a large amount of mitochondria, but seems to lack normal plastids. Most plastids have transformed into large inclusions. Apart from the large inclusions, there are abundant small inclusions and other organelles in the egg cell. During fertilization, pollen tube penetrates into the egg cell at the micropylar end and thereafter the contents are released. Plastid and mitochondrion of male origin are lacking near the fusing sperm-egg nuclei. The second sperm nucleus—not involved in karyogamy—remains at a site near the receptive vacuole. This nucleus is surrounded by large amount of male cytoplasm containing mixed organelles from the sperm cell, tube cell, and egg cell. At the free nuclear proembryo stage, organelles of male and female origin are visible in the perinucleus-cytoplasmic zone. Most of the mitochondria have the same morphological features as those in the egg cell. Some of the mitochondria appear to have originated from the sperm and tube cells. Plastids are most likely of male gametophyte origin because they have similar appearance as those of the sperm and tube cell. Large inclusions in the egg cell become vacuole-like. Paternal plastids have been incorporated into the neocytoplasm of the proembryo. In the cellular proembryo, maternal mitochondria are more abundant. Plastids resembling those of the sperm and tube cell are still present. These cytological results clearly show that in P. tabulaeformis , plastids are inherited paternally and mitochondria bipaternally. The cytological mechanism of plastid and mitochondrion inheritance in gymnosperm is discussed.     

FERTILIZATION IN PLUMBAGO ZEYLANICA: GAMETIC FUSION AND FATE OF THE MALE CYTOPLASM

Developmental phases surrounding the processes of gametic delivery and fusion were examined ultrastructurally in the reduced megagametophyte of Plumbago zeylanica, which lacks synergids. Gametic delivery occurs at the end of pollen tube growth and results in deposition of two male gametes, a vegetative nucleus, and a limited amount of pollen cytoplasm between the egg and central cell. Discharge of these materials from the tube is accompanied by loss of inner and outer pollen tube plasma membranes, loss of sperm-associated cell wall components, and disruption of the formerly continuous cell wall between the egg and central cell. The dispersion of egg cell wall components directly exposes female reproductive cell membranes to the unfused male gametes and pollen tube without disrupting gametic cell plasma membranes. Presence of unfused sperms within the female gametophyte appears to be a transitory phenomenon, lasting less than 5 min at the end of over 8½ hr of pollen tube growth. At the time of gametic deposition, plasma membranes of unfused sperm cells become directly appressed to plasma membranes of both the egg and central cell. Gametic fusion is initiated by a single fusion event between membranes of participating male and female cells, which is rapidly followed by subsequent, secondary fusion events between the same two cells at different locations along their surface. Gametic fusion results in the transmission of male gamete nuclei with co-transmission of nearly the entire sperm cytoplasmic volume and organellar complement, and it is possible to identify heritable male cytoplasmic organelles within both the incipient zygote and endosperm. Paternally originating plastids may be distinguished from maternal plastids by differences in morphology and staining characteristics, whereas paternal mitochondria may be distinguished from maternal mitochondria by populational differences in mitochondrial size which are statistically significant. Such observations further indicate that transmitted paternal mitochondria seem to remain viable, as judged by their ultrastructural appearance, and are transmitted exclusively by sperm cytoplasm rather than discharged pollen cytoplasm. The presence of anucleate, membrane-bounded cytoplasmic bodies between the egg and central cell are identifiable on the basis of their enclosed organelles and indicate that fragmentation of a small amount of the sperm cytoplasm associated with the vegetative nucleus commonly occurs. The presence and identification of sperm cytoplasmic organelles and associated membranes within female reproductive cells following gametic transmission represents strong evidence in support of the cellular basis of nuclear and cytoplasmic transmission during sexual reproduction in Plumbago.     

Cytological evidence of biparental inheritance of plastids and mitochondria inPelargonium

Summary Based on the organelle differences between egg and sperm cells inPelargonium hortorum, the zygote, proembryo, and endosperm were examined under the transmission electron microscope. Plastids and mitochondria in the egg cell are significantly different from those of the sperm cell. Egg plastids are starch-containing and less electron dense. They appear circular, elliptical irregular elongate in sections. Sperm cell plastids are relatively electrondense, mostly cup-shaped or dumbbell and devoid of starch granules. Mitochondria of the egg cell are giant and mostly cup-shaped while sperm mitochondria are smaller and usually circular in section. Double fertilization is completed by 24 h after pollination and the pollen tube can be seen in the degenerated synergid. In the zygote, plastids and mitochondria from male and female gametes can be distinguished by their characteristic differences. Moreover, paternal and maternal organelles appear to be distributed at random in the zygote. Aside from the pollen tube and its released starch granules, there is no enucleated cytoplasmic body in the degenerated synergid. Two days after pollination, the zygote undergoes one transverse division to form a 2-celled proembryo which consists of one larger vacuolated basal cell and one smaller densely cytoplasmic apical cell. Paternal and maternal organelles can be detected in both cells of the proembryo and also in the endosperm at this stage. From these results, it can be concluded that plastids and mitochondria from both male and female gametes have been transmitted into the apical cell of the proembryo and most probably to the following generation.Abbreviations TEM transmission electron microscope - DAPI 4,6-diamidino-2-phenylindole - RFLP restriction fragment length polymorphism     

CYTOLOGICAL BASIS FOR CYTOPLASMIC INHERITANCE IN PSEUDOTSUGA MENZIESII. I. POLLEN TUBE AND ARCHEGONIAL DEVELOPMENT

Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) ovules were used to study the method of pollen tube formation and penetration of the nucellus, the movement of the body cell down the pollen tube and development of the archegonia. No pollination drop forms but nucellar tip cells produce a minute secretion that may initiate pollen tube formation. Pollen tubes penetrate the nucellus causing degeneration of nucellar cells in contact with the pollen tube tip. The body cell becomes highly lobed and the tube cytoplasm forms thin sheets between the lobes. This may be the mechanism by which the large body cell is pulled down the narrow pollen tube. Body cell plastids and mitochondria remain unaltered during pollen tube growth, whereas tube cell organelles show signs of degeneration. The pollen tube penetrates the megaspore wall and settles in the archegonial chamber. During pollen elongation and pollen tube growth the egg matured. Egg cell plastids were transformed into large inclusions which filled the periphery of the egg while mitochondria migrated to the perinuclear zone. The neck cells, ventral canal cell and archegonial jacket cells are described. The significance of the body cell and egg cell ultrastructure is discussed in light of recent restriction fragment length polymorphism studies of plastid and mitochondrial inheritance in the Pinaceae.     

Western white pine (Pinus monticola Dougl.) reproduction: I. Gametophyte development

Male and female gametophyte development are described from light and transmission electron microscope preparations of ovules from first and second year Pinus monticola Dougl. seed cones. In the first year of development, pollen tubes penetrate about one-third the distance through the nucellus. The generative cell and tube nucleus move into the pollen tube. The megagametophyte undergoes early free nuclear division. First-year seed cones and pollen tubes become dormant in mid-July. In the second year, seed cones and pollen tubes resume development in April and the pollen tubes grow to the megagametophyte by mid-June. Early in June the generative cell undergoes mitosis, forming two equal-size sperm nuclei that remain within the generative cell cytoplasm. The generative cell has many extensions and abundant mitochondria and plastids. The megagametophyte resumes free nuclear division, then cell wall formation begins in early July. Cell wall formation and megagametophyte development follow the pattern found in other Pinaceae. Three to five archegonial initials form. The primary neck cell divides, forming one tier of neck cells. Jacket cells differentiate around each central cell. The central cell enlarges and becomes vacuolate; then vacuoles decrease in size and the cell divides, forming a small ventral canal cell and a large egg. Plastids in the central cell engulf large amounts of cytoplasm and enlarge. This process continues in the egg, and the peripheral cytoplasm of the egg becomes filled with transformed plastids. Mitochondria migrate around the nucleus, forming a perinuclear zone. The wide area of egg cytoplasm between these two zones has few organelles. A modified terminology for cells involved in microgametophyte development is recommended. Received: 9 December 1999 / Revision accepted: 30 April 2000     

ULTRASTRUCTURE OF PLASTID INHERITANCE: GREEN ALGAE TO ANGIOSPERMS

1. Plastid inheritance in most green algae and land plants is uniparental. In oogamous species, plastids are usually derived from the maternal parent; even when inheritance is biparental, maternal plastids usually predominate. Only a few species of conifer are known to have essentially paternal plastid inheritance. In spite of the overall strong maternal bias, there exists a spectrum of species in which plastid inheritance ranges from purely maternal to predominantly paternal. 2. Factors that influence the pattern of plastid inheritance operate both before (often long before) and after fertilization. For example, several different mechanisms for exclusion of plastids from particular cells, none of which is completely effective on its own, may operate sequentially during both gametogenesis and embryo-genesis. There appears to exist a general trend such that the more highly evolved the organism, the more numerous the mechanisms employed and the earlier they first come into operation. The pattern of plastid inheritance shown by a species represents the efficiency or lack of efficiency of these combined mechanisms. 3. In the newly-formed zygote of many unicellular algae, the plastids from both gametes are present and there is direct competition between them. Often the plastid from one mating type (usually the ‘invading’ male gamete, where this can be identified) quickly degenerates. Species such as Chlamydomonas are unusual in that the plastids from the two gametes fuse. In spite of this, inheritance of plastid DNA is normally uniparental. How this is accomplished remains unclear. In oogamous algae, the paternal plastids which enter the egg cell are frequently fewer in number and smaller in size than those contributed by the female gamete. The reduced contribution of paternal plastids can result from asymmetrical cell division or from differential timing of cell and plastid division during spermatogenesis. 4. In species ranging from unicellular algae to angiosperms, plastids may be partially or completely debarred from particular cells at critical stages during the reproductive cycle. An important factor in this form of plastid elimination is their postioning with respect to the nucleus prior to a cell division. When plastids closely encircle the nucleus, they are usually incorporated equally into the two daughter cells; when the plastids are concentrated at some distance from the nucleus, they are frequently excluded from one daughter cell. 5. Elimination of plastids from a gamete prior to plasmogamy prevents direct competition between the two types of plastid in the zygote or embryo. Perhaps the most effective method of excluding paternal plastids from the egg cell has been achieved by some lower land plants; the plastids migrate to the posterior part of the spermatozoid, and are discarded from there in a discrete vesicle before the egg is reached. 6. Plastid inheritance in conifers appears to be unique. In those species in which the derivation of plastids in the pro-embryo can be determined, it has been found that they come only from the male gamete. Maternal plastids are positively excluded from the pro-embryo and later degenerate. 7. In most angiosperm species plastid inheritance is maternal; in only a few species is it regularly biparental. The first step towards exclusion of paternal plastids often takes place in the uninucleate pollen grain where the plastids may be concentrated at the pole of the cell farthest from the site of the future generative cell. Any plastids that succeed in entering the generative cell may degenerate before the gametes are released from the pollen tube. Even if paternal plastids reach the egg, they are at a disadvantage because they are (a) entering an environment that is essentially alien, and (b) normally present in much smaller numbers than maternal plastids. Later, when the zygote divides, the few paternal plastids may fail to become incorporated in the small terminal cell which gives rise to the embryo proper. 8. There appears to be no consistent evolutionary progression in the use of more efficient mechanisms to influence plastid inheritance; most of the mechanisms associated with exclusion of paternal plastids in angiosperms, for example, can also be found in one or other species of green alga. The primary factors that influence plastid inheritance appear to be (I) direct competition in the zygote between plastids of the two parental types – the principal mechanism operating in isogamous algae, but also operating in some angiosperms; and (2) the divergent evolution of the two types of gamete - on the one hand a small male gamete with a minimum of cytoplasm which is capable of moving (spermatozoid) or being moved (pollen) efficiently, and, on the other hand, a large egg cell with numerous organelles, which is well able to act as ‘host’ for the future zygote. Many of the additional mechanisms that influence the pattern of plastid inheritance seem to be the more or less ‘accidental’ result of other evolutionary events.     

栽培甜菜花粉发育过程的超微结构

利用透射电镜技术对栽培甜菜（Beta vuigaris）花粉发育过程进行了超微结构观察。结果表明,在小孢子母细胞减数分裂期间,细胞内发生了“细胞质改组”,主要表现在核糖体减少,质体和线粒体结构发生了规律性变化。末期1不形成细胞板,而是在2个子核间形成“细胞器带”。“细胞器带”的存在起到类似细胞板的作用,暂时将细胞质分隔成两部分。四分体呈四面体型,被胼胝质壁包围。小孢子外壁的沉积始于四分体晚期,至小孢子晚期外壁已基本发育完全。单核小孢子时期,细胞核大,细胞器丰富。二细胞花粉发育主要表现在生殖细胞壁的变化上,生殖细胞壁上不具有胞间连丝。成熟花粉为三细胞型,含有1个营养细胞和2个精细胞。精细胞具有短尾突,无壁,为裸细胞,每个精细胞通过2层质膜与营养细胞的细胞质分开。生殖细胞与精细胞里缺乏质体。     

Observations on the Neocytoplasm and Proteid Vacuoles During the Fertilization of Keteleeria evelyniana

Distributions and dynamics of the neocytoplasm and proteid vacuoles during the fertilization of Keteleeria evelyniana were studied by histochemical methods. Before fertilization cytoplasmic sheath surrounding the male and female gametes was indistinct. After fertilization, the dense neocytoplasm appeared around the zygote. Part of the neocytoplasm is invaded by mitochondria of maternal origin which had collected in large numbers in the perinuclear zone. The mitochondria contain electron compact little body which looks like a nucleus in the cytoplasm, but not observed in the rosette tier cell of proembryo and jacket cells. Hence, it was showed that the neocytoplasm participated in the development of embryo by all these observations. By using Feulgen reaction, the staining reaction of neocytoplasm was positive, the egg nucleus or zygote nucleus was weaker in positive reaction, while the proteid vacuoles were negative. When the proembryo developed, there were a few starch grains accumulated in the other three tiers except the upper tier. The Feulgen reaction was in- creased in intensity in the suspensor tier and embryonal cell tier nuclei. When the young embryo developed, Feulgen reaction became normal in the nuclei of the embryo initials. The embryo initials and Suspensor cells showed very weak Feulgen positive reaetion in the proembryo and young embryo. The development of the large proteid vacuoles was from plastid. During the early stage of egg nucleus, contents of large proteid vacuoles were less. When the zygote was formed, they reached the highest. However, after the zygote produced, the proteid vacuoles and egg cytoplasm were getting disintegrated following the course of fission of free nuclei. After the proembryo formed, the proteid vacuoles were wholly disintegrated.     

云南油杉受精过程中新细胞质及蛋白泡的动态观察

云南油杉(Keteleeria evelyniana Mast)在受精前,精核与卵核周围的细胞质鞘不明显。受精后,合子核周围出现细密的新细胞质。应用孚尔根核染色法,可以较清晰地将新细胞质染出,呈现较弱的正反应,而合子的核质及受精前的精核与卵核染色极弱。卵细胞质及其中的蛋白泡均为负反应。原胚形成后,除上层外,其余几层细胞质内开始积累淀粉粒。此时胚原细胞核的孚尔根染色深度有所增加。幼胚形成后,在顶端的胚原细胞群中核的孚尔根染色反应已恢复正常。在原胚及幼胚胚原细胞质中也呈现很弱的正反应。在电镜下,胚原层细胞质及新细胞质中均含有核样电子致密小体或称作染色质小体,而原胚莲座层细胞质及四周套细胞质中的线粒体则不含这种核样小体。因此,大蛋白泡在卵核形成的早期数量不多,当合子形成时含量最高,而随着游离核的分裂进程,蛋白泡以及原卵质均逐渐地解体,在原胚形成后全部消解。     

CAPSELLA EMBRYOGENESIS: THE SYNERGIDS BEFORE AND AFTER FERTILIZATION

The ultrastructure and composition of the synergids of Capsella bursa-pastoris were studied before and after fertilization. The synergids in the mature embryo sac contain numerous plastids, mitochondria, dictyosomes and masses of ER and associated ribosomes. Each synergid contains a large chalazal vacuole, a nucleus with a single nucleolus and is surrounded by a wall. This wall is thickest at the micropyle end of the cell where it proliferates into the filiform apparatus. At the chalazal end of the cell the wall thins and may be absent for small distances. The pollen tube grows into one of the two synergids through the filiform apparatus and extends one-third the length of the cell before it discharges. Following discharge of the pollen tube, mitochondria and plastids of the tube can be identified in the synergid as can hundreds of 0.5 μ polysaccharide spheres liberated by the tube. The method by which the sperm or sperm nuclei enter the egg or central cell is not known although an apparent rupture was found in the wall of the egg near the tip of the pollen tube. The second synergid changes at the time the pollen tube enters the first synergid. These changes result in the disorganization of the nucleus and loss of the chalazal wall and plasma membrane. Eventually this synergid loses its identity as its cytoplasm merges with that of the central cell.     

栽培甜菜花粉发育过程的超微结构

利用透射电镜技术对栽培甜菜(Beta vulgaris)花粉发育过程进行了超微结构观察。结果表明, 在小孢子母细胞减数分裂期间, 细胞内发生了“细胞质改组”, 主要表现在核糖体减少, 质体和线粒体结构发生了规律性变化。末期I 不形成细胞板,而是在2个子核间形成“细胞器带”。“细胞器带”的存在起到类似细胞板的作用, 暂时将细胞质分隔成两部分。四分体呈四面体型, 被胼胝质壁包围。小孢子外壁的沉积始于四分体晚期, 至小孢子晚期外壁已基本发育完全。单核小孢子时期, 细胞核大, 细胞器丰富。二细胞花粉发育主要表现在生殖细胞壁的变化上, 生殖细胞壁上不具有胞间连丝。成熟花粉为三细胞型, 含有1个营养细胞和2个精细胞。精细胞具有短尾突, 无壁, 为裸细胞, 每个精细胞通过2层质膜与营养细胞的细胞质分开。生殖细胞与精细胞里缺乏质体。     

The zygote and proembryo of alfalfa: quantitative,three-dimensional analysis and implications for biparental plastid inheritance

Summary Genetic studies have demonstrated biparental inheritance of plastids in alfalfa. The ratio of paternal to maternal plastids in the progeny varies according to the genotypes of the parents, which can be classified as strong or weak transmitters of plastids. Previous cytological investigations of generative cells and male gametes have provided no consistent explanation for plastid inheritance patterns among genotypes. However, plastids in the mature egg cells of a strong female genotype (6–4) were found to be more numerous and larger than in mature eggs of a weak female genotype (CUF-B), and the plastids in 6–4 eggs are positioned equally around the nucleus. In CUF-B, the majority of plastids are positioned below (toward the micropyle) the mid level of the nucleus, which is the future division plane of the zygote. Since only the apical portion of the zygote produces the embryo proper, plastids in the basal portion were predicted to become included in the suspensor cells and not be inherited. In the present study, we examined zygotes and a two-celled proembryo from a cross between CUF-B and a strong male genotype (301), a cross that results in over 90% of the progeny possessing paternal plastids only. Our results indicate that the distribution of plastids observed in the CUF-B egg cell is maintained through the first division of the zygote. Further, paternal plastids are similarly distributed; however, within the apical portion of the zygote and in the apical cell of the two-celled proembryo, the number of paternal plastids is typically much greater than the number of maternal plastids. These findings suggest that maternal and paternal plastid distribution within the zygote is a significant factor determining the inheritance of maternal and paternal plastids in alfalfa.     

油松细胞质遗传的细胞学机理：Ⅰ.雄配子的超微结构和细胞器类核

证明了油松（Ｐｉｎｕｓ ｔａｂｕｌａｅｆｏｒｍｉｓ Ｃａｒｒ．）雄配子存在质体物线粒体及细胞器ＤＮＡ,提供了油松具父系质体和线粒体遗传基础的确切的细胞学证据,结果与松科植物在遗传学上已确定的父系质体遗传的一般规律是一致的。但精细胞中的线粒体是否能传递至胚,还需要追踪其后的发育过程。另一重要的结果是揭示了油松的雄配子是细胞,这与以前将松科植物雄配子归入雄核（精核）的类型不同。精细胞无壁,仅被质膜包围     

Cytological Mechanism of Cytoplasmic Inheritance in Pinus tabulaeformis: I. Ultrastructural Aspects and Nucleoids of the Male Gametes

The cytological mechanism of plastid and mitochondrion inheritance in Pinus is an interesting research topic with only a limited number of published articles. The results indicate that the sperms of Pinus tabulaeformis Carr. contain abundant plastids, mitochondria and organelle DNA. These data provide reliable cytological evidence of paternal plastid and mitochondrion inheritance in Pinus. The results are in line with the confirmed general rule of paternal plastid inheritance in Pinaceae. But whether mitochondria in sperm cells can be transmitted into the embryos is an issue needs further developmental studies. Another important finding is that contrary to earlier classification of the male gamete of Pinaceae into the male nuclei type, the results reveal that male gametes in Pinus tabulaeformis are actually cells. However, the sperm cells are only surrounded by plasma membranes without cell walls. The larger leading sperm cell in a pollen tube section is long in shape, with a large amount of cytoplasm; while the second sperm cell is smaller, round in shape and contains less cytoplasm. Whether this feature of the male gamete type could be considered as a representative characteristic of the family is discussed and further conclusions await more experimental evidences from studies on plants from different species.     

Exceptional inheritance of plastids via pollen in Nicotiana sylvestris with no detectable paternal mitochondrial DNA in the progeny

Plastids and mitochondria, the DNA‐containing cytoplasmic organelles, are maternally inherited in the majority of angiosperm species. Even in plants with strict maternal inheritance, exceptional paternal transmission of plastids has been observed. Our objective was to detect rare leakage of plastids via pollen in Nicotiana sylvestris and to determine if pollen transmission of plastids results in co‐transmission of paternal mitochondria. As father plants, we used N. sylvestris plants with transgenic, selectable plastids and wild‐type mitochondria. As mother plants, we used N. sylvestris plants with Nicotiana undulata cytoplasm, including the CMS‐92 mitochondria that cause cytoplasmic male sterility (CMS) by homeotic transformation of the stamens. We report here exceptional paternal plastid DNA in approximately 0.002% of N. sylvestris seedlings. However, we did not detect paternal mitochondrial DNA in any of the six plastid‐transmission lines, suggesting independent transmission of the cytoplasmic organelles via pollen. When we used fertile N. sylvestris as mothers, we obtained eight fertile plastid transmission lines, which did not transmit their plastids via pollen at higher frequencies than their fathers. We discuss the implications for transgene containment and plant evolutionary histories inferred from cytoplasmic phylogenies.     

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.     

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.     

小麦受精过程中酸性磷酸酶的超微细胞化学定位

小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）受精前成熟胚囊,除胚囊中央细胞的合点端细胞质中有酸性磷酸酶外,其余部位均未发现酸性磷酸酶。受精时期,以下部位存在酸性磷酸酶活性：卵细胞的细胞核内一部分染色质和细胞质中大部分线粒体;精、卵核融合时两核的核周腔内;退化助细胞合点端细胞质和一些液泡内;进入雌性细胞中的两个精核;胚囊各成员细胞的细胞壁及胚囊周围珠心细胞的细胞壁。二细胞原胚中未见有酸性磷酸酶。早期胚乳游离核染色质上有酸性磷酸酶。小麦受精过程酸性磷酸酶的分布特点可能与卵细胞生理状态的变化和细胞质中线粒体的改组、助细胞的退化、精核的生理状态以及精核与卵核的核膜融合等有关。