Cytoplasmic Inheritance of Sweet Potato: with Respect to the Study of Plastids and Mitochondria and the Existence of Their DNA in Sperm Cells

The mature pollen of sweet potato ( Ipomoea batatas lam. ) was bicellular. After pollination generative cell divided into a pair of sperm cells before its germination. The pair of sperm cells remained in the hydrated pollen was similar in their shape and volume with enriched cytoplasmic plastids and mitochondria. The specific fluorescence of cytoplasm DNA indicated that the sperm cells and the generative cell contained numerous organelle nucleoids. The pair of sperm cells had no significant difference in their numbers of organelle nucleoids. Two kinds of organelle nucleoids existed in the pair of sperm cells. Tile ones as big and strong fluorescent dots appeared to be the plastid nucleoids and the others as tile small and weak fluorescent dots could be the mitochondrial nucleoid. Few of the angiosperms were of biparental or paternal plastid inheritance. The result of this study has provided the cytological evidence for another genus, Ipomoea, which is of biparental or paternal plastid inheritance besides Pharbitis and Calystegia in Convolvulaceae.     

打碗花生殖细胞,精细胞及卵细胞中的细胞质类核

已有不少超微结构的资料阐明被子植物双亲和单亲母系质体遗传的细胞学基础。近年应用DAPI荧光染色的方法,可快速地从检测质体DNA存在的状况确定被子植物中具双亲遗传潜能的种。从质体的类核存在与否判断质体遗传方式为母系遗传或双亲遗传与已有的遗传分析结论基本一致,只有少数种类是矛盾的。DAPI荧光技术可以认为是研究细胞质遗传机理的一个重要手段。我们曾证明旋花科牵牛属植物生殖细胞、精细胞中存在细胞质类核,确定其具双亲或单亲父系质体遗传的潜能,并用RFLP技术进一步确定其为质体父系遗传型。本研究证明旋花科的打碗花属生殖细胞、精细胞和卵细胞中细胞质类核存在的状况与牵牛属的相似,提供了打碗花可能在质体遗传上与牵牛属 具相同的遗传方式的资料。     

Cytoplasmic Nucleoids in the Generative Cell,Sperm Cell and Egg Cell of Calystegia heder acea

Cytoplasmic nucleoids in the generative cell of mature pollens, sperm cells of pollens cultured in vitro and egg cell of mature embryo sac in Calystegia bederacea Wall. were studied by means of the DNA fluorochrome DAPI in conjunction with epitluorescence microscopy for in situ detection of cytoplasmic DNA in cells. Results showed that many cytoplasmic DNA nucleoids were present in the generative cell and speim cells. Two types of nucleoids were observed, one with big and strong fluorescent dots, and the other with small and weak fluorescence. Many dot-shaped and a few circle-shaped nucleoids were randomly distributed in the thin layered cytoplasm of the egg cell. It was suggested that different types of nucleoids might represent plastid DNA and mitochondrion DNA respectively. Results provided cytological data that Calystegia hederaeea had the potential of plastid DNA biparental inheritance, and the mode of which merits further study via molecular biological methods.     

Changes of Plastids Mitochondria and Their DNA in the Egg Cell Before and After Fertilization in Pharbitis

The ultrastructure and cytoplasmic DNA in the egg cell and zygote of Pharbitis purpurea, (L.) Voyght and P. limbata Lindl. which were studied with electron microscopy and DNA epifiuorescence microscopy. The egg cell before fertilization was highly vacuolated with only a few cytoplasmic plastids and mitochondria. Plastids were spherical and/or rod- shaped containing 1 ～ 2 large starch grains. Most of the mitochondria were cup and/or circular. The cytoplasm in the zygote was much more abundant than that in the egg cell. The number of plastids and their electronic density were greatly increased, in most of which containing osmiophilic bodies. The mitochondria were rich and spherical-shaped in the zygote. Two types of cytoplasmic DNA nucleoids were detected in the egg cell, the more abundant one being big and circle-shaped and the other dot-shaped. Only dot-shaped nucleoids were present in the zygote. The content of nucleoids in the zygote was much less than that in the egg cell. Authors propose that some cytoplasmic DNA may degenerate after fertilization. The ultrastructural characteristics of the egg cell and the reduction of cytoplasmic DNA in the zygote may related to the mechanisms of plastid unipaternal inheritance in Pharbitis.     

Male gametophyte development inPlumbago zeylanica: cytoplasm localization and cell determination in the early generative cell

Summary The behavior of the generative cell during male gametophyte development inPlumbago zeylanica was examined by epifluorescence microscopy and electron microscopy with organelle nucleoid as a cytoplasm marker. When the thin sections stained with 4,6-diamidino-2-phenylindoIe (DAPI) were observed under an epifluorescence microscope, two types of fluorescence spots were detected in the cytoplasm of the pollen cells before the second mitosis. The spots emitting stronger fluorescence were confirmed as plastid nucleoids and those emitting dimmer fluorescence were mitochondrial nucleoids. Before the first mitosis, both plastid and mitochondrial nucleoids distributed randomly in the cytoplasm of the microspore. A small lenticular generative cell formed with attachment to the interior of the intine after the mitosis. Small vacuoles were found in the lenticular cell. In the cytoplasm of the lenticular cell, both plastid nucleoids and the small vacuoles were distributed randomly at the very beginning but began to migrate in opposite directions immediately. Plastid nucleoids aggregated to the side of the cell that faces the pollen center and the small vacuoles aggregated to the side of the cell that attaches to the inline. As the result, the lenticular generative cell appeared highly polarized in cytoplasm location soon after the first mitosis. In accordance with the definition of the cytoplasm polarization, the primary wall between the generative and the vegetative cells began to flex and the lenticular generative cell started to protrude towards the pollen center. When the generative cell peeled away from the inline, it was spherical in shape with the pole that aggregated plastids towards the vegetative nucleus. But the cell direction appeared to be transformed immediately. The pole that aggregated small vacuoles turned to the position towards the vegetative nucleus and the pole that aggregated plastid nucleoids turned to the position countering to the vegetative nucleus. A cellular protuberance formed at the edge of the pole that aggregated small vacuoles and elongated into a tapered end that got into contact with the vegetative nucleus. The polarization of the cytoplasm kept constant throughout the second mitosis. The small vacuoles that apportioned to the sperm cell which attached the vegetative nucleus (the leading sperm cell) disappeared during sperm cell maturation. Plastid nucleoids were apportioned to the other sperm cell (the trailing sperm cell) completely. Mitochondrial nucleoids became undetectable after the second mitosis.     

The Cytological Mechanism of Biparental Cytoplasmic Inheritance in Pelargonium hortorum -Ultrastructural and DNA Fluorescence Studies of Male and Female

Electron microscopic investigation has demonstrated that plastids and mitochondria are conserved in the generative cell, sperm cells and egg cell of Pelargonium hortorum Bailey. The plastids in the generative cell which contain starch for a short period, gradually changed to proplastids during the maturation of generative cell. The plastids in the sperm cells are large and numerous the characteristics of dense matrix and an abundant endomembrane systems. These plastids always appear ringlike in cross section. In the generative cell and sperms, the spherical or rod-shaped mitochondria are smaller than the.plastids and remain unchanged during the development process from generative cell to sperm cells. DNA filaments are visualized in the transparent central zone of the mitochondria. In the egg cell, plastids are more abundant than mitochondria. The structures of the plastids and mitochondria are obviously different from those in the sperm cell. Most of the plastids are irregularly rod-shaped and contain starch, the mitochondria are about 3 times larger than those in the sperm cells. Most of them are cup-shaped as proved by successive sections. DNA epifluorescence study demonstrated that DNA nucleoids are present in both plastids and mitochondria of the egg, generative cell and sperm cells. In the sperm cells, there is no ringlike DNA nucleoid as is existed in the egg cell. This study has defined the characteristics of the plastids and mitochondria in both male and female gemates of P. hortorurn. The results are essential contributions for further investigation of the biparental organelle transmission in the zygote and proembryo.     

天竺葵质体和线粒体双亲遗传的细胞学机理——雄性和雌性配子超微结构和DNA荧光的研究

天竺葵（Ｐｅｌａｒｇｏｎｉｕｍ ｈｏｒｔｏｒｕｍ Ｂａｉｌｅｙ）生殖细胞和精细胞在发育中始终存在质体和线粒体。在精细胞中,质体的体积大、数量多,具基质浓厚和在切面上多为环状的特点。线粒体在生殖细胞和精细胞中没有差异,体积较质体小得多,球形或杆状,边缘染色较深。在卵细胞中质体的含量比线粒体丰富,这两种细胞器的结构形态与精细胞的有明显的差异。细胞的质体多呈不规则的棒状和含淀粉粒。线粒体比精细胞的大２—３ 倍,许多为环状。ＤＮＡ 荧光的检测证明了在生殖细胞、精细胞和卵细胞中存在质体和线粒体类核。卵中的环状线粒体类核的形态在精细胞中是不存在的。本研究确定了雄性和雌性配子的质体和线粒体在结构形态上各具特点,可作为鉴别它们的标记,从合子中查明雄性质体和线粒体是否传递,以及在胚胎发育的早期雌雄亲本来源的细胞器的动态     

Quantitative study of organelle nucleoids during the second pollen grain mitosis inOenothera biennis

Summary The behavior of organelle nucleoids in the generative cell was examined at the second (pollen grain) mitosis by epifluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) inOenothera biennis. TheO. biennis generative cell contained a large number of organelle nucleoids distributed randomly in the cytoplasm before mitosis. The epifluorescence images of the nucleoids could be classified distinctly into two groups which corresponded to plastid nucleoids (pt-nucleoids) and mitochondrial nucleoids (mt-nucleoids). Discrimination between pt- and mt-nucleoids was carried out with the aid of DNA immunogold electron microscopy. At metaphase, both pt- and mt-nucleoids migrated to the pole regions of the generative cell. After mitosis, organelle nucleoids in both of the sperm cells scattered in the cytoplasm again. A quantitative examination of pt-nucleoids on 202 pairs of sperm cells showed that the leading sperm cell (Svn) contained 0–39 pt-nucleoids (19.0 ± 7.4) and the trailing sperm cell (Sua) contained 0–40 pt-nucleoids (15.4 ± 6.5). For mt-nucleoids, examination of 28 pairs of sperm cells showed that Svn contained 5–32 mt-nucleoids (14.5 ± 6.8) and Sua contained 6–30 mt-nucleoids (13.4 ±7.5). These results showed that (1) the number of organelle nucleoids per sperm cell varied considerably in the cells studied; (2) quantitative difference in pt- and mt-nucleoids between Svn and Sua could occur in some gametophytes studied; but (3) it was unlikely that there was any pre-differentiational cytoplasm localization and essential sperm heteromorphy with respect to organelle nucleoid content in the gametophyte population.     

Cytoplasmic DNA apportionment and plastid differentiation during male gametophyte development in Pelargonium zonale

In the male gametophyte of Pelargonium zonale, generative and sperm cells contain cytoplasmic DNA in high density compared to vegetative cells. Cytoplasmic DNA was examined using the DNA fluorochrome DAPI (4'6-diamidino-2-phenylindole) and observed with epifluorescence and electron microscopy. The microspore cell contains a prominent central vacuole before mitosis; mitochondria and plastids are randomly distributed throughout the cytoplasm. Following the first pollen grain mitosis, neither the vegetative cell nor the early generative cell display a distributional difference in cytoplasmic DNA, nor is there in organelle content at this stage. During the maturation of the male gametophyte, however, a significant discrepancy in plastid abundance develops. Plastids in the generative cell return to proplastids and do not contain large starch grains, while those in the vegetative cell develop starch grains and differentiate into large amyloplasts. Plastid nucleoids in generative and sperm cells in a mature male gametophyte are easily discriminated after DAPI staining due to their compactness, while those in vegetative cells stained only weakly. The utility of the hydrophilic, non-autofluorescent resin Technovit 7100 in observing DAPI fluorescence is also demonstrated.     

Quantitative ultrastructural analysis of barley sperm

Summary Complete serial ultrathin sections of seven sperm pairs, computer-assisted measurements of cell, nuclear and organelle surface areas and volumes, and three-dimensional imagery were used to demonstrate that a process of cytoplasm and organelle elimination occurs during sperm maturation in barley. The number of mitochondria per sperm cell is reduced by 50%; sperm cell surface area and volume are reduced by 30% and 51% respectively. Mean volume and surface area per mitochondrion are significantly less in mature sperms. No examples of mitochondrial fusion or degeneration were observed within sperm cells. These data, along with observations of plasma membrane apposition and vesiculation within cytoplasmic extensions containing mitochondria, support the proposition that cytoplasm and organelle loss results primarily from the formation of cytoplasmic projections that are subsequently discarded from the sperm cell body. Comparisons of the quantitative data, including the number of mitochondria, indicate that differences between sperm cells of a pair are absent to very slight. Spatial organization within the pollen grain is such that the mature sperms, as well as the sperms and vegetative nucleus, are not in close proximity.     

Cytological Basis of Plastid Inheritance in Phaseolus vulgaris-Investigations of Plastids,Mitochondria and Their DNA Nucleoids During Pollen Development

Electron microscopic and DNA fluorescence microscopic observations of the plastids, mitochondria and their DNA in the developing pollen of Phaseolus vulgaris L. have demonstrated that the male plastids were excluded during microspore mitosis. The formed generative cell was free of plastids because of regional localization of plastids in early developing microspore and the extremely unequal distribution during division. The fluorescence observations of DNA showed that cytoplasmic (plastid and mitochondria) nucleoids degenerated and disappeared during the development of microspore/pollen, and were never presented in the generative cell at different development stages. These results provided precise cytological evidence of maternal plastid inheritance in Phaseolus vulgaris, which was not in accord with the biparental plastid inheritance identified from early genetic analysis. Based on authors' previous observations in a variety of common bean that the organelle DNA of male gamete was completely degenerated, the early genetic finding of the biparental plastid inheritance was unlikely to be effected by genotypic difference. Thus those biparental plastid inheritance might be caused by occational male plastid transmission, and plastid uniparental maternal inheritance was the species character of Phaseolus vulgaris.     

The male germ unit in the pollen and pollen tubes ofPetunia hybrida: Ultrastructural,quantitative and three-dimensional features

Summary The male germ unit ofPetunia hybrida was examined quantitatively and qualitatively at the ultrastructutral level. Three-dimensional reconstructions, the determination of nuclear and cytoplasmic volumes and surface areas, and organelle counts were obtained from serial ultrathin sections and computer analysis. In the mature pollen grain, an elongated generative cell is found in direct physical association with and partially surrounded by the vegetative nucleus. The mature generative cell lacks plastids and has mitochondria equally distributed at both of its tapering ends. In the pollen tube, the sperm cells are physically associated by cytoplasmic connections to each other and to the surrounding vegetative cell membrane. At full style length, the lobed vegetative nucleus and sperm pair are found in close association near the end of the pollen tube. The two sperms of a pair are not strongly dimorphic.     

杜鹃精细胞的超微结构及DNA荧光观察——着重阐明质体双亲遗传的细胞学基础

迎红杜鹃 ( Rhododendron mucronulatum Turcz.)的成熟花粉为二细胞型 ,精细胞在花粉管中形成。花粉管中的两个精细胞及与营养核之间互相联结 ,形成雄性生殖单位。两个精细胞的细胞质中均含有丰富的细胞器 ,包括质体、线粒体、小泡及微管 ,内质网和高尔基体稀少。具正常结构的精细胞质体在切面上多呈环形或哑铃形 ,内膜不发达 ,基质电子密度高。线粒体为球形或棒状 ,基质电子密度较低。 DNA特异性荧光染色显示 ,生殖细胞及精细胞中均含有大量类核 ( nucleoid) ,两个精细胞中的类核数量无明显差异。结果证明了杜鹃精细胞中存在大量具 DNA的可遗传细胞器 ,为杜鹃属植物的双亲细胞质遗传方式提供了细胞学证据。     

Ultrastructural and DNA Epifluorescence Observations of the Sperm Cells of Rhododendron-- with Emphasis on Biparental Plastid Inheritance

The mature pollen grains of Rhododendron mucronulatum Turcz. conform to the 2-celled type. Sperm cells differentiated within the pollen tube about 24 hours after germination in vitro and paired together, one of which being linked with the vegetative nucleus, forming a male germ unit (MGU). Abundance of plastids, mitochondria, microtubules and single-membrane-bounded vesicles could be visualized in each sperm cell, however, endoplasmic reticulum and Golgi apparatus were scarce. The electron-dense plastids with normal structure gave ring-like or dumbbell appearance in sections. Mitochondria were smaller and less electron-dense' in contrast to the plastids. DNA epifluorescence technique revealed that the generative and sperm cells contained numerous organelle nuclei (nucleoids). There was no difference in nucleoid number between the two sperm cells in a pollen tube. The results confirmed the possible existance of cytoplasmic inheritance potential of the male gametes of Rhododendron.     

Heritable paternal cytoplasmic organelles in alfalfa sperm cells: ultrastructural reconstruction and quantitative cytology.

Sperm cells within pollen grains and pollen tubes of alfalfa (Medicago sativa L.) were observed at the ultrastructural level, and their plastid DNA was detected by DAPI (4,6-diamidino-2-phenylindole) staining. One sperm pair within the pollen grain and three sperm pairs within pollen tubes were reconstructed in three-dimensions from serial ultrathin sections. The two sperm cells are linked by cytoplasmic bridges in both pollen grains and tubes, and the vegetative nucleus is closely associated with the sperm cells within the pollen tube. The number of plastids and plastid nucleoids (DNA aggregates) in the sperm cell pair, collectively, is not significantly different from that in the generative cell; however, over 60% of the sperm cell plastids contain no DNA detectable with DAPI. The mean number of mitochondria in sperm cells is reduced from that in the generative cell (from 54 to 17), which suggests that paternal mitochondrial inheritance probably does not occur in the genotype investigated. Sperm cells of a pair may vary in their shape within the pollen grain and tube, but the number of plastids and mitochondria is not significantly different between the sperm cells. Therefore, heterospermy is not a factor determining cytoplasmic inheritance patterns in this species.     

黑麦雄性细胞发育过程中细胞器DNA动态的荧光研究

DAPI(4’,6-diamidino-2-phenylindole)是一种DNA特异结合的荧光染料,可以用于在荧光显微镜下观察和检测各种DNA,尤其是细胞内含量甚微的DNA,包括质体DNA和线粒体DNA,其灵敏性和可靠性是被公认的,并得到了越来越多的Southern杂交实验的证明,而且实验操作简便易行。近几年,DAPI荧光技术已在细胞质遗传的研究领域获得了成功的应用。     

Examination of the cytoplasmic DNA in male reproductive cells to determine the potential for cytoplasmic inheritance in 295 angiosperm species

Mature pollen grains of 295 angiosperm species were screened by epifluorescence microscopy for a marker that denotes the mode of cytoplasmic inheritance. We used the DNA fluorochrome DAPI (4',6-diamidino-2-phenylindole) for pollen cell staining. The presence or absence of fluorescence of cytoplasmic DNA in the generative cell or sperm cells was examined in each species. The species examined represented 254 genera and 98 families, and 40 of these families had not been previously studied in this regard. The cytoplasmic DNA of the generative cell or sperm cells did not fluoresce in 81% of the species examined, from 83% of the genera and 87% of the families examined, indicating the potential for maternal cytoplasmic inheritance in these species. In contrast, the male reproductive cells of 19% of the species, from 17% of the genera and 26% of the families examined, displayed fluorescence of the cytoplasmic DNA, indicating the potential for biparental cytoplasmic inheritance in these species. The results revealed the potential for biparental cytoplasmic inheritance in several species in which the inheritance mode was previously unknown, including plants in the Bignoniaceae, Cornaceae, Cruciferae (Brassicaceae), Cyperaceae, Dipsacaceae, Hydrocharitaceae, Papaveraceae, Portulacaceae, Tiliaceae, Valerianaceae, and Zingiberaceae. Electron microscopy revealed that the sperm cells of Portulaca grandiflora contain both plastid and mitochondrial DNA. However, in the generative cells of Musella lasiocarpa, the mitochondria contain DNA, but the plastids do not. These data provide a foundation for further studies of cytoplasmic inheritance in angiosperms.     

Microgametogenesis in Plumbago zeylanica (Plumbaginaceae). 1. Descriptive cytology and three-dimensional organization

The generative cell is initiated as a small, lenticular, unpolarized cell with a cell wall traceable to two origins: the external segment originates as intine, while an inner callose positive cell wall forms de novo. As the lenticular generative cell begins its migration into the pollen cytoplasm, the generative cell becomes polarized both externally and internally, displaying a characteristic shape and patterns of organelle distribution oriented with respect to the vegetative nucleus and independent of pollen aperture location. Separation of the generative cell from the pollen wall begins at the end opposite the vegetative nucleus and results in an elongating protuberance at the opposite end of the generative cell; this becomes associated with a preformed groove located on the surface of the vegetative nucleus. The generative cell subsequently separates from the intine near the vegetative nucleus and moves progressively toward the opposite end of the cell; during this separation, the edge of the wall facing the intine becomes callose-positive and remains so until separating from the intine. The generative cell becomes a free cell within the pollen, which is in physical association with the vegetative nucleus. Generative cell organization and organelle content become increasingly polarized during maturation, with microtubules evident both in the elongating protuberance of the generative cell and in association with organelles. The generative nucleus migrates away from the vegetative nucleus and toward the plastid-rich end of the generative cell, whereas mitochondria are more generally distributed within the cell. Generative cell polarization is made permanent during mitotic division and cytokinesis, i.e., two sperm cells differing in morphology are formed: the larger cell associated with the vegetative nucleus (S_vn) contains a majority of the mitochondria, and the smaller, unassociated sperm cell (S_ua) receives the plastids.     

玉竹雄配子的发育——着重阐明质体在生殖细胞和营养细胞中的分布和变化

玉竹(Polygonatum simizui Kitag)小孢子在分裂前,质体极性分布导致分裂后形成的生殖细胞不含质体,而营养细胞包含了小孢子中全部的质体。生殖细胞发育至成熟花粉时期,及在花粉管中分裂形成的两个精细胞中始终不含质体。虽然生殖细胞和精细胞中都存在线粒体,但细胞质中无DNA类核。玉竹雄性质体的遗传为单亲母本型。在雄配子体发育过程中,营养细胞中的质体发生明显的变化。在早期的营养细胞质中,造粉质体增殖和活跃地合成淀粉。后期,脂体增加而造粉质体消失。接近成熟时花粉富含油滴。对百合科的不同属植物质体被排除的机理及花粉中贮藏的淀粉与脂体的转变进行了讨论。     

豌豆生殖细胞的发育—着重论述质体母系遗传的细胞学基础

超微结构的研究证明,豌豆（Ｐｉｓｕｍ ｓａｔｉｖｕｍ Ｌ．）生殖细胞自形成直至成熟花粉时期,始终存在少量质体和较多的线粒体。ＤＮＡ 荧光的观察表明,在发育早期的生殖细胞中不含细胞质ＤＮＡ 类核,但在成熟花粉的生殖细胞中有许多的类核。在花粉离体萌发过程中,随着花粉管的生长,生殖细胞中的类核逐渐降解。在花粉培养２４ ｈ 后,生殖细胞的类核全部消失。研究结果确定了豌豆质体母系遗传的细胞学基础,支持遗传分析及ＲＦＬＰ研究的结论,阐明了过去在细胞学上认为是双亲遗传的判断不正确的原因