Cytological Studies of the Cytoplasmic Inheritance in Lilium regale and L. davidii

The distribution and characteristics of plastids and mitochondria in the generative and sperm cells of Lilium regale Wils. and L. davidii Duch. were described. In L. regale there were few plastids and abundant mitochondria in the newly formed generative cell. When the generative cell became free in the vegetative cytoplasm, the plastids degenerated completely within the generative cell. It was further proved by DAPI fluorescent technique that there was no organell DNA in the generative cell within the mature pollen grain or the pollen tube. However, distribution of the plastids was strictly polarizable during the division of the micmspore in L. davidii, resulting the lack of plastids in the newly formed generative cell. Data of RFLP analysis comparable between L. davidii, L. longifiorum and their interspecific hybrid have also proved the plastid inheritance in L. davidii to be of uniparental maternal transmission. Although the mitoehondria were observed both in the generative and sperm cells of L. regale and L. davidii but their DNA was decomposed in the male gametophyte stage. Therefore the mitochondda in the sperm cell could not be transmitted into the offspring. The results provided the detail, cytological evidence that organelles in the microgametophyte are incapable of genetic transmission in the two species of Lilium.     

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.     

Monitoring by epifluorescence microscopy of organelle DNA fate during pollen development in five angiosperm species

The fates of mitochondrial and plastid nucleoids during pollen development in six angiosperm species (Antirrhinum majus, Glycine max, Medicago sativa, Nicotiana tabacum, Pisum sativum, and Trifolium pratense) were examined using epifluorescence microscopy after double staining with 4',6-diamidino-2- phenylindole (DAPI) to stain DNA and with a potentiometric dye (either DiOC7 or rhodamine 123) for visualization of metabolically active mitochondria. From the pollen mother cell stage to the microspore stage of pollen development, mitochondria and plastids both contained DNA detectable by DAPI staining. However, during the further maturation preceding anthesis, mitochondrial DNA became undetectable cytologically in either the generative or the vegetative cell of mature pollen; even in germinated pollen tubes containing hundreds of metabolically active mitochondria undergoing cytoplasmic streaming, vital staining with DAPI failed to reveal mitochondrial DNA. By the mature pollen stage, plastid DNA also became undetectable by DAPI staining in the vegetative cell. However, in the generative cell of mature pollen the timing of plastid DNA disappearance as detected by DAPI varied with the species. Plastid DNA remained detectable only in the generative cells of pollen grains from species known or suspected to have biparental transmission of plastids. The apparent absence of cytologically detectable organelle genomes in living pollen was further examined using molecular methods by hybridizing organelle DNA-specific probes to digests of total DNA from mature pollen and from other organs of A. majus and N. tabacum, both known to be maternal for organelle inheritance. Mitochondrial DNA was detected in pollen of both species; thus the cytological alteration of mitochondrial genomes during pollen development does not correspond with total mtDNA loss from the pollen. Plastid DNA was detectable with molecular probes in N. tabacum pollen but not in A. majus pollen. Since the organelle DNA detected by molecular methods in mature pollen may lie solely in the vegetative cell, further study of the basis of maternal inheritance of mitochondria and plastids will require molecular methods which distinguish vegetative cell from reproductive cell organelle genomes. The biological effect of the striking morphological alteration of organelle genomes during later stages of pollen development, which leaves them detectable by molecular methods but not by DAPI staining, is as yet unknown.     

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。     

Preferential degradation of plastid DNA with preservation of mitochondrial DNA in the sperm cells ofPelargonium zonale during pollen development

Summary In the present study, we studied changes in organellar DNA in the sperm cells of maturing pollen ofPelargonium zonale, a plant typical to exhibit biparental inheritance, by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and by immunogold electron microscopy using anti-DNA antibody. Fluorescence intensities of DAPI-stained plastid nuclei in generative and sperm cells at various developmental stages were quantified with a video-intensified microscope photon counting system (VIMPCS). Results indicated that the amount of DNA per plastid in generative cells increased gradually during pollen development and reached a maximum value (about 70 T per plastid; 1 T represents the amount of DNA in a particle of T4 phage) in young sperm cells at 5 days before flowering. However, the DNA content of plastids was subsequently reduced to about 20% of the maximum value on the day of flowering. Moreover, the DNA content of the plastid further decreased to 4% of the maximum value when pollen grains were cultured for 6 h in germination medium. In contrast, the amount of DNA per mitochondrion did not decrease significantly around the flowering day. Similar results were also obtained by immunogold electron microscopy using anti-DNA antibody. The density of gold particles on plastids decreased during pollen maturation whereas labelling density on mitochondria remained relatively constant. The number of plastids and mitochondria per generative cell or per pair of sperm cells did not change significantly, indicating that the segregation of DNA by plastid division was not responsible for the decrease in the amount of DNA per plastid. These results indicate that the plastid DNA is preferentially degraded, but the mitochondrial DNA is preserved, in the sperm cells ofP. zonale. While the plastid DNA of the sperm cells decreased before fertilization, it was also suggested that the low DNA contents that remain in the plastids of the sperm cells are enough to account for the biparental inheritance of plastids inP. zonale.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system     

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.     

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.     

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.     

Potential for biparental cytoplasmic inheritance in Jasminum officinale and Jasminum nudiflorum

 Mature Jasminum officinale and J. nudiflorum pollen grains were stained with 4′,6-diamidino-2-phenylindole (DAPI) and examined by epifluorescence microscopy. The pollen grains were found to be trinucleate, and the sperm cells in both species contained a large number of epifluorescent spots that corresponded to cytoplasmic DNA aggregates (nucleoids). The nucleoids of J. nudiflorum were observed to be dimorphic under the epifluorescence microscope, indicating that the sperm cells might contain both plastid and mitochondrial DNA. The nucleoids of J. officinale presented a similar appearance when stained with DAPI, but electron microscopic examination of the sperm cells revealed that they contained both plastids and mitochondria. When analyzed by DNA immunogold electron microscopy, gold particles were detected on both plastids and mitochondria. These findings demonstrated the preservation of plastid and mitochondrial DNA in mature sperm cells and thus the potential for biparental cytoplasmic inheritance in J. officinale and J. nudiflorum. Received: 8 August 1997 / Revision accepted: 25 February 1998     

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。     

Populations of plastids and mitochondria during male reproductive cell maturation in Nicotiana tabacum L.: A cytological basis for occasional biparental inheritance

The dynamics of plastid and mitochondrial populations in male reproductive cells of tobacco (Nicotiana tabacum L.) were examined during development using serial ultrathin sections and transmission electron microscopy to reconstruct 58 generative cells and 31 sperm cells at selected stages of maturation from generative cell formation through gametic fusion. The first haploid mitosis resulted in incomplete exclusion of plastids providing an average of 2.81 plastids and 82.7 mitochondria for each newly formed generative cell. During generative-cell maturation, plastid content decreased to an average of 0.48 plastids/generative cell at anthesis owing to autophagy of organelles. Plastids were present in low frequency within generative and sperm cells in the pollen tube and appeared to be transmitted, according to observations immediately prior to fertilization. This forms a cytological basis for genetic reports of occasional biparental plastid inheritance. In contrast, mitochondria were transmitted in larger numbers, and approximately 80 mitochondria per generative cell or sperm cell pair were retained throughout development. This provides a potentially stable source for the transmission of male mitochondrial DNA, if present at fertilization.Abbreviations GC generative cell - SC sperm cell We thank Dr. Frank J. Sonleitner, for helpful suggestions on the statistical calculations and Dr. Bing-Quan Huang for technical assistance in the preparation of embryo sacs during fertilization. This research was supported in part by U.S. Department of Agriculture grant 91-37304-6471. We gratefully acknowledge use of the Samuel Roberts Noble Electron Microscopy Laboratory of the University of Oklahoma.     

Ultrastructural studies on plastids of generative and vegetative cells inLiliaceae 3. Plastid distribution during the pollen development inGasteria verrucosa (Mill.) Duval

Summary This paper describes the development of pollen grains ofGasteria verrucosa from the late microspore to the mature two-cellular pollen grain. Ultrastructural changes and the distribution of plastids as a result of the first pollen mitosis have been investigated using light and electron microscopy. The microspores as well as the generative and the vegetative cell contain mitochondria and other cytoplasmic organelles during all of the observed developmental stages. In contrast, the generative cell and the vegetative cell show a different plastid content. Plastids are randomly distributed within the microspores before pollen mitosis. During the prophase of the first pollen mitosis the plastids become clustered at the proximal pole of the microspore. The dividing nucleus of the microspore is located at the distal pole of the microspore. Therefore, the plastids are not equally distributed into both the generative and the vegetative cell. The possible reasons for the polarization of plastids within the microspore are briefly discussed. The lack of plastids in the generative cell causes a maternal inheritance of plastids inGasteria verrucosa.     

被子植物质体遗传的细胞学研究

植物细胞质遗传涉及细胞质中含DNA的两种细胞器——质体和线粒体从亲代至子代的传递。相对来说线粒体遗传的研究远不及质体的多,这可能是线粒体这种细胞器缺乏合适的表型突变体之故。高等植物质体遗传的研究历史可追溯到本世纪初在杂交试验中对叶色遗传的非孟德尔定律的发现,Baur在马蹄纹天竺葵(Pelargonium zonale)中从叶色突变体(白化体)的杂交遗传分析,发现了双亲质体遗传;而Correns在紫茉莉(Mirabilis jalapa)中则发现了单亲母本质体遗传(见Kuroiwa)。此后,对质体基因组突变性状遗传分析的研究,大量的资料说明了在被子植物中存在双亲质体遗传和单亲母系质体遗传两种类型,而后一种占大多数,仅少数是比较有规律的为双亲质体遗传或偶尔是双亲质体遗传。几十年来应用遗传分析的方法对被子植物质体遗传的研究,着重于揭示不同植物种质体的遗传是单亲母系或是双亲质体传递,以及探索杂种核基因对质体传递方式的影响。     

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.     

菜豆花粉发育中质体和线粒体及其DNA存在的状况——着重阐明质体遗传的细胞学基础

应用电镜和DNA的DAPI荧光检测技术研究了菜豆(Phaseolus vulgaris L.)小孢子/花粉发育中质体和线粒体及其DNA存在的状况。观察表明:在小孢子分裂时质体全部分配到营养细胞中,初形成的生殖细胞已不含质体。线粒体和质体的DNA在花粉发育中也先后降解,生殖细胞从刚形成时发育至成熟花粉时期这两种细胞器DNA均不存在。研究结果为菜豆质体母系遗传提供了确切的细胞学证据。遗传分析的研究曾确定菜豆质体为双亲遗传,对与本研究结论不同的原因进行了讨论。     

Studies of Generative Cell Development in Pisum sativum--With Special Reference to Cytological Basis of Plastid Maternal Inheritance

It was proved by ultrastructural observations that few plastids and abundent mitochondria were ever present in the generative cell of Pisurn sativurn L. from its initiation to maturation. Fluorescence observations of DNA showed that many cytoplasmic DNA nucleoids were present in generative cell of mature pollen, but none in the early developing generative cell. During the germination of mature pollen in vitro, the cytoplasmic DNA nucleoids of the generative cell in the pollen tube degenerated gradually following the growth of the pollen tube and disappered completely 24 h after germination. The results provided a cytological basis for confirming the conclusion of plastid maternal inheritance in P. sativurn obtained from genetic study, and resolved the contradiction between results from cytological observation and genetic or RFLP analysis.     

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     

The male germ unit of Rhododendron: quantitative cytology,three-dimensional reconstruction,isolation and detection using fluorescent probes

Summary The sperm cells of Rhododendron laetum and R. macgregoriae differentiate within the pollen tube about 24 h after germination in vitro. Threedimensional reconstruction shows that the sperm cells are paired together, and both have extensions that link with the tube nucleus, forming a male germ unit. Quantitative analysis shows that the sperm cells in each pair differ significantly in surface area, but not in cell volume nor in numbers of mitochondria or plastids. When isolated from pollen tubes by osmotic shock, the sperm cells became ellipsoidal and surrounded by their own plasma membrane, while a proportion remained in pairs linked by the inner tube plasma membrane. Both generative and sperm cells are visualized in pollen tube preparations by immunofluorescence with anti-tubulin and anti-actin monoclonal antibodies (MAbs) combined with H33258 fluorescence of the nuclei. Video-image processing shows the presence of an axial microtubule cage in the generative cells, and some microtubules are present in the cytoplasmic extensions that clasp the tube nucleus. Following sperm cell division, the extensive phragmoplast between the sperm nuclei is partitioned by the plasma membranes.     

Divergent potentials for cytoplasmic inheritance within the genus Syringa. A new trait associated with speciogenesis

Epifluorescence microscopic detection of organelle DNA in the mature generative cell is a rapid method for determining the potential for the mode of cytoplasmic inheritance. We used this method to examine 19 of the known 22 to 27 species in the genus Syringa. Organelle DNA was undetectable in seven species, all in the subgenus Syringa, but was detected in the 12 species examined of the subgenera Syringa and Ligustrina. Therefore, species within the genus Syringa display differences in the potential cytoplasmic inheritance. Closer examination revealed that the mature generative cells of the species in which organelle DNA was detected contained both mitochondria and plastids, but cells of the species lacking detectable organelle DNA contained only mitochondria, and the epifluorescent organelle DNA signals from the mature generative cells corresponded to plastid DNA. In addition, semiquantitative analysis was used to demonstrate that, during pollen development, the amount of mitochondrial DNA decreased greatly in the generative cells of the species examined, but the amount of plastid DNA increased remarkably in the species containing plastids in the generative cell. The results suggest that all Syringa species exhibit potential maternal mitochondrial inheritance, and a number of the species exhibit potential biparental plastid inheritance. The difference between the modes of potential plastid inheritance among the species suggests different phylogenies for the species; it also supports recent conclusions of molecular, systematic studies of the Syringa. In addition, the results provide new evidence for the mechanisms of maternal mitochondrial inheritance in angiosperms.     

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.