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     

Quantitative cytology of the alfalfa generative cell and its relation to male plastid inheritance patterns in three genotypes

Summary Studies utilizing restriction analysis of plastid DNA, as well as those employing chlorophyll-deficient mutants, have shown a high frequency of paternal plastid transmission in alfalfa. Recent research has also shown that plastid inheritance patterns among alfalfa genotypes and are under genetic control. In a previous study we were unable to detect any correlations between qualitative, three-dimensional ultrastructure of generative cells and male plastid transmission strength in certain genotypes. In the present study we used serial ultrathin sectioning, computerized reconstruction and quantitation, and stereology to further analyze generative cells within mature pollen. Measurements included volumes and surface areas of cells, nuclei, and organelles, as well as organelle number and distribution. Three genotypes were investigated, one that is a strong transmitter of male plastids (genotype 301), one that is a weaker transmitter of male plastids (genotype 7W), and a third that is an even weaker male plastid transmitter (genotype MS-5). Our results show that genotype MS-5 has significantly fewer plastids/generative cell than either of the other genotypes, which may account for it being a relatively poor transmitter of male plastids. However, plastid number does not explain known differences in male plastid inheritance between genotypes 301 and 7W, since plastid number does not differ significantly between these two genotypes. Regarding the other features of generative cells measured in this study, no consistent correlations were found that might account for differences in male plastid inheritance patterns between genotypes. Plastid distribution is equal in each end of the spindle-shaped generative cell in all genotypes studied. Similar relative results were found with regard to mitochondria within generative cells; however, comparative genetic data are not available on mitochondrial transmission patterns in alfalfa genotypes.     

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 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.     

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。     

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     

Microgametogenesis in Plumbago zeylanica (Plumbaginaceae). 2. Quantitative cell and organelle dynamics of the male reproductive cell lineage

Quantitative cell and organelle dynamics of the male gamete-producing lineage of Plumbago zeylanica were examined using serial transmission electron microscopic reconstruction at five stages of development from generative cell inception to sperm cell maturity. The founder population of generative cell organelles includes an average of 3.88 plastids, 54.9 mitochondria, and 3.7 vacuoles. During development the volume of the pollen grain increases from 6,200 μm³ in early microspores to 115,000 μm³ at anthesis, cell volume of the male germ lineage decreases more than 67% from 362.3 μm³ to 118.4 μm³. By the time the generative cell separates from the intine, plastid numbers increase by >600%, mitochondria by 250%, and vesicles by 43 times. A cellular projection elongates toward and establishes an association with the vegetative nucleus; this leading edge contains plastids and numerous mitochondria. When the generative cell completes its separation from the intine, organellar polarity is reversed and plastids migrate to the opposite pole of the cell. Cytoplasmic microtubules are common in association with cellular organelles. Plastids accumulate at the distal end of the cell as a linked mass, apparently adhered by lateral electron dense regions. Before division of the highly polarized generative cell, plastids decrease in number by 16%, whereas mitochondria increase by ∼90% and vacuoles increase by ∼140% from the prior stage. After mitosis, the resultant sperm cells differ in size and organelle content. The sperm cell associated with the vegetative nucleus (S_vn) contains 62.7% of the cytoplasm volume, 87% of the mitochondria, 280.4 vesicles (79% of those in the generative cell), and 0.6% of the plastids. At maturity, the S_vn mitochondria increase by 31% and the cell contains an average of 0.4 plastids, 158.9 vesicles, and 0.36 microbodies. The mature unassociated sperm (S_ua) contains 39.8 mitochondria (up 3.3%), 24.3 plastids (down 31%), 91.1 vesicles (up 54.9%), and 3.18 microbodies. The small number of organelles initially in the generative cell, followed by their rapid multiplication in a shrinking cytoplasm suggests a highly competitive cytoplasmic environment that would tend to eliminate residual organellar heterogeneity. Cell and cytoplasmic volumes vary as a consequence of fluctuations in the number and size of large vesicles or vacuoles, as well as loss of cytoplasmic volume by (1) formation of “false cells” involving amitotic cytokinesis, (2) “pinching off” of cytoplasm, and (3) dehydration of pollen contents prior to anthesis.     

CHANGES IN PLASTID AND MITOCHONDRION CONTENT DURING MATURATION OF GENERATIVE CELLS OF SOLANUM (SOLANACEAE)

A study was made of the number of plastids and mitochondria present in generative cells of Solanum immediately after microspore mitosis, and the fate of these organelles during development of the pollen was determined. Changes were followed via electron microscopy of anthers of S. chacoense and S. tuberosum Group Phureja × S. chacoense. In earliest stages the generative cells were oval and had one surface along the intine and other surfaces in contact with the vegetative cell. As the pollen matured the generative cells elongated, became spindle-shaped, and were completely engulfed in the vegetative cells. At the earliest stages studied, both mitochondria and plastids were present in the generative cell. Plastids of the generative cell were, in contrast to those of the vegetative cells, fewer, smaller, and lacking in starch. Through the maturation stages the content of these organelles in the vegetative cells remained unchanged. While the generative cells retained mitochondria until anthesis, their plastids disappeared completely during maturation. This selective loss during generative cell maturation could lead to transmission of those characteristics encoded in plastid DNA through the pistillate parent only. The mechanism could explain earlier genetic evidence that plastid characters of Solanum were transmitted uniparentally.     

Generative cell composition and its relation to male plastid inheritance patterns inMedicago sativa

Summary Genetic studies have recently shown that plastids are inherited biparentally in alfalfa; yet most crosses produce high frequencies of progenies containing only paternal plastids, and certain genotypes have been characterized as relatively strong or weak transmitters of male plastids. The objective of the present study was to determine whether the structure of generative cells differs among genotypes known to differ in male plastid transmission pattern. Using the techniques of serial ultrathin sectioning and three-dimensional reconstruction, we found that mature generative cells of the genotypes investigated have basically similar morphology, and contain numerous plastids in each end of the spindle shaped cell. Since the morphological variation that does occur is as great within a genotype as it is between genotypes, it does not appear that generative cell structure can be used to predict male plastid transmission behavior in a particular genotype. The number of mitochondria in generative cells, which is much less than that of plastids, varies considerably among genotypes. However, comparable genetic studies between genotypes are not yet available on male mitochondrial inheritance in alfalfa.     

Reduction in amounts of mitochondrial DNA in the sperm cells as a mechanism for maternal inheritance in Hordeum vulgare

It is known that extranuclear organelle DNA is inherited maternally in the majority of angiosperms. The mechanisms for maternal inheritance have been well studied in plastids but not in mitochondria. In the present study we examined the mitochondrial DNA in the male reproductive cells of Hordeum vulgare L. by immunoelectron microscopy. Our results show that the number of anti-DNA gold particles on sections of sperm cell mitochondria decreased by 97% during pollen development. The reduction occurred rapidly in the generative cells and subsequently in the sperm cells, concomitant with a remarkable reduction in mitochondrial volume. It seems that the copy numbers of mitochondrial DNA were reduced in the male reproductive cells, which may be a possible mechanism by which paternal transmission is inhibited. Unlike mitochondria, plastids are excluded from the generative cells during the first pollen mitosis. These data suggest a mechanism for maternal inheritance of mitochondria in angiosperms and for independent control of inheritance of mitochondria and plastids in H. vulgare.     

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.     

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.     

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.     

Transmission of male cytoplasm during fertilization in Nicotiana tabacum

Serially sectioned embryo sacs of Nicotiana tabacum were examined during fertilization events using transmission electron microscopy. After pollen tube discharge, the outer membrane of the sperm pair is removed, the two sperm cells are deposited in the degenerate synergid and the sperm cells migrate to the chalazal edge of the synergid where gametic fusion occurs. During fertilization, the male cytoplasm, including heritable organelles, is transmitted into the female reproductive cells as shown by: (1) the cytoplasmic confluence of one sperm and the central cell during cellular fusion, (2) the occurrence of sperm mitochondria (distinguished by ultrastructural differences) in the zygote cytoplasm and adjacent to the sperm nucleus, (3) the presence of darkly stained aggregates which are found exclusively in mature sperm cells within the cytoplasm of both female cells soon after cell fusion, and (4) the absence of any large enucleated cytoplasmic bodies containing recognizable organelles outside the zygote or endosperm cells. The infrequent occurrence of plastids in the sperm and the transmission of sperm cytoplasm into the egg during double fertilization provide the cytological basis for occasional biparental plastid inheritance as reported previously in tobacco. Although sperm mitochondria are transmitted into the egg/zygote, their inheritance has not been detected genetically. In one abnormal embryo sac, a pair of sperm cells was released into the cytoplasm of the presumptive zygote. Although pollen tube discharge usually removes the inner pollen-tube plasma membrane containing the two sperm cells, this did not occur in this case. When sperm cells are deposited in a degenerating synergid or outside of a cell, this outer membrane is removed, as it apparently is for fertilization.     

Variation in generative cell plastid nucleoids and male fertility in Medicago sativa

Biparental inheritance of plastids has been documented in numerous angiosperm species. The adaptive significance of the mode of plastid inheritance (unior biparental) is poorly understood. In plants exhibiting paternal inheritance of plastids, DNA-containing plastids in the microgametophyte may affect survival or growth of the gametophyte or the embryo. In this study the number of plastids containing DNA (nucleoids) in generative cells and generative cell and pollen volumes were evaluated in a range of genotypes of Medicago sativa (alfalfa). M. sativa exhibits biparental inheritance of plastids with strong paternal bias. The M. sativa genotypes used were crossed as male parents to a common genotype and the relationships between the gametophytic traits measured and male reproductive success were assessed. Generative cell plastid number and pollen grain size exhibited opposing associations with male fertility. Path analysis showed that generative cell plastid number was negatively associated with male fertility. This study provides evidence that there may be a competitive advantage at fertilization afforded sperm that have minimized their organelle content. The apparent lack of strong selection for reduced plastid number in generative cells of M. sativa may be a reflection of the diminished importance of reproductive success due to its perenniality or its long use in cultivation.     

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.     

Quantitative,three-dimensional analysis of alfalfa egg cells in two genotypes: Implications for biparental plastid inheritance

In alfalfa (Medicago sativa L.), plastids are inherited biparentally. Patterns of plastid transmission vary according to the genotypes involved, but there is a strong bias in favor of male plastid transmission. Previous cytological studies on the male gametophyte of this species have not provided an adequate explanation for the differences in plastid transmission frequencies among genotypes. In the present study, we compared egg cells from genotypes classified as strong or weak plastid transmitters to determine whether plastid transmission strength is correlated with egg cell structure before fertilization. We found that plastids in the mature egg cells of the strong female (genotype 6–4) are significantly larger than in mature eggs of the weak female (genotype CUF-B), and that significantly more plastids are positioned in the apical portion of the mature egg cell of genotype 6–4 than in CUF-B. Immature eggs in the two genotypes show the same pattern as mature eggs with regard to plastid number and polarization. Since only the apical portion of the egg cell/zygote gives rise to the functional embryo, these results indicate that the potential input of female plastids, in terms of plastid size and number, may be an important factor in determining the inheritance patterns of these organelles in alfalfa.Support for this work by the United States Department of Agriculture under grant 88-37234-3876, the National Science Foundation under grant DCB-9103658, the Organized Research Fund of Northern Arizona University, and the Arizona Agricultural Experiment Station is gratefully acknowledged. We are indebted to Dr. Craig Caldwell, Northern Arizona University Computer Visualization Laboratory, for his expert help with the computer graphics.     

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