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.     

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.     

Chloroplast DNA inheritance in theStellaria longipes complex (Caryophyllaceae)

Inheritance of chloroplast DNA (cpDNA) was examined in F₁ progenies derived from three crosses and three corresponding reciprocal crosses betweenStellaria porsildii andS. longifolia. Chloroplast DNA restriction fragments were analyzed using methods of nonradioactive digoxigenin-11-dUTP labeling and chemiluminescent detection with Lumi-Phos 530. Distinct interspecific restriction fragment polymorphisms were identified and used to demonstrate the mode of cpDNA inheritance. Mode of cpDNA inheritance differed among crosses. Two crosses in whichS. porsildii, SP2920-21, was the maternal parent exhibited three different types of plastids, maternal, paternal and biparental, among the F₁ hybrids, suggesting a biparental cpDNA inheritance and plastid sorting-out inStellaria.     

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。     

Ultrastructural studies on plastids of generative and vegetative cells in Liliaceae

Summary The behaviour of plastids and mitochondria during the formation and development of the male gametophyte of Chlorophytum comosum has been investigated using electron microscopy. During first pollen mitosis an intracellular polarization of plastids occurs in that the plastids are clustered in the centre of the microspore. The originating generative cell normally lacks plastids. Only in a small number of microspores have plastids been observed near the dividing nucleus of the microspore and later on in the generative cell. These observations agree with the genetic investigations of Collins (1922) on the mode of plastid inheritance which demonstrated a small amount of biparental plastid inheritance in Chlorophytum. The cytological mechanisms underlying plastid polarization during the first pollen mitosis are discussed.     

Exceptional paternal inheritance of plastids in Arabidopsis suggests that low-frequency leakage of plastids via pollen may be universal in plants

Plastid DNA is absent in pollen or sperm cells of Arabidopsis thaliana. Accordingly, plastids and mitochondria, in a standard genetic cross, are transmitted to the seed progeny by the maternal parent only. Our objective was to test whether paternal plastids are transmitted by pollen as an exception. The maternal parent in our cross was a nuclear male sterile (ms1-1/ms1-1), spectinomycin-sensitive Ler plant. It was fertilized with pollen of a male fertile RLD-Spc1 plant carrying a plastid-encoded spectinomycin resistance mutation. Seedlings with paternal plastids were selected by spectinomycin resistance encoded in the paternal plastid DNA. Our data, in general, support maternal inheritance of plastids in A. thaliana. However, we report that paternal plastids are transmitted to the seed progeny in Arabidopsis at a low (3.9 x 10(-5)) frequency. This observation extends previous reports in Antirrhinum majus, Epilobium hirsutum, Nicotiana tabacum, Petunia hybrida, and the cereal crop Setaria italica to a cruciferous species suggesting that low-frequency paternal leakage of plastids via pollen may be universal in plants previously thought to exhibit strict maternal plastid inheritance. The genetic tools employed here will facilitate testing the effect of Arabidopsis nuclear mutations on plastid inheritance and allow for the design of mutant screens to identify nuclear genes controlling plastid inheritance.     

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.     

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。     

Ultrastructural and molecular study of plastid inheritance in Abies alba and some Abies hybrids

 The ultrastructure of egg cells in Abies alba was examined to elucidate the lack of maternal inheritance of plastids. Before fertilization, maternal plastids are absent in the perinuclar zone containing mainly mitochondria and smooth endoplasmic reticulum. During egg cell development the maternal plastids are transformed into large inclusions which are situated mostly towards the periphery of the egg cell, and finally disintegrate. As a consequence, they do not participate in zygote formation. RFLP analysis of cpDNA of parental trees and their F1 interspecific hybrids (A. alba×A. numidica, A. alba×A. nordmanniana, A. nordmanniana×A. Alba) using HindIII and BamHI showed a paternal mode of cpDNA inheritance. Paternal inheritance has also been found with PCR/RFLP analysis of cpDNA from parental trees and their hybrids (A. alba×A. pinsapo, A. pinsapo×A. alba, A. pinsapo×A. numidica) using ApaI and HaeIII digests, as well as in the crosses of A. cephalonica×A. nordmanniana, A. nordmanniana×A. cephalonica, A. cephalonica×A. numidica using TagI digests. Received: 1 June 1998 / Accepted: 14 August 1998     

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.     

Plastid DNA inheritance and plastome-genome incompatibility in interspecific hybrids of Zantedeschia (Araceae)

Plastid DNA (ptDNA) probes were used in RFLP analysis to determine ptDNA inheritance in interspecific hybrids in Zantedeschia. Biparental and maternal ptDNA inheritance was found in albino hybrids between the evergreen species Z. aethiopica and several winter-dormant species. From two albino hybrids, different types of ptDNA were detected in shoots derived from different parts of an embryo. This result indicates that plastids were sorted out during embryo development. Only maternal ptDNA was detected in the hybrids of Z. aethiopica × Z. odorata (a summer-dormant species) but paternal, biparental, and maternal ptDNA were found in the hybrids of the reciprocal cross. Z. odorata × Z. aethiopica. By correlating these ptDNA inheritance patterns with the leaf colour (albino, pale-green, and green) of the hybrids, it is suggested that the Z. odorata plastome is incompatible with the Z. aethiopica genome. The Z. aethiopica plastome is partially compatible with the Z. odorata genome but the development of Z. aethiopica plastids appears to be blocked by the presence of the Z. odorata plastids.     

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.     

Plastid inheritance in the planktonic raphid pennate diatom Pseudo-nitzschia delicatissima (Bacillariophyceae)

Plastid inheritance was followed during sexual reproduction in the raphid pennate diatom Pseudo-nitzschia delicatissima, using rbcL haplotypes as plastid identification tools. Pseudo-nitzschia species are dioecious and show functional anisogamy with 'male' mating type+(PNd(+)) cells and 'female' PNd(-) cells. Vegetative cells possess two plastids. In P. delicatissima, meiosis results in two gametes that both contribute two plastids to the zygote. The latter initially contains four plastids, but during auxospore development two of these four seem to disappear, and the initial cell emerging from the auxospore appears to contain only two. Here we assessed if the plastids are inherited strictly unipaternally, strictly biparentally, or randomly. We traced the source of the plastids in the F(1) generation by using PNd(+) and PNd(-) parental strains with different rbcL genotypes, here denoted AA (homoplastidial, with two plastids of rbcL haplotype A) and BB (homoplastidial; two plastids of haplotype B). Results showed that 16 out of 96 strains raised each from single F(1) cells had retained two paternal (PNd(+)) plastids, 20 had two maternal (PNd(-)) plastids and the remaining 60 had one maternal and one paternal plastid. This pattern is in accordance with the hypothesis that either two of the four plastids are eliminated during auxospore formation, or that all plastids are retained in the auxospore and segregate in pairs joining at random during the first mitotic division of the initial cell. Heteroplastidic F(1)-strains retained the AB genotype throughout the vegetative phase of their life cycle. The finding that 60 out of 96 F(1) strains were heteroplastidial contrasts with an absence of such genotypes in our strains raised from single cells sampled in the Gulf of Naples.     

INVITED SPECIAL PAPER: The hows and whys of cytoplasmic inheritance in seed plants

Cytoplasmic organelles are inherited in a nonMendelian fashion in all eukaryotic organisms investigated. Among the seed plants, plastids can be inherited strictly from the female parent, strictly from the male parent, or biparentally. Most flowering plants studied to date exhibit maternal plastid inheritance, but approximately one-third of the genera investigated display biparental plastid inheritance to some degree. Among the gymnosperms, paternal plastid inheritance is the rule in the conifers, whereas the other groups appear to have maternal plastid inheritance, although they have been less well studied. Mitochondrial inheritance is predominantly maternal in the seed plants, except for a few coniferous families where it is predominantly paternal. The advent of recombinant DNA technology has allowed restriction fragment length polymorphisms to be used as molecular markers, and has stimulated much research in organelle inheritance and its application to studies of population genetics and phylogenetic biology. This report emphasizes the various mechanisms by which organelles are, or are not, transmitted among the seed plants in order that researchers directly or indirectly involved with organelle inheritance may better understand the potential and the limitations of their investigations. A summary and discussion of the possible evolutionary significance of the various patterns of cytoplasmic inheritance among the seed plants are also included.     

Biparental inheritance of chloroplast DNA and the existence of heteroplasmic cells in alfalfa

Summary Mapping of chloroplast DNA (ctDNA) restriction fragment patterns from a chlorophyll deficient mutant and two phenotypically normal alfalfa genotypes (Medicago sativa L.) has demonstrated the existence of a distinct ctDNA genotype from each source. These unique restriction fragment patterns were utilized to identify maternal or paternal origin of ctDNA in hybrid plants from crosses involving the normal alfalfa genotypes as females and the yellow-green chlorophyll deficient sectors as males. Progeny from these crosses expressing the yellow-green sectored phenotypes contained paternal ctDNA in the chlorophyll deficient sectors and maternal ctDNA in the normal sectors, confirming biparental plastid inheritance. The existence of mixed cells containing both mutant and normal plastids at various stages of sorting-out was observed by transmission electron microscopy of mesophyll cells in mosaic tissue from hybrid plants. This observation verified the biparental transmission of plastids in alfalfa.     

Transmission of paternal chloroplasts in Nicotiana

Summary Transmission of paternal chloroplasts was observed in Nicotiana, considered to inherit organelles in a strictly maternal way. Plants carrying streptomycin resistant plastids were used as pollen donors. Cell lines with paternal plastids in the offspring were selected as green (resistant) sectors on calli induced from the seedlings on streptomycin-containing media. The presence of paternal plastids in the regenerated plants was confirmed by restriction analysis. In the Nicotiana plumbaginifolia xN. plumbaginifolia Np(SR1)3 and the N. plumbaginifolia Np(gos)29 xN. tabacum SR1 crosses 2.5% and 0.07% of the offspring were found to contain paternal (tabacum) plastids, respectively. These plants, however, carried maternal mitochondria exclusively. This sexual cybridization method offers a simple way to transfer chloroplasts solely, a goal not accessible by protoplast fusion.     

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     

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.     

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.     

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