Biparental inheritance of plastidial and mitochondrial DNA and hybrid variegation in <Emphasis Type="Italic">Pelargonium</Emphasis>

Plastidial (pt) and mitochondrial (mt) genes usually show maternal inheritance. Non-Mendelian, biparental inheritance of plastids was first described by Baur (Z Indukt Abstamm Vererbungslehre 1:330–351, 1909) for crosses between Pelargonium cultivars. We have analyzed the inheritance of pt and mtDNA by examining the progeny from reciprocal crosses of Pelargonium zonale and P. inquinans using nucleotide sequence polymorphisms of selected pt and mt genes. Sequence analysis of the progeny revealed biparental inheritance of both pt and mtDNA. Hybrid plants exhibited variegation: our data demonstrate that the inquinans chloroplasts, but not the zonale chloroplasts bleach out, presumably due to incompatibility of the former with the hybrid nuclear genome. Different distribution of maternal and paternal sequences could be observed in different sectors of the same leaf, in different leaves of the same plant, and in different plants indicating random segregation and sorting-out of maternal and paternal plastids and mitochondria in the hybrids. The substantial transmission of both maternal and paternal mitochondria to the progeny turns Pelargonium into a particular interesting subject for studies on the inheritance, segregation and recombination of mt genes.     

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     

The selective increase or decrease of organellar DNA in generative cells just after pollen mitosis one controls cytoplasmic inheritance

Organellar DNA in mature pollen grains of eight angiosperm species (Actinidia deliciosa Lindl., Antirrhinum majus L., Arabidopsis thaliana (L.) Heynh., Medicago sativa L., Musa acuminata Colla, Pelargonium zonale (L.) L'Hér, Petunia hybrida Vilm. and Rhododendron mucronatum (Blume) G. Don, in which the modes of organellar inheritance have been determined genetically, was observed by fluorescence microscopy using Technovit 7100 resin sections double-stained with 4′,6-diamidino-2-phenylindole (DAPI) and 3,3′-dihexyloxacarbocyanine iodide (DiOC₆). The eight species were classified into four types, based on the presence or absence of organellar DNA in mature generative cells: namely (1) type “m+p+”, which has both mitochondrial and plastid DNA (P. zonale), (2) type “m+p–”, which only has mitochondrial DNA (M. acuminata), (3) type “m−p+”, which only has plastid DNA (A. deliciosa, M. sativa, R. mucronatum), and (4) type “m−p−”, which has neither mitochondrial nor plastid DNA (A. majus, A. thaliana, P. hybrida). This classification corresponded to the mode of organellar inheritance determined by genetic analysis. The presence or absence of mitochondrial and plastid DNA corresponded to paternal/biparental inheritance or maternal inheritance of the respective organelle, respectively. When organellar DNA was present in mature generative cells (m+ or p+), the DNA content of the organelles in the generative cells started to increase immediately after pollen mitosis one (PMI). In contrast, the DNA content of organelles in generative cells decreased rapidly after PMI when organellar DNA was absent from mature generative cells (m− or p−). These results indicate that the modes of inheritance (paternal/biparental inheritance or maternal inheritance) of mitochondria and plastids are determined independently of each other in young generative cells just after PMI. Received: 22 December 1998 / Accepted: 8 February 1999     

Biparental inheritance of organelles in Pelargonium: evidence for intergenomic recombination of mitochondrial DNA

While uniparental transmission of mtDNA is widespread and dominating in eukaryotes leaving mutation as the major source of genotypic diversity, recently, biparental inheritance of mitochondrial genes has been demonstrated in reciprocal crosses of Pelargonium zonale and P. inquinans. The thereby arising heteroplasmy carries the potential for recombination between mtDNAs of different descent, i.e. between the parental mitochondrial genomes. We have analyzed these Pelargonium hybrids for mitochondrial intergenomic recombination events by examining differences in DNA blot hybridization patterns of the mitochondrial genes atp1 and cob. Further investigation of these genes and their flanking regions using nucleotide sequence polymorphisms and PCR revealed DNA segments in the progeny, which contained both P. zonale and P. inquinans sequences suggesting an intergenomic recombination in hybrids of Pelargonium. This turns Pelargonium into an interesting subject for studies of recombination and evolutionary dynamics of mitochondrial genomes.     

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.     

The foundation of extranuclear inheritance: plastid and mitochondrial genetics

In 1909 two papers by Correns and by Baur published in volume 1 of Zeitschrift für induktive Abstammungs- und Vererbungslehre (now Molecular Genetics and Genomics) reported on the non-Mendelian inheritance of chlorophyll deficiencies. These papers, reporting the very first cases of extranuclear inheritance, laid the foundation for a new field: non-Mendelian or extranuclear genetics. Correns observed a purely maternal inheritance (in Mirabilis), whereas Baur found a biparental inheritance (in Pelargonium). Correns suspected the non-Mendelian factors in the cytoplasm, while Baur believed that the plastids carry these extranuclear factors. In the following years, Baur’s hypothesis was proved to be correct. Baur subsequently developed the theory of plastid inheritance. In many genera the plastids are transmitted only uniparentally by the mother, while in a few genera there is a biparental plastid inheritance. Commonly there is random sorting of plastids during ontogenetic development. Renner and Schwemmle as well as geneticists in other countries added additional details to this theory. Pioneering studies on mitochondrial inheritance in yeast started in 1949 in the group of Ephrussi and Slonimski; respiration-deficient cells (petites in yeast, poky in Neurospora) were demonstrated to be due to mitochondrial mutations. Electron microscopical and biochemical studies (1962–1964) showed that plastids and mitochondria contain organelle-specific DNA molecules. These findings laid the molecular basis for the two branches of extranuclear inheritance: plastid and mitochondrial genetics.     

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.     

Inactivation of pollen and other effects of genome-plastome incompatibility inOenothera

A series of strains of the homozygous speciesOenothera grandiflora (characterized by the genome BB and plastome III) were combined with plastome IV fromO. parviflora (BC-IV) by means of appropriate crosses. An incompatibility between genome B and plastome IV is expressed in the haplo- and diplophase: (1) B-IV pollen, though normally developed, is largely inactive. The extent of the inactivation varies between different strains and shows a seasonal fluctuation as determined by seed set in outcrossing and selfing experiments. (2) In most of the strains lethality of BB-IV embryos is the rule, leading to empty seeds. This can be ameliorated by including another plastome in the zygotes and developing embryos on account of the biparental plastid transmission inOenothera. It can best be demonstrated in crosses with a seed parent having normal green plastids of plastome IV and mutated chlorophyll deficient plastids from a different plastome in the pollen parent, leading to variegated progeny as well as a remainder of empty seeds. (3) In about one-half of the strains the BB-IV plants exhibit a temporary bleaching of thevirescens type. The incompatibily between genome B and plastome IV does not support the earlier assumption that plastome IV is the ancestor of plastomes II, III, and V. Instead, a precursor plastome is postulated from which plastomes II, III, and IV are descended. While plastome I can be derived from II, only plastome V can be descended from plastome IV.Deceased August 28, 1998.     

Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca²⁺-dependent nuclease and a 23 kDa Zn²⁺ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.     

Plastome mutants

Summary Since the first reports seventy-five years ago on the non-Mendelian inheritance of variegation in plants, chloroplast gene mutations have been useful for genetical and physiological investigations. The mutations have been shown to affect the chloroplast translational apparatus, photosystem I, photosystem II, the cytochrome f/b₆ complex, carbon fixation, or the ATP synthase. They arose spontaneously or were induced by mutagens or by the action of nuclear mutator genes. Alterations of chloroplast DNA include point mutations, deletions, duplications, and inversions. In 1909, Correns discovered uniparental transmission of chloroplasts when he observed the maternal inheritance of a chlorophyll deficiency inMirabilis jalapa. At the same time, Baur (1909) reported crosses ofPelargonium zonale in which the offspring inherited chloroplasts from both parents (biparental transmission) with variegated leaves resulting as the green and white plastids sorted out. since the experiments of Baur and Correns, many non-Mendelian mutants have been isolated in both higher plants and algae (for reviews see Hagemann, 1964; Kirk and Tilney-Bassett, 1978; Gillham, 1978). Some of these are mitochondrial traits, including cytoplasmic male sterility in maize and several other plants (Hanson and Conde, 1985; Pring and Lonsdale, 1985). Several other traits have been tentatively identified as mitochondrial since their inheritance pattern differs from that of both nuclear and chloroplast genes, including the deformed leaf (“falsifolia”) syndrome ofOenothera (Stubbe, 1970), non-chromosomal stripe of maize (Coe, 1983), and inChlamydomonas, photoautotropism (Wiseman et al., 1977) and a minute colony phenotype (Alexander et al., 1974). A far larger number of extranuclear mutations affect the plastome (plastid genome). Among the algae,Euglena gracilis (Russell and Lyman, 1982),Scenedesmus obliquus (Bishop, 1982) andChlorella (Galling, 1982) have yielded interesting mutants, but unlikeChlamydomonas, they are not known to undergo sexual reproduction, and thus the Mendelian or non-Mendelian nature of the mutations has not been determined. Most of the plastome mutations which have been characterized have been isolated in higher plant lines or fromChlamydomonas.     

Passiflora plastome sequencing reveals widespread genomic rearrangements

Although past studies have included Passiflora among angiosperm lineages with highly rearranged plastid genomes (plastomes), knowledge about plastome organization in the genus is limited. So far only one draft and one complete plastome have been published. Expanded sampling of Passiflora plastomes is needed to understand the extent of the genomic rearrangement in the genus, which is also unusual in having biparental plastid inheritance and plastome‐genome incompatibility. We sequenced 15 Passiflora plastomes using either Illumina paired‐end or shotgun cloning and Sanger sequencing approaches. Assembled plastomes were annotated using Dual Organellar GenoMe Annotator (DOGMA) and tRNAscan‐SE. The Populus trichocarpa plastome was used as a reference to estimate genomic rearrangements in Passiflora by performing whole genome alignment in progressiveMauve. The phylogenetic distribution of rearrangements was plotted on the maximum likelihood tree generated from 64 plastid encoded protein genes. Inverted repeat (IR) expansion/contraction and loss of the two largest hypothetical open reading frames, ycf1 and ycf2, account for most plastome size variation, which ranges from 139 262 base pairs (bp) in P. biflora to 161 494 bp in P. pittieri. Passiflora plastomes have experienced numerous inversions, gene and intron losses along with multiple independent IR expansions and contractions resulting in a distinct organization in each of the three subgenera examined. Each Passiflora subgenus has a unique plastome structure in terms of gene content, order and size. The phylogenetic distribution of rearrangements shows that Passiflora has experienced widespread genomic changes, suggesting that such events may not be reliable phylogenetic markers.     

The mechanism of the mixed inheritance of chloroplast genes in Pelargonium

Summary The distributions are given of gene frequencies among embryos after G X W and W X G plastid crosses within and between eight Pelargonium cultivars and some of their inbred or hybrid derivatives.Two distinct segregation patterns are recognized. Homozygous type I female parents (Pr₁Pr₁) have a high frequency of progeny with only maternal alleles, are intermediate for biparental and low for paternal offspring. Heterozygous type II female plants (Pr₁Pr₂) have an equally high frequency of maternal and paternal offspring and a generally low biparental frequency. These correspond to L-shaped and U-shaped gene frequency distributions respectively in which the only modes are at 0 per cent (maternal embryos) and 100 per cent (paternal embryos), with no mode corresponding to the population mean and no sign of a Gaussian distribution.The extremely variable plastid gene frequencies are strongly influenced by the maternal nuclear genotype and by the plastid genotype in which the wild-type allele is always more successful than the mutant in strict comparisons.The relative frequencies of maternal and paternal zygotes, and the mean gene frequency among all the zygotes in a cross, are explicable in terms of the input frequencies of genes from the two parents, their degree of mixing, and by some form of selective replication of plastids. This selection is controlled by nuclear and plastid genotypes which may act in the same direction, to increase the frequency of either the maternal or the paternal alleles, or in opposition. But selection alone is inadequate to explain the shapes of the gene frequency distributions. Instead, a model is proposed in which the segregation or replication of plastids appears to have a strong random element, which results in random drift of gene frequencies within a heteroplasmic zygote or embryo.     

Clone bank and physical and genetic map of potato chloroplast DNA

Summary Clone banks of PvuII, BamHI and XhoI fragments were generated of the Solanum tuberosum cv Katahdin plastome. These clone banks, in conjunction with molecular hybridization to tobacco ctDNA probes, were used to construct a physical map of potato ctDNA. The potato plastome was found to be a circular molecule of 155–156 Kbp containing two inverted repeat regions of 23–27 Kbp. The arrangement of restriction sites is very similar to that of other Solanaceae plastomes. Heterologous hybridization to known ctDNA encoded gene probes from tobacco allowed us to establish a genetic map of the potato chloroplast genome. The arrangement of these genes on the potato plastome resembles that on most higher plant ctDNAs.     

Plastid genomes reveal recurrent formation of allopolyploid Fragaria

Premise of the Study

Recurrent formation of polyploid taxa is a common observation in many plant groups. Haploid, cytoplasmic genomes like the plastid genome can be used to overcome the problem of homeologous genes and recombination in polyploid taxa. Fragaria (Rosaceae) contains several octo‐ and decaploid species. We use plastome sequences to infer the plastid ancestry of these taxa with special focus on the decaploid Fragaria cascadensis.

Methods

We used genome skimming of 96 polyploid Fragaria samples on a single Illumina HiSeq 3000 lane to obtain whole plastome sequences. These sequences were used for phylogenetic reconstructions and dating analyses. Ploidy of all samples was inferred with flow cytometry, and plastid inheritance was examined in a controlled cross of F. cascadensis.

Key Results

The plastid genome phylogeny shows that only the octoploid F. chiloensis is monophyletic, all other polyploid taxa were supported to be para‐ or polyphyletic. The decaploid Fragaria cascadensis has biparental plastid inheritance and four different plastid donors. Diversification of the F. cascadensis clades occurred in the last 230,000 years. The southern part of its distribution range harbors considerably higher genetic diversity, suggestive of a potential refugium.

Conclusions

Fragaria cascadensis had at least four independent origins from parents with different plastomes. In contrast, para‐ and polyphyletic taxa of the octoploid Fragaria species are best explained by incomplete lineage sorting and/or hybridization. Biogeographic patterns in F. cascadensis are probably a result of range shift during the last glacial maximum.     

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.     

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.     

Inheritance of chloroplast and mitochondrial DNA in alloplasmic forms of the genus Daucus

The inheritance of mitochondrial (mt) and chloroplast (ct) DNA in the progeny from interspecific crosses between the cultivated carrot (Daucus carota sativus) and wild forms of the genus Daucus was investigated by analysis of mt and ct RFLPs in single plants of the parental and filial generations. We observed a strict maternal inheritance of the organellar DNAs in all interspecific crosses examined. Previous studies on putative F₂ plants from a cross between Daucus muricatus x D. carota sativus suggested paternal inheritance of ctDNA. Our reinvestigation of this material revealed that the mtDNA of the putative F₂ plants differed from the mtDNA of both putative parents. Therefore, our data suggest that the investigated material originated from other, not yet identified, parents. Consequently, the analysis of this material cannot provide evidence for a paternal inheritance of ctDNA.     

Paternal inheritance of plastids in Medicago sativa

Summary Plastids are plant cellular organelles that are generally inherited from the maternal parent in the angiosperms. Many species exhibit biparental inheritance of plastids, but usually with a predominantly maternal influence. In contrast to this, we report strong paternal inheritance of plastids in reciprocal crosses of alfalfa, Medicago sativa, by following restriction fragment length polymorphisms for plastid DNA in two normal green plastids. Mitochondrial inheritance remained exclusively maternal.     

Sequence of the Tomato Chloroplast DNA and Evolutionary Comparison of Solanaceous Plastid Genomes

Tomato, Solanum lycopersicum (formerly Lycopersicon esculentum), has long been one of the classical model species of plant genetics. More recently, solanaceous species have become a model of evolutionary genomics, with several EST projects and a tomato genome project having been initiated. As a first contribution toward deciphering the genetic information of tomato, we present here the complete sequence of the tomato chloroplast genome (plastome). The size of this circular genome is 155,461 base pairs (bp), with an average AT content of 62.14%. It contains 114 genes and conserved open reading frames (ycfs). Comparison with the previously sequenced plastid DNAs of Nicotiana tabacum and Atropa belladonna reveals patterns of plastid genome evolution in the Solanaceae family and identifies varying degrees of conservation of individual plastid genes. In addition, we discovered several new sites of RNA editing by cytidine-to-uridine conversion. A detailed comparison of editing patterns in the three solanaceous species highlights the dynamics of RNA editing site evolution in chloroplasts. To assess the level of intraspecific plastome variation in tomato, the plastome of a second tomato cultivar was sequenced. Comparison of the two genotypes (IPA-6, bred in South America, and Ailsa Craig, bred in Europe) revealed no nucleotide differences, suggesting that the plastomes of modern tomato cultivars display very little, if any, sequence variation. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Rüdiger Cerff]     

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.