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.     

Why does biparental plastid inheritance revive in angiosperms?

It is widely believed that plastid and mitochondrial genomes are inherited through the maternal parent. In plants, however, paternal transmission of these genomes is frequently observed, especially for the plastid genome. A male gametic trait, called potential biparental plastid inheritance (PBPI), occurs in up to 20% of angiosperm genera, implying a strong tendency for plastid transmission from the male lineage. Why do plants receive organelles from the male parents? Are there clues in plastids that will help to elucidate the evolution of plants? Reconstruction of the ancestral state of plastid inheritance patterns in a phylogenetic context provides insights into these questions. In particular, a recent report demonstrated the unilateral occurrence of PBPI in angiosperms. This result implies that nuclear cytoplasmic conflicts, a basic driving force for altering the mode of organelle inheritance, might have arisen specifically in angiosperms. Based on existing evidence, it is likely that biparental inheritance may have occurred to rescue angiosperm species with defective plastids.     

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.     

A one-step organelle capture: gynogenetic kiwifruits with paternal chloroplasts

Androgenesis, the development of a haploid embryo from a male nucleus, has been shown to result in the instantaneous uncoupling of the transmission of the organelle and nuclear genomes (with the nuclear genome originating from the male parent only and the organelle genomes from the female parent). We report, for the first time, uncoupling resulting from gynogenesis, in Actinidia deliciosa (kiwifruit), a plant species known for its paternal mode of chloroplast inheritance. After pollen irradiation, transmission of nuclear genes from the pollen parent to the progeny was inhibited, but transmission of the chloroplast genome was not. This demonstrates that plastids can be discharged from the pollen tube into the egg with little or no concomitant transmission of paternal nuclear genes. Such events of opposite inheritance of the organelle and nuclear genomes must be very rare in nature and are unlikely to endanger the long-term stability of the association between the different genomes of the cell. However, they could lead to incongruences between organelle gene trees and species trees and may constitute an alternative to the hybridization/introgression scenario commonly invoked to account for such incongruences.     

GENETIC EVIDENCE FOR TRACE PATERNAL TRANSMISSION OF PLASTIDS IN LIRIODENDRON AND MAGNOLIA (MAGNOLIACEAE)

Transmission of plastids in Liriodendron and Magnolia was studied by examining inheritance of plastid DNA markers in interspecific crosses. In congeneric hybrids of Liriodendron and Magnolia, 2.9% and 11.1% of progeny, respectively, exhibited uniparental paternal transmission of plastids. This departure from strict uniparental maternal transmission of plastids indicates that mechanisms for the elimination of paternal plastids are not 100% efficient and that any proposed model for plastid transmission must account for a continuum of variation. Our results, and other results reviewed here, show a discrepancy with data obtained by cytological observations and point to the need of confirming the mode of plastid inheritance by genetic analysis. In addition, if paternal plastids are occasionally inherited, intensive sampling of plastid DNA in a putative hybrid population could identify both parents of hybrids and thereby aid in the phylogenetic reconstruction of hybrid zones.     

High transmission of paternal plastid DNA in alfalfa plants demonstrated by restriction fragment polymorphic analysis

Summary A high frequency of paternal plastid transmission occurred in progeny from crosses among normal green alfalfa plants. Plastid transmission was analyzed by hybridization of radiolabeled alfalfa plastid DNA (cpDNA) probes to Southern blots of restriction digests of the progeny DNA. Each probe revealed a specific polymorphism differentiating the parental plastid genomes. Of 212 progeny, 34 were heteroplastidic, with their cpDNAs ranging from predominantly paternal to predominantly maternal. Regrowth of shoots from heteroplasmic plants following removal of top growth revealed the persistence of mixed plastids in a given plant. However, different shoots within a green heteroplasmic plant exhibited paternal, maternal, or mixed cpDNAs. Evidence of maternal nuclear genomic influence on the frequency of paternal plastid transmission was observed in some reciprocal crosses. A few tetraploid F₁ progeny were obtained from tetraploid (2n=4x=32) Medicago sativa ssp. sativa x diploid (2n=2x=16) M. sativa ssp. falcata crosses, and resulted from unreduced gametes. Here more than the maternal genome alone apparently functioned in controlling plastid transmission. Considering all crosses, only 5 of 212 progeny cpDNAs lacked evidence of a definitive paternal plastid fragment.Contribution No. 89-524-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan     

The role of plastids in plant speciation

Understanding the molecular basis of how new species arise is a central question and prime challenge in evolutionary biology and includes understanding how genomes diversify. Eukaryotic cells possess an integrated compartmentalized genetic system of endosymbiotic ancestry. The cellular subgenomes in nucleus, mitochondria and plastids communicate in a complex way and co-evolve. The application of hybrid and cybrid technologies, most notably those involving interspecific exchanges of plastid and nuclear genomes, has uncovered a multitude of species-specific nucleo-organelle interactions. Such interactions can result in plastome-genome incompatibilities, which can phenotypically often be recognized as hybrid bleaching, hybrid variegation or disturbance of the sexual phase. The plastid genome, because of its relatively low number of genes, can serve as a valuable tool to investigate the origin of these incompatibilities. In this article, we review progress on understanding how plastome-genome co-evolution contributes to speciation. We genetically classify incompatible phenotypes into four categories. We also summarize genetic, physiological and environmental influence and other possible selection forces acting on plastid-nuclear co-evolution and compare taxa providing molecular access to the underlying loci. It appears that plastome-genome incompatibility can establish hybridization barriers, comparable to the Dobzhansky-Muller model of speciation processes. Evidence suggests that the plastid-mediated hybridization barriers associated with hybrid bleaching primarily arise through modification of components in regulatory networks, rather than of complex, multisubunit structures themselves that are frequent targets.     

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.     

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.     

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。     

Influence of parental genotype on plastid inheritance in Medicago sativa

Research using chlorophyll-deficient mutants has shown that plastids are inherited biparentally in Medicago sativa L. (alfalfa). Variation in plastid transmission behavior was observed among crosses in earlier studies, but it was not determined whether this variation was under genetic control. In my research, genetic analyses of the frequencies of normal (G), chlorophyll-deficient (CD), and sectored (G and CD) progenies produced from G x CD crosses demonstrated that plastid inheritance patterns in alfalfa are influenced by both maternal and paternal genotypes. A strong paternal bias in plastid transmission existed in the majority of crosses despite the potential developmental disadvantages associated with paternally contributed CD plastids. The high frequencies of uniparental progenies suggest that genetic control of plastid inheritance in alfalfa may be exerted through effects on the number and distribution of maternal and paternal plastids early in embryo development.     

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.     

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.     

Potential cytoplasmic inheritance in Wisteria sinensis and Robinia pseudoacacia (Leguminosae)

We examined pollen cells of Wisteria sinensis and Robinia pseudoacacia (Leguminosae) to determine a possible mode for cytoplasmic inheritance in these species. Epifluorescence microscopy revealed distinct mature generative cells. Mature generative cells of W. sinensis were associated with large numbers of punctuated fluorescent signals corresponding to cytoplasmic DNA aggregates, but no fluorescent signals were observed in the generative cells of R. pseudoacacia. Closer examination showed that the punctate fluorescent signals corresponded to plastid but not mitochondrial DNA. These results suggest a strong potential for paternal transmission of the plastid genome in W. sinensis. Electron microscopy confirmed the presence of plastids in the generative cells of W. sinensis and the absence of plastids in R. pseudoacacia cells due to an unequal distribution of plastids during the first pollen mitosis. Mitochondria were present and intact in the mature generative cells of both species. The lack of fluoresced mitochondrial DNA suggests a very low level of mitochondrial DNA in the cells. Immunoelectron microscopy demonstrated that the labeling of mitochondrial DNA in these cells was reduced by nearly 90% during pollen development. Such a dramatic reduction suggests an active degradation of paternal mitochondrial DNA, which may contribute greatly to the maternal inheritance of mitochondria. In short, we found that W. sinensis exhibits a strong potential for paternal transmission of plastids and that both W. sinensis and R. pseudoacacia appear to share the same mechanism for maternal mitochondrial inheritance.     

Molecular marker systems for Oenothera genetics

The genus Oenothera has an outstanding scientific tradition. It has been a model for studying aspects of chromosome evolution and speciation, including the impact of plastid nuclear co-evolution. A large collection of strains analyzed during a century of experimental work and unique genetic possibilities allow the exchange of genetically definable plastids, individual or multiple chromosomes, and/or entire haploid genomes (Renner complexes) between species. However, molecular genetic approaches for the genus are largely lacking. In this study, we describe the development of efficient PCR-based marker systems for both the nuclear genome and the plastome. They allow distinguishing individual chromosomes, Renner complexes, plastomes, and subplastomes. We demonstrate their application by monitoring interspecific exchanges of genomes, chromosome pairs, and/or plastids during crossing programs, e.g., to produce plastome-genome incompatible hybrids. Using an appropriate partial permanent translocation heterozygous hybrid, linkage group 7 of the molecular map could be assigned to chromosome 9.8 of the classical Oenothera map. Finally, we provide the first direct molecular evidence that homologous recombination and free segregation of chromosomes in permanent translocation heterozygous strains is suppressed.     

The role of plastid competition in the control of plastid inheritance in the zonal Pelargonium

The inheritance pattern of mutant white plastids was studied in W × W crosses, in which one mutant was highly stable (W^s) and the other unstable (W^u) owing to the spontaneous restitution (mutation) of white plastids to a new green form. Thirty-six selfs and crosses were made within and between three nuclear type I cultivars, transmitting the unstable plastids, and three nuclear type II cultivars, transmitting the stable plastids. The allelic frequencies of the restituted plastids among the progeny were subjected to an analysis of variance which showed that within each nuclear type the three cultivars were rather similar except for some heterogeneity after W^s × W^u plastid crosses. The relative average transmission of the two mutant plastids in these W × W crosses was estimated and compared with their individual transmission in reciprocal crosses in which one parent contained green plastids. In the latter crosses, the green plastids were superior to the mutant plastids and the unstable plastid mutant was only slightly more successful than the stable mutant. But when the mutant plastids competed against each other, the unstable mutant became greatly superior to the stable mutant and comparable to a green normal plastid. A model to explain these results is discussed.     

Elimination of plastids during spermatogenesis and fertilization in the plant kingdom

Ultrastructural and genetic investigations involving diverse species of plants have demonstrated that plastids may be transmitted either biparentally or maternally during sexual reproduction. In species in which plastid transmission is maternal, elimination of plastids from the paternal parent may occur in a number of ways: exclusion from the male gamete during spermatogenesis, loss from the motile sperm, exclusion during fertilization, or degradation within the zygote. These diverse ways in which maternal inheritance of plastids is achieved suggest that this inheritance pattern may have evolved independently many times in response to different selective pressures in different phyletic lineages.     

The model plant Medicago truncatula exhibits biparental plastid inheritance

The plastid, which originated from the endosymbiosis of a cyanobacterium, contains its own plastid DNA (ptDNA) that exhibits a unique mode of inheritance. Approximately 80% of angiosperms show maternal inheritance, whereas the remainder exhibit biparental inheritance of ptDNA. Here we studied ptDNA inheritance in the model legume, Medicago truncatula. Cytological analysis of mature pollen with DNA-specific fluorescent dyes suggested that M. truncatula is one of the few model plants potentially showing biparental inheritance of ptDNA. We further examined pollen by electron microscopy and revealed that the generative cell (a mother of sperm cells) indeed has many DNA-containing plastids. To confirm biparental inheritance genetically, we crossed two ecotypes (Jemalong A17 and A20), and the transmission mode of ptDNA was investigated by a PCR-assisted polymorphism. Consistent with the cytological observations, the majority of F(1) plants possessed ptDNAs from both parents. Interestingly, cotyledons of F(1) plants tended to retain a biparental ptDNA population, while later emergent leaves tended to be uniparental with either one of the parental plastid genotypes. Biparental transmission was obvious in the F(2) population, in which all plants showed homoplasmy with either a paternal or a maternal plastid genotype. Collectively, these data demonstrated that M. truncatula is biparental for ptDNA transmission and thus can be an excellent model to study plastid genetics in angiosperms.     

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.