Occurrence of unreduced pollen in diploid blueberry species,Vaccinium sect. Cyanococcus

Summary A total of 1475 individuals belonging to 43 natural populations of seven diploid (2x) blueberry species (Vaccinium section Cyanococcus) and two natural interspecific 2x hybrid populations were evaluated for unreduced pollen production. Significant differences were found in the frequency of unreduced pollen producers between species and within and between populations of the same species. Individuals with 1% or more unreduced pollen were considered unreduced pollen producers. The average frequency of unreduced pollen producers in these diploid species was 13.5%, ranging from 7.4% (V. corymbosum) to 18.4% (V. darrowii). The frequency of unreduced pollen grains in individual clones varied from <-1% to 28.6%. The production of unreduced pollen was not associated with male fertility. The widespread occurrence of unreduced pollen in the diploid species should allow the introgression of this germ plasm to the tetraploid level via unilateral sexual polyploidization.New Jersey Agricultural Experiment Station, Publication No. D-12163-18-91, supported by State and U.S. Federal funds, USDA-ARS Specific Cooperative Agreement No. 58-3615-9-068     

Effect of Sporophytic Heterozygosity on the Male Gametophyte of the Tetraploid Potato (Solanum tuberosum L.)

Quantitative variation for pollen stainability was examinedin both tetraploid x diploid and tetraploid x tetraploid familiesof potato (Solanum tuberosum L.). The diploid parents, whichwere highly heterozygous, produced first division restitution(FDR) 2n pollen. The families used were not affected by cytoplasmic-geneticmale sterility. Significant differences between families werefound. FDR 2n pollen producing diploid clones had progenieswith a significantly higher pollen stainability than those derivedfrom crosses between tetraploid parents. This was related tothe level of heterozygosity in the sporophyte. In FDR 2n pollen,80% of the heterozygosity and a large fraction of epistasispresent in the diploid parent was transmitted to its tetraploidprogeny. This is important for the production of open pollinated,true potato seed, because pollen stainability is significantlycorrelated with fruit set (r = 0·85).Copyright 1994,1999 Academic Press FDR 2n pollen, gametophytic heterozygosity, true potato seed     

Factors Influencing Seed Set in Triploid Musa spp. L. and Production of Euploid Hybrids

Seed set in triploid Musa spp. L. is very low due to high levelsof sterility. This makes cross breeding of plantain and bananadifficult. Nonetheless, several triploid plantain and bananacultivars produce seeds after hand pollination with diploidparents. This paper reports results about the pattern of seedvariation, success of in vitro germination and hybrid productionafter triploid-diploid crosses. Between-and within-clones differencesin seed set were observed. ABB cooking bananas had the highestrates of seed production followed by AAB French plantains. Plantainsproduced more diploid than tetraploid offsprings after triploid-diploidcrosses, which suggests that meiosis resulted in the productionof balanced haploid gametes (n = x) due to normal chromosomebehaviour between homologous genomes. Seed set variation withinclones was influenced by both the production of 2n (= 3x) gametesin the triploid parent and climatic factors. Indeed, seed production,embryo and seed germination success, and euploid and tetraploidhybrid production followed a pattern of seasonal variation reachinga peak in September. High frequency of seed with tetraploidembryos was obtained in the period from January to mid-March.The results demonstrate that production of desirable tetraploidswould be more efficient when hand pollinations are done underhigh temperature, high solar radiation and low relative humidity.These seem to be the most convenient conditions for relativelyhigh production of 2n eggs as compared to haploid gametes.Copyright1995, 1999 Academic Press Banana, fertility, embryo culture, Musa spp. AAA group, Musa spp. AAB group, Musa spp. ABB group, plantain, ploidy, seed set, 2n gametes     

Production of 2n Gametes in Diploid Subspecies of Dactylis glomerata L. 2. Occurrence and Frequency of 2n Eggs

Whereas polyploidy has been found in more than half the Gramineae,the occurrence of 2n gametes, a potentially major mechanismof polyploid production, has been rarely studied. In the presentwork, the frequency of 2n egg production in nine diploid (n= 2x = 14) subspecies of the polyploid complex Dactylis glomeratahas been estimated by determining the ploidy level of the progenyin 98 2X-4X crosses. The 2n egg origin of the 4X offspring wasverified using enzyme markers. The frequency of 2n egg productionin each subspecies was estimated from the number of 4X offspring,the number of florets in the seed plant and the fertility ofthe subspecies. The ploidy level of the progeny was determinedby chromosome counts or nuclear DNA quantification using flowcytometry. Plants producing 2n eggs were detected in 47% ofthe fertile crosses, involving eight subspecies. Variabilitywas related to individuals and not to subspecies. Average 2negg frequency was 0·49% for all crosses, and 1·53%for crosses where 2n egg production was detected. Individualfrequencies did not exceed 3·5%, with the exception of26% in one plant. Triploid block was higher and fertility, frequencyof high 2n gamete producers and average 2n gamete frequencywere lower in the 2X-4X crosses than in the opposite 4X-2X crossesinvolving the same plants, previously studied. Diplogynous anddiplandrous gamete production were not correlated with eachother. However, the widespread occurrence of plants producingboth kinds of 2n gametes in almost all the studied subspecies,as well as the presence of several individuals able to producehigh rates of 2n eggs or 2n pollen, support the hypothesis thattetraploids can be formed anywhere and anytime by sexual polyploidizationin diploid populations of Dactylis glomerata L. 2n eggs, polyploidy, Dactylis glomerata L., flow cytometry, sexual polyploidization     

Molecular Cytogenetic Analysis of Polyploidization in the Anther Tapetum of Diploid and Autotetraploid Arabidopsis thaliana Plants

Like those of most angiosperms, vegetative tissues of Arabidopsisthaliana undergo high levels of endopolyploidization. One suchtissue is the anther tapetum which plays a role in male sporo-and gametogenesis. The degree of polyploidization of the tapetumvaries from species to species. Although the role of this processis not yet fully understood, it may be linked to functioningof the tapetum, increasing the copy number of genes needed forthe synthesis of specific factors required by developing pollenmother cells (PMCs) and pollen grains. The present study focusedon polyploidization during the development of the tapetum ofArabidopsis thaliana. The aim was to outline the mode of tapetumpolyploidization in this model plant species and to establishan efficient method for analysing ploidy levels in differentiatedcells. The course and degree of tapetum polyploidization inArabidopsis was analysed in interphase nuclei using fluorescencein situ hybridization (FISH) with repetitive DNA (45S rDNA).The stages of development of the tapetum were analysed alongsidemeiosis in PMCs. The majority of tapetal cells undergo two,maximally three, rounds of divisions. Tapetal nuclei have usuallydivided by metaphase I of meiosis of PMCs. The pattern of tapetumpolyploidization was similar in diploid and autotetraploid plantsand is thus not affected by increasing amounts of maternal plantDNA. The tapetum of autotetraploid plants exhibits a higherfrequency of additional division than seen in diploid plants.Copyright 2001 Annals of Botany Company Arabidopsis thaliana, autotetraploid, FISH, rDNA polyploidization, tapetum     

Origin of triploid Arachis pintoi (Leguminosae) by autopolyploidy evidenced by FISH and meiotic behaviour

Background and Aims

Polyploidy is a dominant feature of flowering-plant genomes, including those of many important crop species. Arachis is a largely diploid genus with just four polyploid species. Two of them are economically important: the cultivated peanut and A. glabrata, a tropical forage crop. Even though it is usually accepted that polyploids within papilionoid legumes have arisen via hybridization and further chromosome doubling, it has been recently suggested that peanut arose through bilateral sexual polyploidization. In this paper, the polyploid nature of the recent, spontaneously originated triploid cytotype of the tropical lucerne, A. pintoi, was analysed, and thereby the mechanism by which polyploids may arise in the genus.

Methods

Chromosome morphology of 2x and 3x A. pintoi was determined by the Feulgeńs technique and the rDNA sites were mapped by FISH. To investigate whether polyploidization occurred by means of unreduced gametes, a detailed analysis of the microsporogenesis and pollen grains was made.

Key Results

The 2x and 3x plants presented 9m + 1sm and a satellited chromosome type 2 in each haploid genome. Physical mapping revealed a cluster of 18S–26S rDNA, proximally located on chromosome 6, and two 5S rDNA loci on chromosomes 3 and 5. Diploid plants presented 10II in meiosis while trivalents were observed in all triploids, with a maximum of 10III by cell. Diploid A. pintoi produced normal tetrads, but also triads, dyads and monads. Two types of pollen grains were detected: (1) normal-sized with a prolate shape and (2) large ones with a tetrahedral morphology.

Conclusions

Karyotype and meiotic analysis demonstrate that the 3x clone of A. pintoi arose by autopolyploidy. The occurrence of unreduced gametes strongly supports unilateral sexual polyploidization as the most probable mechanism that could have led to the origin of the triploid cytotype. This mechanism of polyploidization would probably be one of the most important mechanisms involved in the origin of economically important species of Arachis, either by triploid bridge or bilateral sexual polyploidization.     

Evidence of 2n microspore production in a natural diploid population of Turnera sidoides subsp. carnea and its relevance in the evolution of the T. sidoides (Turneraceae) autopolyploid complex

Turnera sidoides is a complex of distylous perennial rhizomatous herbs that includes five subspecies. Since polyploidy has played a prominent role within this species (x?=?7), ongoing studies in T. sidoides are focused on the understanding of the mechanisms involved in the origin and the establishment of polyploids. Therefore, aiming to contribute to the understanding of the mode of polyploid formation, in this study we investigated the frequency of unreduced microspores in a natural diploid population of T. sidoides subsp. carnea by analyzing the size range of pollen and the constitution of the sporads. The results showed that some of the individuals studied produced 2n and 4n microspores, both in short- and long-styled floral morphs. The analysis performed documents the production of unreduced microspores in T. sidoides subsp. carnea and provides evidence that support the hypothesis of sexual polyploidization as one of the most probable mechanisms involved in the origin of polyploids within this species complex. The role of unreduced pollen in the establishment and persistence of newly formed polyploids in diploid populations of T. sidoides is also discussed.     

Discovery of nucleotide polymorphisms in the Musa gene pool by Ecotilling

Musa (banana and plantain) is an important genus for the global export market and in local markets where it provides staple food for approximately 400 million people. Hybridization and polyploidization of several (sub)species, combined with vegetative propagation and human selection have produced a complex genetic history. We describe the application of the Ecotilling method for the discovery and characterization of nucleotide polymorphisms in diploid and polyploid accessions of Musa. We discovered over 800 novel alleles in 80 accessions. Sequencing and band evaluation shows Ecotilling to be a robust and accurate platform for the discovery of polymorphisms in homologous and homeologous gene targets. In the process of validating the method, we identified two single nucleotide polymorphisms that may be deleterious for the function of a gene putatively important for phototropism. Evaluation of heterozygous polymorphism and haplotype blocks revealed a high level of nucleotide diversity in Musa accessions. We further applied a strategy for the simultaneous discovery of heterozygous and homozygous polymorphisms in diploid accessions to rapidly evaluate nucleotide diversity in accessions of the same genome type. This strategy can be used to develop hypotheses for inheritance patterns of nucleotide polymorphisms within and between genome types. We conclude that Ecotilling is suitable for diversity studies in Musa, that it can be considered for functional genomics studies and as tool in selecting germplasm for traditional and mutation breeding approaches.     

Cytological,morphological and molecular analyses of controlled progenies from meiotic mutants of alfalfa producing unreduced gametes

A program of sexual polyploidization was carried out in alfalfa using plants from wild diploid species that produced male or female unreduced gametes. Sixteen progenies from 2x-4x and 2x-2x crosses were examined with a combination of morphological, cytological and molecular analyses. The chromosome counts revealed diploid, tetraploid and aneuploid plants. Plants with B chromosomes were also detected. The leaf area of the plants was a useful characteristic for distinguishing tetraploid from diploid plants obtained by unilateral or bilateral sexual polyploidization. Leaf shape and leaf margin were not correlated with the ploidy levels. Plants with supernumerary chromosomes displayed obovate or elliptic leaves which differed markedly from the range of forms typical of diploid and tetraploid alfalfa plants. RAPD markers were investigated in all progeny plants to determine maternal and paternal amplification products. Three alfalfa-specific primers proved to be effective in revealing the hybrid origin of the plants. A combination of cytological, morphological and molecular analyses is essential for a detailed genetic characterization of progenies in programs of sexual polyploidization.     

Inheritance of seed colour in turnip rape (Brassica campestris L.)

Summary A high gene frequency for ps (parallel spindles) is expected in cultivated tetraploid potatoes, S. tuberosum Group Tuberosum, if 2n pollen produced by ancestral diploid plants which were psps was involved in the origin and evolution of the potato. Fifty-six North American cultivars (varieties and advanced selections) were pollinated by diploid clones, either W 5295.7 or W 5337.3 which are homozygous recessive for ps. The segregation ratios in regard to 2n pollen production in derived tetraploid progenies, from 4x×2x crosses, reveal the genotype of ps in the cultivars. Microsporogenesis of 2n pollen producing 4x progeny was observed to avoid an overestimation of the frequency of 2n pollen producing plants due to mechanisms other than parallel spindles. More than 50% of the 56 cultivars are simplex (Pspspsps), since in each of these cultivars about 50% of their progeny produced 2n pollen. The ps gene frequency in the 56 cultivars was estimated as high as 0.69. The high frequency of ps in the tetraploid cultivars clearly supports the hypothesis that 2n pollen produced by plants homozygous recessive for ps have been involved in the origin of cultivated tetraploid potatoes, since a higher frequency of ps in the tetraploid than in the ancestral diploid population can be expected from sexual polyploidization but not from somatic doubling. The importance of meiotic mutants such as ps for the successful evolution of polysomic polyploids is emphasized.     

Microsporogenesis in some Triploid Dactylorchid Hybrids

During meiosis in naturally occurring triploid hybrids betweenthe diploid Orchis fuchsii Druce (2n = 4O) and the two tetraploids,O. purpurella Steph. and O. praetermissa Druce (2n = 8O), thereis a regular formation of 20 bivalents and 20 bivalents. Sincethe two tetraploid species themselves show typical ‘diploid’behaviour in synapsis and fertility, they are considered tobe allopolyploids, and the hybrid pairing to be allosyndetic.The implication is therefore that both tetraploids are amphidiploidsof which O. fuchsii has been one progenitor. It is suggestedthat varieties of the polytypic diploid O. latifolia L. sec.Pugsl. may have been the other progenitors. A feature of interestin the microsporogenesis of both parents and hybrids is theclose synchronization of nuclear events in the pollen massulae,which behave as physiological units throughout meiosis and pollen-mitosis.In the triploids, although numerous dysploid nuclei are produced,none dies prematurely, probably because of mutual compensationwithin what is, in effect, a common cytoplasmic matrix.     

Dioecy and chromosomal sex determination are maintained through allopolyploid speciation in the plant genus Mercurialis

Polyploidization may precipitate dramatic changes to the genome, including chromosome rearrangements, gene loss, and changes in gene expression. In dioecious plants, the sex-determining mechanism may also be disrupted by polyploidization, with the potential evolution of hermaphroditism. However, while dioecy appears to have persisted through a ploidy transition in some species, it is unknown whether the newly formed polyploid maintained its sex-determining system uninterrupted, or whether dioecy re-evolved after a period of hermaphroditism. Here, we develop a bioinformatic pipeline using RNA-sequencing data from natural populations to demonstrate that the allopolyploid plant Mercurialis canariensis directly inherited its sex-determining region from one of its diploid progenitor species, M. annua, and likely remained dioecious through the transition. The sex-determining region of M. canariensis is smaller than that of its diploid progenitor, suggesting that the non-recombining region of M. annua expanded subsequent to the polyploid origin of M. canariensis. Homeologous pairs show partial sexual subfunctionalization. We discuss the possibility that gene duplicates created by polyploidization might contribute to resolving sexual antagonism.     

Polyphyletic allopolyploid origin ofRanunculus cantoniensis (4x) fromR. silerifolius (2x) ×R. chinensis (2x)

Both diploid and tetraploid experimental interspecific hybrids betweenRanunculus silerifolius (2x) andR. chinensis (2x) exhibit normal bivalent pairing. However, microspores of diploid hybrids do not undergo mitosis and their pollen grains are highly sterile, whereas tetraploid hybrids form good pollen grains after microspore division. Evidence is forwarded for the assumption thatR. cantoniensis (4x) has originated by hybridization between these two diploid parental species and by polyploidization of the diploid hybrids. Parallelisms between the different karyotypes ofR. cantoniensis (4x) andR. silerifolius (2x) suggest that the former is a species of polyphyletic origin.     

Use of <Emphasis Type="Italic">2n</Emphasis> gametes for the production of sexual polyploids from sterile Oriental × Asiatic hybrids of lilies (<Emphasis Type="Italic">Lilium</Emphasis>)

Sixteen Oriental and 12 Asiatic cultivars were crossed in 158 different combinations. A total of 708 F₁ hybrids were obtained from 86 of the different combinations of 15 Oriental and 11 Asiatic cultivars. Because the Lilium cultivars (2n=2x=24) used for the production of these hybrids belong to two different taxonomic sections—Archelirion (O) and Sinomartagon (A), respectively—the F₁ hybrids (OA) could be obtained only through embryo, embryo sac rescue, ovary slice or ovule culture. Most of the F₁ hybrids were highly sterile (did not produce viable n gametes) due to the failure of chromosome pairing. However, in a few cases F₁ plants were found that produced viable 2n pollen at variable frequencies. These 2n pollen grains were successfully used for the production of backcross progenies. Using genomic in situ hybridization we found intergenomic recombinant chromosomes in the sexual polyploid progenies. These results indicate that there are effective prospects for combining important horticultural traits from the two main groups of cultivars of lilies through sexual polyploidization.     

Variation in the DNA Content of Glycine Species

Nuclei were isolated from cotyledons of a range of accessionsfrom 14 species of Glycine. These were stained with ethidiumbromide and the relative fluorescence for each genotype wasmeasured by flow cytometry. The DNA content was estimated bycomparison of relative fluorescence with that from nuclei fromseedling leaves of Allium cepa, whose DNA content has been calculatedpreviously by chemical assay. The 4C amounts for diploid Glycineranged from 3.80 to 6.59 pg. Two groups of diploid species appearedfrom the analysis. The first consisted of species with amountsranging from 3.80 to 5.16 pg and included G. canescens (AA),G. argyrea (A₁ A₁), G. clandestina (A²A²), G. microphylla(BB),G. latifolia (B₁B₁), G. tabacina 2n=40 (B²B²), G. tomentella2n=38 (EE) and 2n=40 (DD), G. max and G. soja (GG), G. arenariaand G. latrobeana. A second group had higher DNA contents rangingfrom 5.27 to 6.59 pg, and consisted of G. curvata, G. cyrtoloba(CC), and G. falcata (FF). The polyploid species, G. tabacina2n=80 (AABB, BBB¹B¹), G. tomentella 2n=78 and 2n=80 (AAEE andDDEE, respectively) contained amounts approximating to the sumsof the respective parental diploid species thought to have givenrise to these allotetraploids. Intraspecific variation was detectedin the DNA content of G. canescens. Within the overall distributionof DNA amounts found in A genome species, each genome containeda range of DNA contents specific to that species. This phenomenonwas also detected amongst B genome species.     

Polyploidy‐associated genomic instability in Arabidopsis thaliana

Formation of polyploid organisms by fertilization of unreduced gametes in meiotic mutants is believed to be a common phenomenon in species evolution. However, not well understood is how species in nature generally exist as haploid and diploid organisms in a long evolutionary time while polyploidization must have repeatedly occurred via meiotic mutations. Here, we show that the ploidy increased for two consecutive generations due to unreduced but viable gametes in the Arabidopsis cyclin a1;2‐2 (also named tardy asynchronous meiosis‐2) mutant, but the resultant octaploid plants produced progeny of either the same or reduced ploidy via genomic reductions during meiosis and pollen mitosis. Ploidy reductions through sexual reproduction were also observed in independently generated artificial octaploid and hexaploid Arabidopsis plants. These results demonstrate that octaploid is likely the maximal ploidy produced through sexual reproduction in Arabidopsis. The polyploidy‐associated genomic instability may be a general phenomenon that constrains ploidy levels in species evolution. genesis 48:254–263, 2010. © 2010 Wiley‐Liss, Inc.     

Changes in genomic methylation patterns during the formation of triploid asexual dandelion lineages

DNA methylation is an epigenetic mechanism that has the potential to affect plant phenotypes and that is responsive to environmental and genomic stresses such as hybridization and polyploidization. We explored de novo methylation variation that arises during the formation of triploid asexual dandelions from diploid sexual mother plants using methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. In dandelions, triploid apomictic asexuals are produced from diploid sexual mothers that are fertilized by polyploid pollen donors. We asked whether the ploidy level change that accompanies the formation of new asexual lineages triggers methylation changes that contribute to heritable epigenetic variation within novel asexual lineages. Comparison of MS‐AFLP and AFLP fragment inheritance in a diploid × triploid cross revealed de novo methylation variation between triploid F₁ individuals. Genetically identical offspring of asexual F₁ plants showed modest levels of methylation variation, comparable to background levels as observed among sibs in a long‐established asexual lineage. Thus, the cross between ploidy levels triggered de novo methylation variation between asexual lineages, whereas it did not seem to contribute directly to variation within new asexual lineages. The observed background level of methylation variation suggests that considerable autonomous methylation variation could build up within asexual lineages under natural conditions.     

Polyploidization mechanisms: temperature environment can induce diploid gamete formation in Rosa sp

Polyploidy is an important evolutionary phenomenon but the mechanisms by which polyploidy arises still remain underexplored. There may be an environmental component to polyploidization. This study aimed to clarify how temperature may promote diploid gamete formation considered an essential element for sexual polyploidization. First of all, a detailed cytological analysis of microsporogenesis and microgametogenesis was performed to target precisely the key developmental stages which are the most sensitive to temperature. Then, heat-induced modifications in sporad and pollen characteristics were analysed through an exposition of high temperature gradient. Rosa plants are sensitive to high temperatures with a developmental sensitivity window limited to meiosis. Moreover, the range of efficient temperatures is actually narrow. 36 °C at early meiosis led to a decrease in pollen viability, pollen ectexine defects but especially the appearance of numerous diploid pollen grains. They resulted from dyads or triads mainly formed following heat-induced spindle misorientations in telophase II. A high temperature environment has the potential to increase gamete ploidy level. The high frequencies of diplogametes obtained at some extreme temperatures support the hypothesis that polyploidization events could have occurred in adverse conditions and suggest polyploidization facilitating in a global change context.     

The occurrence and frequency of 2n pollen in three diploid solanums from Northwest Argentina

Summary A group of wild, tuber-bearing species from Northwest Argentina, belonging to the series Tuberosa, Solarium spegazzini Bitt. (spg, 2n=2x=24), S. gourlayi Hawkes (grl, 2n=2x=24 and 2n=4x=48) and S. oplocense Hawkes (opl, 2n=6x=72), and Cuneolata, S. infundibuliforme Phil (ifd, 2n=2x=24), is being used to investigate the mode of origin of polyploids in the genus Solanum. 2n gametes have been detected in the diploid species ifd and spg and in a diploid race of grl, using cytological and breeding approaches. Twenty-two introductions of spg, 8 of grl and 26 of ifd have been tested for 2n pollen; 59%, 63% and 54% of them, respectively, had at least one 2n pollen producing plant. These introductions comprised 238, 76 and 235 plant respectively, of which 20, 16, and 32 plant produced 5% or more 2n pollen. The mechanism of 2n pollen formation was determined in several plant of 2x spg, 2x grl and 2x ifd. All of them were found to form diplandroids via parallel spindles. This mechanism, which gives meiotic products genetically equivalent to first division restitution gametes, is under control of the Mendelian recessive ps. The results suggest that the allele ps is widely distributed in natural populations of the three diploids, and that its frequency is very high. These species are seen as valuable material for population genetic studies, and for the eventual incorporation into a breeding scheme involving sexual polyploidization via 2n gametes.     

Comparison of Embryo Development in Wild and Cultivated Arachis Species

Embryo development following selfing was investigated in twowild diploid peanut species, Arachis batizocoi Krap. et Greg.(coll. K 9484) (2n = 20) and A. duranensis Krap. et Greg. nom.nud. (coll. K 7988) (2n = 20), and one cultivated tetraploidspecies, A. hypogaea L. NC-Ac 18000 (2n = 40). Rates of pegelongation and sequences of embryo development for each specieswere compared. Peg elongation rates were similar for the twowild species, but for A. hypogaea it was only one-third to one-halfthat of the diploid species. Embryos in A. hypogaea showed slightlymore rapid cell division than in the wild species. The observedvariation in reproductive development between the wild and cultivatedspecies indicate that different control mechanisms may governdevelopment in the different species and may be at least partiallyresponsible for failure to produce viable interspecific hybridsat various ploidy levels. The observations are also importantfor determining the time at which embryos of different speciesof Arachis will reach the appropriate stage of development forsuccessful culture on an artificial medium during embryo rescueprocedures. Peanut, Arachis hypogaea, wild species, embryo, peg