2n Gamete Production Research Articles

High Temperature-Induced Spindle Destabilization Results in Aborted Pollen Production in Populus

High temperature can induce the production of 2n gametes and aborted pollen during microsporogenesis in Populus canescens. However, the mechanism by which high temperature induces pollen abortion remains unknown. Here, pollen abortion was induced by exposing male flower buds of P. canescens to 38 and 41 °C; pollen morphology, meiotic abnormalities, defects of the meiotic microtubular cytoskeleton, and tapetum development were characterized. We found that the dominant meiotic stage, temperature, and duration of treatment significantly affected the percentage of high temperature-induced aborted pollen. Damaged spindle microtubules and depolymerized microtubular cytoskeletons were observed, which resulted in many lagging chromosomes at anaphase I and II as well as aneuploid male gametes and micronuclei, thereby generating aborted pollen grains. Tapetum disintegration was also delayed. The anther dehisced normally, and some viable pollen grains were released. However, no sufficient evidence showed that the delayed degradation of the tapetum was responsible for pollen abortion.

The DcPS1 cooperates with OSDLa during pollen development and 2n gamete production in carnation meiosis

BackgroundDeciphering the mechanisms of meiosis has important implications for potential applications in plant breeding programmes and species evolution. However, the process of meiosis is poorly understood in carnation, which is famous for its cut flowers.ResultsWe report that Dianthus caryophyllus parallel spindle 1 (DcPS1) regulates omission of second division like a (OSDLa) during pollen development and 2n gamete production in carnation meiosis. In DcPS1 and OSDLa RNAi lines, an absence of the second meiotic division and the abnormal orientation of spindles at meiosis II might be the main reason for dyad/triad formation, resulting in unreduced gametes. We also found that carnation OSDLa interacted with DcPS1 and DcRAD51D. In the DcPS1 RNAi lines, a decrease in OSDLa and DcRAD51D expression was observed. In the OSDLa RNAi lines, a decrease in DcPS1 and DcRAD51D expression was also observed. We propose that DcPS1 regulates OSDLa expression, allowing entry into meiosis II and the proper orientation of the metaphase II spindle in meiosis II. We also propose that OSDLa regulates DcRAD51D expression, allowing for homologous recombination.ConclusionsThese results suggest a critical role for DcPS1 and OSDLa in diplogamete production during meiosis and open a new pathway for meiosis-related studies.

Genomics-based discrimination of 2n gamete formation mechanisms in polyploids: a case study in nonaploid Diospyros kaki 'Akiou'.

Unreduced gametes (2n gametes), possessing double the haploid genome, whatever ploidy that happens to be, are a common source of ploidy variation in plant populations. First and second division restitution (FDR and SDR) are the dominant mechanisms of 2n gamete production; all else being equal, FDR gametes have a higher degree of heterozygosity, thus they are advantageous in breeding. The discrimination of these mechanisms from the consequence of hybridization is challenging, especially in higher polyploids, and usually requires information on centromere location. In this study, we propose a genotyping-based strategy to uncover the mechanisms of 2n gamete formation in progeny that has a higher ploidy than its parents. Simulation of 2n gamete production revealed that FDR and SDR pathways can be discriminated based on allele transmission patterns alone without information on centromere location. We applied this strategy to study the formation mechanism of a nonaploid Diospyros kaki ‘Akiou', which was bred via hybridization between D. kaki hexaploid cultivars. The result demonstrated that ‘Akiou' was derived from the fertilization of a normal female gamete by a 2n male gamete and that this 2n gamete was produced through FDR. Consequently, the distinct duplex transmission pattern in the FDR gamete enabled us to infer the genomic characteristics of polyploid persimmon. The method could be tested only for the plant being polypoid, which allows for the ability to discriminate causes of 2n gamete formation using allele dosage in progeny, and will be useful in future studies of polyploid genomics.

A Review of Unreduced Gametes and Neopolyploids in Alfalfa: How to Fill the Gap between Well-Established Meiotic Mutants and Next-Generation Genomic Resources.

The gene flow mediated by unreduced gametes between diploid and tetraploid plants of the Medicago sativa-coerulea-falcata complex is pivotal for alfalfa breeding. Sexually tetraploidized hybrids could represent the best way to exploit progressive heterosis simultaneously derived from gene diversity, heterozygosity, and polyploidy. Moreover, unreduced gametes combined with parthenogenesis (i.e., apomixis) would enable the cloning of plants through seeds, providing a unique opportunity for the selection of superior genotypes with permanently fixed heterosis. This reproductive strategy has never been detected in the genus Medicago, but features of apomixis, such as restitutional apomeiosis and haploid parthenogenesis, have been reported. By means of an original case study, we demonstrated that sexually tetraploidized plants maintain apomeiosis, but this trait is developmentally independent from parthenogenesis. Alfalfa meiotic mutants producing unreduced egg cells revealed a null or very low capacity for parthenogenesis. The overall achievements reached so far are reviewed and discussed along with the efforts and strategies made for exploiting reproductive mutants that express apomictic elements in alfalfa breeding programs. Although several studies have investigated the cytological mechanisms responsible for 2n gamete formation and the inheritance of this trait, only a very small number of molecular markers and candidate genes putatively linked to unreduced gamete formation have been identified. Furthermore, this scenario has remained almost unchanged over the last two decades. Here, we propose a reverse genetics approach, by exploiting the genomic and transcriptomic resources available in alfalfa. Through a comparison with 9 proteins belonging to Arabidopsis thaliana known for their involvement in 2n gamete production, we identified 47 orthologous genes and evaluated their expression in several tissues, paving the way for novel candidate gene characterization studies. An overall view on strategies suitable to fill the gap between well-established meiotic mutants and next-generation genomic resources is presented and discussed.

Genomic affinity in hybrids of B-genome Arachis species: new genetic resources toward peanut improvement

Abstract Peanut (Arachis hypogaea L.) is an important oilseed crop and food legume that is highly susceptible to biotic and abiotic factors. Wild Arachis species represent useful genetic resources because of their resistance to pests and diseases. In particular, understanding the affinity between B-genome species is important for broadening the genetic base of A. hypogaea (AABB genome), since only one population of A. ipaensis is known, and relatively few other species have been described. Therefore, we aimed to determine the genomic affinity between A. ipaensis and A. williamsii by developing hybrids. Microsatellite markers verified the hybrid status, and chromosome pairing and pollen viability results revealed that the species share high genomic affinity. Additionally, the presence of dyads detected in sporad and pollen analyses indicated the production of 2n gametes. These results will be useful for performing evolutionary studies and developing strategies for introgression programs to improve the B genome of A. hypogaea.

Unreduced Male Gamete Formation in Cymbidium and Its Use for Developing Sexual Polyploid Cultivars.

Polyploidy plays an important role in crop improvement. Polyploid plants, particularly those produced through unreduced gametes (2n gametes), show increased organ size, improved buffering capacity for deleterious mutations, and enhanced heterozygosity and heterosis. Induced polyploidy has been widely used for improving floriculture crops, however, there are few reported sexual polyploid plants in the floriculture industry. This study evaluated nine cultivars of Cymbidium Swartz and discovered that 2n male gametes occurred in this important orchid. Depending on cultivars, 2n male gamete formation frequencies varied from 0.15 to 4.03%. Interspecific hybrids generally produced more 2n male gametes than traditional cultivars. To generate sexual polyploid plants, seven pairs of crosses were made, which produced five triploid and two tetraploid hybrids. Two triploid hybrids were evaluated for in vitro regeneration and growth characteristics. Compared to the diploid parents, the triploids were more easily regenerated through rhizomes or protocorms, and regenerated plants had improved survival rates after transplanting to the greenhouse. Furthermore, the sexual polyploid plants had more compact growth style, produced fragrant flowers, and demonstrated heterosis in plant growth. Through this study, a reliable protocol for selection of appropriate parents for 2n gamete production, ploidy level evaluation, in vitro culture of polyploid progenies, and development of new polyploid cultivars was established. Our study with Cymbidium suggests that the use of 2n gametes is a viable approach for improving floriculture crops.

Unreduced Megagametophyte Production in Lemon Occurs via Three Meiotic Mechanisms, Predominantly Second-Division Restitution.

Unreduced (2n) gametes have played a pivotal role in polyploid plant evolution and are useful for sexual polyploid breeding in various species, particularly for developing new seedless citrus varieties. The underlying mechanisms of 2n gamete formation were recently revealed for Citrus reticulata but remain poorly understood for other citrus species, including lemon (C. limon [L.] Burm. f.). Here, we investigated the frequency and causal meiotic mechanisms of 2n megagametophyte production in lemon. We genotyped 48progeny plants of two lemon genotypes, “Eureka Frost” and “Fino”, using 16 Simple Sequence Repeat (SSR) and 18 Single Nucleotide Polymorphism (SNP) markers to determine the genetic origin of the progenies and the underlying mechanisms for 2n gamete formation. We utilized a maximum-likelihood method based on parental heterozygosity restitution (PHR) of centromeric markers and analysis of PHR patterns along the chromosome. The frequency of 2n gamete production was 4.9% for “Eureka Frost” and 8.3% for “Fino”, with three meiotic mechanisms leading to 2n gamete formation. We performed the maximum-likelihood method at the individual level via centromeric marker analysis, finding that 88% of the hybrids arose from second-division restitution (SDR), 7% from first-division restitution (FDR) or pre-meiotic doubling (PRD), and 5% from post-meiotic genome doubling (PMD). The pattern of PHR along LG1 confirmed that SDR is the main mechanism for 2n gamete production. Recombination analysis between markers in this LG revealed partial chiasma interference on both arms. We discuss the implications of these restitution mechanisms for citrus breeding and lemon genetics.

Pairing analysis and in situ Hybridisation reveal autopolyploid-like behaviour in Solanum commersonii\xa0\xd7\xa0S. tuberosum (potato) interspecific hybrids

Wild potato relatives are rich sources of desirable traits for introgressive hybridisation into cultivated potato. One of them, Solanum commersonii (2n = 2x = 24, 1EBN, endosperm balance number), is an important species belonging to the potato tertiary genepool. It can be used in potato breeding through bridge crosses and 2n gamete production. Triploid F1 hybrids between S. commersonii (through spontaneous 2n egg formation) and diploid 2EBN Solanum tuberosum Group Phureja were crossed with S. tuberosum Group Tuberosum, resulting in successive backcross 1, 2 and 3 progenies. The main aim of this study was to determine if there are any barriers to homoeologous pairing and recombination in the allotriploid (S. commersonii × potato) hybrids and their backcrosses, and so to predict if S. commersonii chromosomes can be transmitted to the next generation and introgressed into their recipient potato chromosomes. Microscopic observations of spread pollen mother cells suggested no preferential pairing in the triploid hybrids, while chromosome transmission and segregation in further meiotic stages were fairly balanced. Fluorescent in situ hybridisation with BAC probes (BAC FISH) was used to obtain markers to trace the meiotic behaviour of specific chromosome pairs. Moreover, genomic in situ hybridisation (GISH) demonstrated no obvious differences in fluorescence signals between the homoeologues suggesting that repetitive sequences did not diverge much between the parental species. As a consequence, we were not able to trace the course of the S. commersonii chromosomes in the successive introgressive hybridisation backcross generations. Our results strongly point at a high genomic similarity between the homoeologous chromosomes promising high suitability of S. commersonii in introgressive hybridisation breeding of potato.

Frequency and maintenance of unreduced gametes in natural plant populations: associations with reproductive mode, life history and genome size.

Fertilization involving unreduced (2n) gametes is considered the dominant mechanism of polyploid formation in angiosperms; however, our knowledge of the prevalence of and evolutionary mechanisms maintaining 2n gametes in natural populations is limited. We hypothesize that 2n gametes are deleterious consequences of meiotic errors maintained by mutation-selection balance and should increase in species with relaxed opportunities for selection on sexual processes (asexuality), reduced efficacy of selection (asexuality, selfing) and increased genome instability (high chromosome number). We used flow cytometry to estimate male 2n gamete production in 60 populations from 24 species of Brassicaceae. We quantified variation in 2n gamete production within and among species, and examined associations with life history, reproductive mode, genome size and chromosomal number while accounting for phylogeny. Most individuals produced <2% 2n male gametes, whereas a small number had >5% (up to 85%) production. Variation in 2n gamete production was significant among species and related to reproductive system; asexual species produced significantly more 2n gametes than mixed-mating and outcrossing species. Our results, unique in their multi-species perspective, are consistent with 2n gametes being deleterious but maintained when opportunities for selection are limited. Rare individuals with elevated 2n gamete production may be key contributors to polyploid formation.

Unreduced gamete formation in wheat: Aegilops triuncialis interspecific hybrids leads to spontaneous complete and partial amphiploids

Production of unreduced or 2n gametes during meiosis is a common consequence of interspecific hybridization in plants. In the present work, five different bread wheat cultivars were crossed with an accession of Ae. triuncialis and interspecific F1 hybrids (MVT, NAT, OMT, PIT and ZAT) were produced. Meiosis in pollen mother cells (PMCs) of F1 hybrids, pollen viabilities, and seed set ratio were analyzed. Restitution meiosis and consequently unreduced viable pollen were observed. Immunolabeling against phosphorylated histone H3 at serine-10 (phosphoH3S10) suggested that unreduced male gametes were produced by first division restitution (FDR). FDR was more frequent in some hybrids, resulting in higher rates of unreduced male gamete formation and F2 seed set. The F2 embryos were highly variable in chromosome number ranging from 35 to 70 suggesting that unreduced gamete formation led to spontaneous partial and complete amphiploids.

Multilocus half-tetrad analysis and centromere mapping in citrus: evidence of SDR mechanism for 2n megagametophyte production and partial chiasma interference in mandarin cv ‘Fortune’

The genetic structure of 2n gametes and, particularly, the parental heterozygosity restitution at each locus depends on the meiotic process by which they originated, with first-division restitution and second-division restitution (SDR) being the two major mechanisms. The origin of 2n gametes in citrus is still controversial, although sexual polyploidisation is widely used for triploid seedless cultivar development. In this study, we report the analysis of 2n gametes of mandarin cv 'Fortune' by genotyping 171 triploid hybrids with 35 simple sequence repeat markers. The microsatellite DNA allele counting-peak ratios method for allele-dosage evaluation proved highly efficient in segregating triploid progenies and allowed half-tetrad analysis (HTA) by inferring the 2n gamete allelic configuration. All 2n gametes arose from the female genitor. The observed maternal heterozygosity restitution varied between 10 and 82%, depending on the locus, thus SDR appears to be the mechanism underlying 2n gamete production in mandarin cv 'Fortune'. A new method to locate the centromere, based on the best fit between observed heterozygosity restitution within a linkage group and theoretical functions under either partial or no chiasmata interference hypotheses was successfully applied to linkage group II. The maximum value of heterozygosity restitution and the pattern of restitution along this linkage group would suggest there is partial chiasma interference. The implications of such a restitution mechanism for citrus breeding are discussed.

Mutations: effect on the genetic constitution of a population

IMutation rate Consider an allele couple A1 et A2 of frequencies pt and qt respectively in generation t; consider u as the direct mutation rate of A1 towards A2 in each generation and v the reverse mutation rate of A2 towards A1. We define the mutation rate as the probability for a mutation to appear per gamete and per generation. For example, suppose a population only composed of individuals of the genotype A1 A1 which contribute to the production of 2N gametes in the next generation. If u represents the mutation rate of A1 locus towards all the other possible alleles in a diploid population of N individuals, as we have 2N concerned genes, the number of new mutants that we get are 2N x u, in a given generation. In the population, on 2N genes, only one gene among them (1/2N) will be fixed. Thus, the probability for a mutant allele to appear in the generation with a fixed gene, is 2N x u x 1/2N = u. Therefore, the probability for one allele to be substitute by a muted allele is equal to the mutation rate, per gamete and per generation, in a given locus. IIRecurrence law

Production and Identification of Colchicine-derived Tetraploid Vaccinium darrowii and Its Use in Breeding

In Vaccinium L., most tetraploid hybrids between tetraploid cultivated highbush blueberry (V. corymbosum L.) and diploid darrow's evergreen blueberry (V. darrowii Camp) have been produced by exploiting V. darrowii's tendency to produce 2n gametes and a strong triploid block, which greatly reduces the number of triploid hybrids produced. Colchicine-derived tetraploids offer an alternative method of producing V. darrowii plants that will easily cross with tetraploid Vaccinium species. V. darrowii (2n = 2x = 24) seeds were imbibed in 0.2% aqueous colchicine solution for 24 h. The seeds were germinated and seedlings whose morphology suggested colchicine effects were selected at various stages of development. No macromorphological changes were consistently associated with chromosome doubling. However, stomatal guard cells and pollen size increased substantially as a result of chromosome doubling. Several types of plants were identified after colchicine treatment: 1) plants with a doubled LI (epidermal tissues) and LII (internal tissues) plant layers; 2) periclinal chimeras with a doubled LI layer and a normal LII; and 3) periclinal chimeras with a doubled LII layer and a normal LI. Of ≈4000 seedlings that emerged from colchicine-treated seeds, 200 were selected for further examination based on leaf and stem morphology. Of the 200, five appeared to be tetraploid in LI, LII, or both layers based on stomatal guard cell size and pollen size. Crosses between colchicine-derived V. darrowii (4x) plants and southern highbush blueberry (V. corymbosum) cultivars (4x) were successful compared with 4x–2x and 2x–4x crosses using diploid V. darrowii and tetraploid southern highbush blueberry. Stomatal guard cells and pollen screening of the colchicine-treated plants were used as indicators of doubled V. darrowii plants and periclinal chimeras. The results from the crosses between colchicine-treated V. darrowii plants and tetraploid highbush blueberry cultivars confirmed the information obtained by stomata and pollen screening.

Interspecific Crosses and Backcrosses between Diploid Vaccinium darrowii and Tetraploid Southern Highbush Blueberry

Diploid Vaccinium darrowii Camp has been used in breeding tetraploid southern highbush blueberry (Vaccinium corymbosum L.) as a source of reduced chilling requirement, adaptation to hot, wet summers, and resistance to leaf diseases. V. darrowii in Florida is quite variable, but most crosses have involved only one V. darrowii clone, Fla. 4B. The use in breeding of a wider range of V. darrowii accessions would provide beneficial diversity in the blueberry cultivated gene pool. The purpose of this research was to determine the functional 2n gamete frequency of numerous V. darrowii genotypes when crossed with tetraploid V. corymbosum, and to study the pollen fertility and backcross ability of the interspecific (V. darrowii × V. corymbosum) hybrids to V. corymbosum. Crosses between diploid V. darrowii and tetraploid highbush blueberry cultivars had low fruit set compared with the V. darrowii × V. darrowii and highbush × highbush crosses. The unusually high number of hybrids per pollinated flower (HPF) in certain 4x-2x or 2x-4x crosses was attributed to high functional 2n gamete production in certain V. darrowii genotypes. Diploid Vaccinium fuscatum Aiton and diploid V. darrowii × V. fuscatum hybrids, when crossed with southern highbush blueberry cultivars, were equally productive of hybrids whether used as male or female parents. Variation in frequency of functional 2n gametes in V. darrowii, expressed as high HPF, was present within plants (megaspores vs. microspores) and among V. darrowii plants. Of the 114 interspecific (V. darrowii × V. corymbosum) hybrids studied, 106 had pollen stainability &gt;50%. This indicated that most of these hybrids were tetraploid, because triploid blueberries, like most triploid plants, are highly sterile. Twenty-two V. darrowii × V. corymbosum hybrids were backcrossed to tetraploid highbush blueberry cultivars. Fruit set was variable, but large populations of vigorous hybrids were obtained. Lower fruit set was associated with hybrids that had lower pollen fertility. It should be possible to obtain plants of cultivar quality in a few generations of backcrosses.

Overcoming hybridization barriers in potato

AbstractThe cultivated potato is a major crop worldwide. It is a high input crop with complex quality requirements at harvest and during storage. Potato breeders are fortunate to have access to a very diverse and accessible germplasm resource. Wild Solanum relatives provide genetic diversity as well as genes for valuable production and quality traits. In most cases, crossing success can be predicted based on endosperm balance number (EBN), or effective ploidy, of the parents. Crossing barriers between most wild species and the cultivated potato are the consequence of differences in EBN and can be easily overcome using ploidy manipulations and bridge crosses. The most common ploidy manipulations include haploid extraction to reduce EBN and 2n gamete production to increase EBN. Additional methods to produce fertile interspecific hybrids include mentor pollination, embryo rescue, hormone treatments, reciprocal crosses, selection of cross‐compatible genotypes and somatic fusion. Knowledge of crossing barriers and mechanisms to overcome them allows potato breeders access to the rich gene pool in the genus Solanum.

Selection for increasing 2n gamete production in red clover

Three selection cycles were performed in the red clover cultivars Quiniqueli, Redland and Keenland to increase the 2n gamete production. Unreduced gamete production was estimated by the giant pollen grain (GP) production. Plants with more than 1% of GP were selected as progenitors from the original population; plants with at least 2% and 3% of GP, respectively, we re selected and crossed for the second and third cycles. The GP production ranged from 1.4 to 7.5% in the first, 2.0 to 51.0% in t he second, and 3.0 to 46.7% in the third cycle. Considering the three cultivars together, there was a significant increase in GP production from the first (4.0%) to the third (8.9%) cycles. 'Redland' presented higher GP percentages in each cycle and signif icant and steady GP production increases along the three cycles: 4.8, 13.4 and 19.7%, respectively, and should be included in further attempts to obtain sexual polyploids.

Modelling the establishment and spread of autotetraploid plants in a spatially heterogeneous environment

The establishment and spread of autotetraploids from an original diploid population in a heterogeneous environment were studied using a stochastic simulation model. Specifically, we investigated the effects of heterogeneous habitats and nonrandom pollen/seed dispersal on the critical value (micro) of unreduced 2n gamete production necessary for the establishment of autotetraploids as predicted by deterministic models. Introduction of a heterogeneous environment with random pollen/seed dispersal had little effect on the micro value. In contrast, incorporating nonrandom pollen/seed dispersal into a homogeneous environment considerably reduced the micro value. Incorporating both heterogeneous habitats and nonrandom pollen/seed dispersal may lead either to an increase or to a decrease in the micro value compared to that with random dispersal, indicating that the two factors interact in a complex way.

Occurrence, inheritance and use of reproductive mutants in alfalfa improvement

The widespread occurrence of 2ngametes (i.e. gametes with the somatic chromosome number) in the Medicago sativa-coerulea-falcata complex supports the concept that gene flow from diploid to tetraploid species occurs continuously in nature and plays a key role in alfalfa evolution. Breeders realized early that gene transfer between ploidy levels via 2n pollen and 2n eggs would have had potential use in cultivated alfalfa improvement. Cytological investigations provided insights into the types of meiotic abnormalities responsible for the production of 2n gametes. Alterations were defined as genetically equivalent to first (FDR) or second division restitution(SDR) mechanisms. For breeding purposes,data have proven that 2n gametes of the FDR type are more advantageous than those obtained by SDR for transferring parental heterozygosity and retaining epistatic interactions. The use of diploid meiotic mutants that produce 2ngametes is now recognized as one of the most effective methods available for exploiting heterosis and introgressing wild germplasm traits into cultivated tetraploid alfalfa via unilateral (USP) and bilateral sexual polyploidization (BSP) schemes. Both2n egg and 2n pollen producers could be used for direct gene transfer from wild diploid relatives into cultivated alfalfa by means of 2x-4x and4x-2x crosses. Although data have shown that forage yield improvement can be achieved when plants are sexually tetraploidized, problems related to reduced plant fertility and seed production remain largely unexplained. Apomixis has the potential of cloning plants through seed and thus provides a unique opportunity for developing superior tetraploid cultivars with permanently fixed heterosis and epistatic effects. A main goal in alfalfa breeding could be the introduction of functional apomixis (i.e. Apomeiosis and parthenogenesis) in cultivated alfalfa stocks. In the future, the efficiency of alfalfa breeding programs based on the use of reproductive mutants could be improved by direct selection at the genotype level using RFLPs and PCR-based markers. Suitable DNA markers and detailed linkage maps of alfalfa mutants should help to discover apomictic mutants and address basic genetic issues such as the extent of genomicre combination in polyploid hybrids and the effect of sexual polyploidization on heterosis. Molecular markers have recently been used in alfalfa for studying the inheritance of 2n gamete formation and identifying polymorphisms associated to genes involved in meiotic abnormalities. Molecular tagging of 2n egg and 2n pollen formation not only should explain the genetic control and regulation of these traits, but may also be an essential step towards marker-assisted selection of 2n gamete producers and implementation of USP and BSP breeding schemes. Future perspectives include strategies for the map-based cloning of genomic DNA markers,and screening of EST mini-libraries related to flowers at different developmental stages from meiotic mutants and wild-type scan lead to the identification of mRNAs and thus of candidate genes that control 2n gamete formation in alfalfa.

Improving main agronomic characters of dihaploids with 2n gametes by means of sexual hybridisation and anther culture of potato (Solarium tuberosum)

Three hybrid populations were obtained from 4x‐2.x, 2x‐4x, and 2x‐2x crosses by using two tetraploids (Solanum tuberosum L.) and three dip‐loid hybrids of closely related species which can produce 2n gametes. Forty‐three dihaploid plantlets were regenerated from embryoids of anthers of the three tetraploid hybrids by anther culture in vitro. Three of them could produce 2n pollen and five of them could produce 2n eggs. The morphological and agronomic characters of these dihaploids with 2n gametes were compared with those of their diploid and tetraploid parents. Two dihaploid clones with 2n gametes and improved agronomic characters were obtained. The results suggest that ploidy manipulation through sexual polyploidisation and dihaploid induction by anther culture is an effective method to obtain dihaploid clones with 2n gametes in combination with good tuber yield and tuber appearance in potato.

Meiotic mutants of Medicago sativa show altered levels of α- and β-tubulin

We have analysed the level of accumulation of alpha- and beta-tubulin polypeptides in flowers collected from different meiotic mutants of alfalfa (Medicago sativa L.). The H33 mutant previously identified as a producer of male and female gametes with the somatic chromosome number (2n gametes) as a result of defective spindle orientation or, more rarely, abnormal cytokinesis, showed a higher level of alpha- and beta-tubulin compared to control diploid plants and approximately the same level as control tetraploid plants. A higher level of tubulin was likewise observed in diploid plants displaying abnormalities in spindle orientation and cytokinesis, which had gone through 3-4 cycles of phenotypic recurrent selection to increase 2n gamete production. A similar analysis was performed on another class of Medicago meiotic mutants characterized by production of 4n pollen (jumbo pollen, due to the absence of cytokinesis at the end of meiosis) and 2n eggs. Again, the level of alpha- and beta-tubulin was found to be higher in the mutants than in diploid controls. We conclude that meiotic defects, such as abnormal spindle orientation or cytokinesis leading to the formation of 2n gametes, determine an increased level of tubulin, the main constituent of plant microtubules (MTs).