Meiotic restitution mechanisms and 2n pollen formation in a Solanum tuberosum dihaploid and in dihaploid × wild species hybrids*

A Solatium tuberosum dihaploid that proved to be a 2n pollen producer, exhibited two types of meiotic abnormality involved in 2n pollen formation: premature cytokinesis at prophase II and parallel/tripolar spindles in meiosis II. For this reason such a dihaploid was preferentially used as a seed parent in crosses with the diploid species S. tarijense, S. sanctae-rosae, and S. phureja. Diploid interspecific hybrids were evaluated for male fertility, for 2n pollen production and stability over three years and for meiotic nuclear restitution mechanisms. Pollen stainability ranged from 6 to 85% and 2n pollen from 5 to 35% among hybrids. The meiotic mechanism generating 2n pollen was that of spindle abnormality in meiosis II and consequent dyad/triad formation. High correlation was found between fused/parallel spindles and dyads.     

Intra- and intergenomic relationships in interspecific hybrids between Brassica (B. rapa, B. napus) and a wild species B. maurorum as revealed by genomic in situ hybridization (GISH)

Interspecific hybridization plays a crucial role in plant genetics and breeding. The efficiency of interspecific crosses to a considerable extent depends on the genetic relatedness of genomes from parental species. Interspecific hybrids involving Brassica maurorum (2n = 16, MM) and two Brassica crop species, viz B. rapa (2n = 20, AA) and B. napus (2n = 38, AACC), were produced and analyzed for their meiotic chromosome pairings in pollen mother cells (PMCs) by using genomic in situ hybridization (GISH) with the labeled DNA of B. maurorum (MM) as probe. In hybrids B. maurorum × B. rapa (2n = 18, MA), all chromosomes remained unpaired in 28% PMCs, and the maximum of autosyndetic bivalents was two and one among the chromosomes of A and M genomes, with the average per cell being 0.27 and 0.12, respectively. Up to two allosyndetic bivalents between A and M genomes appeared, averagely 0.48 per cell. In hybrids B. maurorum × B. napus (2n = 27, MAC), the maximum of autosyndetic bivalents in M genome was two and the average was 0.11, while the maximum of allosyndetic bivalents between M and A/C genomes was two and the average was 0.78. The 2–7 bivalents formed by A/C-genome chromosomes showed their high homology. The results were compared and discussed with the chromosome pairings in the hybrids of B. maurorum with B. juncea and B. carinata with respect to the genome relationships and the potential for chromosome recombination.     

Meiotic behavior in interspecific hybrids between Brachiaria ruziziensis and Brachiaria brizantha (Poaceae)

The meiotic behavior of two half-sib interspecific tetraploid (2n = 4x = 36) promising hybrids, a sexual and an apomictic one, from crosses B. ruziziensis and B. brizantha, was evaluated. Although chromosome paired predominantly as bivalents, a few tri- and quadrivalents were recorded. Results suggest that B. brizantha and B. ruziziensis are closely related and genetic recombination is expected in hybrids. Introgression of specific target genes from B. ruziziensis into B. brizantha and vice-versa may be foreseen. However, abnormalities such as irregular chromosome segregation, chromosome stickiness and abnormal cytokinesis reported in these hybrids affect pollen fertility. More than 65% of pollen grains are sterile. Since the distinctive cytological feature of these hybrids is abnormal cytokinesis, this fact suggests that both parental genomes are unable to coordinate their activities with regard to this cytological phenomenon. Deployment of such hybrids in the process of developing varieties is discussed.     

Meiotic chromosome pairing in intergeneric hybrids of colchicaceous ornamentals revealed by genomic in situ hybridization (GISH)

Diploid and triploid intergeneric hybrids obtained by crosses among Gloriosa superba ‘Lutea’ (2n = 2x = 22), G. ‘Marron Gold’ (2n = 4x = 44), Littonia modesta (2n = 2x = 22), and Sandersonia aurantiaca (2n = 2x = 24) were analyzed for their meiotic chromosome pairing in pollen mother cells by genomic in situ hybridization (GISH) with digoxigenin-labeled total DNA of one parent as probe. Chromosomes from each parent could be clearly distinguished in pollen mother cells of all the five intergeneric hybrids by GISH. For three diploid hybrids, L. modesta × G. superba ‘Lutea’ (2n = 2x = 22), L. modesta × S. aurantiaca (2n = 2x = 23) and S. aurantiaca × G. superba ‘Lutea’ (2n = 2x = 23), 0.04?0.27 autosyndetic bivalents (intragenomic pairing of non-homologous chromosomes) and 0.13?0.36 allosyndetic bivalents (intergenomic chromosome pairing) were observed per pollen mother cell, indicating that there are some homologous chromosomal regions within each genome and among the genomes of Gloriosa, Littonia and Sandersonia. Differences in the average number of allosyndetic bivalents per pollen mother cell among different genome combinations may reflect the evolutionary distances among the three genera, and Gloriosa and Littonia may be closely related to each other, while Sandersonia may have relatively distant relationships with Gloriosa and Littonia. For two triploid hybrids, L. modesta × G. ‘Marron Gold’ (2n = 3x = 33) and S. aurantiaca × G. ‘Marron Gold’ (2n = 3x = 34), no allosyndetic bivalents were observed. Based on the results obtained in the present study, possible utilization of the diploid and triploid intergeneric hybrids for further breeding of colchicaceous ornamentals is discussed.     

The inheritance of a basal branching type in guar

Summary Eight different genotypes of the F₁ hybrids between Oriental × Asiatic lily (Lilium) hybrids (2n = 2x = 24) were treated with nitrous oxide (N₂O) gas under pressure for 24 and 48 hours. At the time of treatment, all plants possessed early meiotic stages in the anthers of the oldest flower buds. The mature flowers from treated plants were monitored for fertility through pollen germination in vitro as well as by using them in crosses with diploid Asiatic hybrids (2n = 2x = 24) both as male and female parents. In five out of the eight genotypes of OA hybrids there was evidence for the production of 2n pollen which germinated in vitro from either one or both treatments. The 2n pollen from three genotypes was successfully used in crosses. In two cases, the treated plants were successfully used as female parents which indicated the formation of 2n (or 2x) egg cells. From an analysis of 41 sexual polyploid progenies obtained from N₂O treated plants it was shown that they were all euploids consisting of 34 triploids (2n = 3x = 36) and seven tetraploids (2n = 4x = 48). A detailed cytological analysis of 12 progeny plants through genomic in situ hybridization (GISH) proved that N₂O had induced first division restitution gametes in most cases and in two cases they produced gametes through indeterminate meiotic restitution. There was evidence for intergenomic recombination in three cases.     

Barriers to hybridization of Solanum bulbocastanum Dun. and S. Verrucosum Schlechtd. and structural hybridity in their F1 plants

Summary Results of reciprocal crosses between Solanum verrucosum (2n=2x=24) and S. bulbocastanum (2n=2x=24) are described in terms of pollen tube behaviour in styles, of berry and seed set, of fertility and of meiotic behaviour of the F₁ hybrids. Pollen tube growth of S. verrucosum is strongly inhibited in styles of S. bulbocastanum, whereas no inhibition is observed in the reciprocal cross. Therefore S. bulbocastanum x S. verrucosum fails to set berries or seeds, whereas the reciprocal cross produces both berries (54.4% berry set) and seeds (0.3 per berry). Only 14.6% of the seeds germinate. Both the diploid and corresponding tetraploid hybrid plants are vigorous, flower abundantly, have a rather regular meiosis (mainly rod bivalents), but show a high degree of cytoplasmic-genic male sterility. Crossability of the diploid hybrid plants is nil when used as pollen parents and near to zero when used as pistillate parents. The barriers to hybridization of the parent species (unilateral inhibition of pollen tube growth, somatoplastic sterility, cytoplasmic-genic male sterility and structural differences of the parental chromosomes) are discussed and methods are suggested to overcome these barriers.     

Production of an interspecific hybrid between Brassica fruticulosa and B. rapa

An interspecific hybrid between a wild species, Brassica fruticulosa (2n = 16, FF) and a crop Brassica species, B. rapa (2n = 20, AA) was synthesized using sequential ovary‐ovule culture on MS medium supplemented with casein hydrolysate. Morphological, molecular and cytogenetic analysis confirmed the true hybrid nature of the offspring. The F1 plants (2n = 18) were intermediate in morphology, highly pollen‐sterile as well as self‐sterile. A maximum of three bivalents per PMC was recorded, but 14 I + 2 II was the most common meiotic configuration. Normal pollen fertility and regular bivalent (18 II) formation was observed in the amphiploid sectors of the hybrid plants. The F₁ hybrid harboured significantly lower aphid populations than the crop Brassica parent.     

Meiosis and Pollen Viability in Helianthus tuberosus L. and its Hybrids with Cultivated Sunflower

The hexaploid species of Helianthus tuberosus is a potential source of resistance to several sunflower pathogens. But crossed with cultivated sunflower, it produces F, hybrids which have low values of fertility or even full sterility. Pollen viability and meiotic features were studied in 17 populations of the species H. tuberosus and in F¹ hybrids. Significant differences in pollen viability existed between populations (47.1–98.8%). In the 15 FI hybrids, plant fertility ranged from 0 to 100 per combination, while pollen viability ranged from 12.4 to 57.1. Meiosis was almost normal in the analyzed species, and irregular in the F, hybrids. The highest percentage of meiocytes was with bivalents (85.9), but univalents (0.3) and multivalents (13.8) occurred as well. In metaphase and anaphase, the percentage of meiocytes with fast and lagging chromosomes was high. In anaphase, chromosome bridges were detected in 9.9 of the meiocytes.     

Occurrence of 2n gametes in the F1 hybrids of Oriental × Asiatic lilies (Lilium): Relevance to intergenomic recombination and backcrossing

Cytological modes of the origin of 2n gametes were investigated in six different genotypes of F₁ hybrids between Oriental and Asiatic (OA) lilies (Lilium, 2n = 2x = 24). Chromosome pairing between the parental genomes was very low, the average frequency range from 0.3 to 1.2 bivalents per cell among the genotypes. Within a genotype the frequency of bivalents varied from 0 to 6 in some cases. The normally occurring haploid pollen grains were totally sterile. In contrast, in different genotypes, variable percentages of 2n pollen were found and shown to be fertile as estimated from pollen germination. A cytological analysis of Metaphase I and subsequent stages of meiosis using genomic in situ hybridization (GISH) revealed that there was intergenomic recombination between the alien genomes. Following Metaphase I stage, three different types of abnormal cytological events led to the formation of 2n pollen: (i) Post-Metaphase I division (PMI), (ii) Post-Metaphase II division (PMII) and (iii) Asymmetric Cytokinesis of the pollen mother cell followed by chromosome division. All three cytological events led to first division restitution (FDR) gametes. Based on in vitro pollen germination it was proved for two genotypes that 2n pollen was viable only during the first day of anthesis. It was possible to use 2n pollen successfully for backcrossing. Implications of 2n pollen for intergenomic recombination in BC₁ progenies are discussed.     

Novel interspecific hybridization between sweetpotato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.) and its two diploid wild relatives

Interspecific hybridization is an important approach to broaden the genetic base and create novel plant forms in breeding programs. However, interspecific hybridization in Ipomoea is very difficult due to the cross incompatibility. Here we report two novel interspecific F₁ hybrids between I. batatas (L.) Lam. (2n = 6x = 90) and two wild species, I. grandifolia (2n = 2x = 30) and I. purpurea (2n = 2x = 30). Hybridization was stimulated by applying plant growth hormones. Morphological, molecular and cytological tests were conducted to confirm their hybridity. We found that the two hybrids were quite distinctive in leaf color and morphology, and yielded intermediate sizes of storage roots compared to their respective parents. Inter-simple sequence repeat analysis showed that the unique DNA bands from the wild parents could be detected in these two hybrids. The cluster analysis also showed that the two F₁ hybrids were closer to I. batatas in phylogeny relationship. The number of chromosomes of the two hybrids was both 60, indicating that the hybrids were tetraploid. The meiotic configuration analysis of the H₁ of I. batatas × I. grandifolia revealed the occurrence of 17.58I + 14.28II + 1.36III + 2.48IV at metaphase I in average chromosome association per pollen mother cells (PMCs), 4.26I + 18.32II + 2.56III + 3.12IV was average meiotic configuration in the H₂ of I. batatas × I. purpurea. Both hybrids appeared to be polyads and multi-microcytes at tetrad phase and differed in their pollen fertility.     

Intergeneric hybrids and an amphiploid between Elymus canadensis and Psathyrostachys juncea

Summary Fifty four hybrid plants between Elymus canadensis and Psathyrostachys juncea were obtained by handpollination and embryo culture. The average cross compatibility between both species was 31.2 percent. One amphiploid plant was induced by colchicine treatment. The hybrid and amphiploid plants resembled P. juncea in appearance but showed a higher plant height and dry matter yield than the parents. The hybrids showed extremely low pollen stainability and were completely sterile. With the exception of one plant (2n=3x+1=22), all hybrid plants were allotriploids (SHN, 2n=3x=21). The amphiploid plant (SSHHNN, 2n=6x=42) showed 58.9% pollen stainability and 11.6% seed fertility.Mean chromosome associations of the hybrids and amphiploid at metaphase I were 0.02IV+0.06III+2.03II+16.91I and 0.07III+18.00II+5.85I, respectively. Lagging chromosomes, chromosome bridges, abnormal cytokinesis, and micronuclei were occasionally observed at the anaphase, telophase, or tetrad stage.     

Chromosome doubling and fertility study of Alstroemeria aurea × A. caryophyllaea

Self-pollinations of a diploid (2n = 2x = 16) interspecific hybrid Alstroemeria aurea × A. caryophyllaea, resulted in no seeds. Backcrosses of the hybrid with parent A. aurea did not produce any seeds. In an attempt to restore the hybrid fertility, an efficient in vitro procedure has been developed and applied effectively in the chromosome doubling of the diploid hybrid. Forty-one percent of the treated plants were proven to be truly tetraploid by chromosome counts and stomatal measurements after applying 0.2 to 0.6% colchicine for 6 to 24 hours. Over 87.5% of these colchicine-induced tetraploids were stable and retained their tetraploidy after one year of growth. The fertility of the hybrid was not restored although the pollen stainability was increased from 0 to 12% after chromosome doubling. Cytological studies on the pollen mother cells (PMCs) of the sterile diploid hybrids revealed abnormal meiotic behaviors. In addition, aneuploid chromosome numbers, ranging from 2n = 1 to 2n = 18, were observed in over 45% of the PMCs examined. PMCs of the colchicine-induced tetraploids showed that meiotic chromosome pairings were normal in most cases (1.59_I + 15.07_II + 0.05_III + 0.03IV). These results indicate that the sterility of this hybrid is not only caused by parental chromosome differences, but other complex fertility/sterility-regulating mechanisms are involved too. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytogenetic studies on the interspecific hybrid Capsicum baccatum L.× C. frutescens L. and its progeny

Summary Morphological and cytogenetical studies were carried out on the F₁ and F₂ hybrids and backcross derivatives of the interspecific cross Capsicum baccatum L. x C. frutescens L. The F₁ and F₂ hybrids displayed irregular meiosis with a maximum association of eight chromosomes in the former and one quadrivalent in the latter with the appropriate number of bivalents and univalents. It is inferred that C. baccatum differs from C. frutescens (yellow) by at least tow or three interchanges and from the white cultivar by a single interchange. Structural repatterning of chromosomes, erratic meiotic behaviour, genes for pollen sterility, segregational imbalances following intergenomic recombination are believed to be major factors causing sterility in the hybrids. The two species are sympatric and natural hybrids have not been realised.     

Occurrence of numerically unreduced (2n) gametes in Alstroemeria interspecific hybrids and their significance for sexual polyploidisation

The F1 hybrids of seven diploid Alstroemeria species (2n=2x=16) were investigated for the production of numerically unreduced (2n) gametes and their mode of origin. Based on a survey of 17 interspecific hybrid combinations,consisting of 119 genotypes, it was found that the F1 hybrids of Chilean-Brazilian species mostly produced first division restitution (FDR) 2n gametes. These F1 hybrids were self-pollinated in order to obtain F2 seeds, which was an indication that the F1 plants also produced 2neggs simultaneously. All the F2 progeny plants were typical allotetraploids, most of which formed 16 bivalents and a small proportion formed multivalents during metaphase I stages of meiosis. Through genomic in situ hybridisation (GISH) it was proved that multivalent formation in F2plants, derived from A. inodora ×A. pelegrina hybrid, was due to homoeologous recombination but not from reciprocal translocations. In order to test the segregation pattern of the recombinant chromosomes, an F3 population from one genotype, P6C49-6, was investigated. The recombinant chromosomes assorted independently from each other supporting the hypothesis that the segregation of chromosomes in ring quadrivalents did not behave like those in translocation heterozygotes. It was concluded that in allopolyploids of Alstroemeria,bilateral sexual polyploidisation could accomplish genetic recombination by both homoeologous crossing-over as well as through the assortment of chromosomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Stabilization of new types of diploids (2n=22, 24) through selfing of aneuploids (2n=21, 22) derived from crossing of sesquidiploids (2n=29, AAC) and Brassica campestris (2n=20 AA)

Summary Aneuploids with 2n=21 and 2n=22 derived from crossing of sesquidiploids (2n=29, AAC) and Brassica campestris (2n=20, AA) were selfed successively in order to follow the changes in chromosome number of the progenies for three consecutive generations. Progenies with 2n=22, 23 and 24 obtained after selfing of S₀ generation and the succeeding S₁, S₂ and S₃ generations were analyzed in terms of pollen stainability, % seed set as well as cytogenetically based on meiotic behaviour with the aim of determining the possibility of addition of one or more alien chromosomes into n=10 species which may lead to differentiation of single or plural disomic addition lines. The generation of aneuploids with 2n=21 progressed in such a way that most plants seem to revert to the 2n=20 chromosome number of B. campestris after selfing. From 2n=22 aneuploids, however, the succeeding progenies showed high frequency of plants with two additional chromosomes which accounted for 50.6% and 52.9% of total S₃ progenies via 2n=22 and 2n=24 S₂ generations, respectively. The meiotic behaviour of these progenies indicated evidence for a rule governing the frequency distribution of chromosome number among these addition lines and high possibility to breed such disomic plants with 2n=22. A method of selecting stable aneuploids was suggested in addition to the possible role of pollination biology at various processes of such breeding program.     

The mode of seed formation in Allium senescens and two Korean Allium species

The genus Allium includes several important vegetable and ornamental species, and is an economically important crop. The objective of this study was to determine the mode of reproduction in A. senescens L. (2n=6x= 48) and two Allium species collected in Korea. Chromosome numbers of embryo sac mother cells (EMCs) at diplotene of meiosis were 2n = 48 and unreduced in A. senescens. However, normal sexual reproduction was observed at diplotene in both A. senescens var. minor (2n = 2x= 16) and A. nutans L. (2n=4x= 32). Parthenogenetic embryogenesis also occurred in A. senescens at high frequencies among the three Allium species. Antipodal embryos in A. nutans were formed in unpollinated flowers. Normal seeds were not formed in flowers of A. senescens that had been emasculated and kept in sealed conditions. However, normal seeds were formed in the flowers that were pollinated 2 days after anthesis. In conclusion, diplospory, parthenogenesis, and pseudogamy occur in A. senescens.     

Analysis of stability for some characters in Kabuli Chickpea

Summary The meiotic behaviour of a hybrid between Triticum aestivum and the amphiploid Hordeum chilense x T. turgidum conv. durum, was studied using a C-banding staining method. This hybrid has the genome formula of AA BB D Hch with 2n=6x=42 chromosomes. The durum wheat chromosomes (genomes A and B) were easily recognized, whereas the D and Hch chromosomes were recognized as a whole. Meiotic pairing was homologous, as expected (14 bivalents from A and B genomes +14 univalents from D and Hch genomes). However, some pollen mother cells at metaphase-I presented pseudobivalents that could have been caused by either homoeologous or autosyndetic pairing amongst D and Hch chromosomes.     

Analysis of meiotic behavior in selecting potential genitors among diploid and artificially induced tetraploid accessions of Brachiaria ruziziensis (Poaceae)

Sexuality is correlated with diploidy and apomixis with polyploidy in the Brachiaria genus. Brachiaria ruziziensis is a key species in Brachiaria breeding due to its obligate sexuality and intrinsic agronomic qualities. Interspecific crosses in the genus became feasible only when a few diploid accessions of B. ruziziensis were artificially tetraploidized and remained sexual. Hybridization has been done since, using natural tetraploid apomictic accessions of B. brizantha or B. decumbens as pollen donors. Twenty two accessions of B. ruziziensis from the Embrapa Beef Cattle germplasm collection (Campo Grande, MS, Brazil) were cytologically analyzed: 16 are natural diploids (2n = 2x = 18) and six are artificially induced tetraploids (2n = 4x = 36). The meiotic behavior in the 16 diploid accessions varied. The mean of meiotic abnormalities per accession ranged from zero to 24.46%. Meiotic behavior in the induced tetraploid accessions also varied with the mean of meiotic abnormalities ranging from 5.20% to 54.71%. The most common abnormalities observed in both the diploid and the tetraploid accessions, were those related to irregular chromosome segregation. In one tetraploid accession, with a high frequency of those, other irregularities involving chromosome orientation at metaphase plate and chromosome convergence to the poles, a meiotic mutation known as divergent spindle, were recorded. Meiotic behavior should be considered in selecting potential genitors for breeding.     

Pollen storage of Fragaria and Potentilla

Summary Low temperature and humidity were used for the storage of pollen of four species of Fragaria (2n=14, 42 and 56) and two species of Potentilla (2n=14). The stainability of Fragaria pollen was assessed over a number of years by use of aceto-carmine stain and its viability by cross-pollinations; stainability of Potentilla pollen was assessed by acetocarmine. Fragaria pollen so stored remained stainable for seven years and showed the ability to set seed for three years. over the latter period, aceto-carmine staining gave a reasonable prediction of seed set produced by the stored pollen of Fragaria and so is of value in estimating pollen viability in that genus. Potentilla pollen remained stainable for three years.     

Meiotic chromosome behaviour in an allotriploid derived from an amphidiploid × diploid mating in Cucumis

A synthetic amphidiploid species Cucumis hyriviis Chen & Kirkbride (2n = 4x = 38; genome designated as HHCC) has recently been created from an interspecific mating between C. sativus L. (2n = 2x = 14: genome designated as CC) and C. hystrx. Chakr. (2n = 2x = 24; genome designated as HH). This amphidiploid is resistant to root knot nematode, tolerant to low irradiance, and has higher nutritional value than standard processing cucumber cultivars. An allotriploid (2n = 3x = 26; HCC) was derived from a cross between C. sativus L. and C. hytivus Chen & Kirkbride. Diploid meiotic behaviour in C. sativus and C. hystrix involves the development of seven and 12 metaphase bivalents respectively. In the derived allotriploid. univalents. bivalents, and trivalents (at relatively low frequency) were observed at metaphase I indicating that some homeologues from the C and H genomes can synapse. Based on a comparative karyotype analysis of cucumber (i.e. chromosome size and pairing behaviour) and aliotriploid plants, the seven bivalents observed at metaphase I were ascertained to be cucumber homologues, while the 12 univalents were of C. hystrix origin thus confirming the allotriploid karyotypic constitution to be HCC. On average, the frequency of trivalents was 0.24 at diakinesis and 0.22 in 100 meiocytes at metaphase I. indicating the possibility of genetic exchange due to the homoeology between the C and H genomes. After simultaneous cytokinesis, only polyads were observed in pollen mother cells (PMCs) at telophase II, which led to the production of sterile pollen grains. Multi‐polarization of chromosomes was dominant at anaphase II. However. in about 20% PMCs. chromosomes separated to form a 7C + 1‐2H complement, suggesting a possible method for the production of alien addition cucumber‐C hystrix lines through further backcrossing of the allotriploid to diploid cucumber.