Intergeneric hybrid between Brassica napus and Diplotaxis harra through ovary culture and the cytogenetic analysis of their progenies

Brassica napus (2n = 38) and Diplotaxis harra (2n = 26) were used to investigate gene transfer from D. harra to B. napus. Intergeneric F₁ hybrids (dihaploid 2n = 32 chromosomes) were obtained through ovary culture. The chromosome associations in the first meiotic division was (0–2)_III + (2–10)_II + (12–28)_I. Many seeds were harvested in the F₁ hybrid after backcrossing with B. napus, and from open pollination of the F₁ hybrid. Somatic chromosome numbers of BC₁ and hybrid plants varied from 2n = 26 to 52. In the first meiotic division, high frequencies of bivalent association and relatively low pollen fertility were observed. BC₂ plants generated from the BC₁ plants with 2n = 38 chromosomes, 69.6% showed 2n = 38 chromosomes. Many aneuploids with addition and deletion of chromosomes were also obtained. A bridge plant between B. napus and D. harra with 2n = 32 chromosomes should be valuable material for the breeding of brassica crops.     

Production and cytogenetics of intergeneric hybrids between Triticum durum-Dasypyrum villosum amphidiploid and Psathyrostachys huashanica

Summary Intergeneric crosses between Triticum durum-Dasypyrum villosum (2n=42, AABBVV), and Psathyrostachys huashanica (2n=14, N ^h N ^h ) were made, the seed set was 1.67%. Intergeneric hybrid were successfully obtained by means of embryo culture for first time. The average chromosome pairing in the hybrid (ABVN ^h ) was 26.61% univalents, and 0.69 bivalents. The chiasmata per cell was 0.69. The chiasmata was higher than that in Triticum durum dihaploid (AB), and lower than that in T. durum-Dasypyrum villosum trihaploid (ABV). The result indicated that the N ^h genome of Psathyrostachys huashanica has no homology with the V genome of Dasypyrum villosum, and the A and B genomes of Triticum durum. The coenocytism, micronuclei cell and variation in chromosome numbers were also observed. The F₁ hybrid was crossed with Triticum aestivum (AABBDD), and resulted in seed set. The hybrid of T. durum-D. villosum amphidiploid x P. huashanica showed partial fertility. It made the possibility for chromosome manipulation among Triticum aestivum, Dasypyrum villosum and Psathyrostachys huashanica.     

Solanum phureja genes are expressed in the leaves and tubers of aneusomatic potato dihaploids

Summary The expression of leaf isozymes and tuber patatin in dihaploids derived from the Solanum tuberosum cv. Pentland Crown was investigated. Seven of the dihaploids were aneusomatic containing additional chromosomes from the S. phureja dihaploid inducer. Of these, four genotypes expressed leaf isozymes characteristic of the S. phureja dihaploid inducer, and the tubers of three aneusomatic dihaploids contained a S. phureja form of patatin. Aneusomatic dihaploids in which the proportion of cells containing additional S. phureja chromosomes was relatively small (i.e. 1–15%) did not express leaf isozyme markers or patatin bands characteristic of the dihaploid inducer or showed only faint expression of one or two markers. However, those with a high proportion of cells containing additional chromosomes (50–55%) had a range of strongly expressed leaf isozymes that were characteristic of the dihaploid inducer and also expressed the S. phureja tuber patatin.One dihaploid genotype was exclusively euploid (2n<24), yet is expressed a S. phureja leaf isozyme marker and S. phureja tuber patatin, suggesting recombination or chromosome substitution between the genome of the S. phureja dihaploid inducer and the cultivar Pentland Crown.     

Comparison of diploid and tetraploid potato families derived from Solanum phureja x dihaploid S. tuberosum hybrids and their vegetatively doubled counterparts

Summary To determine whether in potatoes the tetraploid level is preferable to the diploid level, especially regarding tuber yield, four diploid (2n=2×=24) Solanum phureja x dihaploid S. tuberosum hybrid parents and their vegetatively doubled, tetraploid (2n=4×=48) counterparts were intermated, which resulted in two F₁ hybrid families at both levels of ploidy. The parents and clones of the F₁ families and their offspring were used in crosses in such a way that in addition Sib₁, Sib₂, F₁×Sib₁, BC₁ and Sib₁×Sib₁ families were produced. Of the first clonal generation of the 12 2 x families and their 12 counterpart 4 x families two tubers per clone were planted in three replications in a field experiment at Sturgeon Bay in 1969; of the parents six tubers were planted in each replication. Data were recorded on 16 characters, including plant height at four different times.The ANOVA's showed significant clone effects within each family for all characters. Computed from all family means as well as from the family means per ploidy level, differences due to family were also significant for all characters except one.As at the 2 x level and at the 4 x level of ploidy the mean phenotypic correlations between characters were of similar magnitude, it is concluded that they are independent of ploidy level.With the exception of eye depth, the mean coefficient of variation was greater at the 2 x than at the 4 x level of ploidy, indicating the greatest response to selection for those characters at the 2 x level. From the differences in family means between the 4 x and the 2 x level of ploidy it was apparent that the 4 x families generally had significantly taller plants, later maturity, fewer tubers, higher mean tuber weight, more tuber yield and more dry matter yield than their counterpart 2 x families. In contrast, the 4 x parents had on the average shorter plants, lower mean tuber weight, much lower tuber yield and lower dry matter yield than their 2 x counterparts.The phenotypic correlation and Spearman's rank correlation between the family means of the 2 x and the 4 x level of ploidy were positive for almost all characters and significant for nearly half of them.From the results it is concluded that 1. in potatoes the 4 x level of ploidy is preferable to the 2 x level, and 2. the performance of 4 x families is predictable from the performance of their counterpart 2 x families.Based on results mentioned in the literature and on the present results, a continued use of S. tuberosum dihaploids in potato breeding needs to be dissuaded.     

Androgenesis as a means of dissecting complex genetic and physiological controls: selecting useful gene combinations for breeding freezing tolerant grasses

The advantages of androgenesis from Lolium × Festuca hybrids as a means towards enhanced gene expression and capture of rare genetic variation are reviewed. New evidence is presented for the technique’s use in combination with introgression-mapping for targeting Festuca-derived genes for enhanced freezing-tolerance. As a starting point, a dihaploid genotype derived by androgenesis from a Lolium multiflorum × Festuca pratensis amphiploid (2n = 4x = 28) hybrid cultivar is used as female parent in a backcross breeding programme with L. multiflorum (2n = 2x = 14). A derivative of the backcross␣breeding programme was a genotype of L.␣multiflorum (2n = 2x = 14) incorporating a F.␣pratensis introgression on chromosome 4 that was more freezing-tolerant than Lolium. New evidence of the importance in Lolium and Festuca species of the adaptive capabilities of Photosystem II (PSII) in relation to subsequent freezing-tolerance, is presented. Non-photochemical quenching (NPQ) mechanisms for expulsion of excess light energy during cold acclimation are found in F. pratensis but not in L. multiflorum. Screens of a backcross population derived from an initial dihaploid genotype (n + n = 14) produced by androgenesis from a L. multiflorum × F.␣pratensis amphiploid, indicate a direct relationship between cold acclimation induced increases in NPQ and freezing-tolerance. Preliminary evidence of a role for genes found on chromosome 4 of F. pratensis for increased NPQ expression, is presented.     

Induction of haploids and aneuhaploids in colchicine-induced tetraploid Solanum chacoense Bitt.

Summary The species Solanum chacoense BITT. (2n=2x=24) is a tuber-bearing, self-incompatible species which is important both for breeding and for genetic research. It crosses readily with most other tuber-bearing Solanum species including the common potato S. tuberosum (2n=4x=48). Gametophytic incompatibility hampers research in and utilization of this species. Doubling the chromosome number by colchicine makes it a self-compatible autotetraploid. By crossing selfed progeny of 4x-S. chacoense with a number of haploid-inducing diploid Solanum species a high yield of different dihaploid S. chacoense individuals (2n=24) could be obtained from one originally diploid clone: S. chacoense CPC 1153. More than 160 haploids showing a large variability were identified. The average haploid frequency was 53.7 per 100 berries. Most hybrid plants (70–100%) from four 4x × 2x crosses studied were tetraploid. The frequency of triploid hybrids was low (0–10%). Haploid-inducing capacity of fifteen male parents used in this study varied from 0–141 haploids per 100 berries. Careful examination of 156 haploids revealed 15 viable aneuhaploids (2n=25, 26, and 27), i.e. 9.4%. The potential value of these aneuhaploids is discussed.About 50% of the haploids were sufficiently male fertile to use them in crossing. A few of them set berries after selfing. Five aneuhaploids including the one with 27 chromosomes were successfully crossed as females with a diploid hybrid clone.     

Occurrence of 2n and jumbo pollen in the Brazilian ginseng (Pfaffia glomerata and P. tuberosa)

An analysis of microsporogenesis was carried out in four populations of Pfaffia tuberosa (2n=4x=68) and in ten populations of P. glomerata(2n=2x=34). Monads, dyads and triads were reported at high frequency in all populations of P. tuberosa. In P. glomerata, on the other hand, only four populations presented monads, and at low frequency. Dyads and triads were observed in both species, albeit at a higher frequency in P. tuberosa populations. Certain abnormalities of spindles in the second division, such as sequential and tripolar spindles, led to triad formation by the rejoining of two chromosome sets. The precise causes of monad and dyad formation have not been found during meiosis, although evidence exists that dyads originated by omission of second meiosis and monads by omission of both meiotic divisions. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Production of 27-, 28-, and 56-chromosome apomictic hybrid derivatives between pearl millet (2n=14) and Pennisetum squamulatum (2n=54)

Summary An interspecific hybridization program designed to transfer gene(s) controlling apomixis from Pennisetum squamulatum Fresen. (2n=6x=54) to induced tetraploid (2n=4x=28) cultivated pearl millet, Pennisetum americanum (L.) Leeke resulted in four offtype plants, two with 27 chromosomes and two with 28 chromosomes. These plants were found among 217 spaced plants established from open-pollinated seed of an apomictic 21-chromosome polyhaploid (2n=21) plant derived from an apomictic interspecific hybrid (2n=41) between tetraploid pearl millet and Pennisetum squamulatum. It appeared that a 21- (or 20-) chromosome unreduced egg from the apomictic polyhaploid united with a 7-chromosome pearl millet (2n=2x=14) gamete to produce a 28- (or 27-) chromosome offspring. Meiotic chromosome behavior was irregular averaging from 3.60 to 4.05 bivalents per microsporocyte in the 27- and 28-chromosome hybrids. The 27- or 28-chromosome hybrids, like the 21-chromosome female parent, shed no pollen, but set from 1.8 to 28 seed per panicle when allowed to outcross with pearl millet. Progeny of the 28-chromosome hybrids were uniform and identical to their respective female parents, indicating that apomixis had been effectively transferred through the egg. In addition, a 56-chromosome plant resulting from chromosome doubling of a 28-chromosome hybrid was identified. Pollen was 68 per cent stainable and the plant averaged 2.3 selfed seeds per panicle. Chromosomes of the 56-chromosome plant paired as bivalents (x=10.67) or associated in multivalents. Three to nine chromosomes remained unpaired at metaphase I. Multiple four-nucleate embryo sacs indicated the 56-chromosome hybrid was an obligate apomict. The production of 27-, 28-, and 56-chromosome hybrid derivatives were the results of interspecific hybridization, haploidization, fertilization of unreduced apomictic eggs, and spontaneous chromosome doubling. These mechanisms resulted in new unique genome combinations between x=7 and x=9 Pennisetum species.     

Genetics of self-compatibility in dihaploids of Solanum tuberosum L. 3. Lethality of S-bearing translocation homozygotes

Summary Self-compatible (sc) plants from an I₁ of the sc dihaploid G254 and from the reciprocal F₁'s of crosses between G254 and another sc dihaploid, B16, were crossed with the self-incompatible (si) dihaploid G609. The 34 progenies thus obtained were tested for self-compatibility. Only 2 progenies consisted merely of sc plants, the remaining ones showing a homogeneous series of 1 : 1 ratios. It is concluded that homozygosity for the S-bearing translocation mostly leads to lethality. Evidence is presented, that lethality takes place in the very early stages of the embryo and endosperm development. On the basis of the results the expected sc : si ratios in G254 × B16 and reciprocal are calculated to be 3.3 : 1. This ratio was found indeed in our experiments. These findings support the hypothesis put forward in earlier publications in this series, that self-compatibility in G254 and B16 is based on the presence of an S-bearing translocation.     

Breeding for quantitative resistance to potato cyst nematode (Globodera pallida) in tetraploid potatoes using dihaploids and unreduced gametes

Summary Dihaploids were produced from tetraploids resistant to potato cyst nematode (Globodera pallida (Stone)). High levels of resistance were found in the dihaploids and three were used to produce tetraploid progenies by crossing them with susceptible tetraploid cultivars. One dihaploid, PDH505, produced more highly resistant offspring than the other two, PDHs 417 and 418. The latter gave progenies whose levels of resistance were similar to those obtained from susceptible dihaploids crossed with resistant tetraploids.The differences between the progenies of the resistant dihaploids were probably due to different modes of unreduced gamete formation (PDH505 producing gametes by first division restitution (FDR) and PDHs 417 and 418 by second division restitution (SDR)) although cytological studies would be necessary to confirm this. The methods by which dihaploids could be utilised in a tetraploid potato breeding programme are discussed in relation to the mode of unreduced gamete formation.     

Female fertility of potato (Solanum tuberosum ssp. tuberosum) dihaploids

Summary Results of testcrosses and seed set data of the dihaploid breeding program at the Potato Research Institute in Gross Lüsewitz, Germany, indicate that the low level of female fertility could hinder success in crossing work. With the objective to reveal major components of female fertility, the behavior of 47 outstanding potato dihaploids (S. tuberosum ssp. tuberosum, 2n=2x=24) was assessed in testcrosses over a period of 3 years. Analysis of variance was carried out for the traits: number of seed per berry (S/B), number of intact seed per berry (IS/B), and number of non-germinable seed per berry (NGS/B). Significant sources of variation included cross combinations, years, and cross combination-year interactions. More than 20 S/B were obtained in 25.4% of all cross combinations performed over 3 years using up to three pollinators. Berry set was not correlated to seed set, but was in general determined by the pollinator used. The number of seedless berries (SLB/F) was related to neither total berry set nor seed production. The trait S/B remained stable over different years, and thus it deemed to be most useful as a measure of the level of female fertility in practical breeding work. Selection for female fertility in early generations of a breeding program is recommended to avoid limitations in the production of variability via sexual recombination.     

The origin of polyploids via 2n gametes in Vaccinium section Cyanococcus

Summary The production of 2n pollen (pollen with the sporophytic chromosome number) was evaluated in 4x and 6x taxa of Vaccinium section Cyanococcus. Mean frequencies of 2n pollen producers were 17.1% and 8.3% in natural 4x and 6x populations, respectively. The frequency of 2n pollen producers in the 4x species ranged from 8.6% (V. angustifolium) to 23.8% (V. pallidum). Level of 2n pollen production was genotypically variable (1% to 37.4%). The widespread occurrence of 2n pollen in 2x, 4x and 6x taxa suggests that sexual polyploidization was widespread and responsible for the origin of the polyploid species found in this genus. The frequency of 2n pollen producers was not significantly different between the 4x species and their putative 2x ancestors. These results support the origin of 4x and 6x taxa as a consequence of sexual polyploidization. Polyploids derived from sexual polyploidization would be expected to have increased fitness and flexibility due to the mode of 2n pollen formation. In blueberry species the predominant mode of 2n pollen formation is genetically equivalent to a first division restitution mechanism (FDR). FDR 2n pollen transmits a high percentage of the heterozygosity and a large fraction of the epistasis from the 2x parent to the 4x offspring.     

The occurrence of 2 n eggs in the dihaploids derived from Rosa hybrida L.

Dihaploid derivatives of 4x Rosa hybrida L. obtained through parthenogenesis induced by irradiated pollen significantly produce 2n eggs,often accompanied by gametes with higher ploidy levels (3x and 4x). The gamete ploidy level distribution depends on the originating genotype. The ploidy level of the male parent affects the maternal /paternal ratio in the endosperm, which can be involved in the counter-selection of triploid genotypes resulting from the crosses made between dihaploid and 4x Rosa hybrida.The observed segregation ratios of flower doubleness in triploid progenies of dihaploids × R. wichuraïanasuggest that the relevant locus could be positioned far from the centromere.     

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.     

A tetraploid hybrid plant from 4x × 2x crosses in Vitis and its origin

To study the origin of unreduced (2n) gametes in diploid Vitis cultivars, we surveyed the occurrence of tetraploid hybrid seedlings from 40 interploid crosses with five tetraploid and seven diploid cultivars. A total of 250 seedlings from the interploid crosses were established through embryo culture and by seed sowing. In 20 2x × 4x crosses, no tetraploid hybrid seedlings were derived from 8,902pollinations. In 20 4x × 2x crosses, two tetraploid hybrid seedlings were obtained from 8,057 pollinations. Investigation of isozyme genotypes of the two tetraploid seedlings using three variable enzyme systems indicated that one of the two seedlings resulted from the union of a diploid egg with a 2n male gamete and that failure of second meiotic division resulted in the formation of the 2n male gamete. The percentage of giant pollen grains in `Muscat Bailey A', a pollen parent of the tetraploid seedling, was relatively high(about 5.9%) and 10.9% of the giant pollen grains germinated on agar medium. These results suggested that 2n pollen of diploid cultivars are useful for breeding tetraploid grape. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Fingerprinting of alfalfa meiotic mutants using RAPD markers

Summary A calendar of female sporogenesis and gametogenesis was made for both apomictic tetraploid (2n=4x=36) Brachiaria brizantha and Brachiaria decumbens and their apomictic F₁ hybrids with sexual tetraploid (2n=4x=36) Brachiaria ruziziensis. Microgametogenesis was used as a reference. Apospory was facultative in both species and hybrids. Environmental conditions had variable effects on the level of apomixis according to each genotype. Ratios of segregation into sexuals and apomicts in the interspecific hybrids suggest an oligogenic determinism with dominant apomixis in the genus Brachiaria. Highly apomictic and partially male fertile hybrids were identified and will be used in an improvement program to transfer genes for apomixis into the sexual species B. ruziziensis.     

Breeding behavior of yield components and hollow heart in tetraploid-diploid vs. conventionally derived potato hybrids

Summary A factorial mating design with six 4x cultivars as stylar parents mated to three groups of pollen parents (4x cultivars, 4x high protein selections, and 2x S. phureja) was used to study breeding behavior of yield, tuber set, marketable yield and hollow heart in potatoes. Mean yield for the 4x-2x hybrids was equivalent to that of their cultivar parents although yield components, tuber set and average tuber size, were greater and less, respectively, than the cultivar parents. High estimates of general combining ability were found in both pollen and stylar parents for yield, tuber set, marketable yield, number of marketable tubers and hollow heart in all three hybrid populations. Specific combining ability estimates were significant only for yield and hollow heart. Frequency of white-fleshed 4x-2x progeny of five heterozygous yellow-fleshed 2x phureja genotypes was used to confirm cytological identification of a first division restitution mechanism operative in the 2x parents. Although three of these phureja genotypes generated high-yielding progeny, two others produced 4x-2x progeny with mean yield similar to that of hybrids among 4x cultivars.Scientific journal series article 11,601 of the Minnesota Agricultural Experiment Station.     

Monohaploid plants from anthers of a dihaploid genotype of Solanum tuberosum L.

Summary Monohaploid Solanum tuberosum plants were produced from the anthers of a dihaploid genotype. From another dihaploid genotype plants containing 36 chromosomes were obtained. For plantlet production anthers containing pollen at the uninucleate microspore stage were inoculated on a Linsmaier and Skoog-based medium supplemented with 1 mg/1 indole-3-acetic acid and 1 mg/1 benzyl aminopurine.Anthers from donor plants grown either in the summer or in the winter responded similarly on a range of media. Anther response in the form of callus induction and root formation was at least partly dependent on the genotype of the donor dihaploid plant.