Assessment of genetic and phenotypic variation among intraspecific somatic hybrids of potato, Solanum tuberosum L.

Intraspecific somatic hybrids have been produced by protoplast fusion in eight combinations involving 10 dihaploids (2n= 2x= 24) in an attempt to provide new material for potato breeding. Cytological analysis revealed extensive variation in chromosome number, such as aneuploid, aneusomatic and mixoploid hybrids. Most of the hybrids represented the expected chromosome number of 48; however, the frequency of aneuploids reached 50% in some combinations. Some hybrids carried structurally rearranged chromosomes and exhibited a high frequency of aberrant anaphases. Isozyme and random amplified polymorphic DNA (RAPD) patterns of the hybrids from the same fusion combination were uniform. In the field, somatic hybrids showed wide phenotypic variation in 20 morphological characters. There was a significant correlation between certain leaf characters and the ploidy level, which may be used to distinguish the tetraploid hybrids from hexaploids and octoploids. Tuber yield and flowering intensity were highest in tetraploid hybrids (2n= 4x= 48). Eighteen of the 73 hybrids reached higher yields than the standard variety ‘Adretta’. Floral development and fertility were restored in hybrids derived from fusions between non-flowering or sterile dihaploids.     

Fertile somatic hybrids of Solanum etuberosum (+) dihaploid Solanum tuberosum and their backcrossing progenies: relationships of genome dosage with tuber development and resistance to potato virus Y

The wild non-tuberous species Solanumetuberosum is resistant to biotic andabiotic stresses, but is very difficult tocross with cultivated potato. Therefore,interspecific somatic hybrids between adihaploid clone of potato S.tuberosum (2n=2x=24, AA genome) and thediploid species S. etuberosum(2n=2x=24, EE genome) were produced byprotoplast fusion. Among the 7 fertilefusion hybrids analysed by genomic insitu hybridisation (GISH), three groups ofplants were found with the genomicconstitution of AAEE, AAEEEE and AAAAEE.Four fusion hybrids had exactly theexpected chromosome composition, while eachof the three aneuploid hybrids had lost twochromosomes of S. etuberosum. Twobackcross progenies were developed, andGISH analysis was applied to analysetransmission of the parental chromosomesinto the sexual generations. BC₁hybrids derived from the crosses of thehexaploid somatic hybrids with tetraploidpotato were pentaploid with thetheoretically expected genomic compositionor with slight deviation from thisexpectation. In the three BC₂ hybridsanalysed by GISH seven to 12 chromosomes ofS. etuberosum were detected in thepredominant S. tuberosum background.No recombinant chromosomes in the hybridswere detected. Genome dosage affects tuberformation in hybrids and their progenies,but has less effect on resistance to potatovirus Y (PVY) in fusion hybrids. Severalgenotypes of the fusion hybrids andBC₁ progeny did not show viralinfection even in the graftingexperiments.     

Expression of nonacclimated freezing tolerance and cold acclimation capacity in somatic hybrids between hardy wild Solanum species and cultivated potatoes

The expression of freezing tolerance was characterized in interspecific somatic hybrids between S. tuberosum (tbr) and two cold-hardy wild species, S. brevidens (brd) and S. commersonii (cmm). The nonacclimated freezing tolerance (NA) and acclimation capacity (ACC, increase in freezing tolerance in response to low nonfreezing temperature), two main genetic components of freezing tolerance, were evaluated separately. In contrast to cmm, which exhibited excellent NA and ACC, the freezing tolerance of brd was mainly due to ACC. However, the ACC of brd was only moderately expressed in the somatic hybrids. The NA of cmm was also suppressed in combination with tbr genomes. However, with acclimation, some of the tbr (+) cmm somatic hybrids achieved freezing tolerance comparable to pure hardy species such as brd used in this study. Analysis of chloroplast DNA type by RFLP markers revealed no significant difference in ACC between somatic hybrids carrying chloroplasts from either tbr or cmm. The reasons for the reduced expression of freezing tolerance from either the brd or cmm parent and the utilization of these somatic hybrids in breeding programs are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Multivariate analysis of variation among Solanum commersonii (+) S. tuberosum somatic hybrids with different ploidy levels

In order to integrate the outcomes of interspecific somatic fusion experiments in breeding schemes, it is important to understand factors involved in the variability observed among regenerated plants. With such a purpose in mind, a population of Solanum commersonii (+) S. tuberosum somatic hybrids was examined by means of discriminant and cluster analyses. Data were collected on 56 hybrids with different ploidies and on the two diploid parents grown in a greenhouse. The ploidy group was used as discrimination criterion. Three significant canonical variables were extracted by discriminant analysis; they were mainly correlated with the number of leaflets and stems, degree of flowering, plant height, and leaf length. After cluster analysis carried out with the significant canonical variables, parental and hybrid clones were grouped in 7 clusters. In the canonical space of reduced dimensions, patterns of morphological variation depended mainly on ploidy level and non-additive gene interactions. Hybrids were in general more similar to the cultivated than to the wild species, suggesting a good chance of fast introgression of useful traits from S. commersonii into the S. tuberosum genetic background.     

Identification of somatic hybrids of dihaploid Solanum tuberosum lines and S. brevidens by species specific RAPD patterns and assessment of disease resistance of the hybrids

Summary Symmetric somatic hybrids were produced by electrofusion of protoplasts of two dihaploid tuber-bearing potato (Solanum tuberosum L.) lines and Solanum brevidens Phil., a diploid non-tuber-bearing wild potato species. A total of 985 plants was obtained. Verification of nuclear hybridity of putative hybrids was based on additive RAPD patterns, general morphological characteristics and chromosome counts. 53 (90%) calli regenerated into plants which were identified as somatic hybrids. Most of the hybrids were aneuploids at the tetraploid (4×) or hexaploid (6×) level. The 20 hybrids tested expressed a high level of resistance to potato virus Y (PVY ^N ) characteristic of the S. brevidens parent. Resistance to late blight (Phytophthora infestans (Mont.) de Bary) varied between hybrids, but was on average better than that of the fusion parents. Resistance of hybrids to bacterial stem rot (Erwinia carotovora subsp. atroseptica (van Hall) Dye) was not superior to that of commercial potato cultivars.     

Production of somatic hybrids between S. tuberosum L. and late blight resistant Mexican wild potato species

Interspecific somatic hybrids between dihaploid breeding clones of potato, S. tuberosum and two accessions of wild Mexican species S. pinnatisectum and the hybrid line S. pinnatisectum × S. bulbocastanum were regenerated following electrofusion of mesophyll protoplasts to combine important agricultural traits of S. tuberosum and a high level of late blight resistance from selected wild accessions. In two fusion combinations 239 calli were regenerated; 162 from 195 calli analysed were identified as hybrids by means of isozyme analysis of peroxidases and, for some hybrid clones, by RAPD analysis. Depending on the fusion combination, 47–89 percent of the somatic hybrids had the expected ploidy level and 7–16 percent were mixoploids. Somatic hybrids were phenotypically intermediate as compared to their parents and some of them were able to be backcrossed sexually with potato. Fertility and crossability depended on combination and ploidy level of the somatic hybrids. In tests with detached leaves the wild partner clones had a high late blight resistance score of 8,6 and 8,9; the susceptible tuberosum-partners of 2,8 and 3,5, respectively. Nearly 25 percent of somatic hybrids had a resistance level of 6 or higher in the first year of assessment. The average resistance value of most somatic hybrids was lower than the average parental level. The reasons for variation in resistance values are discussed in connection with the practical application of fusion hybrids. This revised version was published online in July 2006 with corrections to the Cover Date.     

Breaking the crossability barriers between disomic tetraploid Solanum acaule and tetrasomic tetraploid S. tuberosum

Summary A combination of compatible second pollinations and embryo rescue was applied for systematic production of true tetraploid hybrids from crosses between disomic tetraploid Solanum acaule and tetrasomic tetraploid potato, S. tuberosum. Several genotypes of tetraploid potatoes were pollinated with S. acaule, and the compatible second pollinations were made on the following day, with a genotype of S. phureja, IvP 35 to promote fruit development. Embryo rescue was carried out in 21 families, 14 to 27 days after the first pollination. A total of eight plants were obtained from the embryo rescue and their chromosome numbers were counted in the root tips. Three of the eight plants were identified as tetraploid, and five others as diploid. Morphology, isozyme banding patterns, and pollen stainability, as well as potato spindle tuber viroid (PSTVd) resistance, indicated the hybrid nature of the three plants. This is the first report of successful tetraploid hybrid production between disomic tetraploid S. acaule (4x) and tetrasomic tetraploid potatoes. Seed set from the crosses between one of hybrids and diploid potatoes indicated workable levels of both male and female fertility for introgression of valuable genes from S. acaule into the cultivated potato gene pool. The methodology used may be applied to other disomic tetraploid tuber-bearing Solanum species and with some modifications also to distantly related solanaceous species and genera.     

Identification of the trisomic series in diploid Solanum tuberosum L., group tuberosum. I. Chromosome identification

Summary The morphology of 12 pachytene bivalents of the dihaploid Solanum tuberosum cv. Gineke is described, and the morphological features which are helpful for a critical chromosome identification are illustrated. The difficulties of identifying the individual chromosomes and the extra chromosomes in trisomics are discussed.     

Characterization of morphological variation and cold resistance in interspecific somatic hybrids between potato (Solanum tuberosum L.) and S. brevidens Phil.

Summary Somatic hybrids between Solanum tuberosum L. cv. Gracia (2n=4x=48) and Solanum brevidens Phil. (2n=2x=24) were produced via fusion of mesophyll protoplasts. Selection of the protoplast derived putative hybrid calli was based on their vigorous growth. Additive isozyme patterns and chromosome numbers as well as the expression of parental morphological characters have proved the hybrid origin of the selected regenerants. Extensive chromosome loss during the regeneration process resulted in aneuploid hybrids with high frequency. Genomic instability could not be detected in these plants during the period of vegetative propagation. Regenerants from hybrid tissues exhibited wide morphological variation especially in tuber formation. The detailed morphological analysis based on the use of multivariate method (principal component analysis, PCA) enabled to identify morphological groups among the hybrid clones. The positioning of hybrid clones in the PCA space could not be correlated with chromosome numbers. The genomic ratio represented by the tetraploid and diploid parents influenced the morphology of somatic hybrid population according to the applied analytical system. Two selected hybrid clones have exhibited an intermediate degree of frost tolerance compared to the parents, based on the recovery of plants from lower buds after freezing of potted plants.     

Stability of fingerprints of Solanum tuberosum plants derived from conventional tubers and vitrotubers

The restriction and amplification patterns of potato genomic DNA of eight different cultivars and breeding clones has been studied. The analysis was carried out by polyacrylamide high resolution gels, and subsequent blotting and hybridization to potato DNA probes, and by agarose gel electrophoresis of the amplification products obtained with a variety of random primers. Fingerprinting of the genotypes was possible using two enzyme-probe combinations (Rsal-GP35 and Rsal-CP6) and three random primers (OPA4, OPA19 and OPG12). Based on the same techniques, a number of plants from the cvs. Monalisa and Spunta originated from the sprouting of in vitro-induced tubers (vitrotubers) were analysed to test the reproducibility of RFLP and PCR patterns. No variations were found with RFLP analysis. Some different RAPD patterns were seen, showing putatively vitrotuber-specific variations.     

Isolation and characterisation of somatic hybrids of diploid Solanum tuberosum and Solanum brevidens and the use of amylose-free starch mutation for detection of introgression

Summary Somatic hybrids of diploid amylose-free (amf) Solanum tuberosum and diploid S. brevidens were made by Poly-Ethylene-Glycol (PEG) or electrofusion methods. For the isolation of interspecific hybrids the use of selection markers (kanamycin and hygromycin resistance) was useful but not essential. In this 2x+2x interspecific combination 4x and 6x somatic hybrids were obtained. Seed set was the best in 4x×4x (S. tuberosum) backcrosses, but seed germination was the best in 6x×4x combinations, using in vitro germination of unripe seeds harvested 25 days after pollination. A high degree of pollen stainability (30–40%) was observed in 7 tetraploid hybrids and very low in all hexaploids. After iodine staining, the recessive amf marker was expressed by a red colour instead of blue, visible in starch-containing cells like columella cells of root tips, (micro)tubers or microspores. As expected, complementation was observed in starch-containing cells of the fusion hybrids. Segregation of the amf marker was clearly observed in microspores of 4x and 6x hybrids. Segregation ratios in the 4x hybrids showed variable recombination frequencies. In the backcross progeny of hexaploid F12-5 with a tetraploid amf mutant one amylose-free recombinant among 67 plants was found, indicating the occurrence of meiotic recombination in the megaspore mother cells.     

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.     

Alteration of the genomic composition of Solanum nigrum (+) potato backcross derivatives by somatic hybridization: selection of fusion hybrids by DNA measurements and GISH

Fusion experiments were performed with a first (BC₁‐6738) and a second (BC₂‐9017) generation backcross hybrid of 6x Solarium nigrum (+) 2x potato somatic hybrids with potato cultivars. Because no progeny was obtained from the BC₂ genotypes, alternative approaches were sought to overcome the sexual crossing barrier. Five potato genotypes, one of which contains the hygromycin resistance gene, were used in the fusion experiments. All vigorous regenerants were used for the estimation of nuclear DNA content using flow cytometry. Plants with a DNA content higher than that of the BC₁‐6738 or BC₂ genotypes were considered potential somatic hybrids. Forty‐nine potential somatic hybrids resulted from fusion experiments with BC₁‐6738, from which 20 grew vigorously in the greenhouse and flowered. After pollination with several 4x potato cultivars, eight genotypes produced seeded berries and five genotypes gave seedless berries. In addition, 11 of these 13 somatic hybrids were selected for genomic in situ hybridization (GISH) analysis to determine their genomic composition. Nine had exactly or approximately the expected number of 36 S. nigrum and 60 potato chromosomes. In one genotype, only 22 instead of 36 S. nigrum chromosomes were found and one potato chromosome was possibly missing. Only five potential somatic hybrids were detected among the 79 regenerants from BC₂‐9017 (+) 2x potato fusion experiments that were analysed by flow cytometry. Two of these hybrids were rather vigorous and did flower, but pollinations with potato have not yet set any berries.     

The Use of RFLPs (Restriction Fragment Length Polymorphisms) Detects Germplasm Introgressions from Wild Species into Potato (Solanum tuberosum ssp. tuberosum) Breeding Lines

The germplasm of the potato (S. tuberosum ssp. tuberosum) has been modified since the beginning of this century by breeders who introgressed important agronomic traits, for example disease resistance genes, from several wild and cultivated Solanum species of the Americas. In this paper we show that the Rflp analysis of potato-breeding material can be used to detect chromosomal regions descended from more-distantly related Solanum species. The Rflp patterns of individuals of ten Solanum species, ten breeding lines of S. tuberosum ssp. tuberosum and the cultivated variety ‘Bintje’ were analyzed. Rflp data for each of eight single loci of known genomic position were used for the computation of locus-specific phenograms by distance matrix methods. Several of the potato-breeding lines deviated clearly from the clustered species S. tuberosum, S. stenotomum and S. canasense for one or more of the loci considered. These deviations indicated the presence of “exotic” germplasm at a particular locus. The possibility of detecting such germplasm has implications for mapping the agronomic traits for which the wild species were introgressed and which may still be linked to “foreign” chromosome fragments.     

Somatic hybridization between Solanum tuberosum and species of the S. nigrum complex: Selection of vigorously growing and flowering plants

Fusion experiments were performed between diploid (2n = 2x = 24) or tetraploid (2n = 4x = 48) potato genotypes and four species of the Solanum nigrum complex, namely S. nigrum (2n = 6x = 72), S. villosum (2n = 4x = 48), S. chenopodioides (2n = 2x = 24) or S. americanum (2n = 2x = 24 and 2n = 6x = 72). All five accessions of the S. nigrum-species were successfully hybridized with at least one of the potato genotypes. Somatic combining abilities were influenced by the ploidy level as well as the genotype of the parental species. The use of kanamycin or hygromycin resistance as cell-selectable markersystem had no influence on somatic combining ability, but such markers can be useful to improve efficient selection of somatic hybrids in sufficient numbers. At least 20% of the hybrids of each successful combination performed well in vitro. However, only 60 genotypes out of 761 somatic hybrids were vigorous as well as flowering in the greenhouse. Analysis of the DNA content of somatic hybrids could be used as a criterion for the indirect selection in vitro of hybrids that were vigorous in the greenhouse. Flowering somatic hybrids of S. nigrum (+) 2x potato and S. americanum (+) 4x potato were selected with the aim of introgression of resistance traits after recurrent backcrossing with cultivated potato. This revised version was published online in July 2006 with corrections to the Cover Date.     

Intraspecific somatic and sexual hybridisation between dihaploid lines of Solanum tuberosum L.: evaluation of morphological traits and resistance to late blight Phytophthora infestans (Mont.) de Bary in the foliage

Intraspecific tetraploid somatic and sexual hybrid plants have been resynthesised following protoplast fusion and by sexual crosses between two dihaploid potato (Solanum tuberosum) lines each possessing complementary agronomic traits. The dihaploid PDH 40 possesses good tuber shape and yield but has foliage susceptibility to late blight (Phytophthora infestans). On the other hand, the dihaploid PDH 727 possesses resistance to blight in the foliage but has a low yield of small and irregular shaped tubers. Since it was only possible to use a partial selection strategy based on culture media to facilitate recovery of somatic hybrid plants-further morphological and esterase isozyme based characterisations were performed to identify somatic hybrid plants from amongst the non-hybrid plant material. When the blight resistance of both the intraspecific somatic and sexual hybrid plants was assessed there was no significant difference in the mean resistance value and it was intermediate between those of their parents. However, the range of resistance was much wider among the sexual hybrids than among the plants derived from somatic fusion. An assessment of tuber yield between tetraploid sexual and somatic hybrids showed no significant difference and it was higher than that of either parent value. The implication of these results in the context of potato genetics and breeding is discussed.     

Induction of novel organelle DNA variation and transfer of resistance to frost and Verticillium wilt in Solanum tuberosum through somatic hybridization with 1EBN S. commersonii

Somatic hybridization can be used to induce genetic variability in plastidial and mitochondrial genomes, and transfer multiple uncloned genes across sexual barriers. Somatic hybrids were produced between a dihaploid clone of the common potato, S. tuberosum subsp. tuberosum, and the wild sexually incongruent diploid species S. commersonii. Fusion products were selected on the basis of callus growth and regeneration in vitro. Genome composition of putative somatic hybrids was determined by flow cytometric analysis of nuclear DNA content, RAPD analysis, and Southern analysis with probes specific to organellar DNA. All regenerated fusion products proved to be hybrids based on RAPD analysis. Seventy per cent of somatic hybrids were (near) tetraploids, 22% (near) hexaploids and 8%(near) octoploids. A high correlation was found between the nuclear DNA content determined by flow cytometry and the number of chloroplasts in stomata guard cell pairs. Somatic hybrids inherited the parental plastids in a random manner. On the contrary, they preferentially inherited the mitochondrial DNA fragments of S. tuberosum. The majority of them had a rearranged mitochondrial genome with fragments from both parents. Hybrids were highly vigorous and morphologically more similar to the cultivated than to the wild parent, produced tubers on long stolons under long photoperiod conditions, showed a high degree of flowering, but did not produce pollen. In addition, somatic hybrids were generally more resistant to frost and Verticillium wilt than the cultivated parent, indicating the introgression of relevant resistance genes from the wild species into the genetic background of S. tuberosum. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytogenetic analysis of two interspecific Citrus allotetraploid somatic hybrids and their diploid fusion parents

Microsporogenesis of two interspecific allotetraploid somatic hybrids, i.e.‘Hamlin’ sweet orange +‘Rough Lemon’ (HR) and ‘Key’ lime +‘Valencia’ sweet orange (KV), was examined and compared with their corresponding diploid fusion parents. Meiotic analysis revealed that only 13.2 and 18.0% of the meiocytes exhibited normal tetrad formation in HR and KV somatic hybrid plants, respectively, and irregular chromosome behaviour with univalent or multivalent pairing occurred. Moreover, meiotic abnormalities such as chromosome bridges, chromosomes orientated away from the equatorial plate, especially lagging chromosomes, which resulted in different sizes of pollen grain, were frequently observed in both somatic hybrids. As expected, the percentage of chromosome abnormalities was much lower in their corresponding diploid fusion parents. HR and KV had 15.4 and 7.2% pollen germinability, respectively, intermediate between their corresponding fusion parents. The meiotic behaviour of these somatic hybrids provided valuable information for their practical utilization in a citrus breeding programme.     

Fertility restoration of hybrids between Solanum melongena L. and S. aethiopicum L. Gilo Group by chromosome doubling and cytoplasmic effect on pollen fertility

Reproductive fertility traits were studied in the reciprocal hybrids of the eggplant(Solanum melongena L.) and S. aethiopicum L. Gilo Group, and in synthetic amphidiploids to discover whether fertility in these reciprocal hybrids was restored by chromosome doubling. Isozyme and RAPD analyses confirmed hybridity of the hybrids and amphidiploids. Analyses of chloroplast and mitochondrial DNAs confirmed that the cytoplasm of each of the hybrids and amphidiploids was from the maternal parent. Pollen sterility of S. melongena × S. aethiopicum Gilo Group [F₁ (Mel × Aet)] was restored by chromosome doubling, while the reciprocal hybrid S. aethiopicum Gilo Group ×S. melongena [F₁ (Aet × Mel)]and its amphidiploid did not produce any pollen grains; their microspores degenerated without being released from tetrads. Hence the cytoplasm of S. aethiopicum Gilo Group seems to beresponsible for their pollen-non-formation type sterility of the hybrid. Both the F₁ hybrids did not set any fruits by either selfing or backcrossing, while their amphidiploids set fruits after pollinating with pollen from the amphidiploid of F₁ (Mel × Aet). Seeds obtained from both the amphidiploids germinated normally. Chromosome doubling has been effective in restoring fertility of the hybrids. This revised version was published online in July 2006 with corrections to the Cover Date.