Production of interspecific somatic hybrid plants in the genus Medicago through protoplast fusion

Summary Symmetric somatic hybrid plants have been produced by electrofusion of leaf protoplasts of Medicago sativa and callus protoplasts of Medicago coerulea. The selection of hybrid individuals has been performed at the cellular level by recording the positions of single heterocaryons immobilized in a semisolid culture medium. The hybrid nature of the heterokaryons was assessed in fluorescent light on the basis of their color. Hybrid minicalli were picked up manually and grown first on propagating, and then on regenerating, media. Six putative hybrid calli were selected and two of them regenerated several plants. The hybrid nature of the regenerants was confirmed by cytological and isozyme analysis. Among the several morphological traits taken into account for the characterization of somatic hybrid plants, some were intermediate, some lower, and some higher, with respect to the parents. The somatic hybrid plants were fertile and set seed. The production of somatic hybrid plants in the genus Medicago is discussed in relation to the regenerating capability of parental protoplasts.This research was supported by the National Research Council of Italy, Special Project RAISA, Subproject N. 2 paper N. 347     

Somatic hybridization between birdsfoot trefoil (Lotus corniculatus L.) and L. conimbricensis Willd

Summary Somatic hybrid plants were produced by fusion of birdsfoot trefoil (Lotus corniculatus) cv Leo and L. conimbricensis Willd. protoplasts. Birdsfoot trefoil etiolated hypocotyl protoplasts were inactivated with iodoacetate to inhibit cell division prior to fusion with L. conimbricensis suspension culture protoplasts. L. conimbricensis protoplasts divided to form callus which did not regenerate plants. Thus, plant regeneration from protoplast-derived callus was used to tentatively identify somatic hybrid cell lines. Plants regenerated from three cell lines exhibited additive combinations of parental isozymes of phosphoglucomutase, and L. conimbricensis-specific esterases indicating that they were somatic hybrids. The somatic chromosome number of one somatic hybrid was 36. The other somatic hybrid exhibited variable chromosome numbers ranging from 33 to 40. These observations approximate the expected combination of the birdsfoot trefoil (2n=4x=24) and L. conimbricensis (2n=2x=12) genomes. Somatic hybrid flowers were less yellow than birdsfoot trefoil flowers and had purple keel tips, a trait inherited from the white flowered L. conimbricensis. Somatic hybrids also had inflorescence structure that was intermediate to the parents. Fifteen somatic hybrid plants regenerated from the three callus lines were male sterile. Successul fertilization in backcrosses with birdsfoot trefoil pollen has not yet been obtained suggesting that the hybrids are also female sterile. This is the first example of somatic hybridization between these two sexually incompatible Lotus species.Formerly USDA-ARS, St. Paul, Minn, USA     

Regeneration of intergeneric somatic hybrid plants between Lycopersicon esculentum and Solanum muricatum

Summary Mesophyll protoplasts of tomato (Lycopersicon esculentum) and pepino (Solanum muricatum) were fused by using an electrofusion method and cultured in modified MS medium supplemented with naphthaleneacetic acid and kinetin, in which only pepino and somatic hybrid protoplasts could divide. Somatic hybrid plants showing intermediate characteristics in morphology were regenerated from the calli exhibiting vigorous growth in contrast with those of pepino. The hybrid nature of these plants was confirmed by cytological observation and biochemical analyses of phosphoglucomutase isozymes and the fraction-1-protein. The regenerated somatic hybrids grew to flowering stage and set fruits.     

Somatic hybridization of sexually incompatible top-fruit tree rootstocks,wild pear (Pyrus communis var. pyraster L.) and Colt cherry (Prunus avium x pseudocerasus)

Summary Mesophyll protoplasts of wild pear (Pyrus communis var. pyraster L., Pomoideae) were chemically fused with cell suspension protoplasts of cherry rootstock Colt (Prunus avium x pseudocerasus, Prunoideae), following an electroporation treatment of the separate parental protoplast systems. Fusion-treated protoplasts were cultured, on modified K8P medium, where it had been previously established that neither parental protoplasts were capable of division. Somatic hybrid calli were recovered and, following caulogenesis on MS medium with zeatin and after rooting of regenerated shoots, complete trees were obtained and grown in vivo. Hybridity of these trees was confirmed based on morphological characters, chromosome complement and isozyme analysis. Two separate cloned lines of this intersubfamilial rootstock somatic hybrid (wild pear (+) Colt cherry) were produced. This is the first report of the production of somatic hybrid plants of two woody species, of agronomic value, within the order Rosales.     

Regeneration of somatic hybrid plants formed between Lycopersicon esculentum and L. pennellii

Summary Somatic hybrid plants have been regenerated following polyethylene glycol mediated fusion of leaf mesophyll protoplasts from tomato and protoplasts from Lycopersicon pennellii callus. Three different cultivars of tomato were used as sources of protoplasts: Early Girl, Manapal, and UC82B. Fusions were performed between protoplasts of these tomato cultivars and protoplasts of L. pennellii, and between protoplasts of the cultivars and protoplasts of L. pennellii that had been exposed to 3 or 6 krads of gamma radiation. Somatic hybrid plants were identified on the basis of heterozygous isozyme banding patterns, and leaf and flower morphology. Somatic hybrid plants were regenerated following fusion of tomato protoplasts with either untreated or irradiated L. pennellii protoplasts. All were heterozygous for isozyme loci on five different chromosomes. Regenerated somatic hybrids showed inheritance of either or both parental chloroplast genomes, but predominantly the L. pennellii mitochondrial genome. The regenerated somatic hybrid plants exhibited reduced fertility, less than 20% viable pollen. A total of 34 somatic hybrid calli were identified. Of these, 21 regenerated shoots, and 7 produced seed following manual pollinations.     

Interspecific hybrids between Medicago sativa L. and annual Medicago containing Alfafa weevil resistance

Non-embryogenic protoplasts of Medicago rugosa and M. scutellata were electro-fused with iodoacetic acid-treated protoplasts of M. sativa (alfalfa). Putative somatic hybrid callus were obtained and some plants regenerated from both combinations. Hybridity of regenerants was confirmed by morphology, molecular means and cytological observations. Parental specific bands were recognized in somatic hybrids by Southern analysis. The somatic hybrids were perennial and their morphology was similar to M. sativa. Cytological observations were carried out on the somatic hybrids, their vegetative clones and self-pollinated offspring. Original somatic hybrids were aneuploids (2n=31–59), but during vegetative proliferation, their chromosome numbers reduced to 32. Those clones of hybrids formed seeds from M. sativa (+) M. rugosa by self-crossing. Chromosomal rearrangements within the parental genomes were observed in vegetative clones of hybrids and their S₁ offspring by Genomic in situ Hybridization (GISH). Some of S₁ offspring from M. sativa (+) M. rugosa showed better spring growth than parental M. sativa and tend to be tolerant to Alfalfa weevil. It was considered that these traits were introduced from the genome transferring M.␣rugosa chromosome to M. sativa. The cell fusion may still have a potential in transferring alien chromosomes in order to increase the genetic variation for crop breeding.     

Production and characterization of fertile somatic hybrids of eggplant (Solanum melongena L.) with Solanum aethiopicum L.

Summary In order to produce fertile somatic hybrids, mesophyll protoplasts from eggplant were electrofused with those from one of its close related species, Solanum aethiopicum L. Aculeatum group. On the basis of differences in the cultural behavior of the parental and hybrid protoplasts, 35 somatic hybrid plants were recovered from 85 selected calli. When taken to maturity either in the greenhouse or in the field, the hybrid plants were vigorous, all rapidly overtopping parental individuals. The putative hybrids were intermediate with respect to morphological traits, and all of their organs were larger, particularly the leaves and stems. DNA analysis of the hybrids using flow cytometry in combination with cytological analysis showed that 32 were tetraploids, 1 hexaploid and 2 mixoploids. The hybrid nature of the 35 selected plants was confirmed by a comparison of the isoenzyme patterns of isocitrate dehydrogenase (Idh), 6-phosphogluconate dehydrogenase (6-Pgd) and phosphoglucomutase (Pgm). Chloroplast DNA (ctDNA) restriction analysis using Bam HI revealed that among the 27 hybrid plants analyzed, 10 had S. aethiopicum patterns and the 17 remaining hybrids exhibited bands identical with those of eggplant without any changes. All of the somatic hybrid plants flowered. Both parental plants had 94% stainable pollen, while the hybrids varied widely in pollen viability ranging from 30% to 85%. The somatic hybrids showed high significant variation in fruit production. Nevertheless, there was a tendency for low fertility to be associated often with S. aethiopicum chloroplast type and/or with an abnormal ploidy level, while good fertility was mostly associated with the tetraploid level and eggplant chloroplasts. Interestingly, 2 tetraploid somatic hybrid clones were among the most productive, yielding up to 9 kg/plant. As far as the fertility of the F₁ sexual counterpart was concerned, only 2 fruits of 50 g were obtained. Hybrid fertility in relation to phylogenetic affinities of the fusion partners is discussed.     

Somatic hybrid plants produced by electrofusion between Solanum melongena L. and Solanum torvum Sw

Summary Somatic hybrid plants between eggplant (Solanum melongena) and Solanum torvum have been produced by the electrofusion of mesophyll protoplasts in a movable multi-electrode fusion chamber. Using hair structure as a selection criteria, we identified a total of 19 somatic hybrids, which represented an overall average of 15.3% of the 124 regenerated plants obtained in the two fusion experiments. Several morphological traits were intermediate to those of the parents, including trichome density and structure, height, leaf form and inflorescence. Cytological analyses revealed that the chromosome numbers of the somatic hybrids approximated the expected tetraploid level (2n=4x=48). Fifteen hybrid plants were homogeneous and had relatively stable chromosome numbers (46–48), while four other hybrids had variable chromosome numbers (35–48) and exhibited greater morphological variation. The hybridity of these 19 somatic hybrid plants was confirmed by analyses of phosphoglucomutase (Pgm) and esterase zymograms.     

Somatic hybridization in the gramineae: Pennisetum americanum (L.) K. Schum. (Pearl millet) +Panicum maximum Jacq. (Guinea grass)

Summary Protoplasts from Pennisetum americanum resistant to S-2-amino-ethyl-l-cysteine (AEC) were fused with protoplasts of Panicum maximum utilizing polyethylene glycol-dimethylsulfoxide after inactivation of the Pennisetum protoplasts with 1 mM iodoacetic acid. The iodoacetate treatment prevented division of Pennisetum protoplasts; therefore, only Panicum protoplasts and heterokaryons potentially could give rise to colonies. A second level of selection was imposed by plating 3–4-week-old colonies on AEC medium. Putative somatic hybrid calli were analyzed for alcohol dehydrogenase, 6-phosphogluconate dehydrogenase, aminopeptidase, and shikimate dehydrogenase isozymes. Three somatic hybrid cell lines (lines 2, 3, and 67) were identified which showed two bands of alcohol dehydrogenase activity representing homodimers of P. maximum and P. americanum as well as a novel intermediate band of activity where Panicum-Pennisetum heterodimers would be expected. Aminopeptidase and shikimate dehydrogenase were useful for identifying presumptive hybrid calli but the isozyme patterns were additive-evidence which would not preclude the selection of chimeric callus. A more complex isozyme pattern which varied among the somatic hybrids was observed for 6-phosphogluconate dehydrogenase. In the hybrid calli, the presence of DNA sequences homologous to both P. maximum and P. americanum sequences was confirmed by hybridization of a maize ribosomal DNA probe to XbaI and EcoRI restriction fragments. Growth of hybrid lines on various concentrations of AEC was either similar to the AEC-resistant parent (hybrid line 2) or intermediate between the resistant and sensitive parents (hybrid lines 3, 67).     

Nuclear genomic composition of asymmetric fusion products between irradiated transgenic Solanum brevidens and S. tuberosum: limited elimination of donor chromosomes and polyploidization of the recipient genome

Summary The production of asymmetric somatic hybrid calli after fusion between gamma-irradiated protoplasts from transgenic Solanum brevidens and protoplasts from S. tuberosum are reported. Transgenic (kanamycin-resistant, GUS-positive) S. brevidens plants and hairy root clones were obtained after transformation with Agrobacterium tumefaciens LBA 1060 (pRi1855) (pBI121) and LBA 4404 (pRAL4404) (pBI121), and A. rhizogenes LBA 9402 (pRi1855) (pBI121), respectively. Leaf protoplasts isolated from the transgenic plants or root protoplasts from the hairy root clones were fused with S. tuberosum leaf protoplasts, and several calli were selected on kanamycin-containing medium. The relative nuclear DNA content of the hybrid calli was measured by flow cytometry (FCM), and the percentages of DNA of the S. brevidens and S. tuberosum genomes in the calli were determined by dot blot analysis using species-specific DNA probes. Chromosome-specific restriction fragment length polymorphism (RFLP) markers were used to investigate the elimination of specific S. brevidens chromosomes in the hybrids. The combined data on FCM, dot blot and RFLP analysis revealed that 18–62% of the S. brevidens DNA was eliminated in the hybrid calli and that the RFLP marker for chromosome 7 was absent in seven out of ten calli. The absence of RFLP markers for chromosomes 5 and 11 hardly ever occurred. In most of the hybrids the ploidy level of the S. tuberosum genome had increased considerably.     

Somatic hybridization of sexually incompatible petunias: Petunia parodii,Petunia parviflora

Summary Somatic hybrid plants were regenerated following the fusion of leaf mesophyll protoplasts of P. parodii with those isolated from a nuclear-albino mutant of P. parviflora. Attempts at sexual hybridization of these two species repeatedly failed thus confirming their previously established cross-incompatibility. Selection of somatic hybrid plants was possible since protoplasts of P. parodii would not develop beyond the cell colony stage, whilst those of the somatic hybrid and albino P. parviflora produced calluses. Green somatic hybrid calluses were visible against a background of albino cells/calluses, and upon transfer to regeneration media gave rise to shoots. Shoots and the resultant flowering plants were confirmed as somatic hybrids based on their growth habit, floral pigmentation and morphology, leaf hair structure, chromosome number and Fraction 1 protein profiles. The relevance of such hybrid material for the development of new, and extensively modified cultivars, is discussed.     

Molecular,cytological and morpho-agronomical characterization of hexaploid somatic hybrids in Medicago

Somatic hybrid plants produced by protoplast fusion between tetraploid Medicago sativa (2n= 4x=32) and the diploid species Medicago coerulea (2n= 2x=16) have been RFLP fingerprinted to establish their nuclear composition. Although all of the chromosomes were present, molecular analysis revealed an incomplete incorporation of the alleles of the diploid parent in the fusion products. In the polycross progeny the alleles of both parents segregated in a Mendelian mode. Cytological observations indicated that in the somatic hybrid population minor abnormalities are present; these are restricted mainly to the formation of univalents and lagging chromosomes. Meiosis appeared to be more stable than has been previously reported in the hexaploids of alfalfa. The somatic hybrids grown in the field had a rather vigorous aspect, particularly with respect to the vegetative organs. Forage yield was comparable to that of thmore productive parent. The results are discussed with a view to utilizing the somatic hybrids as starting material for breeding alfalfa at the hexaploid level.This paper was supported by the National Research Council of Italy, Special Project RAISA, Sub-project No.2 paper No. 1911     

Somatic embryogenesis and plant regeneration in Medicago arborea L.

Summary An efficient plant regeneration system employing cotyledons, hypocotyls, petioles and leaves as explants and characterized by continuous and prolific production of somatic embryos, has been developed with Medicago arborea ssp. arborea. The optimal somatic embryogenic response was obtained using a two-step protocol, where explants were incubated under a 16 h photoperiod for 2 mo. on Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D; 9 μM) and kinetin (9 μM), and followed by transfer to kinetin-free MS medium with 2,4-D (2.25 μM). Removal of the cytokinin and a reduction in the concentration of auxin (2.25 μM) in the second step of culture were critical for enhanced production of somatic embryos. The best explants proved to be cotyledons and petioles (i.e. a mean of 18.0±0.70 somatic embryos at 3 mo. for petiole culture). Somatic embryos were converted into normal plantlets (8.0±0.89%) when cultured on basal MS medium with 5 μM indolebutyric acid. No somatic embryos were obtained when thidiazuron was used in the culture media. Using petioles as explants and N⁶-benzyladenine (BA), embryogenesis was induced in the second step of culture when BA was removed from the medium and the concentration of 2,4-D was decreased to 2.25 μM.     

Somatic hybridization between Brassica juncea and Moricandia arvensis by protoplast fusion

Intergeneric somatic hybrids have been produced between Brassica juncea (2n=36, AABB) cv. RLM-198 and Moricandia arvensis (2n=28, MM) by protoplast fusion. Hypocotyl protoplasts of B. juncea were fused with mesophyll protoplasts of M. arvensis using polyethylene glycol. Fusion frequency, estimated on the basis of differential morphological characterstics of parental protoplasts was about 5%. Of the 156 calli obtained, four calli produced shoots intermediate in morphology between the parents. Hybrid nature of the plants was confirmed using wheat nuclear rDNA probe. Hybridization of total DNA with a mitochondrial DNA probe carrying 5s–18s rRNA genes of maize showed that the mitochondria of the somatic hybrids were derived from the wild species M. arvensis. Meiosis in the only hybrid that produced normal flowers revealed the occurrence of 64 chromosomes, the sum of chromosomes of parental species. Inspite of complete pollen sterility, siliquas were produced in this hybrid by back-crossing with B. juncea. These siliquas on in vitro culture produced 12 seeds.     

Asymmetric somatic hybridization between wheat (Triticum aestivum) and Avena sativa L.

Protoplasts from cell suspensions of young-embryo-derived calli, which were nonregenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% of the same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity of less than 10%) irradiated with UV at an intensity of 300 μW/cm² for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts of the recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants.     

Production of somatic hybrids between Daucus carota L. and Nicotiana tabacum

Protoplasts of a kanamycin-resistant (KR, nuclear genome), streptomycin-resistant (SR, chloroplast genome) and chlorophyll-deficient (A1, nuclear genome) Nicotiana tabacum (KR-SA) cell suspension cultures or X-ray-irradiated mesophyll protoplasts of kanamycin- and streptomycin-resistant green plants (KR-SR) were fused with protoplasts of a cytoplasmic male-sterile (CMS) Daucus carota L. cell suspension cultures by electrofusion. Somatic hybrid plants were selected for kanamycin resistance and the ability to produce chlorophyll. Most of the regenerated plants had a normal D. carota morphology. Callus induced from these plants possessed 23–32 chromosomes, a number lower than the combined chromosome number (66) of the parents, and were resistant to kanamycin, but they segregated for streptomycin resistance, which indicated that N. tabacum chloroplasts had been eliminated. Genomic DNA from several regenerated plants was analyzed by Southern hybridization for the presence of the neomycin phosphotransferase gene (NPTII); all of the plants analyzed were found to contain this gene. Mitochondrial (mt) DNA was analyzed by Southern hybridization of restriction endonuclease digests of mtDNA with two DNA probes, PKT5 and coxII. The results showed that the two plants analyzed possessed the mitochondria of D. carota. These results demonstrate that the regenerated plants are interfamilial somatic hybrids.     

Production of a triple mutant,chlorophyll-deficient,streptomycin-, and kanamycin-resistant Nicotiana tabacum,and its use in intergeneric somatic hybrid formation with Solanum melongena

Summary In order to produce a triple mutant, sexual crosses between a chlorophyll-deficient, streptomycin-resistant mutant of Nicotiana tabacum (SA) and a kanamycin-resistant transformant of N. tabacum (KR.) were carried out. From the offspring of this cross, a triple mutant (KR-SA) was selected. In N. tabacum KR-SA, chlorophyll deficiency is due to recessive mutation in the nuclear genome, streptomycin resistance is due to a dominant mutation in the chloroplast genome, and kanamycin resistance is shown to be a dominant nuclear marker. Cell suspension protoplasts of N. tabacum KRSA were fused with callus protoplasts of Solanum melongena by dextran treatment. Somatic hybrid plants were selected for streptomycin resistance and the ability to produce clorophyll in regenerated plants. By using this selection system, green plants were recovered from two colonies. When these green plants were then tested for kanamycin resistance, all analyzed plants carried this trait. In addition, the hybrid nature of these plants was confirmed by investigation of the peroxidase isozyme. The present results show that the use of N. tabacum KR-SA in studies of somatic hybridization makes it possible to select somatic hybrid plants easily and provides information of the N. tabacum genome.Chemical Regulation of Biomechanism, The Institute of Physical and Chemical Research, Wako 351-01, Japan     

Asymmetric somatic hybrid plants between Medicago sativa L. (alfalfa,lucerne) and Onobrychis viciifolia Scop. (sainfoin)

This paper reports on the production of intergeneric somatic hybrid plants between two sexually incompatible legume species. Medicago sativa (alfalfa, lucerne) leaf protoplasts were inactivated by lethal doses of iodoacetamide. Onobrychis viciifolia (sainfoin) suspension-cell protoplasts were gamma-irradiated at lethal doses. Following electrofusion under optimized conditions about 50,000 viable heterokaryons were produced in each test. The fusion products were cultured with the help of alfalfa nurse protoplasts. Functional complementation permitted only the heterokaryons to survive. A total of 706 putative heterokaryon-derived plantlets were regenerated and 570 survived transplantation to soil. Experimentation was aimed at the introduction of proanthocyanidins (condensed tannins) from sainfoin, a bloat-safe plant, to alfalfa, a bloat-causing forage crop; however, no tannin-positive regenerant plants were detected. Most regenerant plants have shown morphological differences from the fusion parents, although, as expected, all resembled the recipient parent, alfalfa. Southern analysis using an improved total-genomic probing technique has shown low levels of sainfoin-specific DNA in 43 out of 158 tested regenerants. Cytogenetic analysis of these asymmetric hybrids has confirmed the existence of euploid (2n=32; 17%) as well as aneuploid (2n=30, 33–78; 83%) plants. Pollen germination tests have indicated that the majority of the hybrids were fertile, while 35% had either reduced fertility or were completely sterile.     

Fertile somatic hybrids between Petunia hybrida and a wild species,Petunia variabilis

Somatic hybrid plants were regenerated following electrofusion between leaf mesophyll protoplasts of P. hybrida (2n = 14) and a wild sexually incompatible species, P. variabilis (2n = 18). The selection of hybrids was based on the hybrid vigour, expressed both in the growth of the callus and at the shoot formation stage, resulting from the combination of parental genomes. Calli exhibiting vigorous growth were selected, and upon transfer to regeneration medium gave rise to shoots. Four regenerated plants from three calli had morphological characteristics intermediate between those of the parents. The hybrid nature of these plants was confirmed by chromosome counts as well as isozyme and DNA analyses. They had amphidiploid chromosome numbers (2n = 32) and were fertile. Following self-pollination and backcrossing with P. variabilis, large numbers of F₂ and BC₁ seedlings were obtained.     

Interspecific hybridization between the cultivated potato Solanum tuberosum subspecies tuberosum L. and the wild species S. circaeifolium subsp. circaeifolium Bitter exhibiting resistance to Phytophthora infestans (Mont.) de Bary and Globodera pallida (Stone) Behrens

Summary Somatic fusions between the cultivated potato Solanum tuberosum and the wild species S. circaeifolium subsp. circaeifolium Bitter were produced in order to incorporate desirable traits into the potato gene pool. Selection of the putative hybrids was based on a difference in callus morphology between the hybrids and their parents, with the hybrids showing typical purple-colored cells in otherwise green calli. In all, 17 individual calli regenerated to plants. Of the nine plants that could be transferred to the greenhouse, eight showed a hybrid and one a parental morphology. Restriction fragment length polymorphism (RFLP) analysis confirmed the hybrid character in the former group. Chloroplast counts in stomatal guard cells and flow cytometric determination of nuclear DNA content showed that four hybrid plants were tetraploid (4x), one was mixoploid (5x–8x), and the others were polyploid (6x; 8x). Three out of four tetraploid hybrids were found to be fully resistant to Phytophthora infestans, and all four hybrids were resistant to Globodera pallida pathotypes Pa2 and Pa3. It was further observed that the type and amount of steroidal glycoalkaloids varied among the tubers of the parents and the hybrids. Using the hybrids as female parents in crosses with S. tuberosum, viable seeds could be obtained. This demonstrates the potential of these hybrids in practical plant breeding.