Nuclear and cytoplasmic genome composition of Solanum bulbocastanum (+) S. tuberosum somatic hybrids.

Somatic hybrids between the wild incongruent species Solanum bulbocastanum (2n = 2x = 24) and S. tuberosum haploids (2n = 2x = 24) have been characterized for their nuclear and cytoplasmic genome composition. Cytologic observations revealed the recovery of 8 (near-)tetraploid and 3 hexaploid somatic hybrids. Multicolor genomic in situ hybridization (GISH) analysis was carried out to study the genomic dosage of the parental species in 5 somatic hybrids with different ploidy. The GISH procedure used was effective in discriminating parental genomes in the hybrids; most chromosomes were unambiguously colored. Two (near-)tetraploid somatic hybrids showed the expected 2:2 cultivated-to-wild genomic dosage; 2 hexaploids revealed a 4:2 cultivated-to-wild genomic dosage, and 1 hexaploid had a 2:4 cultivated-to-wild genomic dosage. Characterization of hybrid cytoplasmic genomes was performed using gene-specific primers that detected polymorphisms between the fusion parents in the intergenic regions. The analysis showed that most of the somatic hybrids inherited the plastidial and mitochondrial DNA of the cultivated parent. A few hybrids, with a rearranged mitochondrial genome (showing fragments derived from both parents), were also identified. These results confirmed the potential of somatic hybridization in producing new variability for genetic studies and breeding.     

Interspecific somatic hybrids between Solanum bulbocastanum and S. tuberosum and their haploidization for potato breeding

Protoplast fusion between incongruent Solanum bulbocastanum and S. tuberosum haploids was accomplished to produce hybrids combining elite traits from both parents. We identified 11 somatic hybrids out of 42 regenerants analyzed through ISSR markers. Some hybrids had loss or gain of fragments compared to the parents, likely due to rearrangements and deletions of chromosome segments after fusion, and/or to somaclonal variation during hybrid regeneration. Increased heterotic vigor for some traits as well as high diversity was observed as the effect of both ploidy and fusion combination. Microsporogenesis analysis indicated the occurrence of multivalent configurations and several meiotic abnormalities, such as chromosomes bridges and various spindle orientations. Since all hybrids were sterile, in vitro anther culture was employed for haploidization as a possible strategy to overcome barriers to hybridizations. Haploids were obtained from all the tetraploid S. bulbocastanum (+) S. tuberosum somatic hybrids tested, although with differences in both the number of embryos per 100 anthers cultured and the number of differentiated green plantlets. This is the first report on the successful production of haploid plants from S. bulbocastanum (+) S. tuberosum hybrids.     

Use of RAPD markers to screen somatic hybrids between Solanum tuberosum and S. brevidens

Summary The identification of somatic hybrids between Solanum tuberosum and S. brevidens can be carried out using polymerase chain reaction (PCR) and arbitrary 10-mer primers to generate random amplified polymorphic DNA (RAPD) markers. Five commercial primers have been tested. Each primer directed the amplification of a genome-specific fingerprint for the fusion parents and S. brevidens. The size of the amplified DNA fragments ranged from 100 to 1800 base pairs. The somatic hybrids showed a combination of the parental banding profiles with four of the five primers surveyed, whereas regenerants from one of the parents had the same or a similar banding pattern to that of the parent. Thus RAPD markers provide a quick, simple and preliminary screening method for putative somatic hybrids.Abbreviations EDTA ethylenediaminetetraacetic acid, - PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphisms - TBE Tris-borate-EDTA buffer - Tris trizma base     

Successful first and second backcrosses of S. nigrum (+) S. tuberosum somatic hybrids with both Solanum parents

Somatic hybrids of Solanum nigrum (+) 2× potato were successfully crossed with S. nigrum and with potato. First and second backcross progeny with S. nigrum could easily be obtained. One of the BC1 genotypes was already self-fertile. Backcrosses with potato had a much lower success rate. Only pollinations with tetraploid potato resulted in seed-containing berries. Two BC1 genotypes were obtained after 4362 pollinations from which 505 ovules were cultured. The first BC1 genotype grew vigorously in vitro and in the greenhouse and flowered abundantly. The second BC1 showed many abnormalities and dropped its flowers before anthesis. The first BC1 was again crossed with tetraploid potato and in this generation also the success rate was low. Over 5000 pollinations resulted in 1750 berries from which over 3000 ovules were obtained. Twelve plants germinated from these ovules and they were not as vigorous in vitro and in vivo as the BC1 parent. Some of the BC2 genotypes were used for further backcrosses but no BC3 plants were obtained. BC1 and BC2 genotypes that resulted from the backcross program with potato were tested for resistance to Phytophthora infestans. The BC1 genotype was as resistant as the S. nigrum fusion parent, but among the eight BC2 genotypes scored six were resistant, whereas two genotypes showing lesions were susceptible. Received: 19 October 1998 / Revision accepted: 20 April 1999     

RFLP analysis of asymmetric somatic hybrids between Solanum tuberosum and irradiated S. brevidens

The nuclear genome composition of five asymmetric somatic hybrids, obtained by fusion of leaf protoplasts from Solanum tuberosum and gamma-irradiated leaf protoplasts from S. brevidens, have been analyzed at the molecular level. An analysis of 21 loci using linkage group-specific restriction fragment length polymorphism (RFLP) was included in the study. All five hybrids contained a complete set of the loci studied from S. tuberosum. The degree of elimination of alleles from the irradiated S. brevidens donor genome ranged from 10–65% in the five asymmetric hybrids analyzed. The detection of incomplete chromosomes, as well as non-parental bands in Southern hybridizations with RFLP markers, revealed extensive chromosome rearrangements in the asymmetric hybrids.     

Genomic instability in Solanum tuberosum x Solanum kurtzianum interspecific hybrids.

The use of interspecific crosses in breeding is an important strategy in improving the genetic base of the modern cultivated potato, Solanum tuberosum L. Until now, it has normally been interspecific Solanum hybrids that have been morphologically and cytologically characterized. However, little is known about the genomic changes that may occur in the hybrid nucleus owing to the combination of genomes of different origin. We have observed novel AFLP bands in Solanum tuberosum x Solanum kurtzianum diploid hybrids; 40 novel fragments were detected out of 138 AFLP fragments analyzed. No cytological abnormalities were observed in the hybrids; however, we found DNA methylation changes that could be the cause of the observed genomic instabilities. Of 277 MSAP fragments analyzed, 14% showed methylation patterns that differed between the parental species and the hybrids. We also observed frequent methylation changes in the BC1 progeny. Variation patterns among F1 and BC1 plants suggest that some methylation changes occurred at random. The changes observed may have implications for potato breeding as an additional source of variability.     

Elimination of Solanum phureja nucleolar chromosomes in S. tuberosum + S. phureja somatic hybrids

Summary The karyotype of the dihaploid SVP1 line of S. tuberosum (2n=2x=24) showed two nucleolar chromosomes with differently sized satellites. The diploid SVP5 line (2n=2x=24) and tetraploid regenerants of S. phureja had larger but similar satellites. Somatic hybrids between the diploid lines of these potato species with genome combinations 4 tub + 2 ph (plants 1–3), 2 tub + 4 ph (plants 4–7) and 4 tub + 4 ph (plant 8) had lost 2 phureja nucleolar chromosomes if 4 phureja genomes were present. One phureja nucleolar chromosome of plants 1–3 and both of plants 5 and 7 had rearranged satellites. Elimination of the two nucleolar chromosomes occurred preferentially, was under genetic control, and probably took place during early callus development. NOR activity resulting in rear-rangements between NORs may have caused the elimination.     

Electrofusion of protoplasts from Solanum tuberosum, S. nigrum and S. bulbocastanum

Leaf protoplasts of two wild species, Solanum nigrum var. gigantea (S. ngr gig) and S. bulbocastanum Dun. (S. blb), were electrofused with leaf protoplasts of two diploid potato clones, H-8105 and ZEL-1136, respectively, in order to confer the late blight-resistance from the wild species to the cultivated potato. The S. ngr gig mesophyll (+) H-8105 mesophyll combination resulted in regenerants of mostly normal ngr phenotype. Two regenerants from this combination were proved to be true hybrids by RAPD analysis but they rooted poorely in vitro and did not survive the transfer to soil. The S. ngr gig (+) H-8105 fusion combination was also performed with H-8105 cell suspension derived protoplasts enabling an easy identification of interspecific fusants on basis of their intermediate morphology. From the S. ngr gig mesophyll (+) H-8105 cultured cell combination, many abnormal shoots were regenerated. The two lines which survived had normal ngr phenotype but the presence of tuberosum (tbr) genome in those regenerants was not confirmed by RAPD analysis. No plants with tbr phenotype were obtained from both of S. ngr gig (+) H-8105 combinations. On the contrary, when S. blb mesophyll protoplasts were electrofused with ZEL-1136 mesophyll protoplasts, all regenerated plants had tbr phenotype, indicating much lower morphogenetic potential of S. bulbocastanum in comparison with that of S. nigrum var. gigantea. However, the hybridity of those regenerants has not been confirmed by RAPD analysis with two different primers. The efficiency of the applied fusion procedure and analysis of the regenerants is discussed.     

Fertility of somatic hybrids from protoplast fusions of Solanum brevidens and S. tuberosum

Summary Two sets of somatic hybrids between Solanum brevidens (2x) and S. tuberosum (2x and 4x) were evaluated for male fertility, meiotic regularity and female fertility. The somatic hybrids were tetraploids from 2x + 2x fusions and hexaploids from 2x + 4x fusions. Pollen stainability ranged from 0 to 83% in tetraploids and from 0 to 23% in hexaploids. The tetraploids had more regular meiosis, lower levels of micropollen and fewer unassociated chromosomes than hexaploids. However, except for a low level of selfing, the pollen of both sets of hybrids was ineffective in pollinations. The tetraploids, as females, crossed poorly with 2x and 4x tester species and selfed only at low levels. The hexaploid fusion hybrids also crossed poorly with the 2x tester species and selfed only to a limited degree; however, they crossed well with 4x testers. Seed set in crosses with S. tuberosum Group Andigena, and S. tuberosum Group Tuberosum cultivars Kathadin and Norland averaged 16.7, 15.6 and 28.6 seeds per fruit, respectively. Progeny from these crosses had 5x or nearly 5x ploidy levels. The results indicate that reasonable levels of female fertility can be obtained in somatic fusion hybrids of S. brevidens and S. tuberosum.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

Early tapetal degeneration and meiotic defects are involved in the male sterility of Solanum commersonii (+) S. tuberosum somatic hybrids

 Somatic hybridization between Solanum commersonii and S. tuberosum resulted in the production of male-sterile hybrid plants, except for one fully male-fertile hybrid. The male-sterile hybrids exhibited a“pollen-less” phenotype, with rare pollen grains which were abnormal in shape and exine sculpture. Microsporogenesis and tapetal development were investigated both in male-sterile and male-fertile somatic hybrids to assess the cytological events that were involved in male sterility. The pattern of male sterility was complex, arising through mechanisms expressed at both sporophytic and gametophytic levels. Various abnormalities occurred first in the tapetum, and later during meiosis-II and cytokinesis. These caused the degeneration of the sporads and of the microspores when they were released. In the male-fertile hybrid, normal development of the tapetum and pollen mother cells was restored. The hypothesis that tapetal breakdown, meiosis-II and cytokinesis defects are related to each other, and depend on nuclear-mitochondrial interactions, is discussed. Because of the formation of multivalent chromosome configurations, it is likely that gene exchange between S. commersonii and S. tuberosum can occur in somatic hybrids, offering potential perspectives for the introgression of useful traits from S. commersonii into S. tuberosum. Received: 10 December 1996/Accepted: 21 March 1997     

Plant regeneration from the protoplasts of Solanum tuberosum, S. nigrum and S. bulbocastanum

The mesophyll protoplasts were isolated from the Solanum tuberosum (S. tbr) clones of different ploidy level (4x Bzura cv., 2x H-8105, and 2x ZEL-1136) as well as from the wild species: S. bulbocastanum (S. blb, 2x) and two accessions of S. nigrum (S. ngr, 6x). Additionally, the protoplasts were isolated from the cell suspensions of Bzura cv. and H-8105 clone. The conditions of protoplast isolation as well as the media for their culturing and regeneration, were selected and optimized for the studied genotypes. For mesophyll protoplasts, the shooting calli were produced by all the cultured protoclones except that of S. bulbocastanum. The shoots excised from the protoplast-derived calli developed into whole plants in all the studied potato clones but only in one accession of S. nigrum, i.e. S. ngr var. gigantea. As for suspension-cell-derived protoplasts, only H-8105 clone produced the regenerative type of calli, though normal shoots could not be obtained. The regenerative capacity of the protoplasts isolated from leaves and cell suspensions is compared and discussed. We regret to report the death of M. Sc. Maria Borkowska after the completion of this work.     

Production of somatic hybrids between frost-tolerant Solanum commersonii and S. tuberosum: characterization of hybrid plants

Somatic fusion of mesophyll protoplasts was used to produce hybrids between the frost-tolerant species Solanum commersonii (2n=2x=24) and dihaploid S. tuberosum (2n=2x=24). This is a sexually incompatible combination due to the difference in EBN (Endosperm Balance Number, Johnston et al. 1980). Species with different EBNs as a rule are sexually incompatible. Fifty-seven hybrids were analysed for variation in chromosome number, morphological traits, fertility and frost tolerance. About 70% of the hybrids were tetraploid, and 30% hexaploid. Chloroplast counts in stomatal guard cells revealed a low frequency of cytochimeras. The frequency of aneuploids was relatively higher at the hexaploid level (hypohexaploids) than at the tetraploid level (hypotetraploids). The somatic hybrids were much more vigorous than the parents, and showed an intermediate phenotype for several morphological traits and moderate to profuse flowering. Hexaploid hybrid clones were less vigorous and had a lower degree of flowering than the tetraploid hybrid clones. All of the hybrids were female fertile but male sterile except for one, which was fully fertile and self-compatible. Many seeds were produced on the latter clone by selfing and on the male-sterile clones by crossing. The somatic hybrid plants showed an introgression of genes for frost tolerance and an adaptability to cold from S. commersonii. Therefore, the use of these somatic hybrids in breeding for and in genetic esearch on frost tolerance and cold-hardening is suggested.     

Mitochondrial DNA rearrangements in somatic hybrids of Solanum tuberosum and Solanum brevidens

Summary Thirty somatic hybrids between Solanum tuberosum and Solanum brevidens were analysed for mitochondrial and chloroplast genome rearrangements. In all cases, the chloroplast genomes were inherited from one of the parental protoplast populations. No chloroplast DNA alterations were evident but a range of mitochondrial DNA alterations, from zero to extensive intra- and inter-molecular recombinations, were found. Such recombinations involved specific recombination hot spots in the mitochondrial genome. Not all hybrids regenerated from a common callus possessed identical mitochondrial genomes, suggesting that sorting out of mitochondrial populations in the callus may have been incomplete at the plant regeneration stage. Sorting out of organelles in planta was not observed.     

Interspecific somatic hybrids between dihaploid Solanum tuberosum L. and the wild species, S. pinnatisectum Dun

Leaf-derived protoplasts of S. tuberosum and S. pinnatisectumwere electrofused. A culture medium-based procedure was usedto select heterok-aryon-derived tissues from which somatic hybridplants were regenerated. Somatic hybridity was confirmed bymorphological, isozyme and restriction fragment length polymorphism(RFLP) analyses. After 22 weeks, four somatic hybrid plantswere produced. These plants had phenotypically abnormal stolon-derivedshoots in which flow cytometry revealed a large degree of mixoploidy.Haploid nuclei were also present in these shoots. One shootexhibited a noticeably higher DNA content in comparison withother adventitious shoots. Key words: Potato, Solatium tuberosum, S. pinnatisectum, somatic hybridization, flow cytometry     

RAPD markers for confirmation of somatic hybrids in the dihaploid breeding of potato (Solanum tuberosum L.)

Summary The applicability and reliability of RAPD markers were evaluated for an examination of the possible use of RAPD markers to confirm hybridity of somatic hybrids between dihaploids of potato (Solanum tuberosum L.). Most of the primers examined detected polymorphism among either tetraploids or dihaploids, and polymorphism was easily detected even among closely related clones. Most of the examples of polymorphism were confirmed as being the result of amplification from the nuclear genome by a comparison of patterns generated by PCR of clones that carried the same cytoplasm. All the bands of dihaploids were transmitted stably to the respective hybrids. In the absence of primers that generated complementary polymorphic bands for both parents, a mixture of two appropriate primers, each of which generated a band specific to one parent, permitted the simple confirmation of hybridity. Hybridity of all the fusion-derived regenerants of 32 fusion combinations was unequivocally confirmed, a result that suggests that RAPD analysis could be universally applicable to the confirmation of hybridity in the dihaploid breeding of potato.     

Characterization of chromosome instability in interspecific somatic hybrids obtained by X-ray fusion between potato (Solanum tuberosum L.) and S. brevidens Phil

Summary Asymmetric somatic hybrids between Solanum tuberosum L. and S. brevidens Phil. have been obtained via the fusion of protoplasts from potato leaves and from cell suspension culture of S. brevidens. The wild Solanum species served as donor after irradiation of its protoplasts with a lethal X-ray dose (200 Gy). Selection of the putative hybrids was based on the kanamycin-resistance marker gene previously introduced into the genome of Solanum brevidens by Agrobacterium-mediated gene transfer. Thirteen out of the 45 selected clones exhibited reduced morphogenic potential. The morphological abnormalities of the regenerated plantlets were gradually eliminated during the extended in vitro culture period. Cytological investigations revealed that the number of chromosomes in the cultured S. brevidens cells used as protoplast source ranged between 28–40 instead of the basic 2n=24 value. There was a high degree of aneuploidy in all of the investigated hybrid clones, and at least 12 extra chromosomes were observed in addition to the potato chromosomes (2n=48). Interand intraclonal variation and segregation during vegetative propagation indicated the genetic instability of the hybrids, which can be ascribed to the pre-existing and X-ray irradiation-induced chromosomal abnormalities in the donor S. brevidens cells. The detection of centromeric chromosome fragments and long, poly-constrictional chromosomes in cytological preparations as well as non-parental bands in Southern hybridizations with restriction fragment length polymorphism (RFLP) markers revealed extensive chromosome rearrangements in most of the regenerated clones. On the basis of the limited number of RFLP probes used, preferential loss of S. brevidens specific markers with a non-random elimination pattern could be detected in hybrid regenerants.     

Characterisation of somatic hybrids between Solanum tuberosum and its frost-tolerant relative Solanum commersonil

 Somatic hybrids between three dihaploid Solanum tuberosum (2n=2x=24) genotypes and the frost-tolerant, diploid, relative Solanum commersonii (2n=2x=24) were analysed for variation in morphological traits, fertility and frost tolerance. The somatic hybrids were more vigorous than their parents and in many ways resembled a tetraploid S. tuberosum. All of the hybrids flowered profusely, although the male fertility was largely dependent on the S. tuberosum genotype used. In one hybrid combination all plants were both male- and female-fertile, while in the other two combinations the majority of plants were male-sterile but female-fertile. The somatic hybrids showed an increase in direct frost tolerance when compared with the dihaploid S. tuberosum parents, and to a varying extent had gained the capacity to cold acclimate. These somatic hybrids will be used in breeding programmes involving repeated cycles of anther culture and somatic hybridisation. Received: 20 May 1997 / Accepted: 12 June 1997     

Chromosome pairing and potential for intergeneric recombination in some hypotetraploid somatic hybrids of Lycopersicon esculentum (+) Solanum tuberosum.

Three somatic hybrids resulting from protoplast fusions of a diploid kanamycin-resistant line of tomato (Lycopersicon esculentum) and a dihaploid hygromycin-resistant transformant of a monohaploid potato (Solanum tuberosum) line were used for a cytogenetic study on chromosome pairing and meiotic recombination. Chromosome counts in root-tip meristem cells revealed two hypotetraploids with chromosome complements of 2n = 46 and one with 2n = 47. Electron microscope analyses of synaptonemal complex spreads of hypotonically burst protoplasts at mid prophase I showed abundant exchanges of pairing partners in multivalents involving as many as eight chromosomes. In the cells at late pachytene recombination nodules were found in multivalents on both sides of pairing partner exchanges, indicating recombination at both homologous and homoeologous sites. Light microscope observations of pollen mother cells at late diakinesis and metaphase I also revealed multivalents, though their occurrence in low frequencies betrays the reduction of multivalent number and complexity. Precocious separation of half bivalents at metaphase I and lagging of univalents at anaphase I were observed frequently. Bridges, which may result from an apparent inversion loop found in the synaptonemal complexes of a mid prophase I nucleus, were also quite common at anaphase I, though the expected accompanying fragments could be detected in only a few cells. Most striking were the high frequencies of first division restitution in preparations at metaphase II/anaphase II, giving rise to unreduced gametes. In spite of the expected high numbers of balanced haploid and diploid gametes, male fertility, as revealed by pollen staining, was found to be negligible.     

Resistance to potato virus Y in somatic hybrids between Solanum etuberosum and S. tuberosum x S. berthaultii hybrid

Somatic hybrids between a potato virus Y (PVY) resistant Solanum etuberosum clone and a susceptible diploid potato clone derived from a cross between S. tuberosum Gp. Tuberosum haploid US-W 730 and S. berthaultii were evaluated for resistance to PVY. All but one of the tested somatic hybrids were significantly more resistant than cultivars Atlantic and Katahdin. However, none was as resistant as the S. etuberosum parent. One hexaploid somatic hybrid, possibly the product of a triple-cell fusion involving one S. etuberosum protoplast and two haploid x S. berthaultii protoplasts, was as susceptible to PVY infection as the cultivars. Tetraploid progeny of the somatic hybrids, obtained from crosses with Gp. Tuberosum cultivars, were neither as resistant as the maternal somatic hybrid parent, nor as susceptible as the paternal cultivar parent. It appears that the introgression of PVY resistance from (1EBN) S. etuberosum into (4EBN) S. tuberosum (EBN-endosperm balance number) will be successful through the use of somatic hybridization and subsequent crosses of the somatic hybrids back to S. tuberosum.     

Molecular,cytogenetic and morphological characterization of somatic hybrids of dihaploid Solanum tuberosum and diploid S. brevidens

Summary Fifty-eight somatic hybrid plants, produced both by chemical (11) and electrical fusion (47) of protoplasts of dihaploid Solanum tuberosum and S. brevidens, have been analysed by molecular, cytological and morphological methods. The potentially useful euploid plants constituted 34% of the total, of which 20% were tetraploid and 14% hexaploid; the remainder were aneuploid at the tetraploid, hexaploid and octoploid levels. Analysis of chloroplast DNA showed that 55% of hybrids contained chloroplasts from S. brevidens and 45% from S. tuberosum. Hexaploids, the products of three protoplasts fusing together, were analyzed with specific DNA probes, and this revealed that nuclear genome dosages could be either 21 S. tuberosumS. brevidens, or vice-versa. Chloroplast types of hexaploids were not influenced by nuclear genome dosage, and all six possible combinations of genome dosage and chloroplast types were found amongst tetraploids and hexaploids. To examine the morphology of the hybrid population and its possible relation to the chromosome number and chloroplast DNA type, 18 morphological characteristics were measured on greenhouse-grown plants and analyzed by principal component and canonical variate analyses. Both analyses showed that nuclear ploidy has the most prominent influence on the overall morphology of the hybrids. Differential parental genome expression in the morphology of the hybrids is discussed. These results provide useful data on the range of genetic combinations that can be expected to occur amongst somatic hybrid plants.