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.     

Interspecific somatic hybrids between wild potato Solanum acaule Bitt. and anther-derived dihaploid potato (Solanum tuberosum L.)

Solanum acaule Bitt. is a disomic tetraploid (4x) wild potato species which is resistant to several potato diseases. Introgression of disease resistance and abiotic stress tolerance to the tetrasomic tetraploid (4x) cultivated potato (S. tuberosum L.) gene pool via crossing has been limited due to the difference in the endosperm balance number. In the present study, protoplast fusion was applied to produce hexaploid (6x) somatic hybrids between the parental lines, tetraploid (4x) S. acaule and two anther-derived dihaploid (2x) lines of S. tuberosum cv. White Lady. One callus (0.4%) of a total of 229 calli obtained regenerated into shoots in the fusion combination S. acaule (+) White Lady 15.dh.8.2.2. All the regenerated shoots were confirmed to be interspecific somatic hybrids using species-specific RAPD markers. In another fusion combination, S. acaule (+) White Lady 7.dh.23.1.1, fifteen calli (5%) regenerated into a total of sixteen shoots from 289 calli. All the analysed somatic hybrids between S. acaule and S. tuberosum were hexaploid. The mean DNA content (2C value) of the combination S. acaule (+) White Lady 15.dh.8.2.2 somatic hybrids (4.55 pg), was approximately the sum (4.69 pg) of the DNA contents of the parental lines, S. acaule (2.95 pg) and S. tuberosum (1.74 pg). In the greenhouse, the two somatic hybrids analysed were normal in their morphological characteristics and more vigorous than their parental lines. Most of the morphological characteristics were closer to the tetraploid S. acaule than to the dihaploid S. tuberosum. The interspecific somatic hybrids are currently being tested for frost tolerance and glycoalkaloid composition. Received: 19 January 1998 / Revision received: 27 March 1998 / Accepted: 20 April 1998     

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.     

Fertile Solanum tuberosum+S. commersonii somatic hybrids as sources of resistance to bacterial wilt caused by Ralstonia solanacearum

 The wild potato relative Solanum commersonii is reported to carry resistance to bacterial wilt disease caused by Ralstonia solanacearum. To overcome sexual incompatibilites due to differences in ploidy and endosperm balance numbers, somatic hybrids were made that combine the S. tuberosum and S. commersonii genomes. The resulting somatic hybrid plants are vigorous, but their disease resistance level and their fertility was unknown. We therefore tested the S. commersonii and S. tuberosum source material cv Superior, potato cv Atlantic and six somatic hybrid lines for resistance to a virulent strain of R. solanacearum (race 3, biovar 2) at 28°C. As expected, S. commersonii was significantly more wilt-resistant than the cultivated potatoes. In five of the six somatic hybrid lines, disease resistance levels were similar to that of the resistant S. commersonii parent. The resistance level of the sixth somatic hybrid was intermediate, significantly different from both S. commersonii and S. tuberosum. In controlled crosses, the somatic hybrids in this study proved both to be male- and female-fertile and were self-compatible. More importantly, the somatic hybrids can be crossed with S. tuberosum to produce viable seeds. Received: 23 June 1998 / Accepted: 13 October 1998     

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.     

The nucleolar cycle in karyomeres ofTetranychus urticae Koch (Acari,Tetranichidae) in relation to replication of fragments of holokinetic chromosomes

Summary Chromosomes and nucleoli in the karyomeres of cleavage eggs of the arrhenotokous spidermiteTetranychus urticae Koch were treated with a silver-staining procedure for nucleolar material to establish the number of nucleoli and of the chromosomes that produce them. Each of the three holokinetic chromosomes of the haploid chromosome complement produces nucleolar material. This is confirmed by observations on the ultrastructure of karyomeres in serial sections of interphase cleavage divisions. These findings are thought to be in agreement with facts from previous radiation research and may help to explain the fate of induced fragments in holokinetic chromosomes.     

Production and identification of somatic hybrids between Solanum tuberosum and S. papita by using the rolC gene as a morphological selectable marker

A successful hybridization of a diploid clone of Solanum tuberosum with a rolC-transgenic, diploid S. papita clone is reported. By using leaf expiants of this S. papita clone, which after transformation expressed kanamycin resistance, intact protoplasts were obtained, but these protoplasts did not develop to microcalli or regenerate to mature plants. However, protoplasts of the S. tuberosum clone showed a high capacity to regenerate plants from isolated protoplasts. On a medium containing Kanamycin only calli regenerated to plants, which revealed a rolC phenotype (reduced apical dominance with a large number of adventitious shoots and a pale green color of leaves) and later on turned out to be true hybrids. Self fusions of S. papita never developed to microcalli and those of S. tuberosum ceased to develop on the kanamycin-containing medium. Identification of somatic hybrids was done by RFLP and RAPD analysis. In the greenhouse, out of four selected hybrids only FK3.1 was successfully crossed with two standard S. tuberosum varieties (Datura, Desirée). Out of all the seeds germinated, only rolC-negative F¹ seedlings were further characterized. Within the seedling population obvious differences were evident in respect of the S. papita and S. tuberosum characteristics.     

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.     

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.     

Interaction between cytoplasmic composition and yield parameters in somatic hybrids of S. tuberosum L.

The nuclear and cytoplasmic composition of five different fusion combinations, consisting of up to 50 hybrid regenerants each, was characterized by RFLP analysis. Simultaneously, the hybrid clones of four fusion combinations were evaluated in field experiments for yield and starch content.Predominantly complete chloroplast segregation was found with a 11 ratio, in all but one fusion combination. Mitochondria, in contrast revealed up to 75% recombination, as proven by the partial addition of parental banding patterns and the altered assignment of the same genotypes with different probes. Newly occuring DNA bands were also indicative of rearrangements in the mitochondrial genome. Correlations between RFLP data and field parameters were calculated. Deviating RFLP patterns of the nuclear genome did not influence yield parameters. Also the assignment of hybrids to different chloroplast genotypes did not affect yield or starch content. However, mitochondrial types could be distinguished with respect to starch content and tuber yield. The more thorough analysis of mitochondrial composition, with different probes homologous to coding regions, revealed a relationship between the homogeneity of the mt genome and the yield level.     

Analysis of the mitochondrial DNA of the somatic hybrids of Solanum brevidens and S. tuberosum using non-radioactive digoxigenin-labelled DNA probes

Summary Mitochondrial (mt) DNAs of somatic hybrids obtained by electrical and chemical fusion of mesophyll protoplasts of S. brevidens and a dihaploid line of S. tuberosum PDH 40 were analysed by Southern hybridization using the digoxigenin-labelled mtDNA sequences nad5 or orf25. In the Southern analysis of the hybrid mtDNA probed with nad5, most of the 19 hybrids analyzed had an RFLP pattern similar, but not identical, to one of the parents, S. tuberosum, PDH40. Nineteen percent of the hybrids had most of the S. brevidens fragments. Five of the hybrids had an identical RFLP pattern to either one of the parents while another two hybrids had novel RFLP patterns. Similar results were obtained by Southern analysis with orf25. These results clearly show that mtDNA rearrangements had occurred at a high frequency in the somatic hybrids. There were no differences in the frequencies of rearrangements observed between the hybrids regenerated from chemical and electrical fusions.     

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.     

Field assessment of dihaploid Solatium tuberosum and S. brevidens somatic hybrids

Summary Following both chemical and electrical fusion of protoplasts of a dihaploid line of potato (Solanum tuberosum), (PDH40), with those of the wild species, Solanum brevidens, 11 and 40 somatic hybrid plants, respectively were obtained. Fifteen of these somatic hybrid genotypes and the two parents were studied further in a small field trial to assess field performance and phenotypic variability. In the UK, somatic hybrid plants are classified as genetically engineered organisms, and the UK Advisory Committee on Genetic Manipulation have imposed various restrictions on field experiments. Examination of the somatic hybrids in the field showed extensive phenotypic variability, and no two genotypes were identical. Some of the variation reflected changes in chromosome numbers, but other factors were also involved. Half the somatic hybrid genotypes produced tubers in the field, although the tubers were smaller and differed morphologically from those of PDH40. The results of the study suggest that the extent of somaclonal variation manifested in somatic hybrids is greater than that found in protoplast-derived plants of potato. The implications of this observation and the current regulations concerning field experiments of somatic hybrid plants in the UK are discussed.     

RAPD analysis of the interspecific somatic hybrids: Solanum bulbocastanum (+) S. tuberosum

The diploid Mexican species S. bulbocastanum (blb) was used as a source of late blight resistance in somatic hybridization with the potato (S. tuberosum, tbr) dihaploid H-8105. The produced 2x blb (+) 2x tbr H-8105 somatic hybrids did not retain the blb parent's characteristic high resistance to P. infestans. The revealed aneuploidy of blb (+) tbr H-8105 hybrids indicated a possible loss of individual blb chromosome(s) carrying the resistance genes. Four hybrid clones differing in terms of their ploidy, morphology and growth potential were analysed for the presence of all twelve blb chromosomes (linkage groups). The RAPD markers assigned to particular chromosomes were selected on the basis of the linkage map of S. bulbocastanum constructed by Naess et al., Mol. Gen. Genom. 265 (2001) 694-704. Of the 86 markers analysed, twelve (14%) were common for blb and H-8105, while 34 (40%) and 40 (46%) markers were specific for the blb and H-8105 genome, respectively; this confirms the differences between the nuclear genomes of the two species. Seventeen markers (20%) present in one or the other parent were absent in the hybrids, and only one new marker was found in the hybrids. The poorly growing, aneuploid and chimeric hybrids had the same band profiles as the well growing, morphologically normal hybrids, except for two bands that were present only in normal hybrids. The presence of eleven blb linkage groups in the blb (+) tbr H-8105 hybrids was confirmed. The markers specific for the second linkage group (chromosome 2) of blb were not present in the RAPD patterns of the somatic hybrids analysed, suggesting a loss or rearrangement of this chromosome in the combined nuclear genome of the hybrids.     

Chloroplast and mitochondrial DNA composition of triploid and tetraploid somatic hybrids between Lycopersicon esculentum and Solanum tuberosum

The chloroplast (cp) DNA type and mitochondrial (mt) DNA composition of 17 somatic hybrids between a cytoplasmic albino tomato and monoploid potato (A7-hybrids) and 18 somatic hybrids between a nitrate reductase-deficient tomato and monoploid potato (C7-hybrids) were analyzed. Thirteen A7-hybrids and 9 C7-hybrids were triploids (with one potato genome); the other hybrids were tetraploid. As expected, all A7-hybrids contained potato cpDNA. Of the C7-hybrids 7 had tomato cpDNA, 10 had potato cpDNA and 1 hybrid contained both tomato and potato cpDNA. The mtDNA composition of the hybrids was analyzed by hybridization of Southern blots with four mtDNA-specific probes. The mtDNAs in the hybrids had segregated independently from the cpDNAs. Nuclear DNA composition (i.e. one or two potato genomes) did not influence the chloroplast type in the C7-hybrids, nor the mtDNA composition of A7- or C7-hybrids. From the cosegregation of specific mtDNA fragments we inferred that both tomato and potato mtDNAs probably have a coxII gene closely linked to 18S+5S rRNA genes. In tomato, atpA, and in potato, atp6 seems to be linked to these mtDNA genes.     

Effects of parental ploidy level and genetic divergence on chromosome elimination and chloroplast segregation in somatic hybrids within Brassicaceae

Summary Chromosome and organelle segregation after the somatic hybridization of related species with different degrees of genetic divergence were studied by comparing the interspecific somatic hybrids Brassica oleracea (CC) (+) B. campestris (AA), B. napus (AACC) (+) B. oleracea (CC) B. napus (AACC) (+) B. nigra (BB) and B. napus (AACC) (+) B. juncea (AABB) with the intergeneric somatic hybrids B. napus (AACC) (+) Raphanus sativus (RR) and B. napus (AACC) (+) Eruca sativa (EE). Within each combination, some hybrids were found whose DNA content was equal to the sum of parental chromosomes, others had a relatively higher DNA content and in most of the cases, some had a relatively lower content. However, the frequency distribution in these three classes differed significantly between the combinations. A positive correlation between the frequency of hybrids with eliminated chromosomes and the genetic distance between the species in each combination was found. Furthermore, by combining species with different ploidy levels we found a significantly higher degree of chromosome elimination compared to combinations of species with the same ploidy level. In the B. napus (+) B. Nigra, B. napus (+) R. sativus and B. napus (+) E. sativa combinations chromosomes from the B, R and E genomes appeared to be preferentially sorted out, as indicated by the fact that some of the nuclear markers from these genomes were missing in 7–46% of the plants, whereas no plants were lacking B. napus nuclear markers. Fertile hybrids were found in all but the B. napus (+) R. sativus fusion combination; the latter hybrids were male sterile, but female fertile. Hybrids between the A and C genomes were more fertile than hybrids obtained between the distantly related AC and B, R or E genomes, respectively. Analysis of the chloroplast RFLP pattern revealed that chloroplasts in the B. oleracea (+) B. campestris hybrids segregated randomly. A slightly biased segregation, favouring B. napus chloroplasts, was found in the B. napus (+) B. oleracea combination, whereas B. napus chloroplasts were strongly selected for in the B. napus (+) B. juncea, B. napus (+) B. nigra, B. napus (+) R. sativus and B. napus (+) E. sativa somatic hybrids.     

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.     

Selection of somatic hybrids between diploid clones of potato (Solanum tuberosum L.) transformed by direct gene transfer

Summary Five diploid potato clones have been transformed by electroporation of protoplasts with different selectable markers. The resulting diploid regenerated plants have been used in somatic hybridization. It has been shown that hybrid cell selection on the basis of antibiotic or herbicide resistances brought by the two parents of fusion is an efficient method for the recovery of tetraploid somatic hybrids.     

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