Analysis of ribosomal RNA genes in somatic hybrids between wild and cultivated Solanum species

Ribosomal RNA genes were exploited as markers to identify somatic hybrids between Solanum tuberosum cv. Brodick and wild diploid Solanum species, S. megistacrolobum, S. sanctae-rosae and S. sparsipilum and DNA methylation as a possible regulatory factor in gene expression was investigated. Specific restriction enzyme/probe combinations revealed useful polymorphisms in the conserved coding and variable intergenic spacer regions of the ribosomal RNA genes. Some intermediate ribosomal RNA gene profiles indicate hybridity whereas others were characteristic of S. tuberosum cv. Brodick. This evidence is suggestive of somatic exchange/re-arrangement between the NOR region of S. sanctae-rosae and S. tuberosum cv. Brodick. Ribosomal RNA gene copy number analysis of the somatic hybrids did not reveal hexaploid values suggesting that these products are not symmetric hybrids derived from the parental diploid and tetraploid plants. The results indicate site-specific methylation of ribosomal RNA gene sequences for the parental plants; while some somatic hybrids display a reduction, others show an increase. The significance of the findings for somatic cell genetics and plant breeding studies is discussed.     

5′-Anchored simple-sequence repeat primers are useful for analysing potato somatic hybrids

Somatic hybrids between Solanum tuberosum cv 'Brodick' and S. sanctae-rosae were selected for having elevated chromosome numbers, intermediate morphology and potato cyst nematode resistance (derived from S. sanctae-rosae). DNA was extracted from this material and subjected to inter-SSR PCR directed by oligonucleotide primers comprising 5′-anchored di- and tri-nucleotide repeat motifs. PCR products were separated by polyacrylamide gel electrophoresis and visualised by silver staining. An average of 30.31 bands per primer were generated in each parent and, on average, 8.19 of these bands were polymorphic. S. sanctae-rosae-specific bands were observed in the hybrid material as was evidence of deletions, substitutions and rearrangements. The relative merits of this technique for the analysis of somatic hybrid material are discussed. Received: 23 July 1996 / Revision received: 22 October 1996 / Accepted: 22 March 1999     

Somatic hybrids between the cultivated potato Solanum tuberosum L. and the 1EBN wild species Solanum pinnatisectum Dun.: morphological and molecular characterization

Interspecific somatic hybrids between the 1EBN-wild species Solanum pinnatisectum (S. pnt) and four different diploid breeding lines of Solanum tuberosum (S. tbr) were produced by electrofusion. S. pnt exhibits resistance to Phytophthora infestans and Erwinia blackleg. Somatic hybrids were identified by RFLP analysis using the oligonucleotide (GATA)₄ as a probe. In three of four combinations all regenerates obtained were somatic hybrids. All 86 somatic hybrids between the breeding line H256/1 and S. pnt were analyzed in detail with respect to morphological and molecular characters; 50% of the somatic hybrids showed normal intermediate leaf morphology. Tubers of somatic hybrid plants grown in the greenhouse as well as in the field were evenly shaped and remarkably similar to those of the S. tbr breeding line. Analysis of relative DNA content by flow cytometry revealed that 75% of the somatic hybrids were tetraploid, some were hypotetraploid and others polyploid or mixoploid. Slotblot and RFLP analyses were carried out using repetitive and some single-copy DNA probes. The genome portion of the S. tbr breeding line was determined by slot-blot analysis using the species-specific repetitive probe pSA287. Obviously, most somatic hybrids contain the complete genomes of both fusion partners. In some of the somatic hybrids, a significantly lower intensity of the S. pnt-specific hybridization signal indicated a certain degree of asymmetry.Dedicated to Prof. Melchers on the occasion of his 90th birthday     

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.     

Novel somatic hybrids (Solanum tuberosum L. + Solanum tarnii) and their fertile BC1 progenies express extreme resistance to potato virus Y and late blight

Solanum tarnii, a wild diploid, tuber-bearing Mexican species belonging to the series Pinnatisecta is highly resistant to Potato virus Y (PVY) and Colorado potato beetle and shows a strong hypersensitive reaction to Phytophthora infestans. Therefore, it could be a potential source of resistance to pathogens for potato breeders. S. tarnii (2n = 2x = 24) is reproductively isolated from tetraploid Solanum tuberosum and hence difficult to include in potato breeding programmes. In this study, interspecific somatic hybrids were produced for the first time by protoplast electrofusion of the cells of potato cv. Delikat (Solanum tuberosum L.) and Solanum tarnii. The hybrid nature of the regenerants was confirmed by simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers and by morphological analysis and flow cytometry. Selected somatic hybrids were successfully backcrossed with cv. Delikat. Parental lines, primary somatic hybrids and BC₁ progeny were assessed for resistance to PVY by mechanical inoculation, grafting and exposure to viruliferous aphid vectors in the field, and resistance to late blight (P. infestans) by detached leaflet and whole tuber tests. The somatic hybrids showed no symptoms of viral infection and most of them displayed high levels of resistance to foliage blight. The BC₁ progenies were highly resistant to PVY and a few were resistant to foliage blight. Selected hybrids and BC₁ clones were evaluated in the field for tuber quality and tuber yield. Some BC₁ clones produced yields of good quality tubers. The results confirm that both the resistance to PVY and to late blight of S. tarnii is expressed in somatic hybrids, and PVY resistance is transferred to BC₁ progeny, whereas blight resistance is harder to transfer. Somatic hybridization again proved to be a valuable tool for producing pre-breeding material with increased genetic diversity.     

Characterization of the multiple resistance traits of somatic hybrids between Solanum cardiophyllum Lindl. and two commercial potato cultivars

Interspecific somatic hybrids between commercial cultivars of potato Solanum tuberosum L. Agave and Delikat and the wild diploid species Solanum cardiophyllum Lindl. (cph) were produced by protoplast electrofusion. The hybrid nature of the regenerated plants was confirmed by flow cytometry, simple sequence repeat (SSR), amplified fragment length polymorphism (AFLP), microsatellite-anchored fragment length polymorphism (MFLP) markers and morphological analysis. Somatic hybrids were assessed for their resistance to Colorado potato beetle (CPB) using a laboratory bioassay, to Potato virus Y (PVY) by mechanical inoculation and field trials, and foliage blight in a greenhouse and by field trials. Twenty-four and 26 somatic hybrids of cph + cv. Agave or cph + cv. Delikat, respectively, showed no symptoms of infection with PVY, of which 3 and 12, respectively, were also resistant to foliage blight. One hybrid of cph + Agave performed best in CPB and PVY resistance tests. Of the somatic hybrids that were evaluated for their morphology and tuber yield in the field for 3 years, four did not differ significantly in tuber yield from the parental and standard cultivars. Progeny of hybrids was obtained by pollinating them with pollen from a cultivar, selfing or cross-pollination. The results confirm that protoplast electrofusion can be used to transfer the CPB, PVY and late blight resistance of cph into somatic hybrids. These resistant somatic hybrids can be used in pre-breeding studies, molecular characterization and for increasing the genetic diversity available for potato breeding by marker-assisted combinatorial introgression into the potato gene pool.     

Organelle DNA and male fertility variation in Solanum spp. and interspecific somatic hybrids

Novel and potentially useful genetic variation in cytoplasmic genomes can be induced by interspecific somatic hybridization in plants. To evaluate such variability and correlate it with nuclear-cytoplasmic interactions leading to male sterility in Solanum spp., we examined progeny of male-sterile and male-fertile somatic hybrids between Solanum tuberosum (tbr), the common potato, and S. commersonii (cmm), a wild species showing sexual incongruity with tbr, for fertility and organelle DNA composition. Uniform male-fertile and male-sterile progenies were obtained by selfing the male-fertile hybrid and crossing the male-sterile ones, indicating maternal inheritance of the fertility phenotype. The two fusion partners were only slightly differentiated in the plastidial genome. MtDNA polymorphism between the species was greater, although its extent varied with the genomic region investigated. All somatic hybrids had non-parental organelle genomes, with reassorted organelles and/or rearranged mitochondria (i.e., cmm-specific bands for some regions and tbr-specific bands for others). Mitochondria reassorted independently from chloroplasts. Most hybrids showed the cmm cpDNA hybridization pattern, indicating non-random transmission of chloroplasts. Most male-sterile hybrids showed preferential inheritance of tbr mtDNA fragments. The male-fertile somatic hybrid clone had predominantly cmm mtDNA fragments. This result suggests that a tbr-derived region involved in nuclear-cytoplasmic incompatibility and male sterility has been lost by rearrangement; however, no clear correlation between a specific mitochondrial region and male sterility has been found so far. Received: 3 January 1999 / Accepted: 20 February 1999     

Interspecific somatic hybrid plants between eggplant (Solanum melongena) and Solanum torvum

Summary Mesophyll protoplasts of eggplant (cv Black Beauty) and of Solanum torvum (both 2n=2x=24) were fused using a modification of the Menczel and Wolfe PEG/DMSO procedure. Protoplasts post-fusion were plated at 1 × 10⁵/ml in modified KM medium, which inhibited division of S. torvum protoplasts. One week prior to shoot regeneration, ten individual calluses had a unique light-green background and were verified as cell hybrids by the presence of the dimer isozyme patterns for phosphoglucoisomerase (PGI) and glutamate oxaloacetate transaminase (GOT). Hybridity was also confirmed at the plant stage by DNA-DNA hybridization to a pea 45S ribosomal RNA gene probe. The ten somatic hybrid plants were established in the greenhouse and exhibited intermediate morphological characteristics such as leaf size and shape, flower size, shape, color and plant stature. Their chromosome number ranged from 46–48 (expected 2n=4x=48) and pollen viability was 5%–70%. In vitro shoots taken from the ten hybrid plants exhibited resistance to a verticillium wilt extract. Total DNA from the ten hybrids was restricted and hybridized with a 5.9 kb Oenothera chloroplast cytochrome f gene probe, a 2.4 kb EcoRI clone encoding mitochondrial cytochrome oxidase subunit II from maize and a 22.1 kb Sal I mitochondrial clone from Nicotiana sylvestris. Southern blot hybridization patterns showed that eight of ten somatic hybrids contained the eggplant cpDNA, while two plants contained the cpDNA hybridization patterns of both parents. The mtDNA analysis revealed the presence of novel bands, loss of some specific parental bands and mixture of specific bands from both parents in the restriction hybridization profiles of the hybrids.Michigan Agricultural Experiment Station Journal Article No. 12545     

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     

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     

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.     

Current status in production and utilization of dihaploids from somatic hybrids between eggplant (Solanum melongena L.) and its wild relatives

The major constrains for practical exploitation of the somatic hybrids between eggplant and its wild relatives have been their sterility and tetraploidy which prevented their incorporation into breeding programs. Here we demonstrate that anther culture was successfully utilized to bring back the ploidy level to the diploid status in tetraploid interspecific hybrids between eggplant and the allied species S. integrifolium and S. aethiopicum gr. gilo. Both the relative species are resistant to Fusarium oxysporum f. sp. melongenae and to some strains of bacterial wilt (Ralstonia solanacearum) which are very destructive diseases of eggplant. Dihaploid androgenetic plants were obtained from the somatic hybrids, from the “double somatic hybrid” obtained by sexual cross of the two somatic hybrids [(eggplant + S. aethiopicum) × (eggplant + S. integrifolium)], and from tetraploid backcrossed plants between the somatic hybrid with S. aethiopicum and eggplant. Phenotypical, molecular, biological and biochemical characterization, and also artificial inoculation with Fusarium oxysporum are consistent with a recombination between the genomes of the species involved in the hybridizations. Dihaploids resistant to Fusarium were successfully backcrossed with eggplant. Besides their utility as potential valuable breeding materials, the introgressed lines obtained may be utilized in genetic and molecular studies about the resistance to Fusarium from S. integrifolium and S. aethiopicum gr. gilo.     

Electrofusion of protoplasts from <Emphasis Type="Italic">Solanum tuberosum</Emphasis>, <Emphasis Type="Italic">S. bulbocastanum</Emphasis> and <Emphasis Type="Italic">S. pinnatisectum</Emphasis>

This paper discusses a number of experiments performed, involving the fusion by an electric field of mesophyll protoplasts from Solanum tuberosum cv. Bintje, S. tuberosum dihaploid clones 243, 299 and the wild tuberous disease-resistant species S. bulbocastanum and S. pinnatisectum. Three fusion experiments (S. bulbocastanum + S. tuberosum dihaploid 243, S. pinnatisectum + S. tuberosum cv. Bintje and S. pinnatisectum + S. tuberosum dihaploid 299) yielded 542 calli, the 52 ones of which produced shoots. Obtained regenerants were estimated by the flow-cytometry (FC) and RAPD analysis to determine hybrid plants.The utilisation of the FC as a useful method for detecting somatic hybrids is also discussed in this paper. The combination S. bulbocastanum + S. tuberosum dihaploid 243 led to the creation of eight somatic hybrids, the combination S. pinnatisectum + S. tuberosum cv. Bintje yielded four somatic hybrids and the combination S. pinnatisectum + S. tuberosum dihaploid 299 resulted in no hybrid regenerants. Morphology in vitro, growth vigour and production of tuber-like structures were evaluated in hybrid plants. Plants were transferred in vivo for further estimation (acclimatization, habitus evaluation and tuberization ability).     

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.     

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     

Somatic hybrids between Solanum etuberosum and diploid,tuber bearing Solanum clones

Electrofusion was used to obtain somatic hybrids between Solanum etuberosum (2n=2x=24) and two diploid potato lines. These hybridizations were conducted to determine if haploidxwild species hybrids are better fusion partners than conventional S. tuberosumGp. Tuberosum haploids. Restriction fragment length polymerase (RFLP) analyses of the putative somatic hybrids confirmed that each parental genome was present. The somatic hybrids between S. etuberosum and a haploid S. tuberosum clone, US-W730, were stunted and had curled, purple leaves. In contrast, somatic hybrids between S. etuberosum and a haploidxwild species hybrid (US-W 730 haploidx S. berthaultii), were vigorous and generally tuberized under field conditions. These hybrids were designated as E+BT somatic hybrids. Analyses of 23 E+BT somatic hybrids revealed a statistically significant bias towards the retention of S. etuberosum chloroplasts. Stylar incompatibilities were observed when the E+BT somatic hybrids were used as pollen donors in crosses with S. tuberosum cultivars. Reciprocal crosses did not show this incompatibility. The progeny were vigorous and had improved tuber traits when compared to the maternal E+BT parent. RFLP analyses of three sexual progeny lines confirmed the presence of all 12 S. etuberosum chromosomes. In two of these lines, RFLPs that marked each of the 24 chromosome arms of S. etuberosum were present. However, RFLP markers specific for regions on chromosomes 2, 7, and 11 were missing from the third clone. Because other markers for these chromosomes were present in the progeny line, these results indicated the likelihood of pairing and recombination between S. etuberosum and S. tuberosum chromosomes.     

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.     

Nuclear DNA content of Solanum species grown in vitro, as determined by flow cytometry

The DNA relative content in nuclei from several Solanum species, which were used as partners for somatic hybridization, were determined using a flow cytometry method. The nuclei were isolated mechanically or via protoplasts from leaves of in vitro grown plants. In the case of S. nigrum as well as S. tuberosum cv. Bzura and dihaploid clone H8105, the nuclei were also obtained from suspension cultured cells by lysis of protoplasts. The source and the method of nuclei isolation affected the pattern of nuclear DNA in the genotypes studied. The mesophyll nuclei showed two distinct peaks on the DNA histograms, whereas the DNA peaks produced by cell suspension nuclei were broad and less distinct. The DNA content in the nuclei, calculated from the DNA histograms of the samples and a DNA standard historgam (Trout Red Blood Cells, having DNA content of 5.05 pg per nucleus), were much lower in mesophyll nuclei than in those obtained from the cell suspension for the same genotypes. The results are discussed in respect of the genetic instability of Solanum genotypes. The usefulness of a flow cytometry approach in somatic hybridization research is also discussed.     

RFLP Analysis of Chromosomal Segregation in Progeny from an Interspecific Hexaploid Somatic Hybrid between Solanum Brevidens and Solanum Tuberosum

Segregation of restriction fragment length polymorphism (RFLP) loci was monitored to determine the degree of homeologous pairing and recombination in a hexaploid somatic hybrid, A206, the result of protoplast fusion between Solanum tuberosum (PI 203900, a tetraploid cultivated potato) and Solanum brevidens (PI 218228), a diploid, sexually incompatible, distant relative harboring several traits for disease resistance. Somatic hybrid A206 was crossed to Katahdin, a tetraploid potato cultivar, to generate a segregating population of pentaploid progeny. Although the clones of the tetraploid S. tuberosum lines PI 203900 and Katahdin were highly polymorphic, the diploid S. brevidens clone was homozygous at all but two of the tested RFLP loci. Thus, homeologous recombination could be detected only when S. tuberosum and S. brevidens chromosomes paired and the S. brevidens homologs then segregated into separate gametes. A bias toward homologous pairing was observed for all 12 chromosomes. At least four and perhaps six chromosomes participated in homologous pairing only; each of 24 progeny contained all S. brevidens-derived RFLP markers for chromosomes 4, 8, 9 and 10. The remaining six chromosomes paired with their homolog(s) about twice as often as expected if hexaploid pairings were completely random. Where detectable with RFLPs, homeologous recombinations (both single and double) occurred at a frequency of 1.31 per chromosome. Cytological observations of meiosis I in the somatic hybrid indicated that homeologous pairing had occurred. Enhanced recombinational activity was observed for chromosome 2. A specific small deletion from chromosome 4 was detected in A206 and 11 other somatic hybrids out of 14 screened. These hybrids represent 13 independent fusion events between the same clones of S. brevidens and S. tuberosum. In one instance, this deletion occurred in one of two plants resulting from the same callus, indicating that the loss occurred in culture after fusion had taken place. It is possible that this deletion contributes to somaclonal variation.     

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.