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.     

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     

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.     

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.     

Production of asymmetric hybrids between Solanum tuberosum and irradiated S. brevidens

Summary Asymmetric somatic hybrids were obtained by fusion of Solanum tuberosum (PDH40) protoplasts with 300- or 500-Gy irradiated protoplasts of S. brevidens. These radiation doses were sufficient to prevent the growth of the S. brevidens protoplasts. Putative hybrids were selected on the basis of phenotype from regenerated shoots and identified with a S. brevidens-specific probe. From these, 31 asymmetric hybrids were confirmed by morphological characteristics, isoenzyme patterns and RFLP analysis. The morphology of the asymmetric hybrids was intermediate between that of S. tuberosum and symmetric hybrids of both species (obtained without irradiation treatment). Chromosome counts from 17 asymmetric hybrids showed that the chromosome number of the hybrids ranged from 31 to 64. The asymmetric hybrids probably had one or two genome complements (i.e. either 24 or 48 chromosomes) from S. tuberosum and 7–22 chromosomes from S. brevidens. There was no clear correlation between the radiation dose and the degree of elimination of the S. brevidens genome.     

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.     

Analysis of chloroplast and mitochondrial DNA in asymmetric somatic hybrids betweenSolanum tuberosum and irradiatedS. brevidens

Organellar DNA of asymmetric somatic hybrids betweenSolanum tuberosum and irradiatedS. brevidens were analysed by DNA hybridization methods using the spinach chloroplast probepSBD, wheat mitochondrial genenad5 and petunia mitochondrial geneorf25. Eight of the 12 asymmetric hybrid plants hadS. tuberosum chloroplast DNA and the remaining fourS. brevidens chloroplast DNA. A novel mitochondrial hybridization pattern was present in eight out of the 17 hybrids tested. In six hybrids, novel combinations of chloroplasts and mitochondria were found, indicating that both organelle types sorted out independently.     

Improved isolation of dihaploidsolanum tubersoum protoplasts and the production of somatic hybrids between dihaploidS. tuberosum andS. brevidens

Summary A modified protoplast isolation technique, applicable to a range of dihaploidSolanum tuberosum genotypes, has been developed. A combination of high calcium and high pH was used to fuse mesophyl protoplasts of dihaploidS. tuberosum (PDH40) and the diploid wild speciesS. brevidens. Large numbers of colonies were obtained after fusion and putative hybrids selected on the basis of phenotype from regenerated shoots. From these, 11 somatic hybrid plants have been identified by their isoenzyme patterns and morphologic characteristics. Four of these hybrids had the expected chromosome number of 48. The approach of mass culture after fusion followed by selection of hybrids from regenerated shoots and the application of somatic hybridization to potato breeding are discussed.     

Resistance to potato leaf roll virus and potato virus Y in somatic hybrids between dihaploid Solanum tuberosum and S. brevidens

Summary Many somatic fusion hybrids have been produced between a dihaploid potato Solanum tuberosum and the sexually-incompatible wild species S. brevidens using both chemical and electrical fusion techniques. S. brevidens was resistant to both potato leaf roll virus (PLRV) and potato virus Y (PVY), the viruses being either at low (PLRV) or undetectable (PVY) concentrations as determined by enzyme-linked immunosorbent assay (ELISA). The S. tuberosum parent was susceptible to both viruses. A wide range of resistance, expressed as a decrease in virus concentration to both viruses was found amongst fusion hybrids, four of which were especially resistant. The practicality of introducing virus resistance from S. brevidens into cultivated potatoes by somatic hybridisation is discussed.     

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.     

Studies of the gene expression in a somatic hybrid of dihaploid Solanum tuberosum and diploid S. brevidens using two-dimensional protein electrophoresis

Two-dimensional protein electrophoretic patterns of leaf, stem and microtuber were compared between a somatic hybrid (Solanum tuberosum + S. brevidens) and parental plants. Polypeptide spots observed in leaf of the somatic hybrid (BT-1) were similar to those of S. brevidens. In the stem of BT-1, the spots characteristic for each parental plants were also observed. Three specific spots (W, X, Y) found in BT-1 were identical to those of S. tuberosum, however their appearance in S. brevidens depended on the culture conditions (observed at 16h daylength regime, but not in the dark with high sucrose concentration). Potato tuber storage protein patatin was observed in small amounts in the microtubers of BT-1. The data indicated that gene expression unique to each parental plants also existed in the somatic hybrid.Abbreviations BAP 6-benzylaminopurine - 2,4-D (2,4-dichlorophenoxy) acetic acid - IAA indole-3-acetic acid - MES 2-(N-morpholino) ethanesulfonic acid - NAA 1-naphthaleneacetic acid - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

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.     

Production of somatic hybrids by electrofusion in Solanum

Summary Conditions are described for large scale electrofusion of mesophyll protoplasts of dihaploid S. tuberosum with those of diploid S. brevidens. Overall fusion frequencies of 20%–30% were achieved, and following fusion, large numbers of protoplast-derived calli were obtained. Putative somatic hybrid plants were selected from the regenerated shoots by examining their morphological characteristics. Twenty-one somatic hybrids were confirmed by isoenzyme analysis and six somatic hybrids were further confirmed by Southern hybridization. Tetraploid hybrids were obtained, but cytogenetic studies indicated that more of the regenerated hybrids were hexaploid than had previously been found following chemical fusion of the same partners. Some advantages of electrofusion over chemical fusion are discussed.     

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.     

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.     

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 hybrids produced by protoplast fusion between S. tuberosum and S. brevidens: phenotypic variation under field conditions

Summary Phenotypic and flowering characteristics of hybrid plants generated by protoplast fusion between a tetraploid S. tuberosum line and diploid S. brevidens were assessed under field conditions. Hybrids were compared to both clonal parental material and protoplast-derived plants of each parent. Almost all of the hybrids were hexaploid. A wide range of variation in morphological characters was observed for hybrids and protoclones. Flowering was markedly reduced in protoclones. The majority of hybrids flowered, had viable pollen and set tubers. Tuber and pollen characteristics of hybrids produced from individual fusion calli also varied. The potential usefulness of fusion hybrids in potato improvement is discussed.     

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     

Fertile somatic hybrids of Solanum species: RFLP analysis of a hybrid and its sexual progeny from crosses with potato

Summary Restriction fragment length polymorphism (RFLP) markers were used to distinguish the chromosomes of Solanum brevidens from those of potato (S. tuberosum) in a fertile somatic hybrid. The hybrid had markers that account for all 24 chromosome arms from each parent, indicating that the hybrid contained at least one copy of each chromosome from each parent. The markers were then used to follow segregation of chromosomes in sexual progeny that resulted from a cross of the somatic hybrid with the potato cultivar Katahdin. Approximately 10% of the sexual progeny lacked one or more of the markers specific to S. brevidens. No one chromosome or marker appeared to be lost preferentially. This infrequent absence of a chromosome marker derived from the wild parent could be explained by intergenomic pairing and recombination. The loss of a marker band for chromosome 8, coupled with the retention of two flanking markers, suggested that a small region of DNA was deleted during regeneration of the somatic hybrid. These results show the value of RFLP analysis when applied to somatic hybrids and their progeny. Clearly, RFLPs will be useful for following the DNA from wild species during its introgression into potato cultivars.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