Mitotic and meiotic irregularities in somatic hybrids of Lycopersicon esculentum and Solanum tuberosum.

Chromosome numbers were determined in metaphase complements of root-tip meristems of 107 tomato (+) potato somatic hybrids, obtained from five different combinations of parental genotypes. Of these hybrids 79% were aneuploid, lacking one or two chromosomes in most cases. All four hybrids that were studied at mitotic anaphase of root tips showed laggards and bridges, the three aneuploids in a higher frequency than the single euploid. Hybrid K2H2-1C, which showed the highest percentage of aberrant anaphases, possessed 46 chromosomes. Fluorescence in situ hybridization with total genomic DNA showed that this hybrid contained 23 tomato, 22 potato, and 1 recombinant chromosome consisting of a tomato chromosome arm and a potato chromosome arm. The potato parent of K2H2-1C was aneusomatic in its root tips with a high frequency of monosomic and trisomic cells and a relatively high frequency of cells with one fragment or telosome. Meiotic analyses of three tomato (+) potato somatic hybrids revealed laggards, which occurred most frequently in the triploid hybrids, and bridges, which were frequently present in pollen mother cells (PMCs) at anaphase I of hypotetraploid K2H2-1C. We observed putative trivalents in PMCs at diakinesis and metaphase I of eutriploid A7-82A and quadrivalents in part of the PMCs of hypotetraploid K2H2-1C, suggesting that homoeologous recombination between tomato and potato chromosomes occurred in these hybrids. All three hybrids showed a high percentage of first division restitution, giving rise to unreduced gametes. However, shortly after the tetrad stage all microspores completely degenerated, resulting in exclusively sterile pollen.     

Homoeologous pairing and recombination in backcross derivatives of tomato somatic hybrids [Lycopersicon esculentum (+) L. peruvianum]

 Genomic in situ hybridization (GISH) was used to examine genome interactions in two allohexa ploid (2n=6x=72) Lycopersicon esculentum (+) L. peruvianum somatic hybrids and their seed progenies originated from subsequent backcrosses to L. esculentum. The ability of GISH to distinguish between chromatin derived from two closely related species, L. esculentum and L. peruvianum (both 2n=2x=24), allowed the precise chromosomal constitution of somatic hybrids and their backcross progenies to be unequivocally established. This enabled the interaction of species genomes to be observed at meiosis, providing clear evidence of strictly regular homoeologous pairing and the high degree of homoeologous recombination in allodiploid plants (2n=2x=24) of the BC₁ generation. In segmental allodiploids of the BC₂ and BC₃ generations, the recombinant chromosomes continued to pair with a homoeologous partner (in the absence of a homologous one), and therefore could be stably incorporated into gametes. Chiasmata were found almost exclusively in more distal, rather subterminal, chromosome segments. A considerable proportion of meiotic recombination was detected in subterminal heterochromatic regions, often involving distal euchromatin, located in close proximity. GISH also supplied information on the extent of the overall sequence homology between the genomes of L. esculentum and L. peruvianum, indicating that despite their different breeding systems, these species may not be differentiated to a high degree genetically. The present study has demonstrated that somatic hybridization between two such closely related, but sexually incompatible or difficult to cross species, provides a way of transferring genes, via homoloeogous crossing-over and recombination, across the incompatibility barriers. Indeed, such hybrids may offer the preferred route for gene transfer, which subsequently results in more stable gene introgression than other methods. Received: 22 July 1996 / Accepted: 23 August 1996     

Cytogenetic analysis of Lycopersicon esculentum (+) Solanum etuberosum somatic hybrids and their androgenetic regenerants

The aim of the study was to characterize genomic relationships among cultivated tomato (Lycopersicon esculentum Mill.) (2n=2x=24) and diploid (2n=2x=24) non-tuberous wild Solanum species (S. etuberosum Lindl.). Using genomic in situ hybridization (GISH) of mitotic and meiotic chromosomes, we analyzed intergeneric somatic hybrids between tomato and S. etuberosum. Of the five somatic hybrids, two plants were amphidiploids (2n=4x=48) mostly forming intragenomic bivalents in their microsporocytes, with a very low frequency of multivalents involving the chromosomes of tomato and S. etuberosum (less than 0.2 per meiocyte). Tomato chromosomes showed preferential elimination during subsequent meiotic divisions of the amphidiploids. Transmission of the parental chromosomes into microspores was also evaluated by GISH analysis of androgenic plants produced by direct embryogenesis from the amphidiploid somatic hybrids. Of the four androgenic regenerants, three were diploids (2n=2x=24 or 2n=2x+1=25) derived from reduced male gametes of the somatic hybrids, and one plant was a hypertetraploid (2n=4x+4=52). GISH revealed that each anther-derived plant had a unique chromosome composition. The prospects for introgression of desirable traits from S. etuberosum into the gene pool of cultivated tomato are discussed. Received: 2 August 2000 / Accepted: 4 December 2000     

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

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

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.     

Chromosome number variation in somatic hybrids between transgenic tomato (Lycopersicon esculentum) and Solanum lycopersicoides

Leaf mesophyll protoplasts of Lycopersicon esculentum were fused with suspension-culture-derived protoplasts of Solanum lycopersicoides by a PEG treatment. Both species have the same chromosome number (2n = 2x = 24). The hybrid calli were selected using the full selection method - kanamycin resistance and culture conditions critical for L. esculentum protoplast divisions. The genomic in situ hybridization analyses indicated a hypo- and hypertetraploid character of the hybrid plant with a majority of S. lycopersicoides chromosomes and a variation in chromosome number from 46 to 53. The hybrids contained a transgene derived from L. esculentum, as shown by Southern blot hybridization and PCR analyses. Their mitochondria were derived from the wild species, S. lycopersicoides. More than 60 regenerated plants were transferred into the greenhouse. They grew very slowly and were not able to flower for almost one year. The main morphological characters of the hybrids included a single shoot and small, dark-green leaves with strongly wrinkled blades. The reasons for nuclear genome asymmetry between hybrids and the possibilities of using them in a genetic and breeding programme are discussed in this paper.     

Production and characterization of intergeneric somatic hybrids through protoplast electrofusion between potato (Solanum tuberosum) and Lycopersicon pennellii

Mesophyll protoplasts of Lycopersicon pennelli Corr., a wild relative of tomato, were electrofused with those from a dihaploid potato clone, cv Nicola, with the objectives of transferring saline tolerance from L. pennellii to cultivated potato. 150 calli were selected from the fusion experiments, finally giving 2 hybrid shoots. Their hybrid nature was verified by examining isoenzyme patterns for esterases (EST), peroxidase (PRX), phosphogluconate dehydrogenase (6-PGD), and glutamate oxaloacetate transaminase (GOT). The hybrid plants had an intermediate morphology, and grew vigorously in vitro. When transplanted to soil, they were less vigorous, due to difficulties in rooting, but were still capable of flowering, and forming short stolons and mishaped tubers, probably resulting from the effects of gene dosage due to the novel association of two genomes from a tuberizing (potato) and a non tuberizing species (L. pennellii). The characteristics of such mishaped tubers provided strong evidence of a hybrid nature for the selected plants. The hybrid plants were highly sterile, producing only 3–7% viable pollen. Tests for salt tolerance showed that the growth of the somatic hybrid plants was reduced by 50% as for L. pennellii, whilst potato did not grow at all under saline conditions.Abbreviations MS Murashige and Skoog basal medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - BAP 6-benzylaminopurine - PEG polyethylen glycol 6,000 - MES 2-(N-morpholino)ethanesulfonic acid - AC alternating current - EST esterases - PRX peroxidase - 6-PGD phosphogluconate dehydrogenase - GOT glutamate oxaloacetate transaminase - FDA fluorescein diacetate     

Species-specific DNA sequences for identification of somatic hybrids between Lycopersicon esculentum and Solanum acaule

Summary Species-specific highly repeated DNA sequences can be used to screen the progeny of protoplast fusions combining different species. Such probes are easy to clone and can be detected by fast methods, e.g., hybridization to total genomic DNA. Furthermore, due to their high copy number, hybridization signals are strong and represent more than one locus, unlike isozymes or resistance markers. After cloning and screening for species-specific DNA sequences we characterized the highly repeated DNA sequences of the solanaceous species Solanum acaule and Lycopersicon esculentum var. gilva. DNA sequencing and hy ridization revealed a prominent, tandemly arranged satellite DNA repeat of 162 bp in Lycopersicon esculentum and a different satellite repeat of 183 bp, also tandemly organized, in Solanum acaule. Each repeat is absent in the respective other species. Therefore, we have used these DNA repeats as markers to distinguish regenerated interspecific somatic hybrids from the respective fusion partners. These hybrids were clearly identified by Southern hybridization and dot-blot assays to the respective ³²P-labelled satellite DNA.     

Allotriploid somatic hybrids of diploid tomato (Lycopersicon esculentum Mill.) and monoploid potato (Solanum tuberosum L.)

Allotriploid somatic hybrids were obtained from fusions between protoplasts of diploid tomato and monohaploid potato. The selection of fusion products was carried out in two different ways: (1) The fusion of nitrate reductase-deficient tomato with potato gave rise only to hybrid calli if selection was performed on media lacking ammonium. Parental microcalli were rarely obtained and did not regenerate. (2) The fusion of cytoplasmic albino tomato with potato gave rise to albino and green hybrid calli and plants. Allotriploids were identified from the two somatic hybrid populations by counting chloroplast numbers in leaf guard cells and by flow cytometry of leaf tissue. Although some pollen fertility of allotriploids and pollen-tube growth of tomato, potato andLycopersicon pennellii into the allotriploid style were observed, no progeny could be obtained. The relevance of allotriploid somatic hybrids in facilitating limited gene transfer from potato to tomato is discussed.     

Asymmetric somatic hybrids between Lycopersicon esculentum and irradiated Lycopersicon peruvianum

Summary Asymmetric somatic hybrids of Lycopersicon esculentum and Lycopersicon peruvianum were analysed for the retention of genes and alleles specific for L. peruvianum. The hybrids were obtained by fusion of protoplasts from L. esculentum with those of L. peruvianum (the donor), the latter having been irradiated before fusion with 50, 300 or 1,000 Gy of gamma-rays. The retention of three different types of genes or alleles was analysed. (1) The gene coding for kanamycin resistance, which is dominant and had been introduced in most of the L. peruvianum donor plants by transformation. It was present at one locus in 16 L. peruvianum donor plants and at two loci in one donor plant. (2) The genes coding for acid phosphatase, locus Aps-1, and glutamate oxaloacetate transaminase (GOT); different alleles of these genes are co-dominant and were detected by isozyme analysis. (3) Eighteen single gene morphological markers for which most of the L. esculentum genotypes used were homozygous recessive. Kanamycin resistance from donor plants with one locus was retained in about 50% of the asymmetric 30H-hybrids (the donor was irradiated with 300 Gy). L. peruvianum specific alleles of Aps-1 and GOT were present in at least 70% of the hybrids; the retention of donor alleles was lower in 30H- than in 5H-hybrids (donor irradiated with 50 Gy). On average, 73% of the L. peruvianum-specific alleles (one or both) of the morphological markers were detected in the 30H-hybrids. Several of the L. esculentum genotypes used were homozygous recessive for two morphological markers on the same chromosome; in 43% of the 30H-hybrids derived from them, only one of these markers was complemented by the L. peruvianum allele. This is an indication of frequent breakage of the L. peruvianum chromosomes. Several hybrid calli regenerated genotypically different shoots. On the whole, this analyses confirms the conclusion drawn from the cytogenetic and morphological analysis of these asymmetric hybrids, namely that irradiation prior to fusion eliminates the L. peruvianum genome to only a limited extent.     

Asymmetric somatic hybrids between Lycopersicon esculentum and irradiated Lycopersicon peruvianum

Summary Asymmetric somatic hybrids of Lycopersicon esculentum and Lycopersicon peruvianum were obtained by fusion of leaf protoplasts from both species after irradiation of protoplasts or leaf tissue of L. peruvianum with 50, 300, or 1,000 Gy of gamma-rays. These radiation doses were sufficient to abolish the growth of the L. peruvianum protoplasts. The hybrids were selected for their ability to regenerate plants; this regeneration capacity derived from L. peruvianum. All asymmetric hybrid plants were aneuploid. The ploidy level, the morphology, and the regeneration rate were analyzed in relation to the radiation dose applied to L. peruvianum. After a low dose (50 Gy), most hybrids had near-triploid chromosome numbers, whereas after a high dose (300 or 1,000 Gy), most hybrids had near-pentaploid numbers. The morphology of the asymmetric hybrids was intermediate between that of L. esculentum and symmetric somatic hybrids of both species (obtained without irradiation treatment), and approached the morphology of L. esculentum to a greater extent after a high dose of irradiation. The asymmetric hybrids regenerated more slowly than the symmetric hybrids and regeneration proceeded more slowly after a high dose than after a low dose of irradiation. The high-dose hybrids also grew more slowly, flowered less, and set fruits less than the low-dose hybrids. No seeds could be obtained from any asymmetric hybrid.     

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.     

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.     

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.     

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     

Subcellular localization of steroidal glycoalkaloids in vegetative organs of Lycopersicon esculentum and Solanum tuberosum

Cell fractions from the major vegetative organs of tomato and potato plants were obtained by homogenization and differential centrifugation. In both species, steroidal glycoalkaloids were found to accumulate mainly in the soluble fraction, with smaller amounts in the microsomal fraction. Alkaloids appeared sporadically in the mitochondrial fractions but were only detected in lower fractions as an artefact.     

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     

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     

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