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

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

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

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

Somatic hybrids <Emphasis Type="Italic">Solanum nigrum</Emphasis> (+) <Emphasis Type="Italic">S. tuberosum</Emphasis>: morphological assessment and verification of hybridity

Somatic hybrids between the cultivated potato diploid hybrid clone, ZEL-1136, and hexaploid non-tuber-bearing wild species Solanum nigrum L. exhibiting resistance to Phytophthora infestans were regenerated after PEG-mediated fusion of mesophyll protoplasts. The objective was to transfer the late-blight resistance genes from the wild species into plants of the cultivated potato clone. From a total of 59 regenerants, 40 clones survived and have been maintained in vitro on hormone-free MS/2 medium. Thirty-two somatic hybrids were identified by their intermediate morphology (leaves of nigrum type and flowers of tuberosum type) and verified by flow cytometry and random amplified polymorphic DNA (RAPD) patterns. The RAPD analysis of nuclear DNA confirmed the hybrid nature of 29 clones. Flow cytometry revealed a wide range of ploidy in the generated hybrids, from nearly the tetra- to decaploid level. Most of the hybrid clones were stable in vitro, grew vigorously in soil, and set flowers and parthenocarpic berries. However, all of the flowering hybrids were male-sterile. Nine hybrid clones produced tuber-like structures in soil. The most vigorous flowering somatic hybrids were selected for assessment of the late-blight resistance.     

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.     

Effect of ploidy increase on transgene expression: example from <Emphasis Type="Italic">Citrus</Emphasis> diploid cybrid and allotetraploid somatic hybrid expressing the EGFP gene

Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from ‘Murcott’ tangor (Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic ‘Valencia’ orange (C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy level conversion can affect transgene expression and citrus diploid cybrid and allotetraploid somatic hybrid represents another example of gene regulation coupled to ploidy.     

The role of irradiation dose and DNA content of somatic hybrid calli in producing asymmetric plants between an interspecific tomato hybrid and eggplant

Highly asymmetric somatic hybrid plants were obtained by PEG/DMSO fusion of gamma-irradiated mesophyll protoplasts of the kanamycin-resistant (KmR⁺) interspecific hybrid Lycopersicon esculentum x L. pennellii (EP) with mesophyll protoplasts of Solanum melongena (eggplant, E). Elimination of the EP chromosomes was obtained by irradiating the donor genome with different doses of gamma rays (100, 250, 500, 750 and 1000 Gy). The selection of somatic hybrid calli was based on kanamycin resistance; EP and E protoplasts did not divide due to the irradiation treatment and sensitivity to kanamycin, respectively. KmR⁺ calli were recovered following all irradiation doses of donor EP protoplasts. The hybrid nature of the recovered calli was confirmed by PCR amplification of the NptII gene, RAPD patterns and Southern hybridizations using potato ribosomal DNA and pTHG2 probes. Ploidy levels of calli confirmed as hybrid were further analyzed by flow cytometry. Such analyses revealed that the vast majority of hybrid calli that did not regenerate shoots were 5–9n polyploids. The three asymmetric somatic hybrid plants obtained were regenerated only from callus with a ploidy level close to 4n, and such calli occurred only when the donor EP had been exposed to 100 Gy. The amount of DNA in somatic hybrid calli, from 100-Gy exposure, was found by dot blot hybridization with the species-specific probe, pTHG2, to be equivalent with 3.1–25.8% of the tomato genome. Thus, DNA contained in 3.8–13.2 average-size tomato chromosomes was present in these hybrid calli. The asymmetric somatic hybrid plants had the eggplant morphology and were regenerated from one hybrid callus that contained an amount of tomato DNA equivalent to 6.29 average-size tomato chromosomes.     

Asymmetric somatic hybridization between tall fescue (Festuca arundinacea Schreb.) and irradiated Italian ryegrass (Lolium multiflorum Lam.) protoplasts

Intergeneric asymmetric somatic hybrids have been obtained by the fusion of metabolically inactivated protoplasts from embryogenic suspension cultures ofFestuca arundinacea (recipient) and protoplasts from a non-morphogenic cell suspension ofLolium multiflorum (donor) irradiated with 10, 25, 50, 100, 250 and 500 Gy of X-rays. Regenerating calli led to the recovery of genotypically and phenotypically different asymmetric somatic hybridFestulolium plants. The genome composition of the asymmetric somatic hybrid clones was characterized by quantitative dot-blot hybridizations using dispersed repetitive DNA sequences specific to tall fescue and Italian ryegrass. Data from dot-blot hybridizations using two cloned Italian ryegrass-specific sequences as probes showed that irradiation favoured a unidirectional elimination of most or part of the donor chromosomes in asymmetric somatic hybrid clones obtained from fusion experiments using donor protoplasts irradiated at doses 250 Gy. Irradiation of cells of the donor parent with 500 Gy prior to protoplast fusion produced highly asymmetric nuclear hybrids with over 80% elimination of the donor genome as well as clones showing a complete loss of donor chromosomes. Further information on the degree of asymmetry in regenerated hybrid plants was obtained from chromosomal analysis including in situ hybridizations withL. multiflorum-specific repetitive sequences. A Southern blot hybridization analysis using one chloroplast and six mitochondrial-specific probes revealed preferentially recipient-type organelles in asymmetric somatic hybrid clones obtained from fusion experiments with donor protoplasts irradiated with doses higher than 100 Gy. It is concluded that the irradiation of donor cells before fusion at different doses can be used for producing both nuclear hybrids with limited donor DNA elimination or highly asymmetric nuclear hybrid plants in an intergeneric graminaceous combination. For a wide range of radiation doses tested (25–250Gy), the degree of the species-specific genome elimination from the irradiated partner seems not to be dose dependent. A bias towards recipient-type organelles was apparent when extensive donor nuclear genome elimination occurred.Abbreviations cpDNA Chloroplast DNA - 2, 4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

Chloroplast segregation in somatic hybrids of Nicotiana plumbaginifolia and N. sylvestris having different ratios of parental nuclear genomes

Summary Fusion of mesophyll protoplasts of haploid Nicotiana plumbaginifolia (P) and N. sylvestris (S) resulted in the production of somatic hybrid plants of various ploidy levels. Analysis of the restriction fragment patterns of chloroplast DNA from 118 plants belonging to genome constitutions PS, PPS, PSS, and PPSS revealed that two had a pattern corresponding to a mixture of parental DNA while all the others had the pattern of either N. plumbaginifolia or N. sylvestris. In the latter case, the ratio of the two parental types fits 1∶1 in all the four genome constitutions studied. Since the protoplasts used in the fusion experiment were physiologically similar and the hybrid cells were not deliberately selected, these results suggest that chloroplast segregation in the somatic hybrids is independent of the chloroplast input of the fusion partners and the nuclear background of the fusion products.     

Introgression of bacterial wilt resistance from eggplant to potato via protoplast fusion and genome components of the hybrids

Key message

Bacterial wilt resistant somatic hybrids were obtained via protoplast fusion between potato and eggplant and three types of nuclear genomes were identified in the hybrids through GISH and SSR analysis.

Abstract

Cultivated potato (Solanum tuberosum L.) lacks resistance to bacterial wilt caused by Ralstonia solanacearum. Interspecific symmetric protoplast fusion was conducted to transfer bacterial wilt resistance from eggplant (S. melongena, 2n = 2x = 24) into dihaploid potato (2n = 2x = 24). In total, 34 somatic hybrids were obtained, and of these, 11 rooted and were tested for genome components and resistance to race 1 of R. solanacearum. The hybrids exhibited multiple ploidy levels and contained the dominant nuclear genome from the potato parent. Three types of nuclear genomes were identified in the hybrids through genomic in situ hybridization (GISH) and simple sequence repeat (SSR) analysis, including (1) the potato type of the tetraploids in which eggplant chromosomes could not be detected by GISH but their nuclear DNA was confirmed by SSR, (2) the biased type of the hexaploids in which the chromosome dosage was 2 potato:1 eggplant, and (3) the chromosome translocation type of the mixoploids and aneuploids that was characterized by various rates of translocations of nonhomologous chromosomes. Cytoplasmic genome analysis revealed that mitochondrial DNA of both parents coexisted and/or recombined in most of the hybrids. However, only potato chloroplast DNA was retained in the hybrids speculating a compatibility between cpDNA and nuclear genome of the cell. The pathogen inoculation assay suggested a successful transfer of bacterial wilt resistance from eggplant to the hybrids that provides potential resistance for potato breeding against bacterial wilt. The genome components characterized in present research may explain partially the inheritance behavior of the hybrids which is informative for potato improvement.     

Genetic characterization of asymmetric somatic hybrids between Bupleurum scorzonerifolium Willd and Triticum aestivum L.: potential application to the study of the wheat genome

In this paper, we describe how Bupleurum scorzonerifolium/Triticum aestivum asymmetric somatic hybrids can be exploited to study the wheat genome. Protoplasts of B. scorzonerifolium Willd were irradiated with ultraviolet light (UV) and fused with protoplasts of common wheat (T. aestivum L.). All cell clones were similar in appearance to those of B. scorzonerifolium, while the regenerated plantlets were either intermediate or B. scorzonerifolium-like. Genotypic screening using isozymes showed that 39.3% of cell clones formed were hybrid. Some of the hybrid cell clones grew vigorously, and differentiated green leaves, shoots or plantlets. DNA marker analysis of the hybrids demonstrated that wheat DNA was integrated into the nuclear genomes of B. scorzonerifolium and in situ karyotyping cells revealed that a few wheat chromosome fragments had been introgressed into B. scorzonerifolium. The average wheat SSR retention frequency of the RH panel was 20.50%, but was only 6.67% in fusions with a non-irradiated donor. B. scorzonerifolium chromosomes and wheat SSR fragments in most asymmetric hybrid cell lines remained stable over a period of 2.5–3.5 years. We suggest the UV-induced asymmetric somatic hybrids between B. scorzonerifolium Willd and T. aestivum L. have the potential for use in the construction of an RH map of the wheat genome.     

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.     

Rare instances of haploid inducer DNA in potato dihaploids and ploidy-dependent genome instability

In cultivated tetraploid potato (Solanum tuberosum), reduction to diploidy (dihaploidy) allows for hybridization to diploids and introgression breeding and may facilitate the production of inbreds. Pollination with haploid inducers (HIs) yields maternal dihaploids, as well as triploid and tetraploid hybrids. Dihaploids may result from parthenogenesis, entailing the development of embryos from unfertilized eggs, or genome elimination, entailing missegregation and the loss of paternal chromosomes. A sign of genome elimination is the occasional persistence of HI DNA in some dihaploids. We characterized the genomes of 919 putative dihaploids and 134 hybrids produced by pollinating tetraploid clones with three HIs: IVP35, IVP101, and PL-4. Whole-chromosome or segmental aneuploidy was observed in 76 dihaploids, with karyotypes ranging from 2n = 2x − 1 = 23 to 2n = 2x + 3 = 27. Of the additional chromosomes in 74 aneuploids, 66 were from the non-inducer parent and 8 from the inducer parent. Overall, we detected full or partial chromosomes from the HI parent in 0.87% of the dihaploids, irrespective of parental genotypes. Chromosomal breaks commonly affected the paternal genome in the dihaploid and tetraploid progeny, but not in the triploid progeny, correlating instability to sperm ploidy and to haploid induction. The residual HI DNA discovered in the progeny is consistent with genome elimination as the mechanism of haploid induction.

A large potato progeny population produced by crossing tetraploid cultivated clones to diploid Phureja lines displays rare instances of haploid inducer chromosomes, which are frequently damaged.     

A cytogenetic and phenotypic characterization of somatic hybrid plants obtained after fusion of two different dihaploid clones of potato (Solatium tuberosum L.)

Summary Somatic hybrid plants of various ploidy levels obtained after chemical fusion between two dihaploid clones of potato Solanum tuberosum L. have been analysed by cytological, morphological and molecular methods. The hybrid nature of tetraploid and hexaploid plants and the genome dosage in hexaploid hybrids were confirmed by Giemsa C-banding. Tetraploid and hexaploid hybrids showed numerical as well as structural chromosome mutations. The latter occurred mainly in the nuclear organizing chromosome. The tetraploid hybrids were more vigorous than the dihaploid parents as demonstrated by an increase in height, enlargement of leaves, increase in the number of internodes, restored potential for flowering and increased tuber yield. The grouping of tetraploid somatic hybrids into various classes on the basis of leaf morphology revealed that plants with a full chromosome complement were more uniform than aneuploids. Many hexaploid somatic hybrids were also more vigorous than the dihaploid parents and could be grouped into two different classes on the basis of floral colour and tuber characteristics, the differences being due to their different dosage of parental genomes. Most of the tetraploid somatic hybrids showed pollen development halted at the tetrad stage as one of the parental clones contained a S. Stoloniferum cytoplasm. However, one tetraploid plant produced pollen grains with high viability. The chloroplast genome in the hybrid plants was determined by RFLP analysis. All of the hybrids had a cpDNA pattern identical to one parent, which contained either S. Tuberosum or S. Stoloniferum cpDNA. A slight preference for S. Tuberosum plastids were observed in hybrid plants. No correlation between pollen development and plastid type could be detected.     

Expressivity of apomixis in 2n?+?n hybrids from an apomictic and a sexual parent: insights into variation detected in Pilosella (Asteraceae: Lactuceae)

Reproductive variation was studied in the tetraploid Pilosella aurantiaca, hexaploid P. rubra (both species with facultative autonomous apospory) and in their 2n + n hybrids, which were obtained by crossing with a sexual pollen parent (tetraploid P. officinarum). The different DNA content in P. aurantiaca and P. officinarum demonstrated the actual 2n + n origin, both spontaneous from the field and through experimental crosses, of their hexaploid hybrids. The octoploid 2n + n progeny were recovered from an experimental cross of P. rubra and P. officinarum. The reproductive pathways operating in two maternal facultatively apomictic species and in the hybrids were quantified using a flow cytometric analysis of seeds obtained from either open-pollinated or emasculated plants. Whereas both maternal species displayed a high penetrance of apomixis, the level of apomixis among the majority of 2n + n hybrids was much lower and variable. Some of the hexaploid hybrids had a reduced seed set. Compared to the respective maternal parents, the decrease in apomixis due to haploid parthenogenesis and/or n + n mating was evident in almost all unreduced hybrids, irrespective of their field/experimental origin and ploidy. Hence, the reproductive behaviour in the apomictic maternal parent was profoundly different from that of the 2n + n hybrids with a sexual parent in spite of the preservation of the complete maternal genome in the hybrids. The regulatory interactions in hybrid genomes, such as effects of modifiers, heterochrony, and epigenetic control, may be consistent with the different expressivity of apomixis observed under different genetic backgrounds.     

An interspecific somatic hybrid between upland cotton (<Emphasis Type="Italic">G. hirsutum</Emphasis> L. cv. ZDM-3) and wild diploid cotton (<Emphasis Type="Italic">G. klotzschianum</Emphasis> A.)

Somatic hybrids were produced through protoplast electrofusion between the tetraploid cotton Gossypium hirsutum L. cv. ZDM-3 and the wild diploid cotton G. klotzschianum. Hybrid plants were generated from 3 out of 24 callus lines that were derived from fused protoplasts. Hybrid plants were initially identified as somatic hybrids by ploidy analysis: the plants from the 3 callus lines had chromosome numbers near to sum of the two parents (78 = 52 + 26). The plants from the 3 lines were subsequently confirmed as hybrids by cytological, molecular, histological and morphological analyses. The morphology of hybrids was distinct from that of the parents, with elongated stigmas and malformed anthers lacking microspores and pollen, leading to male sterility. It is expected that the male sterility resulted from the high number of univalent and irregular multivalent chromosome pairings per meiocyte.     

Allopolyploidy-Induced Rapid Genome Evolution in the Wheat (Aegilops–Triticum) Group

To better understand genetic events that accompany allopolyploid formation, we studied the rate and time of elimination of eight DNA sequences in F1 hybrids and newly formed allopolyploids of Aegilops and Triticum. In total, 35 interspecific and intergeneric F1 hybrids and 22 derived allopolyploids were analyzed and compared with their direct parental plants. The studied sequences exist in all the diploid species of the Triticeae but occur in only one genome, either in one homologous pair (chromosome-specific sequences [CSSs]) or in several pairs of the same genome (genome-specific sequences [GSSs]), in the polyploid wheats. It was found that rapid elimination of CSSs and GSSs is a general phenomenon in newly synthesized allopolyploids. Elimination of GSSs was already initiated in F1 plants and was completed in the second or third allopolyploid generation, whereas elimination of CSSs started in the first allopolyploid generation and was completed in the second or third generation. Sequence elimination started earlier in allopolyploids whose genome constitution was analogous to natural polyploids compared with allopolyploids that do not occur in nature. Elimination is a nonrandom and reproducible event whose direction was determined by the genomic combination of the hybrid or the allopolyploid. It was not affected by the genotype of the parental plants, by their cytoplasm, or by the ploidy level, and it did not result from intergenomic recombination. Allopolyploidy-induced sequence elimination occurred in a sizable fraction of the genome and in sequences that were apparently noncoding. This finding suggests a role in augmenting the differentiation of homoeologous chromosomes at the polyploid level, thereby providing the physical basis for the diploid-like meiotic behavior of newly formed allopolyploids. In our view, this rapid genome adjustment may have contributed to the successful establishment of newly formed allopolyploids as new species.     

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.     

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     

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.