Interspecific Somatic Hybrids Research Articles

Interspecific somatic hybrids Solanum bulbocastanum (+) S. tuberosum H-8105

Interspecific somatic hybrids between a dihaploid potato clone H-8105 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuberizing species Solanum bulbocastanum were generated and analysed. Only ten regenerants displaying the intermediate morphology with dominating characteristics of the wild parent (simple leaves, anthocyanin pigmentation) were produced in 15 weeks after a single PEG-mediated fusion event. The RAPD patterns confirmed the hybridity of all of them. The hybrids rooted poorly and grew slowly in vitro. The cytological analysis revealed a high degree of aneuploidy in the hybrids with morphological and growth anomalies in vitro, while the morphologically normal hybrids were tetraploids. All the S. bulbocastanum (+) H-8105 hybrids were unstable in culture and three of them were consequently lost during three years of propagation in vitro. The possible reasons for instability of somatic hybrids between the distantly related species are discussed.

Infection of plant material derived from Solanum acaule with Clavibacter michiganensis ssp. sepedonicus: temperature as a determining factor in immunity of S. acaule to bacterial ring rot

The development of potato (Solanum tuberosum) cultivars immune (completely free of infective agents) to bacterial ring rot disease, caused by Clavibacter michiganensis ssp. sepedonicus, would be a significant step toward eradication of the disease. Earlier results suggested that the wild potato species Solanum acaule, acc. PI472655 (genotype 7‐8), was immune to C. michiganensis ssp. sepedonicus. The goal of the present study was to investigate the possibility of transferring this desirable trait to cultivated potato through interspecific somatic hybridization. Eight different somatic hybrids between S. acaule and S. tuberosum, with three different genome ratios, were tested for susceptibility to infection in the glasshouse using two C. michiganensis ssp. sepedonicus strains. All the somatic hybrids expressed symptoms of ring rot and were susceptible to infection as determined by IFAS (indirect immunofluorescent antibody staining) tests. The genome compositions of the hybrids influenced bacterial titres. Most of the hybrids with a higher proportion of the S. acaule genome contained lower numbers of C. michiganensis ssp. sepedonicus cells than hybrids with lower proportions of the S. acaule genome. In growth chamber tests, temperature was found to be a determining factor in the expression of immunity in the tetraploid S. acaule 7‐8 line. At 21°C, S. acaule 7‐8 was immune to infection by C. michiganensis ssp. sepedonicus, but at 15°C, plants supported large populations of the pathogen. However, none of the S. acaule plants expressed disease symptoms. Thus, S. acaule exhibits temperature‐dependent immunity to infection by C. michiganensis ssp. sepedonicus and should be considered tolerant rather than immune.

GISH confirmation of somatic hybrids between Solanum melongena and S. torvum: assessment of resistance to both fungal and bacterial wilts

Interspecific somatic hybrids between Solanum melongena L. (2 n = 2 x = 24) and two accessions of Solanum torvum Sw. (2 n = 2 x = 24) were produced in view of transferring resistance to two soil-born pathogens, Ralstonia solanacearum and Verticillium dahliae, from the wild species into the cultivated eggplant. All somatic hybrids were phenotypically homogenous and intermediate between the parents. Their hybrid nature was confirmed by analysis of isozymes and RAPDs. They showed reduced pollen viability, and all but one possessed the chloroplasts from either one or the other parent. As S. melongena and S. torvum chromosomes were morphologically indistinguishable, genomic in situ hybridisation (GISH) was applied to recognise the chromosomes from each parent in the hybrids. As expected, the selected tetraploid plants contained one complete set of chromosomes from each fusion partner. On spread preparations, the two parental genomes were not spatially separated at any time of the cell cycle. Translocation or recombinant chromosomes could not be demonstrated in the mitotic metaphase. Tests for resistance performed in vitro by using suspensions of two strains of R. solanacearum (race 1 and 3) and filtrate of culture medium of one strain of V. dahliae, revealed that S. melongena was susceptible, whereas both accessions of S. torvum had high levels of resistance. Except for two hybrid clones, which were found susceptible to race 3, as was S. melongena, all somatic hybrids tested showed good levels of bacterial and fungal resistance, either intermediate or as high as that of the wild parent.

Somatic hybridisation for improvement of citrus rootstock: production of five new combinations with potential for improved disease resistance

The development of biotechnological tools such as protoplast fusion has provided a means of bypassing natural barriers related to reproductive biology of citrus found in conventional breeding systems. We report the production of 4 interspecific somatic hybrids, Cleopatra mandarin + Volkamer lemon, Ruby Blood sweet orange + Volkamer lemon, Rohde Red Valencia sweet orange + Volkamer lemon, and Rangpur lime + Sunki mandarin, and 1 intergeneric somatic hybrid, Valencia sweet orange + Fortunella obovata (Tanaka), which may be included and evaluated in rootstock improvement programs. Protoplasts were isolated from embryogenic calli and seedling leaves, and chemically fused with polyethylene glycol. Plants were regenerated via somatic embryogenesis and somatic hybridisation was confirmed by foliar morphology, cytological and DNA (RAPD) analyses. Somatic hybrids may combine required rootstock characteristics such as tolerance to citrus blight, tristeza virus, and Phytophthora, and have potential for control of tree size due to polyploidy.

Fertile somatic hybrids of Solanum etuberosum (+) dihaploid Solanum tuberosum and their backcrossing progenies: relationships of genome dosage with tuber development and resistance to potato virus Y

The wild non-tuberous species Solanumetuberosum is resistant to biotic andabiotic stresses, but is very difficult tocross with cultivated potato. Therefore,interspecific somatic hybrids between adihaploid clone of potato S.tuberosum (2n=2x=24, AA genome) and thediploid species S. etuberosum(2n=2x=24, EE genome) were produced byprotoplast fusion. Among the 7 fertilefusion hybrids analysed by genomic insitu hybridisation (GISH), three groups ofplants were found with the genomicconstitution of AAEE, AAEEEE and AAAAEE.Four fusion hybrids had exactly theexpected chromosome composition, while eachof the three aneuploid hybrids had lost twochromosomes of S. etuberosum. Twobackcross progenies were developed, andGISH analysis was applied to analysetransmission of the parental chromosomesinto the sexual generations. BC1hybrids derived from the crosses of thehexaploid somatic hybrids with tetraploidpotato were pentaploid with thetheoretically expected genomic compositionor with slight deviation from thisexpectation. In the three BC2 hybridsanalysed by GISH seven to 12 chromosomes ofS. etuberosum were detected in thepredominant S. tuberosum background.No recombinant chromosomes in the hybridswere detected. Genome dosage affects tuberformation in hybrids and their progenies,but has less effect on resistance to potatovirus Y (PVY) in fusion hybrids. Severalgenotypes of the fusion hybrids andBC1 progeny did not show viralinfection even in the graftingexperiments.

Production of Somatic Hybrid Plants between Japanese Bunching Onion(Allium fistulosum L.) and Bulb Onion(A. cepa L.) via Electrofusion.

Interspecific somatic hybrids between Japanese bunching onion (Allium fistulosum L., 2n=2x=16) and bulb onion (A. cepa L., 2n=2x=16) were produced by the electorofusion of protoplasts. A concentration of iodoacetamide (IOA), suitable for inhibiting the formation of colonies from protoplasts in Japanese bunching onion, was used. IOA-treated protoplasts of Japanese bunching onion and untreated protoplasts of bulb onion were mixed together and fused, with the expectation that only heterokaryons would selectively develop into colonies. Four hundred and seventeen colonies had formed from fusion products after a 45- day culture, of which approximately 80% developed into calli. Plant regeneration was achieved in 33 out of 325 (10.1%) calli. Some regenerants expressed abnormalities, but two were successfully transplanted in a greenhouse. Cytogenetical and DNA analyses revealed these two regenerants to be amphidiploids (2n=4x=32). Furthermore, it was shown that another three regenerants possessed the nuclear genome of Japanese bunching onion, whereas, their chloroplasts were from bulb onion. The application of these hybrids to a breeding program for Japanese bunching onion is discussed.

Somatic Hybrids between Lycopersicon esculentum and Lycopersicon chmielewskii.

Interspecific somatic hybrids were obtained by electric protoplast fusion of cotyledon protoplasts of Lycopersicon esculentum Mill. cv. Kyoryokutoko treated with iodoacetamide (IOA) and suspension-culture-derived protoplasts of L. chmielewskii (LA1330). A multiple-step selection procedure was used in selecting the hybrids depending on IOA treatments and a culture medium that only allows cotyledon protoplasts to regenerate. Plants were regenerated on the calli derived from fused protoplasts. After acclimation, the hybrid plants with fruits through self-pollination were obtained. Identification of the regenerated putative hybrid plants was carried out by morphological, chromosomal and random amplified polymorphic DNA (RAPD) analysis, as well as restriction fragment length polymorphism (RFLP) comparison of parental and hybrid plants. The hybrids showed unique morphologies in leaf, flower and stem compared with that of two fusion partners, 2n = 4x = 48, 47, 69-72 chromosomes, both of the parental-specific RAPD bands, and genomic recombination and elimination occurred in mitochondria level. Cell fusion technique shows its new potential in foreign gene transformation between L. esculentum and L. chmielewskii.

Field variation in a tetraploid progeny derived by selfing a Solanum commersonii (+) S. tuberosum somatic hybrid: A multivariate analysis

The practical utilization of interspecific somatic hybridization in plant breeding depends on intergenomic interactions and their effects on agronomically important traits. In order to characterize the variation that could be recovered by selfing a tetraploid somatic hybrid between a dihaploid clone of Solanum tuberosum (tbr) and the sexually incongruent diploid wild species S. commersonii (cmm), an F2 progeny was analyzed in the field for a number of vegetative, reproductive and productive traits. Variation for such traits was summarized by principal component analysis, and genotypes were clustered on the basis of the most important derived variables. Ten main principal components (PC) explaining 98.9% of total variance for 12 epigeous traits were extracted and used in cluster analysis. They were mainly correlated to leaflet number, size, and shape, degree of pigmentation, pubescence, and flowering, flower shape, and no. of primary stems. Genotypes were clustered in two groups, one including the tbrclone, the somatic hybrid, and the vast majority of selfed progenies, another the cmm clone and the remaining progenies. The analysis of principal components carried out with the four hypogeous traits identified three main derived variables correlated to tuber production, to the mean weight of the tuber, and to stolon length. Genotypes were grouped again in two clusters: the first included the two parental genotypes, the somatic hybrid and about two thirds of the F2 plants, the second the remaining F2 progenies. A large variability and the reassortment of parental traits were seen in the F2 population, suggesting the possibility to recover useful characteristics from the wild species into the genetic background of the cultivated one.

New mitochondrial genome organization in three interspecific somatic hybrids of Medicago sativa including the parent-specific amplification of substoichiometric mitochondrial DNA units

Three somatic hybrid plants produced by protoplast fusion between Medicago sativa and each of the three species Medicago coerulea, Medicago falcata and Medicago arborea have been analysed for the composition of their mitochondrial DNA. Restriction fragment length polymorphism (RFLP) analysis of mitochondrial genes in somatic hybrids and their parental lines showed various degrees of rearrangement. The M. sativa+M. coerulea hybrid retained all of the M. coerulea-specific bands but lost all the major M. sativa- specific bands. The M. sativa+M. falcata hybrid showed only M. sativa-specific bands together with non-parental bands, and the M. sativa+M. arborea hybrid showed a partial incorporation of bands from both parents together with non-parental bands. The three different outcomes were attributed mainly to differences in the genetic distance between the parents of each hybrid. Analysis of the sexual progeny of the M. sativa+M. coerulea hybrid showed that a residual mitochondrial DNA subunit of M. sativa was retained in the hybrid cytoplasm. This subunit was amplified and inherited in a mutually exclusive, allelic-like fashion with its M. coerulea homologous counterpart in the sexual progeny of the hybrid. Possible mechanisms for the partitioning of mitochondrial DNA in the generative lineage of the somatic hybrids are discussed in relation to the creation of new nucleus-cytoplasm assortments otherwise impossible to obtain by a sexual cross in Medicago.

Development of interspecific somatic hybrid cell lines in cultivated jute and their early characterization using jute chloroplast RFLP marker

AbstractA strong sexual incompatibility barrier that exists between the two cultivated jute species, Corchorus capsularis and Corchorus olitorius, limits the scope for improvement through genetic introgression. Protoplast fusion was carried out to generate interspecific hybrid cell lines. Cotyledonary cell protoplasts of C. capsularis and anthocyaninpigmented hypocotyl protoplasts of C. olitorius were used in the fusion experiments, which appeared to be visually useful in the early selection of the fused products. A chloroplast DNA (cpDNA) marker was developed in jute, which showed species‐specific hybridization patterns with EcoRI‐digested total genomic DNA of C. capsularis and C. olitorius. This cpDNA marker was used in the characterization of the somatic hybrid cell lines at their early stages of growth. Evidence for the presence of both types of cpDNA in the hybrid cell lines was obtained when the total genomic DNA of 4‐ to 7‐month‐old hybrid cell lines was challenged with the chloroplast DNA marker through Southern analysis. It was shown that the early segregation of the parental chloroplasts did not occur in jute, although this is common in other plant species. The hybrid nature of the fused cell lines could also be identified through peroxidase isozyme analysis. Isozyme banding patterns were complex and varied among the hybrid cell lines.

The proportions of glycoalkaloid aglycones are dependent on the genome constitutions of interspecific hybrids between two Solanum species ( S. brevidens and S. tuberosum)

Wild Solanum species, related to the cultivated potato ( Solanum tuberosum L. ssp. tuberosum), generally contain high amounts of various glycoalkaloids, which are undesirable characters when expressed in edible potatoes. The aim of the present study was to define the influence of genome constitution alterations on the relative proportions of glycoalkaloid aglycones in a range of interspecific somatic hybrids between wild Solanum species ( S. brevidens Phil.) and cultivated potato. The S. brevidens parental species principally produced tomatidine aglycone and the S. tuberosum line produced solanidine aglycone. Plant material including primary interspecific somatic hybrids, their anther-derived haploids, and ‘second generation’ somatic hybrids were analysed in terms of their genome constitutions by using GISH (genomic in situ hybridisation). The genome constitutions of the primary hybrids were AAEE (tetraploid hybrids) and AAEEEE (hexaploid hybrids), with A-genomes from S. tuberosum and E-genomes from S. brevidens. The anther-derived diploid and triploid (somato)haploids regenerated from somatic hybrids had genome constitutions of AE and AEE. Gas chromatography-mass spectrometry (GC-MS) analysis of glycoalkaloid aglycones in leaf samples showed that the relative proportions of tomatidine aglycones were higher than that of solanidine or demissidine aglycones in all the somatic hybrids and their somatohaploids. Altering the genome constitution of the hybrids by adding S. tuberosum (AA) genome to the 3x somatohaploid (AEE) in order to produce ‘second generation’ somatic hybrids (AAAEE) had a significant effect on the proportions of aglycones. The relative amount of solanidine increased from 10% in the primary somatic hybrids and their somatohaploids up to the level of 29% in the ‘second generation’ somatic hybrids. The increased formation of solanidine was assumed to be based on a higher dose of A-genome in the ‘second generation’ somatic hybrids. This study demonstrated that specific undesirable traits derived from wild Solanum species, such as alien glycoalkaloid expression, could be reduced by production of ‘second generation’ somatic hybrids of potato.

Production and characterization of interspecific somatic hybrids between Primula malacoides and P. obconica

Mesophyll- or cell suspension-derived protoplasts of Primula malacoides cv ‘Lovely Tokyo’ were electrofused with cell suspension-derived protoplasts of P. obconica cv ‘Aalsmeer Giant White’ to produce somatic hybrids. Totally, 271 callus lines were obtained as a result of 11 independent experiments. Adventitious shoots were induced from these calli on gellan gum-solidified MS medium supplemented with 2 mg/l zeatin. The shoots were excised from each callus, rooted on medium containing 0.1 mg/l NAA, and transferred to plant growth regulator-free medium for establishing the plantlets. In total, 18 different lines of plants were confirmed to be hybrids through RAPD, flow cytometric and chromosome analyses. These results also suggest that the elimination of genetic materials or polyploidization had occurred in some of the hybrid plants obtained.

Production of male sterile interspecific somatic hybrids between transgenic N. tabacum (Bar) and N. rotundifolia (NPT II) and their identification by aflp analysis

A protoplast fusion experiment was carried out aiming to obtain somatic hybrid plants of transgenic Nicotiana tabacum (bar) (+) N. rotundifolia (npt II). The bialaphos resistance marker (bar) was introduced into N. tabacum via Agrobacterium tumefaciens using vector pGV1500 carrying the bar gene phosphinothricin acetyltransferase. N. rotundifolia (npt II) was recovered after direct gene transformation of protoplasts by the pGP6 plasmid carrying the npt II gene for neomycin phosphotransferase. Both plasmids possessed 35S CaMV promotors. Hybrid selection was based on dual bialaphos— kanamycin resistance. Amplified fragment length polymorphism (AFLP) analysis of regenerated plants showed the presence of species-specific bands for both parents, which confirmed their hybrid nature. N. tabacum (bar) (+) N. rotundifolia (npt II) hybrids exhibited a great diversity in morphology. Fertile hybrids which possessed N. tabacum or N. rotundifolia morphology were recovered. Flow cytometric analysis revealed that the N. tabacum- and N. rotundifolio-like hybrids had nuclear DNA contents near that of N. tabacum (9.40±0.24pg) or N. rotundifolia (5.29±0.36 pg), respectively, and were highly asymmetric. Other hybrids combined traits from the two species at various levels—N. tabacum habit or branched, similar to N. rotundifolia. Their leaves varied in shape. The flowers of the hybrid plants were of N. tabacum or N. rotundifolia type, or had N. rotundifolia dimensions, pink with N. tabacum corolla or white with curly fused petals. All were self-sterile or male sterile. The nuclear DNA content varied from 8.90±0.30 to 19.57±0.33 pg. The data from the morphological and cytological analysis provided vidence that parental chromosome elimination in the hybrid clones was spontaneous and not species-specific and that diploidization of the tobacco genome might have occurred in some clones during in vitro culture. This reflects the genomic incompatibility between the two species.

Characterization and expression of cold-induced glutathione S-transferase in freezing tolerant Solanum commersonii, sensitive S. tuberosum and their interspecific somatic hybrids

Glutathione S-transferases (GST) form a large family of non-photosynthetic enzymes known to function in detoxification of xenobiotics. We have cloned and characterized a novel, low temperature regulated GST, Solanum commersonii glutathione S-transferase ( Scgst1), from a cold acclimated wild potato species S. commersonii and studied the level of its transcription in freezing tolerant and sensitive Solanum genotypes. Active oxygen species (AOS) were associated with the early steps of Scgst1 regulation since a strong mRNA signal was detected in hydrogen peroxide and salicylic acid treated plants. In experimental conditions where the formation of AOS is known to accelerate, such as excessive light at low temperature, significant accumulation of the transcript was observed in S. commersonii. Under similar experimental conditions, Scgst1 transcript did not accumulate in freezing sensitive S. tuberosum eventhough a single copy of the Scgst1 sequence was present in both species. Thus, Scgst1 in the S. tuberosum genome did not exhibit the same cold-induction properties as in S. commersonii. In comparison with the parental lines, the somatic hybrid SH9A ( S. commersonii (+) S. tuberosum) had an interparental level of Scgst1 accumulation as well as freezing tolerance. The abundance of Scgst1 transcript thus correlated well with the freezing tolerance of the parental lines and the somatic hybrid SH9A. Increased GST enzyme activity was observed in S. commersonii and SH9A after 2 days of cold acclimation whereas the activity declined in S. tuberosum during the same period. Further studies of potato lines (S1) that were derived by selfing the somatic hybrid revealed a more complex relationship between freezing tolerance and Scgst1 expression level. In the S1 genotypes, the regulation of Scgst1 transcription resembled more that of S. tuberosum and was not directly related to their freezing tolerance. This could be due to the interaction of the two genomes in S1 genotypes as well as chromosomal rearrangements during meiosis.

Citrus somatic hybrid leaf essential oil

The essential oil from the leaves of a Citrus interspecific somatic hybrid obtained by symmetric protoplast fusion between ‘Milam’ lemon [purported sexual hybrid of C. Jambhiri Lush (×) C. sinensis] and ‘Femminello’ lemon (C. limon L. Burm f.) has been characterized by steam distillation GC–MS analysis and its composition compared with that of the corresponding oils obtained from the parent plants. The amount (%) of the different classes of substances present in the oils show, in the new hybrid, an overall lowering of the hydrocarbon fraction and an increase of the oxygenated components. The somatic hybrid shows a close similarity in the amounts of terpenes, sesquiterpenes and alcohols with the ‘Milam’ parent and in the amount of aldehydes with the other parent. Remaining oxygenated components appear lower in the hybrid with respect to both parents. Because of their genetic origins, the hybrid cells possess the nuclear genome of both parents and should potentially maintain all their genetic traits. However, the closer similarities found in the composition of the essential oils in the leaves of the somatic hybrid and the ‘Milam’ parent could be interpreted as the dominance, in the nucleus, of the genetic information from this parent, although possible interactions between the nucleus and the mitochondrial genome cannot be at present accounted for since the origin of the latter is uncertain. Copyright © 2000 John Wiley & Sons, Ltd.

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.

Expression of nonacclimated freezing tolerance and cold acclimation capacity in somatic hybrids between hardy wild Solanum species and cultivated potatoes

The expression of freezing tolerance was characterized in interspecific somatic hybrids between S. tuberosum (tbr) and two cold-hardy wild species, S. brevidens (brd) and S. commersonii (cmm). The nonacclimated freezing tolerance (NA) and acclimation capacity (ACC, increase in freezing tolerance in response to low nonfreezing temperature), two main genetic components of freezing tolerance, were evaluated separately. In contrast to cmm, which exhibited excellent NA and ACC, the freezing tolerance of brd was mainly due to ACC. However, the ACC of brd was only moderately expressed in the somatic hybrids. The NA of cmm was also suppressed in combination with tbr genomes. However, with acclimation, some of the tbr (+) cmm somatic hybrids achieved freezing tolerance comparable to pure hardy species such as brd used in this study. Analysis of chloroplast DNA type by RFLP markers revealed no significant difference in ACC between somatic hybrids carrying chloroplasts from either tbr or cmm. The reasons for the reduced expression of freezing tolerance from either the brd or cmm parent and the utilization of these somatic hybrids in breeding programs are discussed.

Generation of whole-chromosome painting probes specific to each chicken macrochromosome

The realization of physical and genetic maps of the chicken genome is dependent on progress in cytogenetic knowledge of its karyotype. To help achieve this goal, we constructed amplified representative DNA samples of the chicken chromosomes 1, 2, 3, 4, 5, 6, 7, 8, Z, W and of the terminal heterochromatin part of Zq by chromosome microdissection and DOP-PCR amplification. These chromosome DNA samples, which represent about 75% of the chicken genome, were used to generate whole chromosome painting probes for FISH. The direct application of these chromosome specific probes is dual FISH localization and characterization of panels of chicken interspecific somatic hybrids. We discuss some aspects of the chicken genome and its repeated sequences.

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

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

Production of somatic hybrids between Diospyros glandulosa and D. kaki by protoplast fusion

Interspecific somatic hybrids between Diospyros glandulosa (2n=2x=30) and D. kaki cv. Jiro (2n=6x=90) were produced by electrofusion of protoplasts. Protoplasts were isolated from calli derived from leaf primordia, fused electrically, and cultured by agarose-bead culture using a modified KM8p medium. Flow cytometry revealed that the nuclear DNA content was the sum of those of D. glandulosa and D. kaki cv. Jiro in 149 of the 166 calli obtained. RAPD analysis showed that the 149 callus lines yielded specific bands for both D. glandulosa and D. kaki cv. Jiro and further confirmed that they were interspecific somatic hybrid calluses. Shoots were regenerated from 63 of the 149 interspecific hybrid calluses. Chloroplast DNA analysis by PCR-RFLP, flow cytometric determination of nuclear DNA content, and RAPD analysis revealed that the 63 interspecific hybrid shoot lines contained the nuclear genomes from both parents but only the chloroplast genome from D. glandulosa. Microscopic observation of root tip cells demonstrated that somatic chromosome number of the interspecific hybrids was 2n=8x=120.