Somatic Hybrid Clones Research Articles

Somatic hybrids between Solanum melongena and S. sisymbrifolium, as a useful source of resistance against bacterial and fungal wilts

By using electrofusion of mesophyll protoplasts, 4 clones of somatic hybrids were produced between Solanum melongena cv. Pusa purple cluster and Solanum sisymbrifolium, a sexually incompatible wild relative resistant to bacterial ( Ralstonia solanacearum) and fungal ( Verticillium dahliae) wilts. All hybrids were morphologically homogeneous, normal and intermediate between the parents. They set flowers and produced fruits with empty seeds. Their hybrid status was confirmed by examining isoenzymes and random amplified polymorphism DNA analysis. Chloroplast-specific-cleaved amplified polymorphism sequence markers provided evidence that the 4 somatic hybrids possessed chloroplast genome from S. sisymbrifolium. Flow cytometry and chromosome countings revealed that all selected hybrids were tetraploids (2n=4x=48 chromosomes). The parental origin of the hybrid chromosomes was determined by using the techniques of genomic in situ hybridisation. Each hybrid contained one complete set of chromosomes from both parents. Neither spatial separation and recombinant chromosomes nor translocation could be demonstrated during mitotic metaphase. In vitro tests for resistance, using suspensions of two strains of R. solanacearum (race 1 and race 3) and the filtrate of culture medium of one strain of V. dahliae revealed that S. melongena was highly susceptible, whereas S. sisymbrifolium had variable levels of resistance. All somatic hybrids tested showed good levels of resistance, intermediate between those of the parents.

Effect of UV dosage on somatic hybridization between common wheat ( Triticum aestivum L.) and Avena sativa L.

Protoplasts of Avena sativa L. (A) irradiated with ultraviolet light (UV) at an intensity of 300 μW/cm 2 for 0 (symmetric fusion), 30 s, 1 min, 2 min, 3 min, 5 min, respectively were fused by the PEG method with protoplasts of two cultivars of wheat (cv. Jinan 177 or cv. Hesheng 3). Two different sources of material were used for isolation of protoplasts of Jinan 177. Type one (T 1) originated from long-term subcultured suspensions which could not be regenerated to plants, while the other (T 2) derived from embryogenic calli was capable of regeneration. Protoplasts of cv. Hesheng 3 (T 3) also originated from embryogenic calli possessing high regeneration capacity. Regenerated clones were produced in all fusion combinations, but only those from combinations I B and I C (irradiating A. sativa for 1 and 2 min) differentiated to albinos. The clones were recognized as somatic hybrids in nature by cytological, isozyme, RAPD and 5S rDNA spacer sequence analysis. Genomic in situ hybridization (GISH) of somatic hybrid clones at mitosis always revealed the presence of recombined and intact A. sativa chromosomes in the cells. The number of intact chromosomes of donor A. sativa decreased, while the chromosome fragments translocated or inserted to wheat chromosome increased as the dosage of UV enhanced.

Restriction of measles virus RNA synthesis by a mouse host cell line: trans-complementation by polymerase components or a human cellular factor(s).

The mouse epithelial MODE-K cell line expressing human CD46 or CD150 cellular receptors was found to be nonpermissive for measles virus (MV) replication. The virus binding and membrane fusion steps were unimpaired, but only very limited amounts of virus protein and RNA synthesized were detected after the infection. In a minigenome chloramphenicol acetyltransferase assay, MODE-K cells were as able as the permissive HeLa cells in supporting MV polymerase activity. The restriction phenotype of MODE-K cells could be alleviated by providing, in trans, either N-P-L or N-P functional protein complexes but not by P-L complexes or individual N, P, and L proteins. Several human x mouse (HeLa x MODE-K) somatic hybrid clones expressing human CD46 were isolated and found to be either nonpermissive or permissive according to their human chromosomal contents. The MV-restricted phenotype exhibited by the MODE-K cell line suggests that a cellular factor(s) can control MV transcription, possibly by stabilizing the incoming virus polymerase templates.

An improved procedure for protoplast microelectrofusion and culture of Nicotiana tabacum intraspecific somatic hybrids : plant regeneration and initial proofs on organelle segregation

Somatic hybrids were produced between haploid Nicotiana tabacum L. cv. Petite Havana (wild type) and haploid streptomycin resistant (SR1) mutant by an improved version of microelectrofusion of preselected pairs of protoplasts and the culture of fusion products in a nurse culture. Resistance of diploid plants regenerated from 20 somatic hybrid clones was tested at low concentration of streptomycin in the light as well as at high concentrations of streptomycin in the dark. In two independent hybrid lines, plants resistant in the light but sensitive in the dark were found. The existence of this plant type indicates a segregation of chloroplasts and mitocondria in somatic hybrid clones. It is suggested that microelectrofusion of preselected pairs of protoplasts combined with a reliable nurse culture might be a good technique for controlled somatic hybridization, cell reconstitution and partial gene transfer to different plant species. It might also be used to follow and analyse organelle segregation in somatic hybrid clones. The possibility that mitochondria might be resistant to streptomycin in the SR1 mutant is also discussed.

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.

Primed in situ (PRINS) labelling with Alu and satellite primers for rapid characterization of human chromosomes in hybrid cell lines.

The primed in situ (PRINS) labelling method was developed as an alternative to classical cytogenetics and fluorescence in situ hybridization (FISH) for the characterization of interspecific somatic hybrids. Full karyotypes were performed by PRINS using Alu-specific primers to generate the painting of all human material associated with R-like banding. The representativity of individual human chromosomes was established using primers specific for discriminent alpha-satellite DNA sequences providing specific signals on the centromeres of the targeted chromosomes and corresponding spots in interphase nuclei. Using this methodology, a somatic hybrid clone was shown to be monochromosomal for the der(11) from a t(11;22) patient.

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.

Production and characterization of fertile somatic hybrids of eggplant (Solanum melongena L.) with Solanum aethiopicum L.

In order to produce fertile somatic hybrids, mesophyll protoplasts from eggplant were electrofused with those from one of its close related species, Solanum aethiopicum L. Aculeatum group. On the basis of differences in the cultural behavior of the parental and hybrid protoplasts, 35 somatic hybrid plants were recovered from 85 selected calli. When taken to maturity either in the greenhouse or in the field, the hybrid plants were vigorous, all rapidly overtopping parental individuals. The putative hybrids were intermediate with respect to morphological traits, and all of their organs were larger, particularly the leaves and stems. DNA analysis of the hybrids using flow cytometry in combination with cytological analysis showed that 32 were tetraploids, 1 hexaploid and 2 mixoploids. The hybrid nature of the 35 selected plants was confirmed by a comparison of the isoenzyme patterns of isocitrate dehydrogenase (Idh), 6-phosphogluconate dehydrogenase (6-Pgd) and phosphoglucomutase (Pgm). Chloroplast DNA (ctDNA) restriction analysis using Bam HI revealed that among the 27 hybrid plants analyzed, 10 had S. aethiopicum patterns and the 17 remaining hybrids exhibited bands identical with those of eggplant without any changes. All of the somatic hybrid plants flowered. Both parental plants had 94% stainable pollen, while the hybrids varied widely in pollen viability ranging from 30% to 85%. The somatic hybrids showed high significant variation in fruit production. Nevertheless, there was a tendency for low fertility to be associated often with S. aethiopicum chloroplast type and/or with an abnormal ploidy level, while good fertility was mostly associated with the tetraploid level and eggplant chloroplasts. Interestingly, 2 tetraploid somatic hybrid clones were among the most productive, yielding up to 9 kg/plant. As far as the fertility of the F1 sexual counterpart was concerned, only 2 fruits of 50 g were obtained. Hybrid fertility in relation to phylogenetic affinities of the fusion partners is discussed.

Regeneration of intergeneric somatic hybrid plants between Atropa belladonna L. and Hyoscyamus muticus L.

In the present experiments, cell suspension derived protoplast of Atropa belladonna L. (2 n = 72) var acuminata and protoplasts of an achlorphyllous mutant cell line of Egyptian henbane, Hyoscyamus muticus (2 n = 28) were fused to obtain intergeneric somatic hybrid plants. The somatic hybrid plants possessed variable chromosome complements. Those plants with a chromosome number of around 100 and with both parental genomes distinguished on the basis of chromosome size were easier to establish in the glasshouse. Plants with multiple complements (chromosome number = 178) were grossly aberrant types and very difficult to maintain. The work involved complementation at the intergeneric level within the same tribe and evidence of chromosome elimination of Atropa chromosomes was obtained. The somatic hybrid plants developed with intermediate types of leaves and altered characteristics reflecting expression of both the genomes. In one of the somatic hybrid clones, SH-15, the pentamerous orientation of the whorls of sepals, petals and anthers in both the parents was altered to a hexamerous form. Further, the parents possess a bilocular ovary and this was altered to a trilocular form in this somatic hybrid. Plants that have responded better in their development in the glasshouse had a near tetraploid chromosome number of H. muticus and a near haploid chromosome number of A. belladonna. The presence of complementary bands of esterases and peroxidases as studied in the line SH-15, affirmed the hybrid nature.

Verticillium Wilt Resistance in Eggplant, Related Solanum Species, and Interspecific Hybrids

Abstract No significant resistance to verticillium wilt (Verticillium dahliae Kleb) was found in 59 eggplant (Solanum melongena L.) accessions, in a sexual hybrid between S. melongena and S. integrifolium, or in eight somatic hybrid clones between S. melongena and S. sisymbriifolium. A relatively high degree of resistance was observed in accessions of S. aculeatissimum, S. scabrum, and S. sisymbriifolium. All 58 accessions of S. gilo tested were susceptible, as were all accessions of S. incanum, S. integrifolium, S. laciniatum, S. macrocarpum, S. mammosum, and S. nodiflorum.

Genetic behavior of somatic hybrids in the genus Nicotiana: N. otophora + N. tabacum and N. sylvestris + N. tabacum

Somatic hybrids have been produced between N. tabacum and two closely related species in the genus Nicotiana, N. otophora and N. sylvestris, to evaluate interclonal variation and genetic behavior of these hybrids. As with the previously reported N. nesophila+ N. tabacum somatic hybrids, we have detected variation for morphological and isoenzyme characters between somatic hybrid clones, despite stability of chromosome number. One clone of N. sylvestris+N. tabacum was marked by variation in leaf spot frequency. The inheritance of this unstable trait was monitored through two sexual generations. Transmission of the Su gene marker was monitored in self-fertilized and back-crossed progeny of the N. sylvestris+N. tabacum somatic hybrids. Segregation ratios were similar to those previously reported for amphiploid N. sylvestris x N. tabacum sexual hybrids.

Comparison of Nicotiana tabacum and Nicotiana nesophila hybrids produced by ovule culture and protoplast fusion

Somatic hybrids were produced between Nicotiana tabacum and N. nesophila, two species incapable of conventional sexual hybridization. Sexual hybrids, though, could be produced between these two species by using ovule culture only when N. nesophila was female. Clones of somatic hybrids were compared with sexual hybrids. Statistically significant variation was observed between clones, but not between sexual hybrids, for pollen viability, flower morphology, leaf morphology, and trichome density. As all clones of somatic hybrids have 96 chromosomes, the variability could not be explained by interclonal variation in chromosome number. Variation between somatic hybrids could be the result of cytoplasmic segregation or recombination, mitotic recombination or small chromosomal rearrangements prior to plant regeneration. Variation between clones could be exploited as these interspecies hybrids are now being used to incorporate disease resistance into cultivated tobacco.

Complementation and Dominant Expression of Amino Acid Analogue Resistance Markers in Somatic Hybrid Clones from Daucus carota After Protoplast Fusion

Summary Fusion of protoplasts from two cell lines of Daucus carota L. resistant to the amino acid analogues S(2-aminoethyl)-L-cysteine (AEC) or 5-methyltryptophan (5MT) produced large numbers of double-resistant clones on a selective medium containing AEC and 5MT. No colonies emerged from the extensive controls (including homologous fusion and co-culture), indicating the absence of cross-resistance and cross-feeding. Fusion products, exhibiting stable resistance to both inhibitors, were selected at various times after fusion and also after preculture of 70 days in non-selective or half-selective (either AEC or 5MT containing) media. Predominantly tetraploid chromosome numbers were found in several of the selected double-resistant lines. Suspension cultures of a selected double-resistant clone had similar growth rates in the presence and absence of both amino acid analogues. When a double-resistant culture was rechallenged in AEC+5MT containing medium after multiple passages in inhibitor-free or half-selective media, only slightly lowered growth rates were observed, indicating the stable maintenance of both resistance markers for at least 110 generations. These experiments indicate the complementation, following protoplast fusion, and dominant phenotypic expression of both amino acid analogue resistance markers provided by the two parental cell lines.

Genetic linkage studies of the human glycosphingolipid ?-galactosidases

The genetic linkage relationships of the human glycosphingolipid beta-galactosidases were determined using human--mouse somatic cell hybrids. A new method was devised for the estimation of human galactosylceramide, lactosylceramide, and GMI-ganglioside beta-galactosidase activities in the presence of their mouse counterparts, which takes advantage of the reproducible specific activity of lysosomal hydrolases under a given set of culture conditions and is based on differences in both pH optima and sensitivity to chloride ion. Human and mouse chromosomes were identified by their characteristic banding patterns obtained after quinacrine staining, and the optimum glycolipid beta-galactosidase activity was determined for three different substrates. A ratio was defined for each activity which was the specific activity at the human pH optimum divided by the specific activity at the mouse pH optimum. Linear regression analysis was used to test for concordant segregation between pH ratios for each enzyme and the frequency of occurrence of different human chromosomes in the man--mouse somatic hybrid clones. The results obtained from two independent series of hybrid clones indicated that human beta-galactosidase activities consistently segregated with human chromosome 12 in these somatic cell hybrids.