Characterization Of Somatic Hybrids Research Articles

Production and characterization of somatic hybrids between rice (Oryza sativa L.) and reed (Phragmites communis Trin.) obtained by protoplast fusion

Production and characterization of somatic hybrids between rice (Oryza sativa L.) and reed (Phragmites communis Trin.) obtained by protoplast fusion

Production and characterization of somatic hybrids between Solanum tuberosum L. and S. pinnatisectum Dun.

Interspecific somatic hybrids between the dihaploid Solanum tuberosum and the wild species S. pinnatisectum Dun. were produced via protoplast fusion. Protoplast isolation, electrofusion, culture of post-fusion products and regeneration of calli/shoots were undertaken following optimized protocols. Regenerants were characterized for hybridity, ploidy and resistance to Phytophthora infestans (Mont.) de Bery, causal fungal pathogen of late blight disease. From a total of 126 regenerated macrocalli, 12 somatic hybrids were confirmed by possessing species-specific diagnostic bands of their corresponding parents as revealed by RAPD, SSRs and cytoplasmic-DNA analyses. Tetraploid status of the 12 hybrids was determined using flow cytometry analysis. Intermediate phenotypes for leaf, flower, and tuber characteristics and high male fertility were observed in field-grown hybrid plants. Hybrids were highly resistant to foliage late blight based on field assessment for two seasons. In contrast, moderate level of resistance to foliage blight was observed in hybrids based on the detached leaf assay under laboratory conditions. Overall, somatic hybrids with moderate levels of resistance to foliage blight were identified, and these will be useful for in situ hybridization in potato breeding efforts.

Production and characterization of somatic hybrids between Brassica napus and Raphanus sativus

Intergeneric somatic hybridization between Brassica napus and Raphanus sativus was carried out to enrich gene pool of B. napus. Twelve somatic hybrids were produced via PEG-mediated protoplast fusion between B. napus and R. sativus. The hybridity was confirmed by morphological observation and molecular marker analysis. Hybrid progenies (BC1) were obtained via backcrosses with B. napus. Behaviour of R. sativus chromosomes in a B. napus background in the F1 and BC1 plants was revealed by genomic in situ hybridization (GISH). The potential of somatic hybridization to enrich the suitable gene pool for rapeseed breeding is discussed.

Intermediate fertile Triticum aestivum (+) Agropyron elongatum somatic hybrids are generated by low doses of UV irradiation.

We report the production and characterization of somatic hybrids between Triticum aestivum L. and Agropyron elongatum (Host) Nevishi (the synonym is Thinopyrum ponticum). Asymmetric protoplast fusion was performed between Agropyron elongatum protoplasts irradiated with a low UV dose and protoplasts of wheat taken from nonregenerable suspension cultures. More than 40 green plantlets were obtained from 15 regenerated clones and one of them produced seeds. The phenotypes of the hybrid plants and seeds were intermediate between wheat and Agropyron elongatum. All of the regenerated calli and plants were verified as intergeneric hybrids on the basis of morphological observation and analysis of isozyme, cytological, 5SrDNA spacer sequences and random amplified polymorphic DNA (RAPD). RFLP analysis of the mitochondrial genome revealed evidence of random segregation and recombination of mtDNA.

Characterization of Somatic Hybrids with Compatible and Incompatible Species by Protoplast Fusion in Genera Pleurotus (Fr.) P. Karst. and Ganoderma P. Karst. by RAPD-PCR Analysis

Characterization of Somatic Hybrids with Compatible and Incompatible Species by Protoplast Fusion in Genera Pleurotus (Fr.) P. Karst. and Ganoderma P. Karst. by RAPD-PCR Analysis

Characterization of Somatic Hybrids between Tetraploid Solanum tuberosum L. and Dihaploid S. acaule

Electrofusion was performed to produce somatic hybrids between Solanum tuberosum (2n=4x=48) and dihaploid S. acaule (2n=2x=24). Among the 18 regenerated plants obtained, 14 were found to be hexaploid hybrids based on RAPD analysis and cytological observation. In order to identify their cytoplasmic genome constitution, RFLP analysis of chloroplast and mitochondrial DNA was carried out. Six and eight hybrids were found to have S. tuberosum and S. acaule chloroplast genomes, respectively, suggesting that the chloroplast genomes of the somatic hybrids segregated randomly. In addition, two regenerated plants derived from the same callus had different chloroplast genomes, indicating that the segregation of the chloroplast genome occurred before shoot formation from callus. Concerning the mitochondrial genome constitution, 13 hybrids harbored the genome of S. tuberosum type and the others the S. acaule type genome. This mode of segregaion is obviously in favor of S. tuberosum mitochondrial genome. Regarding cytoplasmic genome segregation in somatic hybrids, the mitochondrial genome was not associated with the chloroplast genome. For the cytoplasmic genomes of both species, no hybrid was observed with recombinant or heterogeneous genome from both parents. In most of the somatic hybrids, some characters such as plant habit, shape of foliage and shape of tuber, were generally similar to those of S. tuberosum. The high level of resistance to potato virus X (PVX) detected in dihaploid S. acaule and somatic hybrids, suggests that the resistance to PVX is incorporated by fusion with S. acaule.

Characterization of somatic hybrids between Brassica napus and Eruca sativa using species-specific repetitive sequences and genomic in situ hybridization

To analyze inheritance and presence of donor DNA from Eruca sativa in progenies derived from somatic hybrids between Eruca sativa × Brassica napus, two E. sativa specific repetitive DNA sequences were isolated and characterized by DNA sequencing. One of the sequences, ESR64, showed 100% similarity with a part of the E. sativa rDNA intergenic spacer. In situ hybridization to metaphase chromosomes of E. sativa revealed six sites of hybridization, indicating that E. sativa has three rDNA loci. The other clone, ESR92, was shown to be a tandemly repeated element located close to the telomeres on at least ten chromosomes. Analysis of progenies derived from the somatic hybrids, using these two repetitive sequences as probes, revealed the presence of E. sativa DNA. Furthermore, cytogenetic analyses with genomic in situ hybridization (GISH), was performed, using differently labelled total DNA from the two parental species, in combination with a preannealing step to remove common sequences. These experiments showed that labelling in B. napus was restricted to the centromeric regions while a uniform distribution over the chromosomes was found in E. sativa. With the GISH technique it was also revealed that the somatic hybrid progeny contained one or two complete E. sativa chromosomes, but no intergenomic translocations could be detected.

Production and characterization of somatic hybrids between Arabidopsis thaliana and Brassica nigra

Somatic hybrids between the Brassicaceae species A. thaliana and B. nigra have been produced by fusion of intact A. thaliana protoplasts with irradiated B. nigra protoplasts. Sixty-six callus lines could be selected applying the hygromycin resistance present in B. nigra as selection criterion for fusion products. In regard to the total number of PEG-treated A. thaliana protoplasts, this corresponded to a putative fusion frequency of 1.5 × 10 −8, a frequency considerably lower than such obtained, applying the same protocol, by a fusion between two nearly isogenic lines of A. thaliana. Three hybrid callus lines permanently regenerated shoots. By PCR- and Southern-analysis the nearly symmetric character of two hybrid lines with an intermediate morphology, and the highly asymmetric character of one line with a B. nigra morphology could be proven.

Characterization of somatic hybrids of potato by use of RAPD markers and isozyme analysis

Characterization of somatic hybrids of potato by use of RAPD markers and isozyme analysis

Production and characterization of somatic hybrids between the Japanese radish and cauliflower

Somatic hybrids between the Japanese radish and cauliflower (Brassica oleracea) were produced by protoplast electrofusion in order to introduce clubroot disease resistance in the Japanese radish (Raphanus sativus) into Brassica crops. After electrofusion of iodoacetamide-treated cauliflower protoplasts with untreated radish ones, culture was performed under conditions, that allowed only cauliflower protoplasts to regenerate. Out of 40 regenerated plants, 37 were morphologically of a hybrid type and 3 of a cauliflower type. On the basis of isozyme and RFLP analysis, all of the hybrid-type plants tested proved to be true hybrids. Of the 10 true hybrids tested, 9 were found to contain chloroplasts similar to those found in the Japanese radish, while only 1 contained those of the cauliflower. Using two mitochondrial genes as probes, we were able to show that 3 hybrids contained mitochondria of the Japanese radish, with some modification, while 7 hybrids had either parental or new patterns. All of the hybrid-type plants showed resistance to clubroot disease as high as that found in the Japanese radish. Some hybrids were self-fertile. All of the self-fertile hybrids were found to contain 36 chromosomes, indicating that they were amphidiploids. In addition, a few seeds were obtained from a backcross of the self-fertile hybrids to both parents.

Selection and characterization of somatic hybrids between Lycopersicon esculentum and Lycopersicon peruvianum

Somatic hybrids of the cultivated tomato, Lycopersicon esculentum, and a wild species, L. peruvianum, were obtained by fusion of leaf protoplasts from both species in the presence of poly-ethylene-glycol (PEG) or in an electric field. The somatic hybrids were selected on the basis of kanamycin resistance of L. esculentum and the plant regeneration capacity of L. peruvainum. Chromosome counts in root tips and the determination of the number of chloroplasts in guard cell pairs revealed that the majority of these hybrids was tetraploid (2 n = 4 x = 48). The remaining hybrids were at the hexaploid level with chromosome numbers between 64 and 72. The hybrid nature of the regenerated plants was confirmed by analysis of isozyme markers and by their morphology. Most hybrids did flower and set fruits and seeds after selfing. According to RFLP analysis 6 out of the 10 hexaploid hybrids contained two genomes of L. esculentum and four genomes of L. peruvianum. One of these hexaploids had genomes of two different L. peruvianum genotypes and was therefore considered to be derived from a triple protoplast fusion. The hexaploid plants were less fertile than the tetraploids and more resembled L. peruvianum.

Establishment and characterization of somatic hybrids between human differentiated macrophages and mouse myeloma ns1 cells

Human macrophages obtained from circulating monocytes matured in vitro by culture for seven days in hydrophobic flexible teflon bags were successfully fused with murine myeloma NS1 cells. Six of 20 clones, selected for their adherence properties, were further studied. All possessed human chromosomes (mean number ranging from 4 to 14 depending on the clones studied), exhibited non-specific esterases (but no peroxidase activity) and expressed CD14 antigen and C3 receptors (but no MAX-1 antigen). Moreover, the hybridomas retained phagocytic activity and high interferon plus lipopolysaccharide-activable cytolytic activity against tumor cells.

Molecular, cytogenetic and morphological characterization of somatic hybrids of dihaploid Solanum tuberosum and diploid S. brevidens

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 2∶1 S. tuberosum∶S. 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.

Regeneration and characterization of somatic hybrids between Brassica napus and Diplotaxis harra

Stem cortex protoplasts of Brassica napus, inactivated with iodoacetate, were fused with cell suspension - derived protoplasts of Diplotaxis harra by polyethylene glycol (PEG) treatment. PEG at 15–18% (w/v) induced 5–9% heterofusions. The hybrid callus selection was based on morphology: D. harra calli were yellow and very fine, while B. napus protoplasts typically produced green, well defined calli. A number of green, large calli were selected after fusion experiments and flowering plants were regenerated from two of these callus lines. The morphological traits as well as cytological and biochemical analysis of four isoenzymes confirmed the hybrid nature of the regenerants.

Production and characterization of somatic hybrids between Solanum melongena L. and S. sisymbriifolium Lam.

Protoplasts of 6-azauracil (AU) resistant cell lines of Solanum melongena L. were fused with protoplasts of S. sisymbriifolium Lam. to create somatic hybrids between these sexually-incompatible species. Following fusion, colonies were selected which were capable of growth in medium containing 1mM AU. These colonies were placed on medium containing zeatin which had been shown to stimulate anthocyanin production during shoot organogenesis in tissue explants of S. sisymbriifolium but not in S. melongena. A total of 37 anthocyanin-producing colonies were identified from which 26 hybrid plants were regenerated. The morphological traits intermediate to those of the parents included: flower colour, leaf shape, and trichome density. Cytogenetic analysis revealed that all hybrids were aneuploids but their chromosome numbers were close to the expected number of 48. Isozyme analysis revealed that nuclear genes of both parents were expressed in the hybrids. In addition, isoelectric focussing of the large subunit of ribulose 1,5-bisphosphate carboxylase (Rubisco) provided evidence that each hybrid expressed only the S. sisymbriifolium chloroplast genome. All hybrids regenerated thus far have been sterile.