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  • 1
    ISSN: 1432-2242
    Keywords: Hybrid identification ; Molecular evolution ; Repetitive DNA elements ; Satellite DNA ; Solanaceae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Species-specific repetitive DNA probes are a useful tool for the molecular identification of somatic hybrids. Therefore, the distribution of three repetitive DNA elements of Solanum was investigated in Solanum wild species, Solanum breeding lines, and in more distantly related species of the genera Lycopersicon, Nicotiana, and Datura. The clone pSCH15, obtained from S. circaeifolium, represents a new 168-bp repetitive element; it shows 73–79% sequence similarity to repetitive elements of S. brevidens and Lycopersicon species. The 163-bp element in pSBH6, cloned from S. bulbocastanum, turned out to be very similar (95% sequence homology) to the Lycopersicon element pLEG15/TGRI previously regarded to be present only in species of the genus Lycopersicon and in S. lycopersicoides. Lower sequence similarity of approximately 80% was observed to repetitive elements of S. brevidens which are organized differently. The repeats exhibited different degrees of specificity: by Southern hybridization the element represented by the clone pSBH6 could be detected in almost all Solanum species investigated here but only after long exposure to X-ray film. The previously described “Solanum-specific” element represented by the clone pSA287 was also found, although in a very low copy number, in Lycopersicon esculentum. Therefore, detection of the repetitive elements pSA287 and pSBH6 in those species in which the respective repeat is less represented depends on exposure time. In contrast, the element pSCH15 is prominently present only in a small number of Solanum wild species and — to some extent — in the diploid breeding lines as revealed after long exposure. Use of these repeated elements for the identification of specific genomes in protoplast-fusion hybrids between Solanum wild species and Solanum breeding lines, or between two breeding lines, was evaluated.
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  • 2
    ISSN: 1432-2242
    Keywords: Key words Asymmetric somatic hybrids ; Flowcytometry ; RFLP ; Solanum tuberosum ; Wild species
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The objective of this study was to evaluate the suitability of different techniques for a simple and rapid identification of asymmetric hybrids, without the use of selection markers and independent of the fusion partners used. Additionally, the degree of donor DNA elimination was determined. Among 473 viable plants obtained from asymmetric fusion experiments between three di-haploid breeding lines of potato (Solanum tuberosum) and diploid wild species (S. bulbocastanum, S. circaeifolium; X-ray treatment of the wild species) the most promising ones were investigated with three different methods: flow cytometry, RFLP analysis with an oligonucleotide probe (GATA)4, and with single-copy probes. Flow cytometry, which combines a high screening capacity with detailed information about the DNA content and allows a distinction between asymmetric hybrids and chimeras, detected 31 hypo-tetraploid and 42 hypo-hexaploid regenerates among 224 plants. With the oligonucleotide probe (GATA)4 only a few asymmetric hybrids were detected among all regenerates. More than 50% of these asymmetric regenerates were chimeras. Concerning the degree of DNA elimination, the results obtained by RFLP analysis with 17 single-copy probes were correlated with the results obtained by flow cytometry. The maximum DNA elimination of the donor genome was 52%. As a trend, an irradiation dosage of 210 Gy caused a higher DNA elimination in the wild species than a dosage of 70 Gy. No calli were obtained after irradiation of the wild species with 420 Gy.
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  • 3
    ISSN: 1432-2242
    Keywords: 5S rDNA ; Evolution ; Protoplast fusion ; Somatic hybrids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The nucleotide sequences of the 5S rRNA genes (5S rDNA) of two Solanum tuberosum breeding lines (R1 and B15) and of the Mexican wild species S. pinnatisectum were determined and compared with each other and to the 5S rDNA of other Solanaceae species (Lycopersicon esculentum, Nicotiana rustica and Petunia hybrida). The 5S rDNA repeats of the Solanum species are 324–329 bp in length, and they exhibit 91–95% sequence identity. Sequence variability is mainly located in a short region of the spacer separating the 5S rRNA coding regions. A synthetic 28-mer oligonucleotide constructed according to this region can be used as a specific hybridization probe to distinguish symmetric somatic hybrids between S. tubersosum breeding line B15 and S. pinnatisectum produced by protoplast fusion. Interestingly, the two Solanum breeding lines R1 and B15 differ also in this spacer region.
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  • 4
    ISSN: 1432-2242
    Keywords: Flow cytometry ; Protoplast fusion ; RFLP ; Species-specific repetitive elements
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Interspecific somatic hybrids between the 1EBN-wild species Solanum pinnatisectum (S. pnt) and four different diploid breeding lines of Solanum tuberosum (S. tbr) were produced by electrofusion. S. pnt exhibits resistance to Phytophthora infestans and Erwinia blackleg. Somatic hybrids were identified by RFLP analysis using the oligonucleotide (GATA)4 as a probe. In three of four combinations all regenerates obtained were somatic hybrids. All 86 somatic hybrids between the breeding line H256/1 and S. pnt were analyzed in detail with respect to morphological and molecular characters; 50% of the somatic hybrids showed normal intermediate leaf morphology. Tubers of somatic hybrid plants grown in the greenhouse as well as in the field were evenly shaped and remarkably similar to those of the S. tbr breeding line. Analysis of relative DNA content by flow cytometry revealed that 75% of the somatic hybrids were tetraploid, some were hypotetraploid and others polyploid or mixoploid. Slotblot and RFLP analyses were carried out using repetitive and some single-copy DNA probes. The genome portion of the S. tbr breeding line was determined by slot-blot analysis using the species-specific repetitive probe pSA287. Obviously, most somatic hybrids contain the complete genomes of both fusion partners. In some of the somatic hybrids, a significantly lower intensity of the S. pnt-specific hybridization signal indicated a certain degree of asymmetry.
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  • 5
    ISSN: 1432-2242
    Keywords: Key words  Flow cytometry ; Protoplast fusion ; RFLP ; Species-specific repetitive elements
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract   Interspecific somatic hybrids between the 1EBN-wild species Solanum pinnatisectum (S. pnt) and four different diploid breeding lines of Solanum tuberosum (S. tbr) were produced by electrofusion. S. pnt exhibits resistance to Phytophthora infestans and Erwinia blackleg. Somatic hybrids were identified by RFLP analysis using the oligonucleotide (GATA)4 as a probe. In three of four combinations all regenerates obtained were somatic hybrids. All 86 somatic hybrids between the breeding line H256/1 and S. pnt were analyzed in detail with respect to morphological and molecular characters; 50% of the somatic hybrids showed normal intermediate leaf morphology. Tubers of somatic hybrid plants grown in the greenhouse as well as in the field were evenly shaped and remarkably similar to those of the S. tbr breeding line. Analysis of relative DNA content by flow cytometry revealed that 75% of the somatic hybrids were tetraploid, some were hypotetraploid and others polyploid or mixoploid. Slot-blot and RFLP analyses were carried out using repetitive and some single-copy DNA probes. The genome portion of the S. tbr breeding line was determined by slot-blot analysis using the species-specific repetitive probe pSA287. Obviously, most somatic hybrids contain the complete genomes of both fusion partners. In some of the somatic hybrids, a significantly lower intensity of the S. pnt-specific hybridization signal indicated a certain degree of asymmetry.
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  • 6
    ISSN: 1432-2242
    Keywords: Key words Asymmetric hybrids ; Solanum tuberosum ; Wild species ; Genome stability ; RFLPs
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The objective of this study was to investigate if in asymmetric protoplast fusion experiments the ploidy of the recipient line (di-haploid and tetraploid) has an influence on the extent of the asymmetry of the regenerating fusion products. Nineteen different experiments with the wild species Solanum bulbocastanum and Solanum circaeifolium as donors (irradiated with 210 Gy) and different breeding lines (di-haploid and tetraploid) were carried out. The degree of genome elimination was determined by measuring the relative DNA content using flow cytometry. The data showed that the loss of DNA in hybrid plants was significantly higher for 4x, compared to 2x, plants as recipients. In addition, the stability of asymmetry in the fusion products was studied. For this purpose differences in asymmetry in individual shoots originating from the same callus were analysed. A large variation in the DNA content of individual shoots was detected. Of the 4x to 6x shoots 44% had the same DNA content as another shoot originating from the same callus, 19% had a DNA content between 4x and 6x but different from any other analysed shoot originating from the same callus, 2% were chimeras and 35% had a completely different DNA content (eutetraploid, euhexaploid, eupolyploid or asymmetric with a ploidy level above 6x). RFLP-analysis with single-copy probes of 12 regenerates from six calli (two regenerates per callus) confirmed the assumption that the different regenerates of one callus originate from the same single cell. The analysis of selected regenerates cultivated for a period of more than 1 year demonstrated that the genome of asymmetric regenerates might change during cultivation.
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  • 7
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The isolation of six mutants of Agrobacterium tumefaciens which can induce tumors at low temperatures (22°C) but fail to do so at 28°C is described. At the nonpermissive temperature the following characteristics of the mutants are the same as those of the wild type: growth rates in vitro, growth rates in planta, and sensitivity towards agrocin 84, a marker for the presence of the virulence-plasmid. The tumors induced by the mutants at low temperature grow without addition of hormones at both 22°C and 28°C. The induction of the tumors but not the maintenance of the tumorous phenotypes are affected in the mutants isolated.
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  • 8
    ISSN: 1617-4623
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The technique of polyacrylamide gel electrophoresis was applied to demonstrate interaction between proteins of different strains of tobacco mosaic virus. The advantages and limitations of the method for such studies have been indicated. The results are discussed in connection with the role of primary structure of TMV-proteins for mixed aggregations.
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Planta 135 (1977), S. 177-181 
    ISSN: 1432-2048
    Keywords: Cell division ; Cell wall regeneration ; Cellulose synthesis ; Nicotiana ; protoplast culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Addition of cellulose to the culture medium inhibited cell wall regeneration and nuclear and cell division in tobacco (Nicotiana tabacum L.) mesophyll protoplasts. When the activity of celllase was inhibited by cellobiose, the inhibition of nuclear and cell division was overcome. Cell wall formation thus appears to be a prerequisite for nuclear and cell division in tobacco protoplasts.
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