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  • 1
    ISSN: 1432-2242
    Keywords: Cellular manipulation ; Micronucleus ; Microprotoplast ; Flow cytometry ; Chromosome-mediated gene transfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Subprotoplasts with a DNA content of less than the G1 level (microprotoplasts) were isolated from micronucleated cells of transformedNicotiana plumbaginifolia (‘Doba’ line resistant to kanamycin) and characterized cytologically as well as by flow cytometry and Feulgen microdensitometry. Micronuclei were induced upon treatment of the suspension cells with the anti-microtubule drug amiprophos-methyl (APM). Protoplasts were fractionated on a continuous iso-osmotic gradient of Percoll; this resulted in several visible bands. Flow cytometric analysis of fluorescein and nuclear DNA contents after staining with fluorescein and DAPI respectively showed that the main band contained mostly evacuolated, intact (sub)protoplasts. Microprotoplasts contained one or a few micronuclei surrounded by a thin rim of cytoplasm and an intact plasma membrane. A maximum of 40% of the microprotoplasts in the fraction just below the main band had a DNA content less than the G1 level, in other fractions this maximum was 20%. Some of these contained an amount equivalent to that of one or a few chromosomes. The application of microprotoplasts for chromosome-mediated gene transfer in plants is indicated.
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  • 2
    ISSN: 1432-2242
    Keywords: Single ; specific chromosomes ; Transgenes ; Microprotoplast fusion ; Monosomic additions ; Genomic in situ hybridization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Results are reported on the transfer of single, specific chromosomes carrying kanamycin resistance (KanR) and β-glucuronidase (GUS) traits from a transformed donor line of potato (Solanum tuberosum) to a recipient line of the tomato species Lycopersicon peruvianum through microprotoplast fusion. Polyethylene glycol-induced mass fusion between donor potato microprotoplasts containing one or a few chromosomes and normal recipient diploid L. peruvianum protoplasts gave several KanR calli. A high frequency of plants regenerated from KanR calli expressed both KanR and GUS, and contained one or two copies of npt-II and a single copy of gus. Genomic in situ hybridization showed that several microprotoplast hybrid plants had one single potato donor chromosome carrying npt-II and gus genes and the complete chromosome complement of the recipient L. peruvianum (monosomic additions). Several monosomic-addition hybrid plants could be regenerated within the short time of 3 months and they were phenotypically normal, resembling the recipient line. These results suggest that the transfer of single chromosomes is tolerated better than is the transfer of the whole donor genome. The unique advantages of microprotoplast fusion are discussed: these include the direct production of monosomic addition lines for the transfer and introgression of economically important traits in sexually-incongruent species, the construction of chromosome-specific DNA libaries, high-resolution physical mapping and the identification of alien chromosome domains related to gene expression.
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  • 3
    ISSN: 1432-2242
    Keywords: Key words Microprotoplast fusion ; Chromosome transfer ; Alien gene integration ; Chromosome identification ; Sexual transmission
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Results are reported on the integration sites and copy number of alien marker genes neomycin phosphotransferase II (nptII) and β-glucuronidase (uidA), introduced into diploid potato Solanum tuberosum through transformation by Agrobacterium tumefaciens. Also, the transgenic potato chromosomes 3 and 5 harbouring the nptII and uidA genes, which were transferred to tomato (wild species Lycopersicon peruvianum) by microprotoplast fusion, as revealed by genomic in situ hybridization (GISH), were identified by RFLP analysis using chromosome-specific markers. The data revealed three integration sites in the donor potato genome, each containing the uidA gene, and two also harbouring the nptII gene. Analysis of monosomic-addition hybrid plants obtained after microprotoplast fusion showed that each of these three integration sites is located on a different potato chromosome. The microprotoplast hybrid plants contained only the chromosomes that carried the selectable gene nptII. The data on sexual transmission of the donor potato chromosome carrying the uidA and nptII genes were obtained by analysing the first backcross progeny (BC1) derived from crossing a monosomic-addition hybrid plant to tomato (L. peruvianum). The glucuronidase (GUS) assay and PCR analysis using primers for the uidA gene indicated the presence of the potato chromosome in GUS-positive and its absence in GUS-negative BC1 plants. RFLP analysis confirmed sexual transmission of the potato chromosome carrying the nptII and uidA genes to the BC1 plants. A few BC1 plants contained the nptII and uidA genes in the absence of the potato additional chromosome, indicating that the marker genes were integrated into the tomato genome. The potential applications of the transfer of alien chromosomes and genes by microprotoplast fusion technique are discussed.
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  • 4
    ISSN: 1432-2242
    Keywords: Amiprophos-methyl ; Mass induction ; Micronuclei ; Intact specific chromosomes ; Gene transfer
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Mitotic dynamics and the kinetics of mass induction of micronuclei after treatment of Nicotiana plumbaginifolia cell suspensions with the spindle toxin amiprophos-methyl (APM) are reported. The addition of APM to suspension cells resulted in the accumulation of a large number of metaphases. The course of mitosis was strikingly different from normal. Metaphase chromosomes showed neither centromere division nor separation of chromatids. Single chromosomes and groups of 2 or more chromosomes were scattered over the cytoplasm. After 5–6 h of APM treatment, chromosomes decondensed and formed micronuclei. When treatment duration was increased, the frequency of cells with micronuclei as well as those showing lobed micronuclei increased. Similarly, with an increase in APM concentration the frequency of cells with micronuclei increased. After removal of APM, chromosome grouping disappeared, cells showing lobed micronuclei further increased and mitoses with doubled chromosome numbers appeared in the next cell division. Cytological observations and DNA measurements revealed that several sub-diploid micronuclei containing 1 or a few chromosomes can be obtained, and that flow cytometry can detect and sort out these micronuclei. The applications of micronuclei for genetic manipulation of specific chromosomes and gene mapping are indicated.
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  • 5
    ISSN: 1432-2242
    Keywords: Diploid Agrobacterium-transformed Solanum tuberosum ; Nicotiana plumbaginifolia ; Asymmetric somatic hybrids ; Biparental chromosome elimination ; Karyotypic analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Electrofusion was carried out between mesophyll protoplasts from the transformed diploid S. tuberosum clone 413 (2n=2x=24) which contains various genetic markers (hormone autotrophy, opine synthesis, kanamycin resistance, β-glucuronidase activity) and mesophyll protoplasts of a diploid wild-type clone of N. plumbaginifolia (2n=2x=20). Hybrid calli were obtained after continuous culture on selection medium containing kanamycin. Parental chromosome numbers, determined at 2 months after fusion, revealed hybrid-specific differences between the individual calli. On the basis of these differences three categories of hybrids were distinguished. Category I hybrids contained between 8 and 24 potato chromosomes and more than 20 N. plumbaginifolia chromosomes; category II hybrids had between 1 and 20 N. plumbaginifolia chromosomes and more than 24 potato chromosomes; category III hybrids contained diploid or subdiploid numbers of chromosomes from both parents. The hybrids were evenly distributed over the three categories. After a 1-year culture of 24 representative hybrid callus lines on selection medium the karyotype of 10 hybrids remained stable, whereas 8 hybrids showed polyploidization of the genome of one parent, together with no or minor changes of the chromosome numbers of the other parent. Six hybrids showed slight changes in the hybrid karyotype. The elimination of chromosomes of a particular parent was not correlated to their metaphase location. The processes of spontaneous biparental chromosome elimination leading to the production of asymmetric hybrids of different categories are discussed.
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  • 6
    ISSN: 1432-2242
    Keywords: Nicotiana plumbaginifolia ; Solanum tuberosum ; Hybrid cell suspension cultures ; Hybrid callus sublines ; Karyotypic modifications ; Segregation of transformation marker characters
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Several hybrid callus lines were produced through somatic hybridization between the diploid transformed Solanum tuberosum plant clone 413 (2n = 2x = 24) and a diploid wild-type plant clone of Nicotiana plumbaginifolia (2n = 2x = 20). The hybrid callus lines with subdiploid numbers of potato chromosomes were studied for karyotypic evolution as well as for segregation of the transformation marker characters (i.e. hormone autotrophy, opine synthesis, kanamycin resistance and β-glucuronidase activity). Initially, these hybrids (cultured in kanamycin-containing medium) expressed all of the transformation characters. Six callus lines were selected for the establishment of cell suspension cultures; two of these were also used to initiate sublines, one from single cells of a suspension culture, and the other from callus-derived protoplasts. The cell suspension cultures and the sublines were cultured in kanamycin-free medium. After prolonged culture, karyotypic analysis of the various cell suspension lines revealed independent evolution of both parental genomes. Out of the six suspension lines, four showed a considerably reduced number of potato chromosomes as compared to the original hybrid callus lines, whereas the karyotypes of the individual sublines generally reflected the karyotypic diversity of the original cultures. The fate of the marker characters in various suspension cultures and sublines revealed independent segregation of the markers of TL-DNA (hormone autotrophy) and vector T-DNA (kanamycin resistance and β-glucuronidase activity). Loss of the TR-DNA marker (opine synthesis) was observed only in combination with the simultaneous loss of the TL-DNA marker and the vector T-DNA markers. The results on segregation patterns of marker characters are discussed in the light of specific chromosome loss in the hybrid lines and gene linkage relationships.
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  • 7
    ISSN: 1432-2242
    Keywords: Key words Single ; specific chromosomes  ; Transgenes  ;  Microprotoplast fusion  ;  Monosomic additions  ;  Genomic in situ hybridization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Results are reported on the transfer of single, specific chromosomes carrying kanamycin resistance and β-glucuronidase (GUS) traits from a transformed donor line of potato (Solanum tuberosum) to a recipient line of the tomato species Lycopersicon peruvianum through microprotoplast fusion. Polyethylene glycol-induced mass fusion between donor potato microprotoplasts containing one or a few chromosomes and normal recipient diploid L. peruvianum protoplasts gave several calli. A high frequency of plants regenerated from calli expressed both and GUS, and contained one or two copies of npt-II and a single copy of gus. Genomic in situ hybridization showed that several microprotoplast hybrid plants had one single potato donor chromosome carrying npt-II and gus genes and the complete chromosome complement of the recipient L. peruvianum (monosomic additions). Several monosomic-addition hybrid plants could be regenerated within the short time of 3 months and they were phenotypically normal, resembling the recipient line. These results suggest that the transfer of single chromosomes is tolerated better than is the transfer of the whole donor genome. The unique advantages of microprotoplast fusion are discussed: these include the direct production of monosomic addition lines for the transfer and introgression of economically important traits in sexually-incongruent species, the construction of chromosome-specific DNA libaries, high-resolution physical mapping and the identification of alien chromosome domains related to gene expression.
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  • 8
    ISSN: 1615-6102
    Keywords: Transformed potato ; Spindle toxins ; Metaphase blocking ; Micronuclei ; Chromosome doubling
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The data on mitotic blocking, induction of micronuclei, and chromosome doubling after treatments of transformed potato suspension cells with three different anti-microtubule agents oryzalin, amiprophos-methyl (APM) and colchicine are reported. The fast growing cell suspension line 413 with high mitotic activity is used, which contains various T-DNA introduced genetic markers (kanamycin resistance, β-glucuronidase activity, hairy root phenotype, hormone autotrophy, opine production). When compared to colchicine (0.5–5.0 mM), oryzalin and APM (15–32 μM) arrested the cells in metaphase more efficiently, induced micronucleated cells at higher frequencies and yielded a greater number of micronuclei. Flow cytometric determination of nuclear DNA content in interphase cells and the analysis of chromosome numbers in mitotic cells after removal of the chemicals and subculture showed that oryzalin is the most efficient chromosome doubling agent followed by APM and colchicine in that order. The anti-microtubule properties of the spindle toxins and interrelationship of various cellular events are discussed in relation to the mechanisms and factors involved in mitotic blocking, micronucleation and chromosome doubling.
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  • 9
    ISSN: 1615-6102
    Keywords: Perennial ryegrass ; Isolated sperm cells ; Ploidy level ; Cytoplasmic constituents ; Nuclear state ; Intramembrane particles
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The cytoplasmic content and the distribution of intramembrane particles (IMPs) of the plasma membrane of isolated sperm cells of perennial ryegrass (Lolium perenne L.) have been characterized using flow cytometry, transmission electron microscopy, confocal scanning laser microscopy and freeze-fracture studies. The isolated haploid sperm cells contain a variety of cell organelles with the exception of microtubules. Proplastids and plastids with starch were observed, although only rarely. Vacuoles containing remnants of organelles and stacked lamellae of endoplasmic reticulum with cytoplasmic inclusions were observed frequently, indicating that autophagy takes place. The number of mitochondria varies from 11 to 26 with an average of 17. Generally, the nucleus has a lobed shape and displays various interphasic stages of chromatin condensation. The analysis of the number of mitochondria and the nuclear state did not show evidence of sperm cell dimorphism. The cytological variability observed, could be explained by differences in developmental stages already present in vivo at the moment of isolation. No correlation between the number of mitochondria and the nuclear cross-sectioned area and/or the condensation state of the chromatin could be found. The density of intramembrane particles of the plasma membrane on the exoplasmic fracture face is more than twice that on the protoplasmic fracture face. That is the opposite of what was found for sporophytic cells of perennial ryegrass. These results are discussed in relation to the potential use of these cells for biotechnology and developmental studies.
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  • 10
    Electronic Resource
    Electronic Resource
    Woodbury, NY : American Institute of Physics (AIP)
    Applied Physics Letters 69 (1996), S. 1562-1564 
    ISSN: 1077-3118
    Source: AIP Digital Archive
    Topics: Physics
    Notes: The thermal resistance for conduction normal to diamond layers only a few microns thick, deposited at temperatures of about 500 °C on silicon using microwave-plasma-assisted chemical vapor deposition (MPACVD) and electron cyclotron resonance enhanced MPACVD, is measured using a specific laser-heating method. A detailed analysis of the results obtained using this method allows the determination of the effective diamond–silicon boundary resistance and the resistance within the diamond layer independently of one another. The analysis shows that the effective boundary resistance is comparable to or even larger than the resistance within the thin diamond layers, and suggests that it depends on the grain dimensions at the interface. For grain sizes of 10 nm, an average effective boundary resistance of 2.4×10−7 m2 K/W is determined, whereas at 1.2×10−8 m2 K/W it is significantly lower for grains about an order of magnitude larger. The resistance within the diamond shows a similar qualitative behavior with respect to grain size. © 1996 American Institute of Physics.
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