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  • 1
    Electronic Resource
    Electronic Resource
    PEG- and electroporation-induced transformation in Nicotiana tabacum: influence of genotype on transformation frequencies (1989)
    Springer
    Theoretical and applied genetics 78 (1989), S. 287-292 
    Details
    ISSN: 1432-2242
    Keywords: Direct gene transfer ; Electroporation ; Genotype influence ; Kanamycin resistance ; Nicotiana tabacum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Experimental parameters for direct gene transfer with recombinant DNA encoding neomycin phosphotransferase II (NPTII) under control of eukaryotic expression signals were established. The introduced gene was shown by the growth of transformants on media containing kanamycin, by genomic blotting and by assaying NPTII activity. Leaf protoplasts from three green genotypes of varieties xanthii and petit havanna, and from four plastome-encoded albino genotypes of Nicotiana tabacum were analyzed with respect to cell division kinetics and yield of kanamycin-tolerant colonies after direct gene transfer. No clear correlation was found between the time of onset of cell division and transformation frequency.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00288813
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  • 2
    Electronic Resource
    Electronic Resource
    Lipofectin: direct gene transfer to higher plants using cationic liposomes (1991)
    Springer
    Theoretical and applied genetics 83 (1991), S. 1-5 
    Details
    ISSN: 1432-2242
    Keywords: Cationic liposomes ; Direct gene transfer ; Electroporation ; Plant transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary It has recently been shown that lipofectin, a commercially available preparation of cationic liposomes is capable of animal and plant cell line transfection. Here, it is analyzed with respect to its toxicity for higher plant protoplasts and used for transient expression and stable transformation experiments with mesophyll protoplasts of Nicotiana tabacum and Nicotiana plumbaginifolia. Transient expression of the β-glucuronidase gene (GUS) under control of the CaMV-35S-promoter was lower than after introduction of the same gene by polyethylene glycol. By transferring the neomycin phosphotransferase gene (NPTII) and subsequent culture and regeneration under selection with kanamycin, stably transformed plants were recovered after using Lipofectin in various protocols with or without additional application of electroporation. Efficiencies of stable transformation were comparable to those achieved with PEG and/or electroporation. Confirmation of transformants included assaying the enzyme activity of the gene product, genomic blotting, and transfer of the resistant phenotype to the progeny produced from selfed primary transformants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00229218
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  • 3
    Electronic Resource
    Electronic Resource
    PEG-mediated plastid transformation: a new system for transient gene expression assays in chloroplasts (1991)
    Springer
    Theoretical and applied genetics 82 (1991), S. 717-722 
    Details
    ISSN: 1432-2242
    Keywords: Chloroplast transformation ; β-Glucuronidase ; Nicotiana plumbaginifolia ; PEG ; Transient expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Evidence is presented for the introduction of functional copies of the GUS-reporter gene with plastid regulatory signals into chloroplasts after treatment of Nicotiana plumbaginifolia leaf protoplasts with PEG. GUS-activity is found in cells derived from protoplasts treated with PEG in the presence of plasmids harbouring the GUS-gene under the control of plastid promoter and terminator signals (plastid-specific reporter gene constructions). The activity is maintained after chloroplast isolation and incubation with the protease thermolysin under conditions sufficient to completely remove the much higher transient nuclear/cytoplasmic expression of a GUS-gene carrying the CaMV 35S-promoter. Likewise, GUS-activity derived from a plasmid coding for the nuclear/cytoplasmic expression of the reporter gene with a plastid transit presequence is also maintained after these procedures. These results indicate that PEG-treatment is a suitable protocol by which to introduce DNA into chloroplasts for the study of transient gene expression.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00227316
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  • 4
    Electronic Resource
    Electronic Resource
    A cytokinin-sensitive mutant of the moss,Physcomitrella patens, defective in chloroplast division (1989)
    Springer
    Protoplasma 152 (1989), S. 1-13 
    Details
    ISSN: 1615-6102
    Keywords: Physcomitrella protonemata ; Chloroplast division ; Cytoskeleton ; Cell division abnormalities ; Microinjection ; Moss mutant ; Protoplasts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary An X-ray induced mutant (PC22) of the moss,Physcomitrella patens was analysed with respect to its morphology, physiology and suitability for microculture techniques. The mutant protonemata are defective in bud formation and in chloroplast division. As a consequence of the latter, giant chloroplasts are formed which disturb the development of the phragmoplast, the formation of regular cross walls, and cell division. Abnormal cross walls are rich in callose. The actin cytoskeleton was found to be less regularly developed in the mutant than in the wild type. Three-dimensional analysis of the microtubular arrangement with confocal laser scan microscopy demonstrates a close association between spindle- or phragmoplast- and “interphase”-microtubules. The deficiencies in chloroplast division and in bud formation can partly be compensated for by exogeneously applied cytokinin. The suitability of this particular developmental mutant for further studies was shown by regeneration of protoplasts in microculture and microinjection of the fluorochrome Lucifer yellow into the chloroplast.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01354234
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