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  • 1
    Electronic Resource
    Electronic Resource
    An internal standard improves the reliability of transient expression studies in plant protoplasts (1991)
    Springer
    Plant cell reports 10 (1991), S. 401-405 
    Details
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transient expression of foreign genes introduced on a plasmid into isolated plant protoplasts is widely used to study the control of gene expression. Unfortunately, many experimental variables implicated in this technique are difficult or impossible to control, resulting in a disturbing degree of variability between otherwise identical experiments. We have studied the co-expression of two constitutively expressed genes located on the same plasmid. This has allowed us to identify the lot of plasmid DNA as an important source of variation, along with the protoplast lot. Plasmid DNA concentration was found to be of minor importance. Since the variation of expression level of the two genes was identical for the two genes in all experiments, we propose the use of an internal standard in all comparative transient expression studies, which allows the reduction of the variation between experiments by one order of magnitude.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00232611
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  • 2
    Electronic Resource
    Electronic Resource
    Protoplasts from cotyledon and hypocotyl of sunflower (Helianthus annuus L.): shoot regeneration and seed production (1992)
    Springer
    Plant cell reports 11 (1992), S. 632-636 
    Details
    ISSN: 1432-203X
    Keywords: Helianthus annuus ; Protoplasts ; Plant regeneration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Cotyledon and hypocotyl protoplasts of Helianthus annuus inbred line 47 302 bcd were embedded in alginate and plated on L4 medium (Lenée and Chupeau 1986). After one month, the calli were transferred on MSSH regeneration medium (Murashige and Skoog 1962; Schenk and Hildebrandt 1972) where they regenerated shoots (overall efficiency 10−2%). The shoots were elongated on B5 (Gamborg et al. 1968) medium first without hormones, then supplemented with GA3 and BAP (both 0.05 mg/l). In order to overcome the difficulty to induce rooting by classical methods, the elongated shoots were grafted on a sunflower rootstock. The grafted shoots produced flowers and seeds. Different factors have been shown to have an important influence on the capacity to regenerate shoots: the genotype, the physical culture conditions at the callus regeneration step (e.g. protoplasts embedded in alginate), and the media composition.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00236388
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  • 3
    Electronic Resource
    Electronic Resource
    Oat mesophyll protoplasts: their response to various feeder cultures (1990)
    Springer
    Plant cell reports 8 (1990), S. 590-593 
    Details
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Oat (Avena sativa L.) mesophyll protoplasts were recently demonstrated to be capable of dedifferentiation, repeated divisions, and colony formation. Since the development of oat mesophyll protoplasts is decisively influenced by the nature of the used feeder culture (species, variety and concentration), we conducted a systematic study of this parameter. Generally, graminaceous feeders promoted protoplast proliferation, while dicot species repressed protoplast divisions. The beneficial effect of those feeders that promote divisions was counterbalanced by a factor that causes necrosis. The correct balance between promotion of divisions or necrosis depended on the nature of the feeder and its plating density.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00270060
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  • 4
    Electronic Resource
    Electronic Resource
    Visualisation of transgene expression at the single protoplast level (1992)
    Springer
    Plant cell reports 11 (1992), S. 346-350 
    Details
    ISSN: 1432-203X
    Keywords: Beet Necrotic Yellow Vein Virus ; Chenopodium quinoa ; Coat protein ; Frequency of expression ; Nicotiana tabacum ; NPT II ; Protoplast printing
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts are currently used to study the expression of genes following transformation. Expression is followed on a population of protoplasts after total protein extraction by conventional western blotting or measure of the enzymatic activity of the transgenic protein. We describe here a new method, called protoplast printing, allowing easy detection of the fraction of cells expressing a certain protein within a population of protoplasts. It consists of immobilization of the protoplast proteins on a nitrocellulose filter, so as to retain the outlines of the cell, followed by immunological detection of the protein of interest. The only special requirement is an antibody specific for the protein. We have studied the expression of the BNYVV coat protein after electroporation of Chenopodium quinoa protoplasts with viral RNAs, and the expression of the NPT II gene in protoplasts isolated from transgenic tobacco plants as well as after direct transfer of plasmid DNA into tobacco protoplasts. In both cases — infection with viral RNAs and transformation with plasmid DNA — expressing and non-expressing cells can be distinguished as early as 12h after transfer of the transgenes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233363
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  • 5
    Electronic Resource
    Electronic Resource
    Transformation of sunflower (Helianthus annuus L.): a reliable protocol (1994)
    Springer
    Plant cell reports 14 (1994), S. 81-86 
    Details
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A reliable protocol for the transformation of cultivated sunflower (Helianthus annuus L.) has been established, based on microprojectile bombardment of half shoot apices in combination with Agrobacterium tumefaciens coculture. Transgenic shoots have been obtained from 5 inbred lines, although transformation efficiencies varied with the genotype. Plants expressing the transgenes could be recovered from up to 7% of the explants. A minority of plants was shown to be chimaeric for expression of ß-glucuronidase activity while most appeared to be uniformly transformed. Genetic segregation was 3∶1 for both ß-glucuronidase and neomycine phospho transferase in some plants, indicating that the respective mother plants were uniformly transformed. Integration of the foreign genes was also shown by Southern analysis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233766
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  • 6
    Electronic Resource
    Electronic Resource
    Somatic embryogenesis and organogenesis induced on the immature zygotic embryo of sunflower (Helianthus annum L.) cultivated in vitro: role of the sugar (1995)
    Springer
    Plant cell reports 15 (1995), S. 200-204 
    Details
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Immature zygotic embryos of sunflower constitute an experimental system where the change of a single key factor (sucrose concentration) conditions the in vitro morphogenesis to either organogenesis (87 mM sucrose) or somatic embryogenesis (350 mM sucrose). Experiments with a variety of culture media differing in the sugar type and concentration, as well as osmotic pressure, indicate that a minimal threshold level of both, sugar supply and osmotic pressure, are required for somatic embryogenesis, but not organogenesis, to occur. The nature of the sugar used, though, was less important.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00193720
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  • 7
    Electronic Resource
    Electronic Resource
    A simple and general plant tissue extraction procedure for two-dimensional gel electrophoresis (1987)
    Springer
    Plant cell reports 6 (1987), S. 77-81 
    Details
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A fast and simple extraction procedure of plant tissue for two-dimensional gel electrophoresis is presented. The procedure is especially useful for the extraction of plant cell suspension cultures, callus and other plant tissues having a high content of phenol oxidases, polysaccharides, polynucleotides, terpenoids and other substances interfering with isoelectric focusing. Due to the speed of the extraction procedure (about 20 min), large numbers of samples containing only milligram amounts of tissue can be easily processed. The simplicity of the method makes it particularly suitable for the extraction of radiolabeled tissues (35S, 32P). This method is perfectly compatible with silver staining, autoradiography and Western blotting analysis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00269745
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  • 8
    Electronic Resource
    Electronic Resource
    Callus formation from isolated sunflower (Helianthus annuus) mesophyll protoplasts (1989)
    Springer
    Plant cell reports 8 (1989), S. 226-229 
    Details
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Mesophyll protoplasts of the cultivated sunflower,Helianthus annuus, have been consistently found not to divide or regenerate calli, despite the efforts of several groups. In the present report, we describe the conditions for donor plant culture, protoplast isolation, and their culture that were suitable for repeated regeneration of green, nodular, vigorously growing calli from isolated sunflower mesophyll protoplasts. The best conditions for protoplast isolation employed the use of both CAYLA cellulase and CAYLA pectinase. Culture conditions were not much different from those established earlier for sunflower hypocotyl protoplasts. The most startling observation was the great variability of division frequencies between experiments even under strictly controlled, identical experimental conditions. This finding points to an important influence of a variable in the physiological state of the donor plant which is difficult to control.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00778539
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  • 9
    Electronic Resource
    Electronic Resource
    Expression of foreign genes in sunflower (Helianthus annuus L.) — evaluation of three gene transfer methods (1955)
    Springer
    Euphytica 85 (1955), S. 63-74 
    Details
    ISSN: 1573-5060
    Keywords: Agrobacterium tumefaciens ; electroporation ; particle gun ; polyethylene glycol ; regeneration ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Suitable sunflower tissues and cells were transformed either by direct gene transfer into protoplasts, particle bombardment, or Agrobacterium co-culture. While all techniques allowed efficient short-term or transient expression of the introduced gene(s) in the respective tissues, stable transformation was only observed after transformation with Agrobacterium. The latter technique was suitable for the production of transgenic callus from seedling cotyledons and occasional shoots with chimaeric expression of the transgene. Detailed analysis of the interaction of Agrobacterium with this explant showed that infection efficiency was critically dependent on the co-culture conditions, and that the preferentially-transformed cells were not the ones competent for regeneration.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023931
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  • 10
    Electronic Resource
    Electronic Resource
    Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.) shoot apices: transformation patterns (1996)
    Springer
    Molecular breeding 2 (1996), S. 329-338 
    Details
    ISSN: 1572-9788
    Keywords: chimaeric gene expression ; GUS expression ; Helianthus annuus ; meristems
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Apical segments of embryonic axes of sunflower (Helianthus annuus L.) embryos were submitted to co-culture experiments with a disarmed strain of Agrobacterium tumefaciens, harbouring a plasmid coding for the marker enzyme β-glucuronidase. The expression patterns of this marker were analysed at different developmental stages of the regenerated shoots. The results are consistent with the hypothesis that transformed shoots originate from transformation events that have occurred within the existing meristems. Two of the resulting chimaeric plants have been analysed in detail, and some representative gene integration patterns are presented.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00437911
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