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.
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Abbreviations
- BCIP:
-
5-bromo-4-chloro-3-indolylphosphate
- BNYVV:
-
beet necrotic yellow vein virus
- CaMV:
-
Cauliflower Mosaic Virus
- NBT:
-
nitroblue tetrazolium chloride
- NPT II:
-
Neomycin phospho transferase
References
Bouzoubaa S, Scheidecker D (1990) in: Koenig R (ed) Proceding of the first symposium of the international working group on plant viruses with fungal vectors, vol I, Eugen Ulmer GmbH & Co. Stuttgart, pp 45–48
Denecke J, Gosselé V, Botterman J, Comelissen M (1989) Methods in Molecular and Cellular Biology 1: 19–27
Grosset J, Meyer Y, Chartier Y, Kauffmann S, Legrand M, Fritig B (1990) Plant Physiol. 92: 520–527
Laemmli UK (1970) Nature 227: 680–685
Lepetit M, Ehling M, Gigot C, Hahne G (1991a) Plant Cell Reports 10: 401–405
Lepetit M, Ehling M, Chaubet N, Gigot C (1991b) Mol. Gen. Genet, in press
Otsuki Y, Takebe I (1969) Virology 38:497–503
Pröls M, Töpfer R, Schell J, Steinbiss HH (1988) Plant Cell Reports 7: 221–224
Quillet L, Guilley H, Jonard G, Richards K (1989) Virology 172: 293–301
Rottier PJM, Rezelman G, Van Kammen A (1979) Virology 92: 299–309
Spörlein B, Mayer A, Dahlfeld G, Koop HU (1991) Plant Science 78: 73–80.
Töpfer R, Pröls M, Schell J, Steinbiss HH (1988) Plant Cell Reports 7: 225–228
Veidt I, Bouzoubaa SE, Leiser R-M, Ziegler-Graff V, Guilley H, Richards K, Jonard G (1991) Virology 186: 192–200
Watanabe Y, Meshi T, Okada Y (1987) FEBS lett. 219: 65–69
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Communicated by H. Lörz
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Jung, JL., Bouzoubaa, S., Gilmer, D. et al. Visualisation of transgene expression at the single protoplast level. Plant Cell Reports 11, 346–350 (1992). https://doi.org/10.1007/BF00233363
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DOI: https://doi.org/10.1007/BF00233363