Abstract
We describe an optimized protocol for the transient transformation of tobacco protoplasts mediated by polyethylene–glycol (PEG). As expected, the quantitative β–glucuronidase (Gus) activity driven by pCaMVGus was dependent on the amount of plasmid used. Nevertheless, we demonstrate by an immunodetection method that transformation efficiency did not depend on the amount of plasmid used but on the limitation imposed by cell competence. In fact, we obtained the same percentage of transformed cells (about 60%) using a wide range of plasmid concentrations (0.1–10 μg per test). Finally, we show that, when we used two plasmid types in a mixture at a concentration ranging from 0.1 to 10 μg for each, all transformed cells expressed proteins encoded by both plasmids. Transient expression and co-transformation experiments are routinely used methods and, probably, the major results from this work were assumed by many researchers in this field, but our data experimentally support this assumption.
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Abbreviations
- AMCA :
-
Aminomethylcoumarin
- 6–BAP :
-
6-Benzylaminopurine
- CaMV35S :
-
Cauliflower mosaic virus 35S promoter
- Cat :
-
Chloramphenicol acetyltransferase
- DAPI :
-
4,6-Diamidino-2-phenylindole
- FDA :
-
Fluorescein diacetate
- GFP :
-
Green fluorescent protein
- Gus :
-
β-Glucuronidase
- MES :
-
2-[N-Morpholino]ethanesulfonic acid
- MUG :
-
4-Methyl-umbelliferyl-glucuronide
- ORF :
-
Open reading frame
- PEG :
-
Polyethylene glycol
- PS :
-
Plasmolysis solution
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Acknowledgements
We are grateful to the reviewers for their useful critical comments, corrections and suggestions. This research was supported by the Ministry of University, Scientific and Technological Research, Project 9807212169 (Constitutive vs. induced adaptation to stress in plants).
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Locatelli, F., Vannini, C., Magnani, E. et al. Efficiency of transient transformation in tobacco protoplasts is independent of plasmid amount. Plant Cell Rep 21, 865–871 (2003). https://doi.org/10.1007/s00299-003-0593-x
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DOI: https://doi.org/10.1007/s00299-003-0593-x