Efficiency of transient transformation in tobacco protoplast is independent of plasmid amount

We describe an optimized protocol for polyethylene-glycol (PEG)-mediated transient transformation of tobacco protoplasts. As expected, the quantitative â-glucuronidase (Gus) activity driven by pCaMVGus was dependent on the used amount of plasmid. Nevertheless, we demonstrate by immunodetection method that transformation efficiency did not depend on the used amount of plasmid but on the limitation imposed by the cell competence. In fact, we obtained the same percentage of transformed cells (about 60%) using a wide range of plasmid concentrations (0.1-10 ìg/test). Finally, we showed that, when we used two plasmid types in a mixture at a concentration ranging from 0,1 to 10 mg 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, however our data experimentally support this assumption.