Escherichia coli plasmid pBR313 insertion into plant protoplasts and into their nuclei

Abstract

Cowpea mesophyll protoplasts were shown to bind irreversibly up to 3% input radioactive pBR313 plasmid DNA after 15 min of contact. Maximum uptake occurred in the presence of 5 mM ZnSO4 and 5 microgram/ml poly-L-ornithine. Under these conditions about one half of the TCA precipitable radioactivity was associated with the nuclear fraction and behaved as linear plasmid molecules. These could not be chased from the protoplasts upon further incubation with unlabeled plasmid DNA. The presence of donor DNA within the nuclear fraction is most probably not due to an artifactual redistribution of adsorbed plasmid DNA. Prolonged incubation periods resulted in extensive degradation of plasmid in the incubation medium but little degradation occurred in the protoplasts. The donor DNA was not covalently associated with the protoplast nuclear DNA.