Quantitative analysis of the fate of exogenous DNA in Nicotiana protoplasts

Abstract

After a 5-hour incubation of protoplasts of Nicotiana tabacum L. ;Xanthi' with (3)H-DNA (7.26 mug/ml) from N. tabacum L. ;Xanthi nc' 3.5% of the initial radioactivity was found in acid-insoluble substances of the protoplasts. The addition of DEAE-dextran and poly-l-lysine to the incubation medium nearly doubled radioactivity adsorption. The absorption was inhibited by 2,4-dinitrophenol, KCN, and low temperature (0 C); this inhibition could not be reversed by exogenous ATP. About 500 tobacco plants established from protoplasts of a normally tobacco-mosaic virus-susceptible cultivar that had been allowed to absorb DNA prepared from a resistant cultivar did not show transfer of the virus-resistant gene.A detailed analysis was performed of the disposition of exogenous DNA in plant protoplasts, by employing Escherichia coli(3)H-DNA and Nicotiana glutinosa protoplasts. In 5 to 20 hours, about 10% of the (3)H-DNA entered the protoplasts. Competition experiments between the (3)H-DNA and unlabeled DNA or thymidine showed that the entry occurred as undegraded (3)H-DNA. Examination of intraprotoplast fractions revealed that 60 to 80% of the absorbed radioactivity resided in the "soluble" fraction of the cytoplasm and 20% in the nuclear fraction. The mitochondrion fraction also contained measurable radioactivity. Sizing on sucrose density gradients showed that the bulk of the absorbed E. coli DNA had been depolymerized. Of the incorporated radioactivity, 15% was accountable as DNA, exogenous as well as resynthesized, and 15% as RNA, protein, and other cell constituents. DNA/DNA hybridization test indicated that 17.6% of the re-extractable (3)H-DNA retained homology with the E. coli DNA; this was equivalent to 2.6% of the absorbed radioactivity. Resynthesized receptor protoplast DNA was represented by a fraction at least 1.7% of the total absorbed radioactivity. The amount of bacterial DNA remaining in protoplasts suggests that each protoplast retained 2.3 x 10(-15)g donor DNA, or approximately half of the E. coli genome.