PEG mediated DNA uptake and transient GUS expression in carrot, rapeseed and soybean protoplasts

Abstract Transient expression of PEG mediated DNA uptake was monitored in protoplasts of carrot ( Daucus carota L. cv. Apollo, Riso and Navona), rapeseed ( Brassica napus L. cv. Line) and soybean ( Glycine max L. cv. Mandarin) by measuring the GUS activity of a chimeric CaMV 35S-GUS reporter gene inserted in a 5.6-kb pBI 221.1 plasmid. Identity of the GUS enzyme (EC 3.2.1.31) was confirmed by isoelectric focusing. A time course study revealed that expression of the GUS gene could be detected 1.5 h after DNA uptake. Peak activity was obtained in rapeseed after 27 h and in carrot and soybean after 66–72 h. Optimal experimental conditions for protoplast density, PEG concentration and the order of DNA-PEG addition were analysed. GUS activity in transfected protoplasts was determined quantitatively by a spectrofluorimetric assay and qualitatively by X-Gluc staining. A comparison between the spectrofluorimetric assay and the number of blue cells per millilitre was conducted.