PEG MEDIATED DNA UPTAKE AND TRANSIENT GUS EXPRESSION IN CARROT, RAPESEED AND SOYBEAN PROTOPLASTS

Transient expression of PEG mediated DNA uptake was monitored in proto plasts of carrot (Daucus carota L. cv. Apollo, Riso and Navona), rapes eed (Brassica napus L. cv. Line) and soybean (Glycine max L. cv. Manda rin) 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 enzy me (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 a nd in carrot and soybean after 66-72 h. Optimal experimental condition s for protoplast density, PEG concentration and the order of DNA-PEG a ddition were analysed. GUS activity in transfected protoplasts was det ermined quantitatively by a spectrofluorimetric assay and qualitativel y by X-Gluc staining. A comparison between the spectrofluorimetric ass ay and the number of blue cells per millilitre was conducted.