Critical Evaluation of Electromediated Gene Transfer and Transient Expression in Plant Cells

Applied genetics can benefit from direct gene transfer in two important research areas: (1) transformation of protoplasts followed by regeneration procedures resulting in fertile stably transformed plants, and (2) gene transfer to plant protoplasts leading to a transient expression of the marker gene(s). In both cases, very simple plasmids have been used to develop improved techniques. In our laboratory, we preferred a construction made by Jeff Veiten which was designated pCAP 212 by Veiten and Schell (1985). This vector carries a dual plant promoter fragment from the Ti plasmid of Agrobacterium tumefaciens. It is fused, in both orientations: once to the neomycin phosphotransferase (NPT II) gene of the transposon Tn5, and secondly to the chloramphenicol acetyltransferase (CAT) gene. Expression of NPT II and CAT may be readily assayed in plant cells and tissue. Therefore, this vector is very useful for optimizing transformation procedures as well as for measuring transient gene expression. Additionally, we made all our experiments with protoplasts derived from a carrot suspension culture (exception: Fig. 6). The culture conditions (Hain et al., 1985) as well as the parameters of the enzyme assay were kept constant. Only in this way did a comparison of different techniques seem possible.