I’m worried about the 35S promoter interfering with my promoter study.

And so you should be! The use of the 35S promoter driving the plant selection gene (hph for hygromycin resistance, for example) with its strong enhancer domains creates significant transcriptional activity with adjacent open reading frames. GUS activity is readily detectable from constructs with only a minimal promoter in front of the gusA or gusplus gene when the 35S promoter is used to drive the plant selection gene (which obviously is also inside the T-DNA borders).

We strongly recommend that anyone embarking on a promoter study NOT use the pCAMBIA1281Z, 1291Z, 1381Z & 1391Z as they stand. To get the 35S promoter driving the resistance gene away from your Promoter of Interest driving gusA/gusplus you need to use a co-transformation strategy where most transformants will have dual T-DNAs integrated into different regions of the genome.

Instead of using the “Z-vectors” directly you should modify them by first deleting the 35Spromoter-HygR-35Sterminator cassette by XhoI + BstXI digestion (with blunting) and subsequent self ligation. The resulting vector will have no plant selection gene, so you must use a second vector like pCAMBIA1200, 1300, 2200 or 2300 (whose T-DNA consists of not much more than a plant selection gene expression cassette – in this case, again, HygR) in a co-transformation strategy. To perform co-transformation you will need to transform the two vectors (your promoter cloned into a 35S-HygR deleted version of pCAMBIA1281Z, & say pCAMBIA1200) separately into A.tumefaciens (or another species capable of gene transfer, such as TransBacter) then grow them up separately (with hormone induction if necessary for your plant type) and mix them together immediately before applying them to the plant tissue. A one-to-one mixing ration is a sensible starting point but you may find that a higher proportion of transformants carrying both T-DNAs is obtained if you use a higher proportion of the strain carrying the plasmid with the non-selectable T-DNA. And although you can’t select for the T-DNA carrying the Promoter-of-Interest:gusA/gusplus expression construct, in many cases you will be able to screen putative transformants for GUS activity at any early stage without destroying the whole regenerating plantlet.

In our hands this approach in rice has yielded 15-30% of transformed plants which carry both the selection T-DNA and the non-selected T-DNA.