The plasmid vector pGreenII is widely used to produce plant transformants via a process that involves propagation in Escherichia coli. However, we show here that pGreenII-based constructs can be unstable in E. coli as a consequence of them hampering cell division and promoting cell death. In addition, we describe a new version of pGreenII that does not cause these effects, thereby removing the selective pressure for mutation, and a new strain of E. coli that better tolerates existing pGreenII-based constructs without reducing plasmid yield. The adoption of the new derivative of pGreenII and the E. coli strain, which we have named pViridis and MW906, respectively, should help to ensure the integrity of genes destined for study in plants while they are propagated and manipulated in E. coli. The mechanism by which pGreenII perturbs E. coli growth appears to be dysregulation within the ColE1 origin of replication.