Strain-dependent stability of pGreenII-based ternary construct in <i>Escherichia coli</i>

Binary T-DNA plasmids have been used extensively in plant transformation, but their large size negatively impacts their use in construct preparation. Smaller ternary T-DNA vectors were developed, enhancing construct cloning efficiency, but their stability in Escherichia coli can be compromised, resulting in the rearrangement of constructs and chromosomal recombination events. Lowering the incubation temperature of transformed E. coli improves the stability of such ternary vectors but extends the duration. In this work, we compared the stability of a ternary pGreenII-based vector in commercially available E. coli strains JM109, DH5 alpha, and XL10-Gold. The pGreenII vector was used to prepare a construct (pGNK-LeCas9-AtU6psgRNA) containing tomato-codon-optimized Cas9 (LeCas9) and a potato apoplastic invertase inhibitor-specific synthetic guide RNA (sgRNA) cassettes under the control of constitutive promoters. The stability of this large vector was studied using different E. coli strains and incubation temperatures. Our results showed that the pGNK-LeCas9-AtU6psgRNA vector was stable in the XL10-Gold strain, while JM109 and DH5 alpha were not suitable. In addition, incubating the E. coli strains below 30 degrees C was the optimum temperature for the pGNK-LeCas9-AtU6psgRNA. Our study demonstrates that the stability of pGreenII-based ternary T-DNA vectors can be improved by selecting the right strain, like XL10-Gold.