Enhancing Maize Transformation and Targeted Mutagenesis through the Assistance of Non-Integrating Wus2 Vector.

Efficient genetic transformation is a prerequisite for rapid gene functional analyses and crop trait improvements. We recently demonstrated that new T-DNA binary vectors with /G418 selection and a compatible helper plasmid can efficiently transform maize inbred B104 using our rapid -mediated transformation method. In this work, we implemented the non-integrating () T-DNA vector method for -mediated B104 transformation and tested its potential for recalcitrant inbred B73 transformation and gene editing. The non-integrating (NIW) T-DNA vector-assisted transformation method uses two strains: one carrying a gene-of-interest (GOI) construct and the other providing an NIW construct. To monitor co-integration into the maize genome, we combined the maize expression cassette driven by a strong constitutive promoter with a new visible marker , which produces the purple pigment betalain. As a GOI construct, we used a previously tested CRISPR-Cas9 construct pKL2359 for gene mutagenesis. When both GOI and NIW constructs were delivered by LBA4404Thy- strain, B104 transformation frequency was significantly enhanced by about two-fold (10% vs. 18.8%). Importantly, we were able to transform a recalcitrant inbred B73 using the NIW-assisted transformation method and obtained three transgene-free edited plants by omitting the selection agent G418. These results suggest that NIW-assisted transformation can improve maize B104 transformation frequency and provide a novel option for CRISPR technology for transgene-free genome editing.