AgroGem: A Rapid and Scalable Transient Transformation System for Functional Genetics in Multiple Plant Species

Abstract

Plant genetic transformation technologies are essential for functional genomics and genome engineering in plants. While transient expression systems offer a rapid alternative to stable transformation, existing platforms are often constrained by low efficiency, technical complexity, and limited scalability. Here, we developed AgroGem, an efficient Agrobacterium-mediated transient transformation system utilizing a geminiviral replicon-based T-DNA vector for Arabidopsis and Brassicaceae species. AgroGem significantly outperformed existing transient approaches, including AGROBEST and protoplast-based assays, in CRISPR-mediated editing efficiency. Moreover, AgroGem recapitulated the mutation spectra and chromatin accessibility-dependent editing patterns observed in stable transformation across both Cas9 and Cas12a systems, indicating that it captures genome editing outcomes in native chromatin contexts. Leveraging this capability, we performed high-resolution profiling of CRISPR-induced mutation outcomes across a panel of DNA repair mutants and identified distinct repair signatures, including unexpected roles for KU80 and XRCC4 in regulating non-homologous end joining (NHEJ). AgroGem also supported bimolecular fluorescence complementation assays for protein-protein interaction studies in Arabidopsis and was readily adapted to plate-based formats for high-throughput applications. Together, these results establish AgroGem as a robust, scalable, and versatile platform for genome editing, DNA repair analysis, and functional genetics in plants.