Development of super-infective ternary vector systems for enhancing the Agrobacterium-mediated plant transformation and genome editing efficiency

Abstract Agrobacterium-mediated transformation remains a cornerstone of plant biology, fueling advancements in molecular genetics, new genomic techniques (NGTs), and the biotech industry. However, recalcitrant crops and technical hurdles persist as bottlenecks. The goal was to develop super-infective ternary vector systems that integrate a novel salicylic acid-degrading enzyme, GABA, and ethylene-degrading enzymes, targeting the transformation of crops by neutralizing plant defense system on Agrobacterium. Firstly, both the effect and activity of introducing enzymes were validated in EHA105, an important Agrobacterium strain. Our study demonstrates that all ternary vector (Tv) system variants significantly enhance reporter expression in transient assays with Nicotiana benthamiana and Cannabis sativa. Specifically, incorporating a constitutive virG mutation with novel enzyme combinations increased GFP and RUBY expression in C. sativa by 5-fold and 13-fold, respectively. The Tv system, combined with a geminivirus replicon, markedly boosted GUS gene expression in tomato, enhancing genome editing efficiency. Notably, compared to controls, Tv-VS demonstrated up to 18-fold and 4.5-fold increases in genome editing efficiency in C. sativa and tomato, respectively. Additionally, stable transformation rates in tomato and Arabidopsis improved significantly, with Tv-VS showing a remarkable 2.5-fold increase in transformation efficiency compared to control strains. The research marks notable progress in Agrobacterium-mediated plant transformation. The innovative ternary vectors overcome plant defense mechanisms, enabling genetic manipulation in previously challenging plant species. This development is anticipated to broaden the applications of plant genetic engineering, contributing to advancements in crop genome editing.