Agrobacterium rhizogenes-mediated marker-free transformation and gene editing system revealed that AeCBL3 mediates the formation of calcium oxalate crystal in kiwifruit

The transformation and gene editing of the woody species kiwifruit are difficult and time-consuming. The fast and marker-free genetic modification system for kiwifruit has not been developed yet. Here, we establish a rapid and efficient marker-free transformation and gene editing system mediated by Agrobacterium rhizogenes for kiwifruit. Moreover, a removing-root-tip method was developed to significantly increase the regeneration efficiency of transgenic hairy roots. Through A. rhizogenes-mediated CRISPR/Cas9 gene editing, the editing efficiencies of CEN4 and AeCBL3 achieved 55 and 50%, respectively. And several homozygous knockout lines for both genes were obtained. Our method has been successfully applied in the transformation of two different species of kiwifruit (Actinidia chinensis 'Hongyang' and A.eriantha 'White'). Next, we used the method to study the formation of calcium oxalate (CaOx) crystals in kiwifruit. To date, little is known about how CaOx crystal is formed in plants. Our results indicated that AeCBL3 overexpression enhanced CaOx crystal formation, but its knockout via CRISPR/Cas9 significantly impaired crystal formation in kiwifruit. Together, we developed a fast maker-free transformation and highly efficient CRISPR-Cas9 gene editing system for kiwifruit. Moreover, our work revealed a novel gene mediating CaOx crystal formation and provided a clue to elaborate the underlying mechanisms.