RESET: a root-to-shoot excision system for editing in clonally propagated plants

Abstract Many plant species remain recalcitrant to transformation and regeneration, impeding the use of transgenic and gene editing technologies. We present a transformation, editing, excision, and regeneration system called RESET (Root Excision System for Efficient Transformation) that enables the somatic removal of all functional transgenes in the absence of sexual segregation. It is based on hairy root induction using rol genes from Agrobacterium rhizogenes, and heat-induced expression of both Cre recombinase that acts on lox recognition sites near the T-DNA borders, and the shoot-inducing morphogenic regulator genes WUSCHEL and ipt. Using poplar as a model clonal plant and whole genome sequencing, we demonstrate its effectiveness for producing non-chimeric edited and healthy transgene-free plants. Testing of construct permutations showed that the primary control point for shoot regeneration and excision was the nature of the flanking lox sites, where canonical loxP outperformed an engineered variant. Additional important components were the auxin-synthesis genes iaaH and iaaM, and the chromatin modifying gene 6b; these improved the efficiency of hairy root development and shoot regeneration from hairy roots, respectively.