Shockwave-induced DNA-free genome editing in tobacco: targeting the actin depolymerizing factor gene increases drought and salinity tolerance

Abstract DNA-free genome editing involves the direct introduction of ribonucleoprotein (RNP) complexes into cells, but this strategy has rarely been successful in plants. Here we describe a new technique for the introduction of RNPs into plant cells involving the generation of cavitation bubbles using a pulsed laser. The resulting shockwave achieves the efficient transfection of walled cells in tissue explants by the creation of transient membrane pores. RNP-containing cells were rapidly identified by fluorescence microscopy, followed by regeneration and the screening of mutant plants by high-resolution melt analysis. We used this technique in tobacco to target the endogenous phytoene desaturase (pds) and actin depolymerizing factor (adf) genes. Genome-edited plants were produced with an efficiency of 5.6–8.7%. We also evaluated the effects of adf mutations in T2 mutant plants under drought and salinity stress, showing that adf acts as a key regulator of osmotic stress tolerance in plants.