Genome editing in almond: A CRISPR-based approach through hairy root transformation

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technology has revolutionized genome manipulation for crop enhancement, providing a powerful toolkit. However, the tissue culture and plant regeneration steps that are critical to the CRISPR/Cas editing framework are often challenging, especially in some woody plant species that exhibit substantial resistance to these procedures. To address this, we have developed an injection-based protocol for inducing hairy roots in almond ( Prunus dulcis , syn. Prunus amygdalus ), a species known for its recalcitrance to conventional transformation methods. Notably, the hairy root induction method also proved effective in almond x peach hybrids. To evaluate its utility for gene functional analysis, we combined the hairy root transformation system with CRISPR/Cas9 gene editing technology, targeting two transcription factor genes ( ERF74 and GAI ). Our efforts resulted in transformants with target knock-out, suggesting the potential of this genetic transformation technology as a valuable tool for future routine gene function studies in almond.