Generation of lachrymatory factor synthase-suppressed onion (Allium cepa L.) by Agrobacterium-mediated gene transfer for CRISPR/Cas9 genome editing.

INTRODUCTION: Lachrymatory factor, an irritating volatile compound that induces tearing, is produced when onion bulbs are cut or chopped. Lachrymatory factor synthase (LFS)-specific suppression in onion may reduce the lachrymatory effect and enhance flavor and health-functional properties. Genome editing enables precise modification of target genes in plant genomes. However, no reports targeting genes controlling onion-specific traits have been published. Therefore, we aimed to generate onion plants with reduced LFS activity via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (CRISPR/Cas9) genome editing.

METHODS: Calli induced from primary roots were transformed with Agrobacterium tumefaciens carrying expression cassettes for CRISPR/Cas9, guide RNA, green fluorescent protein (GFP), and hygromycin resistance; callus lines with a high frequency of stable GFP expression were selected as "elite callus lines" suitable for transformation. Cleaved amplified polymorphic sequence (CAPS), heteroduplex mobility assay (HMA), and Sanger sequencing confirmed mutations introduced into the LFS gene, and plants were regenerated from the confirmed LFS-edited callus lines.

RESULTS AND DISCUSSION: The LFS enzyme activity was lower in the leaves and bulbs of the LFS-edited plants than in those of control plants, while the LFS-edited plants exhibited severe growth abnormalities and failed to set seed, possibly due to long-term culturing to maintain the elite callus line. The present study demonstrates the feasibility of onion genome editing to modify a specific onion trait, namely, LFS activity. Our findings provide a basis for further development toward the ultimate goal of producing tear-free onions with enhanced health-functional properties.