CRISPR/Cas9-mediated editing of PHYTOENE DESATURASE gene in onion ( Allium cepa L.).

Out of the total 617 co-cultivated calli, 21 (3.4%) regenerated shoots exhibited three distinct phenotypes: albino, chimeric, and pale green; in comparison to the wild-type non-transformed regenerated shoots. Total chlorophyll content was drastically reduced in albino shoots and significantly decreased in chimeric shoots. Out of the six gene PCR-confirmed regenerated shoots, two exhibited the albino phenotype due to insertions/deletions (InDels) and substitution-based mutations in and around the target sites. Deep amplicon sequencing revealed a significantly variable InDel frequency between two sgRNAs, ranging from 1.2% to 63.4%, along with a 53.4% substitution frequency. The mutation of the gene generated a visually detectable albino phenotype, thus confirming the successful editing of the gene. This is the first time a CRISPR/Cas9-mediated genome editing protocol has been successfully established in onion, with the gene serving as an example. This study will provide the necessary momentum for researchers to further basic and applied research on onions.