Development of Protoplast-Based Gene Editing System for Areca Palm

Areca palm (<i>Areca catechu</i> L.) is an economically significant crop in tropical and subtropical regions. However, an efficient transformation and gene editing system for genetic improvement has still not been established. In this study, protoplasts were isolated from juvenile leaves, followed by PEG-mediated transformation and gene editing targeting the areca palm <i>AcPDS</i> via the CRISPR/Cas9 system. High yield (9.08 × 10⁶ cells/g FW) and viability (91.57%) protoplasts were isolated successfully by digestion for 5 h in an enzyme solution. Transformation efficiency (11.85%) was obtained through PEG-mediated transformation (incubation for 30 min in the mixture containing 40% PEG-4000, 400 mM CaCl₂, 30 µg of plasmid DNA, and 100 µL of protoplasts). Furthermore, subcellular localization was established by the cotransformation of GFP and pNLS-mCherry in the protoplasts. Moreover, the editing efficiency (2.82%) of <i>AcPDS</i> using the CRISPR/Cas9 system was detected by Hi-TOM sequencing. This study established an efficient transformation and gene editing system based on protoplasts in areca palm, which will be beneficial for gene function verification and genetic improvement in areca palm and other tropical palm crops.