PEG-mediated transformation and CRISPR/Cas9 gene editing of CnPDS in coconut protoplast

Abstract

Coconut (Cocos nucifera) is a vital economic and ecological crop widely grown in tropical regions. However, efficient transformation and gene editing system for genetic improvement has not yet been established in coconut. In this study, protoplasts were isolated from juvenile coconut plantlets using a gradient of enzyme concentrations, followed by PEG-mediated transformation and CRISPR/Cas9-based gene editing targeting the coconut phytoene desaturase ( CnPDS ) gene. Protoplast isolation resulted in a maximum yield of 6.1 × 10 6 protoplasts/gram of fresh weight (g FW) with a viability rate of 89.77 %, using an enzyme solution comprising 3 % Cellulase, 1.5 % Macerozyme, and 2 % Pectinase, incubated for 5 h at 60 rpm and 28°C in darkness. Optimized PEG-mediated transformation achieved an efficiency of 48.3 % via GFP observation, employing a mixture containing 40 % (w/v) polyethylene glycol (PEG-4000), 0.4 M CaCl 2 , 40 µg plasmid DNA, and 100 µL protoplasts with 1 min heat shock and 30 min incubation. Furthermore, we developed a gene editing system targeting the CnPDS gene, which achieved an editing efficiency of 4.02 % as determined by Hi-TOM sequencing. This is the first report of CRISPR/Cas9-mediated gene editing in coconut protoplast, establishing an efficient protoplast-based transformation and gene editing platform. When combined with ongoing efforts to regenerate plants from protoplasts, this platform offers a promising approach for the genetic improvement of coconut and other tropical palm species. • High yield (6.1 ×10 6 cells/g FW) and viability (89.77 %) protoplasts isolated successfully. • Transformation efficiency (48.3 %) obtained through PEG-mediated transformation. • Editing efficiency (4.02 %) obtained by CRISPR/Cas9 system targeting CnPDS gene.