Mingjun Ma

Coconut genetic improvement remains challenging due to low regeneration efficiency and limited transformation success. We optimized major components of a gene gun-mediated transient transformation system and evaluated explant types to support future establishment of a stable transformation pipeline. Three reporter genes (eGFP, GUS, and RUBY) were compared in coconut callus, and eGFP was selected as the most suitable due to its strong and non-destructive fluorescence. Background interference in GUS staining was reduced by adjusting the methanol–GUS ratio to 4:10. Single-factor optimization using callus tissue identified 0.4 M mannitol, 300–500 μg gold particles, 1.5 μg plasmid DNA, a 6.5 cm target distance, and 7 MPa pressure as effective parameters for biolistic delivery. Among the callus types, spongy callus showed strong transient eGFP expression but displayed loose and watery morphology consistent with non-embryogenic callus. In contrast, crumbly and smooth callus exhibited compact structures previously associated with embryogenic competence, although transient expression levels were lower. Among differentiated tissues, germinated zygotic embryo plumules and distal young leaflets exhibited moderate transient expression, supporting their suitability as transformation targets. These findings provide practical guidance on reporter selection, parameter refinement, and explant choice for future establishment of an efficient genetic transformation system in coconut.

Areca palm (Areca catechu 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 AcPDS 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 AcPDS 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.