Optimization of Gene Gun-Mediated Transient Transformation and Explant Suitability in Coconut.

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.