Olive (Olea europaea L.) is a major perennial crop in the Mediterranean region with high economic and cultural importance. However, its genetic improvement is hindered by a long juvenile phase, high heterozygosity, and the low efficiency of conventional breeding and genetic transformation methods. Protoplast-based systems offer a versatile platform for transient gene expression, functional genomics, and DNA-free genome editing via ribonucleoprotein (RNP) delivery. Here, we report the optimization of a PEG/Ca²⁺-mediated protoplast transformation system in olive using a CaMV 35S-driven EGFP reporter construct and Cas9-GFP fusion protein. Efficient recovery of highly viable protoplasts from embryogenic callus was achieved through a modified enzymatic digestion protocol in combination with optimized osmotic stabilization. Following purification, protoplasts were transfected with the EGFP reporter plasmid and Cas9- GFP fusion protein using a PEG/Ca²⁺-mediated delivery system. Transfection conditions were systematically optimized to achieve high uptake of both DNA and RNPs while maintaining cell viability and integrity. Strong transient EGFP expression was readily detected by fluorescence microscopy 48–72 hours post-transfection, and high transfection efficiency was observed for both the CaMV 35S-driven EGFP reporter construct and the Cas9-GFP fusion protein. Moreover, the optimization of enzymatic digestion, osmoticum composition, and post-transfection culture conditions proved critical for enhancing transformation efficiency in this recalcitrant woody species. Transfected protoplasts were successfully cultured in liquid MS-based medium, leading to cell wall regeneration and the formation of microcalli. This optimized transformation system supports rapid functional gene analysis and precise genome editing approaches in olive, offering a valuable biotechnological tool for improving resilience against emerging pathogens such as Xylella fastidiosa and other environmental stresses.
Advancing olive biotechnology: an optimized protoplast platform for CRISPR/CAS9 genome editing to enhance pathogen resistance
BASHIR M. A.
;DE MARCHIS F.;BELLUCCI M.;MARIOTTI R.;MOUSAVI S.
2026
Abstract
Olive (Olea europaea L.) is a major perennial crop in the Mediterranean region with high economic and cultural importance. However, its genetic improvement is hindered by a long juvenile phase, high heterozygosity, and the low efficiency of conventional breeding and genetic transformation methods. Protoplast-based systems offer a versatile platform for transient gene expression, functional genomics, and DNA-free genome editing via ribonucleoprotein (RNP) delivery. Here, we report the optimization of a PEG/Ca²⁺-mediated protoplast transformation system in olive using a CaMV 35S-driven EGFP reporter construct and Cas9-GFP fusion protein. Efficient recovery of highly viable protoplasts from embryogenic callus was achieved through a modified enzymatic digestion protocol in combination with optimized osmotic stabilization. Following purification, protoplasts were transfected with the EGFP reporter plasmid and Cas9- GFP fusion protein using a PEG/Ca²⁺-mediated delivery system. Transfection conditions were systematically optimized to achieve high uptake of both DNA and RNPs while maintaining cell viability and integrity. Strong transient EGFP expression was readily detected by fluorescence microscopy 48–72 hours post-transfection, and high transfection efficiency was observed for both the CaMV 35S-driven EGFP reporter construct and the Cas9-GFP fusion protein. Moreover, the optimization of enzymatic digestion, osmoticum composition, and post-transfection culture conditions proved critical for enhancing transformation efficiency in this recalcitrant woody species. Transfected protoplasts were successfully cultured in liquid MS-based medium, leading to cell wall regeneration and the formation of microcalli. This optimized transformation system supports rapid functional gene analysis and precise genome editing approaches in olive, offering a valuable biotechnological tool for improving resilience against emerging pathogens such as Xylella fastidiosa and other environmental stresses.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
