Optimized protoplast technology for highbush blueberry: a gateway to precision breeding

The highbush blueberry (Vaccinium corymbosum L.) is an economically important fruit crop and holds increasing commercial importance also in Italy, where it contributes to sustainable use of marginal areas.
While breeding and genetic improvement in blueberries has made extensive use of biotechnological approaches, the potential of protoplast technology associated with genetic engineering remains largely underexplored.
This technique, however, is particularly appealing due to its potential to enable DNA-free genome editing.
This study aimed to establish an optimized protocol for protoplast isolation and transfection, enabling future applications in DNA-free genome editing through direct delivery of CRISPR/Cas9 ribonucleoproteins (RNPs). Protoplasts were isolated from both leaves and calli of ‘Berkeley’; leaves were collected from micropropagated shoots, while calli were induced on WPM medium supplemented with TDZ (10 µM) and NAA (10 µM). The enzyme solution optimized for cell wall digestion contained Cellulase Onozuka RS (1%), Macerozyme R10 (0.5%) and Hemicellulase from Aspergillus niger (1.5%). Callus-derived protoplasts showed higher yield (1.63×10⁶ protoplasts mL^‑1) and purity than leaf-derived protoplasts.
PEG/Ca²⁺-mediated transfection was performed using the plasmid pAVA393 (GFP marker gene). Transfection efficiency exceeded 50% within 24 h, as confirmed by fluorescence microscopy, with transfected protoplasts maintaining spherical morphology and high viability.
Subsequent culture of transfected protoplasts in liquid medium facilitated early cell divisions within 7 days, with microcolony formation observed after 15 days.
This rapid regeneration response highlighted the suitability of highbush blueberry protoplast technology.
This study reports a valid protoplast isolation and transfection protocol for V. corymbosum L. and sets the stage for DNA-free genome editing approaches.
By overcoming the limitations of foreign DNA integration, this method aligns with regulatory requirements and reduces the risk of chimeric plants.
These advancements pave the way for precision breeding strategies in highbush blueberry, addressing challenges in recalcitrant woody crops and contributing to the development of novel cultivars with improved agronomic traits.

International Symposium on Biotechnological Tools in Horticulture

G. Vaia, V. Pavese, A. Moglia, V. Cristofori, C. Silvestri

Vaccinium corymbosum L., protoplast isolation, PEG-mediated transfection, GFP marker gene

https://doi.org/10.17660/ActaHortic.2026.1454.48