First insights into transgene-free genome editing in European hazelnut protoplasts: a step forward in hazelnut breeding

New Plant Breeding Techniques (NPBTs) are emerging as innovative tools for plant breeding, with the CRISPR/Cas9 system being a valuable method for accelerating genetic improvement. Currently, the combination of the CRISPR/Cas9 system with protoplast technology offers a promising approach to producing transgene-free edited plants. In this study, we established the first protoplast isolation protocol in Corylus avellana L., cv ‘Tonda Gentile Trilobata’. Friable calli were obtained from in vitro-derived leaves cultured on a callus induction medium based on a modified Murashige and Skoog medium with half-strength macroelements, supplemented with 0.2 mg/l benzylaminopurine (BAP) and 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4D). For cell-wall digestion, the solution contained Cellulase Onozuka R-10 (2%), Cellulase from Aspergillus niger (2%), Macerozyme R-10 (0.5%), and Pectinase from Aspergillus aculeatus (1%) was used; the concentration obtained was 1,111,000 protoplasts/ml with 99% viability. Among the purification methods tested, filtration through nylon sieves with washing steps (P1 method) provided the best balance between purity and yield, resulting in 1,008,800 protoplasts/ml. We also developed a protoplast transfection protocol using the CRISPR/Cas9 ribonucleoprotein system, designed to edit the phytoene desaturase (pds) gene. Protoplasts were also cultured on semi-solid media, resulting in microcalli formation after 60 days. The microcalli emerged on a modified MS medium with half-strength NH4NO3 and KNO3 (MS1B), supplemented with 0.1 mg/l BAP and 0.01 mg/l 2,4-D. This study represents the first detailed protoplast isolation and transfection protocol from somatic tissues in hazelnut, providing the basis for future breeding approaches in this plant species.