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.
Pavese et al. (Wed,) studied this question.