Development of new biotechnological tools to accelerate breeding in chestnut and hazelnut

The CRISPR/Cas9 technology is a potent tool for expediting plant breeding by precisely editing target genes. Its potential to enhance resilience to climate change by improving plant yield and quality is widely recognized. Traditional breeding methods are less effective for woody plants due to their lengthy biological and reproductive cycles. Hence, New Plant Breeding Technologies (NPBTs), especially the CRISPR/Cas9 system, offer a promising way to accelerate woody plant breeding. In Italy, chestnut and hazelnut are appreciated tree nut species, renowned worldwide for their high-quality fruits. However, limited genetic information are available for these species. Our study aimed to enhance genetic resources and expedite breeding through the application of NPBTs. For the first time, we tested the CRISPR/Cas9 technology on chestnut somatic embryos using the phytoene desaturase (pds) gene, which, when mutated, results in an albino phenotype. The editing efficiency was found to be satisfactory, with approximately 60% of shoots displaying the expected phenotype. Moreover, we explored the use of the CRISPR/Cas9 system as a ribonucleoprotein complex (RNPs) that acts directly on the target gene and is completely degraded, resulting in transgenefree individuals. This approach was applied both to chestnut and hazelnut protoplasts. In chestnut, we isolated protoplasts (4,500,000 protoplasts mL-1, 9 1 % viability) from Castanea sativa embryogenic calli. Calli were digested using an enzymatic solution containing 1% Cellulase R-10 and 0.5% Macerozyme R-10.Hazelnut protoplasts were obtained from somatic calli derived from thin layers of leaves of the Corylus avellana ‘Tonda Gentile Trilobata’ cultivar. The isolation process from hazelnut yielded 1,100,000 protoplasts mL-1 with 99% viability, using an optimized enzymatic solution (2% Cellulase R-10, 0.5% Macerozyme and 1% Pectinase from Aspergillus aculeatus). To validate transfection ability, we used a plasmid containing the Green Fluorescent Protein (GFP) marker gene. Furthermore, chestnut protoplasts were transfected with RNPs containing the gRNA designed for the pds gene. The editing efficiency ranged from 15 to 20%, consistent with data available in the literature. As for hazelnut, the transfection experiment with RNPs is ongoing, focusing on the Terminal flower 1 (tfl1) gene. Mutation of this gene is expected to induce early flowering in vitro, an intriguing characteristic for the development of new breeding programs.