Development of biotechnological tools for hazelnut breeding

Corylus avellana L. is an economically important tree nut species, highly requested by the confectionery industry. In recent years, the surface area planted to this crop has strongly increased but cultivation still relies on a relatively small number of traditional cultivars. The need for improved cultivars in woody species requires innovative plant breeding strategies to shorten the selection time. The availability of a full genome assembly is an important resource to detect genes involved in several biological processes. Recently, through new plant breeding techniques like CRISPR/Cas9, it is possible to act directly on these genes, improving and accelerating breeding programs. A chromosome-scale genome of cultivar ‘Tonda Gentile delle Langhe’ (‘TGdL’) was then constructed in two stages: 10X Genomics Chromium technology for the high-quality assembly, and subsequent reference-driven scaffolding using the cultivar ‘Tombul’ as the reference. Eleven pseudomolecules were obtained and 27,791 genes were structurally and functionally annotated (AED ≤0.4; 92% BUSCO completeness). The ‘TGdL’ genome and knowledge of candidate genes involved in biological processes offers the possibility to further develop the technology of targeted gene modification aimed at obtaining valuable transgene-free cultivars. A transformation protocol using GFP was successfully tested on calli that were induced to regenerate by caulogenesis. Protoplasts are interesting material for DNA-free gene editing because the membrane is highly permeable to DNA and ribonucleoproteins (RNP). A protoplast isolation protocol was set up using somatic calli derived from leaves cultured on MS medium supplemented with BAP. The optimized enzyme solution for cell wall digestion contained cellulase Onizuka R-10 and macerozyme R-10. The yield was 4.25×105 protoplast mL-1. Transfection with RNP and regeneration of protoplasts are current goals of the research.