Genetic improvement of Oenological Interesting Lucanian Grapevine Varieties through CRISPR/Cas9-Mediated Mutagenesis: Somatic embryogenesis and protoplast regeneration

Cultivated grapevine varieties, propagated vegetatively, exhibit intra-varietal genetic variability due to somatic mutations. While this variability has been exploited in the past for clonal selection, current environmental challenges and market demands necessitate more robust methods for genetic improvement. This study investigates somatic embryogenesis and protoplast culture as essential steps for the application of CRISPR/Cas9 genome editing in grapevine. We optimized somatic embryogenesis protocols for six grapevine varieties (Aglianico, Aleatico, Greco bianco, Merlot, Primitivo, Crimson). Optimal hormone ratios were identified for each genotype, leading to efficient induction of embryogenic calli and subsequent development of somatic embryos. Viable protoplasts were isolated from three varieties (Crimson, Greco bianco, Primitivo) and cultured in three different nutrient solutions. Protoplast multiplication was observed, with Crimson and Primitivo exhibiting higher multiplication rates compared to Greco bianco. Efficient and reproducible somatic embryogenesis and protoplast culture protocols are crucial for enabling targeted genetic modifications via CRISPR/Cas9 in grape, thought regeneration of whole plants from protoplast culture requires further investigation.