CRISPR/Cas9 genome editing technology has revolutionized plant breeding by offering precise and rapid modifications. Traditional breeding methods are often slow and imprecise, whereas CRISPR/Cas9 allows for targeted genetic improvements. Previously, direct delivery of Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes to grapevine (Vitis vinifera) protoplasts has been demonstrated, but successful regeneration of edited protoplasts into whole plants has not been achieved. Here, we describe an efficient protocol for obtaining transgene/DNA-free edited grapevine plants by transfecting protoplasts isolated from embryogenic callus and subsequently regenerating them. The regenerated edited plants were comparable in morphology and growth habit to wild-type controls. This protocol provides a highly efficient method for DNA-free genome editing in grapevine, addressing regulatory concerns and potentially facilitating the genetic improvement of grapevine and other woody crop plants.

Efficiency in vitro regeneration is a crucial prerequisite for the application of New Nenomics Techniques (NGTs) in grapevine (Vitis vinifera L.) for improving resistance to biotic and abiotic stresses. This is especially true given that their management must be addressed sustainably, considering the impact of climate change. Unfortunately, in vitro plant regeneration and the establishment of embryogenic calluses are two genotype-dependent processes. Up to now, extensive research has been conducted on major international cultivars, whereas studies on the application of in vitro protocols for autochthonous cultivars remain limited. In this study, protocols for the acquisition of embryogenic calluses were applied on the most relevant Sicilian grapevine cultivars: the red-skinned ‘Frappato’, ‘Nerello mascalese’, and ‘Nero d’Avola’, and the white-skinned ‘Grillo’, ‘Carricante’, and ‘Catarratto’. Stamens and pistils were cultured in two different induction media (PIV and MSII) and at three stages (mother cells in the late premeiotic phase, tetrads, and mature pollen) to induce embryogenic calluses. Five thousand explants per cultivar were cultured, forming calluses in four selected cultivars. Plantlets were successfully generated from calluses of ‘Carricante’, ‘Frappato’, and ‘Nero d’Avola’. Moreover, protoplasts were isolated from ‘Frappato’ and ‘Nero d’Avola’. Our results establish a critical foundation for developing successful regeneration protocols for the future application of NGTs in Sicilian grapevine cultivars.