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.