Establishment of in vitro regeneration protocols and protoplasts isolation techniques for Sicilian grapevine cultivars

Grapevine is considered a crop of strategic economic importance for both the worldwide extended cultivated area and the high value of wine production industry. Nevertheless, this crop is threatened by several biotic stress, whose management must be addressed in the context of the current climatic crisis where the transition toward a more sustainable viticulture is urgently needed. In this context, the application of new genomic techniques (NGTs) offers a promising approach to improve resistance traits in grapes, reducing chemical inputs and promoting environmental sustainability. The application of NGTs is strictly dependent on two genotype-dependent processes: in vitro plant regeneration and the obtainment of embryogenic calli. While reliable protocols exist for several major international cultivars, limited research has been conducted on autochthonous genotypes. In this study, we focused on six of the most relevant Sicilian grapevine cultivars: ‘Carricante’, ‘Catarratto’, ‘Frappato’, ‘Grillo’, ‘Nerello mascalese’ and ‘Nero d’Avola’. Stamens and pistils were cultivated in vitro for inducing the production of embryogenic calli by using two different induction media PIV and MSII and at three distinct developmental stages of the pollen (mother cells, tetrads and mature pollen). A total of 5,000 explants per cultivar were cultivated, resulting in callus formation in all the genotypes. Embryogenic calli from ‘Carricante’, ‘Frappato’ and ‘Nero d’Avola’ were successfully regenerated into whole plants, and ‘Frappato’ and ‘Nero d’Avola’ were also employed for protoplasts isolation.