New in vitro culture techniques for molecular editing programs in grapevine

Abstract

Grapevine breeding programs have traditionally focused on the development of seedless table grape cultivars combining high berry quality with resistance to major pathogens, in response to both market demand and sustainability goals.

Maintaining the competitiveness of Italian viticulture also requires the introduction of innovative cultivars suitable for modern production systems. In this context, new breeding techniques (NBTs), including genome editing, offer promising opportunities for the genetic improvement of grapevine.

The successful application of genome editing approaches relies on efficient in vitro regeneration systems, which remain a major bottleneck for many grapevine genotypes. In particular, the induction of embryogenic callus and somatic embryogenesis from plant explants is a key prerequisite for genetic transformation and genome editing. Among the genes potentially involved in reproductive development, INO (INNER NO OUTER) has been identified as a candidate regulator of ovule development in grapevine.

This study aimed to develop an efficient protocol for callus induction from immature inflorescences and leaf explants of different table grape cultivars to support future genome editing applications targeting genes involved in seed development. Immature inflorescences and leaf explants from several Vitis vinifera table grape cultivars were in vitro cultured to evaluate their capacity for callus induction. Theselected genotypes include commercially important cultivars widely appreciated in the table grape market, making them particularlyrelevant targets for genetic improvement. Different culture media were tested by modifying plant growth regulator combinations based on protocols reported in the literature. Media derived from Nitsch and Nitsch and Murashige and Skoog formulations, together withadditional callus induction media, were compared to identify the most suitable conditions for embryogenic callus development.

Embryogenic responses were obtained from both immature inflorescences and leaf explants. The modified MS medium showed the highest efficiency for callus development from immature inflorescences in the Muscat Timpurium and Bolgar Rezy cultivars, whereas the best embryogenic callus induction from leaf explants was observed on CIM medium in the Regal cultivar. These results provide a useful basis for the development of regeneration systems supporting genome editing approaches aimed at the generation of improved seedlessgrapevine cultivars.