An efficient protocol for genetic transformation and plant regeneration in pepper

Pepper (Capsicum annuum L.) is an economically and nutritionally important crop belonging to Solanaceae family. The recent availability of the pepper genome sequence demands the development of more efficient tools for gene functional analysis. However, pepper is a recalcitrant species for shoot regeneration, and although a few transient and stable transformation protocols have been published, the study of pepper gene function is still a difficult task. In addition, pepper transformation efficiency varies among different cultivars. In this study, a transient genetic transformation protocol has been optimized for Capsicum annuum cv. "Quadrato d'Asti" using a TRV-based VIGS vector and phytoene desaturase (PDS) as reporter gene. Leaves and seedlings have been infiltrated with Agrobacterium tumefaciens strain GV3101 carrying TRV plasmids, which are a bipartite vector system. Photobleaching appeared after approximately eight days and three weeks on detached leaves and seedlings, respectively. It was observed that infiltrated plants showed photobleaching even four months after infiltration and produced photobleached fruits. Molecular analysis showed that CaPDS transcripts were dramatically reduced in the silenced leaf. In order to reduce the time of symptoms appearance, agroinfiltration has also been tested on flowers, inflorescences and fruits at different developmental stages. As concerns stable genetic transformation, an efficient protocol for high-frequency regeneration of cv. "Quadrato d'Asti" has been set up, and A. tumefaciens-mediated transformation using the strain GV3101 is in progress. These transformation methods will be used for functional analysis of genes involved in merceological (fruit shape and size) and technological (cuticle thickness and composition) quality of pepper fruits.