Biotechnological approaches for genetic improvement in Solanaceae species

Solanaceae is one of the most valuable and variable families among vegetable crops and includes some of the most important agricultural species (potatoes, tomatoes, eggplants and peppers) and some crop model plants for research (e.g. tomato and tobacco). In order to meet the changing and increasing needs of agriculture, new biotechnological tools can be exploited to support breeding programs and to facilitate the development of new crop varieties with improved traits. The purpose of this thesis is to explore the potential of CRISPR/Cas9 mediated genome editing for crop improvement in Solanaceae and to implement new techniques for boosting efficiency of editing, in order to meet legislative requirements on GMOs. Chapter I: Downy Mildew Resistance 6 (DMR6) is a susceptibility gene that encodes an enzyme involved in Salicylic Acid (SA) degradation, and its inactivation in other Solanaceae species (tomato and potato) caused an increase SA levels and conferred tolerance to a broad spectrum of pathogens. We identified the two orthologs of this gene in eggplant’s genome and analysed their transcriptional profile. In eggplant only SmDMR6-1 expression significantly increased upon infection by Phytophthora infestans and Phytophthora capsici, suggesting its involvement in the regulation of plant responses to biotic stresses. Therefore, we knocked out SmDMR6-1 gene’s functionality through CRISPR/Cas9 technology and analysed the mutants’ tolerance to P. infestans and P. capsici through pathogen assays that highlighted an increased tolerance to infection by P. infestans and P. capsici in dmr6-1 mutants, if compared to non-edited plants. Chapter II: with the aim of developing new transformation protocols that produce DNA-free genome edited plants, we adapted and improved a protocol for the isolation of protoplasts and subsequent CRISPR/Cas9 mediated editing. We isolated protoplasts from eggplant cotyledons and after 4-5 months of in vitro regeneration we obtained wild-type regenerated plants. Alongside, we tested two different methods for CRISPR/Cas9 ribonucleoproteins (RNPs) delivery into protoplasts with the aim of knocking out SmCHL_H gene, whose inactivation causes a chlorotic phenotype. Polyethylene glycol (PEG) mediated delivery of RNPs was optimised and after regeneration and Sanger sequencing on regenerated calli a high mutation rate and editing efficiency (up to 74% and 96% respectively) were obtained. Lipofection was also tested as RNPs delivery method, but lower efficiencies were recorded. Chapter III: transcription factors belonging to the ethylene-responsive factor (ERF) family are known to play a role in many physiological processes in tomato, such as fruit ripening and response to abiotic stresses. Since the function of SlERF.F4 gene is still poorly understood, we knocked out its functionality through CRISPR/Cas9 technology in tomato cultivar Microtom and we characterised the regenerated mutants from a physiological, molecular and biochemical point of view after a water deprivation regime. Under severe drought stress conditions, erf.f4 mutants showed a slower decrease in soil relative water content and the analysed physiological parameters indicated a behaviour of reduced stomatal conductance, if compared to wild-type controls. Molecular analyses of transcriptional levels of stress-related genes (involved in abscisic acid metabolism and antioxidant response) and biochemical analyses of photosynthetic pigments and antioxidant capacity suggested a higher tolerance to drought stress and overall a more pronounced water saving behaviour.