TOWARDS GENOME EDITING STRATEGIES IN OLIVE

Modern biotechnological approaches have successfully been extended to a growing range of species, including woody plants, which have shown low responsiveness to in vitro propagation and transformation techniques compared to the herbaceous ones. This work explores different approaches for applying New Genomics Techniques (NGTs) in olive (Olea europaea L.), with preliminary findings suggesting the applicability of these tools, though the development and optimization of the protocols are still necessary. The well-known recalcitrance of olive tissues to in vitro manipulation and regeneration of new plants represents the main obstacle to the implementation of NGTs in this species. To overcome these constraints, several methods are under investigation in our lab, including: i) the optimization of embryogenic callus culture and plant regeneration protocols specific for different genotypes; ii) the design of methods for obtaining protoplasts to be used as starting material for transfection with CRISPR/Cas9 editing agents, employing a transient delivery system based on ribonucleoproteins (RNP); iii) the plant gene editing through de novo meristem induction, using a Golden Gate-assembled vector with a CRISPR/Cas9 system. This multi-approach strategy will maximize the possibility of developing an efficient precision gene editing procedure in olive. Further efforts will be focused on enhancing the stability of CRISPR/Cas9 delivery systems, minimizing off-target effects, and optimizing the conditions for boosting the embryogenic competence of the cultures. As progress is made, it is anticipated that these advanced techniques will significantly accelerate the genetic improvement of olive cultivars with desirable traits useful for addressing current challenges, such as climate change-associated stresses and impact of pathogens. In particular, the developed methodologies will be applied to generate olive genotypes with increased tolerance to Xylella fastidiosa, a quarantine pathogen listed as one of the major phytosanitary threats throughout the Mediterranean region.