OPTIMIZATION OF GENETIC TRANSFORMATION AND REGENERATION PROTOCOLS IN OLIVE SOMATIC EMBRYOS AIMING TO OBTAIN XYLELLA FASTIDIOSA RESISTANT GENOTYPES

The intensification of global plant trade and the ongoing effects of climate change are major drivers of the rapid spread of invasive plant pathogens. The emergence of Xylella fastidiosa (Xf) in olive (Olea europaea L.) in southern Italy exemplifies the capacity of an exotic pathogen to colonize new geographic areas, leading to severe disruptions of ecosystems and significant economic losses. The limited occurrence of resistant traits to this pathogen in olive germplasm prompted in recent years several breeding programs, focused on improving host-response to infections, exploiting the few resistant parentals, and combining agronomic, yield performance, and oil quality. However, conventional breeding methods for this species require long-term efforts. While, the application of modern biotechnology tools, such as in vitro cultivation, molecular markers, and genetic transformation, may support the improvement of important traits, such as biotic and abiotic resistance and tolerance. Among the promising approaches, genetic transformation of olive somatic embryos represents a valuable biotechnological tool for screening candidate genes for resistance to Xf in olive genotypes. Nevertheless, the advancement of these biotechnological strategies is hampered by the intrinsic recalcitrance of olive tissues to in vitro regeneration. Previous studies have reported successful Agrobacterium tumefaciens-mediated transformation protocols, involving the use of a binary plasmid system to genetically transform olive somatic embryos derived from radicles of mature zygotic embryos of Spanish cultivar. The goal of this study was to optimise the genetic transformation of olive somatic embryos using GREEN FLUORESCENT PROTEIN (GFP) as marker gene. The transformation was conducted using embryogenic calli of the cultivar Picual using A. tumefaciens strain AGL-1 carrying the recombinant binary plasmid pBIN-61, harbouring the 35S promoter driving GFP expression. The transformation was confirmed via PCR, followed by a regeneration protocol to validate transgene integration in the resulting transformed plants. We transformed a total of 100 somatic embryos, 30 of which survived and grew in selective medium. Eight of these embryos were subjected to DNA extraction, with 6 yielding positive GFPPCR amplification. The other embryos are still growing and will be tested in the future. Given the recalcitrance of some cultivars to the in vitro manipulation, the protocol will be tested with a wider panel of olive genotypes, including several Italian olive varieties to assess the efficiency of genetics transformation. The overarching objective of the project is to develop genomic tools for generating novel Xf-resistant olive genotypes by editing of susceptibility (S) genes already identified through transcriptomic analyses.