RISK ASSESSMENT OF THE NOVEL MINI-SYNPLASTOME AND CONVENTIONAL CHLOROPLAST TRANSFORMATION GENETIC TOOLS IN AGRICULTURE

Novel cutting-edge technologies for plastid genetic engineering have great potential to improve crop traits in agriculture. Genetic engineering of the plastid genome (plastome) can be performed using both conventional homologous recombination (HR) vectors that induce site-specific transgene integration into the plastome, and novel episomal platforms that rely on synthetic plastomes (mini-synplastome) as alternative methods for the expression of transgenes from the backbone of a non-integrating plasmid. Evaluating the risk of both vertical and horizontal gene transfer (HGT) is an important step for the regulatory approval of the environmental release of these novel genetic tools. While plastids are subjected to prevalently maternal inheritance, greatly reducing the risk of transgene escape through pollen, the endosymbiotic origin of plastids from a prokaryotic progenitor may represent a potential risk for HGT to the environmental microbial community. In this study, the naturally competent soil bacterium Acinetobacter baylyi has been used to test the frequency of plant-to-bacterium HGT under laboratory conditions. While plant-to-bacterium HGT can be visualized in vitro as a low frequency event, the mini-synplastome transformation platform does not have an increased risk compared to conventional transformation vectors. After a comprehensive evaluation of risks associated with sequence components of the mini-synplastome (origin of replications and regulatory elements), optimized versions of the mini-synplastome with no residual activity in bacteria have been designed. This study represents a valuable resource for designing transformation platforms for the production of novel synplastomic and marker-free plant varieties with improved environmental biosafety in agriculture.