Characterization and genome-editing for glufosinate tolerance in micro-tom tomato (Solanum Lycopersicum)

Abstract

This study developed a comprehensive strategy to address this limitation through precise genome editing, grounded in three complementary components: experimental characterization of Micro-Tom sensitivity to glufosinate, bioinformatic identification of candidate genes for editing, and experimental validation of genetic transformation. In the first chapter, Micro-Tom sensitivity to foliar glufosinate applications was systematically characterized under controlled greenhouse conditions. Plants were exposed to five herbicide concentrations (0, 0.5, 1.0, 2.0, and 3.0 g L⁻¹), and phenotypic (visual symptoms) and physiological (catalase activity) responses were evaluated. Results revealed a pronounced dose–response relationship, in which even the lowest dose (25% of the recommended rate) led to complete mortality by ten days, indicating the absence of any safety margin of tolerance. Catalase activity displayed a biphasic pattern (an initial 52% induction followed by depletion), highlighting the insufficiency of the cultivar’s endogenous defense mechanisms. In the second chapter, integrated bioinformatic analyses (reciprocal BLASTP, phylogenetic analyses, and structural modeling with AlphaFold 3) identified seven tomato ortholog candidates of rice genes (OsARF18 and OsSPL10) that act as transcriptional repressors of glutamine synthetase genes (GS1 and GS2), thereby modulating glufosinate resistance. Multifactorial evaluation ranked Solyc11g069500 (OsARF18 ortholog) and Solyc10g018780 (OsSPL10 ortholog) as the most promising editing targets, exhibiting conserved gene architecture, a full complement of essential functional domains, and dense patterns of promoter binding sites. In the third chapter, genetic transformation was experimentally validated via Agrobacterium tumefaciens, achieving a transformation efficiency of 13.3% with Golden Gate–assembled CRISPR–Cas9 vectors (60% cloning efficiency) and optimizing the regeneration protocol through cytokinin replacement, yielding successful regeneration of approximately 20 explants. Collectively, these findings position Solyc11g069500 and Solyc10g018780 as viable genes for future genome-editing strategies aimed at conferring glufosinate resistance in tomato, circumventing the limitations of conventional transgenic approaches and contributing meaningfully to global food security and sustainable agriculture.