Improving the water use efficiency of tomato crops using CRISPR/Cas method

Tomato (Solanum lycopersicum) is a globally significant horticultural crop, yet its shallow root system and high transpirational demand render it highly susceptible to water stress, particularly under the increasingly hot and dry conditions characteristic of Mediterranean regions in Turkey. With the rate at which irrigation is putting a strain on Turkey's water reserves, improving the water use efficiency (WUE) of tomato has thus become a necessity in crop improvement strategies with the aim of ensuring yield stability and resource conservation. In this study, the CRISPR/Cas9 gene-editing system was employed to target the SlROP9 gene, a Rho-like GTPase that has been suspected to function in the regulation of stomatal closure through reactive oxygen species (ROS) signaling pathways independent of the regulation of abscisic acid (ABA). A highly specific guide RNA (gRNA) was designed to target exon 4 of SlROP9 and cloned into the binary vector pco-Cas9. The construct was successfully transformed into Agrobacterium tumefaciens strain EHA105 and introduced into the 267 tomato genotype via Agrobacterium-mediated transformation. Although the tomato 267 genotype exhibited low regeneration efficiency and hypersensitivity to glufosinate selection, transgenic lines were recovered by optimizing selection conditions. PCR and Sanger sequencing confirmed the presence of CRISPR-induced mutations in four independent T₀ plants, with indel analysis revealing both biallelic and chimeric mutations that disrupted the SlROP9 coding sequence. Notably, the edited T0 plants have undergone maturity and produced fruit. From T0 plants, both T₁ plantlets obtained via embryo culture of immature fruits and T₁ seeds from mature fruits were successfully generated, demonstrating the viability of both recovery routes. The study provides evidence of successful targeted mutagenesis in SlROP9, laying the groundwork for the development of tomato lines with improved drought tolerance and WUE. Further physiological characterization, multigenerational field trials, and water-use parameters are recommended to validate the functional impact of SlROP9 disruption on water-use efficiency, drought tolerance and overall productivity. KEYWORDS: Agrobacterium-mediated transformation, CRISPR/Cas9, Drought stress, Gene editing, SlROP9, Stomatal regulation, Tomato, Water use efficiency