Gene editing technology (CRISPR) for breeding tomato resistant to broomrape (Orobanche spp., Phelipanche spp.)

Tomato (Solanum lycopersicum) is one of the major agricultural crops with high global production and consumption rates, possessing significant economic value. However, various limiting factors such as environmental conditions, soil properties, nutrient management strategies, irrigation systems, and diverse pathogens lead to yield and quality losses in tomato cultivation. One of the most critical constraints in tomato production worldwide and in Turkey is the infestation of parasitic weeds known as broomrapes, including Orobanche spp. and Phelipanche spp., which maintain their life cycle by fully parasitizing host plants. Under favorable environmental conditions, the seeds of these parasitic weeds germinate in response to strigolactone hormones synthesized and secreted via the carotenoid pathway from host plant roots, ultimately attaching to the tomato plant and causing significant damage. In this study, CRISPR/Cas9 gene editing technology was applied to develop tomato (Solanum lycopersicum) lines resistant to the root parasitic weeds Orobanche spp. and Phelipanche spp., by targeting the SlCCD8 gene, which plays a role in strigolactone biosynthesis. For this purpose, custom-designed guide RNA (gRNA) sequences were constructed and transferred into tomato plants along with the Cas9 protein via Agrobacterium-mediated transformation. The resulting "in-frame" mutations were introduced into the genome, and the T1 and T2 generations were evaluated for broomrape resistance in both hydroponic systems and pot trials, while the T3 generation was tested in hydroponic systems only. According to the results obtained, the absence of broomrape tubercle formation in the mutant D38 genotype demonstrated that the mutation conferred effective resistance against the parasite. Moreover, the D38 line maintained its developmental performance under biotic stress conditions, indicating its potential for direct use in breeding programs. At the molecular level, RT-qPCR-based gene expression analyses revealed that CCD8 gene expression in mutant plants was significantly higher compared to the control group, whereas CCD7 and MAX1 expression levels showed no statistically significant difference. This study presents a targeted and environmentally friendly resistance breeding strategy that goes beyond the limitations of conventional chemical control methods. Additionally, it demonstrates the applicability of the CRISPR/Cas9 system as a functional genetic tool for combating parasitic weeds in tomato. The findings suggest that gene-editing-based resistance mechanisms using CRISPR/Cas9 may contribute significantly to sustainable breeding programs for strategic crops such as tomato in the future.