Induced Mutagenesis in Safflower (Carthamus tinctorius L.) Uncovers High-Oleic Acid Mutants Genetically Distinct from the Canonical CtFAD2-1 Allele

The high-oleic acid content of the safflower (Carthamus tinctorius L.) oil, regulated by the fatty acid desaturase 2-1 (CtFAD2-1) gene, provides superior oxidative stability for applications. To explore alternative genetic sources for this trait, we employed induced mutagenesis with gamma irradiation and ethyl methane sulfonate (EMS) for two safflower cultivars, AKS 207 and PKV Pink. Screening of M2 populations identified several mutants with significantly higher oleic acid content, reaching up to 36.86%. The mutagenized populations also exhibited a wide spectrum of variation for other agronomically important traits, including increased oil content (up to 35.19%), enhanced seed protein (up to 22.51%), and seed size and weight. Correlation and principal component analyses confirmed the antagonistic relationship between oleic and polyunsaturated fatty acids and the positive association among seed size parameters. Molecular profiling using an allele-specific PCR assay targeting the CtFAD2-1 locus revealed that high-oleic mutants did not carry known mutations, suggesting the involvement of alternative alleles, micro-mutations, or other genes regulating oleic acid accumulation. This study provides valuable pre-breeding germplasm with improved agronomic and quality traits and identifies novel genetic sources for high-oleic acid in safflower. These mutants form a new genetic basis for understanding fatty acid biosynthesis and developing next-generation high-stability oil cultivars.