Development of novel imazamox tolerant wheat lines via combined in vivo and in vitro EMS mutagenesis

Acetolactate synthase ( AHAS ) mutations conferring imidazolinone herbicide resistance typically occur at conserved amino acid positions (Ala122, Ser653) in wheat. This study employed ethyl methanesulfonate (EMS) mutagenesis using two complementary approaches: in vivo seed treatment (50,000 M₁ seeds) and in vitro callus-based mutagenesis (126,000 calli) to generate novel AHAS -resistant wheat lines. Multi-stage selection across M₂–M₄ generations using germination-stage and foliar herbicide applications identified four stable mutant lines: ML-1, ML-2, ML-3 (seed-derived), and InVitML-1 (in vitro-derived). Sanger sequencing of the 1,123 bp AHAS fragment identified five distinct SNPs: G→A at position 502, T→C at position 1075, T→A at position 11, A→T at position 386, and a rare adjacent dual substitution (T→C, C→T) at positions 1119–1120. Notably, all five mutations were located outside canonical hotspots (Ala122, Pro197, Ala205, Trp574, Ser653), suggesting the involvement of alternative structural mechanisms for AHAS tolerance. Bradford assay showed mutant lines maintained AHAS protein concentrations of 4.60–4.93 µg/mL under imazamox stress, compared with 1.97 µg/mL in susceptible controls, consistent with target-site resistance. The mutation spectrum predominantly exhibited G: C→A: T transitions, as expected from EMS mutagenesis, confirming the chemical mutagen’s signature. These results provide strong initial evidence that non-canonical AHAS mutations can confer herbicide tolerance in polyploid wheat, extend the known genetic basis of imidazolinone resistance, and demonstrate the effectiveness of dual mutagenesis strategies for generating novel allelic resources for non-transgenic crop improvement programs.