The enzyme at the heart of the story is acetolactate synthase, known as AHAS or ALS, a critical catalyst in the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine in plants. Because AHAS is essential for plant growth, it has become one of the most successful herbicide targets in modern agriculture. Imidazolinone herbicides such as imazamox work by binding to AHAS and shutting it down, starving weeds of the amino acids they need to survive. However, when crops carry specific mutations in the gene encoding AHAS, the herbicide can no longer lock onto the enzyme effectively, allowing the crop to grow while surrounding weeds perish.

Until now, resistance-conferring mutations in wheat had typically been found at conserved amino acid positions known as Ala122 and Ser653, along with other canonical sites such as Pro197, Ala205 and Trp574 characterized in various plant species. These positions are considered the classical hotspots of imidazolinone resistance. What makes the new study remarkable is that none of the five distinct mutations identified in the tolerant wheat lines fall within any of these hotspots. Instead, they appear in unexpected regions of the 1,123 base pair AHAS fragment sequenced by the team, suggesting that alternative structural mechanisms may be capable of shielding the enzyme from the herbicide’s grip.

To generate these novel lines, the researchers deployed ethyl methanesulfonate, or EMS, a chemical mutagen that has served as a workhorse of plant genetics for decades. EMS primarily causes guanine-cytosine base pairs to convert into adenine-thymine pairs, creating a characteristic mutational signature that can be traced through the genome. The team attacked the problem from two complementary directions. In the first approach, they treated 50,000 wheat seeds directly with EMS, growing out the first mutant generation, known as the M1, in the field. In the second, they turned to tissue culture, exposing 126,000 calli, masses of undifferentiated plant cells grown in vitro, to the mutagen. This in vitro route offers the possibility of capturing mutations in cells that can later regenerate into entire plants, potentially broadening the spectrum of genetic variation that survives into mature crops.

The selection process was rigorous and spanned multiple generations. Mutant populations in the M2 through M4 generations were subjected to repeated herbicide screening, first at the germination stage and later through foliar applications of imazamox. Only plants that survived both rounds of chemical pressure, and that passed that tolerance reliably to their offspring, were considered candidates. Out of the enormous starting populations, four stable mutant lines emerged: three seed-derived lines designated ML-1, ML-2 and ML-3, and one line, InVitML-1, recovered from the in vitro callus pathway. The fact that lines from both mutagenesis routes made it through selection suggests that the dual strategy is a viable and productive framework for creating new genetic resources in polyploid crops like wheat, which carries a notoriously complex genome.

Sanger sequencing of the AHAS gene fragment from the tolerant lines revealed the five novel single nucleotide polymorphisms that form the genetic core of the study. These included a guanine-to-adenine substitution at position 502, a thymine-to-cytosine change at position 1075, a thymine-to-adenine transversion at position 11, an adenine-to-thymine substitution at position 386, and a rare adjacent dual substitution in which thymine became cytosine and cytosine became thymine at neighboring positions 1119 and 1120. The dual substitution is particularly intriguing, as simultaneous changes at adjacent bases are an uncommon outcome of EMS mutagenesis and may point to an especially consequential alteration in the enzyme’s structure. The overall mutation spectrum, dominated by G:C to A:T transitions, matched exactly what would be expected from EMS, confirming the chemical origin of the changes and validating the mutagenesis protocol.

To understand whether these unusual mutations actually protect the AHAS enzyme at the biochemical level, the researchers turned to the Bradford protein assay, a standard colorimetric method for measuring protein concentration. Under imazamox stress, the tolerant mutant lines maintained AHAS protein concentrations ranging from 4.60 to 4.93 micrograms per milliliter. The susceptible control plants, by contrast, dropped to just 1.97 micrograms per milliliter when exposed to the same herbicide treatment. This clear biochemical separation between mutants and controls is consistent with what scientists call target-site resistance, a mechanism in which the molecular target of the herbicide, in this case the AHAS enzyme itself, is altered so that the herbicide binds poorly or not at all. In other words, the novel mutations appear to preserve the quantity and functional presence of the AHAS protein even as the herbicide does its worst, allowing the mutant plants to keep synthesizing their essential amino acids.

The implications of finding functional resistance mutations outside the canonical hotspots are considerable. First, they expand the known genetic basis of imidazolinone resistance in wheat, adding new allelic diversity that breeders can draw upon. Different mutations can carry different trade-offs, affecting enzyme efficiency, agronomic performance and the degree of tolerance achieved. A wider palette of resistance alleles gives breeding programs more room to combine herbicide tolerance with high yield, disease resistance and grain quality. Second, because the lines were created through chemical mutagenesis rather than genetic engineering, they fall into the category of non-transgenic crop improvement. Mutagenesis-derived varieties are generally subject to lighter regulatory burdens than transgenic or genome-edited crops in many jurisdictions, which can shorten the path from laboratory to farmers’ fields considerably.

The scale of the screening effort also deserves attention. Generating and evaluating 50,000 mutagenized seeds alongside 126,000 mutagenized calli represents a substantial investment of labor and field capacity, and it underscores a broader truth about mutation breeding: valuable variants are rare, and finding them requires both large populations and carefully staged selection. In a hexaploid crop like bread wheat, which possesses three closely related subgenomes and thus multiple copies of many genes, the challenge is amplified. A mutation must occur in the right gene copy, be inherited stably, and express its effect strongly enough to survive chemical selection. That the team recovered four stable lines from this gauntlet speaks to the effectiveness of combining in vivo and in vitro mutagenesis, since each approach samples the mutational landscape in a slightly different way.

The study was supported by the Scientific and Technological Research Council of Türkiye, TÜBITAK, under project TOVAG 113O940, and the authors gratefully acknowledge the Department of Field Crops at Atatürk University for laboratory and field facilities. The team, comprising Kamil Haliloğlu of Gazi University, Aras Türkoğlu of Necmettin Erbakan University, Murat Aydin of Atatürk University and Hadi Alipour of Urmia University, notes that the germplasm is available for noncommercial research collaborations under a standard material transfer agreement, an arrangement that could accelerate the spread of these new alleles into public breeding programs across wheat-growing regions.

For farmers, the practical promise is straightforward: wheat that can stand up to imazamox gives growers a flexible option for controlling weeds, including closely related grass weeds that are difficult to eliminate with other herbicide classes without harming the crop. Herbicide-tolerant wheat developed through mutation breeding has a track record elsewhere, with imidazolinone-tolerant varieties commercialized in several countries, but the genetics underlying those varieties have largely relied on the canonical mutations. The non-canonical mutations described in this study could broaden the toolkit, offering tolerance that may differ in degree, stability or breeding behavior from existing sources.

For scientists, the findings raise compelling mechanistic questions. If mutations outside Ala122 and Ser653 can confer tolerance, what structural changes are these novel substitutions inducing in the AHAS protein? Are they altering the herbicide-binding pocket indirectly, changing the enzyme’s conformational dynamics, or triggering compensatory mechanisms at the level of protein abundance? Answering these questions will likely require structural biology and enzyme kinetics on the novel variants, work that could ultimately inform the rational design of even better resistance alleles. In the meantime, the study stands as a striking demonstration that even in a crop as intensively studied as wheat, and at a target as well mapped as AHAS, chemical mutagenesis can still uncover biology that no one had placed on the map, and it suggests that the reservoir of useful variation hiding in mutagenized populations is far from exhausted.