Accelerating genetic gain in crop improvement is required to ensure improved yield and yield stability under increasingly challenging climatic conditions. This case study demonstrates the effective confluence of innovative breeding technologies within a collaborative breeding framework to develop and rapidly introgress imidazolinone Group 2 herbicide tolerance into an adapted Australian chickpea genetic background. A well-adapted, high-yielding desi cultivar PBA HatTrick was treated with ethyl methanesulfonate to generate mutations in the ACETOHYDROXYACID SYNTHASE 1 (CaAHAS1) gene. After 2 years of field screening with imidazolinone herbicide across >20 ha and controlled environment progeny screening, two selections were identified which exhibited putative herbicide tolerance. Both selections contained the same single amino acid substitution, from alanine to valine at position 205 (A205V) in the AHAS1 protein, and KASP™ markers were developed to discriminate between tolerant and intolerant genotypes. A pipeline combining conventional crossing and F2 production with accelerated single seed descent from F2:4 and marker-assisted selection at F2 rapidly introgressed the herbicide tolerance trait from one of the mutant selections, D15PAHI002, into PBA Seamer, a desi cultivar adapted to Australian cropping areas. Field evaluation of the derivatives of the D15PAHI002 × PBA Seamer cross was analyzed using a factor analytic mixed model statistical approach designed to accommodate low seed numbers resulting from accelerated single seed descent. To further accelerate trait introgression, field evaluation trials were undertaken concurrent with crop safety testing trials. In 2020, 4 years after the initial cross, an advanced line selection CBA2061, bearing acetohydroxyacid synthase (AHAS) inhibitor tolerance and agronomic and disease resistance traits comparable to parent PBA Seamer, was entered into Australian National Variety Trials as a precursor to cultivar registration. The combination of cross-institutional collaboration and the application of novel pre-breeding platforms and statistical technologies facilitated a 3-year saving compared to a traditional breeding approach. This breeding pipeline can be used as a model to accelerate genetic gain in other self-pollinating species, particularly food legumes.

Lentil (Lens culinarisMedik.) is an important and expanding crop in southern Australia and a significant crop in western Canada. Currently, production in both countries is limited by an inability to effectively control weeds, due in part to a lack of registered safe and effective herbicides. Metribuzin is a broad-spectrum herbicide providing an alternative weed control option to the imidazolinones, but it has low crop safety in lentil. Two methods, germplasm screening using a hydroponic sand assay and field screening of a large mutated population of the Australian cultivar ‘PBA Flash’ were initially used to identify lines with putative metribuzin tolerance over current cultivars. Dose–response experiments showed the germplasm line SP1333 had GR50(the rate required to reduce dry weight 50%) values up to four times higher than PBA Flash. However, the mutation selections M043 and M009 had GR50values more than 25 times higher than PBA Flash. A field study in Canada, under conditions of induced shade and no shade 72 h before POST application of metribuzin, confirmed the intermediate level of tolerance in SP1333 and the high level in the two mutant lines compared with 20 Canadian and Australian genotypes. This relative increase in metribuzin tolerance of the two mutant lines over the parent cultivar is higher than all previous reports in a range of crop species. The development of large mutant populations combined with large M2field screens was a successful method for developing high levels of metribuzin tolerance in lentil. The estimated mutation rate of the mutant lines was 9.4×10−8. All three lines are currently being used as parents in lentil breeding programs.

BACKGROUND Faba bean (Vicia faba L.) is an important crop in Australian farming systems, however, weed control is a major constraint due to a lack of in‐crop broadleaf herbicide options. To address this, we developed acetohydroxyacid synthase (AHAS) inhibitor herbicide tolerance in faba bean using mutagenesis techniques. Dose–response experiments, agronomic field evaluation and DNA sequencing of the AHAS gene were used to quantify and validate tolerance traits. RESULTS Four M2 faba bean single‐plant biotypes (IMI‐1, IMI‐2, IMI‐3 and IMI‐4) at a frequency of 3.63 × 10−6 were successfully recovered. Molecular characterisation of the AHAS gene identified two known target site mutations (resulting in protein substitutions Ala205Val and Ser653Asn) conferring tolerance. Phenotypic characterisation found that both mutations conferred high levels of tolerance to the imidazolinone herbicide imazapyr. However, although the Ala205Val substitution showed improved levels of cross‐tolerance to a range of sulfonylurea chemistries, the Ser653Asn substitution did not. In the field, IMI‐3 showed the highest level of agronomic tolerance across a range of imidazolinone herbicides. CONCLUSIONS Mutagenesis techniques were successful in the development of tolerance to AHAS inhibitor herbicides in faba bean, and could facilitate the first safe in‐crop broadleaf herbicide control option in Australian faba bean production. © 2019 Society of Chemical Industry

BACKGROUND Weed competition is a major limitation to worldwide lentil (Lens culinaris Medik.) production in part due to limited effective safe herbicide options. Metribuzin is a photosystem II inhibiting herbicide that provides broad spectrum weed control, however it causes excessive injury in lentil. Dose response analysis of photosystem II inhibiting herbicides and DNA sequencing of the psbA chloroplast gene occurred to quantify the spectrum and mechanism of herbicide resistance in two ethyl‐methanesulfonate (EMS) induced mutant lentils. RESULTS Compared to susceptible parent PBA Flash, the level of metribuzin resistance was 33‐fold for mutant M043 and 10‐fold for M009. No improvement in resistance occurred in either mutant to bromoxynil, diuron, bromacil and atrazine herbicides. Nucleotide sequencing of the psbA gene of both mutants identified a substitution at position 751 compared to PBA Flash. The resulting deduced amino acid sequence indicated an Ala251Thr substitution as being most likely responsible for the high level of metribuzin resistance. CONCLUSIONS The Ala251Thr substitution discovered in this study is unique in mutagenized higher plants and the first report of an induced psbA target site mutation in higher plants. This target site metribuzin resistance is likely to have a significant impact on lentil production in Australia and worldwide. © 2019 Society of Chemical Industry

Multiple field experiments and a controlled-environment temperature study were conducted to investigate soil and weather conditions responsible for herbicide phytotoxicity in lentil (Lens culinaris Medik.) from post-emergent application of metribuzin. A linear relationship was observed between plant injury (% necrosis) and metribuzin rate in all 12 environments, but in only 11 environments for anthesis dry weight and nine environments for both plant density and grain yield. Grain-yield reduction from label metribuzin rates of 135 g a.i. ha–1 for sand and 285 g a.i. ha–1 for clay ranged from 0% to 32% and 0% to 67%, respectively, across all environments. Principal component analysis of soil and weather factors around the time of herbicide application suggested that metribuzin-induced plant damage in lentil was due to a combination of multiple soil and weather factors. However, heavy rainfall within 10 days of herbicide application, particularly on light-textured soils or where soil moisture was low, was most strongly linked to plant damage. Experiments targeting the impact of reductions in temperature post-metribuzin application showed no effect, and of light intensities pre- and post-metribuzin application showed low effects on plant-damage measures. Because rainfall in the 10 days after application is a major determinant of metribuzin damage in winter-grown lentil in southern Australia, a higher level of selective tolerance to metribuzin than that present in commercial cultivars is needed for its safe post-emergent use. Early and late measures of plant damage will be required to assess accurately plant tolerance to post-emergent metribuzin application in lentil.

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