关键词: 草甘膦抗性, 小麦, EMS诱变, 突变位点, 基因表达, 药效试验

Abstract:

【Objective】This study aimed to develop novel glyphosate-resistant wheat germplasm using EMS mutagenesis to mitigate weed infestation in wheat fields. Resistant mutant plants were selected through field screening, and the mutation profiles of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene as well as optimal application conditions were characterized, offering a practical approach for breeding glyphosate-resistant wheat varieties.【Method】A mutant population was generated by treating newly germinated seeds of Zhenmai 9 with EMS mutagenesis. Resistant mutants were isolated through multiple rounds of glyphosate screening in the field across M₂ and M₃ generations. Promising lines, including GR1, GR19, and GR23, were identified via pedigree selection, combined with yield and resistance phenotype screening. Mutation sites in the EPSPS gene were detected by PCR amplification and sequencing, while expression levels of TaEPSPS-4A, TaEPSPS-7A, and TaEPSPS-7D were analyzed using RT-qPCR. Field trials involving different glyphosate doses and application growth stages were conducted to systematically evaluate herbicide efficacy and determine the appropriate dosage and timing for safe application.【Result】The resistance mutant frequency in the M₂ population was 6.53×10^-6. In the M₃ generation, 43 mutant plants exhibiting tolerance to 4× the recommended glyphosate dose were successfully obtained. Sequencing analysis revealed that resistant lines GR1 and GR19 harbored 5 and 3 mutation sites in TaEPSPS-7D, respectively, whereas GR23 carried 5 mutation sites in TaEPSPS-4A. Expression analysis indicated that glyphosate treatment significantly downregulated most homoeolog genes in the three mutation lines, irrespective of whether those genes carried resistance mutations. Field trials demonstrated 100% weed control efficacy across all glyphosate treatments, significantly superior to isoproturon. As the glyphosate doses increased, wheat seedling height and fresh weight showed a decreasing trend, but most differences with the untreated control were not significant, indicating no substantial adverse effects on growth. Yield analysis revealed that treatment with 1× and 2× doses did not cause significant yield reduction, whereas 4× and 8× doses led to significant reductions of 3.04% and 4.63%, respectively. Growth stage-specific trials further indicated that spraying a 2× dose of glyphosate from seedling to jointing stages had no significant impact on plant growth, but application at the booting stage significantly reduced plant height, fresh weight, and grain yield, resulting in a 6.48% yield loss.【Conclusion】The combination of EMS mutagenesis and field screening successfully generated new glyphosate-resistant wheat germplasm capable of withstanding 4× the recommended glyphosate dose. Multiple point mutations in the non-active center of the EPSPS enzyme confered enhanced glyphosate resistance without compromising yield. For practical application of such resistant varieties, the optimal weed control window is during wheat green-up (early March), using 41% glyphosate isopropylamine salt at 840-1 680 g ae·hm^-2, diluted in 450 L·hm^-2 of water, applied as foliar spray to weeds under rain-free conditions.

Key words: glyphosate resistance, wheat (Triticum aestivum), EMS mutagenesis, mutation site, gene expression, field efficacy trial