【Objective】Soil salinization severely constrains the sustainable development of rice production. The specific goals are to: comprehensively evaluate the salt tolerance of rice core germplasm accessions at the seedling stage, investigate the morphological and physiological characteristics of different rice subspecies under salt stress, clarify the associated variations in their responses, and summarize their adaptive strategies, thereby providing a theoretical foundation for screening and breeding salt-tolerant rice varieties.【Method】The salt tolerance score (STS), plant height, root length, shoot fresh weight, shoot dry weight, root dry weight and SPAD were measured for 276 rice core germplasm accessions after treatment using 125 mmol·L-1 NaCl for 6 days. The relative values of each trait, except for STS and shoot fresh weight were calculated, along with the shoot water content (SWC). T-tests, significance analysis and correlation analysis were used to explore the morphological and physiological differences for salt stress responses among different rice subspecies. Principal component analysis (PCA) and stepwise linear regression were applied to screen key indicators for salt tolerance. The D-value was calculated to identify typical salt-tolerant accessions and salt-sensitive accessions, which were used to elucidate the regulatory patterns and response strategies of salt stress in different subspecies of rice.【Result】Salt stress affected the growth of seedlings of the three subspecies of rice. Compared with japonica rice, indica rice and AUS exhibited milder inhibitory effects from salt stress, and AUS demonstrated greater phenotypic variation. The STS, relative seedling height (RSH), relative root length (RRL), relative root dry weight (RRDW), relative SPAD (RSPAD) and SWC among the three subspecies of rice accessions were not significantly different. However, the relative shoot dry weight (RSDW) of indica rice was significantly higher than that of japonica rice and AUS, and the salt tolerance of temperate japonica rice seedlings was significantly higher than that of tropical japonica rice and admixed japonica rice. Correlation patterns of the seven salt-tolerance-related traits varied between the three subspecies. Three principal components were extracted from japonica rice, indica rice and AUS, with cumulative contribution rates of 82.587%, 80.117%, and 88.700%, respectively. Based on this, the D-values for the comprehensive evaluation of salt tolerance were calculated for each accession, and key parameters for salt tolerance were screened. It was found that RSDW is a common key indicator affecting the salt tolerance of rice seedlings, while RSH and RRDW are shared by japonica rice and AUS, and STS is the common key parameter for indica rice and AUS. In the three subspecies, high-D-value accessions and low-D-value accessions were selected to analyze root characteristics, ion balance, reactive oxygen species accumulation, and osmotic regulation substance content under salt stress. The root total number (RTN), root tip number (RN), total root length (TRL), and root surface area (RSA) of high-D-value accessions in the three subspecies were significantly higher than those of the three categories of low-D-value accessions. Among the three types of high-D-value accessions, the RTN and RN of indica rice were significantly higher than those of japonica rice and AUS. The root average diameter (RAD) of indica rice and AUS was significantly higher than that of japonica rice. AUS had significantly higher surface area and volume of roots in the 0.5-1 mm diameter range than japonica rice and indica rice, while japonica rice had significantly higher root volume in the 0-0.5 mm diameter range than indica rice and AUS. In terms of ion balance, the shoot Na+ content (SNC) of the three types of high-D-value accessions was significantly lower than that of the three types of low-D-value accessions. Among the three types of high-D-value groups, AUS had significantly lower SNC and shoot Na+/K+ (SNK) than that of japonica rice, japonica rice had significantly lower root Na+ content (RNC) than AUS, indica rice had significantly higher root K+ content (RKC) than AUS, and japonica rice and indica rice had significantly lower root Na+/K+ (RNK) than AUS. In terms of reactive oxygen species content, among the three categories of high-D-value accessions, the hydrogen peroxide content of japonica rice was significantly lower than that of indica rice and AUS. In terms of osmoregulatory substance content, among the three types of high-D-value accessions, the soluble sugar content of indica rice and AUS was significantly higher than that of japonica rice, and the proline content of indica rice was significantly higher than that of japonica rice.【Conclusion】Significant differences were observed in the morphological and physiological characteristics of rice germplasm accessions from different subspecies under salt stress. RSDW is a common key indicator affecting salt tolerance of rice seedlings. In response to salt stress, typical salt-tolerant germplasm from japonica, indica, and AUS developed distinct combinatorial profiles of regulatory modes, which varied in their emphasis on four key aspects: root morphological characteristics, ion homeostasis, reactive oxygen species (ROS) scavenging and osmotic adjustment.
【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.
