To investigate the effect of combined action of discharge plasma (DP) and plasma-activated water (PAW) in mutagenesis breeding, this study focuses on Agropyron mongolicum. We utilized high-voltage DC pulsed dielectric barrier discharge for seed treatment, alone and in combination with PAW. The research focused on germination rates, evolution of physicochemical properties of imbibition residual solution, reactive oxygen species (ROS), malondialdehyde (MDA), and volatile organic compounds (VOCs) to assess DP-induced damage and variability in Agropyron mongolicum. Results indicated that after 18h of combined treatment, the germination rate of Agropyron mongolicum dropped to 29.67%, below the LD50 threshold. Treated seedlings exhibited elevated ROS and MDA levels compared to controls. The concentration of reactive nitrogen and oxygen species (RONS) in the imbibition residual solution of the combined treatment group was lower than in freshly prepared PAW, indicating RONS penetration into the seed embryo via water, leading to oxidative damage. Enhanced lateral root differentiation, early tillering, increased biomass, and albino variant plants were observed in the surviving seedlings post-treatment. Transmission electron microscope (TEM) and Gas Chromatography-Ion Mobility Spectrometry (GC-IMS) analysis confirmed that plasma treatment induced oxidative damage in Agropyron mongolicum. In conclusion, high-power, long-duration direct DP treatment caused oxidative damage and reduced germination rates in Agropyron mongolicum, with PAW intensifying these effects. PAW was identified as the main driver of variation and lethality, while DP played a supportive role. Combined DP and PAW treatment induced variations in Agropyron mongolicum, providing experimental evidence and theoretical insights for applying DP treatment in plant mutagenesis breeding.
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