Male sterility induced by a chemical hybridization agent(CHA) is an important tool for utilizing crop heterosis. Leaves, especially the flag leaves, as CHA initial recipients play a decisive role in inducing male sterility. To investigate effects of different treatment times of CHA-SQ-1 used, morphological, biochemical and physiological responses of wheat flag leaves were detected in this study. CHA induced programmed cell death(PCD) as shown in terminal deoxynucleotidyl transferase-mediated d UTP nick end-labelling(TUNEL) and DNA laddering analysis. In the early phase, CHA-SQ-1 triggered organelle changes and PCD in wheat leaves accompanied by excess production of reactive oxygen species( O~-._2and H_2O_2) and down-regulation of the activities of superoxide dismutase(SOD), catalase(CAT) and guaiacol peroxidase(POD). Meanwhile, leaf cell DNAs showed ladder-like patterns on agarose gel, indicating that CHA-SQ-1 led to the activation of the responsible endonuclease. The oxidative stress assays showed that lipid peroxidation was strongly activated and photosynthesis was obviously inhibited in SQ-1-induced leaves. However, CHA contents in wheat leaves gradually reduced along with the time CHA-SQ-1 applied. Young flags returned to an oxidative/antioxidative balance and ultimately developed into mature green leaves. These results provide explanation of the relations between PCD and anther abortion and practical application of CHA for hybrid breeding. Male sterility induced by a chemical hybridization agent (CHA) is an important tool for utilizing crop heterosis. To investigate effects of different treatment times of CHA- SQ-1 used, morphological, biochemical and physiological responses of wheat flag leaves were detected in this study. CHA induced programmed cell death (PCD) as shown in terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and DNA laddering analysis . In the early phase, CHA-SQ-1 triggered organelle changes and PCD in wheat leaves accompanied by excess production of reactive oxygen species (O ~ -._ 2 and H_2O_2) and down-regulation of the activities of superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (POD). Meanwhile, leaf cell DNAs showed ladder-like patterns on agarose gel, indicating that CHA-SQ-1 led to the activation of the responsible endonuclease. The oxidative stress assays showed that lipid peroxidation was strongly activated and photosynthesis was obviously inhibited in SQ-1-induced leaves. However, CHA contents in wheat leaves gradually reduced along with the time CHA-SQ-1 applied. Young flags returned to an oxidative / antioxidative balance and eventually developed into mature green leaves. These results provide explanation of the relations between PCD and anther abortion and practical application of CHA for hybrid breeding.