Fig. 2.

ROS generation is induced by melatonin. Three-day-old alfalfa seedlings were treated with or without 10 μm melatonin (Mel) for the indicated times. (A, B) Then, root tissues were loaded with 20 μm 2′,7′-dichlorofluorescein diacetate (H₂DCFDA; a fluorescent dye for ROS) and regions of root mature zone were detected by LSCM. Scale bar (A) = 100 μm. Six individual samples were randomly selected and measured per treatment. Fluorescence is shown relative to control (Con) samples without added melatonin at 0 h. RU, relative units. (C) H₂O₂ content was determined by spectrophotography. FW, fresh weight. Means and standard errors were calculated from at least three independent experiments with at least three replicates for each. Asterisks denote significant differences in comparison with control (one-way ANOVA followed by Tukey’s multiple range test, *P < 0 .05, **P < 0 .01).