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Ethylene (C₂H₄), although is a simple molecule, play several important roles in plant growth and development. Triple response approach is a great possibility for evaluation of ethylene biosynthesis and signaling in Arabidopsis thaliana. In this research, by using the various mutant plants, we investigate function of several key genes including EIN2, EDR1, JAR1, PAD4 and NahG on plant responses to ethylene. The Arabidopsis seedlings that had some defection in ethylene biosynthesis, perception or signaling may explicate some changes in triple response. Therefore, several mutants including ein2، jar1، NahG، pad4 ،edr1 and their wild type plant, Col-0, have been employed. The seeds have been grown on MS medium consisting of several concentrations (0, 1, 5, 10 and 20 mM) of ACC (1-aminocyclopropane-1-carboxylic acid). The triple response assay performed on seedlings after incubation for 5 days in darkness. According to the obtained results, non of the mutants fall into eto (Ethylene over expression) and his (Less hook) groups. Most of mutant plantlets displayed a significant difference with the wild type, Col-0, and only pad4 showed the same response as the parental genotype, meaning that ethylene biosynthesis and signaling in pad4 seedlings is probably the same as the wild type. Therefore, mutation in key genes involving in various hormone biosynthesis can induce a different response to ethylene in plants. The triple response approach could be very useful manner to predict the responses of plants in mature stage.