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The involvement of antifreeze proteins (AFPs; type I and III) in the germination of low temperature-treated petunia seeds (cv. ‘Mirage Rose’) was investigated. The addition of AFPs (300 or 500 µg/l) in low-temperature treatment significantly promoted the germination of seeds compared with that in which AFPs were not added. Among all treatments, treatment with AFP I added at 300 µg/l showed the highest germination percentage and improved plant growth. The expression levels of antioxidant-related genes such as superoxide dismutase, peroxidase, and proline synthesis were associated with the germination of low temperature-treated seeds. Overall, this study demonstrated that AFP I may potentially function as a cold-protective agent for the germination of low temperature-treated seeds.