In plant breeding programs, one of the oldest methods is mutation breeding. Currently, mutation breeding has became popular among the breeders and scientists again with its use in plant biotechnology and due to some restrictions on the other techniques such as hybridization, cross breeding, and transgenic plants. Physical mutagens (X-rays, UV light, neutrons-alpha-beta particles, fast and thermal neutrons, especially gamma rays) are used more widely than chemical (ethyl methanesulfonate [EMS]) ones to artificially induce mutations (mutagenesis). However, among the physical mutagens, gamma-rays are widely used. During the irradiation of the seeds with ionizing radiation to generate mutants with desirable traits, reactive oxygen species (ROS) or free radicals can generate in cells. Although, these radicals/species generally can be very dangerous for the cell compartments, they can take an important role as a signal molecule activation of genes of antioxidant enzymes and proline, which are defense systems against these radicals in plant cells. In this chapter, usability of gamma-irradiation to provide the permanent gene expression of antioxidant enzymes and proline through the production of reactive oxygen species (ROS) is discussed.