<p>Creating an effective protocol for plant regeneration and genetic transformation is essential for producing transgenic plants with improved and desirable characteristics. Cold plasma is an emerging and promising technology in various aspects of biological and agricultural applications. In addition, it provided a DNA transfer platform for the genetic transformation of bacterial and animal systems. The present study is the first to explore the effects of atmospheric cold plasma (CP) on callus formation, plant regeneration, and genetic transformation in safflower plants. The results indicated that extending the exposure time to CP up to 90&#xa0;s significantly increase callus induction and growth, while also reducing the time required for callus initiation and regeneration compared to untreated explants. The study on the impact of cold plasma, vacuum, sonication, and their combinations on <i>Agrobacterium</i>-mediated genetic transformation in safflower plants demonstrated that cold plasma significantly improves callus induction in the selection medium, showing comparable effectiveness to other single or combined treatments. Cold plasma greatly enhanced the induction of transgenic plants; however, in the present study, the combination of cold plasma, sonication and vacuum infiltration demonstrated the most pronounced impact on callus induction and transgenic plant regeneration. In addition, molecular and histochemical analysis confirmed the presence and function of <i>gus</i> gene in transgenic calli and plants. Cold plasma has been proposed as a method to promote callus induction and plant regeneration by altering the phytohormonal balance within plant cells. Additionally, CP generates reactive oxygen species (ROS), which can increase cell membrane permeability and facilitate the transfer of plasmid DNA into plant cells. In summary, cold plasma is recommended as an innovative approach for callus formation, plant regeneration, and genetic transformation in plants.</p>