The date palm, or Phoenix dactylifera L., is one of the world’s oldest fruit trees. The entire date palm tree is used to produce a variety of goods, including food, clothing, fibre, and shelter. Tissue culture is one of the most recent methods used to multiply date palms and produce disease-free offspring. Plant tissue culture technology has advantages over conventional methods of propagation for the quick and extensive multiplication of significant plants in vitro, regardless of the season, and disease-free. This is because it preserves space and time. For this study, three different cultivars were used (Barhy, Sakkoti, and Shamia). For the three cultivars, ¾ MS medium supplemented with 1 mg/L indole butyric acid IBA and 0.25 mg/l activated charcoal (AC) provided the optimal in vitro culture conditions. Plant gene function research and cultivar development are now both possible thanks to the advancement of plant transformation technologies. Date palm plants were given the AT1G12660 “Thio-60” gene to make them resistant to fungus infection. Utilizing chitosan nanoparticles for genetic transformation, the gene was introduced into three cultivars of dates (Barhy, Sakkoti, and Shamia). Run a conventional PCR to verify genetic fusion into all three cultivars. The fungal infection with Fusarium oxysporum was used to determine the resistance of the transgenic cultivar lines after it was established that the thionin gene had been transferred into transgenic date palm cultivars. Date crop transgenic lines showed strong resistance and a decline in the percentage of fungal infection-induced inhibition.