The objective of this study was to investigate how gamma irradiation affected the embryonic development of calluses and morphological alterations of mutant patchouli plantlets. The establishment of embryonic callus and morphological alterations concurrent with in vitro patchouli shoot and plantlet production were noted at lethal dosages (LD50). In addition to physical mutagens, irradiating the callus inhibited cell differentiation and in vitro plant growth, resulting in the development of new plant varieties. Through mutation engineering, patchouli plants can be improved by these circumstances. The procedure began with cultivating callus on Murashige dan Skoog (MS) media supplemented with 30 g L-1 sucrose, 5.5 g L-1 agar, 0.25 mg L-1 TDZ, and 1.0 mg L-1 NAA. The embryogenic callus was then irradiated with gamma rays at dosages of 0, 15, 30, 45, and 60 Gy before being subcultured on MS medium for four weeks. The percentage of living callus, callus fresh weight, and callus weight gain decreased gradually as the irradiation dose rose as compared to the control. The treatment without irradiation (control) resulted in the highest percentage of callus viability, fresh weight, and weight increase. Treatment with 60 Gy reduced callus viability by 50 %, compared to 94.4 ± 0.06 % (control). The callus' fresh weight was reduced to 39.28 %, compared to 0.28 ± 0.06 % in the 30-Gy treatment. At 60 Gy exposure, the lowest obtained callus weight was 1.96 ± 0.62 %, a 41.67 % decrease from 3.36 ± 1.29 (control). The median lethal dose (LD50) was obtained with an irradiation dose of 49.5 Gy using the regression equation Y = 0.7407x + 13.333. The correlation coefficient (r) is 0.91, whereas the coefficient of determination (r2) is 0.84. A 45 Gy dose of radiation resulted in morphological changes in patchouli mutant plantlets.