The basic medium containing half-strength of MS basal medium, full-strength of vitamin MS, 20 ml l−1 coconut water and 2 g l−1 activated charcoal supplemented with 170 mg l−1 NaH2 PO4 , 10 mg l−1 2,4D and 0.1 mg l−1 TDZ was found to be appropriate for callus formation of Paphiopedilum delenatii and Paphiope- dilum callosum. Additional supplementation with 2.0 mg l−1 BA and 0.1 mg l−1 TDZ in combination with NaH2 PO4 and 50 mg l−1 oligochitosan enhanced PLB multiplication. Shoot bud proliferation of tested orchids was optimally increased in a basic medium supplemented with 0.5 mg l−1 TDZ and 0.1 mg l−1 NAA. The basic medium supplemented with NaH2 PO4 was suitable for plantlet regeneration. In addition, the values of LD50 (lethal dosage of 50% irradiated samples) of protocorm like bodies, shoot buds and in vitro plantlets were determined for P. delenatii (20 Gy, 23.7 Gy and 38 Gy, respectively) and P. callosum (23 Gy, 27.1 Gy and 40.4 Gy, respectively). No variant line was found for both orchid samples irradiated by gamma-rays, whereas 24 variant lines (which are divided among the two tested Paphiopedilum) were screened for samples irradiated by C6+ ion-beams (3 Gy). The genetic relationships among six gener- ated variant lines and wild types were analyzed using RAPD techniques. Irradiation-induced variation is promising for developing new mutant varieties of Paphiopedilum.