Even after three decades of invention, plastid transformation has been very difficult especially for monocot crops. Difficulty in attaining, efficient in vitro regeneration is one of the many factors affecting plastid transformation. Hence, present study was planned to investigate the impact of 2,4-D and casein hydolysate on in vitro callogenesis and subsequent regeneration of different Pakistani rice cultivars. For this purpose, mature seeds of six Pakistani genotypes IR-6, KSK-133, KSK-434, KS-282, PK-386 and Super Basmati were used. Scutellum from mature rice seeds were incubated on MS medium augmented with 2,4-D (1-5mg/L) to study callogenesis and subsequent regeneration response. Maximum callus formation was obtained in variety IR-6 (2mg/L 2,4-D) and callus size was found increasing with increasing 2,4-D concentration. Later, synergistic effect of casein hydrolysate and 2,4-D was investigated in two steps. Firstly, effect of 2,4-D in combination with 6mg/L casein hydrolyste was observed. Maximum callogenesis was found in IR-6 at 2mg/L 2,4-D and 6mg/L casein hydrolysate. Secondly, effect of casein hydrolysate (4-8mg/L) with 2,4-D 2mg/L was examined and maximum callus formation was found in variety IR-6 (casein hydrolysate 4mg/L + 2,4-D 2mg/L). Then, calli were shifted to different combinations of Plant Growth Regulators (PGRs) i.e. NAA + Kinetin and IBA + BAP to optimize regeneration. Differential response of varieties was examined. Maximum regeneration was observed in PK-386 (2.5 plantlets/callus).