An in vitro culture system for achieving high-efficiency regeneration of Begonia rex Putz. was optimised. Leaf explants of B. rex were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 1-Naphthaleneacetic acid (NAA), and Indole-3-butyric acid (IBA) in combination with or without various concentrations of 6-benzyladenine (BA) to assess callus and direct bud induction. The highest mean number of direct bud organogenesis (23.20) per leaf explant was observed on MS medium supplemented with 1.00 mg L−1 NAA + 1.00 mg L−1 BA. Compared with direct regeneration, the rate of indirect bud organogenesis was very low and varied from 0.33 ± 0.33 to 2.25 ± 0.64 in PGR-free (free of plant growth regulators) medium and MS supplemented with 1.50 mg L−1 BA. Single shoots were excised and transferred to proliferation of bud medium. The highest bud proliferation (12.00 ± 1.15 bud per single shoot) was observed in medium supplemented with 0.50 mg L−1 BA. The developed shoots were rooted on PGR-free MS medium with 100% efficiency. Finally, the rooted plantlets were successfully acclimatised into plastic pots that contained pre-autoclaved perlite and peat moss (1:1).