Begonia rex cv. DS-EYWA is an important plant for indoor and outdoor cultivation, and cv. DS-EYWA is a rare unique cultivar with curly, colorful leaves. Due to their importance, applying plant tissue culture techniques for mass and healthy production in a short period of time without seasonal limitation is of immense economic value. Applying several concentrations of silver nitrate (AgNO3) in combination with varied concentrations of cytokinins including 6-benzylaminopurine (BAP), thidiazuron (TDZ) (0, 0.5, 1, 1.5 mgL−1), and 1-naphthaleneacetic acid (NAA) auxin (0, 0.5, 1 mgL−1) via focusing on transverse thin cell layer (tTCL) petiole explants for high-scale production was used to establish an efficient in vitro propagation protocol. Our results showed that even low concentrations (25 mgL−1) can control internal bacterial infection and increase shoot direct regeneration efficiency. A combination of 1.5 mgL−1 BAP, 0.5 mgL−1 NAA, and 25 mgL−1 AgNO3 was the best treatment to increase the number of direct regenerated shoots, and a lower concentration of BAP (0.5 mgL−1) can be suggested for shoot elongation. Elongated shoots were successfully rooted in MS basal medium and acclimatized in a 1:1 peat moss/perlite sterilized pot mixture.