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An efficient in vitro plant regeneration system has been developed using dark preincubated leaf explants of Rhodiola crenulata, a traditional Tibetan medicinal plant. The leaf explants, preincubated in the dark for 5 d, developed an average of 9.1 shoots per explant on a medium containing 15 μM N 6-benzyladenine (BA) and 2.5 μM gibberellic acid (GA3). The biochemical mechanism underlying dark-induced shoot organogenesis was investigated by measuring polyphenol oxidase (PPO) activity. Dark preincubation significantly reduced PPO activity in leaf explants during the initial period of shoot organogenesis and reduced browning compared to explants cultured in the light. Up to 88.4 % of the regenerated shoots formed roots and developed into complete plantlets on a medium containing 5 μM indoleacetic acid (IAA) within 25 d. Approximately 82 % of the regenerated plantlets survived transplantation and grew vigorously in the greenhouse.