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Croomia japonica (Stemonaceae) is an endangered species both in China and Japan. We have developed an efficient regeneration system through adventitious buds organogenesis in C. japonica using rhizome buds as explants. Multiple buds regenerated directly on the explants without calli within 2 months when explants were cultured on Murashige and Skoog’s (MS) medium with 8.88 µmol 6-benzylaminopurine (BAP) and 1.07 µmol α-naphthaleneacetic acid (NAA). The adventitious buds of newly forming were proliferated by subsequent subcultures on MS medium with 2.66 µmol BAP and 2.69 µmol NAA. We evaluated the kinds and concentrations of plant growth regulators on adventitious shoot regeneration and root induction and also inspected the adsorbent (polyvinyl pyrrolidone and activated charcoal) and antioxidant (ascorbic acid, AS) on the inhibition of tissue browning. The results showed that soaking the explants with 1.14 mmol AS was the best approach for controlling browning. The maximum number of stout shoots per explant was achieved on MS medium containing 8.88 µmol BAP. In vitro regenerated shoots were rooted on MS medium supplemented with three different concentrations of auxin. The highest rooting rate (84.0 ± 3.6%) was reached on MS medium with 5.71 µmol Indole-3-acetic acid (IAA). One-step culture was developed when the adventitious buds cultured on the medium were supplemented with 2.66 µmol BAP and 0.54-2.69 µmol NAA. Rooted plantlets were acclimatized to the green house and development with a 87% survival rate. Genetic stability assessment of in vitro plants compared to the wild plants was revealed by simple sequence repeat markers. Similarly, flow cytometric analysis confirmed that the ploidy level of in vitro plantlets was stable. Total alkaloid content in wild and in vitro plants was tested by acid dye colorimetric analysis with tuberostemonine as the reference. Our results showed that alkaloids content in 11-week cultures reached 40.7-88.4% of the content of wild plants. The protocol described here could be employed for effective mass propagation of C. japonica for commercial and conservational purposes. [ABSTRACT FROM AUTHOR]