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Hedychium coronarium J. Koenig is a rhizomatous herb (Zingiberaceae family) and is well known for its uses in traditional systems of medicine for the treatment of various diseases. The plant has been over-exploited and enlisted as a threatened species in India. Thus, there is an urgent attention required for its conservation. Here, a callus-mediated in vitro plant regeneration protocol was developed using leaf sheath of H. coronarium. The optimal medium for callus induction (85.0%) and subsequent proliferation was found to be MS basal medium augmented with 3.0 mg L−1 N6-benzyladenine (BA), 0.5 mg L−1 α-naphthaleneacetic acid (NAA), and 3.0% sucrose, and gelled with 0.5% agar. Optimum callus-mediated shoot organogenesis (78.3%; ca. 11.4 shoots/0.5 g of callus) was obtained on MS medium supplemented with 3.0 mg L−1 BA after 6 wk of culture. All the in vitro regenerated shoots were rooted (8.5 roots/shoot) successfully on plant growth regulator–free MS medium. About 90% plantlets were acclimatized on the planting tray filled with garden soil and sand (1:1). Transfer of these plants to larger pots containing garden soil and subsequent field transfer under full sun resulted in cent-percent survival. Monomorphic banding profile obtained using Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers ascertained the clonal fidelity of the in vitro regenerated plants. Similarly, the biochemical fidelity of the in vitro regenerated plants vis-à-vis mother plant was ascertained by comparing the composition of essential oil through gas chromatography/mass spectrometry (GC/MS) analysis. Furthermore, the antioxidant activities estimated by DPPH and ABTS free radical scavenging assay and anticancer activities evaluated against two cell lines, i.e., MCF 7 and MDA-MB 231, also confirmed comparable effectiveness of in vitro regenerated plants to that of the mother plant. Thus, the study has the potential to provide a platform to achieve sustainability by using the in vitro regenerated H. coronarium in place of naturally available population. [ABSTRACT FROM AUTHOR]