Development of an efficient integrated in vitro propagation system for virus-free Tongling white ginger and assessment of its genetic fidelity

Tongling white ginger, a national geographical indication product of China, has significant economic value in medicine, food, and culinary uses. However, germplasm degradation and the low propagation efficiency of traditional rhizome-based reproduction severely constrain its industrial development. Here, we established an integrated in vitro regeneration and virus elimination system encompassing rhizome-induced adventitious shoot formation, shoot tip culture with virus eradication, basal stem segment propagation, synchronous proliferation-elongation-rooting, transplantation, and genetic stability assessment. For rhizomes, MS medium containing 0.2 mg/L TDZ achieved an 85.55% induction rate (5.93 shoots/explant). For 2.0 mm shoot tips, MS medium fortified with 0.2 mg/L 6-BA resulted in a 44.19% regeneration rate with 100% virus elimination, while a proliferation coefficient of 3.48 was obtained on MS medium containing 0.05 mg/L TDZ and 0.2 mg/L IBA. For basal stem segments, MS medium supplemented with 0.1 mg/L TDZ and 0.05 mg/L NAA supported 100% induction, producing 8.09 shoots per explant. Synchronous culture on MS medium containing 2.0 mg/L 6-BA and 0.2 mg/L IAA yielded robust plantlets with a multiplication coefficient of 2.43 and an average of 9.17 roots per plantlet. Under optimal conditions and assuming loss-free material transfer between stages, the integrated system achieved a theoretical cumulative propagation coefficient of 68.41, with 100% transplant survival. ISSR analysis of the fifth subculture generations revealed no detectable genetic variation at the surveyed loci among the tested regenerants and the mother plant. This protocol provides a technical basis for the potential commercial production of virus-free, genetically uniform seedlings, which is essential for elite germplasm conservation and sustainable cultivation of Tongling white ginger. Establishment of a high-efficiency i n vitro regeneration system for Tongling white ginger. • Efficient virus-free regeneration of Tongling white ginger via shoot tip culture. • Integrated system achieved theoretical cumulative propagation coefficient of 68.41. • ISSR analysis verified no detectable genetic variation in regenerants after five subcultures. • Synchronizing shoot proliferation, elongation and rooting reduces the process from three steps to one.