Aenhenrya rotundifolia is a critically endangered terrestrial jewel orchid. It is monotypic and endemic to evergreen forests of southern western ghats of India. In the present study, identification of this plant species is validated with DNA barcoding using matK and rbcL chloroplast markers. Further, germ-free juvenile axillary bud explants were cultured on Mitra medium supplemented with different kinds of cytokinins like 6-benzyladenine, 6-furfurylaminopurine, N⁶-(Δ²-isopentyl) adenine, thidiazuron, zeatin and meta-topolin as well as auxins such as α-naphthaleneacetic acid, indole-3-acetic acid and indole-3-butyric acid at different concentrations and combinations for successful proliferation and establishment in vitro. After 12 weeks of culture, axillary bud explants produced an average of 30.12 ± 0.71 shoots per explant, 3.87 ± 0.06 cm shoot length, 1671 ± 2.82 mg fresh mass of proliferated shoots with a proliferation frequency of 100% on Mitra medium supplemented with 6.20 µM meta-topolin and 2.25 µM thidiazuron. No root formation was observed in in vitro proliferated microshoots. However, tiny hair like projections were observed in some elongated shoots on Mitra medium pertaining to 5.37 µM NAA. The tiny hair like structure bearing plantlets were hardened and acclimatized with 100% survival rate in the polytunnel chamber. After 8–10 months of establishment ex vitro, flowering was observed. Additionally, the genetic fidelity of in vitro derived plants was tested with ISSR and SCoT marker profiling. The test results revealed that the plants derived from the protocol has 99% genetic similarity to that of the donor mother plant. This study can be applied in forensic interventions of this species, describes the maintenance of germplasm in vitro and establishment of new viable population in its original habitats by restoring existing sites of this critically endangered jewel orchid.

Aenhenrya rotundifolia是一种极度濒危的陆生宝石兰花。它是印度西南部高止山脉的常绿森林的单种和地方性植物。在本研究中，该植物物种的鉴定通过使用mat K 和rbc L 叶绿体标记的DNA 条形码进行了验证。此外，将无菌幼年腋芽外植体在添加了不同种类的细胞分裂素如 6-苄基腺嘌呤、6-糠基氨基嘌呤、N ⁶ -(Δ ²-isopentyl) 腺嘌呤、噻二唑隆、玉米素和间位托蛋白，以及不同浓度和组合的 α-萘乙酸、吲哚-3-乙酸和吲哚-3-丁酸等生长素，可在体外成功增殖和建立。培养 12 周后，腋芽外植体每个外植体平均产生 30.12 ± 0.71 个芽、3.87 ± 0.06 cm 芽长、1671 ± 2.82 mg 新鲜质量的增殖芽，在 Mitra 培养基上补充 6.20 µM 的增殖频率为 100% meta-topolin 和 2.25 µM 噻二唑脲。在体外增殖的微芽中没有观察到根的形成。然而，在与 5.37 µM NAA 相关的 Mitra 培养基上的一些细长枝条中观察到微小的头发状突起。带有小植株的细小毛发状结构在多隧道室中硬化并适应环境，成活率为 100%。成立 8-10 个月后体外，观察到开花。此外，体外衍生植物的遗传保真度通过 ISSR 和 SCoT 标记分析进行了测试。测试结果显示，来自该协议的植物与供体母株具有 99% 的遗传相似性。本研究可应用于该物种的法医干预，描述了通过恢复这种极度濒危的宝石兰的现有地点，在体外维持种质并在其原始栖息地建立新的可行种群。