Transverse thin cell layer (t-TCL)-mediated improvised micropropagation protocol for endangered medicinal orchid Dendrobium aphyllum Roxb: an integrated phytomolecular approach

Clonal propagation is an important biotechnological tool for the conservation of rare, endangered and threatened as well as commercially important plant species of which orchids are an important representative. In the present research, an attempt was made to develop a fast, clonally stable as well as phytochemically enriched regeneration protocol for propagating Dendrobium aphyllum, an important medicinal orchid using transverse thin cell layer (t-TCL) explants. Murashige and Skoog (MS) medium supplemented with 15 µM meta-topolin along with 10 µM TDZ and 10 µM AgNO₃ was found to be most conducive for shoot proliferation. After 8 weeks of culture, an average of 39.41 shoots/explant proliferated; till date this is the best shooting frequency for D. aphyllum and one of the best amongst the orchids. The highest rooting frequency of 82.34% was accomplished in half-strength MS medium supplemented with 15 µM IBA. The plantlets were successfully acclimatized in greenhouse exhibiting normal developmental patterns and flowering occurred after 6 months of acclimatization with 3–4 flowers/stalk irrespective of the flowering season. The genetic homogeneity of the micropropagated plants was ascertained using IRAP and ISSR markers. A comparative assessment of the antioxidant activity (mother and micropropagated plant) was performed using DPPH and FRAP assays which was recorded to be higher amongst the in vitro-regenerated plants. A rapid, genetically stable regeneration protocol coupled with comprehensively higher phytochemical yield is reported in the present study on D. aphyllum. The results of this study confirm the use of in vitro propagation protocol for sustainable commercial utilization as well as conservation of medicinal orchid bio-resources within a limited time period.