Rapid In Vitro Regeneration and Genetic Fidelity Assessment of Regenerated Plants in Ayapana Triplinervis (Vahl) R.M. King & H. Robinson: An Ethnomedicinal and Ornamental Herb

Background: Ayapana triplinervis is a popular ethnomedicinal and ornamental plant species. Due to its high medicinal importance, A. triplinervis was recently documented in the French Pharmacopeia. Objective: Rapid and efficient tissue culture protocol development is crucial for the high production and biotechnological applications of this plant. Methods: In this study, an efficient tissue culture protocol was developed for plant regeneration using nodal explants of A. triplinervis. The nodal explants were treated in Murashige and Skoog’s (MS) medium supplemented with various individual concentrations of cytokinins (BAP and KIN) and auxins (IAA and IBA). The nodal explant was regenerated in three different steps: (1) initial shoot induction, (2) shoot multiplication and elongation, and (3) rooting. Results: The results revealed that all individual concentrations (10, 20, 30, or 40 mg/L) of BAP or KIN responded to induce shoot initiation. The highest shoot multiplication and elongation were achieved in the MS medium with 20 mg/L BAP and 20 mg/L KIN. The regenerated plantlets produced better roots on MS medium containing 1.0 mg/L of each IAA or IBA. The well-established rooted plantlets were maintained in the culture room and greenhouse for better acclimatization and achieved a 100% survival rate. We analyzed the genetic fidelity of in vitro regenerated plants using random amplified polymorphic DNA (RAPD) markers. No genetic polymorphisms were observed in vitro plants compared to the mother plants. Conclusion: This efficient protocol could benefit future biotechnological applications like mass multiplication, genetic transformation and gene editing for improving the bioactive molecules in A. triplinervis.