Indirect Regeneration and Assessment of Genetic Fidelity of Acclimated Plantlets by SCoT, ISSR, and RAPD Markers in Rauwolfia tetraphylla L.: An Endangered Medicinal Plant

Abstract

Rauwolfia tetraphylla L. is an important medicinal plant species which is well known for its pharmaceutically important alkaloids. In the present study, we are reporting about its conservation by in vitro clonal multiplication through the standardized protocol of indirect regeneration by using leaf and stem based callus and assessment of genetic fidelity of acclimated plantlets by start codon targeted (SCoT), inter simple sequence repeats (ISSR), and randomly amplified polymorphic DNA (RAPD) marker based analysis. Initially friable callus was induced in maximum amounts (378.7, 323.8, and 412.8 in mg) from leaf, root, and stem explants on Murashige and Skoog (MS) media supplemented with 5.0 mg/L, 3.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0 mg/L of naphthalene acetic acid (NAA), respectively. Shoot regeneration with the maximum number of shoot buds (25 and 20) was obtained from leaf and stem calluses on MS media supplemented with TDZ (0.25 mg/L) + BAP (2 mg/L). The regenerated shoots were rooted successfully with maximum rooting percentage of 98.0 on full strength MS media amended with IAA (1.0 mg/L) and IBA (1.0 mg/L). The regenerated plantlets were hardened using 2:1 ratio of sterile garden soil and sand, followed by acclimatization in field conditions with 86% of survival. SCoT, ISSR, and RAPD primers based polymerase chain reaction (PCR) analysis was carried out to check possible genetic variations in micro propagated plants in comparison with mother plant. Among the ten SCoT (S), ISSR (R), and RAPD (OPA) primers used, S2, R10, and OPA3 has given good amplification with scorable DNA bands. The results revealed that the regenerated plants did not have any polymorphism with mother plant. Hence, the in vitro regenerated R. tetraphylla plantlets were confirmed as true-to-type.