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.
Highlights
In India, Ayurveda is a traditional medicinal system which has been practiced for thousands of years and remains as one of the most ancient and yet living traditions across the country, which has a sound philosophical and experimental basis [1, 2]
Stem, and root explants of R. tetraphylla cultured on Murashige and Skoog (MS) basal medium did not produce any callus, but when cultured on MS medium supplemented with various concentrations of plant growth regulators (2,4-D, naphthalene acetic acid (NAA), and Kn), copious amounts of friable callus were produced, after 4 weeks of culture (Figures 1(a)–1(d))
DNA bands produced by amplification with randomly amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR), and start codon targeted (SCoT) markers were monomorphic across the micro propagated plantlets and the mother plant (Figures 8, 9, and 10), which confirms the true-to-type and genetically stable nature of acclimated plantlets of R. tetraphylla
Summary
In India, Ayurveda is a traditional medicinal system which has been practiced for thousands of years and remains as one of the most ancient and yet living traditions across the country, which has a sound philosophical and experimental basis [1, 2]. Ex Kurz is indigenous to India [10, 11] and this species is on the verge of extinction [12] due to its overexploitation by ayurvedic practitioners and pharmaceutical industries [13] and Government of India has restricted the exploitation of this endangered species [14] In this scenario the attention of industrialists and medicinal practitioners was diverted to find an alternative plant species as a source of pharmaceutical alkaloids. It is being cultivated widely as both an ornamental and a source of pharmaceuticals This species produces different type of pharmaceutically important alkaloids in their stem bark and roots such as reserpine, deserpidine, ajmalicine, ajmaline, canescine, yohimbine, and recinnamine [19, 20]. In this study we are reporting a protocol for its conservation through high frequency of plantlet production by using combination of cytokinins amended media
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