Efficient in vitro plant regeneration from leaf-derived callus and genetic fidelity assessment of an endemic medicinal plant Ranunculus wallichianus Wight & Arnn by using RAPD and ISSR markers

Abstract

Ranunculus wallichianus is a medicinally important plant and an endemic species to the Western Ghats of South India. An efficient and reliable indirect regeneration protocol system for R. wallichianus was developed from leaf explants in the present investigation. Leaf explants were cultured on both full-strength and half-strength MS (Murashige & Skoog) medium supplemented with different concentrations (0.5 mg L− 1 to 3.0 mg L− 1) of 2,4-D and NAA. Among the different concentrations tested, the highest percentage of yellowish-green compact nodular callus formation was observed on a half-strength MS medium with 2.0 mg L− 1 of 2, 4-D. Then, the in vitro raised organogenic callus was cultured on a half-strength MS medium containing various concentrations (0.5 mg L− 1 to 3.0 mg L− 1) of BA, KIN, and TDZ with 0.5 mg L− 1 NAA and 10 % CW for in vitro shoot regeneration. The highest percentage of regeneration response (97 %) and a maximum number of shoots formation (11.1 ± 0.13 shoots/culture with 9.2 ± 0.21 cm mean shoot length) were obtained from MS medium containing 2.5 mg L− 1 BA with 0.5 mg L− 1 NAA and 10 % CW. The well elongated in vitro raised shoots were effectively rooted in a half-strength MS medium with 2.5 mg L− 1 IBA + 250 mg L− 1 activated charcoal. The well-rooted plantlets were successfully hardened and acclimatized with a survival rate of 94 %. Clonal fidelity of in vitro raised plantlets was assessed by using DNA-based RAPD and ISSR molecular markers. A total of 56 and 47 monomorphic bands were obtained from RAPD and ISSR markers respectively. This present in vitro propagation protocol system could be effective for the conservation of R. wallichianus with their genetic purity and its further investigations.