Indirect organogenesis, ex vitro rooting, and protein profiling of callus cultures of Vincetoxicum spirale: a threatened climber

Vincetoxicum spirale (Forssk.) D.Z.Li, a threatened perennial climber with therapeutic values, belongs to the family Apocynaceae (Subfamily: Asclepiadoideae). Constraints related to seeds and seedling establishment, coupled with wide-spread habitat destruction depleted natural populations of this species. This study presents the first report of an efficient indirect organogenesis protocol using mature leaf-derived callus of V. spirale. Of the four types of explants evaluated (leaf, node, internode and root), leaf explants were found to be the most suitable for culture establishment and de novo organogenesis. Leaf explants cultured on Murashige and Skoog (MS, 1962) medium containing 2.0 mg L−1 of 2,4-dichlorophenoxyacetic acid (2,4-D) showed the highest response (76.7%) for callus induction. Optimum callus proliferation was achieved on MS medium supplemented with 1.0 mg L−1 of 2,4-D and 0.5 mg L−1 of Kinetin (Kin), which produced morphogenic, competent, and light-green-friable callus. Shoot differentiation was observed on MS medium containing various concentrations of 6-benzylaminopurine (BAP) or Kin alone and in combination with Indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA). Of these, MS medium consisted of 0.5 mg L−1 of BAP, 0.25 mg L−1 of Kin, 0.1 mg L−1 of NAA, additives (25 mg L−1 each of adenine sulphate, L-arginine, citric acid and 50 mg L−1 of ascorbic acid) and 100 mg L−1 of activated charcoal (AC) differentiated the highest number of shoots (30.17 ± 1.51) measuring shoot length 6.26 ± 0.82 cm. More than 75% shoots optimally rooted ex vitro after being pulse-treated with 300 mg L−1 of NAA for 5 min. About 70% of them were successfully survived on transplantation in polybags. The Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) protein profiles of non-regenerative (NR) and regenerative (R) calli were compared to determine the responsible polypeptides and their expression levels in these two conditions. The protocol can be employed for the large-scale regeneration, ex situ conservation and biotechnological applications of this important threatened climber.