Meta-Topolin enhanced direct shoot organogenesis and regeneration from leaf explants of Coleus forskohlii (Willd.) Briq

Abstract

A novel and effective direct shoot regeneration technique from stomatal complexes was developed for a highly sought after medicinal plant Coleus forskohlii. Meta-Topolin was more efficient than 6-benzylaminopurine (BAP) in promoting direct organogenesis from leaf surface. Highest direct organogenesis was achieved from in vitro leaf explants on Murashige and Skoog (MS) medium augmented with meta-Topolin at the concentration of 2.0 mg/L. The regenerated leaves were fixed at weekly intervals and sectioned longitudinally to evaluate the anatomical developments. The results of light microscopic analysis suggested that the in vitro developed stomatal complexes showed new altered cell divisions, after a week of incubation, and the rapid periclinal divisions resulted in the formation of a meristematic dome. Distinct regions of meristematic activity were visible in the second week characterized by promeristems with small and densely stained parenchymatous cells. Promeristems lead to the development of a distinct tunica layer, which grew into shoot-like protuberances converting into adventitious shoots after 4 weeks of incubation. A clear three phases of shoot organogenesis viz. induction (7 days), initiation and organization (14 days), and shoot formation (28 days onwards) were identified. The shoots were proliferated (21.8 shoots/ single leaf explant) and elongated (5.0 cm length) using half-MS medium fortified with 0.5 mg/L mT and indole-3 acetic acid (IAA) at 0.1 mg/L. Notable rhizogenesis of the elongated shoots was achieved when cultured on half-MS medium containing indole-3 butyric acid (IBA) at the concentration of 1.5 mg/L. About 92.0% of the plantlets survived upon field transfer. Start Codon Target (SCoT) markers were employed to examine the regenerated plants’ genetic uniformity with the mother plant, similar banding patterns confirming the genetic uniformity. Hence, the developed protocol of direct in vitro regeneration of C. forskohlii using mT can be used for the commercial production.