High-frequency shoot regeneration, assessment of genetic fidelity, and histochemical analysis of forskolin production in Coleus forskohlii Briq.

Abstract

Forskolin, a diterpenoid found in the roots of Coleus forskohlii, has generated significant interest in the medical field due to its various therapeutic uses. This study aimed to establish an effective system for regenerating C. forskohlii plants, ensuring a year-round supply of plant material and forskolin production. We tested different concentrations of cytokinins, either alone or combined with auxin, to see their impact on shoot multiplication and growth. We found that a medium supplemented with 1.5 mg L^-1 of meta-topolin (mT) resulted in the highest number of shoots (~ 12.66) and leaves (~ 20) within about 5 days. When mT (1 mg L^-1) was combined with a low amount of auxin (0.05 mg L^-1 NAA), we obtained an even greater number of leaves (~ 23). The shoot regeneration capacity was consistent over five subculture passages, showing minimal variation in mean shoot length and number. During acclimatization, around 91% of the plantlets grown in vermiculite + sand survived. The photosynthetic pigment concentration in the plantlets modestly increased in the first 10 days and reached its highest level after 30 days. Genetic fidelity assays using inter simple sequence repeats (ISSRs) confirmed the similarity between the in vitro derived plantlets and the mother plant. Micro-morphological features of in vitro and ex-vitro acclimated plantlets also matched those of the mother plant, further confirming genetic accuracy. Histochemical staining with vanillin confirmed the presence of forskolin in the in vitro roots, indicated by the violet coloration in the cells. Forskolin quantification was also validated by HPLC where in vitro derived roots were documented to undergo an almost ~ 1.8-fold in comparison to that of the mother plant. This established protocol can effectively address resource scarcity for commercial-scale forskolin production and sustainable conservation techniques.