Induction, Metabolite Analysis, and Transgenesis of Hairy Roots from Coleus forskohlii
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Coleus forskohlii (Willd.) Briq. (Lamiaceae) is a medicinal herb cultivated in the subtropical and temperate areas of India, Myanmar, and Thailand. It is known for its antihypertensive and anti-obesity properties which are primarily attributed to its major bioactive metabolite: forskolin. Other bioactive metabolites produced by C. forskohlii include 1,9-dideoxyforskolin (anticancer) and genkwanin (anti-inflammatory). All major bioactive labdane diterpenes produced by C. forskohlii are accumulated in its roots, harvesting of which requires uprooting the plant. Further, its production is severely impacted by changes in weather, soil condition, rainfall pattern, availability of water, as well as biotic factors such as nematode infections of root, which significantly reduces the biomass and quantity of metabolites. The biosynthetic pathway of forskolin is now largely understood, and metabolic engineering may be employed to increase the production of forskolin and related labdane diterpenes or even for production of novel metabolites. Agrobacterium rhizogenes induced hairy roots are a unique system that can be exploited for sustainable and continuous production of metabolites as well as for genetic analysis and engineering secondary metabolite pathways through introduction of transgenes or targeted deletions. Hairy roots are characterized by high growth rate, genetic stability, and culture in hormone-free media. In this chapter, we provide protocol optimized in our lab, for induction, transgenesis, and maintenance of hairy root culture of C. forskohlii, along with the optimized protocols for extraction and quantification of key metabolites.
KeywordsColeus forskohlii Forskolin Agrobacterium rhizogenes Hairy root
VLJ carried out the work related to establishment and maintenance of hairy root cultures, PCR-based confirmation, microscopic localization, and TLC. She also prepared the figures and wrote the manuscript. IAR carried out the work related to subcloning of Osmotin gene, preparation of transgenic hairy roots, and confirmation and expression analysis of Osmotin. NK prepared the extracts from roots and hairy roots of C. forskohlii and also assisted VLJ in writing the manuscript and preparation of figures. SGG designed the study, supervised the work, and corrected the final manuscript and figures.
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