Induction, Metabolite Analysis, and Transgenesis of Hairy Roots from Coleus forskohlii

  • Vijay Lakshmi Jamwal
  • Irshad Ahmad Rather
  • Nitika Kapoor
  • Sumit G. GandhiEmail author
Part of the Rhizosphere Biology book series (RHBIO)


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.


Coleus forskohlii Forskolin Agrobacterium rhizogenes Hairy root 


Author Contributions

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.


  1. Ahmad S, Rizwan M, Parveen R, Mujeeb M, Aquil M (2008) A validated stability-indicating TLC method for determination of forskolin in crude drug and pharmaceutical dosage form. Chromatographia 675-6:441–447CrossRefGoogle Scholar
  2. Alasbahi RH, Melzig MF (2010a) Plectranthus barbatus: a review of phytochemistry, ethnobotanical uses and pharmacology–part 1. Planta Med 7607:653–661CrossRefGoogle Scholar
  3. Alasbahi RH, Melzig MF (2010b) Plectranthus barbatus: a review of phytochemistry, ethnobotanical uses and pharmacology–part 2. Planta Med 7608:753–765CrossRefGoogle Scholar
  4. Biswas B, Scott PT, Gresshoff PM (2011) Tree legumes as feedstock for sustainable biofuel production: opportunities and challenges. J Plant Physiol 16816:1877–1884CrossRefGoogle Scholar
  5. Bolton GW, Nester EW, Gordon MP (1986) Plant phenolic compounds induce expression of the Agrobacterium tumefaciens loci needed for virulence. Science 232(4753):983–985CrossRefPubMedGoogle Scholar
  6. Bourgaud F, Gravot A, Milesi S, Gontier E (2001) Production of plant secondary metabolites: a historical perspective. Plant Sci 1615:839–851CrossRefGoogle Scholar
  7. Chen SC, Liu HW, Lee KT, Yamakawa T (2007) High-efficiency Agrobacterium rhizogenes-mediated transformation of heat inducible sHSP18. 2-GUS in Nicotiana tabacum. Plant Cell Rep 26(1):29–37CrossRefPubMedGoogle Scholar
  8. De Souza NJ, Dohadwalla AN, Reden Ü (1983) Forskolin: a labdane diterpenoid with antihypertensive, positive inotropic, platelet aggregation inhibitory, and adenylate cyclase activating properties. Med Res Rev 32:201–219CrossRefGoogle Scholar
  9. Dixon RA, Paiva NL (1995) Stress-induced phenylpropanoid metabolism. Plant Cell 7(7):1085CrossRefPubMedPubMedCentralGoogle Scholar
  10. Fattahi M, Nazeri V, Torras-Claveria L, Sefidkon F, Cusido RM, Zamani Z, Palazon J (2013) A new biotechnological source of rosmarinic acid and surface flavonoids: hairy root cultures of Dracocephalum kotschyi Boiss. Ind Crop Prod 50:256–263CrossRefGoogle Scholar
  11. Godard MP, Johnson BA, Richmond SR (2005) Body composition and hormonal adaptations associated with forskolin consumption in overweight and obese men. Obes Res 138:1335–1343CrossRefGoogle Scholar
  12. Grąbkowska R, Królicka A, Mielicki W, Wielanek M, Wysokińska H (2010) Genetic transformation of Harpagophytum procumbens by Agrobacterium rhizogenes: iridoid and phenylethanoid glycoside accumulation in hairy root cultures. Acta Physiol Plant 32(4):665–673CrossRefGoogle Scholar
  13. Guillon S, Trémouillaux-Guiller J, Pati PK, Rideau M, Gantet P (2006) Hairy root research: recent scenario and exciting prospects. Curr Opin Plant Biol 9(3):341–346CrossRefPubMedGoogle Scholar
  14. Habibi P, Soccol CR, Grossi-de-Sa MF (2018) Hairy root-mediated biotransformation: recent advances and exciting prospects. In: Hairy roots. Springer, Singapore, pp 185–211CrossRefGoogle Scholar
  15. Han L-K, Morimoto C, Yu R-H, Okuda H (2005) Effects of Coleus forskohlii on fat storage in ovariectomized rats. Yakugaku Zasshi 1255:449–453CrossRefGoogle Scholar
  16. Hess KM, Dudley MW, Lynn DG, Joerger RD, Binns AN (1991) Mechanism of phenolic activation of Agrobacterium virulence genes: development of a specific inhibitor of bacterial sensor/response systems. Proc Natl Acad Sci 88(17):7854–7858CrossRefPubMedGoogle Scholar
  17. Holsters M, De Waele D, Depicker A, Messens E, Van Montagu M, Schell J (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genomics 1632:181–187CrossRefGoogle Scholar
  18. Hou P, Li Y, Zhang X, Liu C, Guan J, Li H, Zhao T, Ye J, Yang W, Liu K (2013) Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science 3416146:651–654CrossRefGoogle Scholar
  19. Hu ZB, Du M (2006) Hairy root and its application in plant genetic engineering. J Integr Plant Biol 482:121–127CrossRefGoogle Scholar
  20. Kiselev KV, Turlenko AV, Tchernoded GK, Zhuravlev YN (2009) Nucleotide substitutions in rolC and nptII gene sequences during long-term cultivation of Panax ginseng cell cultures. Plant Cell Rep 28(8):1273CrossRefPubMedGoogle Scholar
  21. Królicka A, Staniszewska I, Bielawski K, Maliński E, Szafranek J, Łojkowska E (2001) Establishment of hairy root cultures of Ammi majus. Plant Sci 160(2):259–264CrossRefGoogle Scholar
  22. Narayanan P, Laddha K, Akamanchi K (2002) Histochemical localization of forskolin and other terpenoids in Coleus forskohlii. Curr Sci 838:945–946Google Scholar
  23. Nishimura A, Aichi I, Matsuoka M (2006) A protocol for Agrobacterium-mediated transformation in rice. Nat Protoc 1(6):2796CrossRefPubMedGoogle Scholar
  24. Pateraki I, Andersen-Ranberg J, Hamberger B, Heskes AM, Martens HJ, Zerbe P, Bach SS, Møller BL, Bohlmann J, Hamberger B (2014) Manoyl oxide (13R), the biosynthetic precursor of forskolin, is synthesized in specialized root cork cells in Coleus forskohlii. Plant Physiol 1643:1222–1236CrossRefGoogle Scholar
  25. Rao SR, Ravishankar G (2002) Plant cell cultures: chemical factories of secondary metabolites. Biotechnol Adv 202:101–153Google Scholar
  26. Rather IA, Awasthi P, Mahajan V, Bedi YS, Vishwakarma RA, Gandhi SG (2015) Molecular cloning and functional characterization of an antifungal PR-5 protein from Ocimum basilicum. Gene 5581:143–151CrossRefGoogle Scholar
  27. Sasaki K, Udagawa A, Ishimaru H, Hayashi T, Alfermann A, Nakanishi F, Shimomura K (1998) High forskolin production in hairy roots of Coleus forskohlii. Plant Cell Rep 17(6–7):457–459CrossRefPubMedGoogle Scholar
  28. Shan Y, Xu L, Lu Y, Wang X, Zheng Q, Kong L, Niwa M (2008) Diterpenes from Coleus forskohlii (W ILLD.) B RIQ.(Labiatae). Chem Pharm Bull 561:52–56CrossRefGoogle Scholar
  29. Srivastava S, Srivastava AK (2007) Hairy root culture for mass-production of high-value secondary metabolites. Crit Rev Biotechnol 27(1):29–43CrossRefGoogle Scholar
  30. Stachel SE, Messens E, Van Montagu M, Zambryski P (1985) Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318(6047):624CrossRefGoogle Scholar
  31. Stiles AR, Liu CZ (2013) Hairy root culture: bioreactor design and process intensification. In: Biotechnology of hairy root systems. Springer, Berlin/Heidelberg, pp 91–114CrossRefGoogle Scholar
  32. Suneetha DRS, Arundhati A, Rao GS, Joshua PV (2009) Differential methylation pattern of RolA, B and C genes of Agrobacterium rhizogenes in Nicotiana glauca and its hybrid. Asian J Plant Sci 8(5):361–367CrossRefGoogle Scholar
  33. Tepfer D (1990) Genetic transformation using Agrobacterium rhizogenes. Physiol Plant 791:140–146CrossRefGoogle Scholar
  34. Tzfira T, Li J, Lacroix B, Citovsky V (2004) Agrobacterium T-DNA integration: molecules and models. Trends Genet 208:375–383CrossRefGoogle Scholar
  35. Xi J, Patel M, Dong S, Que Q, Qu R (2018) Acetosyringone treatment duration affects large T-DNA molecule transfer to rice callus. BMC Biotechnol 18(1):48CrossRefPubMedPubMedCentralGoogle Scholar
  36. Ziegler FE, Jaynes BE, Saindane MT (1985) A C6, C7 oxygen functionalized intermediate for the synthesis of forskolin: stereochemical control in an intramolecular Diels-Alder reaction. Tetrahedron Lett 2628:3307–3310CrossRefGoogle Scholar
  37. Ziegler FE, Jaynes BH, Saindane MT (1987) A synthetic route to forskolin. J Am Chem Soc 10926:8115–8116CrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2020

Authors and Affiliations

  • Vijay Lakshmi Jamwal
    • 1
  • Irshad Ahmad Rather
    • 1
  • Nitika Kapoor
    • 1
  • Sumit G. Gandhi
    • 1
    Email author
  1. 1.Plant Biotechnology DivisionCSIR-Indian Institute of Integrative MedicineJammu CantonmentIndia

Personalised recommendations