Establishment of adventitious root culture in Echinacea purpurea and enhanced accumulation of caffeic acid derivatives by biotic and abiotic elicitors

Document Type : Original research paper


1 Department of Plant Production and Genetics, Faculty of Agriculture, Malayer University, Malayer, Iran

2 Department of Biology, Faculty of Sciences, Malayer University, Malayer, Iran


The present study aimed to develop a protocol for root induction and evaluate the effects of salicylic acid (SA) (0, 80 and 160 mg/l) and yeast extract (YE) (0, 0.75 and 1.5 g/l) on chlorogenic acid, caftaric acid, cichoric acid, cynarin and echinacoside production in Echinacea purpurea adventitious roots. Also, the effect of NH4NO3 (0, 0.25, 0.75, 1.0 X) concentration in MS medium supplemented with indole-3-acetic acid (IAA) (1 and 3 mg/l) on root induction was investigated. The results showed that adventitious root induction in coneflower was significantly influenced by NH4NO3 and IAA concentrations (p≤0.01). The highest percentage of root induction (100%) and average number of roots formed on each explant (14.3 roots) was observed in 1 mg/l IAA×1/4NH4NO3 MS culture medium treatment. The main effect of SA and YE and their interaction effects with exposure time on the measured traits (except for echinacoside) was significant (p≤0.01). The result showed that application of 1.5 g/l YE and 160 mg/l SA when harvested 96 hour post-elicitation are the most effective treatments to elicit caffeic acid derivatives (CADs) content. The highest chlorogenic acid, cichoric acid, caftaric acid, and cynarin production was obtained in 160 mg/l SA at 96 hours post-elicitation that was 2.13, 1.83, 2.39 and 2.97-fold higher compared to control respectively. The heatmap diagram showed that the CADs content in SA and YE treatments was clearly separated from each other and control treatment.


[1] Abbasi, B.H., Saxena, P.K., Murch, SJ. and Liu, C.Z. 2007. Echinacea biotechnology: challenges and opportunities. In Vitro Cellular & Developmental Biology-Plant 43: 481-492. doi: 10.1007/s11627-007-9057-2.
[2] Abbasi, B.H., Stiles, A.R., Saxena, P.K. and Liu, C.Z. 2012. Gibberellic acid increases secondary metabolite production in Echinacea purpurea hairy roots. Applied Biochemistry and Biotechnology 168:2057–2066. doi: 10.1007/s12010-012-9917-z.
[3] Abdoli, M., Moieni, A. and Naghdi Badi, H. 2013. Influence of KNO3, CaCl2 and MgSO4 concentrations on growth and cichoric acid accumulation in hairy root culture of purple coneflower (Echinacea purpurea L.). Journal of Medicinal Plants 12: 75-84.
[4] Ali B. 2020. Salicylic acid: An efficient elicitor of secondary metabolite production in plants. Biocatalysis and Agricultural Biotechnology 31: 101884. doi: 10.1016/j.bcab.2020.101884.
[5] Arceusz, A., Wesolowski, M. and Konieczynski, P. 2013. Methods for extraction and determination of phenolic acids in medicinal plants: a review. Natural Product Communications 8 (12): 1821–1829.
[6] Baenas, N., Garcia-Viguera, C. and Moreno, D.A. 2014. Elicitation: A tool for enriching the bioactive composition of foods. Molecules 19: 13541-13563. doi: 10.3390 / molecules190913541.
[7] Barrett, B. 2003. Medicinal properties of Echinacea: a critical review. Phytomedicine 10 (1): 66–86. doi: 10.1078/094471103321648692.
[8] Brown, P.N. 2011. Determination of major phenolic compounds in Echinacea spp. raw materials and finished products by high-performance liquid chromatography with ultraviolet detection: single-laboratory validation matrix extension. Journal of AOAC International 94 (5): 1400-1410. doi:10.5740/jaoacint.11-142.
[9] Demirci, T., Aras Ascı, Ö. And Göktürk Baydar, N. 2021. Influence of salicylic acid and L-phenylalanine on the accumulation of anthraquinone and phenolic compounds in adventitious root cultures of madder (Rubia tinctorum L.). Plant Cell, Tissue and Organ Culture 144: 313–324.
[10] Ghasemzadeh, A., Jaafar, H.Z., Karimi, E. and Ibrahim, M.H. 2012. Combined effect of CO2 enrichment and foliar application of salicylic acid on the production and antioxidant activities of anthocyanin, flavonoids and isoflavonoids from ginger. BMC Complementary Medicine and Therapies 12: 229. doi: 10.1186/1472-6882-12-229.
[11] Gorni, P.H., Pacheco, A.C., Moro, A.L., Silva, J.F.A., Moreli, R.R., de Miranda, G.R., Pelegrini, J.M., Spera, K.D., Junior, J.L.B. and da Silva R.M.G. 2020. Salicylic acid foliar application increases biomass, nutrient assimilation, primary metabolites and essential oil content in Achillea millefolium L. Scientia Horticulturae 270: 109436. doi:10.1016/j.scienta.2020.109436.
[12] Hedayati, A. and Abdoli M. 2021. Foliar application of yeast extract and salicylic acid affect chemical composition and content of lemon balm (Melissa officinalis L.) essential oil. Journal of Plant Molecular Breeding 9 (1): 12 – 24. doi: 10.22058/JPMB.2022.544981.1248.
[13] Ho, T.T., Murthy, H.N. and Park, S.Y. 2020. Methyl Jasmonate induced oxidative stress and accumulation of secondary metabolites in plant cell and organ cultures. International Journal of Molecular Sciences 21(3): 716.
[14] Jeong, J.A., Wu, C.H., Murthy, H.N., Hahn, E.J. and Paek, K.Y. 2009. Application of an airlift bioreactor system for the production of adventitious root biomass and caffeic acid derivatives of Echinacea purpurea. Biotechnology and Bioprocess Engineering 14:91–98.
[15] Khanam, M.N., Anis, M., Javed, S.B., Mottaghipisheh, J. and Csupor, D. 2022. Adventitious Root Culture-An Alternative Strategy for Secondary Metabolite Production: A Review. Agronomy 12: 1178.
[16] Kochan, E., Szymczyk, P., Kuźma, Ł., Lipert, A. and Szymańska, G. 2017. Yeast extract stimulates ginsenoside production in hairy root cultures of American ginseng cultivated in shake flasks and nutrient sprinkle bioreactors. Molecules 22 (6): 880. doi: 10.3390/molecules22060880.
[17] Kuzel, S., Vydra, J., Triska, J., Vrchotova, N., Hruby, M. and Cigler, P. 2009. Elicitation of pharmacologically active substances in an intact medical plant. Journal of Agricultural and Food Chemistry 57: 7907-7911. doi: 10.1021/jf9011246.
[18] Lakehal, A. and Bellini C. 2019. Control of adventitious root formation: Insights into synergistic and antagonistic hormonal interactions. Physiologia Plantarum, Wiley 165 (1): 90-100 pp.
[19] Lema-Rumińska, J. Kulus, D., Tymoszuk, A., Varejão, J.M.T.B. and Bahcevandziev, K. 2019. Profile of secondary metabolites and genetic stability analysis in new lines of Echinacea purpurea (L.) Moench micropropagated via somatic embryogenesis. Industrial Crops & Products 142: 111851.
[20] Liu, M., Zhang, H., Fang, X., Zhang, Y. and Jin, C. 2018. Auxin acts downstream of ethylene and nitric oxide to regulate magnesium deficiency-induced root hair development in Arabidopsis thaliana. Plant and Cell Physiology 59: 1452-1465.
[21] Mahalakshmi, R., Eganathan, P. and Parida, A.K. 2013. Salicylic acid elicitation on production of secondary metabolite by cell cultures of Jatropha curcas L. International Journal of Pharmacy and Pharmaceutical Sciences 5: 655–659.
[22] Mehrpooya, Zh., Abdoli, M. and Talebian, M.A. 2021. Effect of salicylic acid and yeast extract on caffeic acid derivatives production in Echinacea purpurea L. Journal of Medicinal Plants 20 (78): 36-47. doi: 10.52547/jmp.20.78.36.
[23] Motiee, M. and Abdoli, M. 2021. Changes in essential oil composition of peppermint (Mentha x piperita L.) affected by yeast extract and salicylic acid foliar application. Journal of Medicinal Plants 20 (79): 47-58. doi: 10.52547/jmp.20.79.47.
[24] Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15: 473–497.
[25] Murch, S.J., Peiris, S.E., Shi, W.L., Zobayed, S.M.A. and Saxena P.K. 2006. Genetic diversity in seed populations of Echinacea purpurea controls the capacity for regeneration, route of morphogenesis and phytochemical composition. Plant Cell Report 25:522–532. doi:10.1007/s00299-006-0118-5.
[26] Murthy, H., Lee, E.J. and Paek, K.Y. 2014. Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation. Plant Cell, Tissue and Organ Culture 118: 1-16. doi: 10.1007/s11240-014-0467-7.
[27] Paek, K.Y., Murthy, H.N., Hahn, E.J. and Zhong, J.J. 2009. Large scale culture of ginseng adventitious roots for production of ginsenosides. Advances in Biochemical Engineering/Biotechnology113: 151–176. doi: 10.1007/10_2008_31.
[28] Raman, P., Patino, L.C. and Nair, M.G. 2004. Evaluation of metal and microbial contamination in botanical supplements. Journal of Agricultural and Food Chemistry 52: 7822–7827.
[29] Skandari, S.A., Piri, K.H., Kayhanfar, M. and Hasanloo, T. 2012. Influence of jasmonic acids, yeast extract and salicylic acid on growth and accumulation of hyosciamine and scopolamine in hairy root cultures of Atropa belladonna L. International Journal of Agriculture: Research and Review 2:403-409.
[30] Sivakumar, G, Yu, K.W., Hahn, E.J. and Paek, K.Y. 2005. Optimization of organic nutrients for ginseng hairy roots production in large-scale bioreactors. Current Science 89: 641-649.
[31] Thakur, M., Bhattacharya, S., Khosla, P.K. and Puri, S. 2019. Improving production of plant secondary metabolites through biotic and abiotic elicitation. Journal of Applied Research on Medicinal and Aromatic Plants 12:1–12. doi:10.1016/j.jarmap.2018.11.004.
[32] Udomsuk, L., Jarukamjorn, K., Tanaka, H. and Putalun, W. 2009. Isoflavonoid production in hairy roots culture of Pueraria candollei. Journal of biosciences 64: 687-91. doi: 10.1515/znc-2009-9-1013.
[33] Wang, J., Man, S., Gao, W., Zhang, L. and Huang, L. 2013. Cluster analysis of ginseng tissue cultures, dynamic change of growth, total saponins, and specific oxygen uptake rate in bioreactor and immunoregulative effect of ginseng adventitious root. Industrial Crops & Products 41:57–63.
[34] Wickens, T.D. and Keppel, G. 2004. Design and analysis: A researcher’s handbook (4th ed.). Englewood Cliffs, NJ: Prentice Hall.
[35] Wu, C.H., Murthy, H.N., Hahn, E.J. and Paek, K.Y. 2007a. Improved production of caffeic acid derivatives in suspension cultures of Echinacea purpurea by medium replenishment strategy. Archives of Pharmacal Research 30: 945–949. doi: 10.1007/BF02993961.
[36] Wu, C.H., Tewari, R.K., Hahn, E.J. and Paek, K.Y. 2007b. Nitric oxide elicitation induces the accumulation of secondary metabolites and antioxidant defense in adventitious roots of Echinacea purpurea. Journal of Plant Biology 50: 636–643.
[37] Yan, Q., Shi, M., Ng, J. and Wu, J.Y. 2006. Elicitor-induced rosmarinic acid accumulation and secondary metabolism enzyme activities in Salvia miltiorrhiza hairy roots. Plant Science 170: 853-858.
[38] Zhang, J., Gao, W.Y., Wang, J. and Li, X.L. 2011. Effects of explant types and media salt strength on growth and secondary metabolite accumulation in adventitious roots of Periploca sepium Bunge. Acta Physiologiae Plantarum. 33: 2447-2452. doi:10.1007/s11738-011-0785-x.
Volume 9, Issue 2
December 2021
Pages 24-34
  • Receive Date: 10 October 2022
  • Revise Date: 17 October 2022
  • Accept Date: 17 October 2022
  • First Publish Date: 17 October 2022