Production of phenolic acids in hairy root cultures of medicinal plant Mentha spicata L. in response to elicitors

Document Type : Original article


Department of Plant Bioproducts, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran


In this study, hairy root induction in leaf and stem explants of Mentha spicata using various Agrobacterium rhizogenes strains was established for the first time. Although inoculation of explants by immersion method resulted in tissue necrosis, direct injection of explants by all examined strains (A13,R318,A4,GMI 9534 and ATCC15834) was effective. All different parts of the stem were susceptible to A. rhizogenes infection. However, the middle and lower internodes showed a higher rate of transformation. Among the different strains, the strain A13 exhibited the highest infection efficiency (almost 75% of the explants). A13 and R318-infected hairy roots showed the highest biomass production (close to 60 mg/flask), while infection with GMI 9534 produced the highest content of phenolic acids. Finally, the effect of phytohormone elicitation on hairy root growth and phenolic acid biosynthesis was investigated. A substantial increase in root growth and phenolic acids accumulation was obtained followed by 0.3 mg L-1 IBA and 100µM MeJA treatment, respectively.


1. Ziaková A, Brandšteterová E. Validation of HPLC determination of phenolic acids present in some lamiaceae family plants. J Liq Chromatogr Relat Technol 2003;26:443-453.

2. Ghasemzadeh A, Ghasemzadeh N. Flavonoids and phenolic acids: Role and biochemical activity in plants and human. J Med Plant Res 2011;5:6697-6703.

3. Hao G, Jiang X, Feng L, Tao R, Li Y, Huang L. Cloning, molecular characterization and functional analysis of a putative R2R3-MYB transcription factor of the phenolic acid biosynthetic pathway in S. miltiorrhiza Bge. f. alba. Plant Cell Tiss Org Cult 2016; 124:151-168.

4. Zhang S, Yan Y, Wang B, Liang Z, Liu Y, Liu F, Qi Z.  Selective responses of enzymes in the two parallel pathways of rosmarinic acid biosynthetic pathway to elicitors in Salvia miltiorrhiza hairy root cultures. J Biosci Bioeng 2014;117:645-651.

5. Petersen M, Simmonds MSJ. Rosmarinic acid. Phytochemistry 2003;62:121-125.

6. Petersen M, Abdullah Y, Benner J, Eberle D, Gehlen K, Hücherig S, Janiak V, Kim KH, Sander M, Weitzel C, Wolters S.  Evolution of rosmarinic acid biosynthesis. Phytochemistry 2009;70:1663-1679.

7. Lin Y, Yan Y. Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex. Microb Cell Fact 2012;11:42.

8. Dorman HJD, Koşar M, Kahlos K, Holm Y, Hiltunen R. Antioxidant properties and composition of aqueous extracts from Mentha species, hybrids, varieties, and cultivars. J Agric Food Chem 2003;51:4563-4569.

9. Ay Kee L, Bakr Shori A, Salihin Baba A. Bioactivity and health effects of Mentha spicata. Integr Food Nutr Metab 2017;5:1-2.
10. Kanatt SR, Chander R, Sharma A. Antioxidant potential of mint (Mentha spicata L.) in radiation-processed lamb meat. Food Chem 2007;100:451-458.
11.Bayani M, Ahmadi-hamedani M, Jebelli Javan A. Study of hypoglycemic, hypocholes-terolemic and antioxidant activities of Iranian mentha spicata leaves aqueous extract in diabetic rats. Iran J Pharm Res 2017;16:75-82.
12. Namdeo AG. Plant cell elicitation for production of secondary metabolites: A review. Phcog Rev 2007;1:69-79.
13. Naik PM, Al-Khayri JM. Abiotic and biotic elicitors–role in secondary metabolites production through in vitro culture of medicinal plants. Abiotic and Biotic Stress in Plants, Recent Advances Future Perspectives. IntechOpen 2016;247-277.

14. Md Setamam N, Jaafar Sidik N, Abdul Rahman Z, Che Mohd Zain CR. Induction of hairy roots by various strains of Agrobacterium rhizogenes in different types of Capsicum species explants. BMC Res Notes 2014;7:414.

15. Pistelli L, Giovannini A, Ruffoni B, Bertoli A. Hairy root cultures for secondary metabolites production. Adv Exp Med Biol 2010; 698:167-184.

16. Thwe A, Valan Arasu M, Li X, Park CH, Kim SJ, Al-Dhabi NA, Park SU. Effect of different agrobacterium rhizogenes strains on hairy root induction and phenylpropanoid biosynthesis in Tartary Buckwheat (Fagopyrum tataricum Gaertn). Front Microbiol 2016; 7:318.

17. Sharifi S, Sattari TN, Zebarjadi A, Majd A, Ghasempour H. The influence of Agrobacterium rhizogenes on induction of hairy roots and ß-carboline alkaloids production in Tribulus terrestris L. Physiol Mol Biol Plants 2014;20:69-80.

18. Liang Z, Ma Y, Xu T, Cui B, Liu Y, Guo Z, Yang D. Effects of abscisic acid, gibberellin, ethylene and their interactions on production of phenolic acids in salvia miltiorrhiza bunge hairy roots. PLoS One 2013; 8:e72806.

19. Kim YB, Kim JK, Uddin MR, Xu H, Park WT, Tuan PA, Li X, Chung E, Lee JH, Park SU. Metabolomics analysis and biosynthesis of rosmarinic acid in agastache rugosa kuntze treated with methyl jasmonate. PLos One 2013;8:e64199.

20. Weathers PJ, Bunk G, McCoy MC. The Effect of Phytohormones on growth and artemisinin production in Artemisia annua hairy roots. In VitroCell Dev BiolPlant 2005; 41:47-53.

21. Young Ki Y, Sook Young L, Woo Tae P, Nam Il P, Sang Un P. Exogenous auxins and polyamines enhance growth and rosmarinic acid production in hairy root cultures of ‘Nepeta Cataria’ L. Plant Omics 2010;3:190-193.
22. Xiao Y, Gao S, Di P, Chen J, Chen W, Zhang L. Methyl jasmonate dramatically enhances the accumulation of phenolic acids in Salvia miltiorrhiza hairy root cultures. Physiol Plant 2009;137:1-9.
23. Bauer N, Kiseljak D, Jelaska S. The effect of yeast extract and methyl jasmonate on rosmarinic acid accumulation in Coleus blumei hairy roots. Biol Plant 2009; 53:650–656.
24. Hao X, Shi M, Cui L, Xu C, Zhang Y, Kai G. Effects of methyl jasmonate and salicylic acid on tanshinone production and biosynthetic gene expression in transgenic Salvia miltiorrhiza hairy roots. Biotechnol Appl Biochem 2015;62:24-31.

25. Li B, Wang B, Li H, Peng L, Ru M, Liang Z, Yan X, Zhu Y. Establishment of salvia castanea Diels f. tomentosa Stib. hairy root cultures and the promotion of tanshinone accumulation and gene expression with Ag+, methyl jasmonate, and yeast extract elicitation. Protoplasma 2016;253:87-100.

26. Lee SY, Xu H, Kim YK, Park SU. Rosmarinic acid production in hairy root cultures of Agastache rugosa Kuntze. World J Microbiol Biotechnol 2007;24:969-972.
27. Chen H, Chen F, Zhang YL, Song JY. Production of lithospermic acid B and rosmarinic acid in hairy root cultures of Salvia miltiorrhiza. J Ind Microbiol Biotechnol 1999;22:133-138.
28. Srivastava SH, Conlan XA, Adholeya A, Cahill DM. Elite hairy roots of Ocimum basilicum as a new source of rosmarinic acid and antioxidants. Plant Cell Tissue Organ Cult 2016;126: 19-32.
29. Esmaeili F, Shiran BJ, Fallahi H, Mirakhorli N, Budak H, Martínez-Gómez P. In silico search and biological validation of microRNAs related to drought response in peach and almond. Funct Integr Genomics 2017;17:189-201.
30. Gonçalves S, Anabela R. Production of plant secondary metabolites by using biotechnological tools. Secondary metabolites, sources and applications, IntechOpen 2018;81-99.
31. Ramirez-Estrada K, Vidal-Limon H, Hidalgo D, Moyano E, Golenioswki M, Cusidó RM, Palazon J. Elicitation, an effective strategy for the biotechnological production of bioactive high-added value compounds in plant cell factories. Molecules 2016;21:182.
32. Shakya P, Marslin G, Siram K, Beerhues L, Franklin G. Elicitation as a tool to improve the profiles of high-value secondary metabolites and pharmacological properties of Hypericum Perforatum. J Pharm Pharmacol 2019;71:70-82.
33. Guerriero G, Roberto B, Muñoz-Sanchez JA, Apone F, Abdel-Salam EM, Qahtan AA, Alatar AA, Cantini C, Cai G, Hausman JF, Siddiqui KS, Hernández Sotomayor SMT, Faisal M. Production of plant secondary metabolites: Examples, tips and suggestions for biotechnologists. Genes (Basel) 2018;9:1-22.
34. Mandal SM, Chakraborty D, Dey S. Phenolic acids act as signaling molecules in plant-microbe symbioses. Plant Signal Behav 2010;5:359-368.
35. Bimakr M, Rahman RA, Taip FS, Ganjloo A, Salleh LM, Selamat J, Hamid A, Zaidul ISM. Comparison of different extraction methods for the extraction of major bioactive flavonoid compounds from spearmint (Mentha spicata L.) leaves. Food Bioprod Process 2011; 89:67-72.
36. Jalali-Heravi M, Moazeni-Pourasil RS, Sereshti H. Thorough analysis of Iranian spearmint essential oil: combination of chemometrics and gas chromatography-mass spectrometry. Anal Methods 2014;6:6753-6759.
37. Cirlini M, Mena P, Tassotti M, Herrlinger KA, Nieman KM, Dall’Asta C, Del Rio D. Phenolic and volatile composition of a dry spearmint (Mentha spicata L.) extract. Molecules 2016;21.
38. Malik S. Production of plant derived natural compounds through hairy root culture. Springer 2017.
39. Sahu L, Jena S, Swain SS, Sahoo S, Chand PK. Agrobacterium rhizogenes-mediated transformation of a multi-medicinal herb, Boerhaavia diffusa L.: optimization of the process and anti-microbial activity against bacterial pathogens causing urinary tract infections. Front Life Sci 2013;7:197-209.
40. Tiwari RK, Trivedi M, Guang ZC, Guo GQ, Zheng GC. Genetic transformation of Gentiana macrophylla with Agrobacterium rhizogenes: growth and production of secoiridoid glucoside gentiopicroside in transformed hairy root cultures. Plant Cell Rep 2007;26:199-210.
41. Kuta DD, Tripathi L. Agrobacterium-induced hypersensitive necrotic reaction in plant cells: a resistance response against Agrobacterium-mediated DNA transfer. African J Biotechnol 2005;4:752-757.
42. Tavassoli P, Safipour Afshar A. Influence of different Agrobacterium rhizogenes strains on hairy root induction and analysis of phenolic and flavonoid compounds in marshmallow (Althaea officinalis L.). 3 Biotech 2018;8:351.
43. Park WT, Baskar TB, Yeo SK, Il Park N, Park JS, Park S. Response of different Agrobacterium Rhizogenes strains for in vitro hairy root induction and accumulation of rosmarinic acid production in agastache rugosa. Online J Biol Sci 2017;17:136-142.

44. Kim SJ, Cha MS, Lee EJ, Kim IH, Kwon JE, Kang SC, Park TH. In vitro induction of hairy root from isoflavones-producing Korean wild arrowroot Pueraria lobata. J Plant Biotech 2012;39:205-211.

45. Balasubramanian M, Anbumegala M, Surendran R, Arun M, Shanmugam G. Elite hairy roots of Raphanus sativus (L.) as a source of antioxidants and flavonoids.3 Biotech 2018;8:128.