Biotechnology Letters

, Volume 41, Issue 3, pp 427–442 | Cite as

Isoflavone production in hairy root cultures and plantlets of Trifolium pratense

  • Andressa Reis
  • Stéphanie Boutet-Mercey
  • Sophie Massot
  • Pascal RatetEmail author
  • José Angelo Silveira Zuanazzi
Original Research Paper



The aim of this study was to develop a Trifolium pratense hairy root (HR) production protocol and select HR lines with high isoflavone yield following elicitor treatments.


We obtained 13 independent HR lines, producing approximately three times more isoflavonoids than seedlings (3.3 mg/g dry weight) and in which 27 isoflavonoids were detected. Each HR line had its own isoflavonoid profile. These lines produced as major components daidzein, genistein, formononetin and biochanin A. Sucrose, salicylic acid (SA), yeast extract (YE) and flagellin 22 (flg22) were tested as elicitors. Using SA 140 mg/L, allowed the maximum isoflavonoid production in plantlets (11.9 mg/g dry weight) but reduced root growth, possibly as a result of its toxicity. The highest isoflavone production in HR (27.9 mg/g dry weight) was obtained using sucrose 60 g/L, for 3.5 days.


This work reports the high production of various isoflavonoids with T. pratense elicited HR cultures.


Elicitors Plantlets Red clover Salicylic acid Sucrose 



Thanks to the Coordination for the Improvement of Higher Education Personnel and National Council for Scientific and Technological Development/Brazil for the financial assistance provided through Ph.D. and Scientific Initiation scholarships. J.A.S.Z. thanks CNPq for the researcher fellowship, Programa Iberoamericano CYTED - proyecto BIFRENES 416RT0511 and the National Institute of Science and Technology - INCT BioNat - Grant # 465637/2014-0. Thanks also to Dr. Sandra Beatriz Rech, Dr. Miguel Dall’Agnol, Dr. Amelia Henriques and Marí Castro for the collaboration. Thanks to Dr. Marie Garmier for careful reading and editing of the manuscript. The project benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS).

Supporting information

Supplementary Fig. 1—Colour modification in the T. pratense plantlets flavonoid extracts cultivated in presence of Sucrose plus phytagel as gelling agent. From left to right extracts of cultures grown on 10, 30, 60, 90 and 120 g/L of sucrose.

Supplementary Table 1—Root growth (mm) and isoflavone content (mg/g dry plants) on T. pratense seedlings grown in medium with elicitors at different concentrations during the first seven days after germination.

Supplementary Table 2—Isoflavones and total isoflavones content of the T. pratense HR lines 1HR, 2HR, 4HR and 6HR during the first 5 months of culture.

Supplementary Table 3—Effect of elicitors on the accumulation of major and total isoflavones (mg/g dry plant) in two T. pratense hairy roots line.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10529_2018_2640_MOESM1_ESM.docx (12 kb)
Supplementary material 1 Fig. S1 Colour modification in the T. pratense plantlets flavonoid extracts cultivated in presence of Sucrose plus phytagel as gelling agent. From left to right extracts of cultures grown on 10, 30, 60, 90 and 120 g/L of sucrose (DOCX 11 kb)
10529_2018_2640_MOESM2_ESM.png (428 kb)
Supplementary material 2 (PNG 427 kb)
10529_2018_2640_MOESM3_ESM.docx (16 kb)
Supplementary material 3 (DOCX 16 kb)
10529_2018_2640_MOESM4_ESM.docx (17 kb)
Supplementary material 4 (DOCX 16 kb)
10529_2018_2640_MOESM5_ESM.docx (20 kb)
Supplementary material 5 (DOCX 20 kb)


  1. Anil Kumar M, Sravanthi Pammi SS, Sukanya MS, Giri A (2018) Enhanced production of pharmaceutically important isoflavones from hairy root rhizoclones of Trifolium pratense L. In Vitro Cell Dev Biol Plant. Google Scholar
  2. Arregui LM, Veramendi J, Mingo-Castel AM (2003) Effect of gelling agents on in vitro tuberization of six potato cultivars. Am J Potato Res 80:141–144CrossRefGoogle Scholar
  3. Beach K, Gresshoff P (1986) In vitro culture of legume root tissue transformed by Agrobacterium rhizogenes. In: Proceedings of the VI International Congress of Plant Tissue and Cell Culture, p 155Google Scholar
  4. Chandra S, Chandra R (2011) Engineering secondary metabolite production in hairy roots. Phytochem Rev 10:371–395. CrossRefGoogle Scholar
  5. Cosson V, Durand P, d’Erfurth I, Kondorosi A, Ratet P (2006) Medicago truncatula transformation using leaf explants. In: Wang K (ed) Methods in molecular biology, Agrobacterium protocols, vol 343. Humana Press Inc., Totowa, pp 115–127CrossRefGoogle Scholar
  6. Cui X, Chakrabarty D, Lee E, Paek K (2010) Production of adventitious roots and secondary metabolites by Hypericum perforatum L. in a bioreactor. Bioresour Technol 101:4708–4716. CrossRefGoogle Scholar
  7. De Coninck B, Timmermans P, Vos C et al (2015) What lies beneath: belowground defense strategies in plants. Trends Plant Sci 20:91–101. CrossRefGoogle Scholar
  8. Doran PM (1997) Hairy roots. CRC Press, Boca RatonGoogle Scholar
  9. Du H, Huang Y, Tang Y (2010) Genetic and metabolic engineering of isoflavonoid biosynthesis. Appl Microbiol Biotechnol 86:1293–1312. CrossRefGoogle Scholar
  10. Durango D, Pulgarin N, Echeverri F et al (2013) Effect of salicylic acid and structurally related compounds in the accumulation of phytoalexins in cotyledons of common bean (Phaseolus vulgaris L.) cultivars. Molecules 18:10609–10628. CrossRefGoogle Scholar
  11. Eskandari-Samet A, Piri K, Kayhanfar M, Hasanloo T (2012) Enhancement of tropane alkaloids production among several clones and explants types of hairy root of Atropa belladona L. J Med Plants By-products 1:35–42Google Scholar
  12. Fu C, Zhao D, Xue X et al (2005) Transformation of Saussurea involucrata by Agrobacterium rhizogenes: hairy root induction and syringin production. Process Biochemistry 40:3789–3794. CrossRefGoogle Scholar
  13. Galland M, Boutet-Mercey S, Lounifi I et al (2014) Compartmentation and dynamics of flavone metabolism in dry and germinated rice seeds. Plant Cell Physiol 55:1646–1659. CrossRefGoogle Scholar
  14. Guillon S, Trémouillaux-Guiller J, Pati PK et al (2006) Hairy root research: recent scenario and exciting prospects. Curr Opin Plant Biol 9:341–346. CrossRefGoogle Scholar
  15. Hu Z-B, Du M (2006) Hairy root and its application in plant genetic engineering. J Integr Plant Biol 48:121–127. CrossRefGoogle Scholar
  16. Kanehisa M, Furumichi M, Tanabe M et al (2017) KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res 45:D353–D361. CrossRefGoogle Scholar
  17. Kerhoas L, Aouak D, Cingöz A et al (2006) Structural characterization of the major flavonoid glycosides from Arabidopsis thaliana seeds. J Agric Food Chem 54:6603–6612. CrossRefGoogle Scholar
  18. León P, Sheen J (2003) Sugar and hormone connections. Trends Plant Sci 8:110–116. CrossRefGoogle Scholar
  19. Liang P, Shi HP, Qi Y (2004) Effect of sucrose concentration on the growth and production of secondary metabolites in Pueraria phaseoloides hairy roots. Shi Yan Sheng Wu Xue Bao 37:384–390Google Scholar
  20. Machado and Conceição (2002) Programa estatístico WinStat Sistema de Análise Estatístico para WindowsGoogle Scholar
  21. Maffei ME, Arimura G-I, Mithöfer A (2012) Natural elicitors, effectors and modulators of plant responses. Nat Prod Rep 29:1288. CrossRefGoogle Scholar
  22. Mehrotra S, Mishra S, Srivastava V (2016) Bioreactor technology for hairy roots cultivation. In: Pavlov A, Bley T (eds) Bioprocessing of plant in vitro systems. Reference Series in Phytochemistry. Springer, ChamGoogle Scholar
  23. Millet YA, Danna CH, Clay NK et al (2010) Innate immune responses activated in Arabidopsis roots by microbe-associated molecular patterns. Plant Cell 22:973–990. CrossRefGoogle Scholar
  24. Monteiro N, Queirós L, Lopes D et al (2018) Impact of microbiota on the use and effects of isoflavones in the relief of climacteric symptoms in menopausal women—a review. J Funct Foods 41:100–111. CrossRefGoogle Scholar
  25. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. CrossRefGoogle Scholar
  26. Petit A, David C, Dahl GA et al (1983) Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation. MGG Mol Gen Genet 190:204–214. CrossRefGoogle Scholar
  27. Putalun W, Luealon W, De-Eknamkul W et al (2007) Improvement of artemisinin production by chitosan in hairy root cultures of Artemisia annua L. Biotechnol Lett 29:1143–1146. CrossRefGoogle Scholar
  28. Ramakrishna A, Ravishankar GA (2011) Influence of abiotic stress signals on secondary metabolites in plants. Plant Signal Behav 6:1720–1731. CrossRefGoogle Scholar
  29. Ramirez-Estrada K, Vidal-Limon H, Hidalgo D et al (2016) Elicitation, an effective strategy for the biotechnological production of bioactive high-added value compounds in plant cell factories. Molecules 21:182. CrossRefGoogle Scholar
  30. Saviranta NMM, Anttonen MJ, von Wright A, Karjalaianen RO (2008) Red clover (Trifolium pratense L.) isoflavones: determination of concentrations by plant stage, flower colour, plant part and cultivar. J Sci Food Agric. Google Scholar
  31. Saviranta NM, Julkunen-Tiitto R, Oksanen E, Karjalainen RO (2010) Red clover (Trifolium pratense L.) isoflavones: root phenolic compounds affected by biotic and abiotic stress factors. J Sci Food Agric 90:418–423. Google Scholar
  32. Shinde AN, Malpathak N, Fulzele DP (2009a) Optimized production of isoflavones in cell cultures of Psoralea corylifolia L. Using elicitation and precursor feeding. Biotechnol Bioprocess Eng 14:612–618. CrossRefGoogle Scholar
  33. Shinde AN, Malpathak N, Fulzele DP (2009b) Enhanced production of phytoestrogenic isoflavones from hairy root cultures of Psoralea corylifolia L. using elicitation and precursor feeding. Biotechnol Bioprocess Eng 14:612–618. CrossRefGoogle Scholar
  34. Shinde AN, Malpathak N, Fulzele DP (2010) Impact of nutrient components on production of the phytoestrogens daidzein and genistein by hairy roots of Psoralea corylifolia. J Nat Med 64:346–353. CrossRefGoogle Scholar
  35. Sivesind E, Seguin P (2006) Effects of foliar application of elicitors on red clover isoflavone content. J Agron Crop Sci 192:50–54. CrossRefGoogle Scholar
  36. Smeekens S (2000) Sugar-induced signal transduction in plants. Stress Int J Biol Stress 51:49–81Google Scholar
  37. Solfanelli C, Solfanelli C, Poggi A et al (2006) Sucrose-specific induction of the anthocyanin biosynthetic pathway in Arabidopsis. Plant Physiol 140:637–646. CrossRefGoogle Scholar
  38. Spagnuolo P, Rasini E, Luini A et al (2014) Isoflavone content and estrogenic activity of different batches of red clover (Trifolium pratense L.) extracts: an in vitro study in MCF-7 cells. Fitoterapia 94:62–69. CrossRefGoogle Scholar
  39. Sujatha G, Kumari BR (2012) Establishment of fast-growing in vitro root culture system in Artemisia vulgaris. J Agric Technol 8:1779–1790Google Scholar
  40. Udomsuk L, Jarukamjorn K, Tanaka H, Putalun W (2011) Improved isoflavonoid production in Pueraria candollei hairy root cultures using elicitation. Biotechnol Lett 33:369–374. CrossRefGoogle Scholar
  41. Veramendi J, Villafranca MJ, Sota V, Mingo-Castel AM (1997) Gelrite as an alternative to agar for micropropagation and microtuberization of Solanum tuberosum L. cv. Baraka. Vitr Cell Dev Biol Plant 33:195–199. CrossRefGoogle Scholar
  42. Webb JK, Jones S, Robbins MP, Minchin FR (1990) Characterization of transgenic root cultures of Trifolium repens, Trifolium pratense and Lotus corniculatus and transgenic plants of Lotus corniculatus. Plant Sci 70:243–254CrossRefGoogle Scholar
  43. Wright E, Wang Z-Y (2015) Medicago trunculata transformation using cotyledonary explants. In: Wang K (ed) Agrobacterium protocols. Springer, New York, pp 35–56Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Laboratory of Pharmacognosy, Department of Raw Material ProductionFederal University of Rio Grande do Sul, Porto Alegre - UFRGSPorto AlegreBrazil
  2. 2.Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRSUniversité Paris-SaclayVersaillesFrance
  3. 3.Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRAUniversité Paris-Sud, Université Evry, Université Paris-SaclayOrsayFrance
  4. 4.Institute of Plant Sciences Paris-Saclay IPS2OrsayFrance

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