Enhanced Eugenol Production in Eryngium campestre L. and Eryngium caucasicum Trautv. via Agrobacterium rhizogenes-Induced Hairy Roots

Document Type : Research Paper

Authors

1 Department of Agronomy and Plant Breeding Science, College of Aburaihan, University of Tehran, Tehran-Pakdasht, Iran

2 Department of Genetics and Plant Breeding, Ahv.C., Islamic Azad University, Ahvaz, Iran

10.22034/jmpb.2026.369169.1945

Abstract

Eryngium, an edible and medicinal plant from the Apiaceae family native to northern Iran, is traditionally used to treat inflammation, hypertension, and diabetes. This study investigated the effect of plant species, explant type and bacterial strain on hairy root induction of Eryngium campestre and Eryngium caucasicum via Rhizobium rhizogenes (formerly: Agrobacterium rhizogenes). Leaf, hypocotyl, and cotyledon explants were inoculated with A. rhizogenes (A4, ATCC-15834 and R318 strains). The transformation frequency and the number of roots generated per explant were recorded. The transformation of hairy roots was verified through PCR using gene-specific primers for rolB and rolC. The highest hairy root induction rate (65.8%) was obtained with the ATCC-15834 strain inoculated on the hypocotyls of E. campestre. The shortest time for root induction (7.5 days) was also observed in E. campestre. A high level of eugenol production in hairy roots induced by strain R318 in E. caucasicum shows that this strain has a significant potential to optimize secondary metabolite production in this species. HPLC analysis of the transgenic roots in both Eryngium species showed that the resulting hairy roots contained a significant amount of eugenol.  By transferring desirable genes to these two plant species, the resulting hairy roots contain a significant amount of eugenol. Based on these results, this technique offers an effective, genetically stable, and sustainable alternative to conventional extraction or chemical synthesis methods for large-scale production of eugenol-rich secondary metabolites.

Keywords

Main Subjects

References

  1. Noori S.A.S., Howyzeh M.S., Moradi N. Ajowan Carrots and related Apiaceae crops. CABI Wallingford UK. 2020;231-39.
  2. Al-Khayri J.M., Rashmi R., Toppo V., Chole P.B., Banadka A., Sudheer W.N., Nagella P., Shehata W.F., Al-Mssallem M.Q., Alessa F.M. Plant secondary metabolites: The weapons for biotic stress management. Metabolites. 2023;13(6):716.
  3. Motie F.M., Howyzeh M.S., Ghanbariasad A. Synergic effects of DL-limonene, R-limonene, and cisplatin on AKT, PI3K, and mTOR gene expression in MDA-MB-231 and 5637 cell lines. International Journal of Biological Macromolecules. 2024;136216.
  4. Wörz A., Diekmann H. Classification and evolution of the genus Eryngium L.(Apiaceae-Saniculoideae): results of fruit anatomical and petal morphological studies. Plant Diversity and Evolution. 2010;387-408.
  5. Wörz A. A new subgeneric classification of the genus Eryngium L.(Apiaceae, Saniculoideae). Botanische Jahrbücher für Systematik, Pflanzengeschichte und Pflanzengeographie. 2005;253-59.
  6. Kremer D., Zovko Končić M., Kosalec I., Košir I.J., Potočnik T., Čerenak A., Srečec S., Dunkić V., Vuko E. Phytochemical traits and biological activity of Eryngium amethystinum and E. Alpinum (apiaceae). Horticulturae. 2021;7(10):364.
  7. Pérez-Muñoz E.P., Antunes-Ricardo M., Martínez-Ávila M., Guajardo-Flores D. Eryngium species as a potential ally for treating metabolic syndrome and diabetes. Frontiers in Nutrition. 2022;9:878306.
  8. Zinman B., Wanner C., Lachin J.M., Fitchett D., Bluhmki E., Hantel S., Mattheus M., Devins T., Johansen O.E., Woerle H.J. Empagliflozin, cardiovascular outcomes, and mortality in type 2 diabetes. New England Journal of Medicine. 2015;373(22):2117-28.
  9. Espinoza-Hernández F., Andrade-Cetto A., Escandón-Rivera S., Mata-Torres G., Mata R. Contribution of fasting and postprandial glucose-lowering mechanisms to the acute hypoglycemic effect of traditionally used Eryngium cymosum F. Delaroche. Journal of Ethnopharmacology. 2021;279:114339.
  10. Choi J.Y., Na J.O. Pharmacological strategies beyond statins: ezetimibe and PCSK9 inhibitors. Journal of lipid and atherosclerosis. 2019;8(2):183-91.
  11. Kanagalingam T., Lazarte J., Wong D.K., Hegele R.A. Liver injury associated with ezetimibe monotherapy. CJC open. 2021;3(2):195-97.
  12. Feliciano G.D. Hypolipidemic Effects of Eryngium Caucasicum Leaves Extract on Rats with Induced Diabetes Mellitus. Al-Anbar Journal of Veterinary Sciences. 2024;17(2):1-7.
  13. Afshari M., Mohammadshahi M., Malayeri A.R., Zaheri L. Antidiabetic, hepato-protective and hypolipidemic effects of Eryngium caucasicum extract in streptozotocin-nicotinamide induced type 2 diabetes in male rats. Iraq Medical Journal. 2019;3(1):11-16.
  14. Trejo-Hurtado C.M., Landa-Moreno C.I., la Cruz J.L.-d., Peña-Montes D.J., Montoya-Pérez R., Salgado-Garciglia R., Manzo-Avalos S., Cortés-Rojo C., Monribot-Villanueva J.L., Guerrero-Analco J.A. An ethyl acetate extract of Eryngium carlinae inflorescences attenuates oxidative stress and inflammation in the liver of Streptozotocin-induced diabetic rats. Antioxidants. 2023;12(6):1235.
  15. Esmaili T., Aghaalitafreshi Z., Gharib M.H., Montazeri M. A study on the correlation of serum magnesium with intima-media thickness of carotid in hemodialysis patients. Journal of Nephropharmacology. 2021;10(2):e21-e21.
  16. Bocharova O., Karpova R., Bocharov E., Aksenov A., Kucheryanu V. TUMOR GROWTH ADHESION. Cardiometry. 2023;(29):9-9.
  17. Zari A.T., Zari T.A., Hakeem K.R. Anticancer properties of eugenol: A review. Molecules. 2021;26(23):7407.
  18. Omidi J., Abdolmohammadi S. Introduction of Eryngium Caeruleum Medicinal Plant. 2022;17(4):1-7.
  19. Roudbari M., Barzegar M., Sendra E., Casanova-Martínez I., Rodríguez-Estrada M., Carbonell-Barrachina Á.A. Characterization of the Different Chemical Components and Nutritional Properties of Two Eryngium Species. Foods. 2025;14(1):118.
  20. Rastegar A., Ghaderi H., Jamzad Z., Jalili A., Maroofi H. The conservation status of Eryngium pyramidale, the rarest species of Eryngium in Iran. Iran Nature. 2024;9(1):115-20.
  21. Ghafari E., Ariaii P., Bagheri R., Esmaeili M. Investigating the effect of nanochitosan-Iranian tragacanth gum composite film along with Eryngium campestre essential oil on the shelf life of goat meat. Journal of Food Measurement and Characterization. 2024;18(2):1543-58.
  22. Jalal-Dowlatshahi S., Sadat-Noori S.A., Mortazavian S.M.M., Howyzeh M.S., Esfahani K. The molecular cloning and structural analysis of a cytochrome P450 (CYP71D500) encoding gene from ajowan (Trachyspermum ammi L.) medicinal plant. Iranian Journal of Genetics & Plant Breeding (IJGPB). 2023;12(1):1-10.
  23. Dorafshan M., Soltani Howyzeh M., Shariati V. Identification of triterpene and sesquiterpene biosynthetic pathway genes in Citrullus colocynthis L. fruit using Next Generation Sequencing (NGS) technology. Iranian Journal of Medicinal and Aromatic Plants Research. 2020;36(3):390-403.
  24. Rozwandowicz M., Brouwer M., Fischer J., Wagenaar J., Gonzalez-Zorn B., Guerra B., Mevius D., Hordijk J. Plasmids carrying antimicrobial resistance genes in Enterobacteriaceae. Journal of Antimicrobial Chemotherapy. 2018;73(5):1121-37.
  25. Boccia E., Alfieri M., Belvedere R., Santoro V., Colella M., Del Gaudio P., Moros M., Dal Piaz F., Petrella A., Leone A. Plant hairy roots for the production of extracellular vesicles with antitumor bioactivity. Communications Biology. 2022;5(1):848.

 

  1. Niazian M., Belzile F., Torkamaneh D. CRISPR/Cas9 in planta hairy root transformation: a powerful platform for functional analysis of root traits in soybean. Plants. 2022;11(8):1044.
  2. Zhao Z., Gao S., Hu J., Lei T., Zhou Y., Li Y., Liu C. The hairy root induction efficiency and that plumbagin of Plumbago auriculata L. Plant Cell, Tissue and Organ Culture (PCTOC). 2023;155(1):67-80.
  3. Mahmoudi M., Sadat Noori S.A., Ebrahimi M., Bahmankar M. Optimization of induction of hairy roots in Perilla. Genetic Engineering and Biosafety Journal. 2023;12(1):17-27.
  4. Ajdanian L., Niazian M., Torkamaneh D. Optimizing ex-vitro one-step RUBY-equipped hairy root transformation in drug-and hemp-type Cannabis. BioRxiv. 2023;2023.11. 29.569008.
  5. Brijwal L., Tamta S. Agrobacterium rhizogenes mediated hairy root induction in endangered Berberis aristata DC. SpringerPlus. 2015;4:1-10.
  6. Muthusamy B., Shanmugam G. Analysis of flavonoid content, antioxidant, antimicrobial and antibiofilm activity of in vitro hairy root extract of radish (Raphanus sativus L.). Plant Cell, Tissue and Organ Culture (PCTOC). 2020;140:619-33.
  7. Doyle J. DNA protocols for plants Molecular techniques in taxonomy. Springer. 1991; 283-93.
  8. Aydoğmuş Z., Yıldız G., Yılmaz E.M., Aboul-Enein H.Y. Determination of eugenol in plants and pharmaceutical form by HPLC with amperometric detection at graphene-modified carbon paste electrode. Graphene Technology. 2018;3:1-9.
  9. 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(8):351.
  10. El-Esawi M.A., Elkelish A., Elansary H.O., Ali H.M., Elshikh M., Witczak J., Ahmad M. Genetic transformation and hairy root induction enhance the antioxidant potential of Lactuca serriola L. Oxidative Medicine and Cellular Longevity. 2017;2017(1):5604746.
  11. Bulgakov V.P., Vereshchagina Y.V., Bulgakov D.V., Veremeichik G.N., Shkryl Y.N. The rolB plant oncogene affects multiple signaling protein modules related to hormone signaling and plant defense. Scientific Reports. 2018;8(1):2285.
  12. Kiselev K.V., Kusaykin M.I., Dubrovina A.S., Bezverbny D.A., Zvyagintseva T.N., Bulgakov V.P. The rolC gene induces expression of a pathogenesis-related β-1, 3-glucanase in transformed ginseng cells. Phytochemistry. 2006;67(20):2225-31.
  13. Shilpha J., Largia M.J.V., Kumar R.R., Satish L., Swamy M.K., Ramesh M. Hairy root cultures: a novel way to mass produce plant secondary metabolites Phytochemical genomics: plant metabolomics and medicinal plant genomics. Springer. 2023: 417-45.
  14. Gutierrez-Valdes N., Häkkinen S.T., Lemasson C., Guillet M., Oksman-Caldentey K.-M., Ritala A., Cardon F. Hairy root cultures—a versatile tool with multiple applications. Frontiers in Plant Science. 2020;11:33.
  15. Gupta R., Pandey P., Singh S., Singh D.K., Saxena A., Luqman S., Bawankule D.U., Banerjee S. Advances in Boerhaavia diffusa hairy root technology: a valuable pursuit for identifying strain sensitivity and up-scaling factors to refine metabolite yield and bioactivity potentials. Protoplasma. 2016;253:1145-58.
  16. Thwe A., Valan Arasu M., Li X., Park C.H., Kim S.J., Al-Dhabi N.A., Park S.U. Effect of different Agrobacterium rhizogenes strains on hairy root induction and phenylpropanoid biosynthesis in tartary buckwheat (Fagopyrum tataricum Gaertn). Frontiers in Microbiology. 2016;7:318.
  17. Niazian M., Belzile F., Curtin S.J., de Ronne M., Torkamaneh D. Optimization of in vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation of soybean for visual screening of transformants using RUBY. Frontiers in Plant Science. 2023;14:1207762.
  18. Lloyd G.S., Thomas C.M. Microbial primer: the logic of bacterial plasmids. Microbiology. 2023;169(7):001336.
  19. Zdravković-Korać S., Muhovski Y., Druart P., Ćalić D., Radojević L. Agrobacterium rhizogenes-mediated DNA transfer to Aesculu s hippocastanum L. and the regeneration of transformed plants. Plant Cell Reports. 2004;22:698-704.
Journal of Medicinal plants and By-products
Volume 15, Issue 3
May and June 2026
Pages 321-326

Files

Share

How to cite

Statistics