Research in Plant Metabolites

Research in Plant Metabolites

Improving the production of relative content of terpene compounds through optimization of hairy root induction conditions in the medicinal plant valerian (Valeriana officinalis L.)

Document Type : Original Article

Authors
Razieh Biglari Farash Biglari Farash 1
Azizollah Kheiry 2
Zahra Ghahremani 1
Khadijeh Bagheri 3
Farhang Razavi 1
1 Department of Horticultural Sciences, Faculty of Agriculture, Zanjan University, Zanjan, Iran
2 Department of Horticultural Sciences, Faculty of Agriculture- , University of Zanjan
3 Department of Plant Production and Genetics, Faculty of Agriculture, Zanjan University, Zanjan, Iran
10.22034/jrpsm.2026.577954.1109
Abstract
Background and Objective: Valerian (Valeriana officinalis L.) is one of the most important medicinal plants and a valuable source of terpene compounds. Due to limitations in field production and the decline of natural reserves, the use of hairy roots induced by Agrobacterium rhizogenes has attracted attention. This research was conducted with the aim of optimizing the conditions for hairy root induction and investigating its effect on growth indices and terpene content.
Materials and Methods: This research was carried out as a factorial experiment in a completely randomized design with three replications. Treatments included four levels of acetosyringone (0, 100, 150, and 200 mg/L), two levels of Agrobacterium rhizogenes strain MSU suspension density based on optical density (OD₆₀₀ = 0.5 and 1), and three co-cultivation times (24, 48, and 72 hours). In vitro leaf explants of valerian were immersed in a bacterial suspension containing acetosyringone for 10 minutes and then co-cultivated on MS medium in darkness at 25 ± 2°C. After co-cultivation, the explants were transferred to medium containing cefotaxime to eliminate the bacteria. Hairy root transformation was confirmed by genomic DNA extraction and amplification of the rolB gene using polymerase chain reaction (PCR) with specific primers and a standard temperature program. The measured traits included the percentage and speed of hairy root induction, number of roots per explant, root dry weight, and terpene content. Terpene content was measured using the vanillin-sulfuric acid colorimetric method with absorbance reading at 550 nm. A standard curve was prepared using β-caryophyllene, and the results were expressed as mg β-caryophyllene equivalent per gram of dry weight. Data were analyzed using SAS software, and means were compared using Duncan's multiple range test at the 5% probability level.
Results: The results showed that the highest percentage of hairy root induction (78%), the highest induction speed (9 days), the highest number of roots (16 roots per explant), and the highest root dry weight (64 mg) were obtained in the treatment of 100 mg/L acetosyringone, bacterial density of OD₆₀₀ = 0.5, and co-cultivation time of 48 hours. Increasing the acetosyringone concentration to 200 mg/L caused a significant decrease in all growth indices, which was probably due to oxidative stress and its inhibitory effects on cell division and growth. Furthermore, increasing bacterial density to OD₆₀₀ = 1 reduced the induction percentage, root number, and dry weight, which can be attributed to bacterial overgrowth, competition for nutrients, and triggering of plant defense responses. The co-cultivation time of 48 hours was identified as the most suitable duration, since shorter co-cultivation did not provide sufficient time for effective T-DNA transfer, while longer co-cultivation led to tissue necrosis and reduced explant viability. The highest amount of terpene (0.151 mg β-caryophyllene equivalent per gram of dry weight) was observed in the optimal treatment, while the lowest amount (0.036 mg/g DW) was found in the 200 mg/L acetosyringone treatment. Additionally, the presence of a specific 780 bp band of the rolB gene in the PCR test confirmed the successful transformation of all examined hairy roots.
Conclusion: The results of this study demonstrated that simultaneous optimization of acetosyringone concentration, bacterial density, and co-cultivation time significantly enhanced hairy root induction efficiency and terpene accumulation in valerian. The treatment of 100 mg/L acetosyringone, OD₆₀₀ = 0.5, and 48 hours of co-cultivation was introduced as the optimal condition for producing hairy roots and terpene compounds.
Keywords
Agrobacterium
Transformation
Secondary metabolite
Hairy root
Alpizar, E., Dechamp, E., Lapeyre-Montes, F., Guilhaumon, C., Bertrand, B., Jourdan, C., Lashermes, P., & Etienne, H. (2008). Agrobacterium rhizogenes-transformed roots of coffee (Coffea arabica): Conditions for long-term proliferation, and morphological and molecular characterization. Annals of Botany, 101(7), 929–940. https://doi.org/10.1093/aob/mcn027
Al-Qurainy, F., Nadeem, M., Khan, S., Alansi, S., Tarroum, M., Al-Ameri, A. A., & Gaafar, A. R. Z. (2020). Effect of different Agrobacterium rhizogenes strains for in-vitro hairy root induction, total phenolic, flavonoids contents, antibacterial and antioxidant activity of (Cucumis anguria L.). Saudi Journal of Biological Sciences, 27(11), 2972–2979. https://doi.org/10.1016/j.sjbs.2020.08.050
Amini, S., Fattahi, M., & Nazemiyeh, H. (2024). Optimization of induction and hairy root culture establishment in two mullein species, Verbascum erianthum and Verbascum stachydiforme. Scientific Reports, 14, Article 5636. https://doi.org/10.1038/s41598-024-56331-8
Balasubramanian, M., Anbumegala, M., Surendran, R., Arun, M., & Shanmugam, G. (2018). Elite hairy roots of Raphanus sativus (L.) as a source of antioxidants and flavonoids. 3 Biotech, 8(2), Article 128. https://doi.org/10.1007/s13205-018-1153-y
Biglari Farash, R. B., Kheiry, A., Ghahremani, Z., Bagheri, Kh., & Razavi, F. (2026). Study on the efficiency of Agrobacterium strains (Agrobacterium rhizogenes) and different types of explants on the transformation efficiency and antioxidant capacity of hairy roots of valerian (Valeriana officinalis L). Journal of Research in Plant Metabolites, 4 (1), 35. doi:10.22034/jrpsm.2.026.562414.1102
Biswas, D., Chakraborty, A., Mukherjee, S., & Ghosh, B. (2023). Hairy root culture: A potent method for improved secondary metabolite production of Solanaceous plants. Frontiers in Plant Science, 14, Article 1197555. https://doi.org/10.3389/fpls.2023.1197555
Castellanos-Arévalo, A. P., Estrada-Luna, A. A., Cabrera-Ponce, J. L., Valencia-Lozano, E., Herrera-Ubaldo, H., de Folter, S., Blanco-Labra, A., & Délano-Frier, J. P. (2020). Agrobacterium rhizogenes-mediated transformation of grain (Amaranthus hypochondriacus) and leafy (A. hybridus) amaranths. Plant Cell Reports, 39(9), 1143–1160. https://doi.org/10.1007/s00299-020-02553-9
Considine, M. J., & Foyer, C. H. (2021). Oxygen and reactive oxygen species-dependent regulation of plant growth and development. Plant Physiology, 186(1), 79–92. https://doi.org/10.1093/plphys/kiaa077
De Koning, R., Daryanavard, H., Garmyn, J., Kiekens, R., Toili, M. E. M., & Angenon, G. (2023). Fine-tuning CRISPR/Cas9 gene editing in common bean (Phaseolus vulgaris L.) using a hairy root transformation system and in silico prediction models. Frontiers in Plant Science, 14, Article 1233418. doi: 10.3389/fpls.2023.1233418.
Foti, C., & Pavli, O. I. (2020). High-efficiency Agrobacterium rhizogenes-mediated transgenic hairy root induction of Lens culinaris. Agronomy, 10(8), Article 1170. https://doi.org/10.3390/agronomy10081170
Gelvin, S. B. (2017). Integration of Agrobacterium T-DNA into the plant genome. Annual Review of Genetics, 51, 195–217.https://doi.org/10.1146/annurev-genet-120215-035320
Ghorbani, M., Omidi, M., & Shah Nejat Boushahri, A. A. (2024). Optimization of genetically pRi-transformed roots induction in Dracocephalum kotschyi Boiss. using response surface methodology. Journal of Genetics, 19(3), 159–171. URL: http://mg.genetics.ir/article-1-1804-en.htm
Giri, A., & Narasu, M. L. (2000). Transgenic hairy roots: Recent trends and applications. Biotechnology Advances, 18(1), 1–22. https://doi.org/10.1016/S0734-9750(99)00016-6
Gutiérrez-Valdes, N., Häkkinen, S. T., Lemasson, C., Guillet, M., Oksman-Caldentey, K. M., Ritala, A., & Cardon, F. (2020). Hairy root cultures—A versatile tool with multiple applications. Frontiers in Plant Science, 11, Article 33. https://doi.org/10.3389/fpls.2020.00033
Hussain, M. J., Abbas, Y., Nazli, N., Fatima, S., Drouet, S., Hano, C., & Abbasi, B. H. (2022). Root cultures, a boon for the production of valuable compounds: A comparative review. Plants, 11(3), Article 439. https://doi.org/10.3390/plants11030439
Husain, Z. Z., Warsi, Z. I. I., Khan, S., Mahendran, G., Afroz, S., Chandran, A., Kashyap, P. K., Khatoon, K., Parween, G., Tandon, S., et al. (2024). Metabolic engineering of hairy root cultures in Beta vulgaris for enhanced production of vanillin, 4-hydroxybenzoic acid, and vanillyl alcohol. Frontiers in Bioengineering and Biotechnology, 12, 1435190. https://doi.org/10.3389/fbioe.2024.1435190
Liu, L., Qu, J., Wang, C., Liu, M., Zhang, C., Zhang, X., Guo, C., Wu, C., Yang, G., Huang, J., Yan, K., Shu, H., Zheng, C., & Zhang, S. (2024). An efficient genetic transformation system mediated by Rhizobium rhizogenes in fruit trees based on the transgenic hairy root to shoot conversion. Plant Biotechnology Journal, 22(8), 2093–2103. https://doi.org/10.1111/pbi.14328
Park, N. I., Tuan, P. A., Li, X., Kim, Y. K., Yang, T. J., & Park, S. U. (2011). An efficient protocol for genetic transformation of Platycodon grandiflorum with Agrobacterium rhizogenes. Molecular Biology Reports, 38(4), 2307–2313. https://doi.org/10.1007/s11033-010-0363-0
Mahmoudi, M., Sadat-Noori, S. A., Bahmankar, M., Ebrahimi, M., & Fadavi, A. (2025). Shikonin and luteolin production in hairy root culture of Perilla frutescens L. Research Square. https://doi.org/10.21203/rs.3.rs-7080593/v1
Mahakur, B., Moharana, A., Madkami, S. K., Naik, S. K., & Barik, D. P. (2024). Optimization of factors affecting Agrobacterium-mediated hairy root induction in Vitex negundo L. (Lamiaceae). International Journal of Secondary Metabolite, 11(2), 244–254. https://doi.org/10.21448/ijsm.1368677
Massah, M., & Rabiei, M. (2023). Effect of acetosyringone, sucrose and nutrients on transgenic hairy root induction in Chenopodium quinoa using different Rhizobium rhizogenes strains. Research Square. https://doi.org/10.21203/rs.3.rs-2833983/v1 
Kim, Y. K., Li, X., Xu, H., Park, N.-I., Uddin, M. R., Pyon, J. Y., & Park, S. U. (2009). Production of phenolic compounds in hairy root culture of tartary buckwheat (Fagopyrum tataricum Gaertn). Journal of Crop Science and Biotechnology, 12(1), 53–58. https://doi.org/10.1007/s12892-009-0075-y
Kuta, D. D., & Tripathi, L. (2005). Agrobacterium-induced hypersensitive necrotic reaction in plant cells: A resistance response against Agrobacterium-mediated DNA transfer. African Journal of Biotechnology, 4(8), 752–757. https://doi.org/10.4314/ajb.v4i8.15199
Moehninsi, & Navarre, D. A. (2018). Optimization of hairy root induction in Solanum tuberosum. American Journal of Potato Research, 95(6), 650–658. https://doi.org/10.1007/s12230-018-9671-z
Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15 (3), 473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.
Niazian, M., Belzile, F., Curtin, S. J., de Ronne, M., & Torkamaneh, D. (2023). 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, 14, 1207762. https://doi.org/10.3389/fpls.2023.1207762
Palmer, A. C. V., & Shaw, C. H. (1992). The role of VirA and VirG phosphorylation in chemotaxis towards acetosyringone by Agrobacterium tumefaciens. Microbiology, 138(12), 2509–2514. https://doi.org/10.1099/00221287-138-12-2509
Piatczak, E., Jeleń, A., & Grzegorczyk-Karolak, I. (2021). Optimization of Agrobacterium rhizogenes-mediated transformation of Valeriana officinalis L. and production of valerenic acid in hairy root cultures. Industrial Crops and Products, 168, Article 113589. https://doi.org/10.1016/j.indcrop.2021.113589
Phuong, V. T. B., Anh, N. P., Thang, N. P. Q., Tran, P., & Phuong, Q. N. D. (2023). Study on induction of Allium sativum L. hairy roots with antibacterial activity against Vibrio. Research Journal of Biotechnology, 18(8), 153–162. https://doi.org/10.25303/1808rjbt1530162
Sezen Karaoğlan, E. (2022). Valeriana officinalis L.  in Novel drug targets with traditional herbal medicines: Scientific and clinical evidence, 565-568. Springer. https://doi.org/10.1007/978-3-030-73234-9_39
Sharma, P., Padh, H., & Shrivastava, N. (2017). Optimizing culture conditions for establishment of hairy root culture of Semecarpus anacardium L. 3 Biotech, 7, Article 101. https://doi.org/10.1007/s13205-017-0728-6
Sharma, S., Singh, Y., Verma, P. K., & Vakhlu, J. (2021). Establishment of Agrobacterium rhizogenes-mediated hairy root transformation of Crocus sativus L. 3 Biotech, 11(2), Article 82. https://doi.org/10.1007/s13205-020-02626-2
Singh, P., Labade, D., Chote, M., Deshmukh, P., Panchal, B., Deshpande, J., Phule, A. S., Tabassum, H., & Wani, M. (2025). Efficient regeneration of Stevia rebaudiana Bertoni transformants through hairy root culture technique. Journal of Applied Botany and Food Quality, 98, 22–28. https://doi.org/10.5073/JABFQ.2025.098.003
Sivanandhan, G., Selvaraj, N., Ganapathi, A., & Manickavasagam, M. (2014). An efficient hairy root culture system for Withania somnifera (L.) Dunal. African Journal of Biotechnology, 13(43), 4141–4147. https://doi.org/10.5897/AJB2014.14128
Srivastava, S., & Srivastava, A. K. (2007). Hairy root culture for mass-production of high-value secondary metabolites. Critical Reviews in Biotechnology, 27(1), 29. https://doi.org/ 10.1080/07388550601173918
Sivakumar, G., Liu, C., Towler, M. J., & Weathers, P. J. (2010). Biomass production of hairy roots of Artemisia annua and Arachis hypogaea in a scaled-up mist bioreactor. Biotechnology and Bioengineering, 107(5), 802–813. https://doi.org/10.1002/bit.22892
Suresh, R., Kamdar, Y., Garg, V., Kalathoti, A., & Renganathan, G. (2021). Evaluation of improved and current vanillin based colorimetric quantification methods of triterpenoids. Journal of Student Research, 10(2). https://doi.org/10.47611/jsrhs.v10i2.1442
Tao, J., Tan, R. F., & Li, L. (2006). Genetic transformation of sunflower (Helianthus Annuus L.) mediated by Agrobacterium rhizogenes. Acta Agronomica Sinica, 32(5), 743–748.
Tiwari, M., Mishra, A. K., & Chakrabarty, D. (2022). Agrobacterium-mediated gene transfer: Recent advancements and layered immunity in plants. Planta, 256(2), Article 37. https://doi.org/10.1007/s00425-022-03951-x
Torkamani, M. R. D., Jafari, M., Abbaspour, N., Heidary, R., & Safaie, N. (2014). Enhanced production of valerenic acid in hairy root culture of Valeriana officinalis by elicitation. Central European Journal of Biology, 9, 853–863. https://doi.org/10.2478/s11535-014-0320-3
Xi, J., Patel, M., Dong, S., Que, Q., & Qu, R. (2018). Acetosyringone treatment duration affects large T-DNA molecule transfer to rice callus. BMC Biotechnology, 18, Article 48. https://doi.org/10.1186/s12896-018-0459-5
Yan, Z., Liu, S., Zhang, J., Huang, G., Duan, L., & Ye, Y. (2020). Optimizing hairy root production from explants of Phyllanthus hainanensis, a shrub used for traditional herbal medicine. Frontiers of Agricultural Science and Engineering, 7(4), 513–522. https://doi.org/10.15302/J-FASE-2019272