Research in Plant Metabolites

Research in Plant Metabolites

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)

Document Type : Original Article

Authors
razieh biglari farash 1
Azizollah kheiry 2
zahra ghahremani 2
khadijeh bagheri 3
farhang razavi 2
1 Department of Horticultural Sciences, Zanjan University . Iran
2 Department of Horticultural Sciences, Faculty of Agriculture, University of Zanjan, Zanjan, Iran
3 Department of Plant Production and Genetics, Faculty of Agriculture, University of Zanjan, Zanjan, Iran
10.22034/jrpsm.2026.562414.1102
Abstract
Background and objective: Production of hairy roots through Agrobacterium rhizogenes is an effective and sustainable method for the production of secondary metabolites in medicinal plants. Valerian (Valeriana officinalis L.) is a suitable candidate for biotechnology studies due to its valuable medicinal compounds such as valpotriates and valerenic acids. The aim of this study was to investigate the interaction effect of explant type and different strains of Agrobacterium rhizogenes on the growth indices and antioxidant capacity of hairy roots of this plant.
Materials and Methods: The experiment was carried out as a factorial in a completely randomized design with two factors of explants (roots, stems and leaves) and bacterial strains (MSU), A4, A13 and ATCC). Standard seeds of the plant were cultured in MS medium. Bacterial strains were cultured in LB medium containing rifampicin and the final suspension was prepared with a concentration of 1×10^8 cells/ml in liquid MS medium. One-month-old explants were inoculated for 30 minutes in a bacterial suspension containing 100 mg/l of acetosyringone. After a 48-hour co-cultivation period on solid MS medium, excess bacteria were removed by washing in a medium containing cefotaxime. The parameters of induction percentage, root formation rate, number of roots per explant and dry weight of hairy roots were measured. Root transformation was confirmed by DNA extraction and amplification of the rolB gene by PCR using specific primers and a standard temperature program. Antioxidant activity Methanolic extracts of roots were measured by DPPH radical scavenging method. Data were analyzed with SAS software and means were compared by Duncan test.
Findings: The results showed that the interaction effect of explant and strain on all measured traits was significant. The highest percentage of hairy root induction (%3.99) was obtained in the combination of root explant and MSU strain and the lowest (%7.48) belonged to the stem inoculated with ATCC strain. The fastest emergence of hairy roots (5 days after inoculation) was observed in root explant and MSU strain and the slowest induction (average 26 days) belonged to stem and ATCC strain. MSU strain induced the highest number of roots (maximum 7 in each explant) in all organs. However, the highest dry weight of hairy roots (0.22 g) was in leaf explant and in all three strains MSU, A4 and A13 was obtained. Hairy roots derived from leaves in all strains showed the highest antioxidant activity (64.08% inhibition) and roots derived from root explants showed the lowest activity (36.21%). The presence of a specific band of 780 bp of the rolB gene in the PCR product confirmed the successful transformation of all studied roots.
Conclusion: The results of this study showed that the optimal selection of the combination of explant and bacterial strain is a determining factor in the success of transformation and production of hairy roots in valerian. The MSU strain was identified as the most efficient strain in terms of induction percentage, formation rate and number of roots, while the leaf explant was superior in achieving maximum biomass and antioxidant activity. This study is an effective step towards standardizing the protocol for producing valerian hairy roots as a sustainable and cost-effective alternative for extracting medicinal compounds from this valuable plant.
Keywords
Molecular analysis
Transformation
Tissue culture
Bandyopadhyay, M., Jha, S., & Tepfer, D. (2007). Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of Withania somnifera. Plant Cell Reports, 26 (5), 599–609. https://doi.org/10.1007/s00299-006-0260-0
Banihashemi, O., Khavari-Nejad, R. A., Yassa, N., & Najafi, F. (2015). Induction of hairy root in Atropa komarovii using Agrobacterium rhizogenes ATCC15834. Indian Journal of Fundamental and Applied Life Sciences, 5 (4), 22–31.
Bhat, B., & Sharma, V. D. (2015). In vitro propagation of the Garden Heliotrope, Valeriana officinalis L.: Influence of pre chilling and light on seed germination. Plant Biology, 17 (2), 211–216. https://doi.org/10.1111/plb.12236
Choi, M., Yoon, J., Yang, S. H., Kim, J. K., & Park, S. U. (2023). Production of phenolic compounds and antioxidant activity in hairy root cultures of Salvia plebeia. Plants, 12 (22), 3840. https://doi.org/10.3390/plants12223840
Dasari, R., Gopu, C., Vankudoth, S., Dharavath, S., & Taduri, S. (2020). Enhancement of production of pharmaceutically important anti-cancerous compound; cucurbitacin E via elicitation and precursor feeding of in vitro culture of Citrullus colocynthis (L.) Schard. Vegetos, 33 (2), 323–334. https://doi.org/10.1007/s42535-020-00110-z
Dehghan E, Häkkinen ST, Oksman-Caldentey KM, Ahmadi FS. (2012) .Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.). Plant Cell, Tissue and Organ Culture 110(1): 35-44.
Desmet, S., Dhooghe, E., De Keyser, E., Van Huylenbroeck, J., Müller, R., Geelen, D., & Lütken, H. (2020). Rhizogenic agrobacteria as an innovative tool for plant breeding: Current achievements and limitations. Applied Microbiology and Biotechnology, 104 (6), 2435–2451. https://doi.org/10.1007/s00253-020-10350-3
Dhakulkar, S., Ganapathi, T. R., Bhargava, S., & Bapat, V. A. (2005). Induction of hairy roots in Gmelina arborea Roxb. and production of verbascoside in hairy roots. Plant Science, 169 (5), 812–818. https://doi.org/10.1016/j.plantsci.2005.05.014
El-Esawi, M. A., Elkelish, A., Elansary, H. O., Ali, H. M., Elshikh, M., Witczak, J., & Ahmad, M. (2017). Genetic transformation and hairy root induction enhance the antioxidant potential of Lactuca serriola L. Oxidative Medicine and Cellular Longevity, 2017, Article 2494721. https://doi.org/10.1155/2017/2494721
Farhadi, N., & Moghaddam, M. (2024). Application of Recent Advanced Technologies for the Improvement of Medicinal and Aromatic Plants. In A.K. Hvoslef-Eide & W. Preil (Eds.), Liquid culture systems for in vitro plant propagation (pp. 539–546). Springer.
Fitzgerald, J. A., Könemann, S., Krümpelmann, L., Županič, A., & Vom Berg, C. (2020). Approaches to test the neurotoxicity of environmental contaminants in the zebrafish model: From behavior to molecular mechanisms. Environmental Toxicology and Chemistry, 40 (4), 989–1006. https://doi.org/10.1002/etc.4959
Folgado, A., Serra, A. T., Prazeres, I., Bento-Silva, A., Bronze, M. R., & Abranches, R. (2021). Hairy root cultures of Cynara cardunculus L. as a valuable source of hydroxycinnamic acid compounds. Plant Cell, Tissue and Organ Culture, 146 (2), 245–256. https://doi.org/10.1007/s11240-021-02063-w
Ghadermazi, S., Masoud Tohidfar, M. and Miri, S.M. (2016). The Production of Hairy Roots from (Datura innoxia L.) by Agrobacterium rhizogenes and Effects of Salicylic Acid and Methyl Jasmonate Biological Elicitors on Atropine and Scopolamine Content. Journal of Medicinal Plants, 16(10): 141-156. (In Persian)
Ganjali, A., Zare Hassanabadi, M., Elhoti, M., and Moshtaghqi, N. (2019). Evaluation of different strains of Agrobacterium rhizogenes in inducing hairy roots and producing secondary metabolites in basil (Ocimum basilicum L.). Journal of Horticultural Sciences 34 (2), 273-284.
Georgiev, M. I., Pavlov, A. I., & Bley, T. (2007). Hairy root type plant in vitro systems as sources of bioactive substances. Applied Microbiology and Biotechnology, 74 (6), 1175–1185. https://doi.org/10.1007/s00253-007-0856-5
Jadid, N., Ramadani, M. R. N., Widodo, A. F., Sa’adah, N. N., Ermavitalini, D., Rahmawati, M., Sari, S. A., Soleha, I. D., & Mas’ud, F. (2024b). In silico characterization of GbPAL, GbCHS, GbDFR and GbANS structural genes involved in the biosynthesis of flavonoids in Gynura bicolor DC. South African Journal of Botany, 165, 428–442. https://doi.org/10.1016/j.sajb.2024.01.011
Jadid, N., Soleha, I. D., Rahayu, A. E., Puspaningtyas, I., Sari, S. A., Rahmawati, M., & Hidayati, D. (2024). Exogenous methyl jasmonate (MeJA) altered phytochemical composition and enhanced the expression of PatAACT gene of in vitro culture-derived patchouli var. Sidikalang (Pogostemon cablin Benth.). Journal of King Saud University - Science, 103301. https://doi.org/10.1016/j.jksus.2024.103301
Kaur, G., Prakash, P., Srivastava, R., & Verma, P. C. (2021). Enhanced secondary metabolite production in hairy root cultures through biotic and abiotic elicitors. In T. S. (Ed.), Plant Cell Tissue Differentiation and Secondary Metabolites: Fundamentals and Applications (pp. 625–660). Springer. https://doi.org/10.1007/978-3-030-30185-9_25
Kumar, V., Murthy, K. N. C., Bhamid, S., Sudha, C. G., & Ravishankar, G. A. (2005). Genetically modified hairy roots of Withania somnifera Dunal: A potent source of rejuvenating principles. Rejuvenation Research, 8 (1), 37–45. https://doi.org/10.1089/rej.2005.8.37
Lee S.U., Kimi S.U., Song W.S., Kim Y.K., Park N.I., and Park S.U. (2010). Influence of different strains of 282 Agrobacterium rhizogenes on hairy root induction and production of alizarin and purpurin in Rubiaakane Nakai. Romanian Biotechnological Letters 15(4): 5405-5409.
Marchev, A. S., Yordanova, Z. P., & Georgiev, M. I. (2024). Green (cell) factories for advanced production of plant secondary metabolites. Arabian Journal of Chemistry, 17 (9), 105904. https://doi.org/10.1016/j.arabjc.2024.105904
Mirazayi Delbari, E., Vatandoust, J., Jami Moeini, M., & Kuhban Bagheirkhira, E. (2022). The effect of Agrobacterium rhizogenes strains on hairy root induction in different explants of alfalfa. The Quarterly Scientific Journal of Applied Biology, 35 (3), 127–142.
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.x
Phuong, T. D., Le Thi Mong, V., & Phuong, Q. N. D. (2021). Antioxidant, anti-bacterial activity of Perilla frutescens ethanol extract and induction of hairy roots by Agrobacterium rhizogenes. VNUHCM Journal of Natural Sciences, 5 (1), 975–983. https://doi.org/10.32508/stdjns.v5i1.917
Pirian, K., Piri, K. H., & Ghiyasvand, T. (2012). Hairy roots induction from Portulaca oleracea using Agrobacterium rhizogenes to Noradrenaline’s production. International Research Journal of Applied and Basic Sciences, 3 (3), 642–649.
Saha, K., Lajis, N. H., Israf, D. A., Hamzah, A. S., Khozirah, S., Khamis, S., & Syahida, A. (2004). Evaluation of antioxidant and nitric oxide inhibitory activities of selected Malaysian medicinal plants. Journal of Ethnopharmacology, 92(2-3), 263-267
Sathasivam, R., Choi, M., Radhakrishnan, R., Kwon, H., Yoon, J., Yang, S. H., Kim, J. K., Chung, Y. S., & Park, S. U. (2022). Effects of various Agrobacterium rhizogenes strains on hairy root induction and analyses of primary and secondary metabolites in Ocimum basilicum. Frontiers in Plant Science, 13, 983776. https://doi.org/10.3389/fpls.2022.983776
Sezen Karaoğlan, E. (2022). Valeriana officinalis L. In Novel Drug Targets With Traditional Herbal Medicines: Scientific and Clinical Evidence (pp. 565–568). Springer.
Soleimani, T., Keyhanfar, M., Piri, K. H., & Hasanloo, T. (2012). Hairy root induction in Burdock (Arctium lappa L.). Journal of Medicinal Plants, 11 (44), 73–83. http://dorl.net/dor/20.1001.1.2717204.2012.11.44.17.9
Srivastava, S., Conlan, X. A., Adholeya, A., & Cahill, D. M. (2016). Elite hairy roots of Ocimum basilicum as a new source of rosmarinic acid and antioxidants. Plant Cell, Tissue and Organ Culture, 125 (1), 107–119. https://doi.org/10.1007/s11240-015-0933-x
Srivastava, S., & Srivastava, A. K. (2016). Effect of elicitors and precursors on azadirachtin production in hairy root culture of Azadirachta indica. Applied Biochemistry and Biotechnology, 172 (5), 2286–2297. https://doi.org/10.1007/s12010-013-0664-6
Su, Y., Lin, C., Zhang, J., Hu, B., Wang, J., Li, J., Wang, S., Liu, R., Li, X., Song, Z., & Wang, J. (2022). One-step regeneration of hairy roots to induce high tanshinone plants in Salvia miltiorrhiza. Frontiers in Plant Science, 13. https://doi.org/10.3389/fpls.2022.913985
Sujatha, G., Zdravković-Korać, S., Ćalić, D., Flamini, G., & Kumari, B. R. (2013). High-efficiency Agrobacterium rhizogenes-mediated genetic transformation in Artemisia vulgaris: Hairy root production and essential oil analysis. Industrial Crops and Products, 44, 643–652. https://doi.org/10.1016/j.indcrop.2012.09.007
Tavassoli, P., & Afshar, A. S. (2018). Influence of different Agrobacterium rhizogenes strains on hairy root induction and analysis of phenolic and flavonoid compounds in marshmallow (Althaea officinalis L.). 3 Biotech, 8 (8), 351. https://doi.org/10.1007/s13205-018-1375-z
Tiwari, R. K., Trivedi, M., Guang, Z. C., Guo, G. Q., & Zheng, G. C. (2007). Genetic transformation of Gentiana macrophylla with Agrobacterium rhizogenes: Growth and production of secoiridoid glucoside gentiopicroside in transformed hairy root cultures. Plant Cell Reports, 26 (2), 199–210. https://doi.org/10.1007/s00299-006-0236-0
Vanek, T., Langhansova, L., & Marsik, P. (2005). Cultivation of root cultures of Panax ginseng in different bioreactors and in temporary immersion Comparison of growth and saponin production. In A.K. Hvoslef-Eide & W. Preil (Eds.), Liquid culture systems for in vitro plant propagation (pp. 539-546). Springer.
Vyas, P., & Mukhopadhyay, K. (2014). Development of a rapid and high frequency Agrobacterium rhizogenes mediated transformation protocol for Ocimum tenuiflorum. Biologia, 69 (6), 765–770. https://doi.org/10.2478/s11756-014-0375-7
Zare Hasan Abadi, M., Ganjeali, M., Lahouti, M., & Moshtaghi, N. (2020). Influence of different Agrobacterium rhizogenes strains on hairy roots induction and secondary metabolites production in Ocimum basilicum L. Journal of Horticultural Science, 34 (2), 273–284. https://doi.org/10.22067/jhorts4.v34i2.82167
Zehra M., Banerjee S., Sharma S., and Kumar S. 1999. Influence of Agrobacterium rhizogenes strains on biomass and alkaloid productivity in hairy root lines of Hyoscyamus muticus and H. albus. Planta Medica 64: 60–63.
Zhao, S., & Tang, H. (2020). Enhanced production of valtrate in hairy root cultures of Valeriana jatamansi Jones by methyl jasmonate, jasmonic acid and salicylic acid elicitors. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 48 (2), 839–848. https://doi.org/10.15835/nbha48211872