Optimization of conditions for the induction of Tagetes patula L. hairy roots


L.P. Khlebova, E.S. Brovko, O.V. Bychkova, N.V. Pavlova

The hairy root cultures are promising sources of secondary metabolites of plants, including rare and endangered species. They possess genetic and biochemical stability, unlimited growth rate in free-hormone medium, short doubling times, high biosynthetic activity and ecological purity of plant raw materials. The hairy root cultures of Tagetes patula L. can be used to produce biologically active substances with biocidal activity. The study aimed to determine the virulent strain of Agrobacterium rhizogenes and the most effective period of co-cultivation of T. patula leaf explants with an agrobacterium to induce actively growing hairy root cultures. We used 3 strains (A-4b, 8196RT and 15834). The time of infection ranged from 3 to 33 hours in increments of 3 hours. We found that 24 h is the best time of infection to induce hairy roots with the highest transformation efficiency (92%). The wild strain A. rhizogenes 15834 turned out to be the most virulent when infected leaf explants of spreading marigold. This strain provided the maximum transformation effect, reaching 85.4%. We have identified 5 actively growing clones of hairy roots with intensive branching, the growth indices of which were 64-75. In the future, they will be transferred to a liquid medium for biomass accumulation and scaling.
 Keywords: Tagetes patula; Leaf explants; Agrobacterium rhizogenes; Hairy root culture
Ayub, M. A., Hussain, A. I., Hanif, M. A. et al. (2017). Variation in phenolic profile, b-carotene and flavonoid contents, biological activities of two Tagetes species from Pakistani flora. Chemistry Biodiversity, 14.
Batish, D. R., Arora, K., Singh, H. P., & Kohli, R. K. (2007). Potential utilization of dried powder of Tagetes minuta as a natural herbicide for managing rice weeds. Crop Protect, 26, 566-571.
Belabbassi, O., Khelifi-Slaoui, M., Zaoui, D., Benyammi, R., Khalfallah, N., Malik, S., Makhzoum, A., & Khelifi, L. (2016). Synergistic effects of polyploidization and elicitation on biomass and hyoscyamine content in hairy roots of Datura stramonium. Biotechnologie, Agronomie, Société et Environnement, 20, 408-416.
Bensaddek, L., Villarrea, M. L., & Fliniaux, M. A. (2008). Induction and growth of hairy roots for the production of medicinal compounds. Electronic Journal of Integrative Biosciences 3, 1, 2-9. Buitelaar, R. M., Langenhoff, A. A. M., Heidstra R., & Tramper J. (1991). Growth and thiophene production by hairy root cultures of Tagetes patula in various twoliquid phase bioreactors. Enzyme microbiology and biotechnology, 13, 487-494.
Bychkova, O. V., Khlebova, L. P., Brovko, E. S., & Borsukova, A. I. (2018). Efficient induction of hairy roots in Tagetes patula L. Ukrainian Journal of Ecology, 8(4), 450-453.
Chkhikvishvili, I., Sanikidze, T., Gogia N. et al., (2016). Constituents of French Marigold (Tagetes patula L.) Flowers protect jurkat Tcells against oxidative stress. Oxidative Medicine and Cellular Longevity, 2016, Article ID 4216285.
Dhiman, N., Patial, V., & Bhattacharya, A. (2018). The current status and future applications of hairy root cultures. In: Kumar N. (ed) Biotechnological Approaches for Medicinal and Aromatic Plants. Springer, Singapore.
Dicosmo, F., & Misawa, M. (1995). Plant cell and tissue culture: Alternatives for metabolite production. Biotechnology Advances, 13, 425-453.
Faizi, S., Fayyaz, S., Bano S. et al. (2011). Isolation of nematicidal compounds from Tagetes patula L. yellow flowers: Structureactivity relationship studies against cyst nematode Heterodera zeae infective stage larvae. Journal of Agricultural and Food Chemistry, 59(17), 9080-9093.
Farag, S., & Kayser, O. (2015). Cannabinoids production by hairy root cultures of Cannabis sativa L. American Journal of Plant Sciences, 6, 1874-1884. http://dx.doi.org/10.4236/ajps.2015.611188 Ghosh, A., Chowdhury, N., & Chandra, G. (2012). Plant extracts as potential mosquito larvicides. Indian Journal of Medical Research, 135, 5, 581-598. Giri, A. A, Ravindra, S. T., Dhingra, V. T, & Narasu, M. L. (2001). Influence of different strains of Agrobacterium rhizogenes on induction of hairy root and artemisinin production in Artemisia annua. Curr. Sci., 81, 378-382. Giri, R. K., Bose, A., & Mishra, S. K. (2011). Hepatoprotective Activity of Tagetes erecta against Carbon Tetrachloride-Induced Hepatic Damage in Rats. Acta Poloniae Pharmceutica–Drug Research, 68, 999-1003. Gonçalves, S., & Romano, A. (2018). Production of plant secondary metabolites by using biotechnological tools. In: Secondary Metabolites – Sources and Applications; Vijayakumar, R., Raja, S. S.S., Eds.; InTech. DOI: 10.5772/intechopen.76414.
Gupta, V., Shanker, K., & Rahman, L. (2016). In vitro production of thiophenes using hairy root cultures of Tagetes erecta (L.). African Journal of Biotechnology, 15(17), 706-713. Halder, M., Sarkar, S., & Jha, S. (2019). Elicitation: A biotechnological tool for enhanced production of secondary metabolites in hairy root cultures. Eng Life Sci, 1‐16. https://doi.org/10.1002/elsc.201900058 Hasspieler, B. M., Arnason, J. T., & Downe, A. E. (1988). Toxicity, localization and elimination of the phototoxin, α-terthienyl in mosquito larvae. J Am Mosq Control Assoc, 4(4), 479-484. Isah, T., Umar, S., Mujib, A., Sharma, M. P., Rajasekharan, P. E., Zafar, N., & Frukh, A. (2018). Secondary metabolism of pharmaceuticals in the plant in vitro cultures: Strategies, approaches, and limitations to achieving higher yield. Plant Cell Tissue and Organ Culture, 132, 239-265. DOI: 10.1007/s11240-017-1332-2
Ketel, D. (1986) Morphological differentiation and occurrence of thiophene in leaf callus cultures from Tagetes sp. The effect of growth medium of the plants. Acta Physiol Plantarum, 66, 392. DOI: 10.1111/j.1399-3054.1986.tb05940.x
Khan, S., Siddiqui, M., & Osama, K. (2018). Bioreactors for Hairy Roots Culture: A Review. Current Biotechnology, 7(6), 417-427. Kochkin, D. V., Globa, E. B., Demidova, E. V., Gaisinsky, V. V, Kuznetsov, V. V., & Nosov, A. M. (2019). Detection of taxuyunnanin C in suspension cell culture of Taxus canadensis. Doklady Akademii Nauk, 485, 3, 374-376. DOI:10.31857/S0869-56524853374-376 Kolewe, M. E., Gaurav, V., & Roberts, S. C. (2008). Pharmaceutical active natural product synthesis and supply via plant cell culture technology. Molecular Pharmaceutics, 5, 243-256. DOI:10.1021/mp7001494
Krzyzaniak, L. M., Antonelli-Ushirobira, T. M., Panizzon, G., Sereia, A. L., de-Souza, J. R. P., Zequi, J. A. C., Novello, C. R., Lopes, G. C., de Medeiros, D. C., Silva, D. B., Leite-Mello, E. V. D., & De-Mello, J. C. P. (2017) Larvicidal Activity against Aedes aegypti and chemical characterization of the inflorescences of Tagetes patula. Evidence-Based Complementary and Alternative Medicine, 1-8. https://doi.org/10.1155/2017/9602368
Kuluev, B. R., Vershinin, Z. R., Knyazev, A. V., Chemeris, D. A., Baymiev, An. H., Chumakov, M. I., Baymiev, K., & Chemeris, A.V. (2015) «Kosmatye» korni rastenui – vazhnyi instrymentariy dlya issledovateley i moshchnaya fitohim-biofabrika dlya proizvodstvennikov. Biomeka, 2, 70-120. (In Russian)
Kuzovkina, I. N., & Vdovichenko, M. Yu. (2011). Geneticheski transformirovannye korni kak model izychenia fiziologicheskih i biohimicheskih processov kornevoi systemy cselogo rastenia. Plant physiology, 58 (5), 787-796. (In Russian) Kyo, M., Miyauchi, Y., Fujimoto, T., & Mayama, S. (1990). Production of nematocidal compounds by hairy root cultures of Tagetes patula L. Plant Cell Reports, 9, 393-397.
Lee, S. Y, Kim, S. G., Song, W. S., Kim, Y. K., Park, N. I., & Park, S. U. (2010). Influence of different strains of Agrobacterium rhizogenes on hairy root induction and production of alizarin and purpurin in Rubia akane Nakai. Romanian Biotechnological Letter, 15, 5405-5409.
Malik, S., Rosa, M., & Cusido B. (2011). Production of the anticancer drug taxol in Taxus baccata suspension cultures. Process Biochemistry, 46, 23-34.
Mares, D., Tosi, B., Poli, F., Andreotti, E., & Romagnoli, C. (2004) Antifungal activity of Tagetes patula extracts on some phytopathogenic fungi: ultrastructural evidence on Pythium ultimum. Microbiol Res, 159, 295-304.
Marotti, M., Piccaglia, R., Biavati, B., & Marotti, I. (2004) Characterization and yield evaluation of essential oils from different Tagetes species. Journal of Essential Oil Research, 16, 440-444.
Mikhailova, E. V., Kuluev, B. R., Sibaeva, G. R., & Chemeris A.V. (2017). Sozdanie kul’tur borodatyh korney Withania somnifera i otcenka parametrov ih rosta pri vyrashchivanii na tverdyh i zhidkih pitatel’nyh sredah. Bulletin of physico-chemical biology and biotechnology. Ovchinnikov, 13(2), 40-45. (In Russian) Mir, R. A., Ahanger, M. A., & Agarwal, R. M. (2019). Marigold: From mandap to medicine and from ornamentation to remediation. American Journal of Plant Sciences, 10, 309-338. https://doi.org/10.4236/ajps.2019.102024
Mulabagal, V., & Tsay, H. (2004) Plant cell cultures as a source for the production of biologically important secondary metabolites. Int J Appl Sci Engin, 2, 29-48.
Murashige, T., & Skoog, F. A. (1962). Revised medium for rapid growth and boassays with tobacco tissue cultures. Physiol. Plant, 15, 473-497.
Nosov, A. M. (2012). Application of cell technologies for production of plant derived bioactive substances of plant origin. Applied Biochemistry and Microbiology, 48, 7, 609-624. Ono, N. N., & Tian, L. (2011). The multiplicity of hairy root cultures: prolific possibilities. Plant Sci., 180(3), 439-446. doi: 10.1016/j.plantsci.2010.11.012Orlova, L. V., Globa, E. B., Chernyak, N. D., Demidova, E. V., Titova, M. V., Solovchenko, A. E., Sergeev, R. V., & Nosov, A. M. (2014). Growth and bioartificial characteristics of Taxus baccata suspension culture (cultivation in flasks and bioreactor). Vestnik of Volga State University of Technology. Ser.: Forest. Ecology. Nature Management, 3(23), 86-97. (In Russian)
Park, S-U, & Facchini, P. J. (2000). Agrobacterium rhizogenes-mediated transformation of opium poppy, Papaver somniferum L., and California poppy, Eschscholzia californica Cham., root cultures. J Exp Bot, 347, 1005-1016.
Patra, N., Srivastava, A. K. (2016). Artemisinin production by plant hairy root cultures in gas- and liquid-phase bioreactors. Plant Cell Rep, 35(1), 143-153. doi: 10.1007/s00299-015-1875-9. Politi, F. A. S., Queiroz-Fernandes, G. M., Rodrigues, E. R., Freitas, J. A., & Pietro, R. C. L. R. (2016). Antifungal, antiradical and cytotoxic activities of extractives obtained from Tagetes patula L. (Asteraceae), a potential acaricide plant species. Microbial Pathogenesis, 95, 15-20.
Rajasekaran, T., Madhusudhan, R., & Ravishankar, G. A. (1999). Elicitation of thiophene production by cultured hairy roots of Tagetes patula. Acta Physiol. Plant, 21, 243-247.
Rajasekaran, T., Ravishankar, G. A., & Obul Reddy, B. (2004). In vitro growth of Tagetes patula L. hairy roots, production of thiophenes and its mosquito larvicidal activity. Indian Journal of Biotechnology, 3, 92-96.
Ramachandra Rao S., & Ravishankar. G. A. (2002) Plant cell cultures: Chemical factories of secondary metabolites. Biotechnol Advances, 20, 1-53. Ramachandra Rao, S., Tripathi, U., Suresh B., & Ravishankar G. A. (2001). Enhancement of secondary metabolite production in hairy root cultures of Beta vulgaris and Tagetes patula under the influence of microalgal elicit. Food biotechnology, 15(1), 35-46. DOI: 10.1081/FBT-100103893
Saleh, N. M. & Thuc, L. V. (2009). Assessment of hairy roots induction in Solenostemon scutellarioides leaves by different strains of Agrobacterium rhizogenes. African Journal of Biotechnology, 8(15), 3519-3523.
Saravanakumar, A., Aslam, A., & Shajahan, A. (2012). Development and optimization of hairy root culture systems in Withania somnifera (L.) Dunal for withaferin-A production. African Journal of Biotechnology, 11(98), 16412-16420.
Siddiqui, M. A., & Alam, M. M. (1987). Utilization of marigold plant wastes for the control of plant parasitic nematodes. Biol Wastes, 21, 221-229. Taha, H. S., Osman, H. A., Youssef, M. M. A., El-Gindi, A.M.Y., Ameen, H. H., & Lashein, A. M. S. (2013). Chromatographic analysis of thiophenes in calli and suspension cultures of Tagetes spp. Journal of Life Sciences, 7(5), 483-490. Talov, R. J., Cascone, O., & Giulietti, A. M. (1994). Content of thiophenes in transformed root cultures of Argentinian species of Tagetes. Planta-Medica, 60(3), 260-262.
Vdovichenko, M. Yu., Kuzovkina, I. N., Pitts, H., & Schneider, B. (2007) Kyltiviruemye in vitro korni kopeechnika chainogo i obrazovanie v nih fenol’nyh soedineniy. Plant physiology, 54 (4), 604-613. (In Russian)
Verpoorte, R., Contin, A., & Memelink, J. (2002). Biotechnology for the production of plant secondary metabolites. Phytochem. Rev., 1, 13-25. Vladimirov, I. A., Matveeva, T. V., & Lutova, L. A. (2015). Opine biosynthesis and catabolism genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes. Russian Journal of Genetics, 51(2), 121-129. Wahyuni, D. K., Wahyuni, S., Ermayanti, T. M., Wardoyo, B. E., Purnobasuki, H., & Utami, E. W. (2017). Various infection time of Agrobacterium rhizogenes strain LB510 for hairy root induction on Justicia gendarussa Burm.f. International Journal of ChemTech Research, 10(4), 404-411. Watcharatanona, K., Ingkaninanc, K., & Putaluna, W. (2019). Improved triterpenoid saponin glycosides accumulation in in vitro culture of Bacopa monnieri (L.) Wettst with precursor feeding and LED light exposure. Industrial Crops & Products, 134, 303-308. https://doi.org/10.1016/j.indcrop.2019.04.011
Weaver, D. K., Zettler, L. J., Wells, C. D., Baker J. E., Bertsch, W., & Throne, J. E. (1997) Toxicity of fractionated and degraded Mexican marigold floral extract to adult Sithophilus zeamais (Coleopteran : Curculionidae). Journal of Economic Entomology, 90, 1678-1683.

Share this article