Hairy roots are obtained from the infection caused by Agrobacterium rhizogenes, a gram negative bacterium and are known to produce different complex molecules. Various biochemical pathways and physiological aspects in plants can be understood by means of hairy roots. Being genetically and biosynthetically stable as well as resultant high biomass accumulation and productivity in short period of time, these roots are great alternatives to conventional methods for the production of pharmacologically important compounds. Various biotechnological approaches i.e. culture medium components and their concentration, culture conditions, elicitation etc. are used and optimized to enhance overall yield. To meet up the increasing demand, production on industrial scale has been considered to be an important where concept of bioreactors is involved. This review presents basic idea of development of hairy roots, requirement of the optimum culture conditions and use of bioreactors to increase yield of the bioactive compounds.
hairy roots, Agrobacterium rhizogenes, metabolites production, bioreactor cultivation
Abbasi, B.H., Stiles, A.R., Saxena, P.K. & Liu, C.Z. (2012). Gibberellic acid increases secondary metabolite production in Echinacea purpurea hairy roots. Applied Biochemistry and Biotechnology.168(7),2057–2066.
Almagro, L., Belchí-Navarro, S., Sabater-Jara, A.B., Vera-Urbina, J.C., Sellés-Marchart S., Bru, R. & Pedreño, M.A. (2013). Bioproduction of trans-resveratrol from grapevine cell cultures. In Ramawat, K.G., Merillon, J.M. (Eds.), Handbook of natural products (pp. 1683-1713). Berlin: Springer.
Bais, H.P., Suresh, B., Raghavarao, K.S.M.S. & Ravishankar, G.A. (2002). Performance of hairy root cultures of Cichorium intybus L. in bioreactors of different configurations. In Vitro Cell Dev Biol Plant. 38(6), 573-580.
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. Biotechnol Agron Soc Environ. 20(3),408–416
Bhagwath, S.G. & Hjortso, M.A. (2000). Statistical analysis of elicitation strategies for thiarubrine A production in hairy root cultures of Ambrosia artemisiifolia. Journal of Biotechnology. 80, 159–167.
Carlín, A.P., Tafoya, F., Solís, A.G.A. & Perez-Molphe-Balch, E. (2015). Effects of different culture media and conditions on biomass production of hairy root cultures in six Mexican cactus species. In Vitro Cell Dev Biol Plant. 51(3), 332–339.
Cham, C.L., Tan, A.H. & Tan, W.H. (2016). Design and construction of a mist reactor system. Proceedings of Annual International Conference, TENCON, 3382-5.
Chandra, S. (2012). Natural plant genetic engineer Agrobacterium rhizogenes: role of T-DNA in plant secondary metabolism. Biotechnol. Lett. 34, 407–415.
Chung, I.M., Rekha, K., Rajakumar, G. & Thiruvengadam, M. (2016). Production of glucosinolates, phenolic compounds and associated gene expression proﬁles of hairy root cultures in turnip (Brassica rapa ssp. rapa). Biotech, 6(2),175.
Cragg, G.M. & Newman, D.J. (2013). Natural products: a continuing source of novel drug leads. Biochem Biophysics Acta, 1830, 3670-3695.
Danphitsanuparn, P., Boonsnongcheep, P., Boriboonkaset, T., Chintapakorn, Y. & Prathanturarug, S. (2012). Effects of Agrobacterium rhizogenes strains and other parameters on production of isoﬂavonoids in hairy roots of Pueraria candollei Grah.exBenth.var.candollei. Plant Cell Tissue Organ Cult. 111(3), 315–322
Dehghan, E., Häkkinen, S.T., Oksman-Caldentey, K.M. & Ahmadi, F.S. (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 Organ Cult. 110(1), 35–44
Doran, P., M. (1997). Hairy Roots, Culture and Applications. Amsterdam: Harwood Academic Publishers
Eder, J. & Cosio, E.G. (1994). Elicitors of plant defense responses. In International review of cytology (Vol. 148, pp.1-36). Academic press
Eibl, R., and Eibl, D. (2006). Plant Tissue Culture Engineering. In Gupta, S.D. & Ibaraki,Y. (Eds.). Focus Biotechnol. (pp. 203–227).
Gai, Q.Y., Jiao, J., Luo, M., Wang, W., Ma, W., Zu, Y.G. & Fu, Y.J. (2015a). Establishment of high-productive Isatis tinctoria L. hairy root cultures: a promising approach for efﬁcient production of bioactive alkaloids. Biochem Eng J, 95, 37–47.
Gai, Q.Y., Jiao, J., Luo, M., Wei, Z.F., Zu, Y.G., Ma, W. & Fu, Y.J. (2015b). Establishment of hairy root cultures by Agrobacterium rhizogenes mediated transformation of Isatis tinctoria L. for the efﬁcient production of ﬂavonoids and evaluation of antioxidant activities. PloS One, ,e0119022.
Gangopadhyay, M., Dewanjee, S. & Bhattacharya, S. (2011). Enhanced plumbagin production in elicited Plumbago indica hairy root cultures. Journal of Bioscience and Bioengineering, 111, 706–710.
Gelvin, S. B. (2009). Agrobacterium in the genomics age. Plant Physiol, 150, 1665– 1676.
Georgiev, M.I., Agostini, E., Ludwig-Müller, J. & Xu, J. (2012). Genetically transformed roots: from plant disease to biotechnological resource. Trends Biotechnol, 30, 528–537.
Georgiev, V.G., Weber, J., Kneschke, E.M., Denev, P.N., Bley, T. & Pavlov, A.I. (2010). Antioxidant activity and phenolic content of betalain extracts from intact plants and hairy root cultures of the red beetroot Beta vulgaris cv. Detroit dark red. Plant Foods Hum Nutr, 65(2), 105–111
Goklany, S., Rizvi, N.F., Loring, R.H., Cram, E.J. & Lee-Parsons, C.W. (2013). Jasmonate-dependent alkaloid biosynthesis in Catharanthus roseus hairy root cultures is correlated with the relative expression of Orca and Zct transcription factors. Biotechnol Prog, 29(6), 1367–1376
Halder, M. & Jha, S. (2016). Enhanced trans-resveratrol production in genetically transformed root cultures of peanut (Arachis hypogaea L.). Plant Cell Tiss Organ Cult, 124(3), 555–572
Halder, M., Roychowdhury, D. & Jha, S. (2018). A critical review on Biotechnological Interventions for Production and Yield enhancement of Secondary metabolites in Hairy Root Cultures. In Hairy Roots (pp. 21-44). Springer, Singapore
Hilton, M.G. & Rhodes, M.J.C. (1990). Growth and hyoscyamine production of “hairy root” cultures of Datura stramonium in a modified stirred tank reactor. Appl Microbiol Biotechnol, 33(2), 132-8.
Hsiao, T.Y., Bacani, F.T., Carvalho, E.B. & Curtis, W.R. (1999). Development of a low capital investment reactor system: application for plant cell suspension culture. Biotechnol. Prog, 15, 114–122
Hu, Z. & Du, M. (2006). Hairy root and its application in plant genetic engineering. J Integr Plant Biol, 48(2), 121-7.
Huang, B., Lin, H., Yan, C., Qiu, H., Qiu, L. & Yu, R. (2014). Optimal inductive and cultural conditions of Polygonum multiﬂorum transgenic hairy roots mediated with Agrobacterium rhizogenes R1601 and an analysis of their anthraquinone constituents. Pharmacogn Mag, 10(37),77–82
Huang, S.Y., Hung, C.H. & Chou, S.N. (2004). Innovative strategies for operation of mist trickling reactors for enhanced hairy root proliferation and secondary metabolite productivity. Enzyme Microb Technol, 35(1), 22-32.
Jaremicz, Z., Luczkiewicz, M., Kokotkiewicz, A., Krolicka, A. & Sowinski, P. (2014). Production of tropane alkaloids in Hyoscyamus niger (black henbane) hairy roots grown in bubble-column and spray bioreactors. Biotechnol Lett, 36(4), 843-53.
Jeong, C.S., Murthy, H.N., Hahn, E.J., Lee, H.L. & Paek, K.Y. (2009). Inoculum size and auxin concentration inﬂuence the growth of adventitious roots and accumulation of ginsenosides in suspension cultures of ginseng (Panax ginseng CA Meyer). Acta Physiol Plant, 31, 219–222
Jiao, J., Gai, Q.Y., Fu, Y.J., Ma, W., Peng, X., Tan, S.N. & Efferth, T. (2014). Efﬁcient production of isoﬂavonoids by Astragalus membranaceus hairy root cultures and evaluation of antioxidant activities of extracts. J Agric Food Chem, 62(52), 12649–12658
Jiao, J., Gai, Q.Y., Fu, Y.J., Ma, W., Yao, L.P., Feng, C. & Xia, X.X. (2015). Optimization of Astragalus membranaceus hairy roots induction and culture conditions for augmentation production of astragalosides. Plant Cell Tiss Organ Cult, 120(3), 1117–1130
Kim, Y., Wyslouzil, B.E. & Weathers, P.J. (2001). A comparative study of mist and bubble column reactors in the in vitro production of artemisinin. Plant Cell Rep, 20(5), 451-5.
Kim, Y.H. & Yoo, Y.J. (1993). Development of a bioreactor for high density culture of hairy roots. Biotechnol. Tech, 7, 859–862
Kim, Y.J., Weathers, P.J. & Wyslouzil, B.E. (2002). Growth of Artemisia annua hairy roots in liquid- and gas-phase reactors. Biotechnol Bioeng, 80(4), 454-64.
Kintzios, S., Makri, O., Pistola, E., Matakiadis, T., Ping Shi, H. & Economou, A. (2004). Scale-up production of puerarin from hairy roots of Pueraria phaseoloides in an airlift bioreactor. Biotechnol Lett, 26(13), 1057-9.
Kondo, O., Honda, H., Taya, M. & Kobayashi, T. (1989). Comparison of growth properties of carrot hairy root in various bioreactors. Applied microbiology and biotechnology, 32(3), 291-294.
Krishnan, S.S. & Siril, E.A. (2018). Elicitor mediated adventitious root culture for the large scale production of anthraquinones from Oldenlandia umbellate L. Industrial Crops and Products, 114, 173-179.
Kuzma, L., Bruchajzer, E. & Wysokinska, H. (2009). Methyl jasmonate effect on diterpenoid accumulation in Salvia sclarea hairy root culture in shake flasks and sprinkle bioreactor. Enzyme Microb Technol, 44(6-7), 406-10.
Li, B., Wang, B., Li, H, Peng, L., Ru, M., Liang, Z., Yan, X. & Zhu, Y. (2015). Establishment of Salvia castanea Diels f. tomentosa Stib. Hairy root cultures and the promotion of tanshinone accumulation and gene expression with Ag+, methyl jasmonate, and yeast extract elicitation. Protoplasma. http://doi.org/ 10.1007/s00709–015–0790–9.
Liu, C., Wang, Y., Guo, C., Ouyang, F., Ye, H. & Li, G. (1998). Enhanced production of artemisinin by Artemisia annua L hairy root cultures in a modified inner-loop airlift bioreactor. Bioprocess Eng, 19(5), 389-92.
Liu, L., Yang, D., Liang, T., Zhang, H., He, Z. & Liang, Z. (2016). Phosphate starvation promoted the accumulation of phenolic acids by inducing the key enzyme genes in Salvia miltiorrhiza hairy roots. Plant Cell Rep, 35(9), 1933–1942
Liu, Q., Cui, L., Guo, Y., Ni, X., Zhang, Y. & Kai, G. (2013). Optimization of nutritive factors in culture media for growth and tropane alkaloid production from Anisodus acutangulus hairy roots. J Appl Pharm Sci, 3(1),001–004
Medina-Bolivar, F., Condori, J., Rimando, A., Hubstenberger, J., Shelton, K., O’Keefe, S.F. & Dolan, M.C. (2007) Production and secretion of reveratrol in hairy root cultures of peanut. Phytochemisrty, 68(14), 1992-2003.
Mishra, B.N. & Ranjan, R. (2008). Growth of Hairy Root Cultures in various bioreactors for the production of secondary metabolites. Biotechnol. App. Biochem, 49, 1-10.
Mukherjee, C., Samanta, T. & Mitra, A. (2016). Redirection of metabolite biosynthesis from hydroxybenzoates to volatile terpenoids in green hairy roots of Daucus carota. Planta, 243, 305–320
Murthy, H.N., Lee, E.J. & Paek, K.Y. (2014). Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation. Plant Cell Tissue Organ Culture, 118, 1–16
Namdeo, A.G. (2007). Plant cell elicitation for production of secondary metabolites: a review. Pharmacogn Rev, 1(1), 69-79.
Nayak. P, Sharma, M., Behera, S.N., Thirunavoukkarasu, M. & Chand, P.K. (2015). High-performance liquid chromatographic quantiﬁcation of plumbagin from transformed rhizoclones of Plumbago zeylanica L.: Inter-clonal variation in biomass growth and plumbagin production. Appl Biochem Biotechnol, 175, 1745–1770
Neelwarne, B. & Thimmaraju, R. (2009). Bioreactor for cultivation of red beet hairy roots and in situ recovery of primary and secondary metabolites. Eng Life Sci, 9(3), 227-38.
Osama, K., Pallavi, S., Pandey, A.K. & Mishra, B.N. (2013). Modelling of nutrient mist reactor for hairy root growth using artificial neural network. Eur J Sci Res, 97(4), 516-26.
Patra, N. & Kumar, S.A. (2014). Mass scale artemisinin production in a stirred tank bioreactor using hairy roots of Artemisia annua. Int J Biosci Biochem Bioinforma, 4, 467-74.
Patra, N. & Srivastava, A.K. (2014). Enhanced production of artemisinin by hairy root cultivation of Artemisia annua in a modified stirred tank reactor. Appl Biochem Biotechnol, 174(6), 2209-22.
Patra, N. & Srivastava, A.K. (2014a). Enhanced production of artemisinin by hairy root cultivation of Artemisia annua in a modiﬁed stirred tank reactor. Appl Biochem Biotechnol, 174 (6), 2209–2222
Patra, N. & Srivastava, A.K. (2014b). Mass scale artemisinin production in a stirred tank bioreactor using hairy roots of Artemisia annua. Int J Biosci Biochem Bioinforma , 4(6), 467–474
Patra, N. & Srivastava, A.K. (2015). Use of model-based nutrient feeding for improved production of artemisinin by hairy roots of Artemisia annua in a modiﬁed stirred tank bioreactor. Appl Biochem Biotechnol, 177(2), 373–388
Patra, N. & Srivastava, A.K. (2016). Artemisinin production by plant hairy root cultures in gas- and liquid-phase bioreactors. Plant Cell Rep, 35(1), 143-53.
Patra, N. & Srivastava, A.K. (2017). Mass production of artemisinin using hairy root cultivation of Artemisia annua in bioreactor. In Pavlov, A. & Bley, T. (Eds.) Bioprocessing of plant in vitro systems (pp. 1-17). Springer International Publishing,
Perassolo, M., Cardillo, A.B., Mugas, M.L., Montoya, S.C.N., Giulietti, A.M. & Talou, J.R. (2017). Enhancement of anthraquinone production and release by combination of culture medium selection and methyl jasmonate elicitation in hairy root cultures of Rubia tinctorum. Ind Crops Prod, 105, 124–132
Peret, B., Clement, M., Nussaume, L. & Desnos, T. (2011). Root developmental adaptation to phosphate starvation: better safe than sorry. Trends Plant Sci, 16, 1360–1385
Rahimi, S. & Hasanloo, T. (2016). The effect of temperature and pH on biomass and bioactive compounds production in Silybum marianum hairy root cultures. Res J Pharmacogn, 3(2), 53–59
Ranjan, R., Ahmed, N., Khanna, R. & Mishra, B.N. (2009). Design of an ON/OFF mist duty cycle in mist bioreactors for the growth of hairy roots. Biotechnol Bioprocess Eng, 14(1), 38-45.
Rao, R.S. & Ravishankar, G. (2002). Plant cell cultures: Chemical factories of secondary metabolites. Biotechnol Adv, 20(2), 101-53.
Saravanakumar, A., Aslam, A. & Shajahan, A. (2012). Development and optimization of hairy root culture systems in Withania somnifera (L.) Dunal for withaferin-A production. Afr J Biotechnol, 11(98), 16412–16420
Satdive, R.K., Fulzele, D.P. & Eapen, S. (2007). Enhanced production of azadirachtin by hairy root cultures of Azadirachta indica A. Juss by elicitation and media optimization. Journal of Biotechnology,128, 281–289.
Sauerwein, M., Wink, M. & Shimomura, K. (1992). Influence of light and phytohormones on alkaloid production in transformed root cultures of Hyoscyamus albus. Jounal of plant physiology, 140(2), 147-152.
Shanks, J.V. & Morgan, J. (1999). Plant hairy root culture. Curr. Opin. Biotechnol, 10, 151–155
Sharaﬁ, A., Sohi, H.H., Mousavi, A., Azadi, P., Razavi, K. & Ntui, V.O. (2013). A reliable and efﬁcient protocol for inducing hairy roots in Papaver bracteatum. Plant Cell Tiss Organ Cult, 113(1), 1–9
Shilpa, K., Varun, K. & Laxmi, B,S. (2010). An alternate method of natural drug production: eliciting secondary metabolite production using plant cell culture. J Plant Sci, 5(3), 222-247.
Shinde, A.N., Malpathak, N. & Fulzele, D.P. (2010). Impact of nutrient components on production of the phytoestrogens daidzein and genistein by hairy roots of Psoralea corylifolia. J Nat Med, 64 (3), 346–353
Sivanandhan, G., Dev, G., K., Jeyaraj, M., Rajesh, M., Arjunan, A., Muthuselvam, M., Manickavasagam, M., Selvaraj, N. & Ganapathi, A. (2013). Increased production of withanolide A, withanone, and withaferin A in hairy root cultures of Withania somnifera (L.) Dunal elicited with methyl jasmonate and salicylic acid. Plant Cell Tissue and Organ Culture,114, 121–129.
Srivastava, S. & Srivastava, A.K. (2012). Azadirachtin production by hairy root cultivation of Azadirachta indica in a modified stirred tank reactor. Bioprocess Biosyst Eng, 35(9), 1549-53.
Srivastava, S. & Srivastava, A.K. (2012). In vitro Azadirachtin production by hairy root cultivation of Azadirachta indica in nutrient mist bioreactor. Appl Biochem Biotechnol, 166(2), 365-78.
Srivastava, S. & Srivastava, A.K. (2013). Production of the biopesticide azadirachtin by hairy root cultivation of Azadirachta indica in liquid-phase bioreactors. Appl Biochem Biotechnol, 171(6), 1351-61.
Srivastava, S. & Srivastava, A.K. (2014). Effect of elicitors and precursors on azadirachtin production in hairy root culture of Azadirachta indica. Appl Biochem Biotechnol, 172(4), 2286–2297
Stiles, A.R. & Liu, C.Z. (2013). Hairy root culture: bioreactor design and process intensiﬁcation. In Doran, P.M. (Ed.) Biotechnology of hairy root systems (pp. 91-14) Springer, Berlin Heidelberg
Sujatha, G., Zdravkovic-Korac, S., Calic, D., Flamini, G. & Kumari, B.R. (2013). High-efﬁciency Agrobacterium rhizogenes-mediated genetic transformation in Artemisia vulgaris: hairy root production and essential oil analysis. Ind Crops Prod, 44, 643–652
Sun. J. &Peebles, C.A. (2016). Engineering over expression of ORCA3 and strictosidine glucosidase in Catharanthus roseus hairy roots increases alkaloid production. Protoplasma, 253(5),1255–1264
Suresh, B., Thimmaraju, R., Bhagyalakshmi, N. & Ravishankar, G.A. (2004). Polyamine and methyl jasmonate-influenced enhancement of betalaine production in hairy root cultures of Beta vulgaris grown in a bubble column reactor and studies on efflux of pigments. Process Biochem, 39(12), 2091-6.
Thakore, D., Srivastava, A.K. & Sinha, A.K. (2017). Mass production of ajmalicine by bioreactor cultivation of hairy roots of Catharanthus roseus. Biochem Eng J, 119, 84–91
Thiruvengadam, M., Rekha, K. & Chung, I.M. (2016). Induction of hairy roots by Agrobacterium rhizogenes-mediated transformation of spine gourd (Momordica dioica Roxb. ex. willd) for the assessment of phenolic compounds and biological activities. Sci Hort, 198, 132–141
Vashishtha, M. & Sharma, N. (2015). Nutrient mist reactor : A remarkable new approach. Int J Adv Sci Eng Technol, 3(1), 8-11.
Vinterhalter, B., Krstic-Milosevic, D., Jankovic, T., Pljevljakusic, D., Ninkovic, S., Smigocki, A. & Vinterhalter, D. (2015). Gentiana dinarica Beck. hairy root cultures and evaluation of factors affecting growth and xanthone production. Plant Cell Tiss Organ Cult, 121(3), 667–679
Al-Masry & Waheed, A. (1999). Effect of scale-up on average shear rates for aerated non- Newtonian liquids in external loop airlift reactors. Biotechnol. Bioeng, 62, 494–498
Weathers, P., Liu, C., Towler, M. & Wyslouzil, B. (2008). Mist reactors: Principles, comparison of various systems, and case studies. Electron J Integr Biosci, 3, 29-37.
Williams, G.R.C. & Doran, P.M. (2000). Hairy root culture in a liquid-dispersed bioreactor: Characterization of spatial heterogeneity. Biotechnol Prog, 16(3), 391-401.
Xiaolong, H., Min, S., Lijie, C., Chao, X., Yanjie, Z. & Guoyin, K., (2015). Effects of methyl jasmonate and salicylic acid on tanshinone production and biosynthetic gene expression in transgenic Salvia miltiorrhiza hairy roots. Biotechnology and Applied Biochemistry, 62(1), 24–31.
Zahra, S., Mehrnaz, K., Gholamreza, A. & Mustafa, G. (2015). Improvement of atropine productio by different biotic and abiotic elicitors in hairy root cultures of Datura metel. Turkish Journal of Biology, 39, 111–118.
Zhao, L., Sander, G.W. & Shanks, J.V. (2013). Perspectives of the metabolic engineering of terpenoid indole alkaloids in Catharanthus roseus hairy roots. In Doran, P.M. (Ed.) Biotechnology of hairy root systems (pp. 23-54). Springer, Berlin Heidelberg
Zheng, Z. & Wu, M. (2004). Cadmium treatment enhances production of alkaloid secondary metabolites in Catharanthus roseus. Plant science,166(2), 507-14
Zhu, C., Miao, G., Guo, J., Huo, Y., Zhang, X., Xie, J. & Feng, J. (2014). Establishment of Tripterygium wilfordii Hook. f. hairy root culture and optimization of its culture conditions for the production of triptolide and wilforine. J Microbiol Biotechnol, 24, 823–834