Document Type : Research Paper

Authors

1 M. Sc. Graduate of Horticultural Science, Department of Horticulture, Faculty of Aburaihan, University of Tehran, Tehran, Iran

2 Assistant Professor, Department of Horticulture, Faculty of Aburaihan, University of Tehran, Tehran, Iran

3 Professor, Department of Horticulture, Faculty of Aburaihan, University of Tehran, Tehran, Iran

4 Assistant Professor, Department of Molecular Physiology, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran

Abstract

Abstract
 
Background and Objectives
Garlic (Allium sativum( is one of the most important pharmaceutical and industrial plants with medicinal properties such as lowering blood pressure and cholesterol, fighting against infectious diseases, cancer prevention, anti-diabetic properties, antioxidant and anti-fungal properties. It is a rich source of organosulphur compounds such as allicin (diallyl disulphide oxide) which is produced enzymatically from alliin (S-2- propenyl-L-cystiene sulfoxide) if cells are damaged. Hairy root culture using Agrobactrium rhizogenes is one of the practical methods for studying secondary metabolite pathway. These hairy roots are genetically stable and grow rapidly.
 
Materials and Methods
In this study, the hairy root induction is studied in two separate experiments which were run in a factorial completely randomized design with three replications. In the first experiment, the effects of bacterial strain (ATCC15834 and A4), variety (Gorgon and Ramhormoz), inoculation medium (MS, MS containing 3% sucrose and MS containing 6% sucrose) and bacterial densities (OD600 = 0.6 to 0.9 and 1.2 to 1.5) were examined. In the second experiment, the effects of co-culture medium (MS, MS containing acetosyringone, B5 and B5 containing acetosyringone) and co-culture time (24, 48 and 72 hours) were studied.
 
Results
A systematic study using two strains and densities of Agrobactrium rhizogenes was carried out to determine the best condition for hairy root induction in two varieties of garlic in different inoculation and co-culture media. Results showed that bacterial strain A4, MS inoculation medium containing 6% sucrose, OD between 0.5 and 1, MS co-culture medium containing acetosyringone, and co-culture time 72 hours had the highest percentage of hairy root induction. Hairy roots were approved using polymerase chain reaction for gene rol B. Amplification of the specific gene was noted in the transformant at 780 bp.
 
Discussion
Hairy root cultures have received more attention in recent years and were extensively used to produce important secondary metabolites and also as experimental systems for secondary metabolic pathway elucidation studies. The results of this studymay be very helpfulfor hairy root induction in garlic which could further be useful for studying gene function and consequently, production of secondary metabolites in this plant.

Keywords

Main Subjects

References
Apple, E., Vallon Eberhard, A., Rabinkov, A., Brenner, O., Shin, I., Sasson, K., Shadkchan, Y., Osherov, N., Jung, S. and Mirelman, D. (2010). Therapy of murine pulmonary aspergillosis with antibody-alliinase conjugates and alliin. Antimicrobial Agents and Chemotherapy, 54(2), 898-906.
Baghalian, K., Ziai, A., Naghavi, M. R. and Naghdi Badi, H. (2005). Pre-planting evaluation of allicin content and botanical traits in Iranian garlic ecotypes. Journal of Medicinal Plants, 1(13), 50-59. [In Farsi]
Bivadi, V., Zakaria, R. A., Zare, N. and Yazdani, B. (2014). Effects of different tissue culture conditions in Hairy roots induction in Hypericum perforatum L. International Research Journal of Applied and Basic Sciences, 8(5), 597-604.
Brijwal, L. and Tamta, S. (2015). Agrobacterium rhizogenes mediated hairy root induction in endangered Berberis aristata DC. Springer Plus, 4, 443-453.
Chhabria, S. and Desai, K. (2016). Purification and characterization of alliinase produced by Cupriavidus necator and its application for generation of cytotoxic agent: Allicin. Saudi Journal of Biological Sciences, 25(7), 1429-1438. [In Farsi]
Doyle, J. and Doyle, J. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, 19(1), 11-15.
Gangopadhyay, M., Sircar, D., Mitra, A. and Bhattacharya, S. (2008). Hairy root culture of Plumbago indica as a potential source for plumbagin. Biologia Plantarum, 52(3), 533-537.
Georgiev, M. I., Pavlov, A. I. and Bley, T. (2007). Hairy root type plant in vitro systems as sources of bioactive substances. Appllied Microbiology and Biotechnology, 74(6), 1175-1185.
Gupta, S., Jayathilaka, L., Huang, J. S., Lee, J. and Lee, B. S. (2015). Anti-cancerous/anti bacterial activities of allicin generated in situ from diastereo pure alliins by alliinase. International Journal of Biochemistry Research and Review, 8(2), 1-16.
Jalilian, A., Ismaili, A., Nazarian Firouzabadi, F. and Hosseini, S. Z. (2017). Induction of transgenic hairy roots in medicinal plant poppy (Papaver somniferum) by Agrobacterium rhizogenes-mediated transformation. Journal of Plant Production, 39(4), 1-14. [In Farsi]
Krystal, A. D. and Ressler, I. (2001). The use of valerian in neuropsychiatry. CNS Spectrums, 6(10), 841-847.
Kumar, V., Sharma, A., Narasimha-Prasad, B. C., Bhaskar-Gururaj, H. and Aswathanarayana Ravishankar, G. (2006). Agrobacterium rhizogenes mediated genetic transformation resulting in hairy root formation is enhanced by ultrasonication and acetosyringone treatment. Electronic Journal of Biotechnology, 9(4), 349-357.
Londhe, V. P., Gavasane, A. T., Nipate, S. S., Bandawane, D. D. and Chaudhari, P. D. (2011). Role of garlic (Allium sativum) in various diseases-An overview. Journal of Pharmaceutical Research and Opinion, 1(4), 129-134.
Mallika, T., Omer, E. and Lianfu, Z. (2014). Separation and purification of alliinase and alliin from garlic (Allium sativum). Journal of Academia and Industrial Research, 2(11), 599-605.
Miron, T., Mironchik, M., Mirelman, D., Wilchek, M. and Rabinkov, A. (2003). Inhibition of tumor growth by a novel approach: In situ allicin generation using targeted alliinase delivery. Molecular Cancer Therapeutics, 2(12), 1295-1301.
Ovesna, J., Mitrova, K. and Kucera, L. (2015). Garlic (A. sativum L.) alliinase gene family polymorphism reflects bolting types and cysteine sulphoxides content. BMC Genetics, 16(1), 53-63.
Pakdin, A. and Farsi, M. (2013). Effect of different Agrobacterium rhizogenes strains on hairy root induction inValeriana officinalis L. Continental Journal of Biological Sciences, 6(2), 9-15.
Petrova, M., Zayova, E. and Vlahova, M. (2013). Induction of hairy roots in Arnica montana L. by Agrobacterium rhizogenes. Central European Journal of Biology, 8(5), 470-479.
Ramin, A., Kashi, A. and Etemadi, N. (2002). The effect of hormone combination and explants on garlic callus induction In vitro. The Scientific Journal of Agriculture, 25(1), 1-11.
Shahabzadeh, Z., Heidari, B. and Hafez, R. F. (2013). Induction of transgenic hairy roots in Trigonella foenum-graceum co-cultivated with Agrobacterium rhizogenes harboring a GFP gene. Journal of Crop Science and Biotechnology, 16(4), 263-268.
Sharafi, A., Sohi, H. H., Azadi, P. and Sharafi, A. A. (2014). Hairy root induction and plant regeneration of medicinal plant Dracocephalum kotschyi. Physiology and Molecular Biolology of Plants, 20(2), 257-262.
Sharma, P., Padh, H. and Shrivastava, N. (2013). Hairy root cultures: A suitable biological system for studying secondary metabolic pathways in plants. Engineering in Life Sciences, 13(1), 62-75.
Siahmansour, Sh., Ismaili, A. and Nazarian Firouzabadi, F. (2018). Effect of different elicitor treatments on hairy roots of medicinal plant poppies (Papaver somniferum L.). Journal of Plant Production, 41(1), 29-42. [In Farsi]
Singh, V. K. and Singh, D. K. (2008). Pharmacological effects of garlic (Allium sativum L.). Annual Review of Biomedical Sciences, 10(1), 6-26.
Soleimani, T., Keyhanfar, M., Piri, K. H. and Hasaloo, T. (2012). Hairy root induction in Burdock (Arctium lappa L.). Journal of Medicinal Plants, 4(44), 176-184. [In Farsi]
Strehlow, B., Bakowsky, U., Pinnapireddy, S. R., Kusterer, J., Mielke, G. and Keusgen, M. (2016). A novel microparticulate formulation with allicin in situ synthesis. Journal of Pharmaceutics and Drug Delivery Research, 5(1), 1-6.
Talano, M. A., Oller, A. L. W., Gonzalez, P. S. and Agostini, E. (2012). Hairy roots, their multiple applications and recent patents. Recent Patents on Biotechnology, 6(2), 1-19.
Tzfira, T., Li, J., Lacroix, B. T. and Citovsky, V. (2004). Agrobacterium T-DNA integration: Molecules and models. Trends in Genetics, 20(8), 375-383.
Weber, R. L. M. and Bodanese-Zanettini, M. H. (2011). Induction of transgenic hairy roots in soybean genotypes by Agrobacterium rhizogenes-mediated transformation. Pesquisa Agropecuaria Brasileira, 46(9), 1070-1075.
Yutani, M., Taniguchi, H., Borjihan, H., Ogita, A., Fujita, K. and Tanaka, T. (2011). Alliinase from Ensifer adhaerens and its use for generation of fungicidal activity. AMB Express, 1(2), 1-8.