In vitro production of secondary metabolite using Atropa komarovii Bline&Shal (Solanaceae) hairy root culture via Agrobacterium rhizogenes ATCC15834
Subject Areas : Journal of Medicinal Herbs, "J. Med Herb" (Formerly known as Journal of Herbal Drugs or J. Herb Drug)افیلا بنی هاشمی 1 , رمضانعلی خاوری نژاد 2 , نرگس یاسا 3 , فرزانه نجفی 4
1 - گروه بیولوزی، واحد علوم و تحقیقات، دانشگاه ازاد اسلامی، تهران، ایران
2 - گروه بیولوزی، واحد علوم و تحقیقات، دانشگاه ازاد اسلامی، تهران، ایران
3 - گروه فارماکوگنوزی، دانشکده داروسازی، دانشگاه تهران علوم پزشکی تهران، تهران، ایران
4 - گروه علوم گیاهی، دانشکده علوم زیستی، دانشگاه خوارزمی، تهران، ایران
Keywords: transformation, Medicinal plant, Atropa komarovii, rolB, tropane alkaloids,
Abstract :
Background & Aim:A new sustainable tissue-based system is presented by plant hairy roots, preserving all of the several specialized types of cell with critical roles in allowing bioactive secondary molecules to be synthesized more consistently as usual. The system is also essential for studying the production of alkaloid in culture. Experimental: The Atropa komarovii leaves were wounded and infected with soil gram-negative bacterium Agrobacterium rhizogenes ATCC15834. After three weeks, the transformation roots and control roots without infection, appeared, and for confirming that T-DNA Ri plasmid fragments were transformed and integrated to plant genome, the rolB gene region, was amplified using PCR. HPLC method was then used for assaying how two tropane alkaloids such as atropine (hyosciamine) and scopolamine (hyoscine) were produced in hairy roots,control roots, leaves and roots of plantlet. Results: The data indicated that diagnostic 500bp rol B product amplification was exhibited to be present by all the transformed hairy roots. Scopolamine content in hairy roots was considerably greater than that in control roots but greatest (Hyoscyamine) atropine content was observed in control roots. Analysis of DW, FW and root length showed that fresh and dry root weight increased in hairy roots compared with that in non transformed root.Recommended applications/industries: The present study demonstrated that secondary metabolite production using medicinal plants concerns many researchers worldwide today and hairy root culture is a useful method for producing tropane alkaloids in solanaceae.
Akramian, M., Fakhrtabatabaei, S. and Mirmasoumi, M. 2008. Virulence of different of Agrobacteriumrhizogenes on genetic transformation of four Hyoscyamusspecies. Agriculture and Enviroment Science, 3(5): 759-763.
Ashtiania, F. and Sefidkon, F. 2011. Tropane alkaloids of Atropa belladonna L. and Atropa acuminate Royle ex Miers plants. Journal of Medicine Plant Research, 5(29): 6515-6522.
Bensaddek, L., and Villarreal, M.L. 2008. Induction and growth of hairy roots for the production of medicinal compounds. Electronic Journal of Integrative Biosciences, 3(1): 2-9.
Bulgakov, V.P, Tchernoded, G.K., Ischenko, M., Khodakovskaya, N.P., Glazunov, M.V., Radchenko, V.P., Zvereva, S.V., Fedoreyev, E.V. and Zhuravlev, Y. N. 2002. Effect of salicylic acid, methyl jasmonate, ethephon and cantharidin on antheraquinone production by Rubiacordifolia callus caltures transformed with the rolB and rolC genes. Journal of Biotechnology, 97(3): 213-221.
Dechuaux, C., and Conti, M.B. 2005. A strategy for over accumulation of scopolamine in Daturainnoxia hairy root culture. Acta Biologica Cracoviensia Series Botanica, 47(1): 101-107.
Doyle, J.J. and Doyle J. L. 1987. A rapid DNA isolation procedure for small amount of fresh leaf tissue. Phytochemistry, 5: 574-555.
Giri, M. and Narasu, L. 2000. Transgenic hairy roots: recent trends and applications. Biotechnology Advances, 18(1): 1-22.
Hank, H., I. Laszlo, and I. Balvanyos. 2003. Effect of magnesium on the growth and alkaloid production of hairy root cultures. Acta Horticultureae, 597: 271-274.
Hashimoto, T., Yan, D. and Yamada, Y. 1993. Production of tropane alkaloids in genetically engineered root cultures. Phytochemistry, 32(3):713-718.
Hu, Z.B. and Du, M. 2006. Hairy root and application in plant genetic engineering. Journal of integrative plant Biology, 48: 121-127.
Khatodia, S., Biswas K., and Bhatotia, K. 2013. Induction and establishment of hairy root culture of Solanum xanthocarpum using Agrobacterium rhizogenes. Journal of Bioscience, 1: 59-63.
Kim, Y.K., Xu, H., Park, W., Park, N., Lee, N., Lee, S. and Park, S. 2010. Genetic transformation of buck wheat (Fagopyrum esculentum M.) with Agrobacterium rhizogenes and production of rutin in transformed root culture. Australian Journal of Crop Science, 4: 485- 490.
Lanoue, A., Conti, M., Dechaux, C., Laberche, J., Christen, P. and Norreel, S. 2004. Comparison of growth properties, alkaloid production and water uptake of two selected Datura hairy root line. Acta Biologica Cracoviensia Series Botanica, 46: 185-192.
Liu, X., Yang, C., Chen, M., Li, M., Liao, Z. and Tang, K. 2010. Promoting scopolamine accumulation in transgenic plants of Atropa belladonna generated from hairy roots with over expression of pmt and h6h gene. Journal of Medicinal Plants Research, 4(17): 1708-1713.
Marchey, A., Georgiev, V., Ivanov, I. and Pavlov, A. 2012. Cultivation of diploid and tetraploid hairy roots of Daturastramonium L. in stirred tank bioreactor for tropane alkaloids production. Journal of Bioscience and Biotechnology, 1(3): 211-216.
Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Plant Physiology, 15(3): 473- 479.
Nikravesh, F., Khavari-Nejad, R.A., Rahimian, H. and Fahimi, H. 2012. Study of antioxidant enzymes activity and isozymes pattern in hairy roots and reganarated plants in Nicotianatabacum. Acta Physiologiae Plantarum, 34: 419-427.
Samet, E., Piri, K., Keyhanfar, H. M. and Hasanloo, T. 2012. Influence of jasmonic acids, yeast extract and salicylic acid on growth and accumulation of hyosciamine and scopolamine in hairy root cultures of Atropa belladonna L. International Journal of agriculture Research and Review, 2(4): 403-409.
Sevón, N. and Oksman-Caldentey, I. 2002. Agrobacterium rhizogenes mediated transformation: root cultures as a source of alkaloids. Planta Medicine, 68(10): 859-868.
Song, G., and Walworth, A. 2013. Agrobacterium tumefaciens-mediated transformation of Atropa belladonna. Plant Cell Tissue Organ Culture, 115(1): 2-9.
Suzuki, K., Yan, D.J., Chen, X., Yamada, Y. and Hashimoto, T. 1999. An Atropa belladonnahyoscyamine 6β-hydroxylase gene is differentially expressed in the root pericycle and anthers. Plant Molecular Biology, 40(1): 141-152.
Vasdekis, N., Barres, M., Ravelo, A. and Zarate, R. 2008. Effect of elicitors on tropane alkaloids and gene expression in Atropabaetica transgenic hairy roots. Journal of Natural Products, 12: 2026-2031.
Widorento, W., Pervitazari, R., Aramingtayas, E., Utomo, E., and Wardiati, T. 2012. Genetic transformation of tomato (LycopersicumesculentumMill.) with Agrobacterium rhizogenesand production of 1ycopene in transformed root culture. International Research Journal of Biotechnology, 3: 158-167.
Yong, K., Hui, X., Woo, T.P., Nam, P., Sook, Y.L. and Sang, U.P. 2010. Genetic transformation of buckwheat (FagopyrumesculentumM.) with Agrobacterium rhizogenesand production of rutin in transformed root cultures. Australian Journal of Crop Science, 4(7):485-490.
Yelin, M., Tovkach, A. and Tzfira, T. 2008. Integration of Agrobacterium T-DNA in plant cells. Plant Cell Monograph, 14: 157-186.
Zarate, R., Vazdekis, N.J., Medina B., and Ravelo, A. 2006. Tailoring tropane alkaloid accumulation in transgenic hairy roots of Atropabetica by over-expressing the gene encoding hyoscyamine 6β-hydroxylase. Biotechnology Letters, 28: 1271-1277.
Zhao, B., Foster, A., Ritesh, K.C., Agblevor, A., Ritesh, K. C., John, G., and Jelekso, G. 2012. Enhanced production of the alkaloid nicotine in hairy root cultures of Nicotianatabacum L. Plant Cell Tissue Organ Culture, 113(1): 234-241.