Hairy Roots Formation in Four Solanaceae Species by Different Strains of Agrobacterium rhizogenes

Authors

1 Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, 81746-73441, Isfahan, Iran

2 Department of Pharmacognosy, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

Plants are the important sources of drugs. Secondary metabolites are responsible for therapeutic propertiesin plants. Three compounds including (-)-hyoscyamine, its racemate atropine, and scopolamine (hyoscine) are the most famous tropane alkaloids in the Solanaceae familly. Nowadays, attempts to developthese alkaloids in biotechnological procedures which are principally based on the hairy root cultures using Agrobacterium rhizogenes. In the present study, we showed the percent of induced hairy roots percent in the leaf and root explants of four plants from the Solanaceae family (Atropa belladonna L., Hyoscyamus niger L., Datura stramonium L. and Datura metel L.), that infected with the six strains of Agrobacterium rhizogenes (A4, A7, Ar15834, Ar9534, Ar9402, and Ar318). Hairy roots were appeared from the leaf and roots explants on ½MS medium culture. The presence of T-DNA in the supposedly transformal lines was shown by PCR. The highest transformation yield of 93% was accomplished using leaf explants of D. metel infected by AR15834 and A4 strains. One fastest growing clone of transforming D. metel roots line (induced by AR15834)was selected and the biomass of hairy and natural roots were measured and compared  after 0, 2, 4, 6, 8 and 10 days. The results showed that, the fresh and dry weight of hairy roots was 4.44 and 4.92 times higher than the weights of non-transformed roots respectively after 8- 10 days. These roots are hormone-autotrophic and have the great lateral branches. Therefore, the hairy roots of D. metel and D. stramonium can be used to increase tropane alkaloids production yield in the pharmaceutical industry.

Keywords


1. Kim Y, Wyslouzil BE, Weathers PJ. Secondary metabolism of hairy root cultures in bioreactors. In Vitro Cell Dev Biol Plant. 2002;38:1-10.
2. Palazón J, Navarro-Ocaña A, Hernandez-Vazquez L, Mirjalili MH. Application of metabolic engineering to the production of scopolamine. Molecules. 2008;13:1722-1742.
3. Croteau R, Kutchan TM, Lewis NG. Natural products (secondary metabolites). Biochem Mol Biol Plant. 2000;1250-1318.
4. Khandakar J, Haraguchi I, Yamaguchi K, KitamuraY. A small-scale proteomic approach reveals a survival strategy, including a reduction in alkaloid biosynthesis, in Hyoscyamus albus roots subjected to iron deficiency. Front Plant Sci. 2013;4:331.
5. Ajungla L, Patil P, Barmukh RB, Nikam TD. Influence of biotic and abiotic elicitors on accumulation of hyoscyamine and scopolamine in root cultures of Datura metel L. Indian J Biotechnol. 2009;8:317-322.
6. Palazón J, Altabella T, Cusidó R, Ribó M, Piñol MT. Growth and tropane alkaloid production in Agrobacterium transformed roots and derived callus of Datura. Biol Plantarum. 1995;37:161-168.
7. Giri A, Narasu ML. transgenic hairy roots: recent trends and applications. Biotechnol Adv. 2000;18:1-22.
8. Hashimoto T, Yamada Y. Scopolamine production in suspension cultures and redifferentiated roots of Hyoscyamus niger. Planta Med. 1983;47:195-199.
9. Hartmann T, Witte L, Oprach F, Toppel G. Reinvestigation of the alkaloid composition of Atropa belladonna plants, root cultures and cell suspension cultures. Planta Med. 1986;52:390-395.
10. Bonhomme V, Laurain-Mattar D, Lacoux J, Fliniaux M, Jacquin-Dubreuil A. Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only. J Biotechnol. 2ooo;81:151-158.
11. Jung H, Kanga S, Kang Y, Kang M, Yun D, Bahk T, Yang J, Choi M. Enhanced production of scopolamine by bacterial elicitors in adventitious hairy root cultures of Scopolia parviflora. Enzyme Microb Tech. 2003;33:987-990.
12. Zhang L, Yang B, Lu B, Kai G, Wang Z, Xia Y, Ding R, Zhang H,  Sun X, Chen W, Tang K. Tropane alkaloids production in transgenic Hyoscyamus niger hairy root cultures over-expressing Putrescine N-methyltransferase is methyl jasmonate-dependent. Planta. 2007;225:887-896.
13. Guillon S, Trémouillaux-Guiller J, Pati PK, Rideau M, Gantet P. Hairy root research: recent scenario and exciting prospects. Curr Opin Plant Biol. 2006;9:341-346.
14. Eskandari-Samet A, Piri K, Kayhanfar M, Hasanloo T. Enhancement of tropane alkaloid production among several clones and explants types of hairy root of Atropa belladonnaL. J Med Plant Prod. 2012;1:35-42.
15. Qing CM, Fan L, Lei Y, Bouchez D, Tourneur C, Yan L, Robaglia C. Transformation of Pakchoi (Brassica rapa L. ssp. chinensis) by Agrobacterium infiltration. Mol Breeding. 2000;6:67-72.
16. Pirian K, Piri KH, Ghiyasvand T. Hairy roots induction from Portulaca oleracea using Agrobacterium rhizogenes to Noradrenaline,s production. Int Res J Appl Basic Sci. 2012;3:642-649.
17. Zhou X, Wu Y, Wang X, Liu B, Xu H. Salidroside production by hairy roots of Rhodiola sachalinensis obtained after transformation with Agrobacterium rhizogenes. Biol Pharm Bull. 2007;30:439-442.
18. Doyle JJ, Doyle JLA. Rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull. 1987;19:11-15.
19. Sambrook J, Fritrsch EF, MAniatis T. Molecular Cloning: A laboratory manua manual. Cold spring laboratory press. Cold spring harbor, NY. 1989.
20. Rahnama H, Hasanloo T, Shams MR, Sepehrifar R. Silymarin production by hairy root culture of Silybum marianum (L.) Gaertn. Iran J Biotechnol. 2008;6:113-118.
21. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962;15:473-497.
22. Królicka A, Staniszewska I, Bielawski K, Maliński E, Szafranek J, Łojkowska E. Establishment of hairy root cultures of Ammi majus. Plant Sci. 2001;160:259-264.
23. Tao J, Li L. Genetic transformation of Torenia fournieri L. mediated by Agrobacterium rhizogenes. S Afr J Bot. 2006;72:211-216.
24. Nguyen C, Bourgaud F, Forlot P, Guckert A. Establishment of hairy root cultures of Psoralea species. Plant Cell Rep. 1992;11:424-427.
25. Akramian M, Tabatabaei SMF, Mirmasoumi M. Virulence of different strains of Agrobacterium rhizogenes on genetic transformation of four Hyoscyamus species. Amer-Euras J Agric Environ Sci. 2008;3:759-763.
26. Chandran RP, Potty VP. Induction of hairy roots through the mediation of four strains of Agrobacterium rhizogenes on five host plants. Indian J Biotechnol. 2008;7:122.
27. Depicker A, Herman L, Jacobs A, Schell J, Van Montagu M.  Frequencies of simultaneous transformation with different T-DNAs and their relevance to the Agrobacterium/plant cell interaction. Mol Gen Genet. 1985;201:477-484.
28. Rudrappa T, Neelwarne B, Kumar V, Lakshmanan V, Venkataramareddy SR, Aswathanarayana RG. Peroxidase production from hairy root cultures of red beet (Beta vulgaris). Electron J Biotechnol. 2005;8:66-78.
29. Pawar PK, Maheshwari VL. Agrobacterium rhizogenes mediated hairy root induction in two medicinally important members of family Solanaceae. Indian J Biotechnol. 2004;3:414-417
30. Ionkova I. Biotechnological approaches for the production of lignans. Pharmacogn Rev. 2007;1:57.
31. Ionkova I, Fuss E. Influence of different strains of Agrobacterium rhizogenes on induction of hairy roots and lignan production in Linum tauricum ssp. tauricum. Pharmacogn Mag. 2009;5:14.
32. Chaudhuri KN, Ghosh B, Tepfer D, Jha S. Genetic transformation of Tylophora indica with Agrobacterium rhizogenes A4: growth and tylophorine productivity in different transformed root clones. Plant cell rep. 2005;24:25-35.