Planta Medica, Inhaltsverzeichnis Planta Med 2005; 71(1): 48-53DOI: 10.1055/s-2005-837750 Original PaperPhysiology, in vitro Biotechnology© Georg Thieme Verlag KG Stuttgart · New YorkExpression of Vitreoscilla Hemoglobin Enhances Growth of Hyoscyamus muticus Hairy Root CulturesAnnika Wilhelmson1 , Pauli T. Kallio2 , Kirsi-Marja Oksman-Caldentey1 , Anna Maria Nuutila1 1VTT Biotechnology, Espoo, Finland 2Institute of Biotechnology, ETH-Zürich, Zürich, Switzerland Artikel empfehlen Abstract Artikel einzeln kaufen(opens in new window) Abstract The Vitreoscilla hemoglobin gene (vhb) was introduced into Hyoscyamus muticus with the aim of investigating its effect on growth and alkaloid production of Agrobacterium rhizogenes-induced hairy root cultures. We were able to generate several VHb-expressing hairy root lines with different integration patterns. Substantial somaclonal variation was observed in growth and hyoscyamine production amongst both VHb-expressing lines and controls. Despite this variation, the growth properties of single lines remained stable over time. Expression of VHb was found to improve growth of H. muticus hairy roots in shake-flask cultures. The dry weights of the root cultures expressing Vitreoscilla hemoglobin were on average 18 % higher than those of the controls. VHb expression also increased the volumetric hyoscyamine production, mainly due to the improved growth properties. However, this difference was not statistically significant due to the wide somaclonal variation and fluctuations over time in both VHb and control hairy root lines. Key words Hyoscyamus muticus - Solanaceae - Vitreoscilla hemoglobin - hairy root cultures - growth improvement Volltext Referenzen References 1 Sevón N, Oksman-Caldentey K -M. Agrobacterium rhizogenes-mediated transformation: Root cultures as a source of alkaloids. Planta Medica. 2002; 68 859-68 2 Yu S, Doran P M. Oxygen requirements and mass transfer in hairy-root culture. Biotechnol Bioeng. 1994; 44 880-7 3 Kwok K H, Doran P M. Kinetic and stoichiometric analysis of hairy roots in a segmented bubble column reactor. Biotechnol Prog. 1995; 11 429-35 4 Shiao T, Ellis M H, Dolferus R, Dennis E S, Doran P M. Overexpression of alcohol dehydrogenase or pyruvate decarboxylase improves growth of hairy roots at reduced oxygen concentrations. Biotechnol Bioeng. 2002; 77 455-61 5 Khosla C, Bailey J E. Heterologous expression of a bacterial hemoglobin improves the growth properties of recombinant Escherichia coli . Nature. 1988; 331 633-5 6 Frey A D, Kallio P T. Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology. FEMS Microbiol Rev. 2003; 27 525-45 7 Kallio P T, Kim D -J, Tsai P S, Bailey J E. Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions. Eur J Biochem. 1994; 219 201-8 8 Park K -W, Kim K -J, Howard A J, Stark B C, Webster D A. Vitreoscilla hemoglobin binds to subunit I of cytochrome bo ubiqionol oxidases. J Biol Chem. 2002; 277 33 334-7 9 Frey A D, Farrés J, Bollinger C JT, Kallio P T. Bacterial hemoglobins and flavohemoglobins for alleviation of nitrosative stress in Escherichia coli . Appl Environ Microbiol. 2002; 68 4835-40 10 Farrés J, Kallio P T. Improved cell growth in tobacco suspension cultures expressing Vitreoscilla hemoglobin. Biotechnol Prog. 2002; 18 229-33 11 Häggman H, Frey A D, Ryynänen L, Aronen T, Julkunen-Tiitto R, Tiimonen H, Pihakaski-Maunsbach K, Jokipii S, Chen X, Kallio P T. Expression of Vitreoscilla hemoglobin in hybrid aspen (Populus tremula x tremuloides). Plant Biotechnol J. 2003; 1 287-300 12 Mattanovich D, Rüker F, da Camara Machado A, Laimer M, Regner F, Steinkellner H, Himmler G, Katinger H. Efficient transformation of Agrobacterium spp. by electroporation. Nucleic Acids Res. 1989; 17 6747 13 Gamborg O L, Miller R A, Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res.. 1968; 50 151-8 14 Sevón N, Hiltunen R, Oksman-Caldentey K -M. Chitosan increases hyoscyamine content in hairy root cultures of Hyoscyamus muticus . Pharm Pharmacol Lett. 1992; 2 96-9 15 Sevón N, Dräger B, Hiltunen R, Oksman-Caldentey K -M. Characterization of transgenic plants derived from hairy roots of Hyoscyamus muticus . Plant Cell Rep. 1997; 16 605-11 16 Hamill J D, Rounsley S, Spencer A, Todd G, Rhodes M JC. The use of the polymerase chain reaction in plant transformation studies. Plant Cell Rep. 1991; 10 221-4 17 Murray H G, Thompson W F. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 1980; 8 4321-6 18 Southern E M. Detection of specific sequences among DNA fragments by gel electrophoresis. J Mol Biol. 1975; 98 503-617 19 Oksman-Caldentey K -M, Vuorela H, Strauss A, Hiltunen R. Variation in the tropane alkaloid content of Hyoscyamus muticus plants and cell culture clones. Planta Medica. 1987; 53 349-54 20 Holmberg N, Lilius G, Bailey J E, Bülow L. Transgenic tobacco expressing Vitreoscilla hemoglobin exhibits enhanced growth and altered metabolite production. Nature Biotechnol. 1997; 15 244-7 21 Sevón N, Hiltunen R, Oksman-Caldentey K -M. Somaclonal variation in transformed roots and protoplast-derived hairy root clones of Hyoscyamus muticus . Planta Medica. 1998; 64 37-41 22 Vanhala L, Hiltunen R, Oksman-Caldentey K -M. Virulence of different Agrobacterium strains on hairy root formation of Hyoscyamus muticus . Plant Cell Rep. 1995; 14 236-40 Kirsi-Marja Oksman-Caldentey VTT Biotechnology P.O. Box 1500 FIN-02044 VTT Espoo Finland Telefon: +358-9-456-4459 Fax: +358-9-455-2103 eMail: kirsi-marja.oksman@vtt.fi