Preliminary results

1. Agrobacterium rhizogenes mediated transformation of Rhodiola sp. – an approach to enhance the level of bioactive compounds Uffe Bjerre Lauridsen1, Martin Himmelboe, Josefine Nymark Hegelund, Renate Müller, Henrik Lütken University of Copenhagen, Faculty of Science Department of Plant and Environmental Sciences, Crop Sciences Section Højbakkegård Allé 9-13, DK-2630 Taastrup, Denmark 1ubl@plen.ku.dk a a b • Preliminary results • Upon transformation, up to 50 % of Rhodiola explants developed hairy roots (Fig. 3). • Substantial growth of untransformed roots on controls, except for R. rosea. • 19 % of R. roseatransformedexplants developed hairy roots, with no roots on the controls. • Control roots were difficult to visually distinguish from putatively transformed roots. • Exogenous auxin had a positive effect on the growth of putatively transformed hairy roots (Fig 3). c Fig. 1. Rhodiolaspecies. A: R. rosea; b: R. pachycladosaccession 1; C: R. pachyclados accession 2 • Introduction • Roseroot (Rhodiola sp.) (Fig. 1), a genus distributed in arctic and alpine regions of the Northern hemisphere, has a long history of being used to treat altitude sickness, depression and to give mental strength due to its adaptogenic properties2. Today roseroot is still used for those same reasons. Additionally, some studies have indicated that roseroot contains compounds with other more profound properties (Table 1). Especially R. rosea, contains compounds with the following effects: • Antidepressive • Improving concentration and emotional stability • Fatigue-reducing • Anticarcinogenic • Cardioprotective • Natural populations are declining due to excessive gathering. • Transformation of plants with the soil bacterium Agrobacterium rhizogenes results in the insertion of root-loci (rol) genes into the plant’s genome. These genes lead to a production of hairy roots often accompanied by: • Increased growth compared to normal roots • Upregulated production of secondary metabolites • Transformations with unmodified A. rhizogenes strains can be assumed not to fall under the GMO according to EU legislations1 Table 1. Essential beneficial compounds found in Rhodiolasp. extracts. • Objectives • To obtain hairy roots of Rhodiola sp. containing rol-genes for future sustainable production of valuable bioactive compounds in bioreactors. • To enhance the level of bioactive compounds in planta. • To regenerate plants from transformed roots. a c Fig. 3. Hairyroots of Rhodiola. A: emerginghairyroots on R. rosea; b: hairyroot on R. pachyclados; c: hairyroot of R. rosea growing in auxinaugmentedliquid medium. Fig. 2. Diagram of experimentalwork flow Materials and methods Stems of R. rosea and two accessions of R. pachyclados were sterilized with ethanol and sodium hypochlorite (Fig. 2). The stems were divided and the leaves were removed. Stem segments and leaves were inoculated with Agrobacterium rhizogenes strain ATCC43057 (A4 plasmid) and placed on co-cultivation media, consisting of ½xMS incl. acetosyringone, for 3 days. The explants were then washed in a Timentin solution and placed on ½xMS incl. arginine and Timentin for the hairy root to be produced. The hairy roots were used for testing the effects of exogenous auxin in a liquid medium containing different concentrations of indole acetic acid (IAA) and regeneration of entire transgenic plants on ½MS containing the cytokinin, N-(2-Chloro-4-pyridyl)-N-phenylurea(CPPU). Finally the phenotypes and genotypes of hairy roots and plants will be analyzed biometrically and by PCR and Q-PCR. b References 1: European Union (2001) Directive 2001/18/EC of the European Parliament and the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC Commission Declaration Galambosi, B., Galambosi, Zs., Hethelyi, E., Volodin, V., Poletaeva, I., Iljina, I. 2010. Z Arznei- Gewurzpflanzen15(4) 160-169 3: Evstatieva LN., Revina TA. 1984. JourneesInternationalesd’Etudes 12 127–128 4: Rodin IA., Stavrianidi AN., Braun AV., Shpigun OA., Popik MV. 2012. Journal of Analytical Chemistry 67(13) 1026-1030 5: Panossian A., Wikman G., Wagner H. 1999. Phytomedicine 6 287-300