Transcript an approach to enhance the level of bioactive compounds

FAC U LTY O F SC I EN C E
UN IVE R S ITY O F C OPE NHAG EN
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
[email protected]
Preliminary results
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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. rosea transformed explants 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).
Fig. 1. Rhodiola species. A: R. rosea; b: R. pachyclados accession 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 Rhodiola sp. extracts.
Group
Glycosylated
tyrosol
Rosavinoids
Glucopyranoside
Compound Amount
%/DW in
Rhodiola
Salidroside 0.72-1.55%
Species
Effect /use
References
Several
Anxiolytic,
antioxidant,
3
Rosavin
2.1%
R. rosea
Anxiolytic
4
Rosin
n.d.
R. rosea
Anxiolytic
4
Rosarin
n.d.
R. rosea
Anxiolytic
4
Triandrin
?
?
Possible
stimulant
5
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.
Fig. 3. Hairy roots of Rhodiola. A: emerging
hairy roots on R. rosea; b: hairy root on R.
pachyclados; c: hairy root of R. rosea growing
in auxin augmented liquid medium.
Fig. 2. Diagram of experimental work 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.
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- Gewurzpflanzen 15(4) 160-169
3: Evstatieva LN., Revina TA. 1984. Journees Internationales d’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