F A C U L T Y O F S C I E N C E

U N I V E R S I T Y O F C O P E N H A G E N

Agrobacterium rhizogenes mediated transformation of Rhodiola sp. – an approach to enhance the level of bioactive compoundsUffe 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 SectionHøjbakkegård Allé 9-13, DK-2630 Taastrup, Denmark 1ubl@plen.ku.dk 

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. 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).IntroductionRoseroot (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

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.

Materials and methodsStems 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 DeclarationGalambosi, 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 12 127–1284: Rodin IA., Stavrianidi AN., Braun AV., Shpigun OA., Popik MV. 2012. Journal of Analytical Chemistry 67(13) 1026-10305: Panossian A., Wikman G., Wagner H. 1999. Phytomedicine 6 287-300

Sterilization and cutting of stems

Inoculation with Agrobacterium rhizogenes and co-cultivation

Production of hairy roots

Cultivation in liquid media augmented with IAA

Plant regeneration from transformed roots

Characterization of phenotype and genotype

Group Compound Amount %/DW in Rhodiola

Species Effect /use References

Glycosylated tyrosol

Salidroside 0.72-1.55% Several Anxiolytic, antioxidant,

3

Rosavinoids Rosavin 2.1% R. rosea Anxiolytic 4

Rosin n.d. R. rosea Anxiolytic 4

Rosarin n.d. R. rosea Anxiolytic 4

Glucopyranoside Triandrin ? ? Possible stimulant

5

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

a

b

c

a b

c

Fig. 1. Rhodiola species. A: R. rosea; b: R. pachyclados accession 1; C: R. pachyclados accession 2

Table 1. Essential beneficial compounds found in Rhodiola sp. extracts.

a