1. 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 Sterilization and cutting of plant material Inoculation with Agrobacterium rhizogenes and co-cultivation Production of hairy rootsCultivation of HRs Plant regeneration from transformed roots Characterization of phenotype and genotype AGROBACTERIUM RHIZOGENES-MEDIATED TRANSFORMATION – A PLATFORM FOR DEVELOPING COMPACT ORNAMENTALS AND BOOSTING BIOACTIVE COMPOUNDS IN MEDICINAL PLANTS Henrik Lütken 1, Uffe Bjerre Lauridsen, Martin Himmelboe, Josefine Nymark Hegelund, Renate Müller University of Copenhagen, Faculty of Science Department of Plant and Environmental Sciences, Crop Sciences Section Højbakkegård Allé 9-13, DK-2630 Taastrup, Denmark 1 hlm@plen.ku.dk Introduction An upcoming biotechnological method with very promising results is plant transformation with Agrobacterium rhizogenes (Fig. 1). In this method bacterial T-DNA containing root-loci-genes (rol-genes) is integrated into the host genome causing growth of hairy roots (HR). An important aspect of the HRs is their distinct morphology, which can be used directly in the selection process as a primary indicator of a successful transformation as an alternative to antibiotic resistance marker genes. Compact growth is a desirable trait in ornamental plant production. This feature is currently achieved by application of chemical growth retardants, which are potentially harmful to both the environment and human health. HR cultures often contain a higher content of secondary metabolites compared to the respective wild types. Moreover, plants regenerated from HRs typically display compact growth. Hence, this method both presents a tool for boosting high value compounds in planta and developing compact ornamentals. Roseroot species (Rhodiola sp.) (Fig. 2) have for centuries been used in biomedicine against depression and for improving mental abilities (1). Specifically, the roots of R. rosea, containing the bioactive compounds salidroside and rosavin (Table 1), have been of particular interest. Transformation material and methods Explants were sterilized with ethanol and sodium hypochlorite, washed and 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 and Timentin for production of HRs. Roseroot HRs were used for testing the effects of exogenous auxin in liquid medium containing different concentrations of indole acetic acid (IAA). Entire transgenic plants of Kalanchoë were regenerated on ½MS containing the cytokinin, N-(2-Chloro-4-pyridyl)-N-phenylurea (CPPU). References 1: Galambosi, B., Galambosi, Zs., Hethelyi, E., Volodin, V., Poletaeva, I., Iljina, I. 2010. Z Arznei- Gewurzpflanzen 15(4) 160-169 2: Lütken H., Clarke JL., Müller R. 2012. Plant Cell Rep 31(7) 1141-1157 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 Figure 1. Diagram of experimental work flow Figure 3. Compact Kalanchöe phenotypes induced by rol-genes. T1 rol-line 331 and wild type cultivars ‘Molly’ and ‘Sarah’ (upper panel) Selected plants from the F1 generation derived by crossing line 331 with ‘Sarah’ (middle panel). Selected plants derived from self-fertilisation of F1- 2022 (lower panel). Plants are shown at an age of 102 days. Plants containing rol-genes are underlined. Modified from 2. GroupCompound Amount %/DW in Rhodiola SpeciesEffect /useReferences Glycosylated tyrosolSalidroside0.72-1.55%Several Anxiolytic, antioxidant, 3 RosavinoidsRosavin2.1%R. roseaAnxiolytic4 Rosinn.d.R. roseaAnxiolytic4 Rosarinn.d.R. roseaAnxiolytic4 GlucopyranosideTriandrin??Possible stimulant 5 Table 1. Essential beneficial compounds found in Rhodiola sp. extracts. Key findings A stable transformation platform for developing compact ornamentals, has successfully been developed. In this method rol-genes are inserted as an alternative to hazardous growth retardants. Production of valuable bioactive compounds can be boosted by rol-genes in medicinal plants, e.g. roseroot. Results An optimised Agrobacterium rhizogenes-mediated transformation platform useful for a wide range of ornamentals has been developed. Kalanchoë was the starting point and the effect of the rol-genes has now been followed in three progeny generations, where plants of superior quality have been selected (Fig. 3). Furthermore, increased ethylene tolerance was observed in several lines containing rol-genes. Other ornamental plant species e.g. Campanula containing rol-genes are currently being generated from the same transformation platform. For roseroot, up to 50% of the explants developed HRs. In general, numerous untransformed roots were observed on controls. For R. rosea, 19% of the explants developed hairy roots and no root appeared on the controls indicating that they are transformed (data not shown). Figure 2. Rhodiola rosea