Agrobacterium rhizogenes mediated transformation of Rhodiola sp. – an approach to enhance the level of bioactive compounds.
Martin Møller Hansen1, Uffe Bjerre Lauridsen2, Josefine Nymark Hegelund3, Renate Müller4, Jihong Liu Clarke5, Henrik Lütken6
University of Copenhagen, Faculty of Science
Department of Plant and Environmental Sciences, Crop Sciences Section
Højbakkegård Allé 9-13, DK-2630 Taastrup, Denmark
1twc525@alumni.ku.dk, 2ubl@life.ku.dk, 3jnh@life.ku.dk, 4ren@life.ku.dk 6hlm@life.ku.dk
5Bioforsk - Norwegian Institute for Agricultural and Environmental Research, Ås, Norway
Jihong.liu-clarke@bioforsk.no
Keywords: Natural transformation - rol-genes – roseroot – rosavin - salidroside
Abstract
Introduction Rhodiola rosea commonly known as roseroot has since ancient times been used against depression and for improving mental abilities mainly due to its two bioactive compounds salidroside and rosavin. Due to excessive gathering the natural populations have been declining. Natural transformation with root-loci (rol)-genes from a wildtype Agrobacterium rhizogenes causes hairy roots (HRs) to develop from the site of infection. These HRs often exhibit a higher rate of synthesis of secondary metabolites compared to wild type roots. The purpose of this study is to obtain HRs containing rol-genes from Rhodiola sp. for future sustainable production in bioreactors.
Materials and Methods
Whole stems of R. rosea and two accessions of R. pachyclados were sterilized with ethanol and NaOCl. The stems were then cut into segments and the leaves were separated. Both the leaves and the stem segment were inoculated with A. rhizogenes. The inoculated explants were placed on co-cultivation media consisting of ½xMS containing acetosyringone for 3 days in darkness. The explants were washed in a timentin solution and moved to a new media containing timentin and arginine. The number of HRs was monitored concurrently. Transformation rates were calculated as the percentage of inoculated explants developing putative HRs.
Results
Of the inoculated R. pachyclados only the stem segments developed putative HRs. The percentages of stem segment developing putative HRs were approx. 43% and 36% of accession 1 and 2, respectively. Of the controls the percentages of explants developing hairy roots were approx. 53% and 39% of the stem segment from accession 1 and 2, respectively, and approx. 0% and 1% of the leaves from accession 1 and 2, respectively. The roots were indistinguishable from the roots of the inoculated explants. The percentages of inoculated R. rosea explants developing putative HRs were approx. 17% and 20% of the leaves and stem segments respectively. None of the control explants developed any roots.
Discussion
The large background of untransformed roots in R. pachyclados indicates that there may only be few or none true HRs containing rol-genes. The results are more promising for R. rosea where only inoculated
explants developed roots. The next step is to verify the transformation using PCR. According to our knowledge this is the first report showing rol-transformation of Rhodiola sp. in vivo.
Conclusion
The transformation protocol resulted in putative HRs from R. rosea. It is however uncertain that the obtained roots from R. pachyclados are transformed HRs.