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
– A PLATFORM FOR DEVELOPING COMPACT ORNAMENTALS AND BOOSTING
BIOACTIVE COMPOUNDS IN MEDICINAL PLANTS
Henrik
1
Lütken ,
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
1hlm@plen.ku.dk
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.
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.
Sterilization and cutting of plant
material
Inoculation with Agrobacterium
rhizogenes and co-cultivation
Production of hairy roots
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 F12022 (lower panel). Plants are shown at an age of 102 days. Plants
containing rol-genes are underlined. Modified from 2.
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).
Table 1. Essential beneficial compounds found in Rhodiola sp. extracts.
Cultivation of HRs
Plant regeneration from
transformed roots
Group
Compound
Amount %/DW Species
in Rhodiola
Effect /use
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
Triandrin
?
?
Possible
stimulant
5
Characterization of phenotype and
genotype
Glucopyranoside
Figure 1. Diagram of experimental work flow
Figure 2. Rhodiola rosea
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
References
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127–128
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