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Establishment of Rhodiola kirilowii hairy roots using Agrobacterium rhizogenes LBA 9402
Establishment of Rhodiola kirilowii hairy roots using Agrobacterium rhizogenes
LBA 9402
Monika Zych 1∗ Agnieszka Pietrosiuk 1** , Monika KARASIEWICZ 2 ,
Anna Bogacz 2 , Radosław Kujawski 2 , Przemysław M. Mrozikiewicz 2 ,
ANNA KRAJEWSKA-PATAN 2 , Mirosława Furmanowa 1
1
Department of Biology and Pharmaceutical Botany
Medical University of Warsaw
Banacha 1
02-097 Warsaw, Poland
2
Research Institute of Medicinal Plants
Libelta 27
61-707 Poznań, Poland
* , **corresponding authors:
e-mail: zych_monika@yahoo.de, agnieszka.pietrosiuk@wum.edu.pl
S u m m a r y
For the first time hairy roots of the Rhodiola genus were obtained by infection of the of
Rhodiola kirilowii plantlets with Agrobacterium rhizogenes LBA 9402. First transgenic roots
appeared 11 days after infection. The transformation process was confirmed by the polymerase
chain reaction (PCR). DNA analysis showed that rol B gene and aux 1 gene from Ri
plasmid of Agrobacterium rhizogenes were incorporated into the plant genome of Rhodiola
kirilowii.
Key words: Rhodiola kirilowii, Agrobacterium rhizogenes, hairy roots, rol B gene
Abbreviations
MS: Murashige and Skoog (1962) medium; B5: Gamborg (1978) medium; YMB:
yeast mannitol broth; IAA: β-indolylacetic acid; CTAB: cetyltrimethylammonium
bromide, TBE: Tris-Borate-EDTA buffer
Vol. 54 No 4 2008

M. Zych, A. Pietrosiuk, M. Karasiewicz, A. Bogacz, R. Kujawski, PM. Mrozikiewicz, A. Krajewska-Patan, M. Furmanowa
Introduction
Rhodiola kirilowii (Regel) Regel and Maximowicz (xia je hong jing tian), of the
Crassulaceae family is found in North Asia and China. According to Ohba [1], Rhodiola
kirilowii is a typical mountain plant that grows at 2000–5000 m a. s. l. Salidroside,
tyrosol, daucosterol, lotaustralin [2] and β-sitosterol [3] have been isolated
from the extracts of the roots. The differences between the compounds found in
natural roots of Rhodiola kirilowii growing in Poland and in Asiatic countries were
described by Wiedenfeld et al. [4]. These authors have found epigallocatechin
gallate, rhodiocyanoside A, arbutin and fructopyrano-(1-4)-glucopyranose in the
rhizomes.
Oral administration of R. kirilowii was shown to protect mountain regions inhabitants
from cardiopulmonary disorders caused by high altitude [5]. R. kirilowii
was also found to reduce damage to rat viscera caused by a hypoxic high altitude
environment [6]. In Poland this species is cultivated only in the Botanical Garden
of the Institute of Medicinal Plants in Poznan. The process of the micropropagation
of R. kirilowii with the use of somatic seeds was elaborated by Zych et al. [7].
The same method was used for the encapsulation of Rubus chamaemorus L. shoot
buds [8]. This process has not resulted in the genetic variability of plants developed
from the beads.
There are many species from the Rhodiola genus, which are used in traditional
Chinese medicine. The best known and best elabrated is Rhodiola rosea, widely
used as an adaptogen (Rosenroot, Lentaya, Antystres Forma, Meteo, Rhodiolin,
CelluVita). It also possesses anticancer [9, 10], antimutagenic [11, 12], cardioprotective
and anti-arrhytmic activities [13-15].
The aim of our work was to obtain the hairy roots of Rhodiola kirilowii. These
organs were not obtained in genus Rhodiola till now. To obtain the transformed
roots, T-DNA of Agrobacterium rhizogenes was transferred to the wounded plant
cell and incorporated into the plant genome. The following expression of bacterial
genes is responsible for the development of hairy roots [16]. Transformed
root cultures grow rapidly, are genetically stable and can produce secondary metabolites
[17, 18]. Many interesting natural compounds used in the therapy are
synthetized in hairy roots and their level could be higher than that found in natural
roots [19, 20].
Materials and Methods
Plant material
The plantlets of R. kirilowii were grown on Murashige and Skoog (MS) solid
medium [21] without growth regulators at 24°C and under 40 μmol m -2 s -1 light for

Establishment of Rhodiola kirilowii hairy roots using Agrobacterium rhizogenes LBA 9402
12 h a day for 2-3 weeks. Their leaves and shoots were used for the hairy roots
initiations.
Transformation of plants explants
Agrobacterium rhizogenes LBA 9402 strain grown in liquid YMB medium [22] for
72 h was used to infect R. kirilowii explants. In one experiment 10 young leaves
and 19 internodal shoot segments were directly infected with a sterile needle
containing bacteria. The leaves were wounded in mid-vein. Inoculated explants
were incubated for 7 days in the darkness on MS solid medium supplemented
with 2 mg l -1 acetosyringone and next were transferred to fresh MS solid medium
containing 0.5 g l -1 cefotaxim to eliminate bacteria and subsequently cultured at
24°C in darkness. Eleven days after transformation the first hairy roots appeared
at the infection sites. After 2 passages on media with antibiotics, bacteria-free
hairy roots were transferred to MS solid medium without antibiotics and then,
after next 10 weeks, to MS liquid medium. Hairy roots were maintained in 30 ml
liquid MS for 4 months in 300 ml Erlenmeyer flasks on a rotary shaker (32 rpm)
at 24ºC. The hairy root cultures were initiated from single root developed at the
wounded site at plant explants. The growth of roots in MS medium was poor.
Therefore, the MS medium was changed on B5 medium [23] supplemented with
0.1 mg l -1 IAA (fig. 1), and then on hormone free DCR medium (fig. 2) [24].
Figure1. Hairy roots of Rhodiola kirilowii growing in B5 liquid medium with 0.1 mg/l IAA, third
passage (scale 4:1)
Vol. 54 No 4 2008

M. Zych, A. Pietrosiuk, M. Karasiewicz, A. Bogacz, R. Kujawski, PM. Mrozikiewicz, A. Krajewska-Patan, M. Furmanowa
10
Figure 2. Hairy roots of Rhodiola kirilowii growing in DCR liquid medium, third passage (scale 2:1)
Isolation and amplification
DNA isolation was performed according to the modified method described by
Doohan et al. [25]. DNA was isolated from 0.20 g of hairy roots and from the
untransformed roots frozen at -80ºC. Roots were rubbed in liquid nitrogen and
with 1 ml of CTAB buffer. The proteinase K (10 mg ml -1 , Sigma) in the amount of
20 μl was added to the mixture. Afterwards, the mixture was incubated at 55ºC
for 1 h. After incubation 500 μl of phenol/chloroform/isoamyl alcohol (25:24:1)
were added to the mixture and then centrifuged for 15 min. (12000 rpm) at room
temperature. Supernatants were placed in the Eppendorf tube. The extraction
process was repeated three times. Supernatants were purified with 500 μl of
chloroform/isoamyl alcohol (24:1) mixture and centrifuged for 15 min. (12000
rpm) at room temperature. Supernatants were placed in new Eppendorf ’s tubes
with 0.1 volume of 3 M sodium acetate (pH=5.2). Two volumes of frozen (–20ºC)
95% ethanol were added and the mixture was allowed to precipitate during the
night. After incubation samples were centrifuged for 20 min. at 4°C (12000 rpm).
Sediments were washed twice with frozen (–20°C) 70% ethanol (each time centrifuged
20 min. at 4°C, 12 000 rpm). Sediments were allowed to evaporate for 40 min.
at 37°C. The DNA was dissolved in deionized water at room temperature.
Each PCR reaction was performed according to the AmpliMix protocol (Novazym
Polska) by using primers designed to amplify specific regions of rol B, aux 1, vir D
genes, homologous to the nucleotide’s at definited positions. Primers sequences,
their GenBank accession number and nucleotide’s position were shown in table 1.

Establishment of Rhodiola kirilowii hairy roots using Agrobacterium rhizogenes LBA 9402
Ta b l e 1 .
Primers sequences, GenBank Accession Number and nucleotide’s position used in the study
11
region primer sequence GenBankAccession No. nucleotide’s positions
rol B
vir D
aux 1
Rhodiola kirilowii
5’-ATGGATCCCAAATTGCTATTCCTTCCACGA-3’ X03433 1861-1891
5’-TTAGGCTTCTTTCTTCAGGTTTACTGCAGC-3’ X03433 2611-2641
5’-ATGTCGCAAGGCAGTAAGCCCA-3’ X12867 1827-1851
5’-GGAGTCTTTCAGCATGGAGCAA-3’ X12867 2243-2265
5’-CGTTTCCAGTTGAGGGTTGT-3’ M61151 1136-1152
5’-GGCCTAAGTCTTCCCGTTTC-3’ M61151 1566-1585
5’-CGAAACGAATGAACCCCG-3’ AB088601 148-165
5’-ACGCTCAAAGGCACAAGG-3’ AB088601 543-560
Each PCR reaction reagents concentration were shown in table 2. The reaction
conditions, carried out in thermal cycler (PTC-200 MJ Research), were shown in table 3.
Concentration of the PCR reaction reagents used in the study
Ta b l e 2 .
reagent volume [µl] concentration
AmpliMix 10 2x
primer forward 0.1 0.25 µM
primer reverse 0.1 0.25 µM
genomic DNA 1 100 ng
H2O 8.8
PCR reaction conditions used in the study
Ta b l e 3 .
region rol B aux 1 vir D Rhodiola
PCR step temperature [˚C] time number of cycles
initial denaturation 94 94 94 94 4 min. 1
denaturation 94 94 04 94 40 s
annealing 62 64 62 56 40 s
35
elongation 72 72 72 72 1 min.
final elongation 72 72 72 72 4 min. 1
The amplified sequences were separated by electrophoresis in 2% agarose gel
in TBE buffer. The gel was stained with ethidium bromide and observed under
UV light.
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M. Zych, A. Pietrosiuk, M. Karasiewicz, A. Bogacz, R. Kujawski, PM. Mrozikiewicz, A. Krajewska-Patan, M. Furmanowa
12
Results and Discussion
Transformed roots culture
The present study was concerned with the establishment of transformed hairy
root cultures of Rhodiola kirilowii. The infection of R. kirilowii plant explants
with Agrobacterium rhizogenes LBA 9402, with the presence of acetosyringone
resulted in hairy roots formation. After two passages on MS medium with
cefotaxim, R. kirilowii hairy roots were transferred to MS solid medium and
then to MS liquid medium, both antibiotic-free. Four months after transformation
process the inhibition of hairy roots growth was observed. Therefore,
in further investigation B5 medium supplemented with 0.1 mg l -1 IAA (fig. 1),
and then hormone free DCR medium (fig. 2) were tested. Biomass increase was
evaluated on the basis of fresh weight after 4 weeks of culture as a ratio of harvested
weight to initial inoculum weight. Hairy roots grew better in hormone
free DCR medium than in B5 medium supplemented with 0.1 mg l-1. Approximately,
the ratio of harvested weight to initial inoculum’s weight was 5 for
DCR medium and 1,35 for B5 medium. Similarly, Furmanowa and Syklowska
(19) noticed a satisfying growth of Taxus x media var. Hicksii Rehd. hairy roots
in DCR medium. The doubling time of fresh weight was observed in 11 day
of culture.
The DNA analysis confirmed incorporation of rol B and aux 1 genes into the
plant genome. Gen rol B, as in the case of Nicotiana [26], causes formation of hairy
roots, but according to Di Cola et al. [27] the presence and expression of the
rol B are necessary but not sufficient to trigger rhizogenesis in transformed plant.
According to the experiment carried out by Damiano and Monticelli [28] the
amplification of the vir D region was done in order to avoid false positives. No
band was detected, which means that DNA of Rhodiola kirilowii was bacteria-free.
The transformation process strongly depended on the bacteria strain used. In
this experiment we also searched for the best strain of Agrobacterium rhizogenes.
We infected the explants with A. rhizogenes ATCC 15834, LBA 9402 and NCIB 8196.
Hairy roots were obtained only in the presence of acetosyringone and by transforming
of plant with the strain LBA 9402 (94.7% of infected explants produce hairy
roots). These results were similar to those obtained by Giri et al. [29], where the
frequency of transformation for LBA 9402 strain amounted 100%. Similarly, in the
experiment with Taxus x media var. Hicksii Rehd. [19] only strain LBA 9402 induces
the formation of hairy roots.
In our research, the addition of acetosyringone to the incubation medium was
a critical condition for hairy roots formation. Acetosyringone or other phenolic
compounds have been reported to increase transformation frequencies [29, 30]
by inducing of transcription of vir region. Similarly, the successful transformation
of Ammi majus occurred only in case Agrobacterium were inoculated on medium
supplemented with 0.2 mM acetosyringone [31].

Establishment of Rhodiola kirilowii hairy roots using Agrobacterium rhizogenes LBA 9402
Our studies were focused on establishing of Rhodiola kirilowii hairy root cultures.
Till now, the transformation process of this species using Agrobacterium rhizogenes
failed. Nowadays, the the phytochemical analysis and biological activity of R.
kirilowii hairy roots are in progress.
Detection of rol B, aux 1 and vir D genes
Integration of the bacterial DNA into the R. kirilowii genome was confirmed by
PCR reaction.
The results are presented in figures 3, 4 and 5. They proved that genes rol B,
aux 1 from Ri plasmid of A. rhizogenes were incorporated into plant genome.
M1Kb 1 K 2 K 3 K 4 K
13
Figure 3. Electrophoretic separation of consecutive PCR product of Rhodiola, rol B, aux 1 and vir D
fragments from untransformed Rhodiola kirilowii template (2% agarose gel). M1Kb – 1Kb ladder; lane 1
– Rhodiola; lane 2 – rol B; lane 3 – aux 1 ; lane 4 – vir D; K – control lane
M1Kb 1 K 2 K 3 K 4 K
Figure 4. Electrophoretic separation of consecutive PCR product of Rhodiola, rol B, aux 1 and vir D
fragments from transformed Rhodiola kirilowii template (2% agarose gel). M1Kb – 1Kb ladder; lane 1
– Rhodiola; lane 2 – rol B; lane 3 – aux 1 ; lane 4 – vir D; K – control lane.
Vol. 54 No 4 2008

M. Zych, A. Pietrosiuk, M. Karasiewicz, A. Bogacz, R. Kujawski, PM. Mrozikiewicz, A. Krajewska-Patan, M. Furmanowa
14
M1Kb 1 k 2 K 3 k
Figure 5. Electrophoretic separation of PCR product of rol B, aux 1 and vir D fragments from
Agrobacterium rhizogenes - positive control (2% agarose gel). M1Kb – 1Kb ladder; lane 1 – rol B primers;
lane 2 – aux 1 primers; lane 3 – vir D primers; K – control lane
Figure 3 introduces electrophoretic separation of consecutive PCR products
of Rhodiola, rol B, aux 1 and vir D fragments from untransformed Rhodiola kirilowii
template - 270bp PCR product size visible on lane 1 confirms detection of plant
genomic DNA in analyzed sample. No band of rol B, aux 1 and vir D was detected
which confirms that the sample used in this case was untransformed. Figure 4
introduces electrophoretic separation of PCR products of Rhodiola, rol B, aux 1
and vir D fragments from transformed Rhodiola kirilowii plant. These PCR reactions
have confirmed the integration of bacterial DNA into the Rhodiola kirilowii genome.
Application of Rhodiola primers has demonstrated the presence of 270bp
long PCR product covering of R. kirilowii genomic DNA in expected transgenic
R. kirilowii sample (lane 1). Primers homologous to the sequence of rol B gene of
Agrobacterium rhizogenes amplified the expected fragment of 780bp (lane 2). To
be sure that plant DNA is free from bacterial impurities, vir D primers were added
to the R. kirilowii. Vir D is not transferred to plant genome (lane 4). Application
of primers homologous to aux 1 gene should confirm whether the T R
fragment of
Ri plasmid was also transferred to the plant genome. Lane 3 shows detection of
plasmid aux 1 fragment incorporated into the plant genome.
The size of obtained PCR products sizes of rol B and aux 1 genes from the
transformed Rhodiola kirilowii plant were the same as the PCR products sizes from
Agrobacterium rhizogenes. Application of primers homologous to the region vir D
has enabled detection of vir D (417 bp) amplification product (fig. 5).
Acknowledgments
This work was supported by the Grant No. PBZ-KBN-092/P05/2003.

Establishment of Rhodiola kirilowii hairy roots using Agrobacterium rhizogenes LBA 9402
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Otrzymywanie korzeni włośnikowatych Rhodiola kirilowii z użyciem
Agrobacterium rhizogenes LBA 9402
Monika Zych 1 , Agnieszka Pietrosiuk 1** , Monika KARASIEWICZ 2 ,
Anna Bogacz 2 , Radosław Kujawski 2 , Przemysław M. Mrozikiewicz 2 ,
ANNA KRAJEWSKA-PATAN 2 , Mirosława Furmanowa 1
1
Katedra i Zakład Biologii i Botaniki Farmaceutycznej
Warszawski Uniwersytet Medyczny
ul. Banacha 1
02-097 Warszawa
2
Instytut Roślin i Przetworów Zielarskich
ul. Libelta 27
61-707 Poznań
*,** autorzy, do których należy kierować korespondencję:
e-mail: zych_monika@yahoo.de, agnieszka.pietrosiuk@wum.edu.pl
S t r e s z c z e n i e
Po raz pierwszy korzenie włośnikowate w rodzaju Rhodiola otrzymano na drodze infekcji
siewek Rhodiola kirilowii przy użyciu Agrobacterium rhizogenes LBA 9402. Pierwsze korzenie
transgeniczne pojawiły się na eksplantatach roślinnych 11 dni po infekcji. Proces transformacji
potwierdzono przy użyciu techniki PCR z zastosowaniem specyficznych starterów
dla amplifikacji genów rol B, aux 1 i vir D. Analiza DNA wykazała włączenie do genomu
rośliny genów rol B i aux 1 z plazmidu Ri Agrobacterium rhizogenes.
Słowa kluczowe: Rhodiola kirilowii, Agrobacterium rhizogenes, korzenie włośnikowate, gen rol B