Production of tropane alkaloids in

diploid and tetraploid plants and in vitro

hairy root cultures of Egyptian henbane
(Hyoscyamus muticus L.)

Esmail Dehghan, Suvi T. Häkkinen,

Kirsi-Marja Oksman-Caldentey &
Farajollah Shahriari Ahmadi

Plant Cell, Tissue and Organ Culture

(PCTOC)
Journal of Plant Biotechnology

ISSN 0167-6857
Volume 110
Number 1

Plant Cell Tiss Organ Cult (2012)

110:35-44
DOI 10.1007/s11240-012-0127-8

1 23
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 Author's personal copy
Plant Cell Tiss Organ Cult (2012) 110:35–44
DOI 10.1007/s11240-012-0127-8
ORIGINAL PAPER
Production of tropane alkaloids in diploid and tetraploid plants
and in vitro hairy root cultures of Egyptian henbane
(Hyoscyamus muticus L.)
Esmail Dehghan • Suvi T. Häkkinen •
Kirsi-Marja Oksman-Caldentey •
Farajollah Shahriari Ahmadi
Received: 22 August 2011 / Accepted: 4 February 2012 / Published online: 26 February 2012
Ó Springer Science+Business Media B.V. 2012
Abstract In this study, the effects of ploidy level and
culture medium were studied on the production of tropane
alkaloids. We have successfully produced stable tetraploid
hairy root lines of Hyoscyamus muticus and their ploidy
stability was confirmed 30 months after transformation.
Tetraploidy affected the growth rate and alkaloid accumu-
lation in plants and transformed root cultures of Egyptian
henbane. Although tetraploid plants could produce 200%
higher scopolamine than their diploid counterparts, this
result was not observed for corresponding induced hairy root
cultures. Culture conditions did not only play an important
role for biomass production, but also significantly affected
tropane alkaloid accumulation in hairy root cultures. In spite
of its lower biomass production, tetraploid clone could
produce more scopolamine than the diploid counterpart
under similar growth conditions. The highest yields of sco-
polamine (13.87 mg l-1) and hyoscyamine (107.7 mg 1-1)
were obtained when diploid clones were grown on medium
consisting of either Murashige and Skoog with 60 g/l
sucrose or Gamborg’s B5 with 40 g/l sucrose, respectively.
Although the hyoscyamine is the main alkaloid in the
H. muticus plants, manipulation of ploidy level and culture
conditions successfully changed the scopolamine/hyoscya-
mine ratio towards scopolamine. The fact that hyoscyamine
is converted to scopolamine is very important due to the
higher market value of scopolamine.
E. Dehghan (&)  F. Shahriari Ahmadi
Department of Plant Biotechnology, Faculty of Agriculture,
Ferdowsi University of Mashhad, P.O. Box 9177948978,
Mashhad, Iran
e-mail: es.dehghanshahreza@gmail.com
S. T. Häkkinen  K.-M. Oksman-Caldentey
VTT Technical Research Centre of Finland, P.O. Box 1000,
02044 Espoo, Finland
Keywords Flow cytometry  GC–MS  Hairy root
cultures  Hyoscyamus muticus  Ploidy level 
Tropane alkaloids
Introduction
Tropane alkaloids, especially hyoscyamine and scopolamine,
are widely used in medicine for their mydriatic, antispasmodic,
anticholinergic, analgesic and sedative properties. They are
industrially extracted from various solanaceous plants
belonging to the genera Atropa, Duboisia, Datura and Hyo-
scyamus (Mateus et al. 2000; Oksman-Caldentey and Arroo
2000). Hyoscyamine is usually the main alkaloid in many of
these plants such as Hyoscyamus muticus and Atropa bella-
donna while scopolamine is only produced in small amounts
(Mateus et al. 2000; Oksman-Caldentey and Arroo 2000).
Scopolamine is more valuable being higher in physiological
activity, possessing fewer side-effects and having commonly
lower yields. The world demand for this alkaloid is estimated
to be about 10 times greater than for hyoscyamine and its
racemic form atropine (Hashimoto et al. 1993; Oksman-
Caldentey and Arroo 2000). Accordingly, there has been a
long-standing interest in boosting the scopolamine content of
producing plants and their in vitro cultures. Due to the com-
plexity of the chemical structure and long route of the bio-
synthesis pathway, the synthetic production of these alkaloids
is more expensive than their extraction from plant materials
(Huang et al. 2005) and it can be assumed that in the foresee-
able future the supply of tropane alkaloids would largely
depend on biological methods of production. So it is of sig-
nificance and importance to improve tropane alkaloid pro-
duction, especially the much more valuable scopolamine, to
meet the expansion of clinical needs (Kai et al. 2007). During
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36
the past three decades different approaches based on callus and
suspension cultures, somatic hybridization and root cultures
have been applied for economical in vitro production of tro-
pane alkaloids. As a promising system, hairy root cultures of
H. muticus have been extensively studied by Sevón et al.
(2001); Jouhikainen et al. (1999); Sevón and Oksman-Cal-
dentey (2002), and some other research groups (Zolala et al.
2007; Mateus et al. 2000).
In order for hairy roots-based technologies to be com-
mercially viable, several criteria have to be met, one of which
is to ensure that the yields of the products are high enough. In
most cases, these yields are low and unsatisfactory. There-
fore, integrated approaches for their improvement have to be
developed, including genetic engineering, selection of
highly productive lines, and optimization of variables such as
the nutrient medium, bioreactor design and environmental
conditions. The scope for improving production by ploidy
manipulation (in parallel with the selection of highly pro-
ductive lines) should be investigated during early stages of
the optimization process, but its potential importance is
usually underestimated or completely ignored (Pavlov et al.
2009; Georgiev et al. 2007). Ploidy level manipulation has
been suggested as an interesting approach for increasing both
the plant biomass accumulation, and the production of high-
value plant secondary metabolites (Lavania 2005). It is well
known that polyploidy is frequently accompanied by con-
spicuous changes in morphology (Sun et al. 2011; Lavania
2005) and secondary metabolite production (Kaensaksiri
et al. 2011; Lavania 2005; Dhawn and Lavania 1996). Tro-
pane alkaloids, terpenoids and isoflavonoids are among the
most important medicinal compounds found in plants. Thus,
induction of artificial polyploidy may be proved to be useful
in increasing the production of these important medicinal
compounds (Dhawn and Lavania 1996). Although we have
relatively good information about effects of ploidy manip-
ulation on secondary metabolite production in several
medicinal plants, discussed by Dhawn and Lavania (1996)
and Lavania (2005), unfortunately, little is known about how
ploidy may affect the growth and the production of sec-
ondary metabolites in hairy root systems (De Jesus-Gonzalez
and Weathers 2003). It should be noted that there were only
few reports concerning exploitation of polyploid hairy root
cultures for secondary metabolite production. These reports
included achieving autotetraploid Artemisia annua L. hairy
root lines by means of treateing diploid hairy root cultures by
colchicine (De Jesus-Gonzalez and Weathers 2003) and
achieving autotetraploid D. stramonium L. hairy root cul-
tures by direct transformation of autotetraploid plants
(Berkov et al. 2003; Pavlov et al. 2009).
Although genetic stability is a very outstanding property
of hairy root cultures, transformation process may affect
the ploidy profile of in vitro cultures (Weber et al. 2008;
Pavlov et al. 2009). Thus, ploidy level investigation of
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Plant Cell Tiss Organ Cult (2012) 110:35–44
hairy root cultures by flow cytometry can provide data
about genetic stability and selection of highly producing in
vitro hairy root cultures.
Even though H. muticus (strain Cairo) is a very important
medicinal plant with a capacity to produce high amounts of
tropane alkaloids (up to 6% of the dry weight in the leaves of
mature plant), and it usually behaves better in cell culture
than solanaceous species generally (Jouhikainen et al. 1999),
this plant nearly exclusively produces hyoscyamine. On the
other hand the effects of ploidy manipulation on biomass
production and tropane alkaloid accumulation by this strain
and its transformed hairy root cultures are still not well
known. Our aim was to investigate whether the ploidy
manipulation in this plant was able to change tropane alka-
loid production toward scopolamine. We also studied the
effects of ploidy level, sucrose concentrations and culture
medium on growth and tropane alkaloid production in hairy
root cultures of this plant. Flow cytometry experiments were
also conducted to check the ploidy and genetic stability of
diploid and tetraploid leaves and derived hairy root cultures.
Materials and methods
Plant material
Seeds of diploid and C5 (Colchiploid 5) generation of
induced autotetraploid H. muticus (Shahriari et al. 2009)
were surfaced sterilized using 5% (v/v) sodium hypochlorite
(NaClO) for 10 min, rinsed three times with sterile distilled
water and then germinated on a basal MS medium. After
3 weeks, the small seedlings (10 seedlings of each) were
transferred to greenhouse and maintained at temperature of
25 ± 2°C at a photoperiod of 16 h light/8 h darkness.
Establishment of hairy roots and culture conditions
Agrobacterium rhizogenes agropine strain A4 (VTT Bio-
technology, Finland) was used for the transformation
experiments. Bacteria were grown for 48 h (OD = 0.6–0.8
in 590 nm) in liquid YMB medium at 28 ± 2°C on a rotary
shaker at 100 rpm. Leaf segments (about 1–2 cm length)
isolated from well grown leaves of diploid and tetraploid H.
muticus were used as explants for cocultivation and hairy
root induction. The explants were surface sterilized using 5%
sodium hypochlorite for 10 min rinsed with sterile water
three times. The explants were wounded with a sterile needle
loaded with the bacterial suspension, then were placed on
hormone free MS medium with 3% sucrose and incubated at
25 ± 2°C in a photoperiod of 16 h light/8 h darkness. After
2 days of cocultivation, the explants were transferred to the
fresh medium containing 500 mg/l cefotaxime. The hairy
roots emerging from the inoculated sites were cut off and
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Plant Cell Tiss Organ Cult (2012) 110:35–44
transferred to 10 ml of B5 (Gamborg et al. 1968) liquid
medium supplemented with 3% sucrose, in 100 ml conical
flasks and grown at 25 ± 2°C on a gyratory shaker (Hei-
dolph, Germany) in darkness and subcultures were per-
formed at four week intervals for 30 months.
Sampling of the leaves and hairy root cultures
The completely grown leaves of several diploid and tetra-
ploid plants were collected at flowering stage, about
120 days after germination, lyophilized by freeze dryer
(VaCo5, ZIRBUS technology, Germany) and stored in
-20°C. The effects of culture medium and sucrose con-
centrations on the hairy root growth and tropane alkaloid
accumulation were tested by cultivation of diploid and
tetraploid hairy roots in liquid growth regulator-free MS
and B5 media, supplemented with different amounts of
sucrose (20, 30, 40, 50 and 60 g/l). The pH was adjusted in
all media to 5.6–5.8. Thereafter about 100 mg (FW) of
fresh roots were excised and transferred to 20 ml of the
corresponding fresh liquid media in 100 ml conical flasks
and grown at 25 ± 2°C on a gyratory shaker (90 rpm) in
darkness. After 28 days of cultivation, the roots were
washed with water then lyophilized and stored at -20°C
until analyzed. All conditions were analyzed as triplicates.
Detection of bacterial DNA in hairy roots
Total DNA samples were isolated from 24 h old cultures of
A. rhizogenes strain A4 using a DIAtomTM DNA Prep Kit.
DNA was also isolated from hairy roots and control roots
according to Saghai–Maroof et al. (1984). The polymerase chain
reaction was performed for rolC gene in hairy roots. Oligonu-
cleotide primers for the PCR detection of homologous sequences
to rolC genes were selected as below (Zolala et al. 2007):
50 -CTCCTGACATCAAACTCGTC-30
50 -TGCTTCGAGTTATGGGTACA-30
Each PCR reaction included standard PCR buffer
(Cinnagen Inc., Iran), 1.0 U Taq DNA polymerase (Cinn-
agen Inc., Iran), 0.5 ll dNTP mix.(10 mM), 1.0 ll MgCl2
(50 mM), 8 pmol of each primer and 40–60 ng of the
target DNA (final volume 25 ll). Amplification conditions
were the same as Zolala et al. (2007), except for primer
annealing which was at 55°C. After separation by elec-
trophoresis, amplified fragments were stained with ethi-
dium bromide and observed under UV light.
Flow cytometry analysis
Flow cytometric analysis for ploidy level and genetic sta-
bility of hairy root cultures that were obtained from diploid
and tetraploid leaf explants, was carried out as below:
37
Cell nuclei were extracted from 120 mg fresh samples
by cutting the roots in small pieces with a razor blade in
400 ll of nuclei extraction buffer (Kit A, suggested by
Partec PA, Germany). After filtration through a 30 lm cell-
trice disposable mesh filter (Partec PA, Germany), 1,600 ll
of staining solution containing the dye 4-6-diamino-2-4-
phenylindole (DAPI, provided by Partec PA, Germany as
kit B) was added and a minimum of 5,000 nuclei per
sample were measured by PA flow cytometer (Partec PA,
Germany) equipped with UV mercury arc lamp excitation
source and filter combination for DAPI staining, after 30 s.
Control samples of diploid plants and hairy root cultures
were regularly run, in order to check the stability of
peak position corresponding to the diploid level. The
entire coefficient of variation was between 3.0 and 5.7%.
Experimental conditions for flow cytometry profile analysis
of different organs (root, stem, leaf and flower) from dip-
loid and tetraploid Egyptian henbane plants were similar to
that of the hairy root cultures.
Tropane alkaloid analyses
Tropane alkaloids were analyzed by GC–MS as described
in Häkkinen et al. (2005). The lipids were removed with
petroleum ether and the alkaloids were subsequently
extracted with dichloromethane from basic solution.
Homatropine (Sigma) was used as an internal standard.
Samples were derivatized with MSTFA (N-methylsilyl-N-
trifluoroacetamide, Pierce) before GC–MS analyses. Hyo-
scyamine (Merck), scopolamine-HBr (Sigma) and tropine
(Sigma) were used as reference compounds.
Experimental and statistical analysis
The experiment was carried out in factorial arrangement
based on the randomized completely design (RCD) with
three factors (ploidy levels, nutrition media and sucrose
concentrations) and three replicates. Duncan’s multiple
range test was used to explain the significant difference at
the statistical level of 0.05. Minitab R13 (Minitab Inc.) and
Mstat-C (Michigan State University, East Lansing, MI,
USA) softwares were used for analysis of variance
(ANOVA) and comparison of means value, respectively.
All experiments had three replicates, and each experiment
was conducted twice.
Results and discussion
Hairy root induction and establishment
In diploid and tetraploid explants, hairy roots were com-
monly emerged from the wounded sites after 8–28 days of
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M 1 2 3 4 5 6 7 M
750bP
612 bP
500b
Fig. 1 PCR analysis of rolC gene of A. rhizogenes, (M): Size marker,
(1): Amplified band from the DNA of A. rhizogenes, Positive control,
(2, 3, 6 and 7): Amplified band from the DNA of hairy roots, (4):
Negative control, (5): The band is absent in reaction with wild type
root DNA
bacterial infection. The diploid and tetraploid established
hairy root culture were maintained for 30 months and
routinely sub-cultured on liquid, hormone free medium.
Totally 20 and 15 diploid and tetraploid root clones were
established, respectively. During this period, some of their
characteristics such as stability of growth rate and biomass
production, as well as stability of ploidy level and mor-
phology of the root were studied. The transgenic state of
these hairy root lines were confirmed by PCR for the
amplification of a segment of 612 bp corresponding to the
bacterial rolC gene (Fig. 1).
There was a significant difference between the growth
rate of the hairy root lines (data not shown). One diploid
and one tetraploid clones, with stable growth and mor-
phological characteristics were isolated for further analy-
ses. It was obvious that tetraploid hairy roots were thicker
but no other morphological differences were observed
between them (Fig. 4). The vigorous nature of tetraploid
clones was not a surprising observation since larger plant
organs are often the result of polyploidy and already
observed in other tetraploid plants (Lavania 2005) and
hairy root cultures (Pavlov et al. 2009; De Jesus-Gonzalez
and Weathers 2003).
Genetic stability and flow cytometry analysis
Plant in vitro systems are valuable sources for the production
of biologically active substances. However, changes in
secondary metabolite profiles as well as low and variable
yields, possibly caused by genetic instabilities, complicate
their industrial implementation. Furthermore, although
transformed root cultures are considered to be stable (Baı́za
et al. 1999), a certain amount of heterogeneity can be found
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Plant Cell Tiss Organ Cult (2012) 110:35–44
between them thus DNA profiling of plant in vitro cultures
may provide data for the selection of highly producing in
vitro cultures (Weber et al. 2008). On the other hand, flow
cytometry is a rapid and accurate method, which can be
efficiently used for ploidy determination in plants growing in
fields and greenhouse (Dolezel et al. 1989) or in vitro cul-
tures and in vitro regenerated plants (Stancheva et al. 2011;
Pinto et al. 2010; Obae and West 2010; Pavlov et al. 2009;
Weber et al. 2008). High chromosome number in tetraploid
H. muticus (2n = 49 = 56) (Lavania 1986) and derived
hairy root cultures makes microscopic cytological analysis
of this species unfeasible. Hence we conducted flow
cytometry experiments in order to ensure about ploidy sta-
bility of tetraploid plants (five generation after induction of
tetraploidy) and derived hairy root cultures (30 month after
induction). In this study the values of G1 and G2 peaks of
tetraploid samples were approximately two-times more than
its diploid counterparts (Fig. 2).
Flow cytometry analysis confirmed tetraploidy of hairy
roots and its stability after 30 months subculturing. In some
samples, both diploid and tetraploid leaves of H. muticus
showed an additional peak, containing doubled DNA
content (Fig. 2). The occurrence of nuclei with 8C and 16C
value showed that the diploid and tetraploid leaves of
Egyptian henbane underwent one cycle of endoreduplica-
tion process. However, no additional peaks in diploid and
tetraploid hairy root cultures were observed. Endoredupli-
cation is wide spread in plants, particularly angiosperms,
and may occur in any cell types except the gametes, the
meristematic, and guard cells (Lema-Rumin’ska 2011;
Ochatt et al. 2011; Cebolla et al. 1999).
In this study we observed endoreduplication in diploid
and tetraploid plants, in the other words, there was not any
obvious relationship between the DNA ploidy level and
endoreduplication. This is in conflict with the results of
Weber et al. (2008) where negative correlation between
genome size and endoreduplication was observed.
On the basis of genetic stability of hairy root cultures,
our results are in accordance with other researchers (Baı́za
et al. 1999; Sevón and Oksman-Caldentey 2002) but are in
conflict with Weber et al. (2008) and Pavlov et al. (2009),
in which in vitro hairy root cultures of Datura stramonium
and H. niger, underwent different degrees of endoredupli-
cation after transformation process. In another study
Lavania and Srivastava (1988) observed that 49 genotype
of H. muticus callus culture was relatively more resistant
than its 29 counterpart in terms of frequency and extent of
chromosomal variability. They also showed the attempt of
the 29 genotype to attain the genomic dosage of 49,
possibly as a sequel of stress caused by callus growth under
artificial conditions.
In this study flow cytometry profile of different organs
(root, stem, leaf and flower) from diploid and tetraploid
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Plant Cell Tiss Organ Cult (2012) 110:35–44 39

Fig. 2 Flow cytometry histograms obtained from diploid (a) and tetraploid (b) H. muticus leaves, and corresponding diploid (c) and tetraploid
(d) hairy root cultures in the stationary phase of growth

Fig. 3 Flow cytometry profile of mature leaves near the flowering top of diploid (a) and tetraploid (b) H. muticus. The majority of nuclei were
stopped in G2 phase of cell cycle

Egyptian henbane plants were analyzed during three gen- (Fig. 3). Although this process was not intense in roots,
erations. Our results indicated that the leaf cells of diploid stems, flowers and some leaves, so that the proportion of
and tetraploid plants were mainly arrested in G2 stage G1 and G2 were nearly balanced. However, the leaf near

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the flowering top (inflorescence) of mature plant rigorously
stopped in G2 phase of the cell cycle (Fig. 3). The higher
proportion of 4n nuclei could be due to higher cell cycle
activity or endoreduplication, which often occurs in older
plant tissue. This process was also observed in some old
and differentiated tissues as well as in dormant leaf tissues
and also in some in vitro-cultured plants. It is possible that
cell cycle in a particular tissue of a certain species is
arrested in G2 instead of G1 (Professor J. Dolezel, personal
communications).
Production of tropane alkaloids in diploid and tetraploid
plants
GC–MS analysis was applied for investigation of three
important compounds in the leaves of diploid and tetra-
ploid H. muticus (late flowering stage). Capillary gas
chromatography/mass spectrometry (GC/MS) is the most
suitable and powerful method for rapid separation and
identification of complex mixtures of tropane alkaloids
(Häkkinen et al. 2005; Berkov et al. 2003; Berkov and
Philipov 2002).
The pattern of tropane alkaloid production changed by
tetraploidy. Although tropine production was not affected
by ploidy level manipulation, the hyoscyamine and sco-
polamine production pattern was quite different in diploid
and tetraploid plants. Tetraploid plants produced three
times higher scopolamine and two times less hyoscyamine
than their diploid counterparts (Table 1). In other words,
the conversion of hyoscyamine to scopolamine was suc-
cessfully accelerated by the increase of ploidy level
therefore the scopolamine/hyoscyamine ratio was higher in
tetraploid plants. In another study Lavania (1986) reported
autotetraploid plants of H. muticus with 36% increase in
total tropane alkaloid concentration. However, in this plant,
increasing of scopolamine/hyoscyamine ratio was not
reported yet. Berkov and Philipov (2002) reported 2.63-
(0.15–0.40% DW) and 1.41-fold (0.05–0.08% DW)
increase of scopolamine and hyoscyamine content in the
leaves of D. stramonium L., after induction of artificial
tetraploidy by colchicine treatment. The mean scopol-
amine/hyoscyamine ratio also altered from 3.65 for dip-
loids to 6.58 for tetraploids.
Table 1 Alkaloids identified in the leaves of diploid (29) and tetraploid (49) plants of Hyoscyamus muticus and in diploid and tetraploid hairy
root clones grown in the B5 ? 20 g/l sucrose
Ploidy level Plant tissues Tropine mg/g DW
Diploid Leaves 0.02 ± 0.05
Hairy root 0.44 ± 0.06
Tetraploid Leaves 0.02 ± 0.06
Hairy root 0.12 ± 0.4
Each value is the average of three replicates ± SD
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In our study the increase in scopolamine content and
decrease in hyoscyamine content as a result of tetraploidy
may be due to higher activity of hyoscyamine-6-beta-
hydroxylase (H6H) that catalyzes conversion of hyoscya-
mine to scopolamine. Changing of the metabolic profile in
autopolyploid plants by a simple duplication of the basic
genome was interpreted as a cause of an alteration in the
mechanism(s) which regulates the biosynthesis of individual
compounds. In autopolyploids the basic genetic material
remains the same, but gene dosage is multiplied and there is
potential to modulate gene expression and hence enzyme
activity per unit protein (Lavania 2005). Due to ploidy
induction of A. annua plants significant upregulation of
several key enzymes related to artemisinin biosynthetic
pathway were also reported by Lin et al. (2011).
As represented in Table 1, there are also some differ-
ences in concentrations of tropane alkaloid compounds
between the leaves and in vitro hairy root cultures. In hairy
roots, tropane alkaloid concentrations, especially that of
tropine, were sharply increased; however for both diploid
and tetraploid, the scopolamine/hyoscyamine ratio was
higher in the leaves than in hairy root cultures. This might
be due to higher epoxidation of hyoscyamine to scopol-
amine in aerial parts of plants, where external factors, such
as day-length and light intensity, may play a role (Berkov
and Philipov 2002).
Hairy roots are fast-growing, auxin-independent,
genetically stable, and can synthesize amounts of material
that are comparable to, and sometimes in excess of, the
amounts generated by the original plants (Georgiev et al.
2007; Sevón et al. 2001). Hairy root cultures of H. muticus
were discussed as promising systems for the in vitro pro-
duction of tropane alkaloids, and as shown in our study
(Table 1), there are several reports that show this potential
at levels which are often comparable to, or greater than
those of the intact plants (Sevón et al. 2001; Oksman-
Caldentey and Arroo 2000; Jouhikainen et al. 1999).
Effects of the culture medium and sucrose
concentrations
It is well known that sucrose is the most energetically
advantageous of the carbohydrates used as carbon sources
Hyoscyamine mg/g DW Scopolamine mg/g DW % Sco/hyo
2.02 ± 0.13 0.12 ± 0.13 5.94
5.43 ± 0.32 0.24 ± 0.01 4.41
0.95 ± 0.06 0.36 ± 0.08 37.89
3.59 ± 0.81 0.49 ± 0.07 13.64
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Plant Cell Tiss Organ Cult (2012) 110:35–44
in the cultivation of plant in vitro systems, especially for
the biosynthesis of secondary metabolites. Rothe and
Drager (2002) reported that sugars act as carbon source as
well as signal compounds in root cultures. Although the
effects of sucrose and other carbon resources on biomass
production and hyoscyamine accumulation in hairy root
cultures of H. muticus were studied by Oksman-Caldentey
et al. (1994), the results varied between the clones. On the
other hand, ploidy level manipulation of hairy root cultures
may also change these results. Therefore, in addition to the
MS and B5 medium cultures, the effects of sucrose con-
centrations on growth and tropane alkaloid production of
diploid and tetraploid clones were investigated.
A considerable variation in growth rates was observed
between hairy root clones. Especially tetraploid clones had
a slow growth rate. Since high growth rates are an
important factor in achieving high overall secondary
metabolite productivity, in this study one diploid and one
tetraploid clone with an average growth rate and high
stability were selected. As seen in Fig. 4, tetraploid clone
(similar to its parental plant) was approximately thicker
than diploid one. Although we already observed nearly
17% increase of dry weight (during one generation) in the
parental plants, due to tetraploidy induction, (Shahriari
et al. 2009), induced tetraploid clone showed somewhat
lower growth rate and potential for biomass production
than their diploid counterparts. This is in agreement with
earlier report of De Jesus-Gonzalez and Weathers (2003)
and Pavlov et al. (2009) where tetraploid hairy root clones
of A. annua and D. stramonium had relatively higher
Fig. 4 Diploid (a) and autotetraploid (b) plants and derived diploid
(c) and tetraploid (d) hairy root cultures of Egyptian henbane
41
diameter but lower growth rate than diploid counterparts.
In most plant species, artificial polyploidy increased the
size of cells and on the other hand, reduced number of cell
divisions during growth and development which is a
common result of polyploidy (Lavania 2005). Thus, overall
reduced growth and increased diameter of hairy roots is not
surprising.
Both diploid and tetraploid hairy root clones of H. mu-
ticus had higher growth and biomass production in liquid
B5 medium (Fig. 5). The sucrose concentrations also had a
significant effect on biomass production. For diploid clone
the highest yields of biomass (19.40 and 17.32 g/l) was
respectively obtained in B5 and MS medium supplemented
with 30 g/l sucrose. The highest biomass production of
tetraploid clone was achieved in B5 ? 40 g/l sucrose
(18.77 g/l) and MS ? 50 g/l sucrose (17.26 g/l). In general
diploid clone was more sensitive to increase of sucrose
concentrations and reached the highest of production in
30 g/l sucrose, whereas increasing of the sucrose concen-
tration was tolerated to 50 g/l in MS medium by tetraploid
clone. With increment in concentration of sucrose, both in
diploid and tetraploid clones, biomass production was
enhanced, but the higher amount of sucrose had negative
effect on growth and biomass production. Therefore, at
least supplementation of 30 g/l sucrose is necessary for
optimal production of biomass. The positive effect of
sucrose on the growth of H. muticus hairy root cultures has
been also reported by Oksman-Caldentey et al. (1994). Our
findings about diploid clone support their results about
LBA1S clone of H. muticus, but are in contrast to those of
Christen et al. (1992), who reported that different sucrose
concentrations in B5 medium had little effect on the growth
of H. albus hairy roots.
Culture medium and sucrose concentration had also an
important role in tropane alkaloid production of hairy root
cultures with different ploidy level (Table 2, Fig. 6). The
Fig. 5 Biomass production in diploid and tetraploid hairy roots in B5
and MS medium. Results are a mean of at least three replicates.
Different letters indicate significant statistical difference at the 95%
level using ANOVA (P \ 0.05: Duncan’s multiple range test)
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42 Plant Cell Tiss Organ Cult (2012) 110:35–44

obtained hairy root cultures in this study showed a higher
potential for tropane alkaloid production than the parental
plants (Table 1). In the best condition (MS ? 20 g/l
sucrose) diploid clone could accumulate 8 and 3.3 times
higher scopolamine and hyoscyamine, respectively, than
parental leaf samples. The same results were also observed
when tetraploid clone produced 1.5 and 6 fold higher
scopolamine and hyoscyamine than parental tetraploid
leaves of the plants in MS medium added with 50 and 20 g/
l sucrose, respectively. There was an inverse correlation
between hyoscyamine accumulation (mg/g DW) and
sucrose concentrations (Table 2). We also obtained 27.5
and 26.5 times higher tropine accumulation than parental
plants for diploid and tetraploid hairy root cultures of H.
muticus in MS medium supplemented with 50 g/l sucrose
(Table 2).
The highest yield of scopolamine (13.87 mg/l) and hyo-
scyamine (107.7 mg/l) were obtained when diploid clone
was grown in MS ? 60 and B5 ? 40, respectively.
Although the obtained scopolamine concentration (0.99 mg/
g DW) for the best treatment in our study was higher than
other studies such as Jouhikainen et al. (1999) that reported
about 0.8 mg/g DW scopolamine for h6h-overexpressed
transgenic hairy root lines of H. muticus, however from an
applicational point of view, our true productivity (13.87 mg/
l) was not higher than those reported earlier. Considering the
biomass production, supplementation of 3 and 4% sucrose, in
MS and B5 nutrient medium respectively were the best
conditions for obtaining the highest hyoscyamine yield (mg/
l), both in diploid and tetraploid hairy root cultures (Fig. 6).
However, the hyoscyamine accumulation was reduced by
increasing of sucrose concentration until it reached the
lowest amount in the highest concentration of sucrose
(Fig. 6). This is in contrast with the results of Pavlov et al.
(2009), who obtained the highest production of hyoscyamine
in tetraploid D. stramonium hairy root in MS medium con-
taining 6% sucrose.
Although hairy root cultures of H. muticus had higher
growth rate in B5 liquid medium and could produce more
biomass than those cultured in MS liquid medium, the
potential of scopolamine accumulation was higher in hairy
roots cultured in MS medium. Tetraploid clone could
successfully produce more scopolamine (mg/l) than diploid
counterpart in B5 medium (Table 2). However, the tropine
and hyoscyamine production significantly reduced in this
condition. A higher conversion of tropine and hyoscyamine
to scopolamine was evident for tetraploid clone in B5
medium. The same result was also observed in tetraploid
parental plants of Egyptian henbane (Table 2). Berkov
et al. (2003) reported that percentage of hyoscyamine in the
alkaloid mixture reduced from 78.75 to 56.60% in tetra-
ploid hairy root culture of D. stramonium. Although tet-
raploid plants could produce 200% higher scopolamine
than diploid counterparts, however, this result was not
observed for corresponding induced hairy root cultures.
This is in contrast to the positive effects of ploidy manip-
ulation on secondary metabolites production by hairy root
cultures of A. annua (De Jesus-Gonzalez and Weathers
2003) and D. stramonium (Pavlov et al. 2009).
Our results showed that culture medium, carbon source
and ploidy manipulation can change the tropane alkaloid
production pattern of Egyptian henbane in vitro hairy root
cultures. We obtained higher scopolamine for tetraploid
parental plants and induced hairy root cultures (only in B5
nutrient medium) than the diploid counterparts. It seems
that tetraploidy can obviously change the ratio of S/H
toward scopolamine. This may be the result of the change
of gene expression in tropane alkaloid pathway caused by

Table 2 Tropane alkaloid concentrations of Egyptian henbane in vitro hairy root cultures with different ploidy levels in MS and B5 medium
under different sucrose concentrations
Culture media ? sucrose Diploid % S/H Tetraploid % S/H
concentrations (g/l)
Tropine Hyoscyamine Scopolamine Tropine Hyoscyamine Scopolamine
mg/gDW mg/gDW mg/gDW mg/gDW mg/gDW mg/gDW

B5 ? 20 0.44 ± 0.06 5.43 ± 0.32 0.24 ± 0.01 4.5 0.12 ± 0.4 3.59 ± 0.81 0.49 ± 0.07 13.7
B5 ? 30 0.34 ± 0.06 4.43 ± 0.61 0.20 ± 0.02 4.6 0.05 ± 0.04 2.78 ± 0.52 0.29 ± 0.06 10.5
B5 ? 40 0.37 ± 0.07 6.27 ± 0.48 0.27 ± 0.02 4.4 0.09 ± 0.04 3.04 ± 0.76 0.33 ± 0.07 10.9
B5 ? 50 0.27 ± 0.03 3.76 ± 0.34 0.14 ± 0.01 3.7 0.12 ± 0.02 2.44 ± 0.27 0.20 ± 0.05 8.1
B5 ? 60 0.29 ± 0.05 3.58 ± 0.24 0.20 ± 0.02 5.5 0.06 ± 0.05 2.32 ± 0.31 0.25 ± 0.05 10.7
MS ? 20 0.39 ± 0.08 6.59 ± 0.76 0.99 ± 0.09 15.0 0.36 ± 0.03 5.81 ± 1.28 0.30 ± 0.06 5.1
MS ? 30 0.56 ± 0.09 6.10 ± 0.42 0.53 ± 0.04 8.8 0.35 ± 0.01 4.97 ± 1.13 0.22 ± 0.03 4.3
MS ? 40 0.33 ± 0.09 5.39 ± 0.18 0.45 ± 0.01 8.4 0.49 ± 0.03 4.45 ± 0.96 0.21 ± 0.05 4.6
MS ? 50 0.56 ± 0.11 4.28 ± 0.35 0.51 ± 0.03 12.0 0.53 ± 0.03 3.42 ± 0.88 0.54 ± 0.10 15.8
MS ? 60 0.39 ± 0.07 3.51 ± 0.26 0.95 ± 0.02 27.0 0.48 ± 0.04 2.87 ± 0.69 0.36 ± 0.07 12.7
Each value is an average of three replicates ± SD

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Plant Cell Tiss Organ Cult (2012) 110:35–44
Fig. 6 Scopolamine (a and b) and hyoscyamine (c and d) productivity in B5 and MS culture media. Results are a mean of at least three analytical
replicates
tetraploidy, when with the lower precursors (tropine and
hyoscyamine) higher amount of final compound (scopol-
amine) was achieved. The genomic rearrangement, change
in the gene expression and enzymes activity due to poly-
ploidization has previously reviewed by Yang et al. (2011)
and Lavania (2005). Polyploidy may also alter the avail-
ability of ornithine and phenylalanine, or the concentration
or activity of the enzymes along the biosynthetic pathway
(Oksman-Caldentey 1986).
Acknowledgments The authors wish to thank Professor G. Asghari
(Department of Pharmacognosy, School of Pharmacy, Isfahan Uni-
versity of Medical Sciences) and Dr M. Zare for assistance in flow
cytometry experiments. The authors also appreciate the Deputy of
Research Affairs in Ferdowsi University of Mashhad for their finan-
cial supports provided as Grant No P446.
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