Int. J. Med. Arom.

Plants, ISSN 2249 4340

RESEARCH ARTICLE
Vol. 3, No. 1, pp. 40-46, March 2013
*Corresponding author: (E-mail) nbenbahri <@> yahoo.fr http://www.openaccessscience.com
2013 Copyright by the Authors, licensee Open Access Science Research Publisher. ijmap@openaccessscience.com
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Morphological effects of zinc and its accumulation in micropropagated
plants of Paulownia tomentosa (Thunb.) Steud.
Nada BEN BAHRI*, Bochra LARIBI, Taoufik BETTAIEB
Dpartement dAgronomie et de Biotechnologies Vgtales, Institut National Agronomique de Tunisie. 43, Av.
Charles Nicolle-1082, Tunis, Tunisie
*
Corresponding author, Tel: +216-71 28 71 10, Fax: +216-71 79 93 91
Article History: Received 8
th
August 2012, Revised 20
th
January 2013, Accepted 21
st
January 2013.
Abstract: In vitro essay of Paulownia tomentosa (Thunb.) Steud. was undertaken to determine the capacity of this me-
dicinal plant to accumulate heavy metal in its tissues. In this case, the nodal explants were placed on Murashige and
Skoogs medium (1962), added with 1.0 mg l
-1
indole-3-butyric acid (IBA). The ZnSO
4
was supplemented to the culture
medium at various concentrations 200, 400, 600, 800 and 1000 M. The MS medium free of Zn was served as control.
The height of plantlets, the number of nodes, the number and length of roots and the biomass of the in vitro plants were
determined. The Zn content in plant tissues was measured by an inductively coupled plasma-optical emission spectrome-
ter (ICP-OES, OPTIMA 2000 DV, PerkinElmer, USA). Results showed that highest Zn level (1000 M) induced a sig-
nificant reduction in shoot height, (18.86%) and root number (84.9%) of plantlets in comparison to the control. The high-
est Zn concentration in the culture medium caused also an important decrease in fresh and dry biomass by 22.8% and
50.5%, respectively compared to the control. Furthermore, a high Zn accumulation in the plantlet tissues which varied
from 121g/g dry matter (DM) in the control to 2580 g/g (DM) in the presence of 1000 M Zn was noticed. Conse-
quently, Paulownia tomentosa could be considered as a bio-indicator and Zn accumulator medicinal plant.
Keywords: Micropropagation, Paulownia tomentosa (Thunb.) Steud., medicinal plant, Zn accumulator.
Abbreviations: IBA: indole-3-butyric acid; MS: Murashige and Skoog medium; Zn: Zinc; DM: Dry Matter.
Introduction
Paulownia tomentosa (Thunb.) Steud. is a
perennial tree species which belonging to the
Scrophulariaceae family. It is native to central
and western China but its widely distributed in
Korea, Japan, and China (Jiang et al. 2004).
Parts of the plant P. tomentosa (leaves,
wood, and fruits) have been used in traditional
Chinese herbal medicine for the treatment of
tonsillitis, bronchitis, asthmatic attack, and bac-
terial infections such as enteritis or dysentery. In
fact, the flower is the most important material
used in folk medicine herbs (Kang et al. 1999,
Jiang et al. 2004, mejkal et al. 2007). Howev-
er, extracts of P. tomentosa contained many bi-
oactive compounds such as flavonoids and par-
ticularly Apigenin. This last has been found to
show a variety of pharmacological activities,
including hypotensive (Loizzo et al 2007), anti-
inflammatory (Gerritsen et al. 1995, Ko et al.
2004), antispasmotic (Capasso et al 1991), anti-
oxidant (Cos et al 1998) and vasorelaxant
(Zhang et al 2000). Besides, Apigenin may exert
its anti-tumorigenic effect in vivo not only via
the inhibition of tumor cell proliferation, but
also via the impairment of the invasive potential
of tumor cells (Czyz et al. 2005).
Recently, P. tomentosa has received an in-
creased attention due to its marketable value for
wood and biofuel production thanks to its rapid
growth, high biomass production, and high
stress tolerance. In fact, this species has exhibit-
ed strong transpiration rates and real tolerance
to high metal concentrations in both hydroponic
and field studies (Liu et al. 2007, Doumett et al.
2010). Therefore, the aim of the present study
was to determine for the first time the effects of
increasing Zinc concentrations on the Paulow-
nia tomentosa performance, grown in vitro con-
ditions and the Zn accumulation by plantlets.
41
Int. J. Med. Arom. Plants Effects of zinc in micropropagated plants of Paulownia tomentosa
Ben Bahri et al.
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The results will be important to indicate the po-
tential application of Paulownia tomentosa in
phytoremediation of contaminated sites.
Material and methods
Plants material
Nodal explants were collected from old
plants of Paulownia tomentosa. These explants
were grown and maintained in the greenhouse
of the Department of Agronomy and Vegetal
Biotechnology at the National Agronomic Insti-
tute of Tunisia.
Surface disinfection, sterilization and culture
media
Disinfection of nodal explants was carried
out in the laminar airflow chamber by using
ethanol (few seconds) and soaked in 0.1% (w/v)
HgCl2 for 5 min. Then, the shoot tips were
rinsed with sterile distilled water and surface
sterilized with 10% (w/v) sodium hypochlorite
for 5 min. Thus, sterilized nodal explants were
cultured on Murashige and Skoogs medium
(1962) supplemented with 1 mg l
-1
IBA and 3%
(w/v) sucrose. The medium was gelled with
0.6% (w/v) agar (Sigma) and the pH was ad-
justed to 5.8 with 0.1 N NaOH or HCl before
autoclaving at 120C for 20 min under a pres-
sure of 1.1 kg cm
-
.
Zn treatments and experimental design
The metal salt Zinc Sulphate 7-hydrate
(ZnSO
4
.7H
2
O) was added to the culture medium
at various concentrations: 200, 400, 600, 800
and 1000 M. The MS medium without adding
of Zn was served as control. The experimental
design was conducted as a factorial randomized
block with three replications.
Growth conditions
The cultures were incubated at 23 1C un-
der 16/8h (light/dark cycle) photoperiod and ir-
radiance (36 mol m
-2
s
-1
) provided by cool-
white fluorescent lamps.
Observations were made after four weeks of
culture and focused on plantlet heights, number
of nodes, root number and average length, bio-
mass accumulation and the content of dry matter
Zn.
Zinc analysis
Vitro plants were carefully washed with dis-
tilled water, oven dried at 65
o
C for about 48
hours to stop enzymatic reactions and to obtain
a constant weight, then ground to a fine powder
before analysis.
The zinc content was determined after di-
gesting the plant tissues in a mixture of concen-
trated HNO
3
and H
2
O
2
(2:1 v/v., Sigma-Aldrich,
Italy) using a digester (VELP Scientifica, Italy).
After digestion, the Zn content was measured by
an Inductively Coupled Plasma-optical emission
spectrometer (ICP-OES, OPTIMA 2000 DV,
PerkinElmer, USA). The ICP-OES analytical
standard (AA/ICP calibration/check standards
for environmental analysis, 1 g l
-1
) for Zn was
supplied by Sigma-Aldrich (Italy).
Statistical analysis
Fifteen explants were used per treatment in
triplicates. Data were subjected to statistical
analysis using the program package SAS (SAS
1999). The one-way analysis of variance
(ANOVA) followed by Duncans multiple range
test at the significance level of 5% was used to
compare means.
Results and Discussion
Zn effect on shoot height and nodes number of
plants
The in vitro shoot height of Paulownia
tomentosa in the culture media supplemented
with Zn at different concentrations showed a
significantly difference between the treatments
(Table 1).
The highest shoots were obtained on MS
culture medium free of Zn (control) and no sig-
nificant difference was observed in media with
200 M of Zn. However, the plant height was
significantly reduced by 18.86%, compared to
the control, at 1000 Mof Zn.
42
Int. J. Med. Arom. Plants Effects of zinc in micropropagated plants of Paulownia tomentosa
Ben Bahri et al.
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When the Zn concentration was up to 400
M, the number of nodes decreased slightly but
without a significant difference between treat-
ments. However, no difference was observed
between shoots grown on the control or on me-
dia supplemented with 200 and 400 M of Zn.
The number of nodes was significantly reduced
by 20.63% compared to the control at the high-
est level of Zn (1000 M) in the culture medium
(Table 1). Indeed, the presence of Zn in the cul-
ture media caused a decrease in the aerial parts
growth of plantlets. These findings corroborate
those of Gerard et al. (2000) who observed a
decrease of the biomass produced in cultures of
Lolium perenne in soils contaminated by Zn. In
the same context, Wang et al. (2010) showed
that the germination rate and shoot length of
Paulownia fortunei decreased when Zn concen-
trations are higher at mining sites rich in this
metal. On the other hand, Ruscinska et al.
(1999) showed that the presence of metals can
cause a decrease in biomass, an inhibition of
photosynthesis, respiration disturbances and de-
generation of main cell organelles. In Brassica
napus, Ben Ghnaya (2008) noted that the treat-
ment in the greenhouse at 2000 M Zn caused a
decrease reaching 75% in shoot and root bio-
mass which is depending on the genotype.
Table 1. Effects of ZnSO
4
on vegetative growth and rooting of in vitro plantlets Paulownia
tomentosa (Thunb) Steud.
Mean values within the column followed by the same letter in superscript are not significantly differ-
ent at P<0.5% (Duncan test).
Effect of Zinc on in vitro rooting
In vitro regenerated shoot from nodal ex-
plants were cultured on MS medium supple-
mented with 1 mg l
-1
IBA in combination with
different concentrations (0, 200, 400, 600, 800,
1000 M) of Zn (Figure 1).
A B C AA BB CC
Figure 1: In vitro plantlets Paulownia tomentosa (Thunb.) Steud. cultured in presence of ZnSO
4
. A:
Control; B: 800 M ZnSO4; and C: 1000 M ZnSO
4
.
The results showed that high levels of Zn in
MS medium didnt inhibited root formation.
However, number and root length average var-
ied significantly and is related to Zn concentra-
tion in MS medium (Table 1). The addition of
Zn to the culture medium caused a significant
reduction in the average number of roots from 7
per vitroplant in the control to a single root in
Zn (M) 0 200 400 600 800 1000
Shoots length (cm) 4.766 0.563
a
4.367 0.550
ab
4.133 0.550
bc
4.067 0.563
bc
4.033 0.611
bc
3.867 0.550
c
No. of nodes 5.333 0.724
a
5.267 0.704
a
5.067 0.594
a
4.533 0.743
b
4.467 0.640
b
4.200 0.676
b
Roots length (cm) 2.606 0.426
a
1.749 0.347
b
1.401 0.257
c
1.339 0.277
d
0.977 0.208
d
0.891 0.149
e
No. of roots 7.067 0.884
a
6.267 0.884
b
3.933 0.594
c
3.067 0.458
c
2.667 0.488
d
1.067 0.258
d
43
Int. J. Med. Arom. Plants Effects of zinc in micropropagated plants of Paulownia tomentosa
Ben Bahri et al.
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plants grown on a culture medium supplemented
with 1000 M of Zn.
Furthermore, root length average varied sig-
nificantly in all the treatments (Table 1). Indeed,
the length of roots was significantly reduced by
65.8% compared to the control at 1000 M of
Zn in the culture medium.
The effect of Zn on root quality was studied
in other plants. Schwartz et al. (1999) reported
that in Thlapsi caerulescens, short roots ar-
ranged in aggregates in presence of high con-
centrations of this metal in soil and in uncon-
taminated soil the roots of the same plant are
very thin and long.
In addition, Shi et al. (2011) showed that
root length, root surface and root volume of
Glochidion puberum grown in lead/zinc (Pb/Zn)
mine tailings areas were reduced by 70, 80, and
86%, respectively, compared to that grown in
uncontaminated soil.
Effect of Zinc on fresh and dry biomass
As shown in Figure 2, the addition of Zn to
the culture medium at different concentrations
affected severely the fresh and dry biomass of in
vitro plantlets and this effect is more pro-
nounced with increasing levels of Zn.
Indeed, the fresh biomass decreased from
9.62 g to 7.42 g in the control and with 1000
M Zn concentration, respectively. However,
the fresh biomass of the plantlets treated with
200 and 400 M of Zn is relatively stable (9 g)
(Figure 2).
0
1
2
3
4
5
6
7
8
9
10
B
i
o
m
a
s
s

(
g
)
0 200 400 600 800 1000
Zn concentration (M)
FW (g) DW (g)
Figure 2: Effects of ZnSO
4
on the biomass of in vitro plantlets Paulownia tomentosa (Thunb) Steud.
FW: Fresh weight; DW: Dry weight.
As well, fresh biomass of plantlets treated
with 600, 800 and 1000 M Zn is near to 7.5 g.
A regression by 22.8% and 50.5% on the fresh
and dry biomass under 1000 M Zn concentra-
tion compared to the control respectively was
recorded. These results agree with those found
by Ben Ghnaya (2008) which observed a signif-
icant decrease in dry matter weight and fresh
tissue culture in all the variety of Brassica
napus L. Drakkar by adding 500 and 1000 M
of Zn to the culture medium. However, Mench
et al. (1981) showed that the vegetative growth
of maize seedlings is related with the Zn con-
centration in the solution. On the other hand, the
addition of different concentrations of Zn to a
culture of Solanum nigrum seeds (0, 100, 500
and 1000 mg Zn/kg of sand) affects the growth
of seedlings. Plants grown at 100 mg kg
-1
showed significantly lower leaf, stem, root and
total biomass than control ones and plants grow-
ing at 500 and 1000 mg kg
-1
presented signifi-
cantly the lowest biomass (Marques et al. 2006).
In fact, the abiotic stress caused by heavy
metals, modifies the structure of essential en-
zyme by altering the protein structure and caus-
ing deficiency symptoms in plants. The plasma
44
Int. J. Med. Arom. Plants Effects of zinc in micropropagated plants of Paulownia tomentosa
Ben Bahri et al.
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membrane is particularly sensitive to heavy
metal toxicity since membrane permeability and
thus, its functionality can be affected by altering
the intrinsic membrane protein such that H
+
-
ATPases (Hall 2002). This stress results in a
production of reactive oxygen species (ROS).
Overproduction of these ROS leads to oxidative
damage in plant tissues due to lipid peroxidation
leading to the formation of degradative products
such as alkanes and aldehydes (Ferrat et al.
2003). Therefore, toxic symptoms such as
chlorosis, growth retardation, browning of roots
and a cell cycle arrest can be observed. To fight
this oxidative stress, plants produce defense sys-
tems enzymatic and non enzymatic antioxidants
in order to regulate the concentration of ROS
and thus maintain ion homeostasis within cells
(Clemens 2001, Hall 2002).
Zn levels in vitro plants
The Zn content in the vitro plantlets was
measured at the end of the experiment. As
shown in Fig.3, the Zn levels recorded in vitro
plantlets vary from 121 mg g
-1
in the control to
2580 mg/g in vitro plants grown in the medium
containing the highest concentration of Zn
(1000 M).
0
500
1000
1500
2000
2500
3000
Z
n

l
e
v
e
l
s

(

g
/
g

D
W
)
0 200 400 600 800 1000
Zn concentration (M)
Figure 3. Zn levels (g g
-1
DW) on tissues of vitro plantlets Paulownia tomentosa (Thunb) Steud.
Indeed, Zn was mostly stored in the tissues
plantlets of Paulownia, where the accumulation
was almost linearly related with the Zn concen-
tration in the MS medium. Similar results were
reported by Azzarello et al. (2012) whose de-
tected a linear increase of Zn accumulation in
Paulownia tomentosa plants with the 1000 M
of Zn. At highest zinc concentrations (2000,
3000 and 5000 M), a linear decrease of Zn ac-
cumulation was detected and was clearly linked
to the reduced biomass production.
On the other hand, Marques et al. (2006)
showed that Solanum nigrum accumulated up to
1450, 3240 and 3810 mg Zn kg
-1
in the leaves,
stems and roots, respectively but without any
visual toxicity signs. This confirmed the idea
that Paulownia tomentosa is Zn tolerant and
hyperaccumulator plant.
Conclusion
In order to evaluate the tolerance and the
ability to Zn accumulation in Paulownia
tomentosa grown under in vitro conditions, dif-
ferent morphological parameters as well as
analysis of the Zn content in tissues of plantlets
were determined. Results showed that 1000 M
Zn concentration caused a reduction of about
18.86% and 50.5%, respectively on shoot height
and dry biomass compared to the control. This
decline was more pronounced in the root sys-
tem. However, a significant reduction in the
number of roots was noticed and estimated by
84.9% with 1000 M Zn concentration com-
pared to the control. The high levels of Zn in the
culture medium limited the root formation.
However, the determination of Zn levels
showed good accumulation in the tissues of
plantlets with ranging from 121 g g
-1
DW to
2580 g g
-1
DW. Overall, results showed that
45
Int. J. Med. Arom. Plants Effects of zinc in micropropagated plants of Paulownia tomentosa
Ben Bahri et al.
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Paulownia tomentosa could be considered as a
bio-indicator and Zn accumulator medicinal
plant since it can tolerate and accumulate high
Zn content.
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