O A

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Advances in Environmental Biology, 7(9): 2235-2240, 2013
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Study of effects of electromagnetic fields on seeds germination, seedlings ontogeny,
changes in protein content and catalase enzyme in Valeriana officinalis L.
1
Sara Farzpourmachiani, 2Ahmad Majd, 2Sedigheh Arbabian, 3Davoud Dorranian, 4Mehrdad
Hashemi
1
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Department of Biology, Faculty of Biological Sciences, North-Tehran Branch, Islamic Azad University,
Tehran, Iran.
3
Plasma Physics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran.
4
Department of genetics, Tehran medical Branch, Islamic Azad University, Tehran, Iran.
2
Sara Farzpourmachiani, Ahmad Majd, Sedigheh Arbabian, Davoud Dorranian, Mehrdad Hashemi;
Study of effects of electromagnetic fields on seeds germination, seedlings ontogeny, changes in protein
content and catalase enzyme in Valeriana officinalis L.
ABSTRACT
In this research, the effects of electromagnetic field strengths (1 and 2mT) have been investigated on seeds
germination, seedlings ontogeny, protein content and catalase activity in Valerian seeds (Valeriana officinalis
L.). The dry and wet (soaked for 30min) Valerian seeds were transferred to MS tissue culture and then were
exposed to electromagnetic fields in the coils of electromagnetic fields generator for 30 min per day for 3 days.
For each treatment groups and corresponding control, three replicates with 10 seeds were used. Results showed
that electromagnetic fields treatment increased the speed and percentage of seeds germination, seedlings growth
and also increased fresh and dry weight compare to control for most treatment groups. The electromagnetic
fields changed protein contents significantly and catalase activity especially in the groups of dry treated seeds.
These results show that the electromagnetic fields probably degenerate proteins in the early stages of seedlings
ontogeny and the treated valerian seedlings can increase catalase activity by decreasing free radicals against
electromagnetic fields tension.
Key words: catalase, electromagnetic fields, germination, ontogeny, protein, Valeriana officinalis L.
Introduction
Electromagnetic fields (EMFs) affect biological
systems as the kind of abiotic stress. The
development of modern technologies has applied
EMFs widely because of their positive or negative
effects on living systems including plants [16,22].
EMFs increase percentage of germination in
Cucumis sativus L. seeds [27] however they decrease
speed of germination in Vicia sativa L. [16],
stimulate plantlets development and increase fresh
weight, growth [1], and chlorophyll a, b and total
chlorophyll amount [3], change antioxidant enzyme
such as superoxide dismutase, ascorbate peroxidase,
guaiacol peroxidase, catalase and glutathione
reductase []13,24] and anti tensional proteins activity
[23]. Electromagnetic fields elongate G1 in the
various plants species and G1 and G2 in lentil and
flax, inhibit primary root growth in the early stages
of germination and decrease proliferation of root
meristem cells compare to control [8]. Weak
magnetic fields decrease genome activity before
proliferation [6], change plant ontogeny [12],
configuration of epicotyl of Triticum aestivum L. and
Pisum sativum L. and mutation in Arabidopsis
thaliana L. and Crepis capillaries L. [21]. EMFs
decrease the number of the grains in the spike and
the spike weight [11], chromosomal aberrations
including
fragments,
bridge
and
lagging
chromosomes in the pollen mother cells of wheat
[28]. High electromagnetic field causes increasing
the amount of plastoglobules and changes the
structure and export products of Golgi apparatus [23]
and configuration and size of hypodermal cells [4].
EMFs affect rhizogenesis through impairment of
biochemical process [24], delay the senescence
process [18], change protein synthesis such as
Calmodulin-N6, cmbp and pin [22], increase the
mitotic index and mitotic abnormalities in root
meristematic cells [22], enhance lipid peroxidation
and hydrogen peroxide content [25], enhance
seedlings survival and increase crop capacity [4], and
Corresponding Author
Sara Farzpourmachiani, Department of Biology, Science and Research Branch, Islamic Azad
University, Tehran, Iran.
Tel.: +9888893199; fax: +9888893199.
E-mail: [email protected]
2236
Adv. Environ. Biol., 7(9): 2235-2240, 2013
heat-shock response [19] and maintain cell
membrane integrity [10].
Valeriana officinalis L. is a highly variable
species of the Valerian family (Valerianaceae) that
has a lot of medicinal properties. The roots and
rhizomes of Valerian extract are traditionally used to
treat sedative, anxiety, epilepsy, insomnia, hysteria,
fatigue and menstrual cramps [5]. In this research,
we have investigated the effects of electromagnetic
fields on seeds germination, seedlings ontogeny,
protein content changes and catalase activity in V.
officinalis L. Considering the fact that this plant is
very useful in medicinal purposes and Valerian seeds
have low germination ability, the goal of this study
was to determine that how electromagnetic fields
with low intensities affect the germination and
growth of Valerian and determine a relationship
between low frequency electromagnetic fields and
catalase activity.
percentage of germination of Valerian seeds were
determined from 3th to 11th day after treatment. Root
length, number of lateral roots, petiole length, fresh
and dry weight and leaf area were considered on 40th
day. After 2 months, seedlings were transferred to
pots containing peat and perlite and kept in green
house under a photoperiod of 16h day/ 8h dark at 23⁰
C and 4000 lux. After a month, aerial parts was
harvested for protein content and catalase activity
assays.
Material and Methods
Protein content assay:
Seeds preparation and treatment:
The protein content of the supernatant was
determined by Bradford's method with bovine serum
albumin (BSA) as reference standard. 90 µl
phosphate buffer and 5 ml Bradford's reagent was
added to 10 µl protein extraction and after 5 min, the
absorbance was measured at 595 nm. The protein
concentration was expressed as mg ml-1.
V. officicnalis L. seeds var. Hungary were
supplied by Pakan bazr institute, which guarantees
high seeds quality and homogeneity. The healthy
uniform seeds were selected and divided into wet and
dry seeds groups. In the case of wet treatment, the
seeds were soaked in distilled water for 30 minutes.
Then wet and dry seeds were separately sterilized.
They were washed with dish washing liquid for 5 to
6 min, then were transferred to laminar air flow, they
were sterilized in benomyl solution (0/2 g in 50 ml)
for 10 min and merck ethanol for 30 s. After each
level, seeds were rinsed with distilled water and
placed on solid MS (Murashig and Skoog, 1962)
basal medium containing 3 % sucrose and 0/7 %
agar. Petri dishes containing wet and dry seeds were
separately divided into three groups: control, 1mT
and 2mT treatments. Three replicates were used in
the experiment with 12 seeds in each one. Treatment
groups were separately exposed to 1mT and
2mTduring 3 days for 30 min in each day. Control
seeds were kept under similar condition but without
any EMFs intensity. To generate EMFs, a handmade
cylindrical-shaped coil was used that had made of a
polyethylene tube with 12 cm diameter and 50cm
length. The coil was connected to a 220V AC power
supply (ED-345BM, China), teslameter (516 62,
LEYBOLD, Germany) and ampermeter to generate
electrical current of 60 HZ and showing
electromagnetic field strengths and current
intensities, respectively. EMF intensities were
measured by a B-probe type of Hall Sound. Petri
dishes containing seeds were placed in the middle
part of coil and then treated by 1 and 2 mT
electromagnetic field strengths. Then they were
maintained in incubator under a photoperiod of 16h
day/ 8h dark at 23⁰ C and 4000 lux. Speed and
Preparing of protein extraction:
Fresh leaf of each treated and control samples
(0.15 mg) was ground in 5 ml of 100 M phosphate
buffer (pH 7) under ice-cold condition and then was
centrifuged at 12000 rpm at 4⁰C for 45 min,
separately. The supernatant was used for assaying of
protein content and catalase activity.
Catalase assay (CAT, EC 1.11.1.6):
Reaction mixture (3 ml) contained 50mM
potassium phosphate buffer (pH 7), 20 mM
Hydrogen peroxide and 100 µl enzyme extraction.
CAT activity was determined as the rate of
disappearance of H2O2 at 240 nm, according to
Pereira (2002). The activity of catalase was
expressed as µmol-1min-1mg protein.
Statistical analysis:
The experimental design is completely
randomized. All of the experiments were carried out
three replicates and all of the data were expressed as
the mean ± SE. Means were compared using the post
hoc Tukey’s test at P<0/05, level of significance to
detect differences between treated and control
seedlings by SPSS 16 software.
Results:
Seeds germination:
Electromagnetic fields increased the speed and
percentage of seeds germination in Valerian
especially in the higher electromagnetic field
intensities. The percentage of wet treated seeds
germination increased compare to dry condition in 3,
5 and 7 day. There was a significant difference
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Adv. Environ. Biol., 7(9): 2235-2240, 2013
between wet control and treated seeds with 2mT at
P<0.05. Percentage of seeds germination increased in
dry treated seeds with 2mT compare to wet ones but
there was no significant difference. The speed of
germination of dry treated seeds was more than
control but it was completely converse in the wet
condition (Table 1).
Table 1: The effect of low frequency electromagnetic fields on seeds germination in V.officinalis L.
condition
EMF intensity (mT)
3th day
5th day
7th day
9th day
wet
0
4.32±0.96b
17.59±4.03 b
21.28±3.7 ab
23.14±4.03 a
1
3.7±0.63 b
19.44±2.78 b
24.2±2.64 b
24.99±4.81 a
2
0±0 a
16.98±1.65 ab
16.98±1.65 ab
34.12±4.54 a
dry
0
1.25±0.33 a
5.55±0 a
5.55±1.32 a
8.33±0 a
1
0.92±0.03 a
11.11±1.5 ab
16.66±4.16 ab
25.75±5.81 a
2
0.81±0.36 a
19.44±2.78 b
27.77±5.55 b
33.33±9.62 a
Data are the means ± SE (n=10). Different letters in the column indicate significant difference at P<0.05 level applying post hoc Tukey's
test.
Petiole length and leaf area:
Electromagnetic fields increased the petiole
length and the leaf area of Valerian seedlings. The
dry treated seeds with 2mT had longer petiole length
compare to control and the group of wet treated
samples but the wet treated seeds with 1mT had
longer petiole length compare to the other wet ones.
There was a significant difference between them, the
other treatments and controls. The leaf area of dry
treated seeds with 2mT significantly increased
compare to wet ones, control and the other treated
samples. There was an increase in leaf area of wet
seeds but it was not significant (Table 2).
Root length and number of lateral roots:
The results showed an increase in the root length
for most of electromagnetic fields treatment. Wet
treated seeds had the longest roots compare to dry
ones. There was a significant difference between
them, control and dry samples. The dry treated seeds
with 1mT had the shortest roots compare to the other
groups. There was a significant difference between
this one and the group of dry treated seeds with 2mT.
Electromagnetic fields increased the number of
lateral roots in Valerian. The dry treated seeds with
2mT had more number of lateral roots compare to the
others. There was a significant difference between
them and dry and wet treated seeds with 2mT (Table
2).
Fresh and dry weight:
Electromagnetic fields increased dry and wet
weight of Valerian seedlings. The dry treated seeds
with 1mT had the freshest weight. The wet control
and the wet treated seeds with 1mT had the shortest
roots compare to the others. There was a significant
difference between this one, wet treated seeds with
1mT and wet control. The highest and the lowest of
dry weight were observed in the dry treated seeds
with 1mT and the wet treated seeds with 2mT,
respectively. There was not any significant difference
between them, controls and the other treatment
groups (Table 2).
Table 2: The effect of low frequency electromagnetic fields on seedlings growth, fresh and dry weight in Valeriana officinalis L.
Condition
EMF
Petiole length
Leaf
Root length
Number of
Fresh weight
Dry weight
intensity
(cm)
area(cm2)
(cm)
lateral roots
(gr)
(gr)
(mT)
Wet
0
0.29±0.1
40.14±12.21**
0.81±0.09
0.53±0.12**
0.008±0.003* 0.0007±0.0002
1
0.53±0.04
54.67±21.05**
0.99±0.08*
0.53±0.13
0.01±0*
0.0023±0.0003
2
0.36±0.06
53.86±7.38**
0.98±0.11*
0.69±0.16*
0.019±0.002
0.0015±0.0008
Dry
0
0.14±0.02
59.88±8.14**
0.39±0.09*
0.05±0.006***
0.034±0.013
0.0023±0.0003
1
0.46±0.11
52.8±10.29**
0.3±0.16*
0.88±0.01
0.05±0.01*
0.0056±0.0016
2
0.86±0.1
179.09±25.5**
0.74±0.17
1.88±0.11***
0.031±0.004
0.0046±0.0012
Means ± Standard Error.
*Significant differences: P˂0.05
**Significant differences: P˂0.01
***Significant differences: P˂0.001
Protein content:
Catalase activity:
Electromagnetic fields decreased protein content
in Valerian. Wet samples and treated seeds with 2mT
had more protein content than dry samples and
treated seeds with 1mT. Dry control and wet treated
seeds with 1mT had the most and the least protein
content, respectively (Table 3).
Electromagnetic fields increased catalase
activity in Valerian. Dry treated seeds had the most
catalase activity compare to the other groups and
control. Dry treated seeds with 1mT and wet treated
seeds with 1mT had the most and the least catalase
activity compare to the others (Table 3).
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Adv. Environ. Biol., 7(9): 2235-2240, 2013
Table 3: The effect of electromagnetic fields on protein content and catalase activity in Valeriana officinalis L.
Protein conetent
Catalase activity
(mgml-1)
(µmomin1mg1protein)
1.32±0.014*
0.016±0.006*
0.99±0.04*
0.02±0.008*
1.05±0.024*
0.2±0.011*
1.35±0.008*
0.3±0.05*
0.82±0.026*
0.35±0.07*
0.94±0.02*
0.22±0.08*
Data are presented as the means ± SE with n=10. Bars with different letters are significantly different at P ≤ 0.05, according to post hoc
Tukey's test.
Discussion:
Effect of electromagnetic
germination:
fields
on
seeds
According to the results, electromagnetic fields
enhanced percentage of seeds germination. The
treated Valerian seeds with 2mT increased
germination compare to controls and 1mT. Wet
condition caused to increase germination percentage
compare to dry one, which confirmed the conclusion
of other studies in which the germination of Arachis
hypogaea L. and tomato seeds Lycopersicum
esculentum were increased [2,17]. Florez et al. [7]
reported the positive effects of magnetic field
treatments on germination rate and growth. Garcia
and Arza [9] found that stationary magnetic fields
cause an increase in water absorption in lettuce
seeds. The electromagnetic fields probably affect
germination genes in Valerian and accelerate the
speed and percentage of germination. Expression of
germination genes was enhanced by increasing the
electromagnetic field intensity. Wet condition
increases water absorption by cellulose, pectin and
starches of the seeds. Water absorption increases
gibberellins activity and then induces hydrolytic
enzymes. Electromagnetic fields probably accelerate
this process especially in the group of treated seeds
with 2mT field intensities. In this case, the genes of
cellulose, protease and pectinase synthesis can be
active and so increase the germination percentage
[15].
Effect of
ontogeny:
electromagnetic fields
on
seedlings
The results showed that the effect of
electromagnetic fields on Valerian growth in terms of
root, petiole and number of lateral roots was more
than control. Wet condition increased growth and
development of roots however dry condition
increased growth of leaf and petiole. Vashisth &
Nagarajan [26] observed an increase in the total
length of Helianthus annuus L. by magnetic field.
Electromagnetic fields probably affect the plant
growth regulators like auxin and cytokinin and can
be effective on the plant growth and development.
Electromagnetic fields probably increase auxin rate
and are effective on genes activity which produce
growth proteins in nucleus and so increase the
protein production and lead to growth. Also they can
increase the ATPase pumps activity in cell wall and
peroxides so they can extend the cell wall integrity
and lead to cells growth. They can be effective on
genes regulators like cytokinin and increase mitosis
divisions in shoot and root meristems [15].
Effect of electromagnetic fields on dry and fresh
weight:
Treated seeds had more fresh and dry weight
compare to control. Electromagnetic fields probably
increase mineral elements absorption, water
absorption and enzymes activity so they lead to
increase plants biomass. On the other hand, they
probably affect mRNA, gene expression and cell
division and lead to increase growth, fresh and dry
weight. Florez et al. [7] indicated that
electromagnetic fields increased enzymes activity
and protein contents and led to enhance biomass of
plants. Yinan et al. [27] demonstrated that biomass
of the Cucumis sativus L. increased by
electromagnetic fields. Radhakrishnan & Kumari
[20] reported an increase in the fresh and dry weight
and mineral accumulation by pulsed magnetic field.
Effect of electromagnetic fields on protein content
and catalase activity:
Our results showed that electromagnetic fields
decreased protein content and increased catalase
activity especially in dry condition. In the early
stages of seedlings ontogeny because of higher levels
of growth and germination genes activity,
electromagnetic fields have more effects on the genes
and cause to stop or decrease their activity in the
other stages of Valerian ontogeny and lead to
decrease protein content in Valerian seedlings. These
results agree with the conclusion of other studies.
Radhakrishnan & Kumari [20] reported that pulsed
magnetic field increased protein content and catalase
activity. Electromagnetic fields tension especially in
dry condition increased catalase activity. Plants begin
to enhance oxidative enzymes activity like catalase
against electromagnetic fields tension and free
radicales which has protective role against
electromagnetic fields and detoxification of H2O2.
These results agree with Kursevich and Travkin [14]
and Atak et al. [3] that found magnetic field
treatment increase catalase and peroxidase activity.
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Adv. Environ. Biol., 7(9): 2235-2240, 2013
Higher levels of catalase, peroxidase, and superoxide
dismutase and glutathione reductase in MF-treated
seedlings cause to delay senescence. Catalase stops
H2O2 accumulation in cells and protects plants
against ROS [18].
12.
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