Appl Biochem Biotechnol (2012) 167:2225–2233

DOI 10.1007/s12010-012-9759-8

Silver Nanoparticle-Mediated Enhancement in Growth

and Antioxidant Status of Brassica juncea

Priyadarshini Sharma & Deepesh Bhatt &

M. G. H. Zaidi & P. Pardha Saradhi & P. K. Khanna &
Sandeep Arora

Received: 20 November 2011 / Accepted: 29 May 2012 /

Published online: 13 June 2012
# Springer Science+Business Media, LLC 2012

Abstract Metal nanoparticles can potentially be used as tools for engineering biological
redox reactions. Present study underlines the effect of silver metal nanoparticles (at 0, 25, 50,
100, 200 and 400 ppm) on the growth and antioxidant status of 7-day-old Brassica juncea
seedlings. Fresh weight, root and shoot length, and vigor index of seedlings is positively
affected by silver nanoparticle treatment. It induced a 326 % increase in root length and
133 % increase in vigor index of the treated seedlings. Improved photosynthetic quantum
efficiency and higher chlorophyll contents were recorded in leaves of treated seedlings, as
compared to the control seedlings. Levels of malondialdehyde and hydrogen peroxide
decreased in the treated seedlings. Nanoparticle treatment induced the activities of specific
antioxidant enzymes, resulting in reduced reactive oxygen species levels. Decrease in
proline content confirmed the improvement in antioxidant status of the treated seedlings.
The observed stimulatory affects of silver nanoparticles are found to be dose dependent, with
50 ppm treatment being optimum for eliciting growth response. Present findings, for the first
time indicate that silver nanoparticles promote the growth of B. juncea seedlings by
modulating their antioxidant status.

Keywords Silver nanoparticles . Growth . Antioxidant status . Redox potential . Quantum

efficiency . Reactive oxygen species

P. Sharma : D. Bhatt : S. Arora (*)

Department of Molecular Biology & Genetic Engineering,
G B Pant University of Agriculture & Technology, Pantnagar, Uttarakhand, India
e-mail: plantstress@gmail.com

M. G. H. Zaidi
Department of Chemistry, G B Pant University of Agriculture & Technology, Pantnagar, Uttarakhand,
India

P. P. Saradhi
Department of Environmental Studies, University of Delhi, Delhi, India

P. K. Khanna
Defense Institute of Advance Technology, Pune, India
2226 Appl Biochem Biotechnol (2012) 167:2225–2233

Introduction

Plant growth and development are controlled by internal regulators that respond to environ-
mental conditions. In nature, plants seldom find optimal quantities of essential factors
required for maximal growth and productivity. Therefore, most often, the ‘physiologically
normal type of plant’ is an exception in nature. Under sub-optimal environmental conditions,
plants show perturbations in their biochemical and physiological processes. Redox reactions,
involving electron exchange, are one of the most vulnerable physico-chemical processes
affected by fluctuations in the external environment. Therefore, even under seemingly
favorable environmental conditions, a plant continuously produces reactive oxygen species
(ROS). Accumulation of these ROS is potentially harmful for the growth and development
of plants. Thus, occurrence of oxidative stress, through the generation of free radicals, is an
inevitable by-product of normal plant metabolism [1].
Generally, these ROS are continuously reduced and detoxified by an extensive antioxidant
system. However, the detoxification process consumes essential cellular resources in terms of
energy, carbon skeleton and loss of nitrogen as NH3. Therefore, any treatment that can help
reduce the ROS load would prove beneficial in improving the overall growth and productivity.
Wang et al. [2] have shown that plants produce natural mineralized nanoparticles, which are
required for growth. Specific reports are now available on the bio-synthesis of nanoparticles by
plants [3]. However, ectopic use of engineered nanoparticles is one of the most recent advances in
the field of agricultural biotechnology. Studies indicate that treatment of plants with a mixture of
nano SiO2 and TiO2 can enhance activities of specific enzymes and could be used for improving
seed germination and seedling growth [4]. Tang and Cao [5] have suggested that nano-SiO2
treatment could be related to increased strength, resistance to disease and thus, increased yields in
rice. Zheng et al. [6] have proposed a protective role for nano-anatase in spinach exposed to UV
light stress. However, the mode of action for these nanoparticles has not yet been established.
Metal nanoparticles, by virtue of having extremely large surface area to volume ratio and
an ability to engineer electron exchange, can develop favorable interactions with various
bio-molecules in a cell. Silver nanoparticles are among the most potential candidates for
modulating the redox status of plants, because of their ability to support electron exchange
with Fe2+ and Co3+ [7], the two elements that participate in several biological redox
reactions. Silver nanoparticle clusters show efficient catalytic activity in redox reactions
by acting as electron relay centers, behaving alternatively as an acceptor and donor of
electrons [8]. An effective transfer of electrons is facilitated when the redox potential of
the cluster is intermediate between the electron donor and electron acceptor system. In spite
of the electron channelizing ability of silver nanoparticles, response of biological systems
exposed to silver nanoparticles has been studied mainly with regard to toxicity, and little
attention has been paid to the possibility that silver nanoparticles can possibly help improve
plant’s redox status and growth. Therefore, the present work attempts to elucidate the role of
silver nanoparticles in plant growth promotion under specified conditions.

Material and Methods

Preparation of Silver Nanoparticles

Silver nanoparticles were synthesized through chemical reduction of silver nitrate by tri-
sodium citrate salt, as described by Sileikaite et al. [9]. The synthesized silver nanoparticles
were characterized by transmission electron microscopy and UV–vis spectroscopy.
Appl Biochem Biotechnol (2012) 167:2225–2233 2227

Plant Material and Treatment

Seeds of Brassica juncea (var. pusa jaikisan), obtained from Indian Agriculture Research Institute,
New Delhi (India), were germinated on MS medium [10] supplemented with silver nanoparticles at
0, 25, 50, 100, 200 or 400 ppm concentration. The seedlings were grown at 26±1 °C temperature
and relative humidity of 70 %. Illumination was provided by four 40-W florescent tubes having a
photon flux density of approximately 16 Wm−2 with a 16/8 h day/night cycle. Seven-day-old
seedlings were used for the experiments.

Growth Profile Measurement

Percent seed germination was calculated by dividing the number of seeds germinated by the
total number of seeds inoculated. Shoot fresh weight was recorded by weighing individual
shoots. Shoot and root length of 7-day-old seedlings was recorded with the help of a
standard meter-scale. Vigor index was calculated as:
Vigor index ¼ ðroot length þ shoot lengthÞ  %germination
Chlorophyll contents were determined by the modified Arnon [11] method. Leaf disks of
7 mm diameter were soaked in acetone and DMSO (1:1v/v) solution for 12 h. The Chla,
Chlb and total chlorophyll concentrations (milligram per gram fw) in the leaf tissues were
calculated according to the following equations:
Chla ¼ ½ð12:7  A663 Þ  ð2:63  A645 Þ=ðwt:ing  1;000Þ

Chlb ¼ ½ð22:9  A645 Þ  ð4:48  A663 Þ=ðwt:ing  1;000Þ

 
Totalchlorophyll ¼ ð20:2  A645 Þþ ð8:02  A663 Þ =ðwt:ing  1; 000Þ

Chlorophyll fluorescence in untreated and silver nanoparticle treated leaves was mea-
sured at room temperature (26 °C) and ambient CO2 concentration. Measurements were
done using an actinic light of 3,000 μmol photons m−2 s−1, 4-s flashes, after dark adapting
the leaves for 20 min. The fluorescence parameters evaluated were: F0, initial fluorescence;
Fm, maximal fluorescence; and the Fv/Fm ratio, representing the maximum quantum effi-
ciency of PS-II, where Fv is the variable fluorescence Fm–F0.

Malondialdehyde Content

The procedure of Heath and Packer [12] was followed for measuring malondialdehyde (MDA)
content. Leaf material was homogenized and extracted in 10 ml of 0.25 % TBA (w/v) prepared
in 10 % trichloroacetic acid (TCA). The homogenate was heated at 95 °C for 30 min and
centrifuged at 10,000×g for 30 min. Absorbance of the supernatant was recorded at 532 and
600 nm. Absorbance at 600 nm was subtracted from the absorbance at 532 nm for non-specific
absorbance. The concentration of MDA was calculated by using an extinction coefficient of
155 mM−1 cm−1.

Hydrogen Peroxide Content

Hydrogen peroxide was measured as per the protocol given by Alexieve et al. [13]. Leaf
material was homogenized in 10 ml of 0.1 % (w/v) aqueous TCA and centrifuged at
2228 Appl Biochem Biotechnol (2012) 167:2225–2233

10,000×g for 30 min at 4 °C. 1 ml of supernatant, 1 ml of 0.1 M potassium phosphate buffer

and 4 ml of 1 M KI reagent were mixed. The reaction was allowed to develop for 1 h in dark
and the absorbance was measured at 390 nm. The amount of hydrogen peroxide (H2O2) was
calculated using standard curve of H2O2.

Free Proline Content

Free proline was determined by the method of Bates and Waldren [14]. Leaf tissue extract
homogenized in 3 % sulfosalicylic acid was mixed with equal quantities of glacial acetic
acid and acid ninhydrin reagent and incubated for 1 h at 100 °C. The reaction was terminated
in an ice bath, followed by extraction of the colored chromophore in toluene. The absor-
bance of the chromophore was measured at 520 nm. Concentration of proline in the samples
was computed from a standard curve of L-proline.

Antioxidant Enzyme Assay

Ascorbate Peroxidase (EC 1.11.1.11)

Ascorbate peroxidase activity was determined as described by Joshi et al. [15] with minor
modifications. Fresh leaf material was homogenized in 100 mM phosphate buffer (pH 7.0)
and 0.1 mM EDTA and centrifuged at 12,000×g for 30 min at 4 °C. For enzyme assay, 60 μl
of supernatant was mixed with 1,438 μl of assay buffer [50 mM phosphate buffer (pH 6.0),
0.1 μM EDTA, 0.5 mM ascorbate] and 2 μl of 0.5 M H2O2 was added to start the reaction.
The decrease in absorbance was recorded and enzyme activity was calculated by using
extinction coefficient 2.8 mM−1 cm−1 at 290 nm. Specific enzyme activity was expressed as
enzyme units per milligram of protein.

Guaiacol Peroxidase (EC 1.11.1.7)

Guaiacol peroxidase (GPX) activity was determined as described by Joshi et al. [15]. Leaf
material was homogenized in 3.0 ml of 100 mM phosphate buffer (pH 7) containing 0.1 mM
EDTA and centrifuged at 12,000×g for 30 min at 4 °C. Two millilitre reaction mixture was
prepared by adding 60 μl of enzyme extract to 1,790 μl of assay buffer [100 mM phosphate
buffer (pH 7), 0.1 μM EDTA, 5.0 mM Guaiacol, 15.0 mM H2O2]; guaiacol was added in the
last to start the reaction. Increase in absorbance was recorded at 470 nm and enzyme activity
was quantified using a molar extinction coefficient of 26.6 mM−1 cm−1. The enzyme specific
activity was expressed as enzyme unit’s per milligram protein.

Catalase (EC 1.11.1.6)

Catalase activity was measured according to Joshi et al. [15] with minor modifica-
tions. Leaf material was homogenized in 100 mM phosphate buffer (pH 7) containing
0.1 mM EDTA and centrifuged at 12,000×g for 30 min at 4 °C. 70 μl of enzyme
extract was added to 1,370 μl of assay buffer (100 mM phosphate buffer pH 7.0,
0.1 μM EDTA) and 60 μl of 0.5 M H2O2 was added to start the reaction. Decrease in
absorbance was recorded at 240 nm and enzyme activity was calculated by using the
H2O2 molar extinction coefficient of 36 mM−1 cm−1 and the enzyme specific activity
was expressed as enzyme units per milligram protein.
Appl Biochem Biotechnol (2012) 167:2225–2233 2229

Statistical Analysis

All experiments were carried out three times, with two replicates each. One-way analysis of
variance was carried out to determine significant differences (P≤0.05) between the means.
The experimental data are expressed as mean±SE.

Results and Discussion

Chemically synthesized silver nanoparticles were characterized by UV–visible spectrophotom-

etry. Formation of silver nanoparticles was ascertained by profiling the absorption spectra of the
synthesized particles from 190 to 1,100 nm. The absorption maximum (λmax) was recorded at
425 nm (Fig. 1a). The average size of synthesized nanoparticles, as deduced by transmission
electron microscopy is 29 nm (Fig. 1b and c). Further the uptake of silver nanoparticles from the
media was also confirmed through transmission electron microscopy of tissues from treated
seedlings (Fig. 1d). The uptake of silver nanoparticles by B. juncea has also been shown earlier
[16]; however, these reports did not explore the biological effects of such an uptake.

Growth Profile

A significant enhancement in growth of B. juncea seedlings was recorded at 25 and 50 ppm

silver nanoparticle treatment. The growth profile was measured in terms of shoot fresh

Fig. 1 Characterization of Silver Nanoparticles: a UV–VIS spectrum in aqueous phase, b morphology, c

particle size distribution, d presence of silver nanoparticles in B. juncea
2230 Appl Biochem Biotechnol (2012) 167:2225–2233

weight, shoot and root length, and vigor index. Above 50 ppm the silver nanoparticle
treatment proved detrimental for the growth of seedlings. A maximum of 22 % decline in
percent germination was recorded at 400 ppm silver nanoparticle concentration (Fig. 2a).
Stampoulis and Sinha [17] have also reported a decrease in seed germination at higher levels
of nanoparticle exposure. Silver nanoparticles interfere with the activities of certain hydro-
lytic enzymes, required during germination process [18], resulting in a reduction of per cent
seed germination [17]. Lin and Xing [19] have also highlighted that seed germination is
inhibited at higher concentrations of nanoparticles. A 17 % increase in shoot fresh weight
was recorded at 50 ppm silver nanoparticle treatment. Shoot and root length increased up to
200 ppm of silver nanoparticle concentration in the growth medium. Sharp increases of 15
and 25 % in shoot length and 167 and 277 % in root length were observed at 25 and 50 ppm,
respectively (Fig. 2b). These increases could be mediated via plant growth regulators like
cytokinins and gibberellins, which are involved in cell division and cell elongation, respec-
tively [17]. Roots are the first target tissues which come in direct contact with silver
nanoparticles in the growth medium by penetrating through seed coat and are responsible
for the continuous uptake of mineral nutrients from the medium. Therefore, the effect of
silver nanoparticle treatment is more prominent in roots as compared to shoots. A decrease
in shoot and root length of seedlings treated with higher silver nanoparticle concentrations,
indicates that the recorded phenomenon is dose dependent. Similar observations have also
been reported for TiO2 nanoparticle-treated spinach seedlings [6]. Vigor index represents the
combined effect of various external and internal growth regulating factors. An increase in
vigor index of the treated seedlings was recorded up to 50 ppm silver nanoparticle treatment.

Fig. 2 Response of B. juncea seedlings grown in media supplemented without and with different concentrations
of silver nanoparticles: a percent germination and vigor index; b root and shoot length and shoot fresh weight; c
Chl-a, Chl-b, total chl and Fv/Fm; d malondialdehyde, H2O2 and proline content. Values represent mean±SE
Appl Biochem Biotechnol (2012) 167:2225–2233 2231

Thereafter, the decline in percent germination offset the effect of increased shoot and root
length on vigor index. Results indicate that the percent seed germination up to 50 ppm is
similar to the control values, and after that it declines consistently. An increase in vigor index
of the treated seedlings rules out the restricted availability of water as a possible cause for
decrease in germination. At all the tested concentrations of silver nanoparticles, the vigor
index of treated seedlings was significantly higher than the control seedlings. This indicates
that silver nanoparticle treatment improves the overall growth profile of the treated
seedlings.
An increase in chlorophyll contents was observed at 25, 50 and 100 ppm silver nano-
particle treatment. Maximal increase of 40 % in chlorophyll-a and 25 % in total chlorophyll
content was recorded at 100 ppm silver nanoparticle treatment. Maximum photosystem II
quantum efficiency, as measured through Fv/Fm ratio, was recorded at 50 ppm silver nano-
particle treatment (Fig. 2c). Fv/Fm ratio determines whether or not the imposed treatment
affects the photosystem II efficiency and is a measure of plant’s photosynthetic performance
[20]. Higher quantum efficiency (Fv/Fm), as recorded in the silver nanoparticle-treated
seedlings, indicates that more number of reaction centers are in an ‘open state’ to carry
out light reaction. Presence of higher number of open or oxidized electron acceptors in PS-II
decreases the probability of generation of reactive radicals [20]. Thus, our observations
signify a lower incidence of photo-oxidative damage in the seedlings treated with 25 and
50 ppm silver nanoparticles. Improved quantum efficiency positively correlates with higher
chlorophyll contents in the leaves of treated seedlings. Zheng et al. [6] have also reported
that nano-TiO2 could promote photosynthesis and improve spinach growth.
These observations clearly indicate that silver nanoparticles improve the cellular electron
exchange efficiency in the treated seedlings. An efficient electron exchange mechanism
arrests electron leakage, reducing the formation of reactive oxygen species [21]. An im-
provement in PS-II electron transport also positively correlates with the recorded reduction
in the levels of MDA and H2O2, in the treated seedlings (Fig. 2d).

Antioxidant Status

Levels of malondialdehyde and hydrogen peroxide declined significantly at 25 and 50 ppm

silver nanoparticle treatment, as compared to the controls. Maximum decline of 28.4 % in

Fig. 3 Specific activity of antioxidant enzymes: guaiacol peroxidase, catalase and ascorbate peroxidase
activity in B. juncea seedlings grown in media supplemented without and with different concentrations of
silver nanoparticles. Values represent mean±SE
2232 Appl Biochem Biotechnol (2012) 167:2225–2233

MDA and 64.41 % in H2O2 levels was recorded at 50 ppm silver nanoparticle treatment.
Even at 400 ppm silver nanoparticle treatment, the levels of H2O2 and MDA were lower than
the control (0 ppm) seedlings (Fig. 2d). Decrease in MDA, an index of ROS production, was
significant at 25 and 50 ppm silver nanoparticle treatment, as compared to other concen-
trations (Fig. 2d). Further, the level of H2O2, one of the most damaging forms of reactive
oxygen species, also decreased in the treated seedlings. This decrease in H2O2 is due to the
induction of H2O2 metabolizing enzyme like GPX, as noted in our experiments. H2O2 can
easily cross the biological membranes, because of its high pKa, and can damage various
cellular organelles [15]. A decrease in H2O2 production also indicates that the efficiency of
redox reactions has increased in presence of silver nanoparticles. Higher concentration of
silver nanoparticles, in the growth medium, significantly increased the activity of H2O2-
metabolizing enzymes. Specific activity of guaiacol peroxidase increased continuously with
increasing concentration of silver nanoparticles in the growth medium. A maximum increase
of 335 % in GPX activity was recorded at 400 ppm silver nanoparticle treatment (Fig. 3).
Free proline content decreased drastically in the seedlings treated with varying concen-
trations of silver nanoparticles. Figure 2d shows the decrease in proline content at different
nanoparticle treatments, with a maximal of 85 % decrease being recorded at 400 ppm.
Several studies attribute an antioxidant property to proline, suggesting ROS scavenging
activity and also acting as a singlet oxygen quencher [15]. Proline is an excellent index of the
existing stress experienced by the plant, since proline level declines after relief from stress
[22]. An unprecedented decline in proline levels recorded in the present experiments is one
of the most convincing evidences for improved electron exchange efficiency in the silver
nanoparticle-treated seedlings. The results for the first time demonstrate that presence of
silver nanoparticle in the growth media can improve the growth of B. juncea seedlings by
improving their antioxidant status. It follows that optimized use of silver nanoparticles can
modulate oxidative stress in plants.

Acknowledgments The authors are thankful to the Department of Biotechnology, Govt. of India, for
financial support. We thank, the Head, Department of Genetics, Indian Agricultural Research Institute, New
Delhi for providing seed material.

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