IET Nanobiotechnology

Research Article

Synthesis and characterisation of metal

nanoparticles and their effects on seed
germination and seedling growth in
commercially important Eruca sativa

ISSN 1751-8741
Received on 13th May 2015
Revised on 8th October 2015
Accepted on 27th October 2015
doi: 10.1049/iet-nbt.2015.0039
www.ietdl.org

Mehreen Zaka 1, Bilal Haider Abbasi 1 , Latif-ur Rahman 2, Afzal Shah 2, Muhammad Zia 1
1

Department of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan

Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
E-mail: bhabbasi@qau.edu.pk

Abstract: The synthesis, characterisation and application of metal nanoparticles have become an important and attractive
branch of nanotechnology. In current study, metallic nanoparticles of silver, copper, and gold were synthesised using
environment friendly method (polyols process), and applied on medicinally important plant: Eruca sativa. Effects of
application of these nanoparticles were evaluated on seed germination frequency and biochemical parameters of plant
tissues. Seeds of E. sativa were germinated on Murashige and Skoog (MS) medium incorporated with various
combinations of nanoparticles suspension (30 g/ml). Phytotoxicity study showed that nanoparticles could induce
stress in plants by manipulating the endogenous mechanisms. In response to these stresses, plants release various
defensive compounds; known as antioxidant secondary metabolites. These plants derived secondary metabolites
having a great potential in treating the common human ailments. In the authors study, small-sized nanoparticles
showed higher toxicity levels and enhanced secondary metabolites production, total protein content, total flavonoids
content and total phenolics content.

Introduction

Nanotechnology is a very versatile eld covering almost all existing

branches of science. Nanotechnology is famed as 21st century
science, as it has found applications in physics, chemistry,
biology and many other elds [1]. In the last decade many
advances in nano-biotechnology were noted. Nanotechnology has
the potential to improve the agriculture with new tools and
enhance the plants ability to absorb nutrients. In recent years,
scientists have started focusing on employment of nanoparticles in
agriculture for the betterment of crop quality and also for
increased growth and disease control in plants. Studies on
biological effects of nanoparticles in higher plants are increasing
day-by-day.
Eruca sativa commonly known as rocket plant is the well-known
fast growing herb of family Brassicaceae. Despite of its popular use
as vegetable, rocket plant is also considered as useful medicinal plant
since ancient times [2]. Its phytochemistry is very rich and it is a
good source of bres, avonoids, carotenoids, vitamin C and
glucosinolates [3]. It has shown anti-tumour, anti-ulcer and
hepatoprotective activities [4, 5].
Plant tissue culture is considered as promising alternate to
conventional breeding. It establishes high-frequency regeneration
protocols and improves the quality of economically important
plants. E. sativa has shown adaptability to in vitro regeneration
protocols [6]. Tissue culture techniques well explained the need of
biotechnology by solving food security and agricultural production
issues, and highlighted the use of biotechnological techniques for
having genetically improved varieties [7].
Stress enhanced secondary metabolites content in medicinal
plants. Elicitors are reported to effect production of these
secondary metabolites positively [8]. Recently, exploitation of
nanomaterials is reported to effect biosynthesis of economically
and commercially viable secondary metabolites in medicinal plant
species [9]. An increase in shoot/root ratio was observed by
applying different nanoparticles in soil. As the change in growth

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& The Institution of Engineering and Technology 2016

behaviour was observed after the certain time period, so it was

assumed that nanoparticles do not directly inuence plants but
indirectly alters the mechanisms [10].
As plants possess cell walls as a primary interacting site for
foreign particles, so entry of nanoparticles become difcult. The
mechanism by which nanoparticles enters the plants is still poorly
dened. Yet, nanoparticles have ability to magnify changes in cell
structures and molecules and also the defensive mechanisms.
Effects of nanoparticles depend on its physical and chemical
properties and include solubility of toxic nanoparticles and
production of reactive oxygen species [11].
Chemical composition of nanoparticles is very much responsible
for the toxic effects of metal nanoparticles (MNPs) on plants and
also the stress caused by surface, shape and size of particle [12].
Moreover, toxic nanoparticles may increase the production of
reactive oxygen species and hydroxyl radicals that damage the cell
membranes and as a result permeability is altered. As a result, entry
of nanoparticles into plant cells become easier and stress induced
by the particles results in secondary metabolites production [13].
Up to now, only few studies have been reported on application of
nanomaterials in plant tissue culture. According to Safavi [14],
nanosilver and nano-titanium dioxide can be used as antimicrobial
agent in plant tissue culture medium.
No report is available on application of MNPs in tissue culture
media to evaluate the seed germination frequency and plant growth
in Eruca. Hence, we have evaluated the effect of chemically
synthesised copper (Cu), silver (Ag) and gold (Au) MNPs on
growth parameters and secondary metabolites content in E. sativa.

2
2.1

Materials and methods

Plant source and surface sterilisation

Seeds of E. sativa were provided by Dr. Bilal Haider Abbasi,

Department of Biotechnology, Islamabad, Pakistan. Seeds were

surface sterilised according to the protocol of Abbasi et al. [15], i.e.

seeds were sterilised by immersion in 0.1% mercuric chloride for 1
min followed by three time washing with distilled water.
2.2

seedling vigor index = [root length (cm) + shoot length (cm)]

germination (%)

Metal nanoparticles

MNPs of Au, Cu and Ag were synthesised by environment friendly

polyols process. Reagents used were Cu chloride (CuCl2) (98%), Ag
nitrate (AgNO3) (99%), hydrogen Au chloride (HAuCl4) (99%),
ethylene glycol (C2H6O2) (98%), polyethyleneimine (2%). All of
these chemicals were purchased from Thermo sher scientic Inc.
(USA), except polyethyleneimine that was obtained from Acros
Organics.
2.2.1 Preparation of Cu nanoparticles: About 10 ml
polyethyleneimine (2%) was added to 20 ml of CuCl2 solution
(1 mM). This mixture was purged with argon blow for 30 min. The
solution was heated in oven at 175C for 30 min. The appearance
of bluish black colour showed the formation of Cu nanoparticles
that was conrmed by ultraviolet (UV)visible spectroscopy.
2.2.2 Preparation of Ag nanoparticles: About 10 ml of
polyethyleneimine was added to 20 ml of AgNO3 solution (1 mM)
and was heated at 150C for 15 min, the appearance of blackish
colour shows the formation of Ag nanoparticles that was also
conrmed by UVvisible spectroscopy.
2.2.3 Preparation of Au nanoparticles: For preparation of Au
nanoparticles 10 ml polyethyleneimine was poured to 20 ml of
HAuCl4 (1 mM) solution and was heated at 100C for 15 min.
The appearance of yellowish back colour evidenced the formation
of Au nanoparticles that was conrmed by UVvisible
spectrophotometry.
All the three nanoparticles Cu, Ag and Au were stable for six
months. These nanoparticles were characterised by using
characterisation techniques which include UV spectrophotometer
(Cary 100, Varian, Shimadzu, Tokyo Japan), X-ray diffraction
(XRD) (Brucker SMART APEX diffractometer) and transmission
electron microscopy (TEM) (Philips, Holland Tecnai 20).
2.3

Anderson [18] and expressed as index numbers [17]

Seed germination protocol

MNPs were suspended directly in distilled water using sonication.

30 g/ml suspension was prepared of each MNPs by sonication in
distilled water for 30 min according to the protocol described by
Savithramma et al. [16]. This suspension was added to MS0
medium in concentration of 3 ml/30 ml of MS medium with the
help of a micropipette. Sterilised E. sativa seeds were incubated
into conical asks containing autoclaved solidied MS media.
Seed germination was observed in 24 days and rst data on
seed germination was collected after 14 days of inoculation. Data
was collected after every 2 weeks and the experiment was
conducted for 42 days, i.e. 6 weeks.

2.5

Analytical methods

2.5.1 Determination of free radical scavenging activity by

2,2-diphenyl-1-picrylhydrazyl (DPPH) method: The free
radical scavenging assay (FRSA) of methanolic extracts of
E. sativa was measured in terms of hydrogen donating or radical
scavenging ability using the stable radical DPPH. Protocol of Lee
et al. [19] was followed with some modications according to
which the test extracts were prepared in methanol, so the DPPH
was also prepared in methanol. DPPH solution was added in
sample solution according to the dened calculated concentrations
separately. These solution mixtures were kept in dark for 30 min
(incubation period) at room temperature. After 30 min, the
absorbance was measured at 515 nm using micro-plate reader.
Lower absorbance of the reaction mixture indicated higher FRSA.
Finally, the radical scavenging activity was calculated as
percentage of DPPH discolouration using the equation
% scavenging DPPH free radical = 100 (1 AE/AD)
where AE is the absorbance of the solution, when extract has been
added at a particular level and AD is the absorbance of the DPPH
solution with nothing added (blank, without extract).
2.5.2 Evaluation of total antioxidant capacity (TAC) by
phosphomolybdenum method: The TAC of the methanol
extract was evaluated by the phosphomolybdenum method
according to the procedure described by Prieto et al. [20].
According to this assay Mo (VI) is reduced to Mo (V) by the
extract and green phosphate/Mo (V) complex is formed at acid pH.
A 50 l of extract was combined with 450 l of reagent solution
(0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM
ammonium molybdate). The extract was then put into water bath at
95C for 90 min and then samples were cooled at room temperature.
The absorbance of the reaction mixture was measured at 695 nm
using a spectrophotometer against blank. Methanol was used as the
blank. The antioxidant activity is expressed as the number of gram
equivalent of ascorbic acid [21]. Some of the compounds which are
normally not measured as antioxidants have some chain breaking
antioxidant activity also, so TAC assay is helpful to measure all
these compounds collectively including complex interactions
occurring during chain breaking antioxidants. Generally, TAC is
decreased under oxidative stress condition and administration of
chain breaking antioxidants increases antioxidant capacity [22].

2.4.2 Root and shoot length: Root and shoot length of seeds
were recorded after every 2 weeks starting from inoculation date.
Mean root and shoot length were compared in the form of bar charts.

2.5.3 Determination of total phenolics and total avonoids

content: Total phenolics content (TPC) was determined following
the FolinCiocalteu (FC) method by Singleton and Rossi [23]. Briey
three solutions were prepared for TPC activity. Ten times diluted
solution of FC reagent in distilled water, 6% sodium carbonate
(Na2CO3) solution in distilled water and 4 mg/ml solution of gallic
acid in methanol were prepared. About 25, 20, 15, 10 and 5 /ml
concentrations of gallic acid were used as positive control and 20 l
of Dimethyl sulfoxide (DMSO) as negative control. Absorbance
was measured at 630 nm by using microplate reader.
Total avonoids content (TFC) was determined by following the
protocol of Haq et al. [24]. Stock solutions of 10% aluminium
chloride in distilled water, 1 M potassium acetate in distilled water
and 4 mg/ml quercetin in methanol were prepared. About 20 l of
methanol was used as negative control and 40, 20, 10, 5 and 2.5
g/ml of nal concentration of quercetin as positive control.
Absorbance was recorded at 415 nm using microplate reader.

2.4.3 Seedling vigour index: The seedling vigour index (VI)

was calculated by using the method suggested by Abdul-Baki and

2.5.4 Estimation of total protein content: For total protein

content estimation method of Lowry et al. [25] was used. Three

2.4

Seed germination parameters

2.4.1 Percentage germination frequency: The percentage

germination was recorded after every 2 weeks. Seeds were taken
as germinated when radicle had emerged from seed coat [17]
percentage germination (%) = number of germinated seeds
100/total number of seeds

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reagents, i.e. A [1 g sodiumpotassium (NaK) tartrate 4 H2O, 50

g Na2CO3, 250 ml 1 N sodium hydroxide (NaOH)], B (2 g NaK
tartrate 4H2O, 1 g CuSO4.5H2O, 0.1 N NaOH) and C (FC
reagent ten times diluted with distilled water) were used. About
40 l of protein extract was taken and 36 l of reagent A was
added and then incubated for 10 min at 50C and after cooling the
reaction mixture 4 l of reagent B was added and again incubated
under same conditions. After cooling reaction mixture at 25C,
120 l of reagent C was added. The absorbance was then recorded
at 650 nm using microplate reader. Standard curve of bovine serum
albumin was prepared at series of 25, 50, 75, 100 and 125 g/ml used.
2.5.5 Superoxide dismutase (SOD) and peroxidase (POD)
activities: SOD activity assay was performed according to
method of Ullah et al. [26] with minor changes. Inhibition of
photochemical reduction of nitro blue tetrazolium (NBT) by SOD
is the principle of assay. About 1 mM ethylenediaminetetraacetic
acid, 130 mM methionine, 0.02 mM riboavin, 0.75 mM NBT and
50 mM phosphate buffer (pH 7) was used. Blank was prepared by
mixing all the chemicals except enzyme extracts in the same
quantity. Reaction mixture was exposed to uorescent light for 7
min. optical density (OD) was taken at 560 nm.
Activity of enzyme was calculated applying LambertBeer law
A = ELC
where A = absorbance; = extinction coefcient (6.39 mM1 cm1);
L = length of each wall (0.25 cm); C = concentration of enzyme
(value of C measured in nM/min/mgFW); and FW = fresh weight
of the sample.
For POD activity method of Lagrimini [27] was used. About 27.5
mM hydrogen peroxide (H2O2) (10), 100 mM guaiacol (10),
distilled water, 1% polyvinyl-pyrrolidone. About 50 mM
K-phosphate buffer of pH 7 were used as chemicals. Reaction
mixture of 200 microlitre was prepared using these chemicals.
Blank was prepared by mixing of 60 l K-phosphate buffer, 20 l
guaiacol, 100 l distilled water and 20 l H2O2. Absorbance was
recorded at 470 nm with a gap of 20 s and activity was calculated
according to the formula used for SOD activity.

Results and discussion

The synthesised nanoparticles were characterised by UVvisible

spectroscopy, XRD and TEM. UVvisible spectra conrmed the
formation of nanoparticles showing single Plasmon band having
maximum wavelength closed to the value that have already been
published by Rahman et al. [28]. Fig. 1 shows UVvisible spectra
of Cu (Fig. 1a), Ag (Fig. 1b) and Au (Fig. 1c) nanoparticles. Their
maximum absorbance for 1 mM solution and maximum
wavelengths are shown in Table 1. Such suspension was
centrifuged and the powdered nanoparticles were analysed by
XRD as shown in Fig. 2. XRD helped us in calculating the size of
nanoparticles as well as their crystallinity. In Fig. 2a, 111 is
considered as desired peak. In Fig. 2b, 111, 200 and 210 were
taken as the desired peaks, whereas in Fig. 2c 111 and 200 were
taken as desired peaks. Applying Debye Scherrer equation,
average size of the nanoparticles was calculated as shown in
Table 1. XRD also revealed that nanoparticles are crystalline and
mostly to be cubic. Fig. 3 exhibits the TEM photographs of pure
Cu, Ag and Au nanoparticles. A typical TEM image of Cu
nanoparticles in Fig. 3a are seemed to be non-spherical; however,
shapes of Ag and Au nanoparticles are spherical as shown in
Figs. 3b and c. Interestingly, size shown by these images was
same as calculated from XRD. The size of nanoparticles evaluated
from XRD as well as TEM can be seen in Table 1.
3.1

Fig. 1 UVvisible spectra of

a Cu nanoparticles prepared by polyols process using C2H6O2 as a solvent
b Ag nanoparticles prepared by polyols process using C2H6O2 as a solvent
c Au nanoparticles prepared by polyols process using C2H6O2 as a solvent

unstable chemicals or samples [29, 30]. Seed germination

frequency was calculated at different time intervals such as 14, 28
and 42 days (Table 2). Among all metals, Ag showed maximum
positive response and seed germination frequency was recorded as
Table 1 Data obtained from UVvisible spectroscopy, XRD and TEM
Nanoparticles

Maximum absorbance for 1 mM

solution

lmax

Average size,
nm

0.26
0.68
0.82

550
451
485

15
18
20

Seed germination frequency

Seed germination and root elongation are easy and widely used in
phytotoxicity test and it is simple, low cost and suitable to

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Cu
Ag
Au

Fig. 2 XRD spectra of

a Cu nanoparticles solidied by centrifugation
b Ag nanoparticles solidied by centrifugation
c Au nanoparticles solidied by centrifugation

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Fig. 3 TEM images of

a Cu nanoparticles prepared by polyol process using C2H6O2 as a solvent and solidied by centrifugation
b Ag nanoparticles prepared by polyol process using C2H6O2 as a solvent and solidied by centrifugation
c Au nanoparticles prepared by polyol process using C2H6O2 as a solvent and solidied by centrifugation

73%. As reported by El-Temsah and Joner [31], Ag nanoparticles of

different sizes and in different concentrations effect the seed
germination frequency differently, small-sized particles exert more
inhibitory effects on seed germination. In present study, time
interval was taken as limitation after expecting that with the
progression of time more aggregation of nanoparticles in cell
compartments happens, so there is a possibility of nanoparticles of
same size to impact in an unexpected way. As reported by Lee
et al. [32] Cu nanoparticles are toxic to two species of mung bean
(Phaseolus radiatus) and wheat (Triticum aestivum), so in the
present paper it also showed inhibitory effect and seed germination
was 56%. Au also showed inhibitory effect and germination
frequency was 53% but in case of Au as time increases the
germination frequency was also increased and toxicity of Au
decreased. In previous reports, it is concluded that toxicity of
nanoparticles depends on two different actions, i.e. chemical
composition and release of toxic ions and stress caused by surface
size or shape of nanoparticles [33]. Therefore, in the present paper
it is also observed that Cu of small size and toxic nature both
contributed in the inhibition of seed germination. Ag showed
positive response and results were almost such as the control
seeds. Au responds late, it showed inhibition in start and then
showed positive response after 28 days but as time increased up to
42 days again seed germination was inhibited due to the release of
toxic ions and stress caused by nanoparticles.

days of inoculation, mostly root and shoot lengths were reduced as

compared with control which can be justied by the reason that
with the passage of time more ions from particles were released
and accumulated in roots and shoots more surface ions were
exposed and they were more toxic to plantlets.

3.3 Percentage DPPH radical scavenging activity and

TAC
After exposing the E. sativa seeds to different nanoparticles,
signicant radical scavenging activity was noted in plantlets of
Table 3 Effect of metal nanoparticles on root length, shoot length and
seed VI of E. sativa values are average of three replications SE
Germination
period

Treatments

Root
length, cm

Shoot
length, cm

VI

after 14 days

MS0
Cu
Ag
Au
MS0
Cu
Ag
Au
MS0
Cu
Ag
Au

1.9 0.09
1.7 0.08
2.3 0.11
1.2 0.06
1.5 0.07
1.9 0.09
4.5 0.22
2.3 0.11
5 0.25
2.1 0.10
2.3 0.11
2.1 0.10

1.5 0.07
2 0.1
2.5 0.12
1.2 0.06
2.5 0.12
2.8 0.14
6.3 0.31
4.5 0.22
9.8 0.49
4.3 0.21
6.7 0.33
3.4 0.17

340 17
296 14.8
484 24.2
192 9.6
164 8.2
225.5 11.27
866.6 43.33
498.6 24.93
1480 74
384 19.2
900 45
330 16.5

after 28 days

after 42 days

3.2

Root and shoot elongation and seed VI

It is inferred that Ag nanoparticles stimulated the root and shoot

length and seed VI at different time periods such as 14, 28 and 42
days with exception in root length after 42 days as root length was
decreased again after 42 days (Table 3). Savithramma et al. [16]
reported that Ag period of time at optimum concentration. Reason
maybe Ag nanoparticles penetrated and induce new pores which
helped inux the nutrients in seed for rapid growth. Chemical
composition of Ag nanoparticles and their precise size and shape
does matter in the response showed by these particles.
Cu and Au nanoparticles have reduced the root and shoot length in
E. sativa and showed more stress than Ag nanoparticles. These
results are similar to the previously reported results [32]. After 42
Table 2 Effect of MNPs on in vitro seed germination of E. sativa values
are an average of five replications SE
Serial
number

Treatments

control
(MS0)
Cu
Ag
Au

2
3
4

Percentage
seed
germination
after 14 days

Percentage
seed
germination
after 28 days

Percentage
seed
germination
after 42 days

76.6 3.3

40 2

100 5

56.6 2.8
73.3 3.6
53.3 2.6

46.6 2.3
80 4
73.3 3.6

60 3
100 5
60 3

Fig. 4 Percentage DPPH radical scavenging activity and TAC of E. Sativa

plantlets against different treatments of nanoparticles

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Fig. 5 Total avonoids and TPC after 6 weeks of time interval

Fig. 7 SOD activity and POD activity of fresh matter after 6 weeks of
E. Sativa treated with MNPs

E. sativa. TAC and DPPH radical scavenging activity of the plantlets

treated with nanoparticles is shown in Fig. 4. Correlation was
established between radical scavenging activity and TAC. Ag
nanoparticles have reduced the antioxidant capacity compared with
control and Au nanoparticles again started increasing the capacity
as compared with Ag nanoparticles.
The contradiction observed between DPPH radical scavenging
activity and TAC in Au treatment can have the reason that TAC is
collective value of antioxidants and numerous other compounds
which are involved in some chain breaking antioxidant activity
[22]. As we are applying MNPs and after a long period of
inoculation accumulation of these particles occurs and ions are
released in cell compartments, so DPPH radical scavenging
activity can be lesser in Cu-treated seeds which have released
more ions and these ions already had scavenged the radicals.

3.5

3.4

TFC and TPC

As reported by Krishnaraj et al. [34], Ag nanoparticles enhanced the

total phenol content in plants; however, AgNO3 produced
considerably higher phenolic content. In current paper, total
phenolics and avonoids content were lesser in Ag/Au treated
plantlets than Cu treatment (Fig. 5). It is reported earlier that Cu
nanoparticles are inducing more stress and thus more secondary
metabolites are biosynthesised for protection of plant cells.

Total protein content, SOD and POD

As shown in Fig. 6, total protein content is comparatively lesser in

plantlets treated with Cu nanoparticles and in Ag and Au treated
plantlets total protein content is comparable with control, i.e. MS0.
Higher total protein content correlates with higher SOD and POD
activities. Stress could be the main cause behind this enhanced
total protein content. SOD and POD activities were stimulated by
nanoparticles. However, these parameters were hampered by Cu
ions presence in medium [35]. As shown in Fig. 7, higher levels
of SOD and POD activities indicating their protective role against
Au nanoparticles-induced stress [34].

Conclusions

It is concluded from the current paper that metals at nanoscale

showed different behaviours than metals in bulk form. Chemical
composition, size and shape of nanoparticles matters a lot to affect
plants [33]. Small-sized nanoparticles are more toxic than larger
size nanoparticles and among all the combinations applied Cu
nanoparticles are more stress inducing than Ag and Au
nanoparticles because of the fact that Cu at bulk level is also more
toxic than Au and Ag. These results can be helpful for future
studies on phytotoxicity of various nanomaterials especially
engineered nanoparticles under certain conditions. Up till now
there are very few reports on using nanomaterials in plant tissue
culture as a stress inducing factor. Using the results reported in
this paper further study in domain of ecotoxicity can be conducted
to check environment friendly nanoparticles and nanoparticles with
harmful effects.

Acknowledgments

The authors are grateful to Miss Shagufta Shaque, National Centre

for Bioinformatics and Mr. Tariq Khan, Department of
Biotechnology at Quaid-i-Azam University for their assistance
throughout the manuscript. Mehreen Zaka acknowledges the
support provided by Higher Education Commission, Pakistan.

Fig. 6 Total protein content of fresh matter after 6 weeks of E. Sativa

treated with MNPs

References

1 Lin, D., Xing, B.: Phytotoxicity of nanoparticles: inhibition of seed germination

and root growth, Environ. Pollut., 2007, 150, pp. 243250
2 Mitsuo, M., Takako, M., Kohsuke, K.: Composition of the essential oil from the
leaves of Eruca sativa, Flavour Fragrance J., 2002, 17, pp. 187190
3 Barillari, J., Canistro, D., Paolini, M., et al.: Direct antioxidant activity of puried
glucoerucin, the dietary secondary metabolite contained in rocket (Eruca sativa
Mill.) seeds and sprouts, J. Agric. Food Chem., 2005, 53, pp. 24752482

IET Nanobiotechnol., pp. 17

& The Institution of Engineering and Technology 2016

4 Khoobchandani, M., Ganesh, N., Gabbanini, S., et al.: Phytochemical potential of

Eruca sativa for inhibition of melanoma tumor growth, Fitoterapia, 2011, 82,
pp. 647653
5 Alqasoumi, S., Al-Sohaibani, M., Al-Howiriny, T., et al.: Rocket Eruca sativa: a
salad herb with potential gastric antiulcer activity, World. J. Gastroenterol.,
2009, 15, pp. 19581965
6 Murashige, T., Skoog, F.: A revised medium for rapid growth and bioassays with
tobacco tissue cultures, Physiologia Plantarum, 1962, 15, pp. 473497
7 Oggema, J.N., Kinyua, M.G., Ouma, J.P., et al.: Agronomic performance of
locally adapted sweet potato (Ipomoea batatas (L.) Lam.) cultivars derived from
tissue culture regenerated plants, Afr. J. Biotechnol., 2007, 6, pp. 14181425
8 Pitta-Alvarez, S.I., Spollansky, T.C., Giulietti, A.M.: The inuence of different
biotic and abiotic elicitors on the production and prole of tropane alkaloids in
hairy root cultures of Brugmansia candida, Enzyme Microb. Technol., 2000, 26,
pp. 491504
9 Gomes, S.I.L., Novais, S.C., Gravato, C., et al.: Effect of Cu-nanoparticles versus
one Cu-salt: analysis of stress biomarkers response in Enchytraeus albidus
(Oligochaeta), Nanotoxicology, 2012, 6, pp. 134143
10 Shah, V., Belozerova, I.: Inuence of metal nanoparticles on the soil microbial
community and germination of lettuce seeds, Water Air Soil Pollut., 2009, 197,
pp. 143148
11 Navarro, E., Baun, A., Behra, R., et al.: Environmental behavior and ecotoxicity of
engineered nanoparticles to algae, plants, and fungi, Ecotoxicology, 2008, 17,
pp. 372386
12 Masarovicova, E., Kralova, K.: Metal nanoparticles and plants, Ecol. Chem. Eng.
S, 2013, 20, pp. 922
13 Kim, J.S., Kuk, E., Yu, K.N., et al.: Antimicrobial effects of silver nanoparticles,
Nanomedicine, 2007, 3, pp. 95101
14 Safavi, K.: Evaluation of using nanomaterial in tissue culture media and biological
activity. Second Int. Conf. on Ecological, Environmental and Biological Sciences
(EEBS2012), Bali, Indonesia, 1314 October 2012
15 Abbasi, B.H., Khan, M.A., Mahmood, T., et al.: Shoot regeneration and
free-radical scavenging activity in Silybum marianum L, Plant Cell Tissue
Organ Cult., 2010, 101, pp. 371376
16 Savithramma, N., Ankanna, S., Bhumi, G.: Effect of nanoparticles on seed
germination and seedling growth of Boswellia ovalifoliolata an endemic and
endangered medicinal tree taxon, Nano Vis., 2012, 2, pp. 6168
17 Ushahra, J., Malik, C.P.: Putrescine and ascorbic acid mediated enhancement in
growth and antioxidant status of Eruca sativa varieties, CIBTech J. Biotechnol.,
2013, 2, pp. 5364
18 Abdul-Baki, A.A., Anderson, J.D.: Vigor determination in soybean and seed
multiple criteria, Crop Sci., 1973, 13, pp. 630633
19 Lee, S.K., Zakaria, H.M., Cheng, H.S., et al.: Evaluation of antioxidant potential
of natural products, Comb. Chem. High Throughput Screen., 1998, 1, pp. 3546

IET Nanobiotechnol., pp. 17

& The Institution of Engineering and Technology 2016

20

21

22
23

24

25
26

27
28

29

30

31

32

33

34

35

Prieto, P., Pineda, M., Aguilar, M.: Spectrophotometric quantitation of antioxidant

capacity through the formation of a phosphomolybdenum complex: specic application
to the determination of vitamin E, Anal. Biochem., 1999, 269, pp. 337341
Aliyu, A.B., Ibrahim, H., Ibrahim, M.A., et al.: Free radical scavenging and total
antioxidant capacity of methanol extract of Ethulia conyzoides growing in Nigeria,
Rom. Biotechnol. Lett., 2012, 17, pp. 74587465
Young, I.S.: Measurement of total antioxidant capacity, J. Clin. Pathol., 2001,
54, p. 339
Singleton, V.L., Rossi, J.A.: Colorimetry of total phenolics with
phosphomolybdic-phosphotungstic acid reagents, Am. J. Enol. Viticult., 1965,
16, pp. 144153
Haq, I.U., Ullah, N., Bibi, G., et al.: Antioxidant and cytotoxic activities and
phytochemical analysis of Euphorbia wallichii root extract and its fractions,
Iran. J. Pharm. Res., 2012, 11, pp. 241249
Lowry, O.H., Rosebrough, N.J., Farr, A.L., et al.: Protein measurement with the
Folin phenol reagent, J. Biol. Chem., 1951, 193, pp. 265275
Ullah, N., Haq, I.U., Safdar, N., et al.: Physiological and biochemical
mechanisms of allelopathy mediated by the allelochemical extracts of Phytolacca
latbenia (Moq.) H. Walter, Toxicol. Ind. Health, 2015, 31, pp. 931937
Lagrimini, L.M.: Wound-induced deposition of polyphenols in transgenic plants
overexpressing peroxidase, Plant Physiol., 1991, 96, pp. 577583
Rahman, L.U., Qureshi, R., Yasinzai, M.M., et al.: Synthesis and spectroscopic
characterization of AgCu alloy nanoparticles prepared in various ratios,
Comptes Rendus Chim., 2012, 15, pp. 533538
Munzuroglu, O., Geckil, H.: Effects of metals on seed germination, root
elongation, and coleoptile and hypocotyl growth in Triticum aestivum and
Cucumis sativus, Arch. Environ. Contamination Toxicol., 2002, 43, pp. 203213
Wang, X., Sun, C., Gao, S., et al.: Validation of germination rate and root
elongation as an indicator to assess phytotoxicity with Cucumis sativus,
Chemosphere, 2001, 44, pp. 17111721
El-Temsah, Y.S., Joner, E.J.: Impact of Fe and Ag nanoparticles on seed
germination and differences in bioavailability during exposure in aqueous
suspension and soil, Environ. Toxicol., 2012, 27, pp. 4249
Lee, W., An, Y., Yoon, H., et al.: Toxicity and bioavailability of copper
nanoparticles to the terrestrial plants mung bean (Phaseolus radiatus) and wheat
(Triticum aestivum): plant uptake for water insoluble nanoparticles, Environ.
Toxicol. Chem., 2008, 27, pp. 19151921
Brunner, T.J., Wick, P., Manser, P., et al.: In vitro cytotoxicity of oxide
nanoparticles: comparison to asbestos, silica, and the effect of particle
solubility, Environ. Sci. Technol., 2006, 40, pp. 43744381
Krishnaraj, C., Jagan, E.G., Ramachandran, R., et al.: Effect of biologically
synthesized silver nanoparticles on Bacopa monnieri (Linn.) Wettst Plant growth
metabolism, Process Biochem., 2012, 47, pp. 651658
Nekrasovaa, G.F., Ushakova, O.S., Ermakov, A.E., et al.: Effects of copper (II)
ions and copper oxide nanoparticles on elodea densa planch, Russ. J. Ecol.,
2011, 42, pp. 458463