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In vitro germination and biochemical profiling of Brassica napus in response

to biosynthesised zinc nanoparticles

Article in IET Nanobiotechnology · October 2018

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IET Nanobiotechnology

Research Article

In vitro germination and biochemical profiling ISSN 1751-8741

Received on 13th March 2018
Revised 15th May 2018
of Brassica napus in response to Accepted on 20th July 2018
E-First on 3rd October 2018
biosynthesised zinc nanoparticles doi: 10.1049/iet-nbt.2018.5012
www.ietdl.org

Sohail1, Umme Amara1, Salma Shad2, Noshin Ilyas1, Abdul Manaf3, Naveed Iqbal Raja1, Zia-ur-Rehman
Mashwani1
1Department of Botany, PMAS Arid Agriculture University, Rawalpindi 46300, Pakistan
2Department of Chemistry, Hazara University, Mansehra, Pakistan
3Department of Agronomy, PMAS Arid Agriculture University, Rawalpindi 46300, Pakistan

E-mail: [email protected]

Abstract: With the progression of nanotechnology, the use of nanoparticles (NPs) in consumer products has increased
dramatically and green synthesis is one of the cheapest and eco-friendly methods to obtain non-hazardous NPs. In the current
research zinc (Zn) NPs synthesis was carried out by using the fresh and healthy leaves of Mentha arvensis L. followed by
characterisation through ultraviolet (UV)–visible spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM).
UV–visible spectroscopy confirmed the green synthesis of ZnNPs, while XRD confirmed the size of NPs, which was 30–70 nm.
SEM shows that the shape of ZnNPs was irregular. The effects of green synthesised NPs on two different varieties of Brassica
napus were evaluated. Exposure to ZnNPs (5, 15, and 25 mg/l−1) caused a significant increase in root and shoot length of B.
napus. The application of NPs significantly improved plant germination and triggered the production of secondary metabolite
and antioxidant enzymes. ZnNPs showed a significant increase in chlorophyll, superoxide dismutase, total flavonoid content
(TFC) and antioxidant enzymes while total phenolic content was decreased when TFC increased. Thus, it has been concluded
from the current study that ZnNPs may possibly trigger the production of antioxidant enzymes and various biochemical
compounds.

1 Introduction of several phytochemicals, in addition to vitamins and pro-

vitamins, has been regarded as an immense nutritional concern in
Nanotechnology is regarded as 21st century sciences, owing to its the prevention of many chronic diseases, for instance, cancer,
tremendous applications in biology, physics, chemistry, and various diabetes mellitus, arteriosclerosis, nephritis, ischemic, rheumatism,
other fields [1]. Nanotechnology has tremendous potential to boost and cardiovascular diseases. These compounds also possess anti-
the agriculture with innovative tools as well as perk up the plants aging properties, in which oxidants or free radicals take part [11,
potential to uptake essential nutrients. Nowadays, the application 12].
of nanoparticles (NPs) in agriculture for the increasing crop quality Now, it became a centre point for researchers to exploit the
and for increased growth and disease control in plants is becoming effects of nanomaterials on the biosynthesis of economically as
a focal point for scientists. Numerous studies highlighted the well as commercially viable secondary metabolites in medicinal
biological effects of NPs on higher plants and the research and plant species [13]. The application of NPs in soil led to an increase
development in this regard is increasing day-by-day. in the shoot/root ratio in different plants. These nanomaterials do
Rapeseed (Brassica napus L.) belonging to family Brassicaceae not directly affect plant growth, but they show their impacts on the
is the potential source of vegetable oils. Rapeseed meal mechanisms that lead to change in the plant growth and
encompassing significant available natural resource is regarded as development [14].
a by-product of the oil extraction process. Since [2] studied the The chemical composition, as well as surface, shape, and size of
protein of the rapeseed meal and reported it as a source of novel NPs, play a crucial role in the toxic effects of metal NPs on plant
peptides possessing ACE-inhibitory activity, it emerges to be a growth and development [15]. Furthermore, the toxic levels of NPs
chief source of many other bioactive compounds such as increase the production of reactive oxygen species (ROS) and free
polyphenols, tocopherols and phytosterols all these compounds hydroxyl radicals that cause huge damage to the cell membranes
play a role in the prevention as well as treatment of several leading to change in the permeability of plasma membrane.
diseases [3]. Massive research has been performed to elucidate the Therefore, the entrance of NPs into different plant cells become
important constituents as sources of natural antioxidants from quite easier and stress persuaded by these NPs leads to the
rapeseed meal. To date, many phytochemical studies revealed the production of secondary metabolites [16].
extraction of several phenolic compounds and peptides hydrolysed The current study has been designed to investigate the effect of
from rapeseed oil and protein [4, 5]. green synthesised zinc (Zn) NP on B. napus L. According to the
Brassica species are stated to acquire tremendous cancer curing best of our knowledge, this is the first report from Pakistan that the
properties [6] and these properties are owing to the presence of ZnNPs effect has been investigated on B. napus L. plant.
glucosinolate as well as their derivative products [7]. The presence
of flavonoids and many other phenolics also play a significant role
in anticancer properties [8]. While fundamentally temperate, 2 Material methods
Brassica oleracea varieties are now cultivated in entire regions all 2.1 Collection of plant materials
over the world [9].
Research evidence reveals that the utilisation of fruits and For the synthesis of ZnNPs, fresh leaves of Mentha arvensis L.
vegetables is, overall, quite beneficial to health because of the plants were used as reducing and capping agents. Leaves were air
significant protection offered by the antioxidant compounds which dried under aseptic conditions for 10 days after that the plant
are chiefly and naturally found in them [10]. Indeed, the presence materials were ground into fine powder. About 10 g of powdered

IET Nanobiotechnol., 2019, Vol. 13 Iss. 1, pp. 46-51 46

© The Institution of Engineering and Technology 2018
plant material was subjected to 100 ml of deionised water followed by absorbance at 725 nm by using a Cecil UV–visible
by shaking for 24 h. The resultant solution was filtered twice with spectrophotometer.
Whatman 21 filter paper. The filtrate was kept in a refrigerator at
4°C for further use [17]. 2.4.4 Determination of total flavonoid content
(TFC): Quantification of TFC was carried out by using the
2.2 Synthesis of ZnNPs protocol proposed by Chang et al. [24]; about 10 mg of quercetin
was dissolved in 80% ethanol followed by further dilution.
Synthesis of ZnNPs was carried out by using the M. arvensis L. Dilution involved mixing with 1.5 ml of ethanol, 1 M CH3CO2K,
extract and zinc nitrate hexahydrate (ZnNO3·6H2O) salt. About 1
2.5 ml of water and 0.2 ml Al2Cl3. The reaction mixture was kept
mM solution of zinc nitrate salt was prepared in deionised water.
at room temperature for 60 min. Using a Cecil UV–visible
The solution was boiled on a hotplate and continuously stirred
spectrophotometer, the absorbance of the mixture was recorded by
while adding plant extract until the colour changed to light yellow.
setting wavelength at 415 nm.
The solution was centrifuged at 12,000 rpm for 15 min. The
supernatant was discarded, and the lower pellet was again
suspended in deionised water followed by centrifugation for 5 min. 2.4.5 Determination of soluble protein content (SPC): For the
The process was repeated three times to remove impurities any determination of the SPC, about 0.5 g of fresh leaves were ground
present [18]. in phosphate buffer with the help of pestle mortar. About 0.5 ml of
the above extract was mixed with 0.5 ml of distilled water and 3 ml
of bio red dye. The homogenised mixture was shaken well and then
2.2.1 Characterisation of ZnNPs: The synthesised ZnNPs were
the absorbance of the sample was observed at 650 nm by using the
characterised by ultraviolet (UV)–visible spectroscopy, X-ray
Cecil 2021 spectrophotometer made in Japan [25].
diffraction (XRD) and scanning electron microscopy (SEM).
2.4.6 Superoxide dismutase (SOD) activity: For determination
2.3 Plant cultivation
of SOD, 10 ml sodium phosphate buffer was used to grind 0.5 g
Seeds of B. napus (Faisal canola and Shiralee varieties) were leaf material. After settling down of the solution, a separate set of
obtained from the National Agriculture Research Center, test tubes containing 0.1 ml of the extract was supplemented with
Islamabad, Pakistan. The seeds were sterilised for 24 h with 0.1 ml riboflavin and 3 ml of phosphate buffer. This reaction
hypercaloric acid following the methods proposed by Kouhi et al. mixture was placed under the fluorescent lamp for 8 min to start
[19]. After sterilisation, seeds were cultured on Petri plates having the reaction. The same reaction mixture was prepared for dark
different concentrations of green synthesised ZnNPs (5, 15, and 25 reaction in another set of test tubes. The absorbance of both sets
mg/l−1). was recorded at 560 nm wavelength [26]

2.3.1 Germination parameter: Germination percentage: SOD, μg/ml = absorbance of sample × K value
Germination percentage of seedling of B. napus was recorded after × dilution factor/weight of sample
7 days by using the method of Abdul-Baki and Anderson [20].
2.5 Statistical analysis
2.3.2 Shoot and root length: Root and shoot length of seedling
The whole experiment was performed in triplicate. Results are
was recorded from the date of germination in cm.
presented as mean ± SD. A significant difference (at P ≤ 0.05) and
means comparison were done using one-way analysis of variance.
2.3.3 Seedling vigour index (SVI): SVI was recorded by using Statistical analysis was carried out by using SPSS 20 software.
the method of Abdul-Baki and Anderson [20] and expressed in
terms of means and standard deviation (SD)
3 Result and discussion
SVI = root length + shoot length × germination percentage . UV–visible spectroscopy of green synthesised ZnNPs confirmed
the synthesis of ZnNPs and absorption spectra were 220–450 nm
2.4 Biochemical and physiological attributes already published in [27] (Fig. 1). The plant extract may contain
possible reducing compounds which triggered the synthesis of NPs
2.4.1 Determination of total chlorophyll content (TCC): The [28]. After confirmation by UV–visible spectroscopy, the NPs were
determination of the TCC was done by taking 0.2 g plant tissue that calcined and dried in the electric furnace at 500°C for 24 h to
was briefly ground in cold 80% acetone solution and the remove oxygen and pure ZnNPs were obtained in the form of fine
absorbance was measured at 652 nm using the method proposed by powder. ZnNPs were then subjected to XRD at the Department of
Arnon [21]. Chemistry, Quaid-e-Azam University, Islamabad, Pakistan. The
XRD pattern showed that ZnNPs are irregularly shaped and
2.4.2 Determination of total sugar content (TSC): For the crystalline in nature [29]. SEM of ZnNPs was carried out at the
determination of the TSC, the method proposed by Dubois et al. Institute of Space Technology Islamabad, Pakistan. Technology by
[22] was followed. About 0.2 g fresh leaves were ground in 80% using SIGMA model (MIRA3; TESCAN Brno). The SEM picture
ethanol solution. The mixture was kept in the water bath for 1 h at confirmed the size in the range of 30–70 nm and morphology of
80°C. About 0.5 ml of the mixture, followed by addition of 1 ml of the synthesised ZnNPs was non-spherical, irregular in shape
18% of phenolic solution and mixture was kept for one hour at previously reported by [30].
room temperature. After the addition of 2 ml of sulphuric acid, the
mixture was constantly shacked and absorbance was noted at 490 3.1 Germination percentage of B. napus seedlings
nm.
In the current study, germination percentage was recorded after 7
2.4.3 Determination of total phenolic content (TPC): The days of exposure to different concentrations of 5, 15 and 25 mg/l−1
determination of TPC was carried out following the protocol ZnNPs. Both the varieties showed significantly higher germination
proposed by Veilaogu et al. [23]; the folin–ciocalteu reagent was percentage than control variety as shown in Fig. 1. It can be
used for quantification. About 0.2 g powdered plant material was attributed that the maximum germination was recorded at 15 mg/l
crushed with 80% ethanol and then 100 μl plant extract was mixed −1 of ZnNPs in both V1 and V2 followed by 25 mg/l−1 of ZnNPs as
with 0.75 ml of folin–ciocalteu reagent followed by incubation at compared with control in a dose-dependent manner. Other
21°C for 10 min. The mixture was equipped with 0.75 ml of researchers have already reported that ZnNPs enhanced
sodium bicarbonate solution and kept for 90 min at 21°C followed germination percentage in wheat crop [31] (Fig. 2).

IET Nanobiotechnol., 2019, Vol. 13 Iss. 1, pp. 46-51 47

© The Institution of Engineering and Technology 2018
 cm) and (8.3 cm), respectively. It has been observed that ZnNPs
can stimulate the growth of root and shoot significantly on
augmentation. Our results are in conformity with [32], which
previously reported the effect of NP on root and shoot length of the
Artemisia plant. Maximum seedling vigour index was observed in
both varieties of B. napus V1 (1106.42 ± 3.6) and V2 791.04 ± 7.1
as compared to control shown in Table 1. Shah and Belozerova
also reported an increase in root and shoot growth of Lactuca seeds
under ZnNP applications [33].
Nanoscale Zn showed large root growth of seedling compared
to bulk ZnSO4 and control. Such a promotory effect of nanoscale
SiO2 and TiO2 on germination was also reported in soya bean [34].
Germination means breaking of seed testa and emergence of
seedling up to 1–8 mm [35]. A significant increase was observed in
the root and shoot of the wheat plant by applying the different
concentration of ZnNPs [31].

3.3 Chlorophyll and sugar content

The chlorophyll content is more important to plant species and play
a vital role in photosynthesis. A significant increase was observed
in both the tested varieties. In the current study, the high content of
total chlorophyll was recorded at 15 mg/l of ZnNPs in V1 (Faizal
canola) as compared to V2 (Shiralee) variety, respectively. Overall,
in all concentrations of ZnNPs both the varieties showed a
significant increase in the chlorophyll content as compared to
control. Our results are in agreement with [36], which reported the
increase in the TPC in the leaves of Soybean variety. Raliya et al.
[37] also reported that ZnNPs increase 63% of the chlorophyll
content in the leaves of tomato as compared to control plant. It was
observed that the sugar content of both varieties in response to the
different concentrations of ZnNPs showed reliable results as
compared to control after 7 and 14 days of seedling while after 21
days of seedling no significant increase was observed on any
concentration of ZnNPs. The highest sugar content was observed at
25 mg/l−1 of ZnNPs in V1 (Faisal Canola) as compared with V2
(Shiralee) which was (3.9 µg/ml fresh weight (FW)) and (2.99
µg/ml FW) respectively. The role of green synthesised ZnNPs is
widely reported in triggering plant biochemical responses [38]
(Fig. 3).

3.4 TPC and TFC

After the introduction of green synthesised ZnNP into seedling of
B. napus varieties, the data was recorded at different days interval,
a significant increase was seen in the TPC of B. napus varieties.
The maximum TPC content calculated was (12.97 µg/mg dry
Fig. 1 Characterisation of ZnNPs weight (DW)) after an interval of 14 days and great variation has
(a) UV–visible scheme, (b) SEM analysis of green synthesised ZnNPs, (c) XRD of been seen in both varieties at different days interval. The result of
green synthesised ZnNPs my study is in comparison with [18], which already reported TPC
in Citrus reticulata plant. It has been seen in the current study that
the TPC and TFC in relation to FW were significantly high in
seedlings treated with ZnNPs after 7 days. Maximum production of
TFC was found after 7 days interval, which was (0.7 µg/mg DW)
in B. napus (V1 Faisal canola) followed by V2 (Shiralee) variety as
compared with 14 and 21 days of interval. Comparable results of
broccoli floral leaf were recorded [39]. Furthermore, the
production of TPC with green synthesised NPs showed a positive
correlation with TFC. It can be explained in terms of role green
synthesised ZnNPs in the biosynthesis of TPC and TFC. Abiotic
stresses cause increased production of phenolic compounds in plant
[40] (Fig. 4).

Fig. 2 Germination percentage of B. napus varieties after exposure to

3.5 Protein and SOD
green synthesised ZnNPs Protein and lipids exchange play a key role in plant metabolism
when an exposed stress of any nature. Plants react to different
3.2 Root length, shoot length and SVI stress stimuli by triggering their immune system. Exposure to such
ZnNPs may initiate a series of stress signalling pathways helping in
The effect of green synthesised ZnNPs on root and shoot of B.
the production of different classes of defensive compounds called
napus (Faisal canola and Shiralee) was recorded after an interval of
secondary metabolites [41]. In this study, the total protein and SOD
7 days. The highly varied trend was observed on different
were investigated at different interval of days on different
concentrations of ZnNPs (5, 15 and 25 mg/l−1). The maximum root concentrations of green synthesised ZnNPs. After seven days of
and shoot length was observed at 15 mg/l−1 of ZnNPs in V1 (2.99
48 IET Nanobiotechnol., 2019, Vol. 13 Iss. 1, pp. 46-51
© The Institution of Engineering and Technology 2018
Table 1 Effect of green synthesised ZnNPs on germination parameters of B. napus
Varieties Treatment of Zn NPs Germination, % Germination index Seedling vigour index Shoot length Root length
Faisal canola (V1) Control 60 ± 1.5 19.78 ± 0.9 440.0 ± 5.9 5.8 ± 0.6 1.54 ± 0.8
5 mg/l−1 62 ± 0.9 18.0 ± 0.2 612.56 ± 6.8 7.9 ± 0.8 1.98 ± 0.5
15 mg/l−1 98 ± 1.4 20.56 ± 1.2 1106.42 ± 3.6 8.3 ± 0.5 2.99 ± 1.8
25 mg/l−1 88 ± 0.4 17.98 ± 0.2 766.48 ± 2.9 6.7 ± 0.5 2.01 ± 1.3
Shiralee (V2) Control 65 ± 0.9 18.12 ± 1.5 403.00 ± 3.2 4.3 ± 0.09 1.90 ± 0.5
5 mg/l−1 60 ± 0.8 17.45 ± 1.2 438.00 ± 5.1 5.7 ± 0.7 1.6 ± 0.1
15 mg/l−1 96 ± 0.7 19.67 ± 0.9 791.04 ± 7.1 6.2 ± 0.2 2.04 ± 0.4
25 mg/l−1 80 ± 0.1 17.67 ± 0.4 583.2 ± 4.9 5.3 ± 0.1 1.99 ± 1.3
The data are presented in the form of means and SD, while the P value was kept >0.05.

Fig. 3 Total chlorophyll and sugar content in B. napus varieties in Fig. 4 TPC and TFC in B. napus varieties in response to green synthesised
response to green synthesised ZnNPs ZnNPs at different days interval

harvesting the highest protein content was observed at 15 mg/l−1 of enhanced the SOD, POD and catalase production in B. napus L.
green synthesised ZnNPs (195 µg bovine serum albumin Differences in the pattern is due to the chemical nature, NPs
equivalent/mg FW) in V1(Faisal Canola) as compared with V2 surface and size. The enzymatic role of SOD and POD and the
(Shiralee), respectively. Both varieties showed a significant catalase activities of different medicinal plants are extensively
increase in its protein content as compared to control. This increase reported [45, 46] (Fig. 5).
could be due to the shape and medium-size of green synthesised
ZnNPs, which triggered the production of protein. The nutritional 4 Conclusion
profile of the canola protein would play a pivotal role in From the current experiment, it has been concluded that green
determining its suitability as a food ingredient [41]. As canola synthesised ZnNPs have the potential to improve the germination
proteins are almost exclusively used for animal feed, knowledge of and biochemical aspects of the B. napus L. plant. ZnNPs have also
their nutritional value to humans is quite limited [42]. In the been reported to play a vital role in the production of auxin and
current research, B. napus varieties treated with ZnNPs of 5, 15 many other hormones which are responsible for plant growth and
and 25 mg/l−1 showed increased SOD activity 0.49 µg/ml in V1 yield. The size and surface structure of NPs is of prime importance
and V2 as compared to control, respectively. This may be due to and play a key role in the production of antioxidant enzymes. Once
the chemical and metallic stress of green synthesised ZnNPs. Plant NPs enter the plant they play a vital role in triggering different
defensive secondary metabolites such as SOD, peroxidase, catalase defence mechanisms of the plant. Green synthesis of NPs is the
etc. are commercially and medicinally important as health- cheapest source of obtaining NPs but it is still a mystery and much
promoting compounds [43]. SOD is the first line defence against work is needed to explore which compounds of plant play a key
any ROS. They can convert superoxide anion O2 to low harmful role in the reducing and capping agent.
ROS [44]. However, the production of protein is in correlation to
SOD concentration. The application of ZnNPs in different forms

IET Nanobiotechnol., 2019, Vol. 13 Iss. 1, pp. 46-51 49

© The Institution of Engineering and Technology 2018
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