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The American Journal of Agriculture and Biomedical Engineering
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ABSTRACT
In recent times, nanotechnology is a branch of precision farming and sustainable agriculture where it expresses all
sides of development for better utilization of resources in agriculture. Graphene oxide (GO) nanosheet is a two-
dimensional crystal structure formed by a flat monolayer of sp² hybridized carbon atoms arranged in a hexagonal
arrangement. Marigold (Tagetes erecta L.) and Calendula (Calendula officinalis L.) are commercially explored flower
crops of the family Asteraceae. These flower species contain a sufficient amount of active compounds, used in various
commercial sectors. Graphene oxide nanosheets were produced by the commercial chemical method.
Characterization of GO is done on powder X-ray diffraction (XRD), Fourier transforms infrared (FTIR) & Scanning
electron microscopy (SEM). Seeds were collected from recognized sources and after surface sterilization; seeds were
treated with GO nano solution with different concentrations (0, 20,40,60,80,100,120 and 140mg/l respectively). The
conducted experiment found that GO in the concentration range from 40 to 80 mg/l (T2 to T4) gives positive results
Research Article
EFFECTS OF GRAPHENE OXIDE NANOSHEET ON GERMINATION AND
SEEDLING ATTRIBUTES ON TAGETES ERECTA L. AND CALENDULA
OFFICINALIS L.
Submission Date:
February 19, 2022,
Accepted Date:
March 05, 2022,
Published Date:
March 14, 2022 |
Crossref doi:
https://doi.org/10.37547/tajabe/Volume04Issue03-01
Kunal Adhikary
Ph.D Research scholar, Department of Floriculture and Landscape Architecture, Faculty of Horticulture.
Bidhan Chandra Krishi Vishwavidyalaya, Mohanpur- 741252, Nadia (WB), India
Tapas Mandal
Department of Department of Floriculture and Landscape Architecture, Faculty of Horticulture, Bidhan
Chandra Krishi Vishwavidyalaya, Mohanpur- 741252, Nadia (WB), India
Jayoti Majumder
Assistant professor, Department of Department of Floriculture and Landscape Architecture, Faculty of
Horticulture, Bidhan Chandra Krishi Vishwavidyalaya, Mohanpur- 741252, Nadia (WB), India
Prasenjit Mandal
Ph.D Research scholar, Department of Chemistry, University of Kalyani- 741235, Nadia (WB), India
Journal
Website:
https://theamericanjou
rnals.com/index.php/ta
jabe
Copyright:
Original
content from this work
may be used under the
terms of the creative
commons
attributes
4.0 licence.
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Volume 04 Issue 03-2022
The American Journal of Agriculture and Biomedical Engineering
(ISSN
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VOLUME
04
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Pages:
1-14
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(2020:
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34
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in the maximum of all aspects in both the annuals. The maximum root length of the marigold was recorded in T2
(5.91cm) and shoot length in T5 with 4.63cm. The coefficient of the velocity of germination (CVG) for calendula was
observed maximum in T3 (32.17) and in marigold T2 (46.34). In GO treated seeds were having a dense rooting system
with good seedling growth, early germination rate also observed in treated seeds in comparison to control.
KEYWORDS
GO; Germination; Growth; FTIR; SEM; XRD.
INTRODUCTION
Nanotechnology is a branch of precision farming
where it is exploring its all sides for the development
of sustainable agriculture. Nano particles have only
one of its kind physicochemical properties and the
potential to boost up the plant metabolism (Giraldo et
al. 2014). According to (Torney et al. 2007) engineered
nanoparticles are capable to enter into plants cells and
leaves, and also can transport DNA and chemicals keen
on plant cells, can also arouse plant growth. Nano
particles when applied to the plants can help out them
to cope up the harsh conditions by releasing the ROS
enzyme (Reactive Oxygen Species).
From the resent study it has been found that
application of engineered nanomaterials to agricultural
sciences has been particularly active and exciting (Lin
and Xing 2007, Prasad
et al.
2012). Under given abiotic
stress
condition,
nano-SiO
2
augments
seed
germination. SiO
2
NPs improves and ignite the
photosynthetic rate by synthesis of photosynthetic
pigments and getting better activity of carbonic
anhydrase, as reported by (Siddiqui
et al.
2014 and Xie
et al.
2012). Xie
et al.
2011 reported from their study
that, it also boost up the plant growth and
development by mounting gas exchange and also
enhance few parameters, such as net photosynthetic
rate, photochemical efficiency, electron transport rate.
Zheng
et al
. (2005) observed the ability of nano-TiO
2
from their experiment on the growth of aged spinach
seeds and found that the plant physiological
parameters like plant dry weight appreciably increases
due to the augmented formation of photosynthetic
pigments and few biochemical activities. Hong
et al.
(2005) found from their investigation that TiO
2
NPs take
care of the chloroplast from unnecessary light by
augmenting and modify the activity of antioxidant
enzymes, such as superoxide dismutase. From one
conducted experiment it has been found that,
exogenous application of TiO
2
NPs positively modifying
net photosynthetic rate, also improves transpiration
rate in plants, which is stated by Qi
et al.
2013. From the
resent study it has been found that function of
engineered and artificially made nanomaterials to
agricultural sciences and plant science has been
particularly exciting (Lin and Xing 2007; Prasad
et al.
2012). Lei
et al.
(2007) reported the efficiency of nano-
anatase, which promoted strongly whole chain
electron transport and photoreduction activity of
photosystem II in plant physiology.
In the context of carbon nano tubes, graphene is an
ultra-thin carbon material with similar chemical and
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physical properties (Allen
et al
. 2010) and also have
massive potentials and ability for biological
applications suggested by Dutta and Pati, (2010). On
the other hand, Nair
et al.
(2012) found that graphene-
treated rice seedlings are healthier with well-
developed root and shoot systems as in relation to
controls. Zhang
et al.
(2015) justified that Graphene
was found to penetrate seed husks that might
facilitate water uptake, resulting in faster germination
and higher germination rates. Similar study was
reported by Khodakovskaya
et al.
(2009), observed
that carbon nano tubes penetrate husk of tomato
seeds and amplify the rate of seed germination greatly.
Graphene oxide (GO) nanosheet is a two dimensional
crystal structure formed by a flat monolayer of sp²
hybridized carbon atoms arranged in a hexagonal
arrangement, technically it`s 2D allotropes of carbon,
one which has honeycomb lattice structure, Graphene
oxides nanosheet electronic and optical properties
enable graphene oxide nanosheet to be used in many
field (Geim and Novoselov, 2007). The major concern
about graphene oxide is mainly focused on its chemical
structure, electronic properties, reduction reaction
and chemical functionalization these are different
kinds of oxygen species and bonding to carbon in
graphene layer, such as epoxy, hydroxy carbonyl &
carboxylic groups (Kumar
et al
. 2013). The application
fields of graphene oxides are mainly focused on sensor
(Robinson
et al.
2008, lin
et al.
2010), efficient catalyst,
as a supercapaciter (liu
et al.
2010), conducting
materials (Zou
et al
. 2009, Lu
et al.
2010) and as a gas
storage medium.
Marigold (
Tagetes erecta
L.) and Calendula (
Calendula
officinalis
L.) is commercially exploited flower crops
belong to family Asteraceae. These flowers are native
to Mexico and Mediterranean region respectively.
These species has been cultivated since India times for
its purposed general medicinal qualities and today
both flowers are grown for medicinal and herbal
(Matic
et al
. 2012, Mohammad and Kashani 2012,
Preethi and Kuttan 2009), and ornamental uses (Clark
et al.
2010, Warner and Erwin 2005). These flowers
species contains a sufficient amount of active
substances or compounds, such as: saponosoides,
flavonides, sterols, polysaccharides, sesquiterpene
lactones along with essential oils, because of only this
reason they are exploited in food industries as food
colorant and supplements. Till now no such research
has been conducted on the escalation of these
secondary metabolites of these crops with any
precision farming such as nano technology.
MATERIALS AND METHODS
Experiment was conducted in collaboration between
University of Kalyani, Department of Chemistry and
Department
of
Floriculture
and
Landscape
Architecture, BCKV during the period of one year (2018-
2019).
GO Preparation and Characterization
Experimental
:
Materials
Natural Graphite powder (200 mes) of spectroscopic
purity
was
purchased
from
Gupta
Electric
Manufacturing Co. (Delhi, India). Conc.H
2
SO
4
(98%),
H
3
PO
4
(85%), and NaNO
3
were purchased from Sigma-
Aldrich and hydrogen peroxide (H
2
O
2
, 30%) and KMnO
4
(99%) were purchased from Shijiazhuang Kunmal
Chemical Technology Co. (China). All reagents were of
AR grade, and were used as received without further
purification.
Synthesis of Graphene Oxide nanosheet (GO)
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Graphene oxide was synthesized by modified
Hummer’s method through oxidation of graphite
powder. The method of preparation is briefly as
follows. Graphite powder (1.5 g) and NaNO
3
(0.4g)
were mixed in 45 ml of Conc. H
2
SO
4
(98%) and 15 ml of
H
3
PO
4
(85%) in a 500 ml round bottom flask kept under
ice bath (0-5 °C) with continuous stirring. The mixture
was stirred for 35 minutes at this temperature and
potassium permanganate (7.5 g) was added to the
suspension very slowly. The rate of addition was
carefully controlled to keep the reaction temperature
lower than 11-19°C. The mixture was then diluted with
very slow addition of 120 ml water and kept under
stirring for 1.20 hrs. The ice bath was then removed,
and the mixture was stirred at 35°C for 2 hrs. The
mixture was then refluxed at
85°C for 10-25 min. After 15 min, the temperature was
changed to 40°C, yielding a brown coloured solution.
After another 25 min, the temperature of the mixture
was changed to 28°C, and maintained at this
temperature for 1.00 hrs. The solution was finally
treated with 35 ml H
2
O
2
whereby its colour changed to
bright yellow. 260 ml of water was taken separately in
two separate beakers and equal amount of solution
prepared was added to it with stirring for 2.50 hr. It was
then kept without stirring for 2-3.5 hrs, allowing the
larger particles to settle at the bottom and supernatant
filtered. The filtrate was washed repeatedly after
centrifugation with (5-10) % HCl and then with
deionized (DI) water several times until it forms a gel
like substance (at neutral pH). The gel like substance
was vacuum dried at 50-65°C for more than 3.5 hrs to
obtain GO powder (shown in scheme 1).
Characterization
The powder X-ray diffraction (XRD) of the sample was
carried out on a Rigaku-MiniFlex 600 X-ray
diffractometer with CuKα source (
=1.5418 Å) at room
temperature. Fourier transform infrared (FTIR) spectra
were
obtained
with
Shimadzu
FTIR-8300
spectrophotometer. Scanning electron microscopy
(SEM) was performed on a ZEISS, EVO40 microscope.
Raman spectrum was obtained with a Thermonicolet
FT-Raman spectrometer.
HOOC
HO
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
COOH
HOOC
COOH
COOH
COOH
OH
OH
HOOC
HOOC
HOOC
HO
COOH
COOH
COOH
Graphene
Graphene oxide nanosheet
KMnO
4
,H
2
SO
4
+H
3
PO
4
0-5
o
c
Scheme 1- Chemical structure of graphene oxide
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Seeds Treatments and seeding observation
Experiment was performed in
PG lab of Department of
Floriculture and Landscape Architecture, Bidhan
Chandra Krishi Vishwavidyalaya, Mohanpur, West
Bengal. Nano particle Graphene oxide is synthesied
and characterized in Department of Chemistry,
University of Kalyani, and West Bengal. Seeds were
collected from research farm, Mondouri, Kalyani.
Proper seed treatment protocol was adopted, where
seeds were immersed in a 10% sodium hypochlorite
solution for 10 min for disinfection pourpose and then
they were washed three times with deionized water.
Then, disinfected seeds were next soaked in
suspensions solution with different concentrations of
GO (20. 40, 60, 80, 100, 120 and 140 mg/l). After that,
seeds were placed onto filter papers placed in 10cm
Petri dishes test unit along with 5 mL of respective
concentration of GO, with 10 seeds per dish, then
placed in a dark growth chamber under a constant
temperature of 25
0
C.
From the 1
st
day, after 24 hrs interval germination
parameters were observed and recorded separately
like, GI- germination index, GRI- germination rate
index, MGT- mean germination time, FGP- final
germination percentage %, CVG- coefficient of velocity
of germination, G3- germination % at 3
rd
day.
After continuous growth for 7 days in the water and
different concentrations of GO, the Marigold (
Tagetes
erecta
L.) and Calendula (
Calendula officinalis
L.)
seedlings were gently cleaned with water to remove
the remaining dirt particles and dry them out on filter
paper. Then, MSD- marigold shoots dry weight (gm.),
MRD- marigold roots dry weight (gm.), CSD- calendula
shoots dry weight (gm.), CRD- calendula roots dry
weight (gm.), CRL- calendula shoot length (cm), CRL-
calendula root length (cm), MRL- marigold root length
(cm), MSL- marigold shoot length (cm) of untreated
and treated plants were monitored. All experiments
were performed using a completely randomized
design with three replicates per treatment.
Data were expressed as mean SD of three experiments
in triplicate. Error bars represented the standard
deviation of the mean. Statistical comparisons were
performed by analysis of variances (ANOVA). The
values of p (<0.05 & <0.01) were considered to be
statistically significant (*) and highly significant (**)
respectively.
RESULTS AND DISCUSSION
Characterization of GO
SEM study
The Fig. 1 (a, b) are shows the scanning electron
microscope of synthesised graphene oxide nano-sheet
materials. These images
analysed the morphology of
nanostructural shape and size of sheets.
SEM of
materials shows regular grooved structure, probably
from ordering of sheet like structures of graphene
oxide nanoflakes or graphene oxide nano-sheet.
These
images are also observed that the agglomerated
sheets of layered type of structure. While the scanning
electron microscope image clearly predicated the
synthesised materials are graphene oxide nanosheet.
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Fig. 1 (a, b) SEM image of synthesized graphene oxide nano-sheet
XRD study
Powder x-ray diffraction (p XRD) data of the
synthesized graphene oxide nano-sheet taken at room
temperature is shown in Fig. 2 below. The
characteristic peaks at 2
value of 10.37
o
correspond to
the crystal faces of (001) planes of pure graphene
oxide nano-sheet (JCPDS, GO file no.
75-2078
). Powder
x-ray diffraction analysis was done to know the
synthesized samples graphene oxide nanosheet.
10
20
30
40
0
200
400
600
800
1000
In
te
nsi
ty
(a
.u
.)
2 theta (degree)
(001)
Fig.2.
Powder X-ray diffraction (pXRD) data of the synthesized graphene oxide nano-sheet
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FT-IR spectra study
The FT-IR spectra of graphene oxide (GO) is shown in
Fig. 3 below. The peak at 1714 cm
-1
corresponding to
ν(C=O) of –COOH on the GO. The peak at 1601 cm
-1
corresponding to ν(C=C) of aromatic π bonds. The
peak at 1047 cm
-1
corresponds to ν(C-O) of epoxy
bonds.
Fig.3. FTIR spectra of graphene oxide nanosheet
Effects of GO on germination parameters of calendula
and marigold seeds
As per the conducted experiment, mean germination
(days) is found less in T
3
followed by T
2
(3.09 and 3.15
respectively). Germination rate index (%/days) is more
effective in T
2
with 36.17 and followed by T
4
(35.33) and
T
3
(35.12). Final germination percentage was recorded
on 7
th
day from the experiment started, which is
recorded maximum in T
3
and T
4
with 98.33% followed
by T
2
with 96.67%. Under the study of germination test
of calendula seeds treated with graphene oxide (GO),
CVG is observed maximum in T
3
(32.17) which are
nearby to T
2
and T
4
(31.91 and 31.83 respectively).
Germination on 3
rd
day, noted more in T
4
(98.33%) than
the rest treatment. Conducted experiment reported
that calendula seeds on control are presented less
value of preferences as compared to the other data. In
some treatment above a particular concentration,
germination parameter shows negative impacts over
the seeds as shown in Table 1. According to Lee
et al
.
(2013), particle size of nano material sometime affects
the germination, like smaller the size more absorption
rate and vice- versa. Villagarcia
et al.
2012 and Tiwari
et
al
. 2014 stated that MWCTs induce the water and
essential Ca and Fe nutrients uptake efficiency that
could augment the seed germination.
In the aspects of marigold, from the Table 1,
germination parameters were recorded separately
after the seed treatment in marigold. Similarly with the
calendula, marigold also shows positive results with
seeds treated with GO. Germination index (GI)
recorded maximum in T
2
with 76.67 followed by T
4
and
T
3
(75.33 and 73.47 respectively). Germination rate
index (GRI) is observed minimum in T
7
(32.94) which
shows that high concentration of GO, germination of
seeds were affected, whereas T
2
recorded maximum
value with 49.55. More values of mean germination
time, shows slow germination rate of the seeds. Mean
(a)
(b)
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germination time (MGT) noted minimum in T
2
(2.15days) followed by T
4
and T
3
(2.25 and 2.29 days
respectively), which proves that the GO at 40 to 80mg/l
concentration leads to fast germination of seeds. Final
germination percentage (FGP) observed 100% in T
1
, T
2
and T
4
. Coefficient of velocity of germination (CVG) is
more in T
2
(46.34) and it is recorded less in T
7
(27.85) at
high concentration of Graphene oxide (GO).
Germination percentage at 3
rd
day recorded maximum
in T
4
followed by T
3
and T
2
. Khodakovskaya
et al.
(2009)
observed more germination rate and other desirable
parameters in vegetable seeds treated with GO.
Effects of GO on seedling parameters of calendula and
marigold
In calendula the effects are par with marigold
parameters in some aspects (Table 2). Shoot dry
weight value is recorded maximum in T
2
(0.0188g)
followed by T
5
(0.016g). In the context of root dry
weight the value is 0.0994g in T
3
with maximum and
0.0166g in T
0
in control with minimum value
respectively. After a week of transplanting the seeding
parameters of calendula were recorded and noted
separately (Fig-4). Calendula root length is observed
maximum in T
2
(5.86cm) followed by T
4
and T
5
(4.4cm
and 5.6cm respectively). Shoot length of the treated
and untreated calendula seedling showed higher value
in T
4
with 3.63cm and lower in T
0
control with 0.13cm.
According to previous studies done by various
researchers, the effects of nanomaterials on plants
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depend on the characteristics of the nanomaterials and
the xylem anatomy within plants, which affect the
absorption rate (Begum et al. 2011, Cañas et al. 2008).
Smirnova
et al.
2012 found from their study that
MWCNTs pick up the root and stem growth and
dehydrogenase activity may be due to primary uptake
and accumulation of MWCNTs by roots followed by the
translocation from roots to leaves, which could
encourage genes expression (Khodakovskaya
et al
.
2012, Wang
et al
. 2012a).
Fig. 4 (a, b) Seedlings of marigold and calendula at 3
rd
day after treatment
After the seed germination, when the seeds were
transplanted in the germination tray to observe the
seedling health. These two flower crops marigold and
calendula were planted separately to observe the
respective seedling parameters. From the presented
Table (2), dry weight of the marigold shoot was
recorded maximum in T
4
(0.0064g) followed by T
2
with
0.0052g, while untreated seedlings recorded less
value.
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Similarly, Root dry weight of marigold is observed
maximum in T
4
(0.0193g) followed by T
3
(0.0072g). In
the context of marigold root length, which showed
maximum value in T
4
(6cm) followed by T
3
and T
2
(4.50cm and 5.91cm respectively). Shoot length of
marigold seedling is observed high in T
5
(4.36cm) and
low in T
0
control with 0.27cm (Fig. 5). Begum
et al.
(2011) reported that because of differences in root
systems, the response of some vegetables crops like,
cabbage, tomato, red spinach and lettuce over
graphene vary. Khodakovskaya
et al.
(2013) and Husen
and Siddiqi, (2014) observed that.
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Fig.5 (c, d) 7
th
day healthy seedlings of marigold and calendula with comparative study
Sometime high concentrations of graphene inhibited
the growth of some species, but had no significant
effect on some. This is due to the difference in the
uptake of nanomaterials by distinctive xylem
structures.
CONCLUSION
From the conducted study it can be concluded that
graphene oxide nano sheet really proves beneficial in
the context of seed germination and seed parameters
of winter annuals (Tagetes erecta L.) and (Calendula
officinalis L.). Seed germination is recorded fast in the
seeds treated with GO as compared to control.
Seedlings growth was also healthy in treated seeds.
Graphene generally trigger the metabolic pathway for
fastening the growth and development. Profuse
rooting and good shoot/root length was observed in
GO treated seeds. At the concentration of 40-80 mg/l
GO promote the healthy seedling growth, but at very
much high concentration it may restrict the plant
growth. GO is one of the good promoting agents for
plant growth, it should be explore more in the field of
agriculture.
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