Carbon
nanotubes (CNTs) are known to have many unique characteristics including
diameter, length, atomic configuration, impurities, defects and functionality,
which allow them were widely used in materials, chemicals, food,
bioengineering, medicine, and other ?elds 1, 2. Because of these physical and
chemical features of CNTs have used in agriculture to increase the crop yield,
mainly in the germination process, root growth, and photosynthesis 1. The
positive, negative and natural effects of CNTs on physiological responses can
variable or even opposite among different plant species 3. For example some
studies showed that multi-walled carbon nanotubes (MWCNTs) did not affect the
growth of wheat 5 and inhibited the growth of rice seedlings 6. But significantly,
enhanced the germination rate of tomatoes 4. Khodakovskaya, et al. (2009) have reported that CNTs could
penetrate plant seed coat and dramatically affect seed germination and plant
growth. However, the penetration, uptake and accumulation of CNTs in plant
cells and tissues are not well documented 8. Plants and plant cells showed
high tendencies to accumulate CNTs 11, 12. Recently, Mariya et al .(2014)  showed that MWCNTs can be absorbed by root
system of tomato plants and reach the leaves and the fruits. Lin  et al.
(2009) reported that
adsorption of an extensive amount of MWCNTs on the root surface may suppress
the water in?ux and uptake of nutrients hence inhibiting the plant growth 14.  Investigations have shown that CNTs could
induce phytotoxicity in plant cells and change the gene expression of plants 9.
Biochymical studies of different plants have demonstrated that, the use of CNTs
can induce the production and accumulation of oxygen reactive species such as
superoxide radical anions and hydroxyl radicals. ROS generation can lead to
protein, lipid, and DNA oxidation and to cell death 13. Most previous
researches of CNTs in the biosciences have focused on their influence on animal
and human cells, but to investigate the potential effects of CNTs on the plant
cells in the natural environment is very necessary. Liu Q et al. (2009) reported that CNTs penetrate
inside the cells (16). Insertion of MWCNTs into the wall of epidermal cells and
root hairs observed in wheat seedlings (17). Khodakovskaya (2011) showed that MWCNTs case enhance
the growth of tobacco and regulate cell division via activating water channels
and regulating genes involve d in cell division and extension (18). According
to Serag et al. (2011),
short MWCNTs with short length (in a range of 30 to 100 nm in length) tended to
target the nucleus, plastids, and vacuoles, which further revealed the close
relationship between MWCNTs size and phytotoxicity from the perspective of
plant cell biology 19. Okra (Hibiscus esculents L.) belongs to the family Malvaceae,
and  grown in all parts of the tropics
and during summer in the warmer parts of the temperate regions. Okra is a
popular home garden vegetable and a good 
source  of  many 
nutrients  including  vitamins 
B  and  C, 
fiber,  calcium,  and 
folic  acid (Hegazi and Hamideldin,
2010). In this study, changes of anatomical structure (thickness and diameter
different tissues) and morphological characterizes of two cultivars of  Okra seedling 
plant under  MWCNT treatment
evaluated. Results from this  research
may help to show the responses and behaviors of family Malvaceae plants  to MWCNTs.

 

Materials
and methods

Preparation
of MWCNTs

MWCNTs
were obtained from Nanosany Company (Iranian Nanomaterials Pioneers Company,
Mashhad, Iran). Specifications of this carbon nanotubes were detected by using
scanning electron microscope (SEM) (Hitachi S-4160,Tokyo, Japan), and the X-ray
diffraction (XRD) (Philips-X’Pert MPD X-ray refractometer) technique. Also, Raman spectra
of the MWCNTs with OD less than 50 nm was prepared using UV–Vis
spectrophotometer (T80+ UV–VIS spectrophotometer PG instruments Ltd, UK). The metals
content of MWCNTs were detected by energy dispersive X-ray spectroscopy
analysis.

Seed
germination and seedling growth

Seeds
of two Okra cultivars (bamia and emerald) were purchased from company
Avan mashregh zamin (www.avanmz.ir).
Okra
seeds were surface-sterilized using sodium hypochlorite (10%, 15 min) and soaked in distilled water for 24 h, then
allowed to germinate on moist ?lter paper at
25 °C in the dark for 4 days. The homogenous seedlings
were transferred to
plastic pots, containing 2 Lit Hoagland (pH of the medium was adjusted at 6.8-7
with  HCl or NaOH) and were completely changed every  day. The
seedlings were grown hydroponically in a controlled climate with diurnal regime
of 16 h light at 25 ± 2 °C and 8 h dark at 19 ± 3 °C in the growth room.

Treatment
of seedling

For
the seedling treatment, MWCNTs were suspended using ultrasonication as before
described by Mansour Ghorbanpour. (2014). Brie?y, nanomaterials were added to in
distilled water and dispersed using ultrasonic vibration (420 W, 20 Khz, for a total of 45
min). Stock solutions were diluted with nutrient solution Hoagland
to ?nal
concentrations (0, 50, 100 and 200 mg/lit) right before use. Parvin   Begum (2012). After  12 
days  of  hydroponic culture, seedlings were harvested
for estimation of biomass (fresh and dry weight) and length
of shoot and root seedling. All
experiments
were  performed with three replication.

Measurement
of anatomical parameters

After
harvest, for anatomical studies, roots and shoots samples of  Okra seedlings were immediately fixed in glycerol
 and ethanol (1:2). Cross-sections  of 
stems, roots and leaf  were  taken 
by  hand. Sections were
cleared  in sodium  hypochlorite 
and  stained by  carmine-vest 
(1%  w/v  in 
50%  ethanol)  and methyl green  (1% 
w/v, aqueous). Cross-sections  was
observed  with  an 
light microscope (Zeiss) and photographed by digital camera (SONY,
DSC-W35) (Hajiboland
et al., 2012). All anatomical 
measurements  were  done with 3 repeats for each part.

Statistical
Analysis

 For all variables, two–way analysis of
variance (ANOVA) was performed to test for differences between different Okra
cultivar and  CNTS treatments
interactions, using the GLM procedure in SPSS Duncan’s test was used to
determine the signi?cant di?erence between treatments.

Results

Growth
responses

When
MWCNTs were present
in the nutrient solution, two cultivars of Okra plants exhibited similar growth
responses that were noticeable. In our observation, used of MWCNT in nutrient
solution lead to increases of shoot and root length when compared to the control groups. Signi?cant increase in shoot length was observed at 50 mg/lit MWCNT treatment for two
cultivars of Okra, but signi?cant diminishing of shoot length
was observed at 200 mg/lit in emerald cultivar (Figure 2a). Also, 100 and 200
mg/lit of MWCNTs treatment
lead to a significant increase of root lenght in bamia when in compared with
the control, but root length of bamia was  significantly affected  at 
all  levels  of  MWCNTs
(Figur 2b). According  to Fig 2 the 
highest  shoot and root  lenght 
was obtained  at 50 mg/lit  MWCNTs treatment  in two cultivars.

The
results of MWCNT effect on  fresh weight (shoot and root) and dry weight
(shoot and root) of
two Okra cultivars are depicted in figure 3a and 3b. Statistical 
data  revealed  that 
shoot fresh weight of bamia cultivar was significantly increased at 50
mg/lit level of  MWCNT,  but in two cultivars  were significantly
decreased at 200 mg/lit levels of  MWCNT.
Also, similar results  were 
obtained  when  shoot 
dry weight of two cultivars measured In compared with the control alone
application of 50 mg/lit MWCNTs in
the nutrient solution, lead
to a significant increase in the root fresh weight of two Okra cultivars,
but  it is the  interesting 
to  note  that  bamia
cultivar  showed reduction in root fresh
at 100 and 200 mg/lit  of  MWCNTs 
while  100 and 200 mg/lit  of MWCNTs treatment, lead to an increase in
the root fresh weight of emerald cultivar. Also, similar effect was obtained
when root dry weight of two cultivars measured and compared to the control
groups (Figure 4a, 4b).

Stem
anatomy

The
results related to the anatomical changes of stem show in table 2. In this
research it observed that all MWCNTs level treatment specifically changed the stem
anatomy in two cultivars of Okra plants. Microscopic  results 
showed  that all MWCNTs levels significant increase in the stem diameter  of bamia cultivar, but  in emerald cultivar stem diameter  was significantly increased
 in
50 and 100 mg/lit MWVNTs
when compared with the control plants (Tables 2
and 3). Statistical data  revealed  that
all MWCNTs levels treatment  lead to significant increase in cortex thickness of  two Okra cultivars in compared with the control plants.

 In two cultivars Okra plants central
cylinder diameter of stem showed  a  remarkable 
increase  at  50 and 100 mg/lit  levels. But data revealed that, central
cylinder diameter of two cultivars of Okra plants were significantly reduced when
Okra seedling were treated with 200 mg/lit MWCNTs (Tables 2 and 3).

According  to table 2, thickness of
xylem of  stem in two cultivars
showed  remarkable  decline 
under 200 mg/lit level MWCNTs 
treatment, but in two cultivars significantly increased  of xylem
thickness was observed
 alone in 100 mg/lit MWVNTs when compared
with the control plants  (Table 3).

Microscopic  results  showed 
that  MWCNTs (50 and 100 mg/lit)  induced 
increment in phloem size in emerald cultivar, but  there  was
no  significant  difference 
among  200 mg/lit treatments   and
control plants when compared.

According to table 2, layer numbers of stem significantly increased only in cultivar bamia at 50
and 100 mg/lit MWCNTs levels, but
in emerald cultivar this parameter no significantly changed in MWCNT treatments
when compared with control plants (Table 3).

Leaf
anatomy

The
results indicated that in two cultivars  of Okra plants, MWCNT lead to a significant changes in
leaf thickness, petiole diameter, length of mesophyll cells, Central midrib diameter in compared
with the control plants. The statistical data indicate that leaf thickness (Table
4) of Okra plants generally decreased with an increase in MWCNT level. In bamia
cultivar, leaf thickness no significant increased in 50 mg/ml MWCNTs level, but
significantly decreased at the highest level (200 mg/lit) however in cultivar
emerald showed significant increase in 50 mg/lit MWCNTs level when compared with
control plants (Tables 4 and 5).

Central midrib diameter
significantly increased in cultivars bamia
and emerald only in 50 mg/lit MWCNTs level, but in high
concentration of MWCNT level, this parameter was decreased. Our results showed no significantly
decreased at the highest level (100 and 200 mg/lit) in bamia, however  in cultivar emerald showed significantly decrease  in 200 mg/ml MWCNTs level when compared whit
control plants (Tables 4 and 5).

Thickness of mesophyll layer in both Okra Cultivar showed
significant decrease at the 100 and 200mg/lit MWCNT level, but in two cultivars
no significantly increased of this parameter was observed  alone in 50 mg/lit MWVNTs when compared
with the control plants.  According  to table 3, thickness of spongy layer  of 
leaf  in two cultivars were not  significantly different with control groups (Tables 4 and 5).

In both Okra cultivars, MWCNTs did not impose significant change in the
length of petiole, however the width of petiole significantly decreased in MWCNTs (100 and 200 mg/lit)
levels in bamia, but in emerald this parameter 
increased in 50 mg/lit level when compared whit control groups(Tables 4
and 5). 

Microscopic results showed that stomata length no
significantly change with an increase in MWCNTs levels of the growth medium in
both cultivars. Also, our results showed that stomata index of two cultivars of Okra plants were
increased in  all MWCNTs treatment, but
significant increase of this parameter observed only in emerald cultivar (100
and 200 mg/lit) when compared with control groups (Tables 4 and 5). 

Root
anatomy

The effects of MWCNTs
treatment on root anatomy of two cultivars Okra seedlings are shown in table 6.  The 
statistical  data  showed root diameter in bamia cultivar significant increased in the 100 and
200 mg/lit MWCNTs level, but no significantly increased
 of this parameter was observed  alone in 50 mg/lit MWVNTs when compared
with the control plants. Also, in emerald cultivar significant increase of root
diameter was observed alone in 100 mg/lit MWCNTs level, however we observed
significant different responses of this cultivar to 200 mg/lit treatment  when compared with the control plants (Tables
6 and 7).

In two cultivar Okra plants cortex
thickness of
root showed  a  significant  increase 
at  50
and 100 mg/lit  levels, but data revealed
that, this parameter in two cultivars of Okra plants was reduced significantly when
Okra seedlings were treated with 200 mg /lit MWCNTs.

We observed that central cylinder diameter of bamia cultivar significantly increased at  all 
levels  of  MWCNTs, but in emerald cultivar this
parameter  showed significantly changes
in 50 and 100 mg/lit treatments when compared with control plants (Tables 6 and
7).

According to table 4, xylem diameter significantly
increased in cultivar emerald at 100 and 200 mg/lit MWCNTs levels, but in bamia cultivar this parameter showed
significantly increase alone in 100 mg/lit treatments when compared with
control plants. Also, significantly
decreased of phloem diameter  were
observed in 100 and 200 mg/lit MWCNTs treatments only in bamia cultivar.

The  statistical analysis, showed that  layer numbers of root no significantly
changed in both cultivars of Okra in MWCNTs treatments when compared with control plants (Tables 6
and 7).

Discussion

As can be seen in this
study, the enhancement of both Okra cultivars height and biomass (shoot and
root) were increased  after addition of
CNTs (50 and 100 mg/lit),  these
observations are in agreement with those of, Haghighi et al. (2014), who
exposed four vegetable species to  different concentration of CNTs for two weeks.
Use of CNTs stimulated water flux and uptake of 
ionic nutrients, may be explaining why growth is then stimulated, as
Tiwari et al. (2014) discovered for maize (Zea mays L.).  Liu et al. (2009)  reported that CNTs can act as molecular
channels for water, also Xiuping Wang
(2012) suggested
that o-MWCNTs can signi?cantly enhance root dehydrogenase activity, which in
turn enhances the ability of water uptake of the seedlings. A  number of 
investigations  have  indicated 
that the  expression  of 
genes encoding  an  aquaporin 
protein  considerably  upregulated 
in  plant cells  exposed 
to  multi-walled  carbon 
nanotubes (Khodakovskaya,
2012),
but in this study when a 200 mg/lit of MWCNTs was used, emerald cultivar plants
showed the least height and biomass (shoot and root). Different studies have reported
harmful effects of high MWCNT levels on grow of plant, for example, Haghighi (2014)
reported that use of high level of CNTs 
decreased of fresh and dry weight and seedling length in  radish and turnip, confirming the possibility
of a toxic effect resulting from a high level of CNTs. Parvin  Begum (2012) indicated that high level of
MWNTs caused cell  death  and 
membrane  damage  in 
red  spinach,  lettuce, 
rice,  and cucumber after 15 days
of exposure,  who suggested  that 
MWNTs  may induce  ROS 
formation,  promoting  cell 
death  and  electrolyte 
leakage in  the  different plant organs. Our  results 
suggest  a  direct 
correlation  between shoot and
root growth parameter change and 
different concentration of MWCNTs in both cultivars of Okra plants. Also,
we found that different cultivarS of Okra plants caused different responses to
MWCNTs. Plasticity in anatomical
characteristics of plants can help to their growth and development for
successful  survival in environmental
hazards.

Very little is known in
relation to  the effects of MWCNTs on plants at the
anatomical level. We evaluated the different indices anatomical and
morphological in shoot, root and leaf according
to understand, durability who sections over it organs extra below consequences
concerning MWCNT treatments. In this survey increasing of root
and shoot diameters in both cultivars that treatment by low and moderate MWCNTs
more affected by increasing of  cortex thickness and central cylinder but in
high concentration this parameter were more affected by cortex
thickness increasing. Base of figure 4 prancheme cells size of cortex area have
higher thickness in high treatments. Shihan 
Yan and et al. (2013) observed  SWCNTs 
were  appeared  in 
the  intercellular  space 
and  mainly present  in 
the  root  cortex.

Furthermore, study
of  xylem and phloem diameters  of shoot
showed increasing of diameter in low (50 mg/lit) and
moderate (100 mg/lit) MWCNT treatments, but these parameters were decreased in
high  MWCNT level. Also, in root this
fact was determined that low treatments are cause of increasing of xylem diameter. However  root phloem diameters in MWCNTs treatment had
not significant changes when compared to control plants in both cultivars.
The tissue samples of Blackberry that treated by SWCNTs-COOH (4µg/ml) showed a
vascular cambium with mature xylem tissue and the presence of phloem cells, furthermore
xylem vessels have  developed  completely 
and do not evidence the presence of cytoplasmic content (Dora Flores). Developed
metaxylem vessels in the stem play an important role for better transport of
water and minerals (Steudle, 2000).

It has suggested that
insertion of carbon nanotubes into the plant tissues, causing plant  development changes by  regulating 
gene expression  and  related 
signal  pathways  as 
well  as  physiological effects  S.  Lin C.(2009). Mariya ( showed that MWCNTs affect the expression
of genes regulating cell division and cell wall extension in treated cells,
resulted in faster growth than the unexposed control cells. Khodakovskaya  et  al(2012)
suggested the existence of different molecular mechanisms for cell growth
activation by the nanosized MWCNTs, who founded that the expression of genes
essential for cell-wall assembly/cell growth, such as extension (NtLRX1), and
for the regulation of cell cycle progression, Cyc B significantly and rapidly
induced by MWCNTs in tobacco cells.

The study of leaf anatomy showed that leaf thickness under
low MWCNT treatments was increased, but this parameter was decreased by
increasing of MWCNT (moderate and high levels) 
treatments in both cultivars.

Our
study indicated that  mesophyll and
spongy layer changes consist of  increasing
in low level and decreasing in moderate and high levels of MWCNT
treatments  in both cultivars are similar. Also, stomata
size was increased in low and moderate treatments in the bamia cultivar, however this parameter
was observed alone in low treatment in 
the emerald cultivar. Moreover, stomata index measurement showed that this
parameter was increased in moderate and high levels  only in the emerald cultivar.

Whereas carbon nanotubes
increase water absorption in plants, correlation between the content of the
absorbed water with increasing stomata index and stomata size may enhance
stomata  conductance that cause
greater  transpiration  and 
water-use. According to Melo et al.
(2007), the increase in stomatal density, coupled with the decrease in stomatal
size, would be an alternative to adequate supply of  CO2 for photosynthesis, without
excessive water loss due to stomata with smaller pores. This may be an
adaptation of  plants in response to
toxicity. Henggu ang Yuan (2011)
reported
that SWNTs can enter into intact Arabidopsis mesophyll cells, and then enter
into the organelles such as chloroplast, vacuole, mitochondria and nucleus. So,
it is possible that  changes of mesophyll
and spongy layers of Okra plants have direct correlation with MWCNTs.