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Influence of the morphology of carbon

nanostructures on the stimulated growth of gram
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Cite this: RSC Adv., 2016, 6, 43864

plant†
Shweta Tripathi, Sutanu Kapri, Abheek Datta and Sayan Bhattacharyya*

Received 14th January 2016
Accepted 27th April 2016
DOI: 10.1039/c6ra01163b
www.rsc.org/advances
The growth stimulation of gram plants (Cicer arietinum) by water dispersible carbon nanostructures (CNSs)
is found to be dependent on the latter's morphology. The <10 nm diameter coiled single-walled carbon
nanotubes (SWCNTs) are observed to be better promoters of plant growth than the
180 nm straight
open-ended multi-walled CNTs post 7 days. In comparison, the 150–200 nm thick close-ended
functionalized (CNW1) and annealed (CNW1A) carbon nanowhiskers show lesser growth stimulation.

570 nm CNW2 and
4.5 nm carbon dots (C-dots) show inferior performance. The varying growth rates
suggest a hypothesis that although C-dots and SWCNT can adapt the symplastic pathway to reach the
root's interior, all the CNSs are expected to prefer the apoplastic route and consequently widen the cell
membrane pores due to their high potential gradient. 1D hollow nanostructures with the smallest
diameters are found to be the best growth stimulators.

walled CNTs (MWCNTs) have shown minimal toxicity and

1. Introduction
With a goal to produce future tools to manipulate plant
genomes, produce better fertilizers, herbicides or insecticides
and to isolate natural products, phytology and nanotechnology
need to cross boundaries.1 So far limited reports are available
on this emerging eld of nanoparticle (NP) aided germination
and growth of plants.2–6 Although NPs namely Ag, Cu, Si, ZnO
and TiO2 show a signicant inuence on growth, bulk samples
do not demonstrate any effect.3,4 Due to bioaccumulation and
agglomeration of the NPs, in many cases the NPs such as Cu
were found to be toxic,2 whereas functionalization of the NPs
with dyes and carbohydrates enables their entrance inside the
plant cells with minimal toxicity.3,5 Different NPs have varying
effects on plants such as CeO2 promotes shoot growth but
reduces the growth of root,6 CuO inhibits the seedlings growth,7
and induce oxidative damage to DNA.8 On the other hand, CNSs
are proven attractive candidates for sub-cellular probing appli-
cations because of their advantageous biocompatibility,
mechanical robustness, ease of surface functionalization and
tunable morphologies.9–12 In the eld of plant-nanotechnology,
CNSs such as fullerene, CNTs, graphene oxide, reduced gra-
phene oxide and activated carbon are known to increase the
water retaining capacity, biomass, fruit yield and they also
demonstrate antifungal activities.13,14 Both SWCNTs and multi-
Department of Chemical Sciences, Indian Institute of Science Education and Research
(IISER) Kolkata, Mohanpur-741246, India. E-mail: sayanb@iiserkol.ac.in; Fax: +91 33
25873020; Tel: +91 9051167666
† Electronic supplementary information (ESI) available.
10.1039/c6ra01163b
enhanced growth of roots and the biomass.15–18 Especially the
water-soluble CNSs were observed to stimulate the growth and
biomass of the plants.18,19 However, water-soluble and func-
tionalized fullerenes demonstrated both positive and negative
inuences in the growth process.20,21 The positive inuences on
plant growth are highly dose dependent and largely depend on
surface chemistry of the nanostructures.22,23
It has been demonstrated that plants uptake NPs from the
environment and can transport these NPs to the shoot organs
through the vascular system, however the transport mecha-
nisms of the NPs from the root to the shoot have not been
systematically studied.24 The penetration of CNSs into the plant
roots can be correlated to a similar mechanism applicable to the
nutrients and water around the root. The water soluble nutrient
ions adjacent to the roots in the soil take part in the mass ow
diffusion process along a concentration gradient and cation
exchange through attraction to the charged surface of the cortex
cells in the roots, thus entering the selectively permeable
plasma membrane and tonoplast of the plant cells.25 Water
enters through the root hairs both via osmosis and capillary
action. The capillary forces acting on the diffused soil water
allow the upward movement of the plant water. Thus if the CNSs
are dispersed in the soil water, they can actively participate in
germination of the seeds to plant growth. The CNSs should
move with water across the cortex and endodermis before it
reaches the vascular bundles consisting of the extremely rigid
xylem and phloem, the pathway of least resistance to the plant
leaves. Water ows through both apoplast and symplast to
See DOI:
reach the xylem.26 The CNSs are expected to prefer the apo-
plastic route to reach the root's interior since the intercellular

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spaces are in 1–10 mm range. The C-dots and SWCNTs can travel
through the symplastic and apoplastic pathways to reach the
endodermal cells where the ow is prevented by Casparian
strip. These endodermal cells have several transport proteins,
which can turn the water ow again in apoplastic pathway
leading to the xylem.
It is understood that the nanocarriers such as functionalized
CNTs and fullerenes have a positive inuence of the NPs/CNSs
on the plant nutrition and growth.13 In case of CNT promoted
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plant growth,18 one of the mechanisms that was hypothesized
earlier was that the inner channel of the water soluble CNTs act
as capillaries for water uptake. However it is well known that
water conned inside
200 nm diameter hydrophilic CNTs
undergoes strong interactions with the disordered tube walls
forming a meniscus with contact angles of 5–20 .27 This means
that the hydrophilic CNTs can assimilate the water molecules
easily but the passage from one end of the CNT to the other is
innitely slow. In order to evaluate the role of size, morphology,
surface functionality of CNSs, the growth stimulation of gram
(Cicer arietinum) plant was performed with minimally toxic and
hydrophilic
6 nm diameter C-dots, air-oxidized SWCNTs, 150–
200 nm MWCNTs and solid closed ends CNWs. The hydrophilic
CNSs were compared with annealed and hydrophobic 150–200
nm diameter CNW1A and partly hydrophilic
570 nm CNW2.
The relative effects of these different CNSs were tested on the
stimulated growth of the plants.
2. Experimental section
Materials
Dextrose anhydrous puried (C6H12O6, Merck India), sodium
hydroxide (NaOH, Merck India), hydrochloric acid (HCl, 35%
Merck India), magnesium sulfate anhydrous (MgSO4 98%
Merck India), air-oxidized SWCNTs (>90% carbonaceous purity
and 4–8 wt% metal impurity, Carbon Solutions, Inc.), anodisc
aluminum oxide membranes (13 mm, 200 nm pore size,
Whatman Scientic), mustard oil (Gokul Refoils and Solvent
Ltd.), iron(III) nitrate nonahydrate (Fe(NO3)3$9H2O; Loba
Chemie), polyvinylpyrrolidone (PVP; Loba Chemie, mol. wt
40 000), inorganic membrane lter (Anatop 10, 0.2 mm, What-
man), dialysis membrane (MWCO 3500, Fischer Scientic) and
uorescein isothiocyanate (FITC, $90% HPLC, Sigma Aldrich)
were used as received. The seeds of common gram (Cicer arie-
tinum) were purchased from local market.
Characterization
The synthesis of CNWs was performed in a Carbolite wire-
wound tube furnace – single zone, model MTF 12/38/400 and
annealing of the products were performed in Lindberg/Blue
MSTF55433 tube furnace. MWCNTs were fabricated in the
latter. Transmission electron microscope (TEM) images were
recorded by UHR-FEG-TEM, JEOL, JEM 2100 F model using
200 kV electron source. The particle size of the colloidal
C-dot solution was measured by Malvern Zetasizer Nano ZS
diffuse light scattering (DLS) instrument. Ultraviolet-visible
absorption spectra were collected using a JASCO V-670
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spectrophotometer. Field emission scanning electron micro-
scope (FESEM) images were obtained in Carl Zeiss SUPRA
55VP FESEM. Energy dispersive analysis of X-rays (EDAX)
measurements were performed with the Oxford Instruments
X-Max with INCA soware coupled to the FESEM. Raman
spectra were recorded using a Raman spectrometer of Horiba
Jobin-Yvon LABRAM HR-800 with grating 1800 g mm1, 632.8
nm excitation wavelength of He–Ne laser. The Fourier trans-
form infrared (FTIR) studies were performed with Alpha FTIR
spectrometer. The carbon content in the CNS treated roots was
determined using 2400 series II CHNS/O Analyzer Perki-
nElmer, USA. The uorescent images were recorded using
Olympus Microscope IX-81. The digital images of the pheno-
types of gram plants were obtained using a Nikon Coolpix L320
camera.
Synthesis of CNSs
Synthesis of C-dots. C-Dots were synthesized as reported by
Li et al. with slight modications.28 Aqueous solutions of 1 M
dextrose and 1 M NaOH were prepared separately. Thereaer,
10 mL of dextrose and 5 mL of NaOH solutions were mixed in
a 100 mL round bottom ask. The mixture was stirred for 10
min and was transferred to a 20 mL glass vial followed by
ultrasonic treatment for 2 h. Upon completion of the reaction
a dark brown suspension was obtained. The dark brown
suspension (15 mL) obtained from the reaction between
dextrose and NaOH were rst adjusted to pH 7 with 0.5 M
hydrochloric acid, and 80 mL ethanol was added drop by drop
into the solution under stirring. Thereaer, the solution was
treated by adding 10 wt% of MgSO4, stirred for 20 min and
stored for 24 h to remove the salts and water. The “smaller”
C-dots were collected by centrifuging the larger nanoparticles at
12 000 rpm for 15 min. Solvent was evaporated from the
supernatant by rotary evaporator and the dried C-dots were
dispersed in ultrapure water for further experiments.
Synthesis of MWCNTs. MWCNTs were synthesized as per
a previously reported technique.29 The commercial aluminum
oxide membranes were subjected to chemical vapor deposition
(CVD) in ethylene gas at 670  C for 4 h. The carbon coating
around the
180 nm pores of the membranes resemble the
MWCNT walls. The carbon layer on top of the membranes
blocking the pores was removed by partial air oxidation at 450

C for 50 min. Thereaer the MWCNTs were released from the
aluminum oxide template by reuxing the carbon coated
membranes under constant stirring in 30 mL of 1 M NaOH in
a 100 mL round bottom ask at 90  C for 4 h. The alkaline
solution of pH
12 was removed by ltration through a poly-
ester membrane. During the process of ltration, the MWCNTs
were continuously washed with doubly distilled water several
times. To ensure the complete removal of NaOH from the
product, pH of the ltrate solution was checked aer each
washing. Once the ltrate solution becomes neutral, the
removal of NaOH from the MWCNTs was conrmed. The
MWCNTs were collected and dried for further use.
The three different types of CNWs were synthesized as per
the previous report:30
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CNW1. 1.5 mL of mustard oil and Fe(NO3)3$9H2O catalyst
were lled within a Ham-Let Union (SS316) 3/800 reactor. The
catalyst : mustard oil mass ratio was maintained at 0.02. The
tightly capped reactor was heat treated at 850  C for 10 h, fol-
lowed by slow cooling to room temperature. The grey colored
product was bath-sonicated in ethanol and the dispersion was
washed with 5% HNO3 and deionized water to free CNW1 from
the leover catalyst particles.
CNW1A. 0.02 g of the catalyst free CNW1 was annealed at
1500  C for 2 h under Ar gas to prepare the graphitized CNW1A,
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free of any organic functional groups.
CNW2. In this case, the mustard oil and Fe(NO3)3$9H2O
catalyst inside the Ham-Let Union (SS316) 3/800 reactor was heat
treated at 600  C for 10 h, followed by annealing at 1500  C for
2 h under Ar gas to prepare the graphitized CNW2 with larger
diameter.
CdSe NP coated SWCNT. The aqueous dispersion of CdSe
NPs were synthesized by a reported method.31 The CdSe NPs
were stirred with 0.04 g of PVP in 10 mL de-ionized water and
bath-sonicated for 1 h. Thereaer, 200 mg of commercial
SWCNTs were added to the CdSe–PVP solution and stirred again
at room temperature for 10 h. Aer the reaction was complete,
the product was ltered using syringe lter with pore size 0.2
mm, washed thrice with ethanol and distilled water to remove
excess uncoated CdSe NPs, followed by drying under high
vacuum and re-dispersion in water for further studies.
Synthesis of FITC labeled SWCNTs. To synthesize FITC
labeled SWCNTs, 5 mg of SWCNTs and 0.5 mg FITC was mixed
with 20 mL phosphate buffered saline (PBS) by ultrasonication
for 30 min and the black suspension was mechanically stirred at
room temperature in dark for 4 h. Excess unbound FITC was
removed by dialysis for 2 days, washed with PBS and water
thrice and dried in vacuum and resuspended in water at 4  C for
further studies.
CNS stimulated plant growth
Seeds of Cicer arietinum were sprouted in a dry location under
ambient conditions to monitor the growth rate of gram plants
treated under different concentrations. Before germination the
seed surfaces were sterilized with 70% ethanol for 10 min,
soaked in water overnight and incubated at 25  C.
CNS dispersions in concentrations of 0.43–10 mg L1 were
used for germination in comparison to a blank solution without
CNS. Aer the seeds germinate and arrive at the seedling stage,
they were transferred to 7 mL test tubes. The CNSs were bath-
sonicated for 2 h in water to make well-dispersed suspen-
sions. The aqueous CNS dispersions were sonicated in water
bath for 2–5 h to make well-dispersed suspensions. Sprouted
seeds were then placed at the mouth of test tubes which were
closed with paraffin lm to prevent seeds from entering the test
tube and get in direct contact with the aqueous solution. This
arrangement also minimizes the rate of evaporation and tran-
spiration. Even if evaporation of water could not be stopped, the
conditions will be the same in all the test tubes with different
CNS concentrations, and therefore will not affect the compar-
ative results. To compare the growth of plants with respect to
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root and shoot lengths with different CNSs (C-dot, SWCNT,
MWCNT, CNW1, CNW1A and CNW2) and control, 7 different
sets of 30 vials were used. From the stock solution of 150 mg L1
of each CNS, solutions of 20, 50, 100, 200 and 500 mL were added
in 7 mL distilled water, which correspond to concentrations of
0.43, 1.06, 2.11, 4.16 and 10 mg L1, respectively. All plants were
grown hydroponically which is a subset of hydroculture where
growth is performed in water medium in the absence of soil. In
the rst row-control set, seeds were grown without any CNS i.e.
only grown in 7 mL of double distilled water. In the next 6 sets
with 6 different carbon nanostructures, 30 vials of 5 different
concentrations were arranged for each nanostructure. Double
distilled water was used in all the experiments. A cm scale ruler
was used to measure the root and shoot lengths. For compar-
ison of the root and shoot length, the gram plants were moni-
tored for 7 days. The temperature was maintained at 28–34  C,
relative humidity at 71.5%, photoperiod of 16 h light and 8 h
dark and the seeds were allowed to germinate and grow for 7
days.
CNS stimulated plant growth with aqueous salt solutions
104 M aqueous solutions of NH4NO3, AgNO3, CaCl2 and FeCl3
were prepared and seeds of Cicer arietinum were grown in the
presence of 2.11 mg L1 CNSs by the method detailed above.
Sample preparation for microscopic analysis of embedded
nanostructures inside the plant
Aer 7 days growth, the roots of the gram plants were cut into
small pieces. These pieces were then transferred to Sylgard Petri
dish and dehydration was carried out twice for 20 min each in
30, 50, 70, 90 and 100% ethanol. Aer drying for 1 day, 0.5–1
mm thick sections were cut with automated sharp razor. The
thin sections were mounted on carbon tape for FESEM analyses.
Fluorescence microscopy
For uorescence microscopy, thin longitudinal sections were
mounted on glass slides with cover slip and glycerol. The C-dots
were monitored using the uorophore TRITC (tetramethyl
rhodamine isothiocyanate) with excitation and emission wave-
lengths at 557 and 576 nm, respectively. For detection of
SWCNTs inside the root sections, the concentration of FITC in
SWCNTs was measured by UV-vis spectrometer at absorbance
495 nm aer subtracting the absorbance of SWCNTs at 495 nm.
3. Results and discussion
Characterization of CNSs
Fig. 1 shows the morphology of the CNSs. From the low reso-
lution TEM image and the diameter histogram obtained from
the DLS measurement in Fig. 1a and b respectively, it is evident
that the spherical C-dots are
4.5 nm in diameter. These dots
could not be imaged at higher magnication due to burning
under the electron beam and organic molecules were evidenced
trapped inside the dots by 13C NMR studies (not shown). Also,
concentric graphitic layers were not observed at lower magni-
cation and so these dots are different from carbon nano-
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Fig. 1 (a) TEM image, (b) diameter histogram from DLS measurement and (c) UV-vis absorption spectrum of C-dots. Inset shows the optical
images under ambient (left) and UV (right) light of wavelength 279 nm. FESEM images of (d) SWCNT bundles, (e) MWCNTs, (f) CNW1, (g) CNW1A
and (h) CNW2. (i and j) Room temperature Raman spectra of the carbon nanostructures.

onions.19,32–34 The C-dots could be easily dispersed in water
without the need of further ultra-sonication and provides
a transparent view under ambient light (Fig. 1c). The optical
images of the aqueous dispersion of C-dots under UV light (365
nm) shows a typical blue color.28,35 The UV-visible absorption
spectrum of the C-dots shows the p–p* transition centered at
279 nm originating from the extended p–p conjugation. The
weak hump at 358 nm corresponds to the n–p* transition. The
commercial SWCNTs in Fig. 1d appear to have a diameter less
than 10 nm and SWCNT bundles are coiled forming a network.
The MWCNTs prepared by CVD of C2H4 (g) are straight, 30–40
mm in length and
180 nm inner diameter (Fig. 1e).29 Unlike
SWCNTs and MWCNTs, the CNWs prepared by dry autoclaving
of mustard oil at 850  C are not hollow. CNW1 has a diameter
similar to the outer diameter of MWCNTs.30 Dilute HNO3
washing was effective in removing any Fe–O particles used in
the catalytic growth of CNWs which makes them minimally
cytotoxic to the plant cells. The hydrophilic CNW1 (Fig. 1f) was
annealed to obtain the hydrophobic CNW1A (Fig. 1g) with
shortened length but with a similar diameter as that of CNW1.
The partly hydrophilic CNW2 was obtained by annealing the
initially autoclaved product at 600  C. CNW2 have larger
diameter
570 nm with spiraled carbon layers transversely
oriented along the length (Fig. 1h). It is noteworthy, that CNW1
(before annealing) is branched unlike the annealed CNW1A.
Diameter histograms of the CNSs (Fig. S1†) show a moderate
size distribution. The impurity elements present in the CNSs
were found by EDAX as shown in Table 1. The excess oxygen in
the CNSs is likely due to the organic functional groups as well as
the presence of the metal impurities in the form of their oxides.
Fig. 1i and j show the Raman spectra of the CNSs. The peaks at
1314–1357 cm1 and 1573–1600 cm1 are the characteristic
disordered carbon (D; Raman-inactive A1g mode) and graphitic
carbon (G; Raman active E2g mode) bands.36 The D/G intensity
ratios of C-dots, SWCNT, MWCNT, CNW1, CNW1A and CNW2
are 0.45, 0.42, 0.95, 1.13, 0.98 and 0.93, respectively. C-Dots

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Table 1 EDAX studies of the CNSs. The elemental compositions are in weight% and taken as an average of 5 spots over each sample. Support
represents the Al stub on which the CNSs were dispersed. The elements Al, Si, Fe and Cu originate from the stub

Elements Support C-Dot SWCNT MWCNT CNW1 CNW1A CNW2

C 12.6(0.9) 35.0(0.6) 50.4(0.4) 48.4(0.2) 46.7(0.3) 88.3(0.2) 84.3(0.2)

O 5.7(2.0) 53.8(0.5) 40.5(0.3) 31.3(0.1) 34.6(0.2) 10.0(0.1) 14.0(0.2)
Al 76.4(2.0) 9.1(2.0) 0 0 0.6(0.1) 0 0
Si 0.3(0.2) 0.11(0.04) 0 0 0 0 0
Fe 0.5(0.1) 0 0 0 0 0 0
Cu 4.5(0.3) 1.0(0.4) 0 0 0 0 0
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Na 0 0 4.4(0.3) 7.5(0.3) 9.3(0.4) 0.7(0.1) 0.3(0.0)

K 0 0 0.8(0.4) 0.4(0.2) 3.1(0.5) 0 0
Ca 0 0 2.0(0.2) 1.2(0.1) 0.9(0.1) 0 0.2(0.0)
Co 0 0 0 0.6(0.2) 0.2(0.0) 0.7(0.2) 0.8(0.2)
Mg 0 0 0.6(0.1) 0.6(0.1) 0.2(0.0) 0 0
S 0 0 0 0.3(0.1) 0.5(0.1) 0 0.2(0.0)
F 0 0 0 8.6(0.3) 0 0.6(0.2) 0
Cl 0 1.0(0.4) 1.3(0.1) 1.1(0.2) 3.9(0.1) 0.4(0.1) 0.2(0.1)

contain a signicantly lower fraction of disordered or sp3
hybridized carbon. Interestingly the Raman spectrum of
SWCNT shows signicant crystallinity as evident from the sharp
bands in Fig. 1i and also highly graphitic in spite of them being
air oxidized. On the contrary, MWCNTs being synthesized at
670  C are partly graphitic. The graphitization of the annealed
CNW1A and CNW2 is slightly more as compared to the as-
prepared CNW1 (Fig. 1j). In fact, the increasing presence of
disordered carbon implies a signicant fraction of sp3 hybrid-
ized carbon and more surface functionalization.
The functional groups on the CNSs were investigated by FTIR
spectroscopy (Fig. 2). SWCNT and CNW1A were found to be the
least functionalized. The bands above 3600 cm1 correspond to
the free O–H stretching vibration, from the adsorbed water
molecules in MWCNT and CNW2. The H-bonded O–H stretch-
ing vibrations at 3400–3500 cm1 were observed in C-dot,
MWCNT, CNW1 and CNW2. In fact, CNW2 was expected to
contain minimum organic functional groups due to their
treatment at high temperatures. In C-dot and CNW1, the bands
at 3046–3079 and 2829–2926 cm1 are the ]C–H and –C–H
stretching vibrations, respectively. The 1740 cm1 band in C-dot
is the C]O stretching vibration, which implies the presence of
carboxylic acid group. The band at 1654–1698 cm1 in C-dot,
MWCNT and CNW2 is the C]C stretching vibration whereas
the band of 1510–1586 cm1 in all the CNSs corresponds to the
aromatic C]C stretching. These bands are signatures of the sp2
bonded carbon present in the CNSs. Also, considering the broad
spectrum of CNW1, the bands between 1270 and 860 cm1
consists of –C–C– and ]C] groups. The fractional sp3 carbon
in C-dot is evident from the bands at 1364 and 1080 cm1 which
are due to the C–O–C and C–O stretching vibrations. In all the
spectra in Fig. 2, the closely spaced bands at 2320–2400 cm1
are due to the asymmetric stretching and rotational splitting of
gaseous CO2 present inside the measurement chamber. From
the above data it is clear that all the CNSs should be hydrophilic
due to the presence of –COOH and –OH groups except SWCNT
and CNW1A. Air oxidation is less effective than acid function-
alization, evident from the minimally functionalized SWCNT as
compared to the other unannealed CNSs.

CNS stimulated plant growth

The cytotoxicity of the CNSs were found to be comparable and all
are minimally toxic to the living biological cells.9,30 The roots were
kept in direct contact with the CNSs in well dispersed aqueous
unstirred solutions, without any sedimentation for over
a week.18,19 The impacts of CNSs on the plant germination and
growth are evident from the different root and shoot lengths as
demonstrated in Fig. 3 and 4 and the results tabulated in Tables 2
and S2† with 2.11 mg L1 CNSs. All experiments were performed
seven times with replicates of 10 plants and at one time with
replicates of 30 plants. In both cases the phenotypic effect was
the same. We also performed statistical analysis (ANOVA test) to
make sure that the observed phenotypic effects are signicant.
The p-value # 0.05 is statistically signicant. From Fig. 3a it is
clear that in spite of the smallest size of C-dots, both hollow and
closed 1D structures of carbon provide the best stimuli in
Fig. 2 FTIR spectra of the CNSs. increasing the length of the roots up to the 7th day. Among them,

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Fig. 3 The influence of different CNSs on the growth and development of gram plants up to 7 days. Average lengths of the growing (a) root and
(b) shoot of the plants. The average length was calculated based on summation of all the 30 plants in different concentration upon the number of
replicates. Control indicates the germination in the absence of any nanostructure. Variation of the final (c) root and (d) shoot lengths of the gram
plants as a function of concentration of the carbon nanostructures used for germination. The standard deviations in all cases are in the range
0.6 to 0.9 cm, detailed in Table 2.

Fig. 4 Phenotypes of gram plants after 7 days with (a) 2.11 and (b) 10
mg L1 of different CNSs: (i) control, (ii) C-dot, (iii) SWCNT, (iv)
MWCNT, (v) CNW1, (vi) CNW1A and (vii) CNW2.
SWCNT gives the best result followed by MWCNT and CNW1,
CNW1A and CNW2 follow suit in chronological order. Similar
order is followed in growth of the shoots (Fig. 3b), however in this
case C-dots promote the shoot growth better than CNW2. All
experiments were performed with 2.11 mg L1 CNSs and the
corresponding root and shoot lengths were calculated based on
an average of 10 plants. The representative phenotypes of gram
plants in Fig. 4a show a close match with the above order of CNSs
in increasing the average length of root and shoot. When the
concentration of the CNSs is increased to 10 mg L1, the CNTs
are still the superior nanostructures for plant growth and C-dots
are observed to be better than the CNWs (Fig. 3c and d and 4b).
The growth rates per day with different CNSs over the course of 6
days also show decent consistency in continuous growth with
SWCNTs and MWCNTs. In a nutshell, the CNSs with hollow
tubular structures provide a superior growth stimulus for the root
and shoot, irrespective of their inner and outer diameter. The
smallest C-dots promote root growth better than the closed,
branched and larger CNWs at higher concentrations. At optimum
concentrations, the length of the root sections is observed to be
more with the CNWs than the C-dots. However at all concentra-
tions, C-dots move faster in the transpiration stream than the
annealed CNW1A and CNW2 to result in higher shoot growth.
From Fig. 3 and 4, the following inferences can be drawn.
With 2.11 and 10 mg L1 concentration of CNSs, both SWCNT

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Table 2 Average lengths of the growing root and shoot of the plants with 2.11 mg L1 CNSs up to 7 days and lengths of roots and shoots as
a function of concentration (conc.) of the CNSs on the 6th day. Control indicates the germination in the absence of any nanostructure

Day Control C-Dot SWCNT MWCNT CNW1 CNW1A CNW2

Average root length (in cm)

1 1.4(0.6) 1.6(0.7) 1.8(0.6) 1.8(0.6) 1.8(0.7) 1.7(0.6) 1.9(0.6)
2 1.8(0.9) 2.0(0.9) 2.7(0.9) 2.8(0.8) 2.4(1.0) 2.2(0.8) 2.0(0.7)
3 2.2(0.8) 2.3(0.8) 3.3(0.8) 3.2(0.9) 2.8(0.8) 2.6(0.9) 2.4(0.8)
4 2.5(0.9) 2.6(0.8) 3.5(0.8) 3.4(0.9) 2.9(0.8) 2.6(0.9) 2.8(0.8)
5 2.7(0.9) 2.7(0.8) 3.7(0.8) 3.5(0.9) 3.3(0.8) 2.7(0.9) 2.8(0.8)
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6 2.7(0.9) 2.7(0.8) 3.7(0.9) 3.5(1.0) 3.3(0.7) 3.0(0.9) 2.8(0.9)

Average shoot length (in cm)

1 0.6(0.4) 1.0(0.4) 0.6(0.5) 0.8(0.4) 0.7(0.4) 0.6(0.3) 0.6(0.5)
2 1.5(0.6) 2.0(0.4) 1.9(0.4) 1.8(0.6) 1.6(0.4) 1.5(0.3) 1.7(0.4)
3 2.4(0.6) 3.2(0.5) 3.5(0.5) 3.4(0.9) 2.9(0.5) 2.4(0.5) 2.6(0.4)
4 2.9(0.8) 3.4(0.6) 4.1(1.0) 3.8(1.0) 3.6(0.7) 3.4(0.8) 2.9(0.8)
5 3.0(0.9) 3.5(0.4) 4.4(0.8) 3.8(1.0) 3.6(0.6) 3.7(0.9) 3.0(0.9)
6 3.0(0.8) 3.5(0.4) 4.4(0.9) 3.8(1.0) 3.6(0.6) 3.7(0.9) 3.0(0.8)

Conc. (mg L1) Control C-Dot SWCNT MWCNT CNW1 CNW1A CNW2

Root length on 6th day (in cm)

0.43 — 2.9(0.5) 3.1(0.5) 3.2(0.9) 4.1(0.5) 2.2(0.1) 3.7(0.3)
1.06 — 2.9(0.2) 3.2(0.5) 3.3(0.5) 4.2(0.1) 2.3(0.1) 3.8(0.1)
2.11 — 1.9(0.3) 4.4(0.1) 3.4(0.3) 3.2(0.1) 3.5(0.3) 3.2(0.2)
4.16 — 2.8(0.5) 4.5(0.5) 3.3(0.5) 2.1(0.1) 3.4(0.4) 2.4(0.1)
10.0 — 3.4(0.3) 5.9(0.2) 5.2(0.3) 2.0(0.2) 2.1(0.3) 2.4(0.6)

Shoot length on 6th day (in cm)

0.43 — 3.5(0.2) 4.0(0.4) 4.1(0.5) 4.3(0.6) 3.1(0.3) 3.2(0.3)
1.06 — 3.5(0.3) 4.0(0.4) 4.7(0.5) 4.3(0.6) 3.1(0.5) 3.2(0.4)
2.11 — 3.7(0.2) 4.0(0.3) 3.9(0.3) 4.1(0.5) 3.8(0.3) 2.9(0.4)
4.16 — 4.4(0.2) 4.4(0.4) 4.7(0.5) 3.8(0.4) 4.0(0.5) 2.8(0.4)
10.0 — 4.5(0.2) 5.7(0.5) 5.1(0.4) 2.1(0.4) 3.9(0.5) 2.4(0.3)

and MWCNT enter the root in larger quantities in a given time.
On the contrary, with both concentrations of C-dots, the entry
rate into the roots is comparable, but introducing higher
concentration of C-dots result in longer shoots. Among the
whiskers, CNW1A has the highest rate of entry into the roots
than the branched CNW1 and the thicker and annealed CNW2.
The least functionalized CNW1A perform next to the CNTs in
promoting the root and shoot growth. However, at a concen-
tration of 10 mg L1, the performance of C-dots rises next to the
CNTs. Fig. 5 gives the microscopic view of the 4.16 mg L1 CNS
treated root and shoot sections aer 7th day germination, where
the C-dots, SWCNT, CNW1, CNW1A and CNW2 embedded
inside the root sections are shown by arrows. Except MWCNT,
none of the CNSs could be clearly evidenced inside the shoots.
In addition, the uorescence microscopy images of the C-dot
treated root sections in Fig. 6 show how C-dots invade the
xylem vessels from a few hours to the 5th day of germination.
Aer 4 h of germination, it is observed that the C-dots diffuse
into the xylem in a cluster and aer 24 h, they spread inside the
root. Aer the 5th day (Fig. 6f), the C-dots could be seen
uniformly distributed throughout the root. Similar experiments
were performed with FITC uorescent dye labeled SWCNTs,
where the increase in uorescent patches from 2 to 4 h of
growth indicates the internalization of the SWCNTs inside the
root sections (Fig. 6g–l). To further conrm the entry of
SWCNTs inside the roots, the plants were grown in their
aqueous dispersions of CdSe NP coated SWCNTs. Elemental
mapping of the root section of these plants in Fig. 5 shows the
presence of both cadmium and selenium which conrms the
entry of SWCNTs inside the roots. To exclude the possibility of
any free CdSe NPs in the mapping, any excess of CdSe NPs were
repeatedly washed by bath sonication, ltered through 0.2 mm
pores, dried and redispersed in water. The absence of any
precipitate from the CdSe NP coated SWCNT dispersion over
two months demonstrated its stability, further conrmed by
reproducible absorption spectra with the peak maximum at 535
nm (Fig. S3†).
Mechanism of CNS transport
The above experimental results demonstrate that morphology
of the CNSs play a major role than the extent of chemical
functionality of the CNSs in promoting the plant growth. The
growth of the roots and shoots obviously depend on the trans-
port of water and mineral nutrients mostly K+, Ca2+, Mg2+, NH4+,
H+, PO43, NO3, SO42 and organic matter through the
xylem.37,38 When the plants were grown with the CNSs in the
aqueous salt solutions, the root and shoot lengths were slightly

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Fig. 5 FESEM images from areas of the thin sections of root and shoot
after the 7th day germination with 2.11 mg L1 of different CNSs.
Elemental mapping of root section of a plant treated with SWCNTs
coated with CdSe NPs.
higher as tabulated in Tables S4 and S5† and shown in Fig. 7.
NH4NO3, AgNO3, CaCl2 and FeCl3 supply the necessary ions
such as NH4+, Ag+, Ca2+, Fe3+, NO3 and Cl ions which play
essential roles in several physiological processes. Although, the
salts incorporated in the aqueous media vary the growth rate of
the roots and shoots, the order of their performance varies with
different ions irrespective of the presence or absence of surface
functional groups on the CNSs. Hence, whether these ions bind
to the CNSs for plant growth is not evident. Moreover, the CNTs
seldom act as capillaries for transport of necessary ions through
Fig. 6 (a–f) Fluorescence microscopy images of the control and C-dot treated longitudinal section of the roots visualised from outer layer
epidermis to inner layer vascular bundle after (a and b) 4 h, (c and d) 1st day and (e and f) 5th day growth. (g–l) Fluorescence microscopy images of
the control and FITC labeled SWCNT treated longitudinal root sections after (g and h) 2 h, (i and j) 1st day and (k and l) 4th day growth.
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the inner CNT channels since the entry and exit mechanisms of
water require an injected force through the straight open-ended
MWCNTs. Also the SWCNTs are coiled and the narrow channels
create barriers at the nanotube ends.39 Fig. 8 shows the
proposed hypothesis for the translocation pathway of CNSs
inside the root which can adapt either the apoplastic or sym-
plastic pathways to reach the vascular bundle. The chosen
pathway depends on the size of the CNSs. If dimensions of the
pores in the root system are comparable to the average diameter
of the CNSs, the CNSs will have a high chance to block those
openings. Rather for efficient transport of the CNSs, the diam-
eter of the intercellular spaces and connections should be 10
times more than the size of the CNSs. Whenever the CNSs enter
through the root hair from the soil, the symplastic and trans-
membrane pathways can be followed only by C-dots and
SWCNT since the intercellular connections are 50–60 nm wide
and cell membrane pores are 10–150 nm wide. As it is well
known that water ows through the apoplast and symplast to
reach the xylem, the CNSs in this study are expected to prefer
the apoplastic route to reach the root's interior since the
intercellular spaces are in the 1–10 mm range.40 Aer reaching
the endoderm, the ow of CNSs is prevented by Casparian strip.
However, the endodermal cells have transport proteins which
facilitate the ow of water again in the apoplastic pathway to
reach the xylem. Due to their potential gradient, the CNSs can
increase the pore size on the cell membrane and force their way
inside the roots (Fig. 8). The rupturing and hence widening of
the 10–150 nm pores is the best possible speculation for the
stimulated root growth by MWCNTs, CNW1 and CNW1A.
The differing growth rates with SWCNTs and MWCNTs as well
as the stimulated growth by the CNWs point towards an entry
mechanism of the CNSs into the root interior. The CNTs and
CNWs have 1D morphologies. The MWCNTs are mostly found
aligned inside the root section in Fig. 5 whereas the CNWs exist
as aggregates. The close proximity of the 1D CNSs except C-dots
can lead to a directional ow faster than the isotropic particles
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Fig. 7 Growth of gram plants up to 7 days in the presence of intentionally added ions. Average lengths of the growing shoots in (a) NH4NO3, (b)
AgNO3, (c) CaCl2 and (d) FeCl3, and roots in (e) NH4NO3, (f) AgNO3, (g) CaCl2 and (h) FeCl3. Control indicates the germination in the absence of
any nanostructure. The standard deviations are in the range 0.1 to 0.9 cm and detailed in Table S4.†
Fig. 8 Schematics based on a proposed hypothesis showing the
anatomical and translocation pathways inside the root and the sym-
plastic and apoplastic pathways for the CNSs to reach the vascular
bundle.
such as C-dots. Also, the close proximity of the CNTs and CNWs
can create a complex network and the interconnected space in
between the aligned 1D CNSs can act as a capillary to ow to the
root interior. The situation is however different at high concen-
tration of the CNSs. At a CNS concentration of 10 mg L1 (Fig. 3c
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and d and 4), a large collection of CNWs essentially block the
pores and in this situation, C-dots being the smallest in dimen-
sions seems to stimulate the growth process faster than the
CNWs. The CNTs however still retain their performance owing to
the smaller size of coiled SWCNTs and the straight open-ended
MWCNTs. Also, it is noteworthy that in spite of CNW1A being
lesser surface functionalized than CNW1, stimulate the plant
growth faster since the latter is branched unlike the former,
which in turn can block the pores in the apoplastic pathway at
higher concentrations.
4. Conclusions
The stimulated growth of gram plants was found to depend on
morphology of the CNSs. The 1D CNSs promote growth of the
roots and shoots faster than the
6 nm spherical C-dots. One of
the least functionalized and highly crystalline SWCNTs stimulate
the growth of roots and shoots more than the
180 nm straight
open-ended 30–40 mm long MWCNTs, followed by the 150–200
nm thick CNW1 and CNW1A. The least penetration inside the
roots was observed for
570 nm thick CNW2. The experimental
observations show that while C-dots and SWCNT can adapt the
symplastic pathway, all the CNSs are likely to enter the xylem
vessels aer travelling through the apoplastic path. Thereaer
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due to a potential gradient, the CNSs likely rupture and widen the
10–150 nm pores in the cell membrane to reach the vascular
bundles. The complex network formed by the aligned 1D CNTs
and CNWs, thus providing a pseudo-capillary effect allows their
superior entry over the spherical “small” C-dots. Our work
demonstrates that 1D bio-compatible hollow nanostructures are
excellent promoters of plant growth and describes a tentative
transport mechanism of nanomaterials across the xylem. The
optimization of the structural features of nano-stimulators will
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be useful in agriculture under arid conditions.
Acknowledgements
The authors thank Ms Ganga Halder for supplying the aqueous
colloidal solution of CdSe NPs. The authors thank DST-SERB for
the nancial support under sanction no. SR/S1/PC-28/2011. ST
thanks Department of Science and Technology – Science &
Engineering Research Board (DST-SERB), Government of India
under grant no. SR/FT/LS-04/2011 for her fellowship. SK and AD
thank Council of Scientic and Industrial Research (CSIR) and
University Grants Commission (UGC), New Delhi for their
fellowships, respectively.
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