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Ecological Engineering 90 (2016) 2534

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Ecological Engineering
journal homepage: www.elsevier.com/locate/ecoleng

Vetiver grass: An environment clean-up tool for heavy metal


contaminated iron ore mine-soil
Ritesh Banerjee a,1 , Priya Goswami a,1 , Khanindra Pathak b , Anita Mukherjee a,
a
Cell Biology and Genetic Toxicology Laboratory, Centre of Advanced Study, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road,
Kolkata 700019, India
b
Department of Mining Engineering, IIT, Kharagpur, 721302 India

a r t i c l e i n f o a b s t r a c t

Article history: Aims: Vetiver grass Chyrsopogon zizanioides (L.) Roberty is a known plant tolerant to heavy metals and
Received 31 July 2015 its use as an alternative method for rehabilitation of iron ore mine-soil has been investigated.
Received in revised form 5 November 2015 Methods: A pot experiment was performed over a period of three months and the performance of vetiver
Accepted 26 January 2016
grass on heavy metal-rich soil from iron ore mine was assessed. The presence of the following heavy
Available online 11 February 2016
metals Fe, Mn, Zn, Cu, Pb, Ni, Cr and Al were assessed in mine-soil and their uptake by vetiver grass
system was evaluated. In addition, oxidative and genotoxic stress were used to monitor the changes over
Keywords:
the period.
Genotoxicity
Heavy metals
Results: Physico-chemical characterization and metal analysis revealed that vetiver plant is a good
Iron ore mine-soil phytostabilizer. The plant is a potential accumulator of heavy metals and the root parts of the plant
Phytoremediation accumulated higher metal concentrations than the shoot. The high proportions of metals inhibited
Vetiver system the chlorophyll content of leaves, but stimulated the accumulation of proline and lipid peroxidation.
Enhanced activities of catalase (CAT), guaiacol peroxidase (GPOD) and glutathione (GSH) implied that
different mechanisms existed to detoxify reactive oxygen species (ROS) in the shoot of the plants. Geno-
toxicity assays demonstrated the absence of genetic instability or DNA damage in the plants.
Conclusion: Vetiver can be used as an excellent candidate for remediation and restoration of iron-ore
mine spoil-dumps.
2016 Elsevier B.V. All rights reserved.

1. Introduction the broken areas near mining site. The iron ore spoil-dump often
poses a major management problem due to its volume and the pres-
India is one of the leading countries in the production of iron ore. ence of pollutants, mainly heavy metals like Fe, Mn, Cr, Cd, Pb, Zn,
Surface mines are the major source of iron ore in India. Over the last Ni, Cu etc. (Verma et al., 2012).
four decades, as a result of mining activity, a large amount of iron Phytoremediation technology is one of the best and ecofriendly
mine spoil-dumps are deposited in these mines. A number of mines alternative management to vegetate the dumpsite area. This can
are now on the verge of completing their mining life. Thus after the serve the purpose of both stabilization of mine spoil-dump site as
closure of the mine, generation of alternative economic activities in well as provide a pleasing landscape. Selection of appropriate plant
the mining areas and enhancing the environmental condition near species for successful site reclamation and in phytoremediation
the mining site is a demanding problem to be addressed under the efforts is important. Plants are known to remove toxic heavy metals
corporate social responsibility. and other elements from contaminated soil via their accumulation
Various steps for reclaiming the mine sites have been under- in harvestable shoot parts (i.e. phytoextraction) or to immobilize
taken by the environmental management. However, there are still heavy metals in soils or sediments by root uptake and transloca-
certain problems like stabilization of spoil-dump slope, control of tion into shoots (i.e. phytostabilization) (Ahmad et al., 2010; Gupta
soil erosion, sedimentation loads on the catchment areas of the and Sinha, 2009; Pedron et al., 2011).
river system near the mining area and enhancing the soil quality of Vetiver grass is regarded as one of the most versatile crops
of the millennium based on its innumerable novel and mar-
velous qualities (Khan, 2006). Vetiver grass Vetiveria zizanioides L.
Corresponding author. recently reclassied as C. zizanioides (L.) Roberty, belonging to the
E-mail address: anitamukherjee28@gmail.com (A. Mukherjee). family Poaceae is a dense, bunch-type grass with stiff stem, and
1
These authors contributed equally to this work. an extremely strong root system (up to 4.6 m deep), and grows to

http://dx.doi.org/10.1016/j.ecoleng.2016.01.027
0925-8574/ 2016 Elsevier B.V. All rights reserved.
26 R. Banerjee et al. / Ecological Engineering 90 (2016) 2534

the height of over 2 m (The Wealth of India, 1976). India is the pri- 2.4. Phytoextraction ability
mary center of origin and dispersion of vetiver (Lavania, 2000). It
grows on all continents in the tropical and subtropical regions, tol- Two important parameters, translocation factor (TF) and
erates a wide range of soil pH and low fertility. The plant is highly bioaccumulation factor (BF) were used for evaluating the phytoex-
tolerant to elevated levels of heavy metals such as arsenic, cad- traction ability of vetiver according to the method of (Qihang et al.,
mium, copper, chromium, lead, mercury, nickel, selenium and zinc 2011).
(Roongtanakiat et al., 2007; Truong and Baker, 1998; Truong and
Hart, 2001). Vetiver plants growing on y-ash soil amendments (0, TF = [Metal]shoot /[Metal]root
25, 50 and 100%), over a period of 18 months, were capable of sta-
bilizing the metals lead, arsenic and cadmium in the roots (Ghosh BFshoot = [Metal]shoot /[Metal]soil
et al., 2015).
The present study is a part of a eld experiment to restore and BFroot = [Metal]root /[Metal]soil
revegetate the iron ore mine site using biological interventions. It
involves the use and assessment of Vetiver system in phytore-
mediating iron ore mine-soil in a controlled laboratory condition. 2.5. Performance of the vetiver plants under the inuence of mine
For better understanding and interpretations of results, elemental soil
analysis, uptake of heavy metals by vetiver grass system, oxidative
and genotoxic stress were carried out. The performance of the vetiver plants under the inuence
of mine-soil was assessed by estimating the total chlorophyll
content and anatomical changes in the root and shoot of the
2. Materials and methods plants.
Total chlorophyll content was determined according to the
2.1. Collection of sample method of Arnon (1949). Fresh leaves (200 mg) were cut and kept
overnight in 80% acetone at 4 C. The extract was centrifuged at
Soil sample was collected from the spoil-dumpsite at Joda East 10,000 g for 5 min. Absorbance of the supernatant was read at
Iron mine (22 0 11 N, 85 26 28 E, Fig. 1), Odisha, India, in plastic 645,663 nm using a spectrophotometer. Total chlorophyll content
drums, dried under sunlight and stored in the laboratory at room was calculated by the following formula:
temperature (28 1 C). Vetiver plants (variety CIMAP Khus-40)
were collected from the Central Institute of Medicinal and Aromatic Total chlorophyll mg/g tissue = [20.2(A645 ) + 8.02(A663 )]
Plants (CIMAP), Lucknow, India, and grown in the experimental
[V/(1000 W )]
garden of the department of Botany, University of Calcutta, for
acclimatization and multiplication. where A = absorbance at specic wavelength; V = nal volume of
chlorophyll extract in 80% acetone; W = fresh weight of tissue
extracted.
2.2. Treatment conditions
Transverse sections of leaf and root were processed to observe
the anatomical changes and localization of iron under light micro-
Disease free, healthy vetiver plants were grown on mine soil in
scope. Perls stain was used to locate the uptake of iron in the root
earthen pots (size diameter 20 cm; height 17 cm) for a period of
and leaf tissue according to the method of Stacey et al. (2008). The
three months (July to September, 2014). Plastic vessels were placed
transverse sections were vacuum inltrated with equal volumes of
below the earthen pots to collect the water that seeped out from
4% (v/v) HCl and 4% (w/v) K-ferrocyanide (Perls stain solution) for
the pots.
15 min and incubated for 30 min at room temperature. Excess stain
Vetiver plants grown on garden soil served as a control set. Two
was removed by washing with distillated water the sections were
plants were planted per pot and three sets were prepared for each
observed under light microscope.
treatment. Plants were allowed to grow under normal environmen-
tal conditions.
2.6. Biochemical stress markers

2.3. Estimation of heavy metals in mine and garden soil; roots 2.6.1. Lipid peroxidation
and shoots of vetiver plants Lipid peroxidation was measured as the amount of MDA pro-
duced by the thiobarbituric acid (TBA) reaction as described by
Air-dried sample of mine soil was ground and passed through a Dhindsa et al. (1981). Briey, fresh leaves were homogenized in
2 mm sieve. Five grams of soil sample were digested in HNO3 (70%) a reaction mixture containing 20% (w/v) trichloroacetic acid (TCA),
in a microwave digestion system (Ethos D Microwave Labstation, and 0.5% (w/v) TBA. The homogenate was incubated at 95 C for
Milestone Inc., USA) and analyzed for Fe, Mn, Zn, Cu, Pb, Ni, Cr and 30 min. The sample was centrifuged at 13,000 g for 10 min. The
Al by Inductively Coupled Plasma Atomic Emission Spectrom- absorbance of the resulting supernatant was recorded at 532 and
eter (ARCOS, Spectro, Germany). The metal concentrations were 600 nm. The non-specic absorbance at 600 nm was subtracted
expressed as mg/kg. from that recorded at 532 nm. The concentration of MDA was cal-
At the end of three months, the plants were taken out of the culated using an extinction coefcient ( = 155 mM1 cm1 ) and
pots, washed thoroughly in running tap water, EDTA and deminer- expressed in mol g1 FW (fresh weight).
alized water. Five grams of plant tissue was cut into small pieces,
and ash was prepared at 500 C after drying in oven at 80 C. The 2.6.2. Activities of antioxidant enzymes
ash was digested in HNO3 (70%) in a microwave digestion system Catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPOD, EC.
(Ethos D Microwave Labstation, Milestone Inc., USA) and analyzed 1.11.1.7) and reduced glutathione (GSH) enzymes were analyzed
for Fe, Mn, Zn, Cu, Pb, Ni, Cr and Al by Inductively Coupled Plasma in the leaves.
Atomic Emission Spectrometer (ARCOS, Spectro, Germany). Metal CAT Fresh leaves (100 mg) were homogenized in 50 mM Tris
concentrations in the plant sample were calculated as g/g dry buffer (50 mM Tris, 1 mM EDTA, 1 mM magnesium chloride, 1.5%
weight. PVP; pH 7.8). The homogenate was centrifuged at 10,000 g for
R. Banerjee et al. / Ecological Engineering 90 (2016) 2534 27

Fig. 1. (a) Geographical location of the collection site (b) iron mine spoil-dump site.

10 min at 4 C. Catalase activity was measured according to the 2.6.3. Proline content
method of Aebi (1984). Briey, enzyme extract was added to a Proline content was determined according to the method of
3 ml reaction mixture containing 50 mM sodium phosphate buffer Bates et al. (1973). Leaves were chopped and transferred to 0.5%
at pH 7.0 and 10 mM of H2 O2 . The decrease in absorbance as aqueous toluene. After an hour, the extract was ltered. An equal
a result of H2 O2 ( = 39.4 mM1 cm1 ) degradation was recorded volume of ltrate, ninhydrin, and glacial acetic acid (1:1:1) was
at 240 nm and expressed in mol of H2 O2 oxidized min1 g1 added and incubated at 100 C for 1 h. The reaction was arrested in
protein. an ice bath and the chromophore was extracted with 4 ml toluene.
GPOD activity was determined in the enzyme extract by the The absorbance was read at 520 nm using a spectrophotometer. The
method of Chance and Maehly (1955). The formation of tetra gua- proline content was determined from a standard curve and calcu-
iacol was measured in a 3 ml reaction mixture containing 50 mM of lated. The values were expressed as mol g1 FW (fresh weight).
sodium phosphate buffer (pH 7.0), 10 mM H2 O2 , and 0.5 mM gua-
iacol. The activity was calculated as the increase in the absorbance 2.6.4. Protein extraction
at 470 nm expressed in mol of H2 O2 reduced min1 g1 protein. One gram of leaf was homogenized with 2 ml of 50 mM TrisHCl
GSH activity was determined in the enzyme extract by the buffer at pH 7.2, containing 0.1 mM ethylenediamine-tetra acetic
method of Smith et al. (1990). acid (EDTA), and 1% (w/v) polyvinyl-polypyrrolidone at 4 C. The
Fresh leaves (100 mg) were homogenized with 0.02 M EDTA on homogenate was centrifuged at 10,000 g for 15 min at 4 C and
ice. The reduced glutathione (GSH) content of the homogenates the resultant crude supernatant was collected and stored at 20 C
were measured using Elmans reagent 5, 5 -dithio-bis-(2- for the estimation of protein. Soluble protein content in the super-
nitrobenzoic acid) (DTNB). Briey, 5 ml of the homogenate was natant was determined according to the method of Bradford (1976)
mixed with 4 ml of distilled water and 1 ml of 50% TCA. After with bovine serum albumin as the standard.
incubating for 15 min on ice, the samples were centrifuged at
10,000 rpm for 15 min. The reaction mixture was prepared by 2.7. Assessment of genomic instability
adding 2 ml of supernatant, 4 ml of 0.4 M Tris buffer (pH 8.9) and
0.1 ml of DTNB. Absorbance of the reaction mixture was read within 2.7.1. Determination of DNA damage by the alkaline comet assay
5 min at 412 nm ( = 14,150 M1 cm1 ) and expressed as nmol mg1 The DNA damage studies were carried out on nuclei isolated
protein. from the leaves of vetiver following the method of Mukherjee and
28 R. Banerjee et al. / Ecological Engineering 90 (2016) 2534

Gichner (2009) with modications (Chakraborty and Mukherjee, Table 1


Physico-chemical characteristics of mine-soil.
2010). For each slide, 25 randomly chosen nuclei were analyzed
from 3 slides per experimental set. A computerized image analysis Parameters Values
system (Komet version 5.5, Kinetic Imaging Ltd., Andor Technology, Conductivity 174.70 S
Nottingham, UK) was employed. For analysis the median values pH 6.04
of comet parameter-tail DNA (%) were scored from each slide and Total dissolved solids (TDS) 165.60 mg/l
expressed as means for each treatment group. The parameter of Salinity (NaCl content) 162.60 mg/l
Resistivity 3.03 
percentage of tail DNA was particularly selected because it was
Fe 1725.28 48.32 mg/kg
considered the most meaningful and easy to conceptualize among Al 627.99 23.85 mg/kg
the parameters available for study (Kumaravel and Jha, 2006). Cu 45.25 10.04 mg/kg
Zn 24.49 9.01 mg/kg
Cr 7.61 1.93 mg/kg
2.7.2. DNA fragmentation assay
Mn 26.48 3.89 mg/kg
DNA was isolated from vetiver leaves using a modied CTAB Ni 2.43 0.08 mg/kg
method (Doyle and Doyle, 1987). Purity was determined by mea- Pb <0.01 mg/kg
suring the absorbance of the diluted DNA solution at 260 and Values are mean of 3 samples SD.
280 nm. The isolated DNA samples were resolved on 2.5% agarose
gel in 1 TAE (Tris-acetate-EDTA) buffer at 100 V, for 90 min at 4 C.
Lane A was loaded with 100 bp DNA ladder for reference. DNA was
stained with aqueous solution of ethidium bromide, visualized and
photographed under a gel documentation system (E-gel Imager, slightly acidic (6.04). Amongst the metals estimated, concentration
Life Technologies, California, USA). of Fe was highest, followed by Al, Cu, Zn, Cr, Mn and Ni.

2.7.3. Random Amplied Polymorphic DNA (RAPD) analysis


A total of 20 random primers were used for RAPD analysis. The 3.2. Performance of the vetiver under the inuence of mine-soil
PCR reaction was performed in a Veriti 96-Well Thermal Cycler
(Applied Biosystems, Carlsbad, CA, USA). Initial denaturation was As compared with the control (garden soil) a signicant decrease
performed at 94 C for 5 min. This was followed by 40 cycles of in shoot and root length was observed in vetiver plants grown on
1 min at 94 C, 1 min at 37 C and 2 min at 72 C. The nal exten- mine-soil (Fig. 2a). The average shoot length of vetiver grown on
sion was performed at 72 C for 10 min. The amplied products garden soil and mine-soil was 140.01 11.81 and 116.25 7.16 cm
were resolved in 1.5% agarose gel, stained with aqueous solution of and that of roots were 91.84 4.95 and 67.97 4.29 cm respec-
ethidium bromide and photographed in gel documentation system tively. To observe the effect on root system, the plants were
(E-gel Imager, Life Technologies, California, USA). taken out of the pot at the end of the experiment. The dense
mesh-like growth of roots held the mine-soil together which
2.8. Statistical analyses is considered as an ideal character of a good phytostabilizer
(Fig. 2b, c).
Statistical analyses of the data were performed using the statis- The anatomical changes in the leaves and roots of vetiver plants
tical program SigmaStat 3.0 (SPSS Inc., Chicago, IL, USA). Students growing on mine-soil were studied by preparing their transverse
t-test was done for the comet parameter percentage of tail DNA, sections (Fig. 3). The root and leaf sections of plants grown on mine-
lipid peroxidation and different antioxidant enzymes between soil do not show any abnormalities. Distribution of Fe in roots and
exposed and control sets. The level of signicance was established leaves of the plant was detected by Perls iron staining procedure
at P 0.05. (Stacey et al., 2008). The epidermal and cortical region of roots and
lower epidermal region and vascular bundle of leaves show blue
3. Results deposition of iron.
Chlorophyll content correlates to the photosynthetic potential,
3.1. Physico-chemical characterization and metal content physiological and metabolic health of the plant (Gaborcik, 2003). A
signicant decrease in the total chlorophyll content was observed
The physico-chemical parameters and heavy metal content in in the leaves of vetiver grown on mine-soil than that grown on
iron ore mine-soil are presented in Table 1. The pH of the soil was garden soil (Fig. 4).

Fig. 2. (a) Shoot and root length of vetiver plants after three months of exposure; [*P 0.05]; (b) root system entangling the soil; (c) dense, mesh-like growth of roots after
removal of the soil.
R. Banerjee et al. / Ecological Engineering 90 (2016) 2534 29

Fig. 3. Transverse sections of shoots of vetiver plant grown on: (a) garden soil (b) mine-soil; roots of vetiver plant grown on: (c) garden soil, (d) mine-soil. Arrows point the
localization of iron; scale bar = 50 m.

3.3. Phytoextraction of heavy metals values <1 (Table 3). The bioaccumulation factor (BF) reveals
efcient uptake of the metals by root and shoot of the
The phytoextraction or phytoaccumulation of the heavy metals plant. It is expressed as the ratio of [Metal]shoot /[Metal]soil or
was estimated. Metals like Fe, Al, Cu, Zn, Cr, Mn, Ni and Pb in shoot Metal]root /[Metal]soil . In general, the root part of the plant accu-
and root of vetiver plants were estimated by ICP-AES (Table 2). The mulated higher concentrations of metal than the shoot part. The
amount of Fe was highest, followed by Al, Cu, Mn, Zn, Cr and Ni in acidic pH of the soil may have helped for the high Al, Fe, and Cr
the roots and that in shoots Cu was the second highest of the metals accumulation in the roots.
in the plants grown on mine-soil.
To study the transfer of heavy metals from the soil to the plant, 3.4. Biochemical stress markers
bioaccumulation and translocation factors (BF and TF respectively)
are very useful parameters. The translocation factor (TF) is the 3.4.1. Changes in proline content
phytoavailabilty of metal from root to shoot part within a plant A signicant increase in proline content was observed in the
and expressed by the ratio of [Metal]shoot /[Metal]root (Maiti and leaves of vetiver plants grown on mine-soil (Fig. 5).
Nandhini, 2006). The data of the present study indicate that, except
Cu, the other metals (Fe, Al, Zn, Cr, Mn and Ni) accumulated by the
3.4.2. Changes in lipid peroxidation
plants were largely retained in the roots, as shown by general TF
Oxidative damage to membrane and cellular lipids were esti-
mated by lipid peroxidation assay. Lipid peroxidation expressed
in terms of malondialdehyde (MDA) formed in leaf tissues of the
plants increased (41%) signicantly when compared to the vetiver
plants grown on garden soil (Fig. 6a). Increased MDA content indi-
cated imbalance of ROS equilibrium as a result of stress-induced by
mine-soil.

3.4.3. Changes in antioxidant enzymes


The activities of catalase (CAT), guaiacol peroxidase (GPOD) and
reduced glutathione (GSH) were estimated in shoots and are pre-
sented in Fig. 6bd. A signicant increase in CAT and GPOD activity
was observed in vetiver plants grown on mine-soil. The CAT values
were 1.309 0.011 and 0.453 0.007 mol/g protein for vetiver
grown in mine-soil and in garden soil, respectively. The GPOD val-
ues increased from 0.244 0.006 mol/g protein in control plants
to 0.485 0.009 mol/g protein in the shoots of the plants grown
on mine-soil. The GSH activity of vetiver leaves was 5.107 0.237
Fig. 4. Total chlorophyll content in shoots of vetiver plants after three months of
and 4.299 0.107 nmol mg1 protein in plants grown on mine-soil
exposure; [*P 0.05]. and garden soil respectively (Fig. 6d).
30 R. Banerjee et al. / Ecological Engineering 90 (2016) 2534

Table 2
Estimation of metal contents (g/g) in shoot and root of vetiver plants grown on garden soil and mine-soil.

Iron Aluminum Copper Zinc Chromium Manganese Nickel Lead

Garden soil
Shoot 5.13 1.94 4.40 1.08 1.92 0.85 1.73 0.67 2.52 1.74 1.70 0.10 1.90 0.23 <0.01
Root 8.45 3.91 6.81 1.41 1.97 0.92 6.99 0.55 3.28 1.89 8.22 0.06 1.41 0.05 <0.01
Mine soil
Shoot 88.26 12.58 34.52 4.37 53.29 9.77 4.07 0.61 6.02 0.96 4.19 0.21 1.05 0.15 <0.01
Root 504.34 83.42 179.5 22.83 32.85 2.8 9.46 1.48 7.20 1.26 13.10 2.18 2.27 0.58 <0.01

Values are mean of 3 samples SD.

Table 3
Relative translocation and bioaccumulation of heavy metals in vetiver plants grown on mine-soil.

Iron Aluminum Copper Zinc Chromium Manganese Nickel

TF 0.17 0.19 1.62 0.43 0.83 0.32 0.46


BFshoot 0.05 0.05 1.17 0.17 0.79 0.16 0.43
BFroot 0.29 0.28 0.73 0.39 0.95 0.49 0.93

3.5. Assessment of genomic instability of 118 RAPD fragments of 1501800 bp in molecular size were
recorded (Table 4). RAPD proles neither showed alterations in
In addition to reclamation and phyto-stabilization, phytoreme- band intensity nor any gain or loss of bands. This conrmed strong
diation can be used in reducing toxic potential of contaminants genomic DNA template stability (GTS).
including complex mixtures of heavy metals, and hence, in addition
to metal estimation, genotoxicity was used to monitor the changes
4. Discussion
over the study period.

In the present study, the efcient use of vetiver system growing


3.5.1. Genotoxicity on iron ore mine-soil was monitored over a period of 3 months.
3.5.1.1. Comet assay. The shoots of vetiver plants grown on mine- The different parameters studied provide a comprehensive under-
soil show an increase in DNA strand breaks leading to greater DNA standing of the potential of vetiver grass as an alternative mean to
migration out of the nucleus into the tail of the comet. The increase vegetate metal-contaminated soil.
in Tail DNA (%) scored was 7.87 2.69 in plants grown on mine-soil The physico-chemical parameters and heavy metal content in
whereas in the control (garden soil) it was 4.82 0.84%. The values iron ore mine-soil demonstrated that the concentration of Fe was
were not statistically signicant (P 0.05) (Fig. 7a). highest, followed by Al, Cu, Zn, Cr, Mn and Ni. The overburden (OB)
and tailings from iron ore mines located in the state of Jharkhand,
3.5.1.2. DNA fragmentation assay. DNA fragmentation assay was in India revealed that the OB samples showed high concentration
carried out to assess the changes in the genomic DNA of the plants. of Fe along with Mn, Pb and Zn as a typical characteristic of iron
The results showed undamaged genomic DNA represented by a ore (Verma et al., 2012). We have taken the advantage of Perls iron
thick band on the agarose gel in both control and experimental staining procedure to detect Fe distribution in roots and leaves of
plants (Fig. 7b). the plant (Stacey et al., 2008). The epidermal and cortical region
of roots and lower epidermal region and vascular bundle of leaves
3.5.2. Random Amplied Polymorphic DNA (RAPD) analysis show a blue deposition of iron. This conrmed the uptake of iron
To study the genetic stability of the vetiver plants grown by the roots that was translocated to the leaves of vetiver plants
on mine-soil RAPD analysis was carried out. 20 arbitrary RAPD growing on mine-soil.
primers were initially screened, among which 16 produced clear After a period of 3 months the vetiver plants grown on mine-
and scorable amplication products (Fig. 8a, b). A total number soil manifested a signicant decrease in shoot and root length and
a decrease in chlorophyll content. There was a signicant increase
in proline and malondialdehyde (MDA) levels in leaf tissues of the
plants. Accumulation of proline in plant tissues is a clear marker of
environmental stress (Jaleel et al., 2007; Routley, 1966), believed
to play an adaptive role in plant stress tolerance (Verbruggen and
Hermans, 2008). Similar increases in proline concentrations were
observed in both leaves and roots of vetiver plants growing on met-
alliferous mine wastes (Pb/Zn tailings) (Pang et al., 2003). Increased
MDA content indicated imbalance of ROS equilibrium as a result of
stress-induced by the mine-soil. Previous studies by Patnaik et al.
(2013) in onion, Rai et al. (2004) in Ocimum tenuiorum and Reddy
et al. (2005) in horsegram and Bengalgram have shown an increase
in the levels of lipid peroxidation under heavy metal stress.
The activities of antioxidant enzymes catalase (CAT), guia-
col peroxidase (GPOD) and reduced glutathione (GSH) were
estimated in shoots. A signicant increase in CAT, GPOD and
GSH activity was observed in vetiver plants grown on mine-soil.
Our results are in agreement with those reported by Pang et al.
Fig. 5. Proline content in shoots of vetiver plants after three months of exposure; (2003) that Pb/Zn mine tailings could enhance the activities
[*P 0.05]. of POD, SOD and CAT, activities in vetiver. It is well known
R. Banerjee et al. / Ecological Engineering 90 (2016) 2534 31

Fig. 6. (a) Lipid peroxidation (LPO); (b) catalase (CAT); (c) guaiacol peroxidase (GPOD); (d) total glutathione (GSH) activity in shoots of vetiver plants after three months of
exposure; [*P 0.05].

that exposure of plants to heavy metals induces the generation mutagenic or genotoxic alterations in the genome may often lead to
of reactive oxygen species (ROS), which is harmful to plants genomic instability or genotoxic stress (Saha et al., 2015). Although
(Zenk, 1996). Rennenberg (1995) reported that GSH plays an most DNA damages are repaired, error-prone DNA repair or DNA
active role in protecting membranes against free radical dam- damage beyond repair triggers programmed cell death (Bray and
age. Major enzymes that are responsible for participating in West, 2005). Earlier report from our laboratory Chakraborty and
mechanisms to scavenge the toxic ROS, play an important role Mukherjee (2010), conferred that the DNA damage in vetiver plants
in plant defense mechanism (Anderson and Davis, 2004). In the grown on y ash for three months was not signicantly different
present study the adverse effects of mine-soil enhanced the activ- from that of the plants grown in garden soil. In the present study,
ities of major antioxidant enzymes in vetiver plants to detoxify the shoots assessed for genotoxicity, induced a 2-fold increase in
ROS. DNA strand breaks in plants grown on mine-soil when compared
Excess of ROS can damage cellular components, such as DNA, to that of plants grown in control (garden soil). The values were
proteins, and lipids (Lopez et al., 2006). All primary compo- however not statistically signicant (P 0.05). Absence of dam-
nents of DNA (sugar residues, phosphodiester linkages, purine and age in the nuclear DNA of vetiver implies its long-term survival on
pyrimidine bases) can suffer from damage. The resulting DNA- the mine soil. In addition, DNA fragmentation assay and the RAPD
lesions that vary from innocuous molecular changes to highly proles of the genomic DNA in vetiver plants grown on mine-soil

Fig. 7. DNA damage in shoots of vetiver plants after three months of exposure: (a) comet assay; (b) agarose gel electrophoresis of DNA isolated from shoots (Lane L 100 bp
DNA ladder, Lane A garden soil, Lane B mine-soil).
32 R. Banerjee et al. / Ecological Engineering 90 (2016) 2534

Table 4
List of primers, their sequences and size of the amplied fragments generated by 16 RAPD (Sl. No. 116) primers.

Sl. No Primer Primer sequence (5 -3 ) G + C content (%) No of scorable bands Size range (bp) approx.

1 OPY-15 AGTCGCCCTT 60 8 4001100


2 OPY-18 GTGGAGTCAG 60 12 3001800
3 OPZ-6 GTGCCGTTCA 60 8 2801400
4 OPH-4 GGAAGTCGCC 70 2 350700
5 OPH-5 AGTCGTCCCC 70 4 3501400
6 OPH-6 ACGCATCGCA 60 8 4001600
7 OPH-18 GAATCGGCCA 60 10 2801200
8 OPI-12 AGAGGGCACA 60 7 2501500
9 OPI-13 CTGGGGCTGA 70 12 2001200
10 OPI-14 TGACGGCGGT 70 10 4001200
11 OPI-18 TGCCCAGCCT 60 8 3201200
12 OPA-7 GAAACGGGTG 60 2 150600
13 OPA-9 GGGTAACGCC 70 10 2801400
14 OPA-13 CAGCACCCAC 70 7 4001550
15 OPL-14 GTGACAGGCT 60 5 300800
16 OPL-17 AGCCTGAGCC 70 5 4501400

Total 118 1501800

established genomic stability without any alterations in band ideal character of a good phytostabilizer. This is particularly impor-
intensity nor any gain or loss of bands. Recently, molecular tech- tant because the dense root system and vegetative cover may also
niques have proven to be a potential tool for eco-toxicological help to retard the formation of hazardous leachate from the soil
studies (Wolf et al., 2004). According to the previous reports, DNA (Chakraborty and Mukherjee, 2010).
ngerprinting offers a useful biomarker assay in the toxicology of Besides performance, phytoextraction of metals by the plant is
soil. Changes in RAPD banding patterns induced by pollutants like an important criterion for the success of phytoremediation. When
heavy metals can be regarded as changes in genomic DNA tem- plants are grown in a heavy metal-contaminated soil, the roots are
plate stability (GTS) that can be directly correlated with alterations the primary site of heavy metal accumulation. Plants are capable of
in other parameters (Atienzar et al., 2000; Liu et al., 2005). RAPD absorbing and accumulating metals in their tissues from contami-
analysis is capable of detecting temporary DNA changes that may nated soils, sediments and water at high concentrations (Porter and
not nally manifest themselves as mutations and that is why it is Peterson, 1975). Such a process is called phytoextraction or phy-
preferred over classical genotoxicity assays (Labra et al., 2003). toaccumulation (USEPA, 2000). In our study, a considerable amount
To observe the effect on the root system, the plants were taken of heavy metals was absorbed by the roots of vetiver but the same
out of the pot at the end of the experiment. The dense mesh-like was not translocated to the shoot system above. The amount of Fe
growth of root that held the mine-soil together is considered as an was highest followed by Al, Cu, Mn, Zn, Cr and Ni in the roots and
in shoots Cu was the second highest of metals in the plants grown
for 3 months on mine-soil. Roongtanakiat and Chairoj (2001) con-
ducted pot experiments to investigate the uptake potential of three
ecotypes of vetiver grass. They found that the uptake and retention
of Mn, Zn, Cu, Cd and Pb in shoot and root were dependent on the
period and ecotype of the vetiver grass harvested. In contrast to
the shoot, vetiver grass harvested at 120 days had higher heavy-
metal root concentration than the 60-day harvest. At the 60-day
harvest, for all three vetiver ecotypes, heavy-metal concentrations
were found to be higher in shoots than in roots.
Bioaccumulation and translocation factors (BF and TF respec-
tively) are very useful parameters to study the soil to plant transfer
pattern of heavy metal accumulation (Rezvani and Zaefarian, 2011;
Scragg, 2005). The translocation factor (TF) is the phytoavailabilty
of metal from root to shoot part within a plant and expressed by the
ratio of [Metal]shoot /[Metal]root (Maiti and Nandhini, 2006). BF and
TF values greater than unity are indicative of the fact that the plant
is a potential heavy metal accumulator and is effective in transloca-
tion of the metals from root to harvestable parts (Tu and Ma, 2002)
respectively. Thus BF and TF determine the overall phytoremedi-
ating potential of the plant. The data of the present study indicate
that except Cu, the other metals (Fe, Al, Zn, Cr, Mn and Ni) accumu-
lated by the plants growing on the mine-soil were largely retained
in the roots, as shown by general TF values <1. The bioaccumulation
Fig. 8. (a) Random Amplied Polymorphic DNA (RAPD) amplication pattern factor (BF) reveals efcient uptake of the metals by root and shoot
obtained from plants grown on garden soil (1a, 2a, 3a, 4a, 5a, 6a, 7a) and mine-soil of the plant. It is expressed as the ratio of [Metal]shoot /[Metal]soil
(1b, 2b, 3b, 4b, 5b, 6b, 7b) generated by 7 primers viz. 1. OPY-15, 2. OPY-18, 3. OPZ-6, or [Metal]root /[Metal]soil . In general, the root parts of the plant
4. OPH-4, 5. OPH-5, 6. OPH-6, 7. OPH-18, L. 100 bp DNA ladder. (b) Random Ampli- accumulated higher metal concentrations than the shoot. A sim-
ed Polymorphic DNA (RAPD) amplication pattern obtained from plants grown on
ilar trend in heavy metal accumulation and translocation in root
garden soil (1a, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a) and mine-soil (1b, 2b, 3b, 4b, 5b, 6b,
7b, 8b, 9b) generated by 9 primers viz. 1. OPI-12, 2. OPI-13, 3. OPI-14, 4. OPI-18, 5. and shoot has been observed in vetiver grown on iron ore mine
OPA-7, 6. OPA-9, 7. OPA-13, 8. OPL-14, 9. OPA-17, L.100 bp DNA Ladder. tailings (Roongtanakiat et al., 2008).
R. Banerjee et al. / Ecological Engineering 90 (2016) 2534 33

5. Conclusion Jaleel, C.A., Gopi, R., Sankar, B., Manivannan, P., Kishorekumar, A., Sridharan, R., Pan-
neerselvam, R., 2007. Studies on germination, seedling vigor, lipid peroxidation
and proline metabolism in Catharanthus roseus seedlings under salt stress. S. Afr.
In the present study vetiver plants growing on mine-soil act as a J. Bot. 73, 190195.
good phytostabilizer. The dense mesh-like growth of root that held Khan, A.G., 2006. Developing sustainable rural communities by reversing land degra-
the mine-soil together can prevent from erosion and also help to dation through a miracle plant vetiver grass. In: Rural Futures Conference
Proceedings: The RuralCitizen: Governance, Culture and Wellbeing in the 21st
retard the formation of hazardous leachate from the soil. The plant Century, pp. 57.
is a potential accumulator of heavy metals and the root parts of the Kumaravel, T.S., Jha, A.N., 2006. Reliable Comet assay measurements for detecting
plant accumulated higher metal concentrations than the shoot. The DNA damage induced by ionising radiation and chemicals. Mutat. Res. 605,
716.
adverse effect of mine soil in vetiver plants induced a decrease in
Labra, M., Di Fabio, T., Grassi, F., Regondi, S.M.G., Bracale, M., Vannini, C., Agradi, E.,
photosynthetic activities with enhanced activities of major antiox- 2003. AFLP analysis as biomarker of exposure to organic and inorganic genotoxic
idant enzymes to detoxify the ROS generated under stress. Absence substances in plants. Chemosphere 52, 11831188.
Lavania, U.C.,2000. Primary and secondary centers of origin of vetiver and its dis-
of damage in the nuclear DNA of vetiver implies its long-term sur-
persion. In: The 2nd International Vetiver Conference Proceedings. TVN, Thiland,
vival on the mine-soil. pp. 424426.
The results of the present study points to the success of uti- Liu, W., Li, P.J., Qi, X.M., Zhou, Q.X., Zheng, L., Sun, T.H., Yang, Y.S., 2005. DNA changes
lizing vetiver plants in phytoremediating iron ore mine-soil in a in barley (Hordeum vulgare) seedlings induced by cadmium pollution using RAPD
analysis. Chemosphere 61, 158167.
controlled laboratory conditions. For the restoration and revegeta- Lopez, E., Arce, C., Oset-Gasque, M.J., Canadas, S., Gonzalez, M.P., 2006. Cadmium
tion of iron ore mine site further study should be carried out on induces reactive oxygen species generation and lipid peroxidation in cortical
site as this will help in addressing a challenging problem under the neurons in culture. Free Radic. Biol. Med. 40, 940951.
Maiti, S.K., Nandhini, S., 2006. Bioavailability of metals in y ash and their bioaccu-
corporate social responsibility. mulation in naturally occurring vegetation: a pilot scale study. Environ. Monit.
Assess. 116, 263273.
Mukherjee, A., Gichner, T., 2009. Plant bioassays: comet assay in higher plants. In:
Conict of interest Pietro, S. (Ed.), Research Methods in Plant Sciences: Allelopathy. Studium Press,
LLC, Houston, Texas, USA, pp. 97108.
The authors declare that there is no conict of interest. Pang, J., Chan, G.S.Y., Zhang, J., Liang, J., Wong, M.H., 2003. Physiological aspects of
vetiver grass for rehabilitation in abandoned metalliferous mine wastes. Chemo-
Ritesh Banerjee and Priya Goswami contributed equally in all sphere 52, 15591570.
experiments performed. Patnaik, A.R., Achary, V.M.M., Panda, B.B., 2013. Chromium (VI)-induced hormesis
and genotoxicity are mediated through oxidative stress in root cells of Allium
cepa L. Plant Growth Regul. 71, 157170.
Acknowledgements Pedron, F., Petruzzelli, G., Barbaeri, M., Tassi, E., Ambrosini, P., Patata, L., 2011.
Mercury mobilization in a contaminated industrial soil for phytoremediation.
Commun. Soil Sci. Plant Anal. 42, 27672777.
The authors would like to acknowledge the Council of Scien- Porter, E.K., Peterson, P.J., 1975. Arsenic accumulation by plants on mine waste
tic and Industrial Research, New Delhi, Govt. of India for nancial (United Kingdom). Sci. Total Environ. 4, 365371.
support (Sanction No. 38 (1367)/13/EMR-II dt. 01.10.2013 Inves- Qihang, W., Wang, S., Thangavel, P., Qingfei, L., Zheng, H., Jun, B., Qui, R., 2011.
Phytostabilization of Jatropha curcas L. in polymetalic acid mine tailings. Int.
tigation on iron ore mine site restoration and species performance J. Phytoremediation 13, 788804.
in spoil dumps slope stabilization with vetiver system technology). Rai, V., Vajpayee, P., Singh, S.N., Mehrotra, S., 2004. Effect of chromium accumulation
on photosynthetic pigments, oxidative stress defense system, nitrate reduc-
tion, proline level and eugenol content of Ocimum tenuiorum L. Plant Sci. 167,
References 11591169.
Reddy, A.M., Kumar, S.G., Jyothsnakumari, G., Thimmanaik, S., Sudhakar, C., 2005.
Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121126. Lead induced changes in antioxidant metabolism of horsegram (Macrotyloma
Ahmad, A., Ghufran, R., Wahid, Z.A., 2010. Metals Cd, As, Cu, and Zn transfer through uniorum (Lam.) Verdc.) and bengalgram (Cicer arietinum L.). Chemosphere 60,
dry to rehydrated biomass of Spirulina platensis from wastewater. Pol. J. Environ. 97104.
Stud. 19, 674682. Rennenberg, H., 1995. Processes involved in glutathione metabolism. In:
Anderson, J.V., Davis, D.G., 2004. Abiotic stress alters transcript proles and activity Wallsgrove, R.M. (Ed.), Amino Acids and Their Derivatives in Plants
of glutathione S-transferase, glutathione peroxidase, and glutathione reductase Biosynthesis and Metabolism. Cambridge University Press, Cambridge, pp.
in Euphorbia esula. Physiol. Plant. 120, 421433. 155171.
Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Rezvani, M., Zaefarian, F., 2011. Bioaccumulation and translocation factors
Beta vulgaris. Plant Physiol. 24, 115. of cadmium and lead in Aeluropus littoralis. Aust. J. Agric. Eng. 2,
Atienzar, F.A., Cordi, B., Donkin, M.E., 2000. Comparison of ultraviolet-induced 114119.
genotoxicity detected by random amplied polymorphic DNA with chlorophyll Roongtanakiat, N., Chairoj, P., 2001. Uptake potential of some heavy metals by
uorescence and growth in a marine macroalgae. Aquat. Toxicol. 50, 112. vetiver grass. Kasetsart J. (Nat. Sci.) 35, 4650.
Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for Roongtanakiat, N., Tangruangkiat, S., Meesat, R., 2007. Utilization of vetiver grass
water-stress studies. Plant Soil 39, 205207. (Vetiveria zizanioides) for removal of heavy metals from industrial wastewa-
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of micro- ters. ScienceAsia 33, 397403, http://dx.doi.org/10.2306/scienceasia1513-1874.
gram quantities of protein utilizing the principle of protein-dye binding. Anal. 2007.33.397.
Biochem. 72, 248254. Roongtanakiat, N., Osotsapar, Y., Yindiram, C., 2008. Effects of soil amendment on
Bray, C.M., West, C.E., 2005. DNA repair mechanisms in plants: crucial sensors growth and heavy metals content in vetiver grown on iron ore tailings. Kasetsart
and effectors for the maintenance of genome integrity. New Phytol. 168, J. (Nat. Sci.) 42, 397406.
511528. Routley, D.G., 1966. Proline accumulation in wilted ladino clover leaves. Crop Sci. 6,
Chakraborty, R., Mukherjee, A., 2010. Technical note: vetiver can grow on coal y 358361.
ash without DNA damage. Int. J. Phytoremediation 13, 206214. Saha, P., Mukherjee, A., Biswas, A.K., 2015. Modulation of NaCl induced DNA damage
Chance, B., Maehly, A.C., 1955. Assay of catalase and peroxidase. Methods Enzymol. and oxidative stress in mung bean by pretreatment with sublethal dose. Biol.
2, 764775. Plant. 59, 139146.
Dhindsa, R.S., Plumb-Dhindsa, P., Thorpe, T.A., 1981. Leaf senescence: corre- Scragg, A.H., 2005. Environmental Biotechnology, 2nd edition. Oxford University
lated with increased levels of membrane permeability and lipid peroxidation, Press, New York.
and decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 32, Smith, I.K., Polle, A., Rennenberg, H., 1990. Glutathione. In: Alscher, R.G., Cumming,
93101. J.R. (Eds.), Stress Responses in Plants: Adaptation and Acclimation Mechanisms.
Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small quantities of Wiley-Liss, New York, pp. 201215.
fresh leaf tissue. Phytochem. Bull. 19, 1115. Stacey, M.G., Patel, A., McClain, W.E., Mathieu, M., Remley, M., Rogers, E.E.,
Gaborcik, N., 2003. Relationship between contents of chlorophyll (a + b) (SPAD val- Gassmann, W., Blevins, D.G., Stacey, G., 2008. The Arabidopsis AtOPT3 protein
ues) and nitrogen of some temperate grasses. Photosynthetica 41, 285287. functions in metal homeostasis and movement of iron to developing seeds. Plant
Ghosh, M., Paul, J., Jana, A., De, A., Mukherjee, A., 2015. Use of the grass, Vetiveria Physiol. 146, 589601, http://dx.doi.org/10.1104/pp.107.108183.
zizanioides (L.) Nash for detoxication and phytoremediation of soils contami- . The Wealth of India, vol. X. Publication & Information Directorate, CSIR, New Delhi
nated with y ash from thermal power plants. Ecol. Eng. 74, 258265. (India), pp. 451456.
Gupta, A.K., Sinha, S., 2009. Growth and metal accumulation response of Vigna radi- Truong, P.N., Baker, D., 1998. Vetiver Grass System for Environmental Protection.
ata L. var PDM 54 (mung bean) grown on y ash-amended soil: effect on dietary Pacic Rim Vetiver Network. Ofce of the Royal Development Projects Board,
intake. Environ. Geochem. Health 31, 463473. Bangkok, Thailand, Technical Bulletin No. 1.
34 R. Banerjee et al. / Ecological Engineering 90 (2016) 2534

Truong, P.N., Hart, B., 2001. Vetiver System for Wastewater Treatment. Pacic Rim Verma, S.R., Chaudhari, P.R., Satyanaranyan, S., 2012. Impact of leaching from iron ore
Vetiver Network. Ofce of the Royal Development Projects Board, Bangkok, mines on terrestrial and aquatic environment. Int. J. Environ. Sci. 2, 23782386.
Thailand, Technical Bulletin No. 2. Wolf, H.D., Blust, R., Backeljiau, T., 2004. The use of RAPD in ecotoxicology: a review.
Tu, C., Ma, L.Q., 2002. Effects of arsenic concentrations and forms on arsenic uptake Mutat. Res. 566, 249262.
by the hyperaccumulator ladder brake. J. Environ. Qual. 31, 641647. Zenk, M.H., 1996. Heavy metal detoxication in higher plants: a review. Gene 179,
USEPA, 2000. Introduction to Phytoremediation, in EPA/600/R-99/107. 2130.
Verbruggen, N., Hermans, C., 2008. Proline accumulation in plants: a review. Amino
Acids 35, 753759.