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and Industrial Health
Toxic effects of heavy metals (Cd, Cr and Pb) on seed germination and growth and DPPH-scavenging
activity in Brassica rapa var. turnip
Maryam Mehmood Siddiqui, Bilal Haider Abbasi, Nisar Ahmad, Mohammad Ali and Tariq Mahmood
Toxicol Ind Health 2014 30: 238 originally published online 7 August 2012
DOI: 10.1177/0748233712452605
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Article
Abstract
Toxicity of heavy metal is a wide spread environmental problem affecting all life forms including plants. In the
present study the toxic effects of heavy metals, cadmium (Cd), chromium (Cr) and lead (Pb) on seed germination
rate (%), germination index (G-index) and growth (mm) of Brassica rapa var. turnip have been investigated. The
seeds were soaked either in distilled water (control) or in aqueous solutions of Cd, Cr and Pb (1 g/l, 2.5 g/l and 5 g/l)
at 4 C in dark for 24 hours. Prior to inoculation onto MS0 medium, the soaked seeds were either washed with
sterile distilled water or inoculated without washing on solidified MS0 medium at 25 + 2 C with 16/8-hour
photoperiod in a growth chamber to germinate in vitro. Such stress conditions revealed that by increasing the
concentration of heavy metals, the germination rate (%), G-index value and growth (mm) decreased significantly,
suggesting their toxic effect on B. rapa var. turnip. This study further revealed that experiment with seed washing
resulted in less toxicity of selected heavy metals on germination and growth of B. rapa var. turnip, as compared to
experiment without washing. However, the resulting toxicity order of the selected heavy metals remained the
same (Cd > Cr > Pb). Significant decrease has been observed in seed viability and germination potential and
finally heavy metals completely ceased further growth and development of plants. The 2, 2-diphenyl-1picrylhydrazyl (DPPH)-scavenging activity revealed that significantly higher activity was observed in control
plants without heavy metals treatment. Furthermore, the Cd-treated plants showed decreased antioxidant
activity. Cr and Pb were less toxic as compared to Cd (control > Pb > Cr > Cd). This study revealed that
selected heavy metals not only affected plant development but also disturbed plant metabolic pathways.
Keywords
Brassica rapa var. turnip, heavy metals, toxicity, seed germination, DPPH
Introduction
In todays world of fast pacing technology and industrialization, heavy metal toxicity has become a global
threat to all life forms: plants, animals and ultimately
humans. The undesirable accumulation of toxic heavy
metals, particularly due to various anthropogenic
activities is not only causing worldwide devastation
of agricultural soils and products but also posing serious food safety issues, health risks and disruption of
ecosystems. As a result, unexpected consequences
and ecological crisis are anticipated with heavy metal
pollution (Amico et al., 2008; Heidari and Sarani,
2011; Kachout et al., 2009; Leon et al., 2005; Malik
et al., 2010; Yadav et al., 2009).
Siddiqui et al.
239
affecting their growth, development and/or other physiological processes (Fargasova, 2001; Heiss et al.,
2003; Kupper et al., 1998; Peralta et al., 2001). In contrast to this, some plant species bear a remarkable potential to tolerate and grow in heavy metal contaminated
sites. Such plants can do so through metal exclusion,
accumulation or indication depending upon the plant
species and type of metal (Peralta-Videa et al., 2009;
Xiong and Wang, 2005). Because of long residence
time, toxicity, nonbiodegradability, irreversible nature
of contamination and accumulation of heavy metals in
food chain, the existing technologies are not very useful
to decontaminate heavy metal polluted sites (Malik et
al., 2010; Munir et al., 2010). So far, phytoremediation
that is a new emerging technology that uses green plants
to extract, sequester or detoxify heavy metal pollutants
seems promising to do away heavy metals from the
environment in a more effective, less expensive and
environmental friendly manner than conventional technologies (Bah et al., 2010; Kachout et al., 2009).
Effect of heavy metals on early growth stages of a
plant is a widely studied subject by the researchers,
because plants early growth stages such as seed
germination serve as an important indicator in determining the toxicity effects of heavy metals on plants
(Kuriakose and Prasad, 2008; Salvatore et al.,
2008). Besides this, such studies seem helpful in the
identification of new and better heavy metaltolerant
plant species as we are living in an age where it has
become inevitable to phytoremediate toxic heavy
metals from the polluted sites.
Therefore, the current study has been done to
assess the effects of Cd, Cr and Pb on the seed germination and growth of Brassica rapa var. turnip.
Turnip is a winter season food crop of worldwide economic significance, belonging to a well-known plant
family called Brassicaceae (or mustard family;
Abbasi et al., 2011a). Many plant species from this
family are known to have remarkable heavy metal
stress tolerance such as B. napus, mustard, etc.
(Amico et al., 2008; Meng et al., 2009; Xiong and
Wang, 2005). The inhibitory effects of these and other
heavy metals on different plant species have been
immensely reported in previous studies (Peralta
et al., 2001). Similarly, the inhibitory effects of some
heavy metals (Be, Ni, T1, and V) has already been
reported on seed germination of six plant species
including turnip (Carlson et al., 1991). However, this
study is particularly aimed at assessing the effect of
Cd, Cr and Pb (as they are heavy metals of international concern) on a local turnip variety (Bah et al.,
2010). Thus, this study supposed to be useful in determining heavy metal stress tolerance to grow on Cd, Cr
and/or Pb polluted sites or its subsequent phytoremediation potential to clean up the contaminated sites by
cultivating B. rapa.
Sterilization of seeds
Seeds were surface sterilized according to the method
of Abbasi et al. (2011b), with some modifications.
Briefly, seeds were immersed in 0.1% HgCl2 for 1
min. Subsequently, they were washed three times
with sterile distilled water to avoid any fungal/ bacterial contamination and then air-dried on dry filter
papers prior to their inoculation on medium.
240
Figure 1. Experiment without washing demonstrating a 7-day-old experiment on inhibitory effect of Cd, Cr and Pb on
seed germination and growth of Brassica rapa var. turnip ((a) to (l)), upon increase in concentration of these heavy metals in
their respective seed soaking solutions.
Siddiqui et al.
241
Figure 2. Seed soaked germination experiment without washing prior to inoculation: germination rates attained over
7 days of seed incubation in Brassica rapa var. turnip upon exposure to different concentrations of Cd. The data shown
here are an average of three replicates with standard error.
Table 1. Experiment without washing: effect of various concentrations of Cd, Cr and Pb on G-index of Brassica rapa var.
turnip.
Cd
Cr
Pb
Control (dH2O)
1.0 g/l
2.5 g/l
5.0 g/l
4.93 + 0.09
4.94 + 0.08
4.89 + 0.17
2.89 + 1.65
4.58 + 0.39
4.56 + 0.51
1.47 + 1.07
2.05 + 0.60
3.18 + 1.78
0.28 + 0.49
0.71 + 0.62
3.13 + 1.78
Table 2. Experiment without washing: effect of various concentrations of Cd, Cr and Pb on seedling growth (average) of
Brassica rapa var. turnip.
Cd
Cr
Pb
Control (dH2O)
1.0 g/l
2.5 g/l
5.0 g/l
8.04 + 0.36
8.14 + 0.21
8.22 + 0.30
1.1 + 1.08
6.7 + 0.67
8.08 + 0.79
0.00 + 0.00
1.4 + 0.96
4.7 + 0.84
0.00 + 0.00
0.6 + 0.42
1.32 + 0.73
Determination of 2, 2-diphenyl-1-picrylhydrazyl
(DPPH)-scavenging antioxidant activity
The DPPH-scavenging activity was determined
according to the method of Ahmad et al. (2010b) and
Abbasi et al. (2011b) with slight modification. DPPH
242
Figure 3. Seeds soaked germination experiment without washing prior to inoculation: germination rates attained over
7 days of seed incubation in Brassica rapa var. turnip upon exposure to different concentrations of Cr. The data shown here
are average of three replicates with standard error.
Data analyses
The average and standard error of three replicates for
each treatment were calculated using Microsoft Excel
software for seed germination rate (%), germination
index (G-index) and growth (mm). For statistical
analysis, three replicates were conducted for DPPHscavenging activity and the experiments were
repeated twice. Analysis of variance (ANOVA) and
Duncans Multiple Range Test (DMRT) was used for
comparison among treatment means.
Siddiqui et al.
243
Figure 4. Seed soaked germination experiment without washing prior to inoculation: germination rates attained over
7 days of seed incubation in Brassica rapa var. turnip upon exposure to different concentrations of Pb. The data shown
here are average of three replicates with standard error.
244
Figure 5. Seed soaked germination experiment with washing prior to inoculation: germination rates attained over 7 days
of seed incubation in Brassica rapa var. turnip upon exposure to different concentrations of Cd. The data shown here are
average of three replicates with standard error.
Figure 6. Seed soaked germination experiment with washing prior to inoculation: germination rates attained over 7 days
of seed incubation in Brassica rapa var. turnip upon exposure to different concentrations of Cr. The data shown here are
average of three replicates with standard error.
Siddiqui et al.
245
Figure 7. Seeds soaked germination experiment with washing prior to inoculation: germination rates attained over 7 days
of seed incubation in Brassica rapa var. turnip upon exposure to different concentrations of Pb. The data shown here are
average of three replicates with standard error.
minerals and water balance, upsets in hormonal status, and so on (Peralta-Videa, 2009; Sharma and
Dubey, 2005). Like Yang et al. (2010) showed a significant inhibition to seed germination and seedling
growth in a wheat variety, called Xihan 2, subjected
to high Pb (NO3)2 concentrations and exogenous
H2O2. According to the results, Pb showed minimum
inhibition to seed germination and growth in B. rapa
var. turnip, compared to Cd and Cr. Similarly, Mami
et al. (2010) have also reported the less toxic effect of
Pb on seed germination and growth indices of the
selected tomato varieties and was in the order:
Cu2 > Fe2 > Pb2 and Cu2 > Pb2 > Fe2, respectively, at five different doses (0, 0.001, 0.01, 0.1 and
1%) of the selected metals. So the least toxicity of
Pb in this study can be due to less solubility and mobilization to uptake by B. rapa var. turnip or may be a
high-tolerance potential of a plant to adapt to Pb
stress, probably due to its high chlorophyll stability
(Peralta-Videa, 2009; Sudhakar et al., 1992).
246
Figure 8. Experiment with washing demonstrating a 7-day-old experiment on inhibitory effect of Cd, Cr and Pb on seed
germination and growth of Brassica rapa var. turnip (A-L), upon increase in concentration of these heavy metals in their
respective seed soaking solutions.
Table 3. Experiment with washing: effect of various concentrations of Cd, Cr and Pb on G-index of Brassica rapa var.
turnip.
Cd
Cr
Pb
Control (dH2O)
1.0 g/l
2.5 g/l
5.0 g/l
4.93 + 0.09
4.94 + 0.08
4.89 + 0.17
2.85 + 1.13
4.84 + 0.15
4.89 + 0.08
1.04 + 0.92
3.37 + 1.35
4.04 + 0.43
0.33 + 0.57
1.75 + 0.64
3.09 + 0.44
Table 4. Experiment with washing: effect of various concentrations of Cd, Cr and Pb on seedling growth (average) of
Brassica rapa var. turnip.
Cd
Cr
Pb
Control (dH2O)
1.0 g/l
2.5 g/l
5.0 g/l
7.9 + 0.42
8.14 + 0.21
8.22 + 0.30
4.6 + 1.56
7.76 + 0.51
8.04 + 0.29
2.3 + 2.17
7.68 + 0.97
7.98 + 0.32
0.00 + 0.00
0.48 + 0.35
8.0 + 0.35
Siddiqui et al.
247
Heavy metal
20
40
60
80
100
a
Control
Pb-100 M
Pb-300 M
Pb-500 M
Pb-0.5 mM
Pb-1 mM
Cr-100 M
Cr-300 M
Cr-500 M
Cr-0.5 mM
Cr-1mM
Cd-100 M
Cd-300 M
Cd-500M
Cd-0.5 mM
Cd-1mM
ab
ab
bc
c
bc
c
cd
d
e
c
c
cd
d
de
0
20
40
60
80
100
the toxic effects of these heavy metals on seed germination and growth of B. rapa var. turnip (Figures 5 to 8
and Tables 3 and 4). However, washing with water has
resulted in a slight increase in germination rates, Gindex values and growth, as compared to the results
obtained without washing of seeds (Figures 1 to 4;
Tables 1 and 2). Furthermore, despite the fact that
seeds washing with water resulted in less toxicity of
selected heavy metals, it can be clearly seen that negative effect of these heavy metals on seed germination
and growth was the common observation in both the
experiments, with increase in concentration of Cd, Cr
or Pb in soaking solutions as compared to control
seeds. Hence, this shows that even though the soaking
treatment has been over after a certain time period (24
hrs) and soaked seeds have also been washed with
water, heavy metals conglutinated on the surface
of seeds were still effective in inhibiting seed germination and subsequent seedling growth of B.
rapa var. turnip, particularly in the case of Cd as
shown in Figures 1 and 8 (Wei et al., 2009).
Thus, this study clearly shows that seed soaking in
Cd, Cr and Pb had deteriorated the germination potential of turnip seeds. This means that B. rapa seeds
were badly influenced by these heavy metals and lost
their viability and growing potential with increase in
heavy metal concentration in the soaking solutions.
This toxic (inhibitory) effect of these heavy metals
on turnip seed germination can be linked to various
metabolic adjustments, leading to modulation of different enzymes involving heavy metal-induced structural alterations, leading to cell death, interference
248
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