32 Toxic effects of heavy metals (Cd, Cr and Pb) on seed germination and growth and DPPH-scavenging...

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DOI: 10.1177/0748233712452605

2014 30: 238 originally published online 7 August 2012Toxicol Ind HealthMaryam Mehmood Siddiqui, Bilal Haider Abbasi, Nisar Ahmad, Mohammad Ali and Tariq Mahmood

turnipvar.Brassica rapaactivity inxic effects of heavy metals (Cd, Cr and Pb) on seed germination and growth and DPPH-scavengin

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Article

Toxic effects of heavy metals(Cd, Cr and Pb) on seed germinationand growth and DPPH-scavengingactivity in Brassica rapa var. turnip

Maryam Mehmood Siddiqui1, Bilal Haider Abbasi1,Nisar Ahmad1, Mohammad Ali1 and Tariq Mahmood2

Abstract

Toxicity of heavy metal is a wide spread environmental problem affecting all life forms including plants. In thepresent study the toxic effects of heavy metals, cadmium (Cd), chromium (Cr) and lead (Pb) on seed germinationrate (%), germination index (G-index) and growth (mm) ofBrassica rapavar.turniphave been investigated. Theseeds 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 4C in dark for 24 hours. Prior to inoculation onto MS0 medium, the soaked seeds were either washed withsterile distilled water or inoculated without washing on solidified MS0 medium at 25 + 2C with 16/8-hourphotoperiod in a growth chamber to germinate in vitro. Such stress conditions revealed that by increasing theconcentration of heavy metals, the germination rate (%), G-index value and growth (mm) decreased significantly,suggesting their toxic effect onB. rapavar.turnip. This study further revealed that experiment with seed washingresulted in less toxicity of selected heavy metals on germination and growth ofB. rapavar.turnip, as compared toexperiment without washing. However, the resulting toxicity order of the selected heavy metals remained thesame (Cd > Cr > Pb). Significant decrease has been observed in seed viability and germination potential andfinally heavy metals completely ceased further growth and development of plants. The 2, 2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging activity revealed that significantly higher activity was observed in control

plants without heavy metals treatment. Furthermore, the Cd-treated plants showed decreased antioxidantactivity. Cr and Pb were less toxic as compared to Cd (control > Pb > Cr > Cd). This study revealed thatselected 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 indus-

trialization, heavy metal toxicity has become a global

threat to all life forms: plants, animals and ultimatelyhumans. 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 seri-

ous 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).

Heavy metals such as arsenic, cadmium, chromium,

mercury and lead are potentially toxic and have no

known biological function in plants (Peralta-Videa et

al., 2009). Usually an unwanted exposure of heavy

metal/metals produces detrimental effects on plants,

1Department of Biotechnology, Quaid-i-Azam University, Islama-

bad, Pakistan2Department of Plant Sciences, Quaid-i-Azam University, Islama-

bad, Pakistan

Corresponding author:

Bilal Haider Abbasi, Department of Biotechnology, Quaid-i-Azam

University, Islamabad 45320, Pakistan.Email: [email protected]

Toxicology and Industrial Health

2014, Vol. 30(3) 238249

The Author(s) 2012

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affecting their growth, development and/or other phy-

siological processes (Fargasova, 2001; Heiss et al.,

2003; Kupper et al., 1998; Peralta et al., 2001). In con-

trast to this, some plant species bear a remarkable poten-

tial to tolerate and grow in heavy metal contaminated

sites. Such plants can do so through metal exclusion,

accumulation or indication depending upon the plantspecies 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 andenvironmental friendly manner than conventional tech-

nologies (Bah et al., 2010; Kachout et al., 2009).

Effect of heavy metals on early growth stages of aplant is a widely studied subject by the researchers,

because plants early growth stages such as seed

germination serve as an important indicator in deter-

mining 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 heavymetals from the polluted sites.

Therefore, the current study has been done to

assess the effects of Cd, Cr and Pb on the seed germi-

nation and growth of Brassica rapa var. turnip.

Turnip is a winter season food crop of worldwide eco-

nomic significance, belonging to a well-known plantfamily 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 andWang, 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 interna-

tional concern) on a local turnip variety (Bah et al.,

2010). Thus, this study supposed to be useful in deter-

mining heavy metal stress tolerance to grow on Cd, Cr

and/or Pb polluted sites or its subsequent phytoreme-

diation potential to clean up the contaminated sites by

cultivatingB. rapa.

Materials and methods

Seeds and seed soaking solutions of selectedheavy metals

Seeds of B. rapa var. turnip were obtained from

National Agricultural Research Council (NARC),

Pakistan. These were stored in a clean plastic bag and

kept in dark at room temperature (15-16 C) until

further use. To assess the effects of selected heavy

metals (Cd, Cr and Pb) on the seed germination and

subsequent seedlings growth of B. rapa var. turnip,

their respective salts that is cadmium chloride(CdCl2; MW 183.32 g/mol), chromium chloride(CrCl3.6H2O; MW 266.47 g/mol) and lead acetate

(Pb (CH3COO)2.3H2O; MW 379.369 g/mol), respec-

tively, were selected (UNI-CHEM1). Then the seed

soaking solutions of three different concentrations

(1, 2.5, 5 g/l) of these tested heavy metals were pre-

pared in autoclaved distilled water.

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/ bacter-

ial contamination and then air-dried on dry filter

papers prior to their inoculation on medium.

Seed germination experiments

According to previous reports, direct exposure

method that is placing seeds on filter paper, previously

moistened with heavy metal solution, is one the most

frequently used method to study the effects of heavymetals on the early growth stages in plants (Kuriakose

and Prasad, 2008). Recently, some scientists found

that it was more appropriate to perform such toxicity

tests on agar (solidified medium) than on filter paper

(Salvatore et al., 2008). Thus, based on previous

reports and our lab experience, a novel approach has

been devised to understand the effect of different con-

centrations of selected heavy metals on seed viability,

its germination potential and growth, over a specific

time period (i.e. 24 hours seed soaking in respective

Siddiqui et al. 239

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salt solutions of Cd, Cr and Pb). In this study, the basic

idea for the experiments has been adapted from the

work of Wei et al. (2009). In short, surface sterilized

seeds were first soaked in test solutions at 4C in dark

for 24 hours to get heavy metal treatment followed by

their inoculation on MS0 medium (Murashige and

Skoog, 1962). In this study, seeds soaked in autoclaved

distilled water were used as controls.

Germination of heavy metals-treated seeds without

washing prior to inoculation. This experiment was per-

formed in triplicate for seven days. Out of the seeds

soaked in respective concentrations (1.0, 2.5, 5.0

g/L) of Cd, Cr or Pb against control (i.e. seeds soaked

in distilled water), five seeds were inoculated in a

100-ml Erlenmeyer flask containing 30 ml of solidi-

fied MS medium (pH 5.5) in each flask withoutwashing with distilled water prior to inoculation.

After inoculation, flasks were tightly plugged and

incubated in growth chamber to allow seed germina-

tion at 25+2C with 16/8-hour photoperiod. The ger-

minated seeds were counted for seven days after seed

inoculation and scores were recorded. Seeds were

scored as germinated when the breakage of seed coat

was visible.

Germination of heavy metals-treated seeds with washing

prior to inoculation. This experiment was performed in

the same way as the above mentioned experiment

(section on Germination of heavy metals-treated

seeds without washing prior to inoculation) with the

only difference that the soaked seeds were washed

with distilled water prior to their inoculation on to the

medium.

At the end of each experiment, average germina-

tion rate (%), germination index (G-index; according

(l) Pb-5 g/l

(h) Cr-5 g/l(g) Cr-2.5 g/l

(k) Pb-2.5 g/l(j) Pb-1 g/l

(b) Cd-1 g/l

(f) Cr-1 g/l(e) dH2O/Cr 0 g/l (Control)

(i) dH2O/Pb 0 g/l (Control)

(c) Cd-2.5 g/l (d) Cd-5 g/l(a) dH2O/Cd 0 g/l (Control)

Figure 1.Experiment without washing demonstrating a 7-day-old experiment on inhibitory effect of Cd, Cr and Pb onseed germination and growth ofBrassica rapavar.turnip((a) to (l)), upon increase in concentration of these heavy metals intheir respective seed soaking solutions.

240 Toxicology and Industrial Health 30(3)



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to the method of Akinci and Akinci, 2010) and

seedling growth (mm) were determined at the given

concentrations of Cd, Cr and Pb.

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

powder, 1.25 mg. was dissolved in 40 ml ( 4) ofethanol to prepare stock solution. With 0.5 ml of

sample solution, 1 ml of DPPH solution was added

separately. These solution mixtures were kept at room

temperature in dark for 30 min (incubation period). Its

absorbance was measured at 517 nm. Lower absor-

bance of the reaction mixture indicated higher free

radical scavenging activity using the equation:

%DPPHscavengingactivity 100 1AE=AD

Figure 2. Seed soaked germination experiment without washing prior to inoculation: germination rates attained over7 days of seed incubation in Brassica rapa var. turnip upon exposure to different concentrations of Cd. The data shownhere 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 ofBrassica rapavar.turnip.

Control (dH2O) 1.0 g/l 2.5 g/l 5.0 g/l

Cd 4.93 + 0.09 2.89 + 1.65 1.47 + 1.07 0.28 + 0.49Cr 4.94 + 0.08 4.58 + 0.39 2.05 + 0.60 0.71 + 0.62

Pb 4.89 + 0.17 4.56 + 0.51 3.18 + 1.78 3.13 + 1.78Cd: cadmium; Cr: chromium; Pb: lead.

Table 2.Experiment without washing: effect of various concentrations of Cd, Cr and Pb on seedling growth (average) ofBrassica rapa var. turnip.


Cd 8.04 + 0.36 1.1 + 1.08 0.00 + 0.00 0.00 + 0.00Cr 8.14 + 0.21 6.7 + 0.67 1.4 + 0.96 0.6 + 0.42Pb 8.22 + 0.30 8.08 + 0.79 4.7 + 0.84 1.32 + 0.73

Cd: cadmium; Cr: chromium; Pb: lead.

Siddiqui et al. 241



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AE is absorbance of the solution when extract has

been added at a particular level, and AD is the absor-

bance of the DPPH solution with nothing added

(blank, without extract). Whereas the percentage of

free radical scavenging activity (% FRSA) in ethanol

extract of these plant materials was recorded after the

incubation period of 30 min.

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 DPPH-

scavenging activity and the experiments were

repeated twice. Analysis of variance (ANOVA) and

Duncans Multiple Range Test (DMRT) was used for

comparison among treatment means.

Results and discussion

Germination of heavy metals-treated seedswithout washing prior to inoculation

This experiment clearly demonstrates that seed soak-

ing in the given concentrations (1, 2.5 and 5 g/l) of

selected heavy metals (Cd, Cr and Pb) had deteriorat-

ing effects on B. rapa var. turnip seeds which either

resulted in a marked reduction or inhibition of seed

germination (about 793% of control) and subsequent

seedlings growth (Figure 1). According to results,

*100% seed germination took place in seeds soaked

indH2O (control) after 1 day of seed incubation. How-

ever, either subsequent delay or inhibition to seed ger-

mination and growth occurred with increase in heavy

metal concentration, depending upon the heavy metal

used over the period of 7 days of study (Figures 2 to

4). In addition to this, inhibitory effect of these heavy

metals was more pronounced on seedling growth than

on seed germination. These results were found in great

agreement with a number of previously reported

results. Peralta et al. (2001) have also reported reduc-

tion in seed germination and growth of alfalfa as the

metal concentration in the growing media was

increased. They reported that exposure to 40 ppm Cd

and Cr (IV) had significantly inhibited seed germina-tion (45 and 55%, respectively) and shoot growth as

compared to other heavy metals used. Similarly,

Fargasova (1994) and Li et al. (2005) have also

reported in their studies that seedling growth is more

sensitiveto heavy metals (Hg2, Pb2, Cu2 andZn2)

than seed germination, whereas inhibition was more

pronounced for both, germination and growth, in the

presence of Cd2.

Likewise, according to the results, Cd displayed thehighest inhibition in the seeds soaked in different Cd

Figure 3.Seeds soaked germination experiment without washing prior to inoculation: germination rates attained over7 days of seed incubation inBrassica rapavar.turnipupon exposure to different concentrations of Cr. The data shown hereare average of three replicates with standard error.




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concentrations (1, 2.5 and 5 g/l) and seed germination

rates were reduced from 100% (control) to 60, 33 and

7%, respectively (Figure 2). Like germination rate,

G-index values and seedlings growth (mm) also

depicted strong inhibition to seed germination and

growth of B. rapa var. turnip at all of the given Cd

concentrations and were significantly smaller than

control (Tables 1 and 2).

In a number of previous studies on Cd toxicity in

plants, Cd has been reported as the one determining

the highest toxic effects on different species

(Li et al., 2005; Peralta et al., 2001; Salvatore et al.,

2008). Similarly, Kiran and Sahin (2006) have

demonstrated inhibitory effect of Cd on seed germina-

tion, growth and increase in mitotic abnormalities,

with the increase in CdCl2 concentration (0.0625,

0.125, 0.250, 0.500 and 1.00 mM). Response to toxicCd is quite variable among different plant species as

has been reported by Salvatore et al. (2008) that let-

tuce was the plant that suffered more among the tested

vegetable plants. Similarly, Jun-Yu et al. (2008) have

reported Xiushui 110 (a rice variety) has a higher Cd

tolerance than Xiushui 11, a rice variety with low Cd

tolerance. In most of the studies, decrease in germina-tion, growth or enzymes activity can be seen with

increase in Cd concentrations but an increase in the

total chlorophyll content has been reported in tomato

plants, treated with Cd (10, 20, 30 and 40 mg) whereas

growth, plant biomass, leaf number and leaf area were

found to be negatively correlated with the concentra-

tion of Cd (Rehman et al., 2011). Thus, these contrast-

ing findings suggest that Cd is highly toxic to affect

different vital processes in plants, but its toxicity

range varies in different plant species, depending

upon their ability to tolerate Cd.

Theseresults also show that Cr wasless toxic than Cd

to affect seed germination and growth in turnip. In case

of Cr, significantly less difference was found between

control and seeds soaked in low Cr concentration

(Cr 1 g/l). However, considerable inhibitory effect was

seenat itsmedium(2.5 g/l) andhigh concentrationvalue

(5 g/l; Figures 1 and 3).Hence, theresulting germination

rates found at applied Cr concentrations were 100, 73

and 20%, respectively (Figure 3). Similarly, G-indexvalues and growth (mm) for Cr concentrations were less

than the control but larger than those found at the corre-

sponding Cd concentrations (Tables 1 and 2).

In nature, Cr exists either in Cr (III) and Cr (IV)

oxidation states as the most stable forms of Cr such

that Cr (IV) is more toxic than Cr (III) (Peralta-

Videa et al., 2009; Zayed and Terry, 2003). Effects

of both Cr (III) and Cr (IV) on plants have been

widely reviewed, depicting detrimental effects of

Cr on plants growth, development and other

Figure 4. Seed soaked germination experiment without washing prior to inoculation: germination rates attained over7 days of seed incubation in Brassica rapa var. turnip upon exposure to different concentrations of Pb. The data shownhere are average of three replicates with standard error.

Siddiqui et al. 243



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physiological processes (Peralta-Videa et al., 2009;

Shanker et al., 2005; Zayed and Terry, 2003). Gener-

ally, plants exposure to low Cr levels is known to

have stimulating effects on plant growth and yield,

whereas its synergistic effect is possible upon

increase in Cr concentration (Akinci and Akinci,

2010; Peralta-Videa et al., 2009; Zayed and Terry,

2003). Similar findings have also been reported by

Zou et al. (2006) which showed that Cr (VI) had a sti-

mulating effect on root growth ofAmaranthus viridis

Figure 5.Seed soaked germination experiment with washing prior to inoculation: germination rates attained over 7 daysof seed incubation in Brassica rapavar.turnipupon exposure to different concentrations of Cd. The data shown here areaverage of three replicates with standard error.

Figure 6.Seed soaked germination experiment with washing prior to inoculation: germination rates attained over 7 daysof seed incubation inBrassica rapavar.turnipupon exposure to different concentrations of Cr. The data shown here areaverage of three replicates with standard error.




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exposed to 105 M Cr (IV), but it had inhibitory effect

at 104 M and 103 M Cr (IV). On the other hand,

some scientists have conclusively argued the nonessen-

tiality of Cr to plants by showing that if Cr is required

for normal plant growth, the required concentrations in

plant tissues ought to be much lower than the levels ofany known essential nutrient (Zayed and Terry, 2003).

This experiment also reveals that the least inhibi-

tory effect was shown by Pb as compared to Cd and

Cr and both germination and growth were quite sim-

ilar to control. For Pb, the germination rates obtained

at the given Pb concentrations were 93, 80 and 73%,

respectively, which shows that seed germination rates

were almost similar to control (100%) at the given Pb

concentrations (Figure 4). However, at low Pb con-

centration (1 g/l), the corresponding G-index values

and growth lengths (mm) were also very similar to

control but larger than Cd and Cr, respectively; butwith increase in Pb concentration, decrease in corre-

sponding values was found, particularly in case of

growth (Tables 1 and 2). This shows that in case ofPb, seedling growth was more sensitive to various

Pb concentrations as compared to seed germination.

Several reports already exist on Pb phytotoxic

effects showing that excess Pb is toxic and leads to

a number of toxicity symptoms in plants for example

inhibition to seed germination, stunted growth,

chlorosis, blackening of root system, changes in

minerals and water balance, upsets in hormonal sta-

tus, and so on (Peralta-Videa, 2009; Sharma and

Dubey, 2005). Like Yang et al. (2010) showed a sig-

nificant inhibition to seed germination and seedling

growth in a wheat variety, called Xihan 2, subjected

to high Pb (NO3)2 concentrations and exogenousH2O2. 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, respec-

tively, 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 mobi-

lization to uptake by B. rapa var. turnipor may be a

high-tolerance potential of a plant to adapt to Pbstress, probably due to its high chlorophyll stability

(Peralta-Videa, 2009; Sudhakar et al., 1992).

Germination of heavy metals-treated seeds withwashing prior to inoculation

This experiment demonstrates that prior washing of

seeds soaked in the given concentrations of Cd, Cr and

Pb, with distilled water, before their inoculation on

MS0 medium did not significantly reduce or attenuate

Figure 7. Seeds soaked germination experiment with washing prior to inoculation: germination rates attained over 7 daysof seed incubation in Brassica rapavar.turnipupon exposure to different concentrations of Pb. The data shown here areaverage of three replicates with standard error.

Siddiqui et al. 245



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(a) dH2O/Cd 0 g/l (Control)

(e) dH2O/Cr 0 g/l (Control)

(i) dH2O/Pb 0 g/l (Control)

(b) Cd-1 g/l

(f) Cr-1 g/l

(j) Pb-1 g/l

(c) Cd-2.5 g/l

(g) Cr-2.5 g/l

(k) Pb-2.5 g/l

(d) Cd-5 g/l

(l) Pb-5 g/l

(h) Cr-5 g/l

Figure 8.Experiment with washing demonstrating a 7-day-old experiment on inhibitory effect of Cd, Cr and Pb on seedgermination and growth ofBrassica rapa var. turnip (A-L), upon increase in concentration of these heavy metals in theirrespective 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 4.93 + 0.09 2.85 + 1.13 1.04 + 0.92 0.33 + 0.57Cr 4.94 + 0.08 4.84 + 0.15 3.37 + 1.35 1.75 + 0.64Pb 4.89 + 0.17 4.89 + 0.08 4.04 + 0.43 3.09 + 0.44


Table 4. Experiment with washing: effect of various concentrations of Cd, Cr and Pb on seedling growth (average) ofBrassica rapa var.turnip.


Cd 7.9 + 0.42 4.6 + 1.56 2.3 + 2.17 0.00 + 0.00Cr 8.14 + 0.21 7.76 + 0.51 7.68 + 0.97 0.48 + 0.35Pb 8.22 + 0.30 8.04 + 0.29 7.98 + 0.32 8.0 + 0.35





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the toxic effects of these heavy metals on seed germi-

nation and growth ofB. 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, G-

index 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 neg-

ative effect of these heavy metals on seed germination

and growth was the common observation in both theexperiments, 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 ger-

mination 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 inCd, Cr and Pb had deteriorated the germination poten-

tial 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 metalson turnip seed germination can be linked to various

metabolic adjustments, leading to modulation of dif-

ferent enzymes involving heavy metal-induced struc-

tural alterations, leading to cell death, interference

with the functions of metal ion cofactors, inhibition

to enzyme activity or their posttranslational processing,

growth retardation, and so on (Kuriakose and Prasad,

2008; Li et al., 2005; Meng et al., 2009). Similarly, a

number of reasons can be anticipated with the inhibi-

tion of growth in B. rapa var. turnip, induced by the

tested heavy metals: inhibition of mitosis, the reducedsynthesis of cell wall components, damage to the Golgi

apparatus, changes in the polysaccharide metabolism,

breakdown in photosynthesis (because of substitution

of Mg, the central atom of chlorophyll, by heavy

metals), decrease in the water potential (causing lossof turgor and wilted growth), and so on (Heidari and

Sarani, 2011; Kupper et al., 1998).

Another noticeable finding of this study was the

great difference in the relative toxicity of tested

heavy metals, with the resulting toxicity order as

Cd > Cr > Pb. This difference in the relative toxicityof Cd, Cr and Pb can be due to their variable selec-

tive permeability across the seed barriers, particu-

larly, seed coat and embryonic tissues. This

permeability of heavy metals across the seed coat

is mainly governed by a relationship between metal

intake by the seed and media water status. Hence,

greater the concentration of heavy metal in the soak-

ing solution, greater will be its influx into the plant

system and vice versa, leading to a multitude ofinhibitory reactions, manifested as reduced

germination and growth retardation (Akinci and

Akinci, 2010; Kuriakose and Prasad, 2008; Liet al., 2005; Wierzbicka and Obidzinska, 1998).

Toxic effect of heavy metals on DPPH-scavengingantioxidant activity

Plants produce complex secondary metabolites during

metabolism (Ahmad et al., 2010a, 2011a). Some of

these metabolites have potential to detoxify free radi-

cals. But during stress conditions the scavenging

power of the metabolites decreases (Abbasi et al.,

2011b; Ahmad et al., 2011b, 2011c). In the presentinvestigation, heavy metals (Cd, Cr and Pb) are sup-

plemented to MS-medium and theB. rapaseeds were

allowed to germinate. After germination, thein vitro

plantlets were collected for DPPH-scavenging activ-

ity. Analysis of the assay revealed that significantly

higher activity (87.058%) was observed in control

plants without heavy metals treatment (Figure 9). It

has been observed that cadmium was the most toxic

heavy metal on antioxidant activity (37.7451

55.372%). However, chromium was less toxic than

0 20 40 60 80 100

0 20 40 60 80 100

e

ded

d

cdc

cd

c

c

cbc

bcb

abab

Cd-1mMCd-0.5 mMCd-500MCd-300 MCd-100 M

Cr-1mMCr-0.5 mMCr-500 MCr-300 M

Cr-100 MPb-1 mM

Pb-0.5 mMPb-500 MPb-300 MPb-100 M

DPPH-Scavenging activity (%)

Heavy

metal

Control a

Figure 9. 2, 2-Diphenyl-1-picrylhydrazyl (DPPH)-scaven-ging antioxidant activity in cadmium-, chromium- andlead-treated (different concentrations) plants in Brassica

rapavar. turnip.

Siddiqui et al. 247



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cadmium (31.86259.627%). In comparison with Cd

and Cr, the Pb-treated plant exhibited higher antioxi-

dant activity but lower than control (Control

87.058% > Pb 52.94175.549% > Cr 31.862

59.627% > Cd 37.745155.372%). From the current

observation, it has been concluded that these heavy

metals especially cadmium not only inhibit the plantgrowth but can also affect the antioxidant activity.

Funding

This research received no specific grant from any funding

agency in the public, commercial, or not-for-profit sectors.

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