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Enhanced Phytoremediation of Cadmium-ContaminatedSoil by Parthenium hysterophorus Plant: Effect ofGibberellic Acid (GA3) and Synthetic Chelator, Aloneand in CombinationsFazal Hadi a , Nasir Ali a & Ayaz Ahmad ba Department of Biotechnology , University of Malakand , Chakdara , Khyber Pakhtunkhwa ,Pakistanb Department of Botany , University of Malakand , Chakdara , Khyber Pakhtunkhwa ,PakistanPublished online: 09 Jan 2014.

To cite this article: Fazal Hadi , Nasir Ali & Ayaz Ahmad (2014) Enhanced Phytoremediation of Cadmium-Contaminated Soilby Parthenium hysterophorus Plant: Effect of Gibberellic Acid (GA3) and Synthetic Chelator, Alone and in Combinations,Bioremediation Journal, 18:1, 46-55, DOI: 10.1080/10889868.2013.834871

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Bioremediation Journal, 18(1):46–55, 2014Copyright ©c 2014 Taylor and Francis Group, LLCISSN: 1088-9868 print / 1547-6529 onlineDOI: 10.1080/10889868.2013.834871

Enhanced Phytoremediation ofCadmium-Contaminated Soil by Parthenium

hysterophorus Plant: Effect of GibberellicAcid (GA3) and Synthetic Chelator, Alone

and in Combinations

Fazal Hadi,1 Nasir Ali,1

and Ayaz Ahmad2

1Department of Biotechnology,University of Malakand,Chakdara, KhyberPakhtunkhwa, Pakistan2Department of Botany,University of Malakand,Chakdara, KhyberPakhtunkhwa, Pakistan

ABSTRACT The roles of gibberellic acid (GA3) and ethylenediaminete-traacetic acid (EDTA) in phytoremediation of cadmium (Cd)-contaminatedsoil by Parthenium hysterophorus plant was investigated. GA3 (10−9, 10−7, and10−5 M) was applied as a foliar spray. EDTA was added to soil in a singledose (160 mg/kg soil) and split doses (40 mg/kg soil, four split doses). GA3 andEDTA were used separately and in various combinations. P. hysterophorus was se-lected due to its fast growth and unpalatable nature to herbivores to reduce theentrance of metal into the food chain. The Cd phytoextraction potential of theP. hysterophorus plant was evaluated for the first time. Cd significantly reducedplant growth and dry biomass (DBM). GA3 alone increased the plant growthand biomass in Cd-contaminated soil, whereas EDTA reduced it. GA3 in combi-nation with EDTA significantly increased the growth and biomass. The highestsignificant DBM was found in treatment T3 (10−5 M GA3). All treatments ofGA3 or EDTA significantly enhanced the plant Cd uptake and accumulationcompared with control (C1). The highest significant root and stem Cd con-centrations were found in the combination treatment T11 (GA3 10−5 M +EDTA split doses), whereas in leaves it was found in the EDTA treatments. Cdconcentration in plant parts increased in the order of stem < leaves < roots.The combination treatment T9 (GA3 10−7 M + EDTA split doses) showed thesignificantly highest total Cd accumulation (8 times greater than control C1,i.e., only Cd used). The GA3 treatments accumulated more than 50% of thetotal Cd in the roots, whereas the EDTA treatments showed more than 50%in the leaves. Root dry biomass showed a positive and significant correlationwith Cd accumulation. GA3 is environment friendly as compared with EDTA.Therefore, further investigation of GA3 is recommended for phytoremediationresearch for the remediation of metal-contaminated soil.

KEYWORDS cadmium, EDTA, contaminated soil, Parthenium hysterophorus, phytoex-traction

Address correspondence to Fazal Hadi,Plant Biotechnology Laboratory,Department of Biotechnology, Facultyof Biological Sciences, University ofMalakand, 18000 KhyberPakhtunkhwa, Pakistan. E-mail:dr.fhadi@uom.edu.pk

Color versions of one or more of thefigures in the article can be foundonline at www.tandfonline.com/bbrm.

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INTRODUCTIONHeavy metal-contaminated soil is a potential threat

to the environment due to the high level of durabil-ity of metals and their toxicity to biota (Alkorta et al.2004). The nondegradable nature of metals makes thempermanent pollutants in the environment (El-Nadyand Atta 1996; Mubeen, Naeem, and Taskeen 2010).Mainly the metals enter the soil through anthropogenicactivities such as mining, industrial discharge, sewageeffluents, pest and disease control agents, and waste dis-posal (Adewole, Sridhar, and Adeoye 2010; Mazhariaand Homaeeb 2012; Singh et al. 2003). Cadmium (Cd)is one of the heavy metals of great concern due to itshighly toxic nature (Saberi and Shahriari 2011). Plantscan easily absorb cadmium due to their strong bioaccu-mulative capacity (Alkorta et al. 2004). Consequently,it reaches human and animal bodies through the foodchain (Jayakumar, Jaleel, and Vijayarengan 2009; Ade-wole, Sridhar, and Adeoye 2010) and causes serioushealth problems such as kidney and liver damage, whereit can be stored for a long time and may lead to impair-ment of kidney function and other chronic toxicities(Liu et al. 2005). Cadmium is a potential mutagenicand carcinogenic agent (Raikwar et al. 2008) and alsocan interact with essential minerals such as Ca and Pand can affect other aspects of bone metabolism (Liuet al. 2005).

The removal of Cd from soil and water is ofgreat importance and needs an effective and afford-able technological solution. Many techniques suchas physical, chemical, thermal, and electrokineticmethods have been employed for the restoration ofmetal-contaminated soil (Khan et al. 2000). However,the recent discovery of some plants capable of accu-mulating large quantities of heavy metals from soil hasled to the development of a new emerging technologyknown as phytoremediation (Chiang et al. 2006). Phy-toremediation is advantageous over other remediationtechnologies. It is aesthetically pleasing, environmentfriendly, economical, solar driven, and providesground covering to protect communities from therisk of metal exposure while reducing erosion by windand water and preserving the soil’s physical properties(Danika and Terry 2005; Hadi, Bano, and Fuller 2010).The term phytoremediation (“phyto” meaning plant,and the Latin suffix “remedium” meaning to clean orrestore) is used for a diverse collection of technologies(such as phytoextraction, rhizofiltration, phytostabi-

lization, and phytovolatilization) based on the use ofeither naturally occurring or genetically engineeredplants to clean contaminated soil and water (Revathi,Haribabu, and Sudha 2011; Jadia and Fulekar 2009).

Parthenium hysterophorus, a member of family Aster-aceae, is native to the tropics and subtropics of Amer-ica (Parsons and Cuthbertson 1992). It is a commoninvasive species in Australia, India, Pakistan, and someparts of Africa that invades all lands, including pastures,farms, and roadsides (Dhawan and Dhawan 1996). P.hysterophorus was selected for the present investigationdue to its fast growth rate, stress tolerance, and unpalat-able nature to herbivores in order to help in preventingentrance of metal into the food chain. Previously thisplant was tested for lead (Pb) phytoextraction abilityand showed encouraging results (Hadi and Bano 2009).Plants with a high metal-accumulating capacity oftenhave a slow growth rate and produce limited amountsof biomass in metal-contaminated soil (Denton 2007).Previous literature encourages the use of plant growthhormones for restoration of growth rate and biomassaffected by heavy metals (Falkowska et al. 2011). Gib-berellic acid (GA3) increases plant growth and biomass(Tassi et al. 2008). In the present study, GA3 was usednot only to restore the growth and biomass of the plantin contaminated soil but also to investigate its rolein Cd phytoextraction and to compare it with the ef-fect of a synthetic chelator, ethylenediaminetetraaceticacid (EDTA). EDTA enhances metal bioavailability inthe soil and facilitates its uptake by plants, but conse-quently, due to high metal concentration in the plant,growth is reduced and ultimately the phytoextractionefficiency of the plant decreases (Bruno-Fernando et al.2007).

Therefore, this study was carried out with an objec-tive to evaluate the effect of a plant growth regulator(gibberellic acid, GA3) and a chelating agent (EDTA) ei-ther alone or in different combinations (synergy) on (1)growth and biomass of the P. hysterophorus plant in Cd-contaminated soil and (2) Cd uptake and accumulationin different parts of the plant.

MATERIALS AND METHODSSoil Preparation

Soil was collected from fertile agricultural fields nearthe University of Malakand main campus at Chakdara.The soil was dried in the sunlight and ground into

47 Remediation of Cd-Contaminated Soil

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powder. Maximum water holding capacity of the soilwas measured (300 ml water/kg soil ± 3) using the Keenmethod (Keen 1931). Plastic pots (18 × 15 cm) wereused, and each pot was filled with 1 kg soil. The soilwas basic (pH 8 ± 0.4, n = 6), so it was adjusted to 6.5 ±0.3 using acetic acid (50% v/v) solution. Mostly at lowpH do heavy metals mobilize and become availableto plants. Cadmium was added at a rate of 100 mgCd/kg of soil in the form of cadmium acetate dihydrate(CH3COO)2Cd·2H2O (Merck, Darmstadt, Germany).A stock solution was prepared by dissolving 17.06 g ofthe cadmium acetate dihydrate in 7.21 L of distilledwater, and from stock solution 100 ml was poured intoeach pot except the control (C) pots.

Seedlings Transplantationand Growth

The pots were watered 24 h before transplantation.Uniform seedlings (height: 3 in) were transferred toeach pot (single plantlet/pot). Six replicate pots wereused for each treatment and control. Pots were arrangedin a completely randomized design in a glass houseunder natural conditions with day/night temperaturesof 35/25◦C. All pots were watered twice a week with tapwater according to the water holding capacity of soil.No additional fertilizers were applied to the soil duringthe experiments.

Treatments Used during PotExperiments

Treatments used during pot experiments are summa-rized in Table 1.

Exogenous Application of GibberellicAcid (GA3)

Preparation of GA3 Stock Solution and TestSolutions (10−5, 10−7, and 10−9 M Solutions)

A stock solution of GA3 was prepare by dissolving2.516 × 10−3 g (calculated amount) of GA3 in 100 μlof ethanol (EtOH), and then the volume was raised to200 ml (0.2 L) by adding distilled water. The concen-tration of the stock solution was 3.6363 × 10−5 M andtest solutions were made by dilution using the formulaC1V 1 = C2V 2. Test solutions of GA3 were applied asfoliar spray (each dose 10 ml solution/plant) in fourdoses (each dose at 10-day intervals). The first treat-ment was made 15 days after transplantation. DuringGA3 application, the soil in the pots was covered withpolythene bags to avoid the entrance of GA3 into theroot zone.

Ethylenediaminetetraacetic Acid (EDTA)Addition into Soil

Aqueous solutions of EDTA were used in 2 ways,single dose (160 mg EDTA/kg soil, once) and four splitdoses (40 mg EDTA/kg soil, each dose). The EDTA(single or split dose) first treatment was made 15 daysafter seedling transplantation, and the remaining splitdoses were applied at 10-day intervals. The volume ofEDTA solution was made according to the water hold-ing capacity of soil.

TABLE 1 Treatments Used during Pot Experiments

Treatment Treatment code Treatment Treatment code

Control without Cd C Cd + GA310−9 M in four doses + EDTA160 mg/kg soil in single dose

T6

Control with Cd C1 Cd + GA310−9 M in four doses + EDTA40 mg/kg soil in four split doses

T7

Cd + GA3 10−5 M (four treatments) T1 Cd + GA3 10−7 M in four doses +EDTA 160 mg/kg soil in single dose

T8

Cd + GA3 10−7 M (four treatments) T2 Cd + GA3 10−7 M in four doses + EDTA40 mg/kg soil in four split doses

T9

Cd + GA3 10−9 M (four treatments) T3 Cd + GA3 10−5 M in four doses +EDTA 160 mg/kg soil in single dose

T10

Cd + EDTA 160 mg/kg soil (once) T4 Cd + GA3 10−5 M in four doses + EDTA40 mg/kg soil in four split doses

T11

Cd + EDTA 40 mg/kg soil in four splitdoses

T5

Note. Control C was compared with control C1 to find the effect of Cd on growth, whereas C1 was compared with all other treatments.

F. Hadi et al. 48

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GA3 and EDTA in Different Combinations

Different concentrations of GA3 (10−9, 10−7, and10−5 M) were applied in combination with EDTA.The EDTA was applied in both single dose (160 mgEDTA/kg soil) and split doses (40 mg EDTA/ kg soil,in four doses). In combination treatments, both theGA3 and EDTA applications were made as mentionedearlier.

Plant Growth Parameters AnalysisThe effect of different treatments on plant height

and internode numbers was measured on a weekly basisusing centimeter ruler. Plants were harvested 2 monthsafter transplantation (vegetative stage) and then washedwith tap water and each plant separated into three parts(roots, stems, and leaves). The roots were further washedwith a solution containing 5 mM Tris·HCl pH 6.0 and5 mM EDTA, and they were then rinsed with distilledwater in order to remove the surface-bound metal ions(Genrich et al. 2000). Plant height and root length weremeasured with a centimeter ruler from the root andshoot joint to the apices. The fresh and dry biomassof each part (roots, stems, and leaves) was measuredusing an analytical balance. For dry biomass, differentplant parts were dried in an oven at 80◦C for 48 h, andthen the dry biomass of the roots and shoots (stems +leaves) was noted.

Cadmium (Cd) Analysis in DifferentPlant Parts

Oven-dried samples of roots, stems, and leaves wereground into fine powder using a commercial blenderand were stored in small polyethylene bags until usedfor acid digestion by the Allen (1974) method. Eachsample (powdered) was dissolved separately by adding0.25 g powdered sample to 50-ml flasks, and 6.5 mlof mixed acid solution (nitric acid, sulfuric acid, andperchloric acid in a ratio of 5:1:0.5 v/v/v) was addedand kept on electric hot plates until completely dis-solved. The dissolved samples were transferred into 50-ml volumetric flasks, and the volume was raised upto 50 ml with deionized H2O (dH2O). Filtered thesamples and the filtrates were kept in small plasticbottles for further analysis of cadmium (Cd) concentra-tion in plant parts. For Cd analysis, the atomic absorp-tion/flame spectrophotometer (Hitachi model Z-8000;xx Japan) at the Pakistan Council of Science and In-dustrial Research (PCSIR) laboratories at Peshawar wasused.

Statistical AnalysisThe data were subjected to analysis of variance

(ANOVA), and the mean values were compared by us-ing Tukey’s honestly significant difference test, at p <

.05. Minitab 15, SPSS 16, and MS Excel 2007 softwarewere used for the analysis of data.

FIGURE 1 Effect of different treatments of GA3 and EDTA on the root and shoot length of Parthenium hysterophorus plant in cadmium-contaminated soil (100 mg Cd/kg soil). Treatments used: C (control without Cd), C1 (control with Cd), T1 (GA3 10−5 M), T2 (GA3 10−7 M),T3 (GA3 10−9 M), T4 (EDTA 160 mg/kg), T5 (EDTA 40 mg/kg), T6 (GA3 10−9 M + EDTA 160 mg/kg), T7 (GA310−9 M + EDTA 40 mg/kg), T8(GA3 10−7 M + EDTA 160 mg/kg), T9 (GA3 10−7 M + EDTA 40 mg/kg), T10 (GA3 10−5 M + EDTA 160 mg/kg), and T11 (GA3 10−5 M + EDTA40 mg/kg). GA3 was applied in four split doses, and 160 mg/kg EDTA was added in a single dose, whereas 40 mg/kg EDTA was given infour doses.

49 Remediation of Cd-Contaminated Soil

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TAB

LE2

Eff

ect

of

GA

3an

dE

DTA

,Alo

ne

and

inC

om

bin

atio

ns,

on

Par

then

ium

hyst

ero

ph

oru

sP

lan

tG

row

nin

Cad

miu

m-C

on

tam

inat

edS

oil

Mea

np

lan

tg

row

th(P

G)

(cm

)±

SDM

ean

fres

hb

iom

ass

(FB

M)

(g)±

SDM

ean

dry

bio

mas

s(D

BM

)(g

)±

SDM

ean

tota

lwat

erco

nte

nt

(TW

C)

(g)±

SDTr

eatm

ent

Ro

ot

len

gth

Sho

ot

len

gth

Ro

ot

Stem

Leav

esR

oo

tsSt

emLe

aves

Ro

ots

Stem

Leav

esC

17.1

9±

1.11

ab26

.16

±1.

25b

c1.

80±

0.26

fg3.

74±

0.37

bc

7.84

±3.

31g

0.54

±0.

04c

0.71

±0.

01cd

1.20

±0.

03c

1.26

±0.

30g

h3.

03±

0.36

bc

6.64

±3.

92fg

C1

7.80

±0.

45e

17.0

0±

0.50

ef1.

35±

0.11

gh

3.46

±0.

23cd

6.85

±1.

13g

hi

0.42

±0.

01d

e0.

62±

0.03

e0.

91±

0.02

e0.

94±

0.13

gh

2.85

±0.

25cd

5.95

±1.

14fg

T117

.37

±0.

47a

27.0

0±

2.00

bc

3.84

±0.

10b

7.76

±3.

70b

8.47

±0.

80d

0.70

±0.

01a

0.86

±0.

03b

1.37

±0.

03b

3.15

±0.

11b

c6.

90±

3.69

bc

7.10

±0.

79d

T217

.36

±0.

45a

29.3

3±

1.60

ab3.

24±

0.18

c7.

84±

3.48

b9.

32±

0.83

c0.

72±

0.03

a0.

86±

0.05

b1.

58±

0.04

a2.

52±

0.16

d6.

98±

4.45

b7.

74±

0.85

d

T316

.30

±0.

36ab

c36

.67

±6.

25a

2.62

±0.

38d

e7.

80±

4.34

ab8.

42±

3.49

b0.

73±

0.04

a0.

98±

0.02

a1.

64±

0.03

a1.

89±

0.34

ef6.

82±

4.41

b6.

78±

3.48

c

T47.

34±

2.05

e16

.17

±1.

04ef

1.34

±0.

10g

h7.

80±

0.28

f5.

85±

0.07

hi

0.24

±0.

01f

0.26

±0.

02g

0.57

±0.

02g

1.10

±0.

11g

h4.

94±

0.21

gh

5.13

±0.

25fg

T56.

45±

0.32

e12

.67

±2.

08f

1.09

±0.

15h

4.34

±4.

16f

4.15

±1.

61i

0.21

±0.

01f

0.24

±0.

03g

0.60

±0.

02g

0.88

±0.

15h

4.10

±4.

14h

3.55

±0.

59g

T614

.22

±0.

22d

28.3

3±

1.60

bc

3.79

±2.

64cd

7.42

±4.

90e

5.75

±1.

60g

h0.

48±

0.02

cd0.

67±

0.02

de

0.75

±0.

02f

3.31

±2.

64d

e6.

76±

4.89

ef5.

00±

1.58

f

T715

.98

±0.

47ab

cd24

.60

±4.

40b

cd3.

62±

1.54

ef5.

07±

2.44

e5.

47±

1.89

i0.

57±

0.03

bc

0.74

±0.

05cd

0.81

±0.

01f

3.05

±1.

56fg

4.33

±2.

42fg

4.66

±1.

89g

T815

.07

±0.

22cd

26.0

0±

1.00

bcd

6.50

±0.

14a

10.2

7±

2.12

a9.

55±

0.64

a0.

66±

0.05

ab0.

76±

0.02

c1.

02±

0.07

d3.

74±

3.82

a6.

24±

1.57

a8.

51±

0.40

a

T915

.77

±0.

25ab

cd21

.50

±0.

50cd

e4.

19±

0.27

b4.

79±

0.64

ab7.

75±

1.94

a0.

65±

0.05

ab0.

80±

0.02

bc

1.36

±0.

03b

3.54

±0.

23b

3.99

±0.

63ab

6.39

±1.

95b

T10

17.0

6±

0.41

ab18

.67

±1.

52d

ef4.

39±

1.23

cd8.

55±

5.13

de

8.56

±2.

33e

0.39

±0.

02d

e0.

49±

0.04

f0.

73±

0.04

f4.

00±

1.25

cd8.

06±

5.10

de

7.83

±2.

33e

T11

15.4

1±

0.18

bcd

15.3

3±

1.52

ef3.

42±

0.68

d5.

54±

2.81

e5.

74±

0.63

f0.

35±

0.05

e0.

47±

0.04

f0.

59±

0.02

g3.

07±

0.71

de

5.07

±2.

80d

ef5.

15±

0.64

e

Not

e.D

iffer

ent

lett

ers

insu

pers

crip

tsh

owsi

gnifi

cant

diff

eren

ce(T

ukey

’ste

st,p

≤.0

5).

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RESULTS AND DISCUSSIONEffect of Various Treatments on Plant

Growth and BiomassThe effect of GA3 and EDTA treatments on growth

(shoot height, root length, and biomass) of plantsgrown in Cd-contaminated soil is presented in Figure 1

and Table 2. The results demonstrated the negative ef-fect of cadmium on the growth and dry biomass of theP. hysterophorus plant when C (control without Cd) wascompared with C1 (control with Cd only), as shown inTable 2. Similar effects of cadmium were reported byHaouari et al. (2012) on Lycopersicon esculentum, Sheirdilet al. (2012) on Glycine max, Bavi, Kholdebarin, and

FIGURE 2 Mean cadmium concentration (ppm) in (A) root, (B) stem, and (C) leaves. Mean Cd accumulation (mg/DBM) by (D) root, (E)stem, (F ) leaves, and (G) whole plant. Bars indicate standard error. Different letters show significant difference (Tukey’s test).

51 Remediation of Cd-Contaminated Soil

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Moradshahi (2011) in Pisum sativum plant, John et al.(2009) on Brassica juncea, Varalakshmi et al. (2009) onAmaranthus tricolor cultivars, Kaznina, Laı̌dinen, andTitov (2006) on Hordeum vulgare, and Ahmad et al.(2001) on Brassica species. The decrease in growth anddry biomass might be due to a change in the functionof some of the key enzymes involved in metabolism(John et al. 2009) or the inhibition of chlorophyll syn-thesis and consequently the process of photosynthesis(Padmaja, Prasad, and Prasad 1990). On the other hand,no significant effect of cadmium-contaminated soil onfresh biomass and water contents of plants was recordedwhen comparing C with C1 (Table 2).

GA3 (foliar spray) significantly increased plantgrowth and biomass in Cd-contaminated soil comparedto control C1 (Table 2). This increase in shoot heightand root length might be due to the ability of GA3 topromote cell division. Other important effects of GA3

on DNA, RNA, and protein synthesis (Broughton andMcComb 1971; Benjerano and Lips 1970) and riboseand polyribosome multiplication (Evins 1972) wouldcontribute to higher biomass production of vegetativeparts. The highest shoot length (36.67 ± 6.25 cm) wasshown by GA3 at a lower concentration (10−9 M),whereas the highest root length (17.37 ± 0.47 cm)was recorded for GA3 applied at high concentration(10−5 M). These findings indicate that shoot length pos-sessed an inverse relation whereas root length showeda direct relation with the concentration of exogenousGA3 (foliar spray) applied to the shoot (Bidadi et al.

2009). On the other hand, the EDTA addition to soilsignificantly reduced the root length (6.45 ± 0.32 cm)and shoot length (12.67 ± 2.08 cm) as compared withC1 (7.8 ± 0.45 and 17 ± 0.5 cm, respectively) (Ta-ble 2). This decrease in growth rate might be due to theincrease in Cd bioavailability by EDTA in soil, thus in-creasing the Cd absorption/accumulation by the plantand consequently reducing plant growth and biomassdue to its toxic effect (Elkhatib, Thabet, and Mahdy2001). GA3 applied in combination with EDTA signif-icantly increased plant growth and biomass comparedwith EDTA alone treatments, which suggests that GA3

compensates for the negative effect of EDTA on growthand biomass. Fresh biomass and total water content ofthe plant showed the highest increase when treated withGA3 (10−7 M) and EDTA (160 mg kg-1 once) in com-bination treatment T8, as shown in Table 2.

Effect of Treatments on Cd Uptakeand Accumulation in Plant Parts

The effect of different treatments of GA3 and EDTAalone and in various combinations on cadmium uptakeand accumulation in plants is shown in Figure 2A–G.All the treatments showed a significant increase in cad-mium concentration (ppm) in roots (Figure 2A), stems(Figure 2B), and leaves (Figure 2C) when comparedwith control C1 (only Cd). Subsequently, the total Cdaccumulation (mg/total dry biomass) was found sig-nificantly higher than in the control (C1) in all parts

TABLE 3 Effect of GA3 and EDTA on Percent Increase of Cd Accumulation Compared with C1 and Also on the Percent Cd DistributionAmong Plant Parts within Plant

Fold increase in Cd accumulation compared to C1aPercent (%) Cd distributionamong different plant parts

Entire plantTreatment Root Stem Leaves (roots + stem + leaves) Roots Stem Leaves

C1 0.053 mg 0.0031 mg 0.085 mg 0.141 mg 37.40 2.19 60.40T1 3.17 2.52 1.75 2.30 51.60 2.41 46.00T2 4.50 9.13 2.51 3.40 49.60 5.89 44.60T3 5.61 14.32 2.65 4.02 52.30 7.81 39.90T4 3.45 9.80 2.50 3.02 42.82 7.12 50.06T5 3.57 8.91 2.84 3.24 41.20 6.02 52.80T6 5.73 26.98 2.17 4.05 53.00 14.60 32.40T7 7.12 31.43 3.12 5.24 50.86 13.14 36.00T8 12.04 23.59 2.86 6.75 66.71 7.66 25.63T9 12.48 26.62 4.23 7.81 59.79 7.48 32.73T10 7.67 21.00 1.50 4.24 67.69 10.86 21.45T11 7.54 23.66 1.40 4.19 67.33 12.38 20.29

aFor C1, actual values of extracted Cd (mg) are given.

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of the plant such as in roots (Figure 2D), stems (Fig-ure 2E), and leaves (Figure 2F). Although GA3 (foliarspray) treatments (T1, T2, T3) showed a significant in-crease in Cd accumulation compared with control (C1),the Cd concentration in different parts of the plantdecreased with increasing GA3 concentration, suggest-ing that high concentrations of exogenous GA3 havenegative effects on Cd absorption by roots (Figure2A, D), stems (Figure 2B, E), and leaves (Figure 2C,F). EDTA treatments (T4, T5) significantly reducedthe plant growth and biomass, as mentioned earlier(Table 2), but increased the Cd uptake and accumula-tion significantly in all parts of the plant (Figure 2A–F)when compared with control (C1). EDTA as a chelat-ing agent has positive effects on bioavailability of heavymetals in soil, thereby increasing the amount of met-als concentration in the plants (Lai and Chen 2005;Luo, Shen, and Li 2005; Jean et al. 2008; Sun et al.2011). Cadmium concentration in different parts ofthe plant was in order of roots > leaves > stems, whichis in agreement with the work of Sun, Zhou, and Diao(2008). The highest Cd concentration in roots (1124.67± 4.5 ppm) (Figure 2A) and stems (154.33 ± 7.5 ppm)

(Figure 2B) was recorded for combination treatmentT11 (GA3 10−5 M + EDTA split doses), whereas inleaves (375 ± 4.5 and 400 ± 2 ppm) it was shownby the treatment of EDTA alone (T4 and T5) (Fig-ure 2C). Since GA3 (foliar spray) increased the plantdry biomass and the EDTA addition to soil enhancedthe Cd phytoextraction, their combination treatments(GA3 + EDTA) showed the most significant effect oncadmium accumulation in different parts of the plant.The highest significant cadmium accumulation in roots(0.65 ± 0.04 mg/g dry weight) (Figure 2D) and leaves(0.35 ± 0.03 mg/g dry weight) (Figure 2F) was shown byT9 (GA3 10−7 M + EDTA split doses), whereas in stems(0.097 ± 0.003 mg/DBM) (Figure 2E) it was demon-strated by the treatment T7 (GA3 10−9 M + EDTA splitdoses).The maximum accumulation of Cd in the entireplant was found in T9 (Figure 2G). The T9 treatmentsshowed a 12.48-fold increase in root Cd accumulationand a 4.23-fold increase in leaf Cd accumulation com-pared with control (C1) (Table 3), whereas in the steman increase of 31.43-fold was found in treatment T7, asshown in Table 3. Plant stems possessed the lowest Cdaccumulation percentage as compared with roots and

FIGURE 3 Correlations between dry biomass and cadmium accumulation in (A) root, (B) stem, and (C) leaves.

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leaves in all the treatments. GA3 alone or in combi-nation treatments with EDTA accumulated more than50% of its total plant Cd in the roots, which indicatesthat GA3 can increase the Cd uptake into roots butnot translocate it into aerial parts. The EDTA-treatedplants showed 50% or more of its total Cd in its leaves(Table 3).

The root dry biomass showed a strong positive cor-relation (R2 = .898) with Cd concentration (Figure 3A),whereas the plant stem (Figure 3B) and leaf (Figure 3C)dry biomass showed a weak negative correlation (rootR2 = .267, leaf R2 = 0.328) with the Cd concentration(Figure 3).

ConclusionsThe GA3 treatments increased the growth and

biomass of the plant in Cd-contaminated soil, andEDTA reduced the growth and biomass. However,GA3 compensated for the negative effect of EDTA ongrowth and biomass when applied in combinations.GA3 significantly increased the Cd accumulation inplants, but response to EDTA was better than GA3 inrespect to Cd translocation into aerial parts. GA3 is en-vironmentally friendly as compared with EDTA, andthe present results showed that GA3 increases Cd inroots, whereas EDTA increases Cd translocation intoaerial tissues, which might be a cause of Cd entranceinto the food chain. Therefore, the use of GA3 forphytoextraction purposes is better than EDTA. Furtherinvestigation on GA3 effects on metal phytoextractionis recommended.

FUNDINGHigher Education Commission (HEC) of Pakistan is

acknowledged for providing partial financial support.

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