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Advances in Environmental Biology, 5(4): 559-565, 2011
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Phytotoxicity of Cadmium on Seed Germination, Early Growth, Proline and
Carbohydrate Content in Two Wheat Verities
1
M.R. Asgharipour, 2M. Khatamipour and 3M. Razavi-Omrani
1
University of Zabol, Zabol, Iran.
University of Zabol
3
Islamic Azad University-Quchan Branch
2
M.R. Asgharipour, M. Khatamipour and M. Razavi-Omrani; Phytotoxicity of Cadmium on Seed
Germination, Early Growth, Proline and Carbohydrate Content in Two Wheat Verities
ABSTRACT
Human activities all over the earth have increased environmental pollution by heavy metals in agricultural
soil. The present study was designed to examine the toxicological effects of different concentrations of
cadmium (Cd) on the germination, early-growth parameters, and accumulation of Proline and carbohydrates
in two wheat varieties, namely Roshan and Omid. Seeds of these plants were exposed to five different
concentrations of Cd in an increasing fashion (5, 20, 50, 100 and 200 mg L-1 of Cadmium Chloride). The
endpoints of wheat seedlings, including seed germination percentage, mean germination time, seedling dry
weight, root length and shoot height, all decreased along with increasing the Cd concentrations. Cd-treated
seedlings, on the contrary, showed an increase in Proline and carbohydrate when compared to control.
Significant differences in seed germination, biomass, root length, shoot height and the accumulation of Proline
and carbohydrates were observed through the treatments and between the two varieties. In the two varieties,
Roshan was found to be more resistant to Cd phytotoxicity. The sensitivity of wheat early-growth to the
toxicity of the Cd contamination was in the following sequence: root elongation > shoot elongation >
germination.
Key words: Toxicology; Cadmium; Wheat Triticum aestivum, Varieties, seedling.
Introduction
The ever-increasing environmental pollution in
agricultural soil caused by heavy metals due to
application of sewage sludge, city refuse, and heavy
metals containing fertilizers or pesticides, is
becoming a major problem in modern agriculture
[30,5].
Cadmium is one of the toxic heavy metals in
contaminated crop environments. In natural soils, Cd
concentration in soil solution is estimated to be
around 0.04–0.32 mM. Nevertheless, the soil solution
with 0.32 to nearly 1 mM Cd can be considered
polluted or toxic[8]. The mobility of this metal in
soil–plant system allows its easy take-up in excess
by plant so that it will directly or indirectly inhibit
the physiological processes like respiration,
photosynthesis, transpiration, cell elongation, plantwater relationship, mineral nutrition, nitrogen, and
carbohydrate metabolism, leading to poor growth and
low biomass[15,28].
The seed germination and early-seedling growth
are important stages in the whole process of plantgrowth and due to being the most sensitive stage in
the plants changing of their environment, have been
widely used in environmental bio-monitoring. In this
study, we looked at the ecotoxicological effects of
Cd in small-scale toxicity tests using seed
germination assay of wheat (Triticum aestivum L.) as
a model crop. Not only is wheat one of the most
Corresponding Author
M.R. Asgharipour, University of Zabol, Zabol, Iran,
Phone No.: +989153167234
Email: [email protected].
Adv. Environ. Biol., 5(4): 559-565, 2011
important food crops, it is also easier to culture, to
maintain in the laboratory conditions, and even to
use in toxicity tests for examining the effects of
cadmium, especially on the germination of seeds and
the growth of early seedlings which are more
sensitive to heavy metals [26,23]. Many plants at
seed germination and seedling stages are sensitive to
environmental factors. Therefore, the change of plant
growth at the germination and seedling stage under
heavy metal stress is often regarded as an important
index to evaluate plant tolerance to heavy metals[29].
Cd stress leads to protein degradation and
increased levels of Proline [32,12]. Therefore, Proline
accumulation, accepted as an indicator of
environmental stresses [2]. Proline is also considered
to have important protective roles through osmotic
adjustment [2].
Different plant species and varieties show a wide
range of plasticity in Cd tolerance, varying from the
high degrees of sensitivity to the hyper accumulating
phenotype of some tolerant higher plants [40,21]. It,
moreover, brings the possibility to develop crop
varieties with low accumulation and high tolerance of
heavy metals, which could be planted in slightly to
moderately contaminated soils. Two popular wheat
varieties, Roshan and Omid, are being widely
cultivated all over Iran. Therefore they were chosen
for examining the toxicological effects of Cd on seed
germination, and root and shoot elongation.
Yet, most of the experiments so far have mainly
concentrated on oxidative stress only in adult plants
during Cd toxicity [34,39] and only a limited number
of studies has been dedicated to the germinating
stage of plants exposed to Cd stress [42]. Previous
experiments indicated that Cd can cause significant
reduction in the germination rate in Triticum and
Cucumis [27] or will serve to inhibit the germination
and growth of Arabidopsis embryos [20].
There is little information available about the
sensitivity and physiological responses of wheat
seedlings to Cd toxicity, eventhough wheat ranks as
the third most important cereal in terms of planting
area and production (FAO "Faostat", 2007). The
main objective of the present study is to examine the
effects of cadmium on the seed germination, early
seedlings biomass, and root and shoot elongation of
two wheat seedling varieties differing in Cd toxicity
resistance by a laboratory experiment. The
availability of carbohydrates (soluble sugars, glucose
and fructose) and Proline changes occurring in
germinating seeds following cadmium exposure to
achieve a better understanding of the Cd toxicity has
been investigated as well.
Materials and Methods
The experiment was carried out in April 2010 at
the Biotech Research Center of the University of
Zabol, Zabol, I.R. Iran. Two wheat (Triticum
560
aestivum) varieties, Roshan and Omid, being widely
planted all over Iran, were used in this study. Seeds
were kindly provided by Zabol Agricultural Research
Center.
Prior to germination, the seeds of wheat were
surface-sterilized with 3% Formaldehyde for 10
minutes and washed 3 times with re-distilled water.
The seeds were then germinated in sterilized Petri
dishes, 100mm in diameter, on Whatmann filter-paper
moistened with 10 mL of either double-distilled
water (control) or Cadmium test solution. The test
was performed on 25 wheat seeds exposed to
increasing concentrations (5, 20, 50, 100 and 200 mg
L-1) of Cadmium Chloride (Sigma, St. Louis, MO).
Concentration in terms of weight-by-volume is
defined in the reference protocol of the Inter
calibration Action in molar terms, 200 mg L!1
corresponds to 1.1 mM of Cadmium. Petri dishes
were subsequently kept in the dark, at 25 NC, for a
span of 7 days. The solutions were renewed after 3
days. The experiment was laid out as a split-plot
design with Cd concentration as the main plot and
verities as the sub-plot, together with four replicates.
After 7 days, 10 seedlings of each petri were
sampled with an aim to measure the root length and
shoot height using a ruler (against a black
background). Dry weight was also evaluated after
drying the specimens (10 seeds) for 72 hours at 76
NC. During the experiment, germinated seeds were
counted on a daily. Seed germinability was assessed
by the final cumulative percentage of germination at
the end of the tests. Here, germination was
considered only when the radicles were longer than
2 mm. FGP and D 50 germination was calculated
based on Soltani et al. [38].
Proline content was determined using a
colorimetric method modified from Li [19] with
minor modifications. The fine powder of freeze-dried
plant tissues (0.2 g) was treated with 5ml of 3%
Sulphosalicylic acid and maintained at 100 NC for 10
minutes. The supernatant (2ml) was added to a
solution of 2 ml of glacial Acetic plus 2 ml of 2.5%
(w/v) acidic Ninhydrin, and kept at 100 NC for 25
minutes. After the liquid was cooled down, it was
added to 4 ml of Toluene. The photometric
absorbance of the Toluene extract was read at 520
nm. Contents were calculated to ηg g-1 dry matter.
For determination of soluble Carbohydrate
contents, embryonic tissues were ground in 80%
Ethanol, boiled for 30vminutes at70 °C, and then
centrifuged at 8000g for 10 minutes at 41°C. The
supernatants were used as samples to determine total
soluble Carbohydrate glucose and Fructose [9].
Calibration curves were obtained using Sigma
standards.
The experiments were repeated twice and the
pooled mean values were separated on the basis of
Duncan Multiple Range Test (DMRT) at a
probability level of 0.01.
561
Adv. Environ. Biol., 5(4): 559-565, 2011
Results:
Two-way ANOVA exhibited that all assessed
parameters were significantly affected by Cd
concentration and verities (Table 1). It was also
confirmed that statistically significant differences of
Cd treatment by verities exist on these parameters.
Toxicity of Cd on Wheat Seed Germination and Early
Developmental Stages:
The response of final germination percentage
(FGP), days to 50% germination (D 50), seedling dry
weight, radicle length, shoot height, and ratio of
radicle length to shoot height to Cd levels are
summarized in Table 2.
The mean FGP (i.e. the average of both
varieties) over control (without Cd) decreased
significantly along with increasing the concentrations
of Cd. The FGP was 96.0, 64.7, 48.0, 32.0 and
26.0% at 0, 5, 20, 100 and 200 mg L-1 of Cd,
respectively. One the other hand, the effect of Cd on
D 50 germination is dependent on its concentration
and the D 50 was 18.7, 21.1, 28.6, 35.0 and 37.6
hour at 0, 5, 20, 100 and 200 mg L-1 of Cd,
respectively.
There were significant differences in FGP and D
50 for different wheat varieties with the treatments of
Cd. In both varieties, the FGP and D 50 decreased
with increasing the Cd. Seed germination in Omid
showed more resistance to the highest concentration
of Cd (200 mg L-1). For the treatment with 200 mg
L-1 of Cd, the FGP was 30.7 and 21.3% while the D
50 was 34.0 and 41.3 hours for Roshan and Omid,
respectively.
The seedling dry weight of both verieties
decreased significantly with increasing the
concentration of Cd. The mean seedlings dry weight
was 0.191, 0.117, 0.094, 0.073 and 0.058 g at 0, 5,
20, 100 and 200 mg L-1 of Cd, respectively.
Differential responses were observed among the
varieties in terms of seedling dry weight; Omid
showed more resistance to Cd contamination, so that
the seedling dry weight for Roshan and Omid at 200
mg L-1 of Cd were 0.054 and 0.062 g, respectively.
The mean radicle length was 6.02, 0.93, 0.65,
0.30, and 0.10 at the Cd concentrations of 0, 5, 20,
100, and 200 mg L-1 of Cd, respectively.
The radicle length for both varieties decreased
significantly with the increase of Cd concentrations.
Differential responses in radicle length were noted
for different Cd concentrations and between the two
varieties. The radicle lengths in Roshan and Omid
for the treatment with 200 mg L-1 of Cd were 0.11
and 0.10, respectively.
Significant reduction in shoot height was
observed with the increase of Cd concentrations. The
mean shoot height was 12.17, 5.94, 4.94, 3.15, and
2.17 cm at the treatments with 0, 5, 20, 100, and
200 mg L-1 of Cd, respectively. The shoot heights in
Roshan and Omid, for the treatment with 200 mg L-1
of Cd were 2.07 and 2.27, respectively.
The difference in radicle and shoot height
indicated that the resistance of wheat varieties to Cd
was greater in Omid compared with that of Roshan.
In our experiments, radicle length was more
affected than radicle length and shoot height. On an
average of two genotypes, radicle length was reduced
by 84, 89, 95 and 98% in 0, 5, 20, 100, and 200 mg
L -1 of Cd treatments, respectively, while,
correspondingly, the reductions in shoot height were
11, 45, 59, 74, and 82%, respectively. Thus, the
increase in the ratio of radicle length to shoot height
observed in the treated plants was mainly due to a
decrease of the radicle length while the shoot height
was less affected. The effect of Cd on ratio of
radicle length to shoot height was dependent on its
concentration and differed with genotypes.
Toxicity of Cd on Proline and Carbohydrate Content:
Proline content and total Carbohydrates in
germinated seeds with different concentrations of Cd
treatment are presented in Table 3.
The Proline content was higher in the seedlings
treated with Cd than in the control (Table 3).
Therefore, the Proline contents were respectively
0.082, 0.092, 0.106, 0.126, and 0.244 at 0, 5, 20,
100, and 200 mg L-1 of Cd which, in comparison to
the control, increased by respectively 12, 28, 54 and
198%,
In addition, the treatment of wheat seedlings
with Cd significantly increased the content of
Carbohydrate reserves in seeds from which the
seedlings had developed. Cadmium-treated seedlings
with 200 Cd mg L-1 exhibited the greatest
Carbohydrate content, while the seedlings in control
had the least Carbohydrate content.
On an average of the two genotypes, the
Carbohydrate contents of 0, 5, 20, 100, and 200 mg
L-1 of Cd treatments were 1.035, 1.249, 1.421, 1.552,
and 1.725, respectively. Between the two varieties,
distinct differences existed regarding the response of
Proline and Carbohydrate contents to Cd treatment
and the accumulation of Proline was always greater
in Roshan than in Omid regarding the response to Cd
stress, while Carbohydrates content was crelatively
greater in Omid.
Discussion:
Germination and Early Seedling Growth:
Germination and early seedling development
assay has been regarded as a basic experiment for
evaluating the toxicity effect of any kind of metal or
chemical on plants [1,25,24]. Germination inhibition
is among the best-known effects of toxic impact of
Adv. Environ. Biol., 5(4): 559-565, 2011
562
Table 1: Results of ANOVA testing the effect of Cd concentration and varieties on final germination percentage (FGP), days to 50%
germination (D 50), Seedling dry weight, Radicle length, Shoot height, ratio of radicle length to shoot height, Proline and Total
soluble carbohydrate content.
SS
SOV
df
FGP
D 50
Seedling dry
Radicle
Shoot
ratio of radicle Proline
Total soluble
(hour)
weight
length
height
length to shoot content
carbohydrate
height
(mg g-1 DW) content
(mg g-1 DW)
Conc.
4
**21.594 **0.0024
**0.0163
**37.269 **92.187 **319.863
**0.0261
**0.427
1st Error
10
0.0328
0.00002
0.000021
0.078
0.097
1.323
0.0000068
0.00018
Var.
1
1.659** **0.00023 **0.0099
**1.81
**25.502 *8.945
**0.00215
**0.43
Var. × Conc. 4
0.14*
0.000057ns **0.0015
*0.31
**2.835
**7.903
**0.00008
**0.0173
Error
10
0.342
0.0002
0.000051
0.0682
3.384
1.045
0.0000038
0.00015
Errorns: not significant; (*) and (**) represent significant difference over control at p<0.05 and p<0.01, respectively.
Table 2: Influence of various Cd concentrations on Final germination percentage (FGP), Days to 50% (D 50) mean, Seedling dry weight,
Radicle length, Shoot height and ratio of radicle length to shoot height of two wheat varieties.
Concentrations
FGP (%)
D 50 (hour)
Seedlings dry
Radicle length
Shoot height
ratio of radicle
of Cd (mg L-1)
weight (g)
(cm)
(cm)
length to shoot
height
---------------------- --------------------------------------------------------------- --------------------- -----------------Roshan Omid
Roshan Omid
Roshan
Omid
Roshan
Omid
Roshan Omid
Roshan Omid
0
94.7a
97.3a
18.7c
18.7c
0.236a
0.146a
6.60a
5.45a
14.07a 10.27a 1.95d
1.88e
5
62.7b
66.7b
20.6b
21.7c
0.138b
0.096b
1.25b
0.61b
7.13b
4.77b
5.68c
7.80d
20
44.0c
52.0c
23.6b
33.7b
0.106c
0.082c
0.92bc
0.39bc 5.88c
4.00b
6.38c
9.49c
100
29.3d
34.7d
36.7a
33.3b
0.082d
0.064d
0.37cd
0.23cd 3.63d
2.66c
9.80b
11.47c
200
21.3e
30.7e
34a
41.3a
0.062e
0.054e
0.10d
0.10d
2.07c
2.27e
22.67a 20.67a
* Values followed by the same letter within the same columns do not differ significantly at p =1% according to DMRT.
Table 3: Influence of various Cd concentrations on Proline and Total soluble carbohydrate content of two wheat varieties.
Concentrations of Cd (mg L-1)
Proline content (mg g-1 DW)
Total soluble carbohydrate content (mg g-1 DW)
---------------------------------------------------------------------------------------------------------Roshan
Omid
Roshan
Omid
0
0.086e
0.078e
0.967e
1.103e
5
0.100d
0.084d
1.127d
1.372d
20
0.115c
0.096c
1.350c
1.493c
100
0.133b
0.119b
1.414b
1.690b
200
0.258a
0.231a
1.526a
1.924a
* Values followed by the same letter within the same columns do not differ significantly at p =1% according to DMRT.
heavy metals [10]. Under heavy metal stress, the
processes of germination, like embryo growth, will
be depressed [1]. Some researchers [31] have
reported the reduction of germination rate and
seedling growth of different crops by heavy metals
toxicity. The present research also showed that the
wheat germination percentage and time are
considerably affected by Cd.
With the increasing the concentrations of Cd in
the growth media, the root length reduced more
significantly than the other parameters and the
reduction was in the following order: root length >
shoot height > seedling dry weight > germination.
More sensitivity of root length to Cd could be
described by the fact that a plant root is the first
point to contact with the toxicants in the growth
media. Cadmium can easily penetrate the root cortex
[41], consequently the roots are more likely to
experience the Cd damage before any other part [8].
The previous work by Blum [6] also showed that
root was the most sensitive part to Cd treatment.
The wheat varieties showed different levels of
sensitivity to Cd toxicity. The data of inhibition rates
of germination, seedling dry weight, root length, and
shoot height all showed that Omid was more tolerant
to Cd contamination. High resistance to Cd could be
achieved by: 1) complexated the Cd by such peptides
as SH-groups [25]; 2) enhanced the production of
antioxidants that detoxify free reactive oxygen
species (ROS) produced in response to Cd [14]; and
3) depressed the total ion activity in the solution
caused by the Cd [22].
Toxicity of Cd on Proline and Carbohydrate Content:
As shown in table 3, increasing the
concentrations of Cd in the growth medium resulted
in a pronounced increase in Proline. In many plants,
unfavorable environmental effects bring about the
accumulation of Proline, which is, by itself, one of
the most universal poly-functional stress-protective
substances [3]. Proline is known to accumulate under
heavy metal exposure and is considered to be
involved in the particular stress resistance [7,11]. The
Proline accumulation in Cd- treated seedlings can be
regarded as one of the most sensitive responses to
water deficiency and osmotic stress [4]. The
capability of plants for a heavy-metal induced Proline
accumulation could be brought about not only by a
direct effect of Cd ions, but also by water deficiency
563
Adv. Environ. Biol., 5(4): 559-565, 2011
[36]. This deficiency develops in the plant tissues
under the conditions of Cd stress due to damage to
the water-absorbing capacity of roots.
Schat et al. [33] considered that Proline
accumulation is mainly induced by the water stress
component of Cd toxicity while Kastori et al. [18]
argued that Proline accumulation occurred as a result
of Cd toxicity independent of any water-stress
component. From an experimental point of view,
however, causes and consequences are quite difficult
to distinguish. Besides its putative impact on osmotic
adjustment processes, Proline was shown to protect
enzymes and cellular structures against heavy metal
damages as a consequence of the formation of
Cd–Proline complexes [35] or against maintenance of
the glutathione redox state, thus indirectly acting as
an antioxidant [37]. So, greater Proline content in
Omid may be a major factor involved in the
comparatively higher degree of resistance of this
variety.
Heavy metals also modified the Carbohydrate
accumulation in wheat seedlings (Tables 3).
Increasing the Cd significantly increased the
Carbohydrate levels measured in the seedlings. The
present results contrast with those of Greger and
Bertell [13] since they found that Cd decreased the
Carbohydrate levels in both shoots and roots from
sugar beet whereas the nutrient medium and the
metal ion were supplied in a similar manner to those
of ours. However, the same authors observed that Cd
increased the carbohydrate levels when plants were
cultivated with an exponential supply of nutrients.
Seed germination relies almost exclusively on
seed reserves for the supply of metabolites for
respiration as well as other anabolic reactions. Starch
is quantitatively the most abundant storage material
in seeds and available evidence indicates that, in
germinating seed, starch is degraded predominantly
via the amylolytic pathway [17]. Our results showed
that Cd treatments reduce the seedling growth
corroborating the results previously found by others
[8,31,1]. The higher Carbohydrate levels observed in
the Cd-treated plants can be explained by less
utilization of Carbohydrate for growth subjected to
Cd stress.
study that Roshan is more suitable for cultivation in
soils with Cd contaminations.
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