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Introduction
Since the mid 1960s, progressive symptoms
of forest decline have been observed in Europe.
It has been caused mainly by emissions of indus
-
trial gases harmful to plants, like SO
2
,NO
X
,CO
and HF, as well as by precipitation of heavy metals
contained in industrial dusts. In Poland strong
signs of forest degradation were first observed at
the end of 1970s. It is estimated that nowadays
only 20% of forests in Poland are free from signs
of harmful industrial pollution (Wawrzoniak
2002; Zwoliñski 2002). The air and soil contami
-
nations cause direct damage of the vegetative and
generative organs of plants as well as influence
physiological processes (De Kok et al. 1998). Gas
-
eous air pollutants, by acidification of soil, induce
changes in its chemical and mechanical properties
and release harmful metal ions, which in turn dam
-
age the soil microflora and mycorrhizal fungi
(Godbold et al. 1998; Cairney and Meharg 1999;
Nielsen and Winding 2002). These adverse
changes affect the vitality and fertility of trees.
This is manifested in the case of Pinus sylvestris in
decreased biomass increments, linked to a signifi
-
cant economical loss, decrease in seed production,
and their diminished germination (Oleksyn et al.
1992, 1994). When stress caused by the pollution
exceeds a certain level, then individual trees
and whole tree stands start to decline. The ob
-
served differential mortality of trees within the
population raises a question whether it is a sto
-
chastic process or the trees are subject to some se
-
lection, based on genetic structure. The existence
of distinctly pollution-tolerant and pollu
-
tion-sensitive individuals suggests genetics back
-
ground of the phenomenon. This was confirmed
by a high rate of heritability of resistance to indus
-
J Appl Genet 47(2), 2006, pp. 99–108
Effects of heavy metal pollution on genetic variation
and cytological disturbances in the Pinus sylvestris L. population
Wies³aw Prus-G³owacki
1
, Ewa Chudziñska
1
, Aleksandra Wojnicka-Pó³torak
1
, Leon Kozacki
2
,
Katarzyna Fagiewicz
2
1
Department of Genetics, Institute of Experimental Biology, Adam Mickiewicz University, Poznañ, Poland
2
Institute of Physical Geography and Environmental Planning, Adam Mickiewicz University, Poznañ, Poland
Abstract: This isoenzymatic and cytogenetic study has shown significant differences in genetic composition
between two groups of Pinus sylvestris trees: tolerant and sensitive to heavy metal pollution. Total and mean
numbers of alleles and genotypes per locus were higher in the pollution-sensitive group of trees, but
heterozygosity (H
o
) was lower in this group. Fixation index (F) indicates that trees tolerant for pollution were in
the Hardy-Weinberg equilibrium, while the sensitive group had a significant excess of homozygosity.
Cytological analyses demonstrated numerous aberrations of chromosomes in meristematic root tissue
of seedlings developed from seeds collected from trees in the polluted area. The aberrations included
chromosome bridges and stickiness, laggards, retarded and forward chromosomes, and their fragments.
The mitotic index was markedly lower in this group of seedlings, as compared to the control. Both isoenzymatic
and cytological analyses showed a significant influence of heavy metal ions on the genetic structure of the Pinus
sylvestris population.
Key words: chromosome aberrations, genetic structure, heavy metals, isoenzymes, Pinus sylvestris, pollution.
Received: November 1, 2005. Accepted: January 3, 2006.
Correspondence: W. Prus-G³owacki, Department of Genetics, Institute of Experimental Biology, Adam Mickiewicz
University, Miêdzychodzka 5, 60-371 Poznañ, Poland; prusw@amu.edu.pl
trial pollution of some individuals (Geburek et al.
1987; Hertel and Paul 2001; Mejnartowicz 2001).
The introduction of isoenzymatic markers to the
studies on diversity allowed demonstrating differ
-
ences in genetic structure of pollution-tolerant and
pollution- sensitive trees, definitely confirming
the genetic background of the resistance
(Mejnartowicz 1983; Bergmann and Scholz 1987;
Geburek et al. 1987, Müller-Starck 1987, 1989;
Prus-G³owacki and Nowak-Bzowy 1992,
Korshikov et al. 2002). The studies on
isoenzymatic diversity allowed not only to deter
-
mine differences in genetic structure of trees but
also permitted to demonstrate the type and direc
-
tion of processes of adaptation to pollution at the
population level. The cytogenetic studies con
-
firmed the influence of pollution on P. sylvestris
and on other plants, manifested in aberrations of
chromosomes and in values of mitotic index
(Starova et al. 1994; Müller and Grill 1996;
Kristen 1997; Dixon and Buschena 1998; Müller
et al. 1998; Kovalchuk et al. 1998; Kirkland 1998;
Wonisch et al. 1999). The aim of this study was to
determine the genetic structure of tolerant and sen-
sitive groups of trees growing in an environment
heavily polluted due to the activity of a zinc
smelter, by isoenzymatic and cytogenetic analy-
ses. The population is located in a direct vicinity of
the smelter, which since the 1960s has been emit-
ting notable amounts of heavy metal ions and in-
dustrial dusts. This pine population results from
natural regeneration and consists of trees that
show adverse effects of long-term industrial stress
as well as trees without any visible signs of injury
(Prus-G³owacki and Godzik 1991; Prus-G³owacki
and Nowak-Bzowy 1992; Prus-G³owacki et al.
1998). This situation provides a good model for
the studies and, together with determination of
the extent of air and soil pollution, it opens per
-
spectives for reliable monitoring of the adaptive
strategy in the group of trees that tolerate the stress
and for examination of heavy metal influence on
the process of mitosis and development of chro
-
mosome aberrations.
Material and methods
Collection of the material
The material was collected from Scots pine trees
(Pinus sylvestris L.) that grow 500–800 m NE
of the Zinc Smelter in Miasteczko Œl¹skie (Upper
Silesia, SW Poland). The trees selected for
the study, aged 15–20 years, originated from
natural regeneration of the pine forest that
bordered with the studied area. The material,
including winter buds, two-year-old needles
and seeds, was collected there from two groups of
40 trees each. The first group represents trees
showing signs of damage, such as needle necrosis
and chlorosis, atypical habit and poor growth.
In the second group of trees, no such signs of
damage were manifested. During collection
of the material, height of the individual trees was
measured. For the cytological study, control
samples were collected in the National Park of
Wielkopolska (NPW), which is free from heavy
metal pollution.
Analysis of the environmental pollution
The status of the natural environment in the region
of zinc smelter was characterized by determining
of levels of air pollution with industrial gases,
SO
2
,NO
2
, CO, and deposition of industrial dusts
containing heavy metals like Zn, Pb, Cd, Cu, Cr,
Ni, Mn and Co (data from the Engineer- Con-
sulting Office ‘EKOKOKS’, Zabrze).
The amounts of heavy metals (Cd, Cr, Cu, Ni, Pb
and Zn) in the soil were estimated by taking 5 soil
samples every 50 meters along 4 NE-SW
transects. The results were means for 4 samples.
The analyses were conducted by absorptive atom
spectrometry in the Department of Subterranean
Water Analysis, Faculty of Chemistry, Adam
Mickiewicz University, Poznañ. Soil pH was mea
-
sured by the potentiometric method in the 1M KCl
suspensions.
Analysis of biological material
In order to determine the genetic structure of
the pollution-tolerant (T) and pollution-sensitive
(S) trees, electrophoresis was used (according to
the procedure described by Rudin and Ekberg
1978; Muona and Szmidt 1985; Prus-G³owacki
and Nowak-Bzowy 1992).
Variability of 18 loci within 11 enzymatic sys
-
tems was studied. These enzymatic systems in
-
cluded: fluorescence esterase (Fest, 2 loci) EC
3.1.1.1; glutamate-oxalacetate transaminase
(GOT, 3 loci) EC 2.6.1.1; glucose-6-phosphate
dehydrogenase (6PGD, 1 locus) EC 1.1.1.49;
shikimate acid dehydrogenase (ShDH, 2 loci) EC
1.1.1.14; malate dehydrogenase (MDH, 2 loci)
EC1.1.1.37; alcohol dehydrogenase (ADH, 1 lo
-
100 W. Prus-G³owacki et al.
cus) EC 1.1.1.1.1; glutamate dehydrogenase
(GDH, 1 locus) EC 1.4.1.3; isocitrate
dehydrogenase (IDH, 1 locus) EC 1.1.1.42;
diaphorase (Dia, 2 loci) EC 1.6.99;
phosphoglucoisomerase (PGI, 2 loci) EC 5.3.1.9;
and malic enzyme (ME, 1 locus) EC 1.1.1.40.
In the studied groups of trees, frequencies of al
-
leles and genotypes, mean numbers of alleles
(A/L) and genotypes (G/L) per locus, observed
heterozygosity (H
o
) and expected heterozygosity
(H
e
), genotype polymorphism index (P
g
) and fixa
-
tion index (F) were estimated. For evaluation of
the genetic parameters, the GEN and the PopGEN
programs were used (Bzowy and Nowak-Bzowy,
unpublished; Yeh et al. 1999). Statistic signifi
-
cance of differences in genetic parameters was
evaluated by using t and c
2
tests.
For the cytogenetic analysis, cones were collected
from 65 cone-bearing trees; 34 from pollu
-
tion-tolerant (T), 21 from pollution-sensitive (S)
trees, and 15 from the control population (NPW).
The best results of germination were observed
if seeds were soaked for 24 hours in water and then
incubated at 27
o
C for 5–7 days on wet filter paper
in the dark. The proportion of germinating seeds
was estimated for all three groups of trees.
The seeds were incubated for2hincolcemide and
fixed overnight in ethanol and acetic acid (3:1),
at room temperature. The meristematic tissue was
macerated in the enzyme mix (pectinase, cellulase
and pectoliase) for1hat37
o
C, squashed in 45%
acetic acid, and stained by using DAPI or modi
-
fied Giemsa C-banding (Chudziñska 2002).
For studied trees the mitotic index and chromo
-
somal aberration frequency were determined in 10
seedlings obtained from 5 trees of each group: T,
S and control.
Results
Environmental status of the study site
Data processed by the Silesian Sanitary and Epi
-
demiological Station (2001) demonstrated that
dust sediments in the direct vicinity of the smelter
in Miasteczko Œl¹skie amounted to 52 g m
–2
per
year and did not exceed the Polish norms estab
-
lished by the Ministry of Environment Protection
(Regulation 2002). However, the analysis of metal
contents of the dust documented that they ex
-
ceeded 3.5-fold the norms for lead (365 g m
–2
per
year) and for cadmium (0.01 g m
–2
per year). In the
case of gaseous contaminations, in 2002 the levels
of sulfur dioxide and nitrogen dioxide did not ex
-
ceed the norms, but they were markedly exceeded
in the past. In 1972, the SO
2
levels in the air were
on average 50-fold higher than in 2002.
Soil analysis revealed markedly exceeded levels
of zinc, lead and cadmium in the tested area (Ta
-
ble 1). In the direct vicinity of the smelter, at the dis
-
tance of 100 m, 500 m and 1000 m, zinc content of
the soil ranged between 1560.43 mg kg
–1
and 1260.28 mg kg
–1
, i.e., it proved to be about
90-fold higher than the average level in Poland.
In the case of lead, the levels ranged between
2972 mg kg
–1
and 1685 mg kg
–1
, i.e. they were
about 200 times higher than the average level in Po
-
land. Also cadmium and zinc levels in the soil were
elevated, amounting to 10.3–22.7 mg kg
–1
and 1289–2946 mg kg
–1
, respectively, and exceeded
markedly the norms for forests (Table 1). In view of
the very high cadmium, lead and zinc content of the
soil, it should be classified as very strongly pol
-
luted, representing class V of soil contamination
with lead and cadmium ions and class IV of con
-
Heavy metals effects on plants fluetic material 101
Table 1. Polish norms for heavy metal contamination of the soil and its levels in the study site
Pollution
Data for MS mg kg
–1
of soil
Polish norm mg kg
–1
of soil
P1 P2 P3 P4 C D E
Cd
22.7 15.3 20.1 10.3 4–8 4.0 15
Cr
2.5 2.1 2.1 1.4 5–10 150 500
Cu
28.2 20.2 28.0 17.0 40–80 150 600
Ni
2.2 2.3 2.3 0.8 5–10 35 300
Pb
2972 2273 2228 1685 200–400 100 600
Zn
1480 2946 1337 1289 400–800 300 1000
(pH)
4.66 4.75 4.74 4.04 6.7–7.4 – –
tamination with zinc ions on a five-grade scale
(Kabata-Pendalis and Pendalis 1999; Kabata-
Pendalis and Pendalis 2001). Soil acidity tests
demonstrated that the soil was very acidic, with
pH ranging between 4.04 to 4.75, which promoted
the uptake and accumulation of heavy metal ions
in plants, so it represented an unfavorable habitat
for growth and development of trees.
Height and habit of trees
Height of sensitive and tolerant trees (Table 2),
measured as an indicator of their viability
and resistance to pollution, was significantly
lower for the trees with signs of damage, such as
needle necrosis or chlorosis and untypical; bushy
and dwarf habit. On the other hand, the variance
and standard deviation were higher in the group of
trees with no signs of injury. The range of tree
height values was much wider in the group
of affected trees.
Genetic structure
Comparison of genetic parameters between
the two groups of trees demonstrated significant
differences in their genetic structure. In the group
of sensitive trees, 43 alleles were found, while in
the group of tolerant trees, 40 alleles were
detected. Mean number of alleles per locus (A/L)
proved to be higher in the group of sensitive trees
(Table 3). Rare alleles (frequency < 0.05) were
more numerous in the group of pollution-tolerant
trees. Frequencies of alleles in the studied 18 loci
are listed in Table 5.
In the
c
2
test, the compared groups of trees
manifested no statistically significant differences
in frequencies of individual alleles. Even if their
significance was not confirmed by the statistical
test, evident differences were observed in fre
-
quency of some alleles. This applied to Fest A 2,
Fest B 3 and GOT B 2 alleles. In the tolerant
group, 6 alleles were not detected: 6 PGD 3 and 4,
Dia B 4, GOT B 4, MDH A 2, ShDH B 3 alleles.
In the sensitive group, only 2 alleles were noted
which were not seen in tolerant trees, including
Fest B 5 and GOT A 3.
102 W. Prus-G³owacki et al.
Table 2. Statistics for height (m) of sensitive
and tolerant pine trees. 0 = mean S
2
= variance;
SD = standard deviation; R = range
Trees n 0 S
2
SR
S 39 116.6 2.98 1.72 0.35–2.70
T 39 600.51 4.00 6.32 2.70–10.0
Table 3. Total numbers of alleles and genotypes,
number of rare alleles, numbers of alleles per locus
(A/L) and genotypes per locus (G/L) in sensitive
and tolerant groups of pine trees
Trees Total
number
of alleles
Number
ofrare
alleles
A/L Number of
genotypes
G/L
S 43 10 2.4 56 3.19
T 40 8 2.2 52 2.9
Table 4. Genetic parameters for pine trees sensitive (S)
and tolerant (T) to pollution: H
e
= heterozygosity
expected; H
o
= heterozygosity observed; F = Wright
fixation index; P
g
= genotype polymorphism index
Locus Trees He Ho F Pg
6PGD
6PGD
S
T
0.447
0.420
0.513
0.450
–0.146*
–0.071*
0.614
0.566
DIA A
DIA A
S
T
0.393
0.375
0.385
0.450
0.023
–0.200*
0.556
0.521
DIA B
DIA B
S
T
0.226
0.237
0.256
0.275
–0.132*
–0.159*
0.393
0.399
FEST A
FEST A
S
T
0.332
0.266
0.079
0.050
0.762*
0.812*
0.398
0.310
FEST B
FEST B
S
T
0.421
0.517
0.395
0.575
0.062*
–0.113*
0.619
0.704
G6PD
G6PD
S
T
0.050
0.072
0.051
0.075
–0.026
–0.039
0.097
0.139
GDH
GDH
S
T
0.426
0.462
0.410
0.375
0.037
0.189*
0.584
0.626
GOT A
GOT A
S
T
0.000
0.025
0.000
0.025
0.000
–0.013
0.000
0.049
GOT B
GOT B
S
T
0.453
0.513
0.308
0.450
0.320*
0.122*
0.625
0.676
GOT C
GOT C
S
T
0.405
0.453
0.256
0.487
0.367*
–0.076*
0.563
0.584
MDH A
MDH A
S
T
0.074
0.000
0.077
0.000
–0.040
0.000
0.142
0.000
MDH C
MDH C
S
T
0.436
0.447
0.385
0.375
0.117*
0.161*
0.598
0.611
ME
ME
S
T
0.278
0.219
0.282
0.250
–0.015
–0.143*
0.440
0.375
PGI B
PGI B
S
T
0.206
0.284
0.227
0.333
–0.106*
–0.174*
0.368
0.469
SHDH A
SHDH A
S
T
0.359
0.317
0.308
0.325
0.144*
–0.027
0.521
0.480
SHDH B
SHDH B
S
T
0.144
0.072
0.154
0.075
–0.071*
–0.039
0.267
0.139
Mean
Mean
S
T
0.258
0.260
0.255
0.286
0.012
–0.099*
0.377
0.369
*significant differences (P < 0.05) between sensitive and tolerant
groups
Frequencies of genotypes in the two groups are
shown in Table 6. In total, 63 genotypes were
found, of which all were seen in the sensitive
group and only 52 in the tolerant group. Mean
number of genotypes per locus (G/L) was 3.1 in
the group of sensitive trees and 2.9 in the other
group (Table 3). At individual loci significant
differences between studied tree groups were
noted in frequencies of Dia B1/2, Fest B 1/1, Fest
B 1/3, GOT B 1/1, GOT C1/1, GOT C 1/2,
and ShDH B 1/1 genotypes. In the group of
tolerant trees, most of the listed loci represented
heterozygous genotypes (Table 6).
The values varied between individual loci.
The most heterozygous proved to be 6PGD locus
and the least heterozygous was locus GOT A.
The average expected heterozygosity (H
e
) for 16
loci in the group of tolerant trees proved to be
Heavy metals effects on plants fluetic material 103
Table 5. Frequencies of alleles in sensitive (S)
and tolerant (T) groups of trees
Locus Allel S T
6PGD
6PGD
6PGD
6PGD
DIA A
DIA A
DIA B
DIA B
DIA B
FEST A
FEST A
FEST A
FEST B
FEST B
FEST B
FEST B
G6PD
G6PD
GDH
GDH
GOT A
GOT A
GOT B
GOT B
GOT B
GOT B
GOT C
GOT C
IDH
MDH A
MDH A
MDH C
MDH C
ME
ME
PGI A
PGI B
PGI B
PGI B
SHDH A
SHDH A
SHDH A
SHDH A
SHDH B
SHDH B
SHDH B
1
2
3
4
1
2
1
2
4
1
2
3
1
2
3
5
1
2
1
2
1
3
1
2
3
4
1
2
1
1
2
1
2
1
2
1
1
2
3
1
2
3
5
1
2
3
0.69
0.27
0.03
0.01
0.73
0.27
0.87
0.12
0.01
0.80
0.14
0.05
0.74
0.11
0.16
0.00
0.97
0.03
0.69
0.31
1.00
0.00
0.67
0.32
0.00
0.01
0.72
0.28
1.00
0.96
0.04
0.68
0.32
0.83
0.17
1.00
0.89
0.02
0.09
0.78
0.17
0.03
0.03
0.92
0.06
0.01
0.70
0.30
0.00
0.00
0.75
0.25
0.86
0.14
0.00
0.85
0.09
0.06
0.65
0.13
0.21
0.01
0.96
0.04
0.64
0.36
0.99
0.01
0.56
0.41
0.03
0.00
0.65
0.35
1.00
1.00
0.00
0.66
0.34
0.88
0.13
1.00
0.83
0.02
0.15
0.81
0.15
0.03
0.01
0.96
0.04
0.00
Table 6. Frequencies of genotypes in sensitive (S)
and tolerant (T) groups of trees
Locus Genotyp S T
6PGD
6PGD
6PGD
6PGD
6PGD
DIA A
DIA A
DIA A
DIA B
DIA B
DIA B
FEST A
FEST A
FEST A
FEST A
FEST A
FEST B
FEST B
FEST B
FEST B
FEST B
FEST B
FEST B
G6PD
G6PD
GDH
GDH
GDH
GOT A
GOT A
GOT B
GOT B
GOT B
GOT B
GOT B
GOT B
GOT C
GOT C
GOT C
IDH
MDH A
MDH A
MDH C
MDH C
MDH C
ME
ME
ME
PGI A
PGI B
PGI B
PGI B
SHDH A
SHDH A
SHDH A
SHDH A
SHDH A
SHDH A
SHDH A
SHDH A
SHDH B
SHDH B
SHDH B
1
1
1
1
2
1
1
2
1
1
1
1
1
1
2
3
1
1
1
1
2
2
3
1
1
1
1
2
1
1
1
1
1
1
2
2
1
1
2
1
1
1
1
1
2
1
1
2
1
1
1
1
1
1
1
1
2
2
2
3
1
1
1
1
2
3
4
2
1
2
2
1
2
4
1
2
3
2
3
1
2
3
5
2
3
3
1
2
1
2
2
1
3
1
2
3
4
2
3
1
2
2
1
1
2
1
2
2
1
2
2
1
1
2
3
1
2
3
5
2
3
5
3
1
2
3
0.44
0.44
0.05
0.03
0.05
0.54
0.38
0.08
0.74
0.23
0.03
0.76
0.08
0.00
0.11
0.05
0.55
0.13
0.24
0.00
0.03
0.03
0.03
0.95
0.05
0.49
0.41
0.10
1.00
0.00
0.51
0.28
0.00
0.03
0.18
0.00
0.59
0.26
0.15
1.00
0.92
0.08
0.49
0.38
0.13
0.69
0.28
0.03
1.00
0.77
0.05
0.18
0.64
0.26
0.00
0.03
0.03
0.00
0.03
0.03
0.85
0.13
0.03
0.48
0.45
0.00
0.00
0.08
0.53
0.45
0.03
0.73
0.28
0.00
0.83
0.03
0.03
0.08
0.05
0.38
0.18
0.35
0.03
0.03
0.03
0.03
0.93
0.08
0.45
0.38
0.18
0.98
0.03
0.35
0.40
0.03
0.00
0.20
0.03
0.41
0.49
0.10
1.00
1.00
0.00
0.48
0.38
0.15
0.75
0.25
0.00
1.00
0.67
0.04
0.29
0.68
0.25
0.03
0.00
0.00
0.03
0.03
0.00
0.93
0.08
0.00
slightly higher (H
o
= 0.286) than in the group of
trees sensitive to pollution (H
o
= 0.255). Analysis
of
F values revealed deviations from
the Hardy-Weinberg equilibrium in most of
individual loci. On average, the tolerant group
of trees demonstrated some excess of
heterozygotes while the group of sensitive trees
was in the equilibrium, with a slight excess
of homozygotes. Coefficients of genotype
polymorphism (P
g
) showed no significant
differences between the 2 groups of trees. In the 9
pollution-sensitive loci (6PGD, Dia A, Fest A,
Fest B, G6PD, GOT B, GOT C, PGI B, ShDH B)
(Table 6), significant differences in observed
heterozygosity were documented between tolerant
and sensitive trees and values of the parameters
were up to 20% higher in the tolerant trees.
The latter group of trees is in the Hardy-Weinberg
equilibrium (F = 0.010), while the sensitive trees
manifested a significant homozygosity
and deviated from the genetic equilibrium;
F = 0.168 (Table 4 and 7).
Cytological analyses
Germination rate reached 81% for seeds originat
-
ing from the unpolluted area (NPW), used as the
control, while only 54.5% for seeds from sensitive
trees in the polluted area and 69.2% for seeds from
tolerant trees in the polluted area. Viable seedlings
were obtained from seeds of 23 pollution-tolerant
104 W. Prus-G³owacki et al.
Table 7. Genetic parameters for 9 pollution-sensitive
loci in the groups of sensitive and tolerant pine trees.
Description of genetic parameters as in Table 4
Trees He Ho F Pg
Sensitive (S)
0.316 0.263 0.168 0.456
Tolerant (T)
0.330 0.327 0.010 0.456
Figure 1. Some types of aberrations observed in P. sylvestris root tip cells (a) single
chromosome fragments, (b) retarded chromosome, (c) unoriented pole at anaphase,
(d) sticky prophase, (e) lagging chromosome, (f) single bridge at anaphase
trees per 34 trees from which cones were collected
and from 17 sensitive trees per 27 examined trees.
All trees originating from the control population
yielded viable seedlings. The cytological analyses
documented numerous anomalies in meristematic
tissues of seedlings originating from seeds col
-
lected in the polluted area, such as chromosomal
stickiness, chromo- some bridges, laggards, re
-
tarded and forward chromosomes, and also frag
-
ments of chromo- somes. Examples of such
aberrations are shown in Figure 1. The mean pro
-
portions of cells in which such aberrations were
detected are listed in Table 8. In seedlings devel
-
oping from seeds of sensitive trees, chromosomal
aberrations were noted in 17.8% of dividing cells
and in 12.3% of dividing cells of seedlings devel
-
oping from seeds of tolerant trees. In the control
seedlings, only 3.7% cells contained chromo
-
somal anomalies. Chromosomal stickiness was
most common, accounting for 35% aberrations,
followed by anaphase bridges (26.5%), laggards
(13.3%), forward chromosomes (13.3%), and in
-
dividual chromosomal fragments (12.1%). The
most frequent cell divisions were observed in the
apical root meristems. Values of mitotic index
(MI) differed between studied groups of seedlings.
Seedlings originating from seeds in the polluted
area demonstrated a markedly lower mitotic index
as compared to control seedlings originating from
areas not polluted with heavy metals (Table 8).
In the control the average mitotic index amounted
to 5.5%, as compared to 3.1% for seedlings of tol
-
erant trees and 2.5% for seedlings of sensitive
trees.
Discussion
The results of the performed study have demon
-
strated that the stress resulting from both gaseous
pollution and contamination of the soil with heavy
metals, exerts a significant effect on phenotype
Heavy metals effects on plants fluetic material 105
Table 8. Mitotic index and frequency of aberrations (mean values of 10 seedlings) in root tips from different
groups of pine trees
Tree
number
Number of
analyzed cells
Number of cells in Mitosis
Mitotic index
(%)
P M A T Total Interphase
WNP
1 329 6 5 7 5 23 306 6.9
2 315 5 7 3 1 16 299 5.1
3 319 4 5 4 3 16 303 5.0
4 221 3 2 0 5 10 211 4.5
5 312 8 2 2 7 19 293 6.1
Mean
299.2 5.2 4.2 3.2 4.2 16.8 282.4 5.52
Aberration
Total P M A T Mitotic cells Aberrant cells Aberration %
1496 1 0 2 0 84 3 3.7
Tolerant
1 268 3 4 2 1 11 257 4.1
2 275 5 1 1 2 9 266 3.3
3 279 5 1 1 2 9 270 3.2
4 301 3 1 2 1 7 294 2.3
5 251 4 1 1 1 7 244 2.8
Mean
274.8 5 1.6 1.4 1.4 8.6 265.4 3.14
Aberration
Total P M A T Mitotic cells Aberrant cells Aberration %
1374 1 1 3 0 43 5 12.3
Sensitive
1 270 4 1 1 2 8 262 2.9
2 271 2 2 1 1 6 265 2.2
3 300 2 0 1 3 6 294 2.0
4 230 1 1 3 0 5 225 2.2
5 272 4 1 2 2 9 263 3.3
Mean
268.6 2.6 1 1.6 1.6 6.8 261.8 2.52
Aberration
Total P M A T Mitotic cells Aberrant cells Aberration %
1343 1 1 3 1 34 6 17.8
of individuals and on genetic structure of popula
-
tions inhabiting such an environment. The effect
has been expressed also by structural chromosome
aberrations and in mitotic activity.
The differences in genetic structure between
the group of pollution-sensitive individuals
and individuals tolerant to the pollution may point
to the adaptive strategy of the population, linked to
selection processes. The chromosomal aber- ra
-
tions, on the other hand, represent direct effects of
pollution on the genetic material. Our study has
documented differences in tree growth rate, habit
and proportion of germinating seeds between pol
-
lution-sensitive and pollution-tolerant trees. The
sensitive trees showed the habit untypical for the
Scots pine and were clearly smaller. Germination
rate of seeds of pollution-sensitive trees was
around 16% lower, as compared to the group of
pollution-tolerant trees. Thus pollution clearly af
-
fects viability of pines both at the level of adult in
-
dividuals and of embryos in the studied
population. Effects of pollution, such as decreased
annual ring width, lower diameter of branches,
needle survival time, germination energy and low-
ered pollen quality have already been documented
by Oleksyn et al. (1994); Prus-G³owacki and
Godzik (1991); Staszak et al. (2004); Cha³upka
(1998); Wolf (2001). The differences in genetic
structure between pollution-sensitive and pollu-
tion-tolerant trees in this study were expressed by
lower mean numbers of alleles (A/L) and of geno
-
types per locus (G/L) in the tolerant trees and also
by lower numbers of rare alleles. Mean observed
heterozygosity (H
o
) and mean fixation index (F)
calculated for 9 loci, which reacted to pollution
(Table 7), also differed between the 2 groups. Ob
-
served heterozygosity (H
o
) was around 20%
higher in the group of pollution-tolerant trees,
while the F index attested to deviation from
the Hardy-Weinberg’s equilibrium. A marked ex
-
cess of homozygotes in the group of sensitive trees
(40% as compared to tolerant trees) was noted.
The obtained results have pointed to the adaptive
strategy of the population, and a similar tendency
was observed also in Norway spruce populations,
where the isozyme gene markers indicated a simi
-
lar trend of genetic differentiation (Bergmann and
Hozius 1996). On the other hand, the report by
Riegel et al. (2001) did not provide any evidence
for such an excess of homozygosity in a tolerant
group of Norway spruce trees. In the discussed
case of Scots pine, some of homozygotic geno
-
types were eliminated. This is linked to an increase
in population heterozygosity but at the same time
to loss of genetic richness due to elimination of
some alleles. Therefore, the genetic cost of adapta
-
tion has involved impoverishment of the popula
-
tion’s gene pool.
Cytological investigations have demonstrated
an interesting relationship between the level of
chromosomal aberrations and mitotic index on
the one hand and sensitivity of trees to the environ
-
mental stress on the other. In seedlings obtained
from seeds of trees growing in conditions of envi
-
ronmental stress, increased frequencies of chro
-
mosomal aberrations and lowered mitotic indices
(by about 50%) were noted, as compared to the
control group (Table 8). The frequency of aberra
-
tions and level of mitotic index has differed also
depending on whether the seeds originated from
pollution-sensitive or pollution- tolerant trees.
Thus, the different genetic structure of the
stress-sensitive and the stress-tolerant trees dem-
onstrated by isoenzymatic methods has been trans-
lated also into the extent of chromosomal
aberrations and values of mitotic index. Decreased
frequency of cell divisions represents an example
of inhibitory effects of heavy metal ions (Powell
et al. 1986; Liu et al. 1995; Ishido and Kunimoto
2000). The observed aberrations have reflected
the influence of zinc, lead and cadmium ions,
whose concentrations were markedly higher in
the study site as compared to the area colonized
by the control population. The activity of heavy
metal ions, inducing chromosomal aberrations in
plants in controlled conditions, has been demon
-
strated by, e.g., El-Ghamery et al. (2003); Liu et al.
(1995); Pahlsson (1990); Duan and Wang (1995);
Kristen et al. (1997); Das et al. (1998); Grant and
owens (2001). Our study is one of few reports
demonstrating chromosomal aberrations and dis
-
turbed mitotic divisions in plants and linking the
phenomena to genetic structure of the individuals
in nature.
Acknowledgements. This research was partly
supported by Adam Mickiewicz University grant
PI-II/1 2003.
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