Considerable efforts have been made recently tocreate an expedient system for monitoring the state ofnatural ecosystems. The traditional approach to thisproblem involves air, water, and soil sampling and rou-tine laboratory analysis of the samples by chemical andphysical methods. However, such estimations entail toomuch uncertainty. For example, this approach does notensure the necessary accuracy in estimating the totalradiation dose from all sources or the expected toxicand mutagenic effects of several adverse factors actingsimultaneously (Ulsh
et al.
, 2003).
Most methods of chemical analysis are based onquantitative estimation of the concentrations of certainelements and their compounds, which makes it neces-sary to know a priori the composition of environmentalpollutants. Moreover, however accurate the chemicaland physical methods for the analysis of technogenicimpact on the environment might be, there are alwaysmany more environmental pollutants, both existing andnewly formed, than the available monitoring methodscan identify.
Another approach is based on the analysis of the insitu responses of animals, plants, and microorganismsto environmental factors. Methods developed withinthe framework of the bioindication approach allow spe-
cialists to obtain direct information on the total hazardto the biota created by all factors, including those notdetected by the agencies responsible for monitoringtechnogenic environmental pollution.
From the early 1970s, several authoritative interna-tional organizations have recommended higher plantsas indicators for mutation screening and the monitoringof genotoxic substances (Grant, 1999). Most protocolsrecommend transfer indicator species (
Tradescantia
spp.,
Vicia faba
, etc.) to the test grounds where they areexposed to the technogenic factors studied. Althoughthis method is very sensitive, it also has some draw-backs (Lazutka
et al.
, 2003). To use plants from naturalpopulations is the best approach to the assessment andprognosis of the state of natural ecosystems. Biologicalmonitoring deals with characteristics of an inherentlystochastic process; therefore, it is advisable to analyzethe genetic consequences of technogenic environmen-tal pollution at the population level.
Countries that possess nuclear technologies are nowfacing the problem of storage and processing of theconstantly increasing amount of radioactive waste.Facilities for radioactive waste storage are one of themain potential sources of hazard to humans and thebiota related to the use of nuclear energy. In this study,
Bioindication-based Estimation of Technogenic Impact on
Pinus sylvestris
L. Populations in the Vicinity of a Radioactive Waste Storage Facility
S. A. Geras’kin*, D. V. Vasil’ev*, V. G. Dikarev*, A. A. Udalova*, T. I. Evseeva**, N. S. Dikareva*, and V. L. Zimin***
*All-Russia Research Institute of Agricultural Radiology and Agroecology, Russian Academy of Agricultural Sciences, Obninsk, Kaluga oblast, 249020 Russia
**Institute of Biology, Komi Research Center, Ural Division, Russian Academy of Sciences, ul. Kommunisticheskaya 28, Syktyvkar, 167982 Komi Republic, Russia
***Khlopin Radium Institute, St. Petersburg, Russia
Received August 24, 2004
Abstract
—The spectrum and frequency of cytogenetic aberrations in the reproductive (seeds) and vegetative(foliage) organs of Scotch pine (
Pinus sylvestris
L.) have been studied in the vicinity of the LSK Radon facilityfor radioactive waste storage and processing and in a 30-km zone around the Chernobyl Nuclear Power Plant.The results indicate that the pine populations of these regions are exposed to mutagenic factors. In contrast tothe 30-km Chernobyl zone, the increased environmental mutagenicity in the vicinity of LSK Radon and in thecenter of the city of Sosnovyi Bor is mainly accounted for by chemical factors. The results of additional acute
γ
-irradiation have shown an increased radiation resistance of Scotch pine seeds from the LSK Radon and Sos-novyi Bor populations. Regression analysis demonstrated a significant increase in the cytogenetic aberrationrate in plants from the experimental plots throughout the study period (1997–2002).
L.) are an expedient tool for estimating theenvironmental situation in the vicinity of a facility forradioactive waste storage and processing. We used theanalysis of the frequency and spectrum of cytogeneticaberrations in the reproductive and vegetative organs ofScotch pine to test whether relatively low levels of tech-nogenic pollution actually had biologically significanteffects on natural populations and, if so, to quantita-tively evaluate the strength of the effect. Thus, weraised several key questions concerning the long-termeffects of technogenic pollution on natural plant popu-lations; the results of our study contribute to our knowl-edge of adaptation processes under these conditions.
MATERIAL AND METHODS
Object.
We used the Scotch pine (
Pinus sylvestris
L.),the main forest-forming species of northern Eurasia, asa test object for estimating the possible environmentalimpact from LSK Radon, a facility for radioactivewaste storage and processing. In natural communities,the Scotch pine is an edificator species that determinesthe general pattern of the phytocenosis and has a sub-stantial effect on the life of other plants. Data indicatingthe high radiosensitivity of conifers were obtained inBrookhaven National Laboratory (United States) in theearly 1960s (Sparrow and Woodwell, 1962) and con-firmed in the framework of the Ecos large-scale exper-iment (Karaban’
et al.
, 1979). Similar radiosensitivityof pine and human cells, the wide geographic range ofpine, and the informativeness, efficiency, and sensitiv-ity of testing methods have made
P. sylvestris
one of themain natural test objects for ecological genetic moni-toring.
The studies on
P. sylvestris
in the zone of the Cher-nobyl accident, the Eastern Ural radioactive trace, andthe Semipalatinsk nuclear weapons test range made itpossible to find test systems for indicating the effects oflow-dose exposure (Kozubov and Taskaev, 1994;Sidorov, 1994; Kal’chenko
et al.
, 1995; Kal’chenkoand Fedotov, 2001). The reproductive organs of coni-fers, characterized by a complex organization and along generative cycle, are most sensitive to damagingfactors. While the reproductive cycle of mostangiosperms is several months, the period between theinitiation of generative organs and seed ripening in pineexceeds two years (Kozubov and Taskaev, 1994).Because of this long developmental cycle, unspeciali-zed initial cells of seeds exposed to technogenic factorsaccumulate enough DNA lesions manifested in mitoticaberrations (mostly during the first mitosis; Geras’kin
et al.
, 2003b) to serve as indicators of the effects ofexternal factors. Therefore, this test system is a goodbioindicator of the cumulative technogenic impact.Recording cytogenetic lesions in the foliage is also atest suitable for biological monitoring, because woodyplants, including conifers, are characterized by a highcapacity for accumulating numerous air pollutants and
a low slow self-purification of their aboveground phy-tomass from them.
Study region.
The center of the nuclear powerindustry near the city of Sosnovyi Bor (Fig. 1) includesthe Leningrad Nuclear Power Plant with four RMBK-1000 units, the Research Technological Institute, LSKRadon (a regional facility for radioactive waste storageand processing), and ZAO Ekomet-S (a plant for thedecontamination and processing of metal waste con-taining radioactive substances that was founded in1995). LSK Radon is responsible for the collection,processing, and storage of low- and medium-activityradioactive waste in northwestern Russia.
Throughout the period of operation of this nuclearpower complex (beginning in the 1970s), the exposureof the biota and humans to technogenic radionuclideshas never exceeded the maximum allowable doses indi-cated in hygienic guidelines (Blinova, 1998). However,after LSK Radon was put into operation, the instancesof substantial increase in the concentrations of radionu-clides and other technogenic pollutants, includingheavy metals, in the ground air layer, snow, foliage, andmoss in the vicinity of the enterprise were recordedmore often. This made it necessary to assess the possi-ble ecological consequences of the operation of LSKRadon (Blinova, 1998).
Sampling.
In the period from 1997 to 2002, coneswere collected in autumn in the local populations ofScotch pine (Fig. 1) growing on the territory of LSKRadon (plot 3), in Sosnovyi Bor (plot 2), and in theBol’shaya Izhora village (plot 3) located 30 km awayfrom Sosnovyi Bor, beyond the zone presumablyaffected by the nuclear power industrial complex. Theaverage
γ
-radiation exposure dose rates during theperiod of study were 12.6, 12.8, and 18.8
µ
R/h in plots1, 2, and 3, respectively.
The collected cones were stored at room tempera-ture and humidity until they opened and shed seeds;then the wings were detached manually. The seedsplaced in Petri dishes onto filter paper soaked in dis-tilled water germinated in a thermostated cabinet at24
°
C. As with most wild plants, pine seed germinationand the development of seedlings were extremely het-erogeneous. When determining the number of rootletswith the largest numbers of dividing cells during thefirst mitosis, we took into account that, at the first stageof germination, the size increases due to differentiationand elongation of the cells of the root rudiment that hadexisted even before the embryo was formed. A specialexperiment (Geras’kin
et al.
, 2003b) demonstratedthat, under these conditions, the first and second mitosispeaks were observed in 8- to 14-mm and 20-mm orlonger rootlets, respectively.
Some of the seeds collected in 1999–2001 in plots1
−
3 were exposed to acute
γ
-irradiation at a dose of15 Gy (dose rate 0.6 Gy/min) from a
60
Co source at roomtemperature. The seeds were placed in the thermostat for
BIOINDICATION-BASED ESTIMATION OF TECHNOGENIC IMPACT 251
germination immediately after the irradiation. We ana-lyzed 23–55 seedlings per experimental variant.
In the period from 1998 to 2002, we collected young20- to 30-mm shoots in spring in plots 1–3 and near thefence surrounding the LSK Radon territory (plot 4). Ineach plot, the samples were collected from 10–15 treeswithin a homogeneous forest stand with a high propor-tion of pines.
Cytogenetic preparations.
Seven- to 15-mm root-lets of the seedlings and foliage of young shoots of theScotch pine were fixed with a 1 : 3 acetic acid–alcoholmixture and stained with acetoorcein. Temporarysquashed specimens were made and encoded. We ana-lyzed all anaphase and telophase cells (2700–16500 cellsper variant) in each preparation and calculated the pro-portion of cells with chromosome aberrations. Whenanalyzing the aberration spectrum, we distinguishedchromatid (single-stranded) and chromosome (double-stranded) bridges and fragments, multipolar mitoses,and chromosome lags. Note that the anaphase methodallowed us to detect, in root meristem cells, the lesionsthat appeared in the period between the formation ofgametes and the ripening and collection of seeds,because the chromosome rearrangements that were
induced at the vegetative stage (before gamete forma-tion) were eliminated during meiosis (except symmet-ric translocations and inversions, which were notdetected by this method anyway).
Statistical treatment.
We used the method for thestatistical analysis of empirical distributions (Geras’kin
et al.
, 1994) to determine the sample size that was opti-mal for estimating the test parameters with a fixed rela-tive error at a given confidence probability. The experi-mental data were screened for outliers, which wereexcluded from subsequent analysis. To estimate the sig-nificance of differences between mean values, Stu-dent’s
t
-test was used. Changes in cytogenetic aberra-tions with time were estimated using regression analy-sis (Dreiper and Smith, 1986).
RESULTS
Aberrant cell frequency.
The frequencies of cyto-genetic aberrations in the reproductive (seeds) and veg-etative (foliage) organs of Scotch pine were signifi-cantly higher in affected plots (2–4) than in the control(plot 1) (table) in all cases except foliage samples fromplot 2 in 1998. The frequency of aberrant cells in the
St. Petersburg
Gulf of Finland
Bol’shaya Izhora
SosnovyiBor
N
Leningrad Nuclear Power Plant
LSK Radon (radioactive waste storage facility)
Sampling site
15 km
Fig. 1.
Map of the study region in Leningrad oblast (1997–2002).
252
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No. 4
2005
GERAS’KIN
et al
.
intercalary meristem of the foliage was higher than inthe root meristem of seedlings in both control andaffected samples. Earlier, we observed similar ratiosbetween the frequencies of cytogenetic aberrations inthe leaf and root meristems when studying cultivatedplants (Geras’kin
et al.
, 2003a) and analyzing the radio-ecological consequences of the Chernobyl accident.
The detected cytogenetic aberrations were anepiphenomenon of global structural and functionalgenomic rearrangements induced by external factors(Geras’kin, 1995). Therefore, it is important to have aclear idea of the long-term consequences of mutationsin the cells of vegetative and reproductive organs for thepopulation as a whole. The former alter the viability ofadult plants, whereas the latter are fewer (Cervantes
et al.
, 2002; Geras’kin
et al.
, 2003a) but affect futuregenerations. Thus, both types of damage alter the
genetic structure of the exposed populations, althoughthe cytogenetic aberrations are formed by differentmechanisms.
Compare these data with the results of earlier anal-ysis of the variation in the cytogenetic parameters of thereproductive organs of Scotch pine from a 30-km zonearound the Chernobyl Nuclear Power Plant (NPP)(Geras’kin
et al.
, 2000). In 1995, cones were collectedfrom Scotch pine trees from three populations (Fig. 2)located near the asphalt–concrete plant (ACP) in thezone of sublethal contamination (Kal’chenko andFedotov, 2001), where the absorbed dose in the year ofthe Chernobyl accident (1986) was 10–20 Gy; near theCherevach village in a relatively “clean” place in the30-km zone; and near the city of Obninsk in the Kalugaoblast (background contamination). The
γ
-radiationexposure dose rates in the ACP, Cherevach, and Obn-
Aberrant cell frequency in the seedling root meristem and foliage intercalary meristem of
P
.
sylvestris
Year Plot
Seedlings Foliage
total cellnumber aberrant cells, % total cell
number aberrant cells, %
1995 Obninsk 1994 0.60
±
0.17
Cherevach 2011 2.04
±
0.32*
ACP 2049 4.34
±
0.45*
1997 Bol’shaya Izhora 14643 0.60
±
0.06
Sosnovyi Bor 12342 1.19
±
0.10*
LSK Radon 7927 1.53
±
0.14*
1998 Bol’shaya Izhora 12217 0.53
±
0.07 10156 0.97
±
0.10
Sosnovyi Bor 12832 1.30
±
0.10* 12084 1.36
±
0.11
Fence around LSK Radon 11376 1.60
±
0.12*
LSK Radon 9437 1.73
±
0.13* 5274 2.73
±
0.22*
1999 Bol’shaya Izhora 16482 0.57
±
0.06 5549 0.81
±
0.12
Sosnovyi Bor 8302 1.36
±
0.13* 3724 1.67
±
0.21*
Fence around LSK Radon 4026 1.49
±
0.19*
LSK Radon 5613 1.73
±
0.17* 3943 2.21
±
0.23*
2000 Bol’shaya Izhora 9885 0.66
±
0.08 11206 0.88
±
0.09
Sosnovyi Bor 3517 1.73
±
0.22* 5798 1.64
±
0.17*
Fence around LSK Radon 5361 1.79
±
0.18*
LSK Radon 2674 2.28
±
0.29* 4374 2.29
±
0.23*
2001 Bol’shaya Izhora 13807 0.70
±
0.07 6823 0.89
±
0.11
Sosnovyi Bor 5229 1.84
±
0.19* 3663 1.69
±
0.21*
LSK Radon 4355 2.18
±
0.22* 3191 2.76
±
0.29*
2002 Bol’shaya Izhora 13790 0.70
±
0.07 10868 0.88
±
0.09
Sosnovyi Bor 5929 1.75
±
0.17* 5333 1.78
±
0.18*
LSK Radon 4912 2.16
±
0.21* 5441 2.21
±
0.20*
* Differences from the control are significant at
p
< 5%.
RUSSIAN JOURNAL OF ECOLOGY
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BIOINDICATION-BASED ESTIMATION OF TECHNOGENIC IMPACT 253
insk experimental plots during sampling were 2690,250, and 12.6
µ
R/h.The root meristems of seedlings from the two con-
trol populations (Obninsk, 1995 and Bol’shaya Izhora,1997–2002) did not differ significantly in cytogeneticaberration rate (table), which was within the spontane-ous variation range; hence, these data could be pooled.In contrast to the plant populations from the 30-kmzone of the Chernobyl NPP characterized by consistentgrowth in cytogenetic damage rate with increasingexposure dose, the increased frequency of aberrantcells in both vegetative and reproductive organs of pinetrees from plots 2–4 cannot be explained by irradiationalone. For example, the
γ
-radiation exposure dose ratein plot 2 did not considerably exceed the backgroundlevel throughout the period of study; however, the fre-quency of cytogenetic aberrations in the seedling root-lets and foliage intercalary meristem was significantlyhigher than the spontaneous value in all samples exceptthe foliage collected in 1998. Thus, the mutageniceffect was observed not only in the Scotch pine popula-tion located near LSK Radon, but also in the populationfrom Sosnovyi Bor.
The variation of the responses of many biologicalindicators to weak environmental factors is a more sen-
sitive parameter than the difference between mean val-ues (Handy
et al.
, 2003; Tsytsugina and Polikarpov,2003). Therefore, the variation of the recorded parame-ters was also studied in plants from all experimentalplots. The results (Fig. 3) indicate that the variation ofthe frequency of aberrant cells in the vegetative andreproductive organs of Scotch pine from the affectedpopulations significantly exceeded that in the controlpopulation.
Analysis of the cytogenetic aberration spectrum.
The spectrum of cytogenetic aberrations provides addi-tional information on the factors that make the maincontribution to the increase in mutational variation(Evseeva
et al.
, 2003; Tsytsugina and Polikarpov,2003). In this connection, the presence of tripolar mito-ses, which are attributed to damage of the mitotic spin-dle (Alov, 1972; Micieta and Murin, 1998), in plantsfrom plots 2 and 3 (Fig. 4) and their absence in all con-trol variants and samples from the zone of the Cherno-byl accident deserve special attention. Bessonova(1992) demonstrated that the increasing yield of tripo-lar mitoses in
Syringa vulgaris
L. and
Armeniaca vul-garis
Lam. is related to contamination with heavy met-als from the soil on which the affected populations ofthese plants grew (in control samples, there were no
10 km
ACPChernobyl
Chernobyl
Cherevach
30 km
Sampling site
NPP
Fig. 2. Map of the study region in the 30-km zone around the Chernobyl Nuclear Power Plant (1995).
such aberrations). Similar results were obtained in astudy on the spectrum of cytogenetic aberrations inthree species of the genus Pinus (including P. sylves-tris) growing in the vicinity of two integrated metallur-gical works in Slovakia (Micieta and Murin, 1998).Indeed, heavy metals rarely have a direct effect onDNA (Valverde et al., 2001), but they are strong induc-ers of various mitotic abnormalities (Seoane andDulout, 2001). Combined with the results of dosimetry,these data suggest that chemical factors strongly affectScotch pine populations from the Sosnovyi Bor region.
Acute g-irradiation of seeds. Analysis of theresponse of organisms to acute irradiation reveals theirhidden genotypic variation. Therefore, some of theseeds collected in 1999–2001 were exposed to acuteγ-irradiation. Figure 5 shows the results of this experi-
ment, which indicate that the seeds from technogeni-cally affected populations, which were characterized byan increased cytogenetic aberration frequency, weresignificantly more resistant to acute γ-radiation than thecontrol seeds. Kal’chenko and Fedotov (2001) obtainedsimilar results when studying the acute γ-irradiationresistance of P. sylvestris seeds collected in the 30-kmzone around the Chernobyl NPP in 1997. Together withthe considerable difference between the control andaffected populations with respect to the variances of theparameters studied (Fig. 3), these results indicate thatthe affected Scotch pine populations were adapting toanthropogenic impact.
Changes in the frequency of cytogenetic aberra-tions with time. Since we monitored the pine popula-tions for six years, we can estimate the trend of theobserved changes. If they followed a regular patterninstead of being stochastic, simplified estimates ignor-ing the changes in parameters with time would yieldincorrect predictions of subsequent changes in the stateof the populations studied.
Figure 6 shows how the aberrant cell frequency inthe rootlets of P. sylvestris seedlings changed with time(1997–2002). A linear model fits the experimental datawell. The control population significantly (p < 0.05)differed from the two affected populations (SosnovyiBor and LSK Radon) with respect to the acceleration ofthe increase in cytogenetic aberration frequency esti-mated by the slopes of regression lines (0.03 ± 0.01,0.14 ± 0.03, and 0.15 ± 0.04% per year, respectively).In the affected populations, the steady rise of cytoge-netic aberration frequency with time may be related tothe technogenic effects of the nuclear industries locatednear them; however, the increase in mutation rateobserved in the control population is most probablyrelated to global environmental changes.
50
01997
Variance
1
2
3
1998 1999 2000 2001 2002
100
150
200
Fig. 3. Changes in the variance of aberrant cell frequency inthe pine seedling root meristem with time in the P. sylvestrispopulation of the Sosnovyi Bor region (Leningrad oblast).Sampling plots: (1) Bol’shaya Izhora, (2) Sosnovyi Bor,(3) LSK Radon.
Tripolar mitosis frequency, %12
9
6
3
01997 1998 1999 2000 2001 2002
1
2
3
Fig. 4. Frequency of tripolar mitoses in the P. sylvestris pop-ulation of the Sosnovyi Bor region (Leningrad oblast). Sam-pling plots: (1) Bol’shaya Izhora, (2) Sosnovyi Bor, (3) LSKRadon.
8
01999
Aberrant cell frequency, %
2000 2001
6
4
21 2 3 1 2 3 321
Fig. 5. Aberrant cell frequency in the root meristems ofseedlings obtained from pine seeds collected in the controland technogenically affected P. sylvestris population of theSosnovyi Bor region (Leningrad oblast) after acute irradia-tion at a dose of 15 Gy. Sampling plots: (1) Bol’shayaIzhora, (2) Sosnovyi Bor, (3) LSK Radon.
BIOINDICATION-BASED ESTIMATION OF TECHNOGENIC IMPACT 255
The change in cytogenetic aberration frequencywith time in the intercalary meristem of the foliage fol-lowed a fundamentally different pattern. As with seed-lings, this parameter in the affected populations washigher than the control value but had no significant ten-dency to decrease (Fig. 7). Essential differences in thebiology of the test system most likely account for this.When analyzing seedlings, aberrations are recordedduring the first mitosis, when most lesions accumulatedduring seed formation become manifest. Thus, thisparameter reflects the total exposure of the plant duringseed formation. When analyzing the foliage intercalarymeristem, we dealt with an asynchronously dividingcell population, and the recorded frequency of cytoge-netic aberrations reflected a dynamic balance betweenthe induction of cytogenetic aberrations, their repair,and the elimination of aberrant cells from the popula-tion. Therefore, this parameter gives a “snapshot” of theirradiation effect. In addition, the pathways and dura-tions of the action of technogenic pollutants on the stateof the test system may be different.
DISCUSSION
The increase in the frequency of mutations in thereproductive and vegetative organs of P. sylvestrisgrowing close to the radioactive waste storage facilityindicates that they are exposed to mutagenic environ-mental factors. Long-term monitoring performed in aregion of Japan where five nuclear power plants werelocated also demonstrated a significant increase inmutation frequency in hairs of the anther filaments ofTradescantia (Ichikawa, 1981). Although the mutationrate was considerably higher than that expected on thebasis of the estimated external dose of inflorescenceirradiation, it was supposed that the ionizing radiationwas the leading factor of the increase in mutation fre-quency. In our study, the radiation levels were also toosmall to account for the observed effect; however, theanalysis of the spectrum of cytogenetic aberrations
shows that chemical factors make the main contributionto environmental pollution near LSK Radon. Qualita-tively similar results were obtained in studies on thebioindicator-based estimation of the radioactive andchemical contamination of waters near a storage facil-ity for radium mining waste (Evseeva et al., 2003) andon the genetic variation and reproductive parameters inGambusia affinis populations living in radioactivelycontaminated waters in Oak Ridge (United States)(Theodorakis et al., 1997).
Plants are most susceptible to technogenic factors,because they are sedentary and have to adapt to anadverse environment. The founders of evolutionary the-ory did not attach much importance to anthropogenicstress as a factor in evolution and adaptation. Althoughadaptation to environmental conditions was the centralidea in Darwin’s evolutionary theory, Darwin (1859)considered intra- and interspecific competitions to playconsiderably more important roles in evolution thanextreme environmental conditions do. The role ofmicroevolutionary processes in the response of naturalpopulations to low-dose technogenic factors is notentirely clear; however, it has been conclusively dem-onstrated that exposure to these factors may alter thegenetic structure of populations (Dukharev et al., 1992;Shevchenko et al., 1992; Wurgler and Kramers, 1992;Theodorakis et al., 1997; Prus-Glowacki et al., 1999;Bickham et al., 2000). Therefore, we may assume thatlong-term exposure to factors related to the nuclearindustries located near Sosnovyi Bor, the effect ofwhich has been reliably detected by cytogenetic tests,has changed the genetic structure of P. sylvestris popu-lations.
Genetic polymorphism with respect to the resistanceto various factors is observed in natural populations (Mac-nair, 1987; Pitelka, 1988; Bickham et al., 2000). Anincrease in variation is one of the main adaptive responsesto stress (Shevchenko et al., 1992; Wurgler and Kramers,1992; Geras’kin, 1995; Theodorakis et al., 1997),
01997
Aberrant cell frequency, %
1
2
3
1998 1999 2000 2001 2002
1
2
3
Fig. 6. Changes in aberrant cell frequency in the P. sylvestrisseedling root meristem with time. Sampling plots:(1) Bol’shaya Izhora, (2) Sosnovyi Bor, (3) LSK Radon.
0
Aberrant cell frequency, %
1
2
3
1998 1999 2000 2001 2002
2
3
4
1
Fig. 7. Changes in aberrant cell frequency in the P. sylvestrisfoliage intercalary meristem with time. Sampling plots:(1) Bol’shaya Izhora, (2) Sosnovyi Bor, (3) LSK Radon.
256
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GERAS’KIN et al.
because a population or species cannot exist after adrastic change in environmental conditions unless atleast some individuals can survive and reproduce underthe new conditions. Selection of the variants that are themost fit for the new conditions results in changes in themean values of quantitative traits. Thus, an increase invariation indicates that adaptive processes occur in thepopulation, and the difference in mean values is thequantitative measure of the changes that have beencaused by selection pressure. Therefore, the signifi-cantly higher cytogenetic variation compared to thecontrol value that we observed in anthropogenicallyaffected populations of P. sylvestris indicates activeadaptation in the latter.
Increase in population average radiation resistanceis one of the consequences of the chronic irradiation ofnatural populations (Cherezhanova and Aleksakhin,1975; Shevchenko et al., 1992): it has been termed theradioadaptation phenomenon. The results of experi-ments with repair inhibitors and analysis of the dose–effect curves for densely and rarely ionizing radiation,as well as the measurement of unscheduled DNA syn-thesis and the efficiency of the repair of single-strandDNA breaks, led to the conclusion that populationdivergence with respect to radioresistance was relatedto selection for repair efficiency (Sergeeva et al., 1985;Shevchenko et al., 1992). Therefore, the appearance ofpermanent environmental factors of natural or techno-genic origin in the plant habitat activates the geneticmechanisms that alter the plant sensitivity to these fac-tors, which we observe as an increased seed radioresis-tance in experiments with acute irradiation (Fig. 5).Thus, the adaptive processes that occur in pine popula-tions and result in the increased variation and radiore-sistance observed in our study have not been com-pleted, and selection for the fit genotypes goes on.
Most studies that have demonstrated the evolutionof the systems determining resistance to environmentalfactors were performed on plants with relatively shortlife cycles growing on mining waste dumps rich inheavy metals (Antonovics et al., 1971; Macnair, 1987;Pitelka, 1988), in zones of large radiation accidents,and in areas where technogenic factors had increasedthe natural radiation background (Aleksakhin et al.,1990; Cherezhanova and Aleksakhin, 1975; Pozolotina,2003; Shevchenko et al., 1992). These studies demon-strated that some species of plants can evolve into resis-tant forms very rapidly, in fact, within several genera-tions. A short life span in combination with intenseselection may rapidly lead to changes in populationgenetic structure. However, most natural ecosystemsare dominated by long-lived woody and herbaceousplants.
The number of P. sylvestris generations that havepassed since the foundation of LSK Radon and theappearance of the Chernobyl radioactive trace is obvi-ously insufficient for classical natural selection for theefficiency of repair systems. Even if all mutations are
favorable, it is hardly imaginable that this will lead torapid (within several years) adaptation of a plant popu-lation, even at the high mutation rate that we observed.However, our data indicate that pine populations veryrapidly adapt to pollutants even under these conditions.One possible explanation of this phenomenon(Kal’chenko and Fedotov, 2001) is related to the sam-pling of radiosensitive cells, which are replaced bymore radioresistant cells; another, with hereditary epi-genetic variability, i.e., changes in the spectrum offunctionally active genes that is reproduced in a seriesof cell generations. This hypothesis is confirmed by thedata (Kovalchuk et al., 2003) indicating that the DNAof pine irradiated after the Chernobyl accident is hyper-methylated. It is known that changes in methylationpattern cause changes in the expression of both stress-induced genes and household genes. Earlier, we consid-ered the possible molecular mechanisms of this switch-ing (Geras’kin, 1995; Geras’kin and Sarapul’tsev,1995). Further studies are necessary to determine therole of epigenetic mechanisms in the formation of theresponse of natural populations to chronic exposure totechnogenic factors.
The results of this study indicate that the analysis ofthe spectrum and frequency of cytogenetic aberrationsin the reproductive and vegetative organs of P. sylves-tris may be used to evaluate the ecological state of coni-fer forests not only in zones distinctly deteriorated bytechnogenic factors, but also in tree stands character-ized by weak or visually indiscernible technogenicimpact. The approach proposed here permits not onlythe efficient diagnosis of various types of anthropo-genic pollution, but also the formulation of well-grounded hypotheses on their nature and the study ofthe directions and trends of adaptation processes inplant populations.
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