Changes of reliability and efficiency of micronucleus
bioassay in
Vicia faba
after exposure to metal
contamination for several generations
Changqun Duan
a,*, Bin Hu
a, Tao Guo
a, Mingbo Luo
a, Xiaoyong Xu
a,
Xuexiu Chang
a, Chuanhao Wen
a, Ling Meng
a, Liang Yang
b,
Huanxiao Wang
aaDepartment of Biological Sciences,Yunnan Uni6ersity,52N Greenlake St.,Kunming,Yunnan 650091, PR China bYunnan Center for En6ironmental Monitoring,Yunnan 650232, China
Received 12 February 2000; received in revised form 18 February 2000; accepted 22 April 2000
Abstract
Mitotic root micronucleus (MCN) frequency inVicia fabaas a bioassay, is primarily based on the extent of the sentinel Vicia response in terms of cytogenetic damage quantitatively or qualitatively to indicate the presence of mutagenic contaminants. This paper describes an investigation designed to assess changes in MCN frequencies of Vicia fabafrom three generation plants obtained, respectively from a reference site (RS) and a metal-contaminated experimental field (EF) in the bioassay of mutagenic Cd2+ and NaN
3. The background value, dose – response to
Cd2+and to NaN
3in three generation (F1, F2and F3) plants of the EF and the initial (F0) plants were determined
in terms of MCN frequencies. With more generations of growingViciaplants in the EF, a higher background value of MCN frequency, a lower slope value in the regression equation, a smaller ratio of MCN frequency between the control and treatment in the same generation and larger perturbation values were observed. This denotes that the decreased reliability and efficiency are represented inViciaplants from the EF if the plants are used as sentinels in the bioassay of mutagenic Cd2+ and NaN
3. It was concluded that the ViciaMCN bioassay should be used as an
endpoint biomarker acceptable in biomonitoring environmental mutagens when the sentinel plants were collected from clean areas. Because no place is absolutely without pollutants, it is suggested that several seed stock centers should be established for providing sentinelViciaifViciaMCN bioassay is used as a biomarker to identify the global environmental status. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Bioassay reliability; Environmental mutagens; Micronucleus test; Resistance;Vicia faba
www.elsevier.com/locate/envexpbot
1. Introduction
The micronucleus (MCN) test in the root tip of
Vicia fabahas been widely used in biomonitoring
* Corresponding author. Tel.:+86-871-5032753; fax:+ 86-871-5153832.
E-mail address:[email protected] (C. Duan).
genotoxicity in water and soils. Some countries and international institutions, including China, United States of America and United Nations Environmental Program (UNEP) have approved this biomarker technique as a bioassay for the detection of environmental hazardous matter (Ma, 1982; CEPA, 1990; Ma et al., 1995). The
ViciaMCN bioassay was improved by Ma et al. (1995) through scoring MCN frequency from the F1cells region of the root tip rather than from the meristematic region. Good reliability, short turn-around time and low cost enableViciaMCN test to have a promising future in biologically uncov-ering the global environmental status, especially in the detection of environmental mutagens (Grant et al., 1992; Duan et al., 1994; Duan and Wang, 1995; Duan et al., 1999).
It has been well recognized that the mitotic root
V. faba MCN bioassay as a biomarker, is the measurement of cytogenetical damage that quan-titatively indicates the presence of mutagenic con-taminants on the basis of the extent of theV.faba
response to mutagens (Ma, 1982; Ma and Harris, 1985; Grant et al., 1992; Butterworth, 1994; Grant, 1994; Duan and Wang, 1995). Thus, the sensitivity to mutagens inV.faba, which is depen-dent on the response of test plants to mutagens in term of MCN induction, is crucial to the reliabil-ity and efficiency of theVicia MCN test applica-ble to biomonitoring mutagens. Many researchers have revealed that a lot of plants have certain potentials to evolve resistance sufficient not to present physiological or genetic damages after they have experienced polluted environments for a certain time (Ernst, 1976; Shaw and Albright, 1990; Dickinson et al., 1991, 1992; Prasad, 1995; Prus-Glowacki and Godzik, 1995; Duan, 1995, 1996). In another words, these plants may de-crease their sensitivities to mutagens if they have been exposed. So we hypothesised: V. faba may become less sensitive to chemical mutagens after it has been exposed to mutagen-stressed environ-ments, and the reliability and efficiency of Vicia
MCN test will be reduced if a bioassay is per-formed by use of such an already mutagen-ex-posed sentinel V. faba.
To prove this, a 4-year field cultivation was conducted to obtain different generation plants
derived from the same variety ofV.fabathat had been successively planted in the experimental field (EF); three-generation seeds from the EF, to-gether with the seeds harvested from the reference site (RS), were used to determinate the back-ground values of MCN frequencies and the dose effects between MCN frequencies and mutagenic chemical treatments; the reliability and efficiency of ViciaMCN bioassay observed in the plants of the different generations were compared.
2. Materials and methods
2.1. Material preparation
2.1.1. Characteristics of the experimental field
(EF) and the reference site (RS)
The EF of 20×20 m2 was located within the plantation that had been polluted due to an acci-dental overflow of industrial waste water from the northern suburb of Kunming City of Yunnan Province for several years. In order to meet the demands of this investigation, the RS was chosen within a stock seed base that was without any pollution and about 1 km away from the EF.
A comparative soil analysis had been per-formed between the EF and the RS, and the relevant results had been reported in our pub-lished papers (Duan et al., 1997a; Duan, et al., 1997b). Some properties of the soil relevant to this paper in the EF and the RS are summarized as follows: the soil type in both the EF and the RS was a clay sand paddy soil and there were no differences in general properties of the soil, in-cluding pH, cation exchange capacity, total nitro-gen, total organic matters, except for cadmium and lead between the RS and the EF. Total contents of lead and cadmium were, respectively 23998.6 and 3.6290.2 mg kg−1
2.1.2. Culti6ation of V. faba for obtaining three-generation seeds in the RS and the EF
V. faba, a widely distributed bean crop, has long been used as a model plant in cytogenetical studies due to few (2n=12) large chromosomes,
whose MCN test has been adopted as a
biomarker for environmental mutagens in the Gene – Tox Program (Ma, 1982). In this study,V.
faba seeds, which was provided by Yunnan Insti-tute of Bean Crops, was a cultivar named as Dayu. The V. faba (F0) seed were obtained from the stock seed base for improving crop yield, in which V. faba had been planted for 3 years. Nothing unusual was observed during the plant was grown.
The EF and the RS were selected in their corresponding sites from which the soils were as uniform as possible. The cultivation in both the EF and the RS started on the second week of October and ended on the second week of May in accordance to the local farming season (Kunming has a subtropical climate). F0seed was planted in the well-prepared EF. Average 40 seedlings per square meter were preserved for developing seed after the seedlings grew to a height of 25 cm by randomly removing additional seedlings. All these seeds (F1) derived from F0 in the EF were har-vested, dried under sunlight and stored at 4°C in a dry and dark place. In the next growing season of the next year, F1seeds were planted in the EF in the same way as that of the F0, and F2 seeds were obtained. F3 seeds were the offspring of F2 seeds planted in the EF. Correspondingly, the reference seeds of F1, F2 and F3 were obtained through planting the seeds from the previous gen-eration in the RS. The initial seed (F0) planted in the RS was the same one as initially planted in the EF.
The field management of RS and EF was con-ducted as identically as possible. No obvious damages from the stress of weather and pests were observed during the growing periods of each year. The cultivation was carried out from 1988 to 1991.
2.2. Micronucleus frequency analysis
2.2.1. The background micronucleus frequency in the different generation
Thirty seeds of V. faba from each generation were randomly taken to soak in tap water for 24 h. The soaked seeds were germinated in moist perlite for 5 days at 22°C. After removal of the primary roots, the seedlings were aerated in water tanks at 22°C for 4 days. Hereafter one secondary root tip of each seedling was cut off and fixed with 3:1 ethanol:glacial acetic acid for 24 h. Slide preparation and scoring were performed following the method put forward by Ma et al. (1995). About 1000 – 1200 cells were scored from each of ten slides per experimental group. The average and standard deviations were derived from about 10 000 – 12 000 cells and expressed in terms of MCN 1000 cells−1. Analysis of variance (ANOVA), Dunnett’s t-test or Student’s t-test was used to determine significant differences between F0 and the other generations, and between the reference and experimental groups at the 0.05 level of significance.
2.2.2. Dose–responses of the different generation plants of V. faba to Cd2+
and NaN3 through MCN frequency
In general, the procedure was the same as in the background analysis for the MCN frequency, with differences as follows: the seedlings with well developed secondary roots were treated with CdCl2 solution or NaN3 solution for 6 h in the dark at 22°C and then the treated seedlings were transferred into tap water for 40 – 45 h to recover. Hereafter the coming steps were the same as the former one for the background value study.
2.3. Perturbation analysis
changed by contribution factors. So all the tems can be regarded as serial states of one
sys-tem. Perturbation Value (PV) is used to
quantitatively measure the differences between the systems. PV can be obtained from any system (no less than two systems) only if each of them shares the parameters induced by common contributing factor(s). The lower PV denotes fewer differences among systems.
Perturbation analysis was conducted among the reference plants (F0) and the three generation plants of V. faba growing in the EF. MCN fre-quency was exclusively used in calculation of the PV, based on background value, data obtained from the treatment of NaN3 and Cd
2+ at
differ-ent dose levels. GivenFm(m=1, 2, 3) is the state of initial F0 plants that have experienced metal pollution for different series of exposure years (m); contributing parameter set (f) for evaluating the difference ofV.faba from four different gen-erations included one group of background, six groups of data obtained from NaN3 treatments with six different concentrations, and six groups of data from Cd2+ treatments with six different concentrations. So we have 13 contributing parameters (i.e. f=f1, f2,... fj,... f13) to calculate PV. PV can be determinated by the following formula:
PVm=% r
j=1
[1/2×(1/r+aj)]
×
1−Min(P0(fj),Pm(fj))Max(P0(fj),Pm(fj))
n
where, PVm: relative value of PV of Fm; r: total numbers of contributing factors (r=13); aj: desig-nated weighted coefficient ofjcontributing factor (Saj=1. As a rule, all weighted coefficients is usually designated equal in biological studies be-cause it is hard to say which contributing factor
should be more important than any others); P0(fj): parameter of F0 as to fj contributing fac-tors;Pm(fj) parameter of Fmas to fjcontributing factors. In general, the lowest background value and the largest increased dose-effects was set as optimum criteria for evaluation of the reliability and efficiency of different generations of V.faba. As a result, the lower value of PV in this study indicates the higher reliability and efficiency ofV.
faba to be used as sentinel plants for biomonitor-ing mutagens.
Calculation for PV was performed with soft-ware programmed with FORTRAN 77 language.
3. Results
The background values of the MCN frequen-cies of different Vicia fabageneration plants con-tinuously growing in both the RS and the EF are given in Table 1.
There was no difference in the background values of MCN frequency ofV.faba between the generations that had been planted in the RS. However, significantly different background MCN frequencies were observed between F0 and the other generation plants from the EF. Among the different generation in EF, MCN frequency in F0was significantly lower than that in root tips of F1, F2 and F3. With increasing generations of experiencing metal-contaminated soil,V.fabahad a decreased background MCN frequency. The sequence of background MCN frequency in the plants obtained from EF was F3BF2BF1.
According to these results, the root tips MCN frequencies of V.faba growing in RS represented no differences between the generations. This fact may indicate that background Vicia MCN fre-quency will be constant if the test Vicia plants
Table 1
Background MCN frequency in four generations ofV.fabaContinuously planted in RS and EFa
F2 F3
F0 F1
Generation
7.4691.29b In the RS MCN 1000 cells−1 7.3291.48b 7.0892.31b 7.5691.63b
7.4091.56b 12.7193.74d 10.4891.76cd 8.5490.87c In the EF MCN 1000 cells−1
Fig. 1. Dose effect relationship between Cd2+treatment and MCN frequency in theV.fabaamong different generation plants in
EF (bars indicate standard deviations)
have not been exposed to chemicals. On the ba-sis of this observation, we may take the initial F0 plants as the reference ones that have never been experienced environmental mutagens in or-der to compare the others that have grown in metal-contaminated soil for different years. If not specifically mentioned in the following text, F1, F2 and F3 are referred, respectively to the F1, F2 and F3 generation that are obtained from metal-contaminated EF.
Dose – response curves in V. faba from differ-ent generations to Cd2+ treatments are given in Fig. 1. The dose-effect curve of F0 was typical in toxicology, which increased MCN frequencies with increasing Cd2+ level up to 20 mg l−1
, and decreased slightly when Cd2+ was over 20 mg l−1
. However, as shown in Fig. 1, the dose-effect curves for the following three generations in response to Cd2+ treatment were obviously different, just representing increased MCN fre-quencies with increased Cd2+
concentration, but at a lower incremental MCN level.
Regression analysis between Cd2+
dosage and MCN frequency in different generation plants
are given in Table 2. Because good linear regres-sions between Cd2+ concentrations and MCN values were observed in F1, F2 and F3, even if the relationship in F0 is not well linearly corre-lated, we may choose all the linear regression slope to contrast with different generations in order to make these data sets comparable. In general, the declined regression slope was ob-served in V. faba plants growing in the EF for more generations. The slope values of F0, F1, F2 and F3 are respectively 0.069, 0.058, 0.048 and 0.034.
In response to NaN3 treatment at different dose levels, MCN frequencies in root tips of Vi
As shown in Fig. 2, the ViciaF0 plants, which had never experienced contamination showed the lowest background value and constantly increas-ing MCN value with increasincreas-ing NaN3level. With regard to F1 and F2 generation, they shared a high background MCN value, keeping increase with NaN3 up to 20 and 10 mg l−1, respectively.
Judging from the regression slopes, F0plants kept the largest in RIV, and F3 plants (0.23) had a lower RIV than F0’s (0.28); F2(0.15) plants had a higher RIV than F1’s (0.14).
Perturbation analysis results are given in Table 3. From Table 3, the sequence of perturbation value among generations was F3\F2\F1.
Table 2
Regression betweenViciaMCN frequency (Y, MCN 1000 cells−1) and mutagenic treatment (X, mg l−1) of Cd2+and NaN
3in root tip ofV.fababetween different generations from EF
Vicia Cd2+ NaN
3
Regression equation Correlation cooefficient Regression equation
Generation Correlation cooefficient
Y=0.28X+9.69 0.931** F0 Y=0.069X+13.26 0.56*
0.652**
Y=0.14X+14.68 0.86**
F1 Y=0.058X+13.55
Y=0.15X+12.83 0.557* F2 Y=0.048X+10.93 0.99**
0.90**
Y=0.034X+9.30 0.975**
F3 Y=0.23X+8.21
*PB0.05. **PB0.01.
Table 3
Perturbation values in different generation plants ofV.fabafrom EF
F0
Vicia generation F1 F2 F3
0.133 0.214
Perturbation value 0.102 0.303
Sensitivity to mutagens judging from perturbation value F0\F1\F2\F3
4. Discussion
The differences of background values of MCN frequencies in the same generation plants between the RS and the EF should attribute to the con-tamination of cadmium and lead. Soil analysis showed that the key differences of the environ-ments between the RS and the EF resulted from total and soluble contents of cadmium and lead (Duan et al., 1997a,b). Several investigators have proved that it is not possible to remove heavy metals and they are constant in the soil so that metal-contaminated soil sites have long been used as ideal stands to study eco-genotoxicological ef-fects of long-termed pollution on tester plants (Macnair, 1993; Jules, 1994). Judging from the increased background MCN frequencies in Vicia
plants in the EF (see Table 1), we may confirm that the Vicia plants had experienced metal con-tamination, otherwise there could not be a signifi-cant difference between F0plants and other plants that were from the EF. This result corresponded to previous studies, which showed both Cd2+ and Pb2+ significantly increased MCN frequency in root tips ofV.faba(Wang and Tang, 1987; Duan and Wang, 1995; Vachkova-Petrova et al., 1997). This method proved that cadmium and lead from certain concentration onward were mutagens (De-grassi and Rizzoni, 1982; Duan and Wang, 1992; Grant, 1994). Actually cadmium and lead were both in the 1997 list of US EPA top 20 hazardous substances (Internet at the website: http://
atsdr1.atsdr.cdc.gov.8080/cxcx. html).
As observed in Fig. 2, MCN frequencies of F0 plants significantly increased when plants were treated with NaN3, indicating that NaN3 is an effective mutagen. Grant et al. (1992), Grant (1994), Ma (1994) and Ma et al. (1994a,b) have drawn a similar conclusion from previous studies with theTradescantia test.
Judging from the declined background MCN frequency in F1,F2and F3 plants from the EF as shown in Table 1 and the decreased slope values of the Cd2+ dose – response regression equations as shown in Table 2, it may be concluded that the
V.fabaplants had evolved the higher resistance to cadmium when they were grown in the EF for a longer time. The lower background MCN values in coming generation plants than those in the previous ones from the EF means that the plants had less cytogenetic damages. The decreased slopes under this situation represent that the metal-experienced Vicia plants were not sensitive to cadmium so that MCN frequencies were no longer so positive to increased cadmium dosage as F0 plants do. The order of the sensitivities of different generation plants to Cd2+ was F1\ F2\F3. There have been many reports on plant pollutant resistance after the experience of pollu-tant exposure through morphological and physio-logical studies (Ernst, 1976; Shaw and Albright, 1990; Prasad, 1995) and ecological and genetical investigations (Ernst et al., 1990; Holloway et al., 1990; Dickinson et al., 1991, 1992; Macnair, 1993; Duan, 1997; Krokje et al., 1997).
environmental pollution exists in the environ-ments (Barrett and Bush, 1990). Thus the later generations will become more resistant to pollu-tants than the previous ones. In this study, V.
fabaplants resistant to cadmium were in a certain sense resistant to NaN3, but they were somehow different in resistance between the two kind of pollutants. Whether the plants that have evolved resistance to a pollutant are also resistant to another pollutant reminds further investigation.
The increased background value ofViciaMCN frequency, which took place in F1, F2 and F3 plants from the EF, may lead to misrepresenting the genotoxicity of chemical mutagens if these plants are used in bioassay of environmental mu-tagens. As demonstrated in Fig. 1, F1 plants had background value as high as 12.7 MCN 1000−1 cells, which is almost equal to F0’s value induced by 5 mg l−1 Cd2+. That is to say, even if there are no any mutagens, F1 plants will give a wrong result that is equivalent to the mutagenicity of 5 mg/l−1Cd2+.
If Vicia plants have been exposed to environ-mental mutagens, and the plants become less sen-sitive to mutagens, the efficiency of Vicia MCN test will decrease if the plants are used in bioassays. For instance, F3plants were more resis-tant to cadmium as illustrated in Fig. 1, and they would produce 9.291.2 and 9.591.9 MCN 1000−1cells at 5 and 10 mg l−1cadmium, respec-tively, but F0 plants would produce 12.1590.58 and 16.892.3 MCN 1000−1cells, respectively. In this case, the relative increment ratio (RIR, RIR=MCN frequency ratio obtained at two treatments/corresponding treatment concentration ratio) in F3 is 0.52, and RIR in F0 is 0.69. It means that in terms ofVicia MCN frequency F3 plants have lower efficiency than F0 in bioassay. A similar conclusion will be drawn if we do a comparative analysis in another way. The ratio of MCN frequency between the treatment and the control (0 dosage treatment) (RTC) was dramati-cally decreased when the same generation plants were used in the comparison. As exemplified un-der treatment of 20 mg l−1Cd2+, RTC is 2.63 in F0plants, and RTC is 1.29 in F1plants, 1.17 in F2 plants and 1.27 in F3 plants.
The reduced reliability for detecting Cd2+ mu-tagens can be identified when the Vicia plants growing in the EF are used to do a bioassay in terms of MCN frequency. If we take 20 mg Cd2+ l−1 treatment as mutagen dosage, in terms of
ViciaMCN 1000−1cells showing its mutagenicity (see Fig. 1), the value of F0 plant was as high as 19.5, and F1, F2 and F3 plant is 16.4, 12.3 and 10.8, respectively. Judging from the baseline of F0’s, the mutagenicity of 20 mg l−1Cd2+ may be underestimated if F1, F2and F3plants are used to do the bioassay. In another way of comparison from Fig. 1, the mutagenicity of 20 mg l−1 generation from the EF, the plants will underesti-mate Cd2+ mutagenicity in most cases of this experiment.
The decreased reliability and efficiency will also be presented if already-metal-contaminated plants are applied to detect NaN3 mutagenicity. As shown in Fig. 2, there was a very good linear relationship between MCN frequency and NaN3 treatment in F0 plants, but most part of dosage-effect curves of F1’s and F2’s were above F0’s. F3’s was under the curve of F0’s. Because background values of MCN frequencies in F1, F2 and F3 plants are higher than that in F0, F1, F2 and F3 plants will misrepresent mutagenicity if there are not any mutagens in environments. If we take F0’s curve as a standard one in dosage effect, it is probable that overestimated mutagenicity will be recorded if F1and F2plants are used to test NaN3 mutagenicity, and correspondingly underesti-mated mutagenicity will be observed in F3. All in all, F1, F2 and F3 plants, which have experienced metal contamination, will lose their reliability in a bioassay for NaN3 mutagenicity. As discussed above, the decreased regression slopes (Table 2) in F1, F2 and F3 plants show the reduced efficiency in NaN3 bioassay.
al., 1994a; Cotell et al., 1997). Thus, the results of perturbation analysis could be explained in this way: the lower value of PV denoted the higher efficiency and reliability of the generation of V.
faba to be used as sentinel for biomonitoring mutagens. It was clear that the most ideal sen-tinels should be F0 plants; and the longer the plants had grown in metal-contaminated soil, the lower the reliability and efficiency of the plants was in detecting mutagenic Cd2+ and NaN3 concentration.
According to this investigation, theViciaMCN bioassay in environmental mutagens needs careful reconsideration. Although V. faba, whose mi-cronucleus has been used as an effective cytoge-netical endpoint, is one of the sentinels that have been known for being highly sensitive to muta-gens (Degrassi and Rizzoni, 1982; Grant et al., 1992; Cotell et al., 1997; Duan et al., 1999), a lot of data obtained by different investigators were incomparable with each other (Wang and Tang, 1987; Vachkova-Petrova et al., 1997). The reason for this may result from sentinel plants, that had a different historic status of exposure to muta-genic pollutants.
TheViciaMCN test has well been documented in world-widely biomonitoring environmental mu-tagens (Grant, 1994; Ma et al., 1995; Duan and Wang, 1995), but it may be suggested thatVicia
MCN test be used as an acceptable endpoint of biomarker only if the sentinel plants are collected from sites where there has never been any chemi-cal exposure. In order to eliminate misdescription resulted from sentinels of Vicia plants that are with different historic status of chemical exposure, there is a need to further understand if already-ex-perienced-pollutionViciaplants influence the reli-ability and efficiency of Vicia MCN bioassay. Because almost everywhere on the earth may be a contamination by any pollutants, it is necessary to establish several international centers of stock seed for providing non-toxic-chemical-exposed sentinel Viciaif we are to applyVicia MCN as a biomarker to identify global environmental status with guarantee of reliability and efficiency.
Acknowledgements
We express our deepest appreciation to Dr Te-Hsiu Ma for his valuable comments and dis-cussion on preparing this manuscript. All the faculty members of the broadbean group of Yun-nan Institute of Bean Crops are also indebted for their generously providing the RS in this investi-gation. This work is in part supported by China National Science Foundations (No. 39760022 and No. 39970142), Yunnan Foundations for Basic and Applied Science (No. 96C006Q) and Yunnan Provincial Scholarship for Outstanding Young Scientists (97C010G).
References
Barrett, S.C.H., Bush, E.J., 1990. Population process in plants and the evolution of resistance to gaseous air pollutants. In: Taylor, G.E., Pitelka, L.F., Clegg, M.T. (Eds.), Ecolog-ical Genetics and Air Pollution. Springer – Verlag, New York, pp. 137 – 166.
Butterworth, F.M., 1994. Introduction to biomonitors and biomarkers as indicators of environmental changes. In: Butterworth, F.M., Corkum, L.D., Guzman-Rincon, J. (Eds.), Biomonitors and Biomarkers as Indicators of Envi-ronmental Changes. Plenum Press, New York, pp. 1 – 8. CEPA, 1990. Protocols on Environmental Monitoring, China
Environmental Sciences Press, Beijing, pp.456-468 (in Chinese).
Cotell, S., Masfaraud, J.F., Ferard, J.F., 1997. Ecogenotoxic-ity of contaminated soil samples assessed by theAllium/Vi
-ciaroot tip test. Mut. Res. 379, S104.
Degrassi, F., Rizzoni, M., 1982. Micronucleus test in Vicia faba root tips to detect mutagen damage in fresh water pollution. Mut. Res. 97, 19 – 33.
Dickinson, N.M., Turner, A.P., Lepp, N.W., 1991. How do trees and other long-lived plants survive in polluted envi-ronments? Funct. Ecol. 5, 5 – 11.
Dickinson, N.M., Turner, A.P., Watmough, S.A., Lepp, N.W., 1992. Acclimation of trees to population stress: cellular metal resistance traits. Ann. Bot. 70, 569 – 572. Duan, C.Q., 1995. Plant adaptation to environmental
pollu-tion and its microevolupollu-tion. Chin. J. Ecol. 14, 43 – 50 In Chinese, with English abstract.
Duan, C.Q., 1996. Effects of environmental pollution on the evolution of organisms and the new perspectives in envi-ronmental biology. In: Hou, B.Z. (Ed.), On Envienvi-ronmental Issues from Chinese scholars. Chinese Environmental Sci-ences Press, Beijing, pp. 767 – 770.
Duan, C.Q., Wang, H.X., 1992. Effects of heavy metals on the contents of nucleic acids and nuclease activities in root tip of Vicia faba. Environ Sci 13, 31 – 36 In Chinese with English abstract.
Duan, C.Q., Wang, H.X., 1995. Cytogenetical toxical effects of heavy metals onVicia fabaand inquires into theVicia -MCN. Acta Bot. Sin. 37, 14 – 24.
Duan, C.Q., Hu, B., Yang, J.B., Lu, X.P., 1994. Screening appropriate broadbean (Vicia faba) varieties for Vicia -MCN test and application in monitoring water pollution in the Dianchi Lake of China. J. Yunnan Uni. 16, 250 – 256 In Chinese with English abstract.
Duan, C.Q., Wang, H.X., He, X.F., 1997a. Fuzz clustering to the effects of heavy metals on the quantitative traits of broadbean in four generations and perturbation analysis. Acta Sci. Circum. 16, 450 – 460 In Chinese with English Abstract.
Duan, C.Q., Wang, H.X., Jiang, H.Q., 1997b. Differentiation of quantitative characters of Vicia faba contaminated with heavy metals. Acta Ecol. Sin. 17, 133 – 144 In Chinese with English Abstract.
Duan, C.Q., Hu, B., Jiang, X.H., Wen, C.W., 1999. Genotox-icity of the water samples of Dianchi lake detected byVicia fabamicronucleus test. Mut. Res. 426, 121 – 125. Ernst, W.H.O., 1976. Physiological and biochemical aspects of
metal tolerance. In: Mansfield, T.A. (Ed.), Effects of Air Pollutants on Plants. Cambridge University Press, Cam-bridge, pp. 115 – 133.
Ernst, W.H.O., Schat, H., Verkleij, J.A.C., 1990. Evolutionary biology of metal resistance inSilene6ulgaris. Evol. Trends Plants 4, 45 – 51.
Grant, W.F., 1994. The present status of higher plant bioassays for the detection of environmental mutagens. Mut. Res. 310, 175 – 185.
Grant, W.F., Lee, H.G., Logan, D.M., Salamone, M.F., 1992. The use ofTradescantiaandVicia fababioassays for the in situ detection of mutagens in an aquatic environment. Mut. Res. 270, 53 – 64.
Holloway, G.J., Sibly, R.M., Povery, S.R., 1990. Evolution in toxin-stressed environments. Funct. Ecol. 4, 289 – 294. Jules, E.S., 1994. Adaptation to metal-contaminated soils in
populations of the moss, Ceratodon pirpureus: vegetative growth and reproductive expression. Am. J. Bot. 86, 791 – 797.
Krokje, A., Bingham, C., Ostby, C., 1997. Genetic effects of high and low exposure to heavy metals. Mut. Res. 379, S112.
Ma, T.H., 1982. Vicia cytogenetic tests for environmental mutagens: a report of the US environmental protection agency gene-tox program. Mut. Res. 99, 259 – 271. Ma, T.H., 1994. Tradescantia plants as biomonitors of the
genotoxicity of environmental pollutants. In: Butterworth, F.M., Corkum, L.D., Guzman-Rincon, J. (Eds.), Biomoni-tors and Biomarkers as IndicaBiomoni-tors of Environmental Changes. Plenum Press, New York and London, pp. 207 – 216.
Ma, T.H., Harris, M.M., 1985. In situ monitoring of environ-mental mutagens: hazard assessment of chemicals. Curr. Dev. 4, 77 – 106.
Ma, T.H., Cabreea, G.L., Wasilewska, A.C., et al., 1994a.
Tradescantia micronucleus bioassay. Mut. Res. 310, 221 – 230.
Ma, T.H., Cabreea, G.L., Wasilewska, A.C., et al., 1994b.
Tradescantia stamen hair mutation bioassay. Mut. Res. 310, 211 – 220.
Ma, T.H., Xu, Z., Xu, C., et al., 1995. The improvedAllium/
Vicia root tip micronucleus assay for clastogenicity of environmental pollutants. Mut. Res. 334, 185 – 195. Macnair, M.R., 1993. The genetics of metals resistance in
vascular plants. New Phytol. 124, 541 – 559.
Mao, Y.G., He, X.F., Zhuang, Z., 1990. Modern Regional Planning: Methodology. Yunnan University Press, Kunming In Chinese.
Prasad, M.N.V., 1995. Cadmium toxicity and resistance in vascular plants. Environ. Exp. Bot. 35, 525 – 545. Prus-Glowacki, W., Godzik, S., 1995. Genetic structure of
Picea abiestrees tolerant and sensitive to industrial pollu-tion. Silvae Genet. 44, 62 – 65.
Shaw, A.J., Albright, D.L., 1990. Potential for the evolution of heavy metal resistance in Bryum argenteum, a moss. II: generalized resistances among diverse populations. Bryolo-gist 93, 187 – 192.
Vachkova-Petrova, R., Kappas, A., Filipov, S., Tyagunenko, E., Ivanova, N., 1997. Use of biomarkers in risk assess-ment for exposure to environassess-mental heavy metals. Mut. Res. 379, S111.
Wang, Y., Tang, D., 1987. Pollution detected by Vicia faba
root tip cell micronucleus test and comparison with other index. Chin. Environ. Sci. 7, 45 – 52 In Chinese with En-glish Abstract.
