a © ü a щ m 0 w © [? © B G a G В © D ® $ s 0 © m © 0 ©
20 0 8 ---Folia Biologica et O ecologica 4: 3 7 - 4 9
(Acta Univ. Lodz., Folia Biol. et Oecol.)
Be a t a Ko ś m i d e r 1’, Re g i n a Os i e c k a1 & Ja n T . Si c i ń s k i2
' ' D ep a rtm en t o f C y to g e n e tics an d P lan t M o le c u la r B io lo g y , U n iv er sity o f Ł ó d ź, 12 /16 B a n ac h a Str.. 9 0 -2 3 7 Ł ód ź , P o lan d
2 D e p ar tm en t o f B o tan y , U n iv ers ity o f Ł ó d ź, 12/1 6 B a n ac h a S tr.. 9 0 - 2 3 7 Ł ó d ź, Po lan d e- m a il: b e atak o s m id er @ in teria.p l
APPLICATION OF MICRONUCLEUS ASSAY IN PLANTS TO MONITOR ENVIRONMENTAL POLLUTION IN SITU
A b stra c t: Sam ples o f 5 vascular plant species: A chillea millefolium L., A rtem isia vulgaris L.,
Festuca gigantea (L.) VILL., Ranunculus acris L. s. str. and Vicia angustifolia L. were collected
near Fuel Storage Station, Chem ical Fibre Factory “W istom ” in T om aszów M azowiecki and Sewage Treatm ent Plant in Sulejów. Genotoxicity o f environm ental pollution was analysed using micronucleus assays in pollen m other and root tip cells in com parison with plant m aterial collected in the Tatra and Babia G óra National Parks. Abnorm alities in m orphology o f plants growing in polluted areas were also found. T he cytogenetical analyses revealed positive correlation betw een the percentage o f m icronuclei and the presence o f heavy m etals in soil. Pollen m other cells (germ line) were found m ore susceptible to DN A dam age as com pared with root tip cells (som atic line).
K ey w ord s: m onitoring in situ, environm ental pollution, m icronuclei, pollen m other cells, root tip cells.
1. IN T R O D U C T IO N
The negative effects caused by anthropogenic factors e.g. industrial pollution, expansion of urban-industrial agglomerations, use o f chemical compounds in agriculture, deposition of dangerous refuses, exhaustion o f natural resources, pollution o f ecosystems and even radioactive contamination o f natural environ-ment are considered one of the major problems of the present time. This situation disturbs homeostasis of ecosystems and exerts influence on all or-ganisms which are connected with one another by food chains.
Monitoring system is one of the elements of the interdisciplinary program used to control and prevent further contamination of human environment. It registers simultaneously emission of pollution and its consequences for
ecosys-terns. Physico-chemical methods are used to define concentrations of various factors in natural environment as well as biological research is conducted to
reveal their influence on living organisms and determine pollution effects (ClEPAŁ
1992). Methods of monitoring in situ are used among others to analyze morphological and anatomical changes in plants and an impact of various pollutants on the structure and organization of plant genomes. DNA damage can be detected by including genotoxicity assays to methods of environmental pollution monitoring in situ.
T h e m icr onu cleu s assay is usefu l in b iom on ito ring and e c o t o x ic o lo g y as a se n sitiv e m etho d to d etect chro m o som a l abn orm alities ind uced by m utagens both in vivo and in vitro (STOPPER, MÜLLER 1997). M ic ron u clei are form ed through lo ss o f w h o le ch r om o so m e s or their fragm en ts during c ell divisio n . Th e m icr on u cleu s assay is sim p le and ea sy to perform and b elo n g s to the standard cy to g e n e tic s a ssay s recom m end ed in d ifferent m o nitorin g sy stem s and e c o to x ic o lo g y (Ma, Gr a n t 1982).
The aim of this research was to analyze frequency of micronuclei occurrence in pollen mother and root tip cells of plant species collected from polluted areas and from the National Parks. These experiments were carried out in order to evaluate the usefulness of micronucleus assay to monitor environm ental pollution in situ.
2. M A T E R IA L S AN D M E T H O D S
The plant species Achillea millefolium L., Artemisia vulgaris L., Festuca
gigantea (L.) VILL., Ranunculus acris L. s. str. and Vicia angustifolia L. were
collected in the polluted areas (Fuel Storage Station, Chemical Fibre Factory “W istom ” in Tomaszów Mazowiecki and Sewage Treatment Plant in Sulejów) and from Tatra and Babia Góra National Parks in 1998. Whole inflorescences and root tips were cut for cytogenetic analyses. Moreover, plants for herbarium material were brought.
Approximately 50 individual plants were observed in each studied area.
Stereomicroscope and botanical key were used to identify plant species (Sz a f e r
et. al. 1986). Additionally, abnormalities in their morphological structures were also observed. For cytogenetical analyses the root tips and flower buds (Ma 1982) were fixed in Camoy solution ( 1 :3 icy acetic acid : 96% ethanol) and
stored in 70% ethanol at 4°C (GRANT 1982). Various staining methods (Feulgen
method, aceto-carmine, M ayer’s hematoxylin) were used. The dry-ice method was applied. Four preparations were made from pollen mother cells and root tips of each species. The percentage of the micronuclei was determined in 1000 root tip cells per slide and in 100 pollen mother cells. The standard deviations and Student test were also applied.
2.1. Characteristics of studied area
2.1.1. Fuel Storage Station
Although the high concentrations of heavy metals and ether-extractable substances (Tables 1, 2) indicating organic-laden water in this station and its surrounding were known to be well above-average, the actual content of organic substances has not been determined there (unpublished data). Aromatic hydro-carbons, lead compounds, mineral oils and wastes from alkaline fuel purification which are released to soil and water from the Fuel Storage Station were found on the list of dangerous wastes (Dz.U. 2001; Dz.U. 2002).
T a b le 1: M ean values o f chem ical analyses of sewage from Fuel Storage Station and Sewage Treatm ent Plant in Sulejów (unpublished data from these stations). The highest threshold limit values o f pollution in sewage inflow to water and into soil (Dz.U. 1991). B O D - biochem ical oxygen dem and; C h O D - chem ical oxygen demand; n t - not tested. Values exceeding lim its are given in bold
Specific ation
Fu el S to rag e
S ta tion
Sewage T re a tm e n t P lan t in Sulejów
L im it value Unit inflow sewage outflow s ludg e b o d5 33.7 32 12 nt 50 mg/1 Cadm ium nt nt nt 3.51 0.2 mg/1 Chloride 9.16 30 42 nt 1 000 mg/1 ChOD 133.6 90 39 nt 250 mg/1 Cooper nt nt nt 124.45 0.5 mg/1 Lead nt nt nt 90.78 0.5 mg/1 R eaction 6.86 7.3 7.9 6.9 6.5-8.5 pH Sulphates nt 41.8 36.2 nt 500 mg/1 T otal nitrogen 4.06 nt nt 50.35 30 mg/1 Total suspension 7.6 51 13 nt 35 mg/1 Zinc nt nt nt 2 192.15 2 mg/1
T ab le 2: Soil contam ination near and in Fuel Storage Station (1 - tankage facilities, 2 - tank cleaning departm ent, 3 and 4 — station boundaries, 5 — 50 m away from station, unpublished data from Fuel Storage Station). T he highest threshold lim it values o f pollution in sewage inflow to water and into soil (Dz.U. 1991).
In d ic a to r 1 2 3 4 5 L im it values Unit Ether-extractable substances Lead Reaction 117 < 6 7.37 5 220 15.5 7.32 165 < 6 7.69 106 < 6 4.87 118 < 6 4.7 50 0.5 6 .5 -9.0 mg/1 mg/1 pH
2.1.2. Sewage Treatm ent Plant in Sulejów
Domestic and municipal wastes inflow to this sewage treatment plant (Table 1). Localization of sewage treatment plants is important because sub-stances harmful for water environment are usually present there. These include wastes containing among others: cadmium, lead, mercury, phosphorus and compounds of chromium, zinc and copper (Dz.U. 2001).
2.1.3. Chemical Fibre Factory “W istom” in Tomaszów Mazowiecki
Until 1991 industrial wastes were disposed directly on this dumping site, which meant with probability of landfill leachate occurrence, but after
moder-nisation of the waste site wastes were deposited in a rational way (SADURSKI,
Li s z k o w s k a 1993). However, in spite of the fibre production stoppage this
waste site is still dangerous for the environment (Table 3). Its negative en-vironmental impact is not exactly known but soil degradation was detected
within 200-m radius (DANIELAK et al. 1993). There are still stable substances
which have negative impact on oxygen balance in water (e.g. nitrites), copper compounds and cyanides (Dz.U. 2001; Dz.U. 2002).
T ab le 3: M ean values o f chem ical analyses o f water extracts from dum ping site o f Chem ical Fibre Factory “ W istom ” in Tom aszów M azowiecki ( Si u t a et al. 1991) and the highest threshold lim it values of pollution in sewage inflow to water and into soil (Dz.U. 1991). Values exceeding limits are given in bold
In d ic a to r
C o n te n t in w aste L im it
values
Unit
fib res plastics viscoses
Cooper <0.1 <0.1 <0.1 0.5 mg/1 Nitrates <0.1 <0.1 <0.1 30 mg/1 Reaction 1.54 1.34 13.8 6.5-8.5 PH Sulphates 12 000 550 40 500 mg/1 Total nitrogen <1 <1 <1 30 mg/l Zinc 117 4 5 2 mg/l
2.1.4. Tatra and Babia Góra National Parks
They are located far from industrial centres and should be potentially unpolluted. However, increased level of sulphur dioxide, nitric oxides and heavy metals were detected here. Precipitation that increases with the height above sea-level also increases pollution inflow. Strong winds cause an air-mass mixing and facilitate dust settlement in a moist soil zone. Lasting low temperature increases gases solubility, causes plant mechanical damages and facilitates pollution infiltration ( G r o d z i ń s k a , S z a r e k 1996).
3. R E SU L T S
3.1. Abnorm alities in morphology of plants collectcd in the studied area
3.1.1. Fuel Storage Station
Achillea millefolium L.: single leaves at the stem base while in the upper part
they were abnormally numerous and growing in whorled arrangement; lingual flowers were not developed.
Artemisia vulgaris L.: excessive thickness o f roots.
Festuca gigantea (L.) VILL.: above-average growth; leaf blade thickened
con-siderably; larger sizes of leaves.
Vicia angustifolia L.: some leaves abnormal bigger; variability in their sizes;
leaf developm ent disturbed e.g. splitting o f leaf tips.
3.1.2. Chemical Fibre Factory “W istom” in Tomaszów Mazowiecki
Achillea millefolium L.: abnormally numerous leaves in nods; inflorescences
untypical growing from the middle part of a steam; scanty flowers.
Vicia angustifolia L.: deformation o f a whole plant; untypical variability in
sizes of leaves which are very narrow and small; abnormal distribution of flowers in leaf corners; S-shaped pods; disturbances in leaf developments e.g. splitting o f leaf tips.
3.1.3. Sewage Treatment Plant in Sulejów
Ranunculus acris L. s. str.: fasciated stems; leaves of abnormal shapes and
3.2. Micronuclci analysis
3.2.1. Pollen mother cells
Figure 1 shows examples of micronuclei in pollen mother cells observed in plant species collected in the studied area. The percentage o f the micronuclei in pollen mother cells of Ranunculus acris L. s. str. in relation to the area of its growing were presented in Figure 2. Their lowest number was noticed in the material from Tatra (2%) and Babia Góra National Parks (4.3%). On this ground a suggestion could be drawn concerning the degree o f contamination o f the studied area. Greater number of these structures (10%) in pollen mother cells of plants growing on drying beds in Sewage Treatment Plant in Sulejów as compared with its adjacent (5.8%) was also observed which can be connected with the high concentration of heavy metals in the form er place (Table 1). The high percentage o f micronuclei was observed both in Achillea millefolium L. (47.3%) growing on the dumping site o f Chemical Fibre Factory “ Wistom” and in Artemisia vulgaris L. (15.5%) from Fuel Storage Station (Fig. 3).
Fig. 1: M icronuclei in pollen m other cells: ( l ) - Ranunculus acris L. s. str. (2) - Achillea
Fig. 2: T he percentage o f m icronuclei in pollen m other cells o f R anunculus acris L. s. str. collected in the studied area: 1 - Tatra National Park; 2 - Babia Góra National Park; 3 - area o f Sewage T reatm ent Plant in Sulejów; 4 - drying beds in Sewage T reatm ent Plant in Sulejów. (*) statistical significance (Student test; p < 0.05).
14% 12% ® 10% и I 8% E 'S 6% г? 4% 2% 0% 1 2 3 4 Ranunculus acris L. s. str.
Fig. 3: The percentage o f m icronuclei in root tip cells (solid grey) and pollen m other cells (dotted) in: 1 - A rtem isia vulgaris L. (Fuel Storage Station), and 2 - A chillea millefolium L. (dum ping site o f Chem ical Fibre Factory “ W istom ” in T om aszów Mazowiecki).
3.2.2. Micronuclei analysis in root tip cclls
The num ber of micronuclei in root tip cells in 4 plant species collected near Fuel Storage Station was compared (Fig. 4). The percentage of these structures occurrence was similar in 3 plant species: Achillea millefolium L.,
Festuca gigantea (L.) VILL. and Vicia angustifolia L. However, in Artemisia vulgaris L. this value was twice higher. Note, that differences in the percentage
of micronuclei in root tip cells between these species were smaller (Fig. 4) as compared to pollen mother cells (Fig. 3).
Fig. 4: The percentage o f m icronuclei in root tip cells o f plant species collected near Fuel Storage Station 2.0% 1.5% '5 ö 3 с 0.0% --- И м и --- --- --- ii&Ma---,--- ... A. m illefo liu m F. g igantea V. ang ustifolia A. vu lgaris
Plant s p ec ie s
Moreover, samples of flower buds and root tips of Artemisia vulgaris L. and
Achillea millefolium L. were also collected near Fuel Storage Station and Chemical
Fibre Factory “Wistom” in Tomaszów Mazowiecki (Fig. 3). In both cases distinct differences in the percentage of micronuclei between pollen mother and root tip cells suggest greater sensitivity of germ line to pollution than somatic one.
4. D ISC U SSIO N
Higher plants provide valuable genetic assay systems for screening and
monitoring environmental pollutants (Gr a n t 1994). Natural environment is
particular phytocoenosis. Because of that finding the same plant species in different places in area poses some problems. However, plants collected from
different areas were also analysed for in situ studies ( D a n i e l et. al. 1997).
Moreover, pollution can affect the distribution of plants. It was reported that the abundance of plants strongly depends on actual conditions e.g. increased
soil acidity causes decrease in species population ( D a n i e l et. al. 1997; B a n â -
s o w ä , Ś u c h a 1998).
In the system of in situ monitoring control species distributed in natural environment have to be included. Assays can be undertaken employing flora
from test and control areas (M iC lE T A , M u r i n 1996). G r a n t and Z u r a (1982)
also recomm ended the use in situ weed communities for the detection and monitoring of mutagens.
There are several advantages in using microspore mother cells and tetrads as bioindicators of mutagenicity e.g. high sensitivity conditioned by haploid state, when mutations effecting the development of pollen are immediately evident. In situ studies in polluted regions and control (in botanical garden) were carried out using haploid cells as indicators of mutagenicity. Therefore by using plants living in the polluted environment is possible to evaluate the
effectiveness of ecological factors including pollution components (M lC IE T A ,
M u r i n 1996).
In the Tatra and Babia Góra National Parks some pollution is also observed but it is mainly a result of common gaseous emission. In Ranunculus acris L. s. str. collected in the Tatra National Park micronuclei num ber (2%) in pollen mother cells was lower than from the Babia Góra National Park (4.3%; Fig. 2). However, these results are comparable to those obtained for pollen mother cells o f Tradescantia in which spontaneously formed micronuclei number is
5% in pollen mother cells (M Ü L L E R , S t r e f f e r 1994). In the dumping site of
Chemical Fibre Factory “W istom” in Tomaszów Mazowiecki the amount of heavy metals and other chemical compounds exceeding accepted standards was detected (Table 3). It is important that there is lack o f soil sorption complex that can bind heavy metals and act as a buffer which causes inflow of toxic substances into plant tissues. Abnormalities in morphology o f plants growing on soil with excess concentration of toxic mineral com pounds could result from their high accumulation in plants as well as from secondary deficiency of
macro- and microelements (FA B ISZ EW SK I et al. 1983).
Nickel, cyanide, cadmium, chromium and lead compounds were proved to
have toxic and genotoxic effect on plant cells (SO M A SH E K A R , A r e k a l 1983;
V i l l a l o b o s - P i e t r i n i 1987; G a b a r a , G o ł a s z e w s k a 1992; G a b a r a et al. 1992;
G w ó ź d ź 1996; K a c p e r s k a 1998). It was shown that heavy metals which cause
D N A damage and cancer in animals and humans such as cadmium, chromium,
nickel, arsenic were highly genotoxic in Tradescantia and Vicia micronucleus
1998), whereas metals which pose no or minor hazards to man e.g. antimony
and copper caused no significant dose-effects in this system (KNASMULLER et
al. 1998). Moreover, STEINKELLER et al. (1998) ordered the heavy metals
P b > C d > Z n > C u according to decreasing potential genotoxicity in the mic-ronucleus assay in pollen mother cells in Tradescantia and in root tip cells in
Allium сера and Vicia faba.
Cadm ium was found to reduce the D N A content in the meristem atic
zone of Pisum sativum and its action in meristem could be attributed to
the reduction in the transcriptional activity (Wo j t y l a - K u c h t a , G a b a r a
1991). Note, that in pollen mother cells of Ranunculus acris L. s. str. collected on drying beds in Sulejów, where the excess o f lead, zinc and cadmium was noticed (Table 1), the highest percentage of micronuclei (10%)
was also observed (Fig. 2). S o m a s h e k a r and A r e k a l ( 1 9 8 3 ) showed toxic
influence of pollutants with high nickel and cyanide contents on Allium
сера root tip cells. They observed chromosome breaks, micronuclei, chromatid
bridges, dicentric chromosomes and binuclear cells induction, disintegration and fragm entation of nuclei, disturbances in m itotic spindle organization and function.
S m a r d a k i e w i c z and W o ź n y (1995) reported that heavy metal absorption
in monocotyledonous species was lower than in dicotyledonous. This is in agreement with our observations in Festuca gigantea (Fig. 4). Achillea
mil-lefolium and 70% of grasses are polyploidal plants (M IZ IA N T Y 1995). Poly- ploidization can be advantageous in occupation of new ecological niches as
well as it can facilitate of adaptation to new environmental conditions (K O N O -
NO W IC Z 1989).
In disturbed habitats microevolution processes resulting in the development
of plants resistant to heavy metals present in soil can be observed. F a l i n s k a
(1996) reported that sometimes these reactions have an adaptive character and then resistant ecotypes appear. It was demonstrated that species growing in soil naturally rich in metallic elements inherited resistance mechanisms. It was also observed that plant tolerance can be induced and stabilized by gradual intro-duction of species to polluted areas but attention must be paid not to exceed their ecological resistance.
Existence of plants in polluted environments can be connected with e.g the presence of enzymes able to function despite high levels of heavy metals ( S c h n e i d e r 1995; G w ó ź d ź 1996).
The harmful effects o f aromatic hydrocarbons in petroleum derivative pro-ducts, e.g. toluene, n-hexane and n-heptane on plants were also confirmed. They are soluble in lipids and can damage lisosome membranes and DNA can be degraded by DN-ase. In Vicia fab a root tip cells these compounds caused induction of micronuclei, C-mitosis, chromosomal aberrations and chromosomes with inactive centromers. It was proved that centromere was inactivated already
after 1 h incubation with these substances. This caused an increase in chromo-some number with inactive or damaged centromeres that did not participate in
anaphase and appeared as micronuclei ( G o m e z - A r r o y o et al. 1986). Low
percentage of micronuclei number in root tip cells (Fig. 4) suggests the occurrence of detoxification mechanism and tolerance.
In conclusion the correlation between the highest number o f micronuclei and the presence o f heavy metals in soil was noted. Moreover, we found higher number of these structures in germ line in comparison with somatic one which suggests higher sensitivity of pollen mother cells than root tip
cells to pollution. This tendency was also reported by K NA SM U L LE R et al.
(1998) and ST E IN KE L L E R et al. (1998). The presented results confirm
use-fulness of micronucleus assay in plants to monitor environmental pollution
in situ.
5. R E F E R E N C E S
Ba nAs o w â, V., Śu c h a, V. 1 9 9 8 . Degradation of grassland after strong soil acidification. Ekologia (Bratislava) 17: 28-38.
Ci e p a ł, R. 1 9 9 2 . Przenikanie S, Pb, Cd, Zn, Cu i Fe do biom asy oraz gleby ekosystem u leśnego (na przykładzie wschodniej części województwa katowickiego). Z naczenie bioindykacyjne: 10. Uniwersytet Śląski, Katowice.
Da n i e l, P., Ko v a c s, B., Pr o k i s c h, J., Gy o r i, Z. 1997. Heavy m etal dispersion detected in soils and plants alongside roads in Hungary. Chem . Spec. B ioavailability 9: 83-93.
Da n i e l a k, D ., Eb e l t, M ., Le n a r t, W ., Li s z k o w s k a, E ., Pa r t e k a, T ., Sa d u r s k i, A ., Św i ą t e k, H ., Ty s z e c k i, A ., Wa r c h a ł o w s k i, A . 1 9 9 3 . O c e n a o d d z i a ły w a n ia n a ś ro d o w is k o Z W C h „ W i s -t o m ” w T o m a s z o w i e M a z o w i e c k im . R a p o r-t k o ń c o w y . F u n d a c ja E c o b a l -ti c , G d a ń s k , p p . 1 -7 5 . Dz.U. 1991, nr 116, poz. 503. Dz.U. 2 0 0 1 , nr 112, poz. 1206. Dz.U. 2 0 0 2 , nr 2 1 2 , poz. 1 7 9 9 .
En n e v e r, F. K ., An d r e a n o, G ., Ro s e n k r a n z, H. S. 1988. T he ability o f plant genotoxicity assays to predict carcinogenicity. Mutat. Res. 205: 99-105.
F a b i s z e w s k i , J ., Br e j, T ., Bi e l e c k i, К. 1 9 8 3 . Fitoindykacja wpływu huty m iedzi na środowisko biologiczne. W ydawnictw o Polskiej Akadem ii Nauk, W rocław, pp. 1-110.
Fa l i ń s k a, K . 1 9 9 6 . E kologia roślin. PWN, W arszawa, p p . 176-177.
Ga b a r a, B ., Go ł a s z e w s k a, E . 1992. Calcium effect on m itotic activity and frequency o f m itotic disturbances in the cortex cells o f Pisum sativum L . roots treated with heavy m etals. Bull. Pol. Acad. Sc. Biol. Sc. 40: 97-103.
Ga b a r a, В., Wo j t y ł a- Ku c h t a, B „ Ta r c z y ń s k a, M. 1992. T he effect o f calcium on DNA synthesis in pea (Pisum sativum L .) roots after treatm ent with heavy m etals. Folia Histochem . Cytobiol. 30: 69 -74.
G o m e z - A r r o y o , S ., Ca s t i l l o- Ru i z, P ., Vi l l a l o b o s- Pi e t r in i, R . 1 9 8 6 . Chrom osom al alterations induced in Vicia fa ba by different industrial solvents: thinner, toluene, benzene, n-hexane, n-heptane and ethyl acetate. Cytologia 51: 133-142.
G r a n t , W . F . 1982. Plant m utagen assays based upon chrom osom e m utations. In: E. J.
Kl e k o w s k i, J r (ed.). Environm ental m utagenesis, carcinogenesis and plant biology. Vol. 2. Praeger Press, New York, pp. 1-24.
Gr a n t, W . F., Zu r a, K. D. 1982. Plant as sensitive in situ detectors o f atm ospheric mutagens. In: J. A. Heddle (ed.). M utagenicity: new horizonts in genetic toxicology. Academ ic Press, New York, pp. 407-434.
Gr a n t, W . F. 1994. The present status o f higher plant bioassays for the detection o f environ-mental m utagens. Mutat. Res. 310: 175-185.
Gr o d z i ń s k a, K., Sz a r e k, G . 1996. Skażenie obszarów górskich - uwarunkowania przyrod-nicze. In: R. So j a, P. Pr o k o p (eds.). Zintegrowany m onitoring środow iska przyrodniczego. M onitoring geosystem ów górskich. Państwowa Inspekcja Ochrony Środow iska, Warszawa, pp. 6 4-77.
G w ó ź d ź , E . A. 1996. M olekularne podstawy odpowiedzi roślin na stresy środowiskowe. In: J. Ba r c i s z e w s k i, K. Ła s t o w s k i, T . Tw a r d o w s k i (eds.). Nowe tendencje w biologii molekularnej i inżynierii genetycznej oraz m edycynie. T . 2. Sorus, Poznań, pp. 469-492.
Ka c p e r s k a, A . 1998. R eakcje roślin na czynniki stresowe. In: J . Ko p c e w i c z, S . Le w a k (eds.). Podstawy fizjologii roślin. PWN, W arszawa, p. 622.
Kn a s m u l l e r, S ., Go t t m a n, E., St e i n k e l l e r, H., Fo m i n, A., Pi c k l, C., Pa s c h k e, A., Go d, R„
Ku n d i, M. 1998. Detection o f genotoxic effects o f heavy m etal contam inated soils with plant bioassays. M utat. Res. 420: 37-48.
Ko n o n o w i c z K . K . 1 9 8 9 . Cytochem iczne aspekty odporności roślin na zasolenie in vitro. W ydawnictw o U niwersytetu Łódzkiego, Łódź, pp. 109-110.
Ma, T . H. 1982. Tradescantia cytogenetic tests (root-tip m itosis, pollen m other-cell m eiosis).
A report o f the U.S. Environm ental Protection Agency Gene-Tox Program . Mutat. Res. 99: 293-302.
M a , T . H , G r a n t , W . F . 1 9 8 2 . The Tradescantias - adventurous plants. The Herbarist 48: 36-44.
M a , T . H., Ca b r e r a, G . L., Ch e n, R., Gi l l, B. S ., Sa n d h u, S . S ., Va n d e n b e r g, A. L., Sa l a m o n e,
M. F . 1 9 9 4 , Tradescantia m icronucleus bioassay. Mutat. Res. 310: 221-230.
Mi c i e t a, K., Mu r i n, G. 1996. Microspore analysis for genotoxicity o f a polluted environm ent. Environ. Exp. Botany 36: 21-27.
Mi z i a n t y, M . 1995. T raw y - grupa roślin, która odniosła ew olucyjny sukces. W iad. Bot. 39: 59-70.
Mü l l e r, W . U., St r e f f e r, C . 1994. Micronucleus assays. In: G. Ob e (ed.). Advances in m uta-genesis research. Springer-Verlag, Berlin, Heidelberg, New York, pp. 1-63.
Sa d u r s k i, A., Li s z k o w s k a, E . 1993. W stępna ocena oddziaływania ZW C h „W istom ” w To-m aszow ie M azowieckiTo-m na środow isko wód podzieTo-m nych. Fundacja E cobaltic, Gdańsk, pp. 1-35.
Sc h n e i d e r, J . 1995. W pływ ołowiu na kwasy nukleinowe. In: A. Wo ź n y (ed.). Ołów w kom ór-kach roślinnych - pobieranie, reakcje, odporność. Sorus, Poznań, pp. 83-92.
S i u t a , J . , Wa s i a k, G., Si e n k i e w ic z, R. 1 9 9 1 . Opracowanie koncepcji gospodarki odpadam i technologicznym i w ZW Ch „Wistom ” . Organizacja Badań i Projektów „Inżynieria” Ekologiczna, W arszawa, pp. 3 -4.
S m a r d a k i e w i c z , S ., W o ź n y , A . 1 9 9 5 . Pobieranie, translokacja i rozm ieszczenie ołowiu w roślinie. In: A. Wo ź n y (ed.). Ołów w kom órkach roślinnych - pobieranie, reakcje, odporność. Sorus, Poznań, pp. 21-33.
So m a s h e k a r, R . K ., Ar e k a l, G . D. 1983. Chrom osom al aberrations induced by electroplating waste water. Cytologia 48: 621-625.
St e i n k e l l e r, H ., Mu n-Si k, K ., He l m a, C ., Ma, T . H ., Ho r a k, О ., Ku n d i, M ., Kn a s m u l l e r, S.
1998. Genotoxic effects o f heavy metals: com parative investigation with plant bioassays. Environ. Mol. Mutagen. 31: 183-191.
St o p p e r, H ., Mü l l e r, S . O. 1997. M icronuclei as a biological endpoint for genotoxicity: a m inireview. Toxicology In Vitro I I: 661-667.
Sz a f e r, W ., Ku l c z y ń s k i, S ., Pa w ł o w s k i, В. 1 9 8 6 . Rośliny Polskie. Opisy i klucze do oznaczania wszystkich gatunków roślin naczyniowych rosnących w Polsce bądź dziko, bądź też zdziczałych lub wcześniej hodowanych. Т. 1, 2. PWN, W arszawa, pp. 1-1019.
VILLALO BO S-PIET RIN I, R ., Go m e z-Ar r o y o, S ., Fl o r e s-Ma r q u e z, A. R„ Ci s n e r o s, A. 1 9 8 7 . cantia - M icronucleus test on potassium dichromate. Sem inar on som atic m utation in Trades-cantia as a m odel system in radiobiology and environm ental studies. Institute of Nuclear Physics, Kraków, pp. 1-68.
Wo j t y ł a- Ku c h t a B ., Ga b a r a, В . 1 9 9 1 . Changes in the content o f DNA and NYS-stained nuclear, nucleolar and cytoplasm ic proteins in cortex cells o f pea (Pisum sativum cv. De Grace) roots treated with cadm ium , Biochem. Physiol. Pflanzen 187: 67-76.
