ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 1 : 27 - 33 
Sprejeto (accepted): 2003-10-30 
Impact of air pollution on the mitotic activity in meristematic cells in shallot 
(Allium cepa L. var. ascalonicum) 
Vpliv onesnaženega zraka na mitotsko aktivnost v meristemskih celicah šalotke 
(Allium cepa L. var. ascalonicum) 
Erika GLASENČNIK1, Dieter GRil.V, Maria MULLER3, Cvetka RIBARIČ-LASNIK1 & Franc BATIČ2 
11 ERICo Velenje; Environmental Research & Industrial Co-operation Institute, Koroška 58, 3320 
Velenje, Slovenia, E-mail: Erika.Glasencnik@erico.si 
21 Biotechnical Faculty, Agronomy Department, Applied Botany, Ecology and Plant Physiology, 
Jamnikarjeva 101, 1000 Ljubljana, Slovenia. 
31 Institut fiir Pflanzenphysiologie, Universitat Graz, SchubertstraBe 51, A-8010 Graz, Austria. 
Abstract. Test shallot plants Allium cepa L. var. ascalonicum were exposed to 
field conditions in research plots in the most polluted areas in Slovenia in the 
vegetation season in 1999. The intention of this research was to evaluate the influence 
of air pollution on mitotic activity in meristematic tissues of root tips of bioindication 
plants. At each sampling site the mitotic activity was determined under field conditions 
and in a pot experiment. The maturated bulbs were collected from the field and after 
winter dormancies cytogenetic analyses were run on them in a lab. Root tips were 
fixed in Clark's fixative and afterwards stained with Schiff's reagent. 
Significant differences in the mitotic activity in different sampling plots in pot 
experiments were found. The results showed the presence of cytotoxic substances at 
chosen sampling sites which caused the decrease of mitotic cell division. 
Key words: Allium cepa L. var. ascalonicum, cytotoxicity, mitotic index, 
environmental pollution. 
Izvleček. Izbrane testne rastline šalotke (Allium cepa L. var. ascalonicum) so 
bile v času vegetacijske dobe izpostavljene naravnim razmeram na vzorčna mesta v 
bližini virov onesnaženja na ozemlju Slovenije. Namen raziskave je bil oceniti vpliv 
onesnaženega zraka na mitotsko aktivnost v meristemskih celicah koreninskih 
vršičkov bioindikatorskih rastlin. Vpliv onesnaženega okolja smo analizirali pri 
rastlinah izpostavljenih v lončnem in poljskem poskusu. 
Posušene in očiščene čebulice so bile po zimski dormanci izpostavljene 
v laboratoriju, koreninski vršički so bili fiksirani v Clarkoven fiksativu in obarvani s 
Schiffovim reagentom. 
Ugotovljeno je bilo, da se pojavljajo statistično značilne razlike med posameznimi 
lokacijami pri rastlinah izpostavljenih v lončnem poskusu. Rezultati kažejo, da so 
na izbranih lokacijah citotoksične substance, ki vplivajo na mitotsko aktivnost celic 
izpostavljenih rastlin. 
28 
Introduction 
Acta Biologica Slovenica, 46 (1), 2003 
Ključne besede: Allium cepa L. var. ascalonicum, citotoksičnost, mitotski indeks, 
onesnaženost okolja. 
Besides the analysis of chromosomal aberrations to evaluate genotoxicity a mitotic index is 
most frequently used as a parameter to evaluate cytotoxicity to show effect of chosen substances in 
cytogenetic bioindication. Mitotic activity decreases while the influence of environmental stress is 
stronger (FlSKESJ0 1994, PARADIŽ 1996, 1998, PARADIŽ & al. 1996, SMAKA-K.!NCL & al. 1996, BAVCON 
& al. 1999, FREISZMUTH & al. 2000, Mi.iLLER & al. 2000, PAVLICA & al. 2000, PARADIŽ & DRUŠKOVIČ, 
2001). In the Alliaceae family shallot is not commonly used for bioindication (PAVLICA & al. 1997, 
2000, ZOLDOŠ & al. 1997). 
Test shallot plants (Allium cepa L. var. ascalonicum) were exposed to field conditions at research 
plots of differently polluted areas of Slovenia in 1999: in the Šalek Valley the most polluted sites are 
either those which are very close to the thermal power plant (Veliki Vrh, ash dump of the Šoštanj 
Thermal Power Plant) or those which are highly polluted due to climatic conditions and topography 
but more distant from the thermal power plant (Zavodnje) or sites with relatively clean air in relation 
to the wind direction from the thermal power plant (Arnače, Škale); in the Zasavje region (Kovk) 
and in the Upper Meža Valley (Žerjav) two very polluted sites were taken for one vegetation period. 
At each sampling plot plants were growing under field conditions and pot experiment (see Material 
and methods for a short description of growing conditions). Mitotic activity was later determined in 
a laboratory. 
In this study we attempted to determine the influence of air pollutants on plants (pot) and indirect, 
accumulated effects in soil exposed to environmental pollution on the cytogenetic material of the 
shallot. The aims of the study were to make a comparison among the sampling plots regarding the 
mitotic activity to define cytotoxic effect arising from environmental pollution and to confirm the 
applicability of chosen method for relatively fast, simple and cost-effective determination of 
cytotoxicity of polluted air. In comparison with the pots experiments (where plants were exposed 
primarily to air pollution) we expected decrease of mitotic activity in plants in field conditions, 
since these shallots were exposed to stress due to air and soil pollution. 
Materials and methods 
For every sampling site we used approximately equal-sized shallot bulbs (Allium cepa L. var 
ascalonicum Escalote de Jersey' , 2n=l6) . 
The choice of sampling sites and sampling procedure was carried out as described by GLAsENČNIK 
& al. (2002) and the method for cytogenetic analyses was performed as described by FrsKEsJo (1994) 
and detailed by GLASENČNIK & al. (2002). 
In the pot experiment plants were exposed in 15-litre self-watering pots (five per site, three 
bulbs per pot) and for experiment under field conditions plants were put clirectly in field soil (six 
bulbs per site) near the pots in the end of May 1999. After maturation all plant material was removed 
from sampling plots and bulbs were stored under dry conditions (4°C) for two months. 
The soil from pots and field soil were analysed. The analysis of soils was carried out after 
homogenisation (ISO 11464) and digestionation (ISO 11466) using the methods for spectroscopy 
(ICP-MS, ET-AAS, CV-AAS). 
Cytogenetic analyses were run after winter dormancies. Root tips were treated with using Feulgen 
"squash" technic. A mitotic activity was determined in five bulbs per sampling site, one slide per 
bulb and 2000 cells per slide. A mitotic index (MI) is a percentage of dividing cells scored. 
E. Glasenčnik, D. Grili, M. Milller, C. Ribarič-Lasnik, F. Batič: Irnpact of air pollution on the ... 29 
All statistical treatments were performed using the Statistica for Windows 5.5 software package 
(STATS0Ff 1999). Differences among sampling sites were tested using Kruskal-Wallis ANOV A. Cross 
comparisons between means were performed using the Mann-Whitney U test. 
Results 
Significant differences were found in MI among the sampling sites for shallots grown in pots 
(Kruskal-Wallis ANOVA: H <6, 47l = 19,49, p = 0,003; Fig. lb), while for plants grown in field soils no 
significant differences could be found (H <5•41> = 7,68, p = 0,17; Fig. la) . Detailed statistics of pair-
wise comparisons using Mann-Whitney U test are presented in Table 1. 
The analysed plants material in pot experiment showed that the MI was between 3 and 14 %. 
The lowest value of MI was determined in Arnače near Velenje (3,7±2,5 %), a very low value was 
also at Veliki Vrh and at Kovk (6,1±2,6 % and 6,1±1,6 %), the highest was at Žerjav (10,93±3,5 %). 
The results of shallot grown under field conditions showed that the value of MI was between 6 
and 10 %, the lowest was determined at Veliki Vrh (6,4±3,7 %), the highest at the sampling site of 
Žerjav (10,93±3,5 %). 
A:Soll 
22 
20 
18 
16 
14 
~ 12 
~ @~~~® :ii 10 
4 
Arnače Ash dump Velik Vrh Zerjav • Mean 
Kovk Vnajnarje Zavodnje Skale D±SE 
Sampllng site 
I±1,96'SE 
B: Pots 
22 
20 
~ 
18 
16 
14 
-- ~ ~ 12 :ii 10 1® 
,@~ ~ 
~ 
Arnače A5h dump Velik Vrh Zerjav c Mean 
Kovk Vnajnarje Zavodnje Skale D ±SE 
Sampllng site 
::C ±1 .96'SE 
Figure la, b: Mitotic activity (%) in root tip cells of shallot, exposed to field conditions in soils (A) 
and in pots (B) at eight different localities in Slovenia in the vegetation season in 1999. 
30 Acta Biologica Slovenica, 46 (1), 2003 
Table 1: Significance of differences in mitotic activity between sampling sites (Mann-Whitney 
U-test). 
Soil 
1999 ARNAČE KOVK ASH ŠKALE VNAJNARJE VELIKI ZAVODNJE ŽERJAV 
MI DUMP VRH 
ARNACE / / / / / / / 
KOVK * / NS NS NS NS NS 
ASHDUMP NS NS / / I I I 
SKALE ** ** NS NS * NS NS 
VNAJNARJE * * NS NS NS / NS 
VELIKI VRH NS NS NS * * NS NS 
ZAVODNJE I / / / I I NS 
ZERJAV * I NS NS NS NS * 
***: p<0.001; **: p<0.01 , *: p<0.05; NS: not significant. 
Discussion 
The inhibition of mitotic activity was often used for tracing cytotoxic substances. Cytotoxicity 
was defined as a decrease in the mitotic index and as an increase in the frequencies of chromosomal 
aberrations (FrsKESJb 1994). The analysis of chromosomal aberrations for evaluation of genotoxicity 
was already done (for results see Gt.ASENČNIK & al. 2002). In our study a decrease in the mitotic 
index of sballot root meristems was found depending on the chosen location in pot experiment with 
probability p<0,01. We used the same soil for all pots and therefore plants grown in pots were 
exposed only to air pollutants. Analysis of chromosomal aberrations showed no significant differences, 
but the frequency of chromosomal aberrations was at least two-fold higher in comparison to published 
<lata for a control environment (3%, e.g. VIDAKOVIČ & al. 1993). Therefore air pollution had a strong 
influence on mitotic activity and also on the frequency of chromosomal aberrations. These results 
are in accordance with the results of fumigation experiments of a research group in Graz: ozone 
treatments consistently increased the rate of chromosomal aberrations, the Ml remained unchanged, 
with fumigation with SO
2 
and ~S the MI was lower and the chromosomal aberrations were also 
consistently increasing (MDLLER & al. 2000). 
Arnače sampling plot showed, against expectation, a very small mitotic activity, though the 
finding was in accordance with the recent results from other research fields (SVETINA 1999, KuooNIČ 
& STROPNIK 2001 ), which revealed elevated levels of Co, Cr and Ni in meadow soil and higber Jevels 
of heavy metals in vegetables and forage were found in comparison with other sampling sites in the 
bottom of the Šalek Valley. The highest frequency of chromosomal aberrations (for details see 
Gt.ASENČNIK & al. 2002) in our pot experiment confirmed the presence of stress inducing factor at 
this sampling site. 
Since the cleaning device was built on the Šoštanj Power Plant in 1995, the emissions have been 
drastically reduced, which has reflected in an improvement of ecosystem conditions (RIBARIČ-LASNIK 
& al. 1999b). This is nota case at some sites, which are stili polluted due to their position or due to 
accumulation of pollutants in the past. At the sampling site of Veliki Vrh just facing power plant 
chimneys and the prevailing wind direction a high concentration of air pollutants is directly brought 
from chimneys to this highly exposed site. This was confirmed by severa! studies (e.g. high levels of 
Cd in soil and vegetables KuooNIČ & STROPNIK 2001, high levels of Cd, As and Hg in fungi AL 
SAYEGH-PETKOVŠEK & al. 2002, the most acid precipitation (pH<5,6) SEVŠEK & al. 2000). The low 
value of MI deterrnined and the highest frequences of chromosomal aberrations (see Gt.ASENČNIK & 
al. 2002) at that sampling site were in agrement with the above mentioned studies. 
E. Glasenčnik, D. Grili, M. Miiller, C. Ribarič-Lasnik, F. Batič: Impact of air pollution on the ... 31 
Considering the extreme pollution with heavy metals in the Upper Meža Valley (see RIBARIČ­
LASNIK & al. 1999a and references therein) the high value of MI in both experimented plots in 
žerjav were surprising because we expected a Iow value of MI at this site due to extreme value of 
heavy metals in the surroundings (e.g. 4,99±6,92 mgkg·1Cd, 1496±1071,02 mgkg·1 Pb, 498,22±546,0 
mgkg·' Zn KuooNIČ & STROPNIK 2001). More investigations are needed at this research plot to verify 
the results. · 
Contrary to our expectation no significant differences in the mitotic activity could be found 
among Iocalities in the experiment under field conditions. We found significant differences in the 
frequency of chromosomal aberrations in different sampling sites conceming shallot grown in the 
field (see GLASENČNIK & al. 2002). The results are in accordance with the statement of FlsKESJ• 
(1997) saying that when chromosomal aberrations occur, there are almost always some growth 
restrictions. These results indicated that the influenece of chronic exposure (soil) is far more genotoxic 
while the acute exposure (pots) is more cytotoxic. Results obtained with our investigation were 
confirmed also with the results of an assessment of cytogenetic hazard for plants caused by highway 
traffic: the mitotic activity decrease and the frequency of chromosomal aberrations increase with 
the duration oftraffic influence in the onion (Allium cepa L.) exposed in short-lasting in situ exposure 
near highways in Slovenia (PARADIŽ & DRUŠKOVIČ 2001 ). In spruce and other perennial plants growing 
spontaneously on experimental sites near the highways as bioindicators of long-lasting exposure, 
cytogenetic damages increased in correlation with the duration of traffic, but only at sites with more 
than 20 years' traffic influence (ibid). 
Nevertheless, we also found out that the values of MI in the field conditions and in the pot 
experiment were lower than the average values from the literature for the control (10-15 %; e. g. 
Oud & RICKARDS 1999; 5-10%; e. g. PARADIŽ 1996) and these results showed a decrease in mitotic 
activity and a cytotoxic effect of chosen sites. 
Considering the present results we can conclude that the polluted air does have a cytotoxic 
effect on mitotic activity with the decrease of MI and that more polluted sites have a higher effect on 
mitotic activity. We can also say that the polluted air and soil together probably had a stronger effect 
at the majority of investigated plots. Therefore we conclude that the mitotic activity is the appropriate 
testing system for determination of MI in shallot root tip meristematic cells for quick, easy and 
useful estimation of cytotoxic effect of environmental pollution. 
Acknowledgements 
This work was financially supported by the Šoštanj Therrnal Power Plant and by the Ministry of 
Education, Science and Sport of the Republic of Slovenia. We owe a deep debt of gratitude to ali 
colleagues who helped us with many very useful comments and suggestions or assisted us at field 
work: Karin Savinek, Melita Stropnik, Meta Zaluberšek, Leopolda Pačnik, Andreja Bienelli, Stane 
Vanovšek, Nataša Kopušar, Boštjan Pokomy for statistical advice (ERICo Velenje), Gabrijel Leskovec 
and Tomaž Sinkovič (Biotechnical Faculty, Department for Applicative Botany). Colleagues from 
the Institute for Plant Physiology, Graz, introduced us to the Allium test. Finally, we are grateful to 
Marta Pačnik, who helped us linguistically, and to my family for support. 
32 Acta Biologica Slovenica, 46 (1), 2003 
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