Caryologia

International Journal of Cytology, Cytosystematics and Cytogenetics

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Chromosome damage studies in the onion plant

Allium cepa L.

Peter Firbas & Tomaž Amon

To cite this article: Peter Firbas & Tomaž Amon (2014) Chromosome damage studies in the onion
plant Allium�cepa L., Caryologia, 67:1, 25-35, DOI: 10.1080/00087114.2014.891696

To link to this article: https://doi.org/10.1080/00087114.2014.891696

Published online: 24 Mar 2014.

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Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics, 2014
Vol. 67, No. 1, 25–35, http://dx.doi.org/10.1080/00087114.2014.891696

Chromosome damage studies in the onion plant Allium cepa L.

Peter Firbasa* and Tomaž Amonb
a
Private Laboratory for Plant Cytogenetics, Ljubljanska c.74. SI – 1230 Domžale, Slovenia; bCenter for Scientific Visualization,
Gorazdova ul. 3, SI – 1000 Ljubljana, Slovenia

The onion plant (Allium cepa L.) is a suitable indicator plant for the determination of potential genotoxic agents in the
samples taken from the environment. The genotoxic level of the agent under study is reflected by structural changes of
the chromosomes and their changed numbers. The chromosomes under study are taken from the meristem cells of the
young growing onion roots. A healthy normal onion cell has 16 (2n = 16) chromosomes. They are relatively large and
so very appropriate for the detection of morphological changes. Prior to the chromosome study the root tip cells were
immersed in a 0.1% aquatic solution of colchicine which stopped the mitotic cycle continuing beyond metaphase. The
changes in morphology varied from a single distortion of a single chromosome up to several morphological changes
observed on many chromosomes. We identified 15 categories of morphological aberrations which are classified into three
groups: chromatid damage (CtD), centromere damage (CmD) and chromosome damage (CsD). CtD includes: single
break chromatid, double break chromatid, isochromatid break, multiple break chromatid, gap chromatid, centric ring
chromatid, acentric ring chromatid and triradial chromosomes. CmD includes: break centromere, gap centromere, single
break centromere, double break centromere and multiple break centromere. CsD includes: ring chromosomes and dicen-
tric chromosomes. Sometimes also the chromosome number changed which occurred as aneuploidy with monosomy 2n
= 15 (2n =16 – 1) and euploidy – increased number of the basic chromosome number (2n = 6x – 8x). We identified also
the translocation: t(3p–; 5p+).
Keywords: Allium cepa L.; karyotype; chromosome damage; chromatid damage

Introduction Monitoring chromosome morphology abnormalities

The use of chemicals that accumulate in the environ-
ment is increasing in Europe. These pollutants can enter
spring and underground water used as drinking water.
Although chemical analysis provides accurate measure-
ments at the moment of sampling, as the repetition of
sampling is often limited, we do not obtain a realistic
impact on organisms over a longer period of time.
Onion (Allium cepa L.) root meristem cells are very
sensitive to genetic damage by chemicals, and the
Allium test, involving the length of the root and chro-
mosome aberration (CA) measurements, proved to be a
suitable model system for measuring the environmental
cytogenetic potential of pollutants (Al-Sabti 1989). The
Allium test is very good indicator for analysing antipro-
liferative effects on plant medicinal extracts (Frescula
et al. 2012).
Seven higher plant bioassays were reviewed by the
US Environmental Protection Agency (EPA) Gene-Tox
program in 1980. The Allium root tip CA assays were
adopted by the International Program on Plant Bioassays
(IPPB) for monitoring or testing environmental pollutants
(Ma 1999; Ma et al. 2005). The metaphasic chromosome
anomalies as detected in the Allium test procedure are
not excluded to occur also in the human chromosomes
when exposed to similar pollutants. There are many
reports on the excellent correlation of the plant system
with the mammalian system (Grant and Salamone 1994).
in order to detect the degree of water toxicity is an old
technique. In 1928 Stadler reported the effect of
chemicals and physical agents on chromosome damage
(Mulaszynska and Juchimiuk 2005). After the effect of
colchicine on the mitosis of Allium had been discovered
(Levan 1938) it was possible to determine the number of
chromosomes and their morphology in A. cepa L. (Levan
et al. 1964). The Allium test (Levan 1938, 1949) is based
on the chromosome study of the meristem cells of the
apical root cells of A. cepa L. in order to determine the
influences of genotoxic substances in, for instance,
benzene, naphalen, phenanthren and benzopyren. More-
over, the A. cepa system provides important information
to evaluate the clastogenic and/or aneugenic effects of an
agent on the genetic material (Leme and Marin-Morales
2009).
C-mitosis (without colchicine application) is stimu-
lated by chemicals such as methyl mercury and nickel
salts, and by many organic chemicals (Fiskesjö 1993).
After such treatment a more detailed study of chromo-
some morphology is possible (Fiskesjö 1993). The
diluted cytostatic chemical colchicine is used to stop the
cell division process exactly in its metaphase. Thus in
c-metaphase chromosome or chromatid break can be
studied in detail (Fiskesjö 1993). So the colchicine
pre-treatment enables the study of structural metaphase
CAs. Metaphase chromosomes are chromosomes with

*Corresponding author. Email: peter.firbas@gmail.com

© 2014 Dipartimento di Biologia Evoluzionistica, Università di Firenze

26 P. Firbas and T. Amon
the most condensed chromatin and can be therefore
studied with an optical microscope. Allium cepa L. is an
important bioindicator for environment poisons which
influence mitosis (Fiskesjö 1985; Rank 2003; Bakare
et al. 2012) and damage metaphase chromosomes
(Al-Sabti and Kurelec 1985; Al-Sabti 1989; Kumar and
Panneerselvam 2007; Ragunathan and Panneerselvam
2007; Panneerselvam et al. 2012).
The centromere is observed as a constriction in the
chromosome and stains lighter than other parts of the
chromosome. Its location on the chromosome is most
important in determining the morphology of chromo-
somes, which are characterized as metacentric (m), sub-
metacentric (sm), subtelocentric (st) or telocentric (t).
The location of the centromere is described by the cen-
tromeric index using the rules of Levan et al. (1964) and
the Chicago Conference (1966).
The complete sequence of stages from one cell divi-
sion to the next is: G0, G1, S, G2 and M phase. The
administration of genotoxic agents during the G0 and G1
phases of the cell cycle results in chromosome or
chromatide damage prior to the mitosis. This induces the
formation of the dicentric or ring chromosome;
meanwhile administration in the S or G2 phase usually
provokes the breakage of one or both chromatids.
Onion (Allium cepa L.) cells have eight pairs of rel-
atively large chromosomes. This allows an easy detec-
tion of possible chromosome damage. In addition the
chromosome morphology is very easily changed by
chemicals. The Allium metaphase test, involving mea-
surements of the length of the root and chromosome
damage, proved to be a suitable model system for
measuring the environmental cytogenotoxic potential of
pollutants (Al-Sabti and Kurelec 1985). Therefore
chromosome damage has become a relevant testing
method. This biotest even introduces a new quality in
the assessment study: chromosomes constitute the pri-
mary target for cytogenotoxically induced irreversibly
damaged chromosome, damage or disturbance of cell
division.
Onion is suitable for genotoxic studies because: (i)
the root growth dynamics is very sensitive to pollutants;
(ii) the mitotic phases are very clear in the onion; (iii) it
has a stable chromosome number and stable karyotype;
(iv) there is diversity in the chromosome morphology;
(v) there is a clear and fast response to the genotoxic
substances; and (vi) spontaneous chromosomal damages
occur rarely. In this study we describe various chromo-
some and chromatid damages in the root meristem cells
of onion, which represent biomarkers for different types
of environmental pollution.
Material and methods
Onion preparation
Small onion (Allium cepa L.) bulbs of the same uniform
size, weighing about 3–3.5 g, were denuded by
removing the loose outer scales and scraped so that the
root primordia were immersed into the different tested
liquids (drinking quality water, river and lake water,
industrial wastewater, rain water, snow melt water, river
and lake sediments, soil, condensed water obtained from
the atmosphere, and known chemicals).
Experimental procedure
The exposure time of the onion bulbs in each experiment
was 72 hours at 22°C; they were protected against direct
sunlight. In order to eliminate the influence of daylight
rhythms the plants were exposed to contain artificial light
of middle intensity. In an alternative version of our
Allium metaphase test, the five onion bulbs were placed
directly in the experimental water containers. The water
sample under investigation was divided into three por-
tions which were successively applied to the onion roots
for 24-h periods. So each 24 h the roots obtained a fresh
bath of the sample solution. After 72 hours the experi-
ment was terminated and followed by macroscopic and
microscopic tissue morphology investigation.
Chromosome preparations
The squash technique for onion root described by
Al-Sabti and Kurelec (1985) and Al-Sabti (1989) was
used for the preparation of chromosomes. Chromosome
preparations were set up from root meristems containing
actively growing cells by the following method: develop-
ing roots with bulbs were pre-treated with an 0.1% aqua-
tic solution of colchicine for 3 hours at 21°C. After
washing in distilled water for 20 min the terminal devel-
oping roots (2 mm) were fixed for 1 h in methanol:pro-
pionic acid mixture (3:1 or 1:1). Then they were
macerated and stained in order to obtain a cellular sus-
pension. This sample was stained with 0.5% aceto-car-
mine for 4–5 min at 60°C without hydrolysis, and
squashed in aceto-carmine (Firbas and Al-Sabti 1995).
The optical microscope used in the investigation was the
Olympus–BX 41 (Olympus, Japan) with the PM 10 SP
photo system. Typical magnifications used were 400×
and 1000×.
Macroscopic parameters
Macroscopic parameters include the root length and
other parameters such as usual root form, number, colour
and turgidity after cultivation for 72 h.
Microscopic parameters
Therefore, additional colchicine treatment should only be
used for the specific study of chromosome and chromatid
(breaks) damage. and the study of of the chromosome
shapes in their methaphasic state.
 Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics
Level of genotoxicity
The level of genotoxicity is defined by the percentage of
metaphase cells with damaged cells with damaged
chromosomes related to all the cells inspected (typically
200).
Chromosome morphology, centromere position and
karyotype
Chromosomes are categorized according to the position
of the centromere. Arm ratio can be calculated by
measuring the length of the short arm (p) and of the long
arm (q) of the chromosome, and the centromeric index is
as described by Levan et al. (1964) and Chicago
Conference (1966). For numerical characterization of the
karyotypes the parameters were calculated as in Genc
et al. (2013).
Control inhibition test
Toxicity and genotoxicity assays were performed with
six concentrations of the test methyl methanesulphonate
(MMS 4016, Sigma, USA) chemicals as control.
Results
Distribution genotoxicity (induction of chromosome
damage in root cells) and general toxicity (root growth
inhibition and malformation) in Allium cepa L. exposed
to 100–0.001 ppm MMS are shown in Table 1 and
Figure 1A–F. Results for 10 mg l−1 MMS varied from
18.4% to 28.6% aberrant cells (Rank 2003). The normal
metaphases and different types of chromosomal deviation
are shown in Figures 2A, B, 3, 4A–U, 5, 6A, B, 7,
8A–D and Tables 2–4. Samples were taken in rivers,
springs, tarns, lowland lakes and tap water in Slovenia.
1. Chromosome number and karyotype
Allium cepa L. has 16 monocentric chromosomes
(2n =16) with basic number x = 8 (Figure 2A). Its
karyotype contains six metacentric (m), eight submeta-
centric (sm) and two subtelocentric (st) chromosomes
(Figure 2B). Two subtelocentric chromosomes contain
satellites (Figure 3), which are not always seen under a
microscope. Telocentric chromosomes are not present.
2. Types of metaphase damage chromosome
The aberrations seen at metaphase are of three types:
chromatid damage, chromosome damage, and chromatid
type damage in the centromere region.
Table 1. Growth retardation toxicity and genotoxicity effects of MMS after 72 h of treatment at different concentrations.
Concentration of MMS 4016 Sigma (ppm) 100 10
Length of root testing plant (mm) 0–2 8–13
Level of genotoxicity (%) 0 25.5–31.0
27
2.1. Chromatid damage
Single break chromatid (SBCt). SBCt can appear
anywhere on the chromatid. The broken fragment can
remain aligned with the chromatid (Figure 4A) or get
displaced from it (Figure 4).
Double break chromatid (DBCt). DBCt involves two
breaks, with the elimination of the chromosomal material
between them (Figure 4C).
Isochromatid break (IctB). IctB is the parallel breakage
of the chromatid which is composed of two breaks in
the same chromatid region. The broken fragments can
also get displaced from the chromatid axis (Figure 4D).
Multiple break chromatid (MBCt). In MBCt the broken
fragments typically accumulate in a small group
(Figure 4E).
Gap chromatid (GCt). GCt is the result of the chromatid
breakage and appears under the microscope as an
unstained point in the chromatid. There can also appear
several such unstained points. If gaps exist in both chro-
matids they are called isochromatid gaps (Figure 4K, 4P,
4T) and multiple gaps (Figure 8D, arrows).
Centric ring chromatid (CRCt). CRCt is formed when
the ends of the broken chromatid join and chromatid
becomes circular in shape, containing the centromere
(Figure 4F).
Acentric ring chromatid (ARCt). ARCt appears when the
displaced fragment of the chromatid becomes circular. It
contains no centromere (Figure 4G).
Triradial chromosomes (TCs). TCs are chromosomes
with one centromere and three terminal chromatid ends
(Figure 4H).
2.2. Chromosome damage
Dicentric chromosomes (DCs). DCs appear with the
simultaneous breakage of two chromosomes. These
chromosomes join into the dicentric chromosome which
contains two centromeres and an acentric fragment
(Figure 4I).
Ring chromosomes (RCs). RCs appear after the double
breakage of the long and short chromosome part. The
broken ends fuse, thus forming a cyclic chromosome and
an acentric fragment (Figure 4J).
1 0.1 0.01 0.001 Tap water reverse osmosis
22–25 28–32 38–40 40–42 40–44
19.5–23.5 8.5–9.5 5.5–6.5 3.0 2.0–2.5
28 P. Firbas and T. Amon
Figure 1. Examples of series of onions cultivated for 72 h in different concentrations of MMS: (A) 100 ppm; (B) 0.001 ppm;
(C) 10 ppm; (D) 0.01 ppm; (E) 1 ppm; (F) 0.1 ppm.
2.3. Centromere damage
Damages to the centromere belong in the chromatid
damage class and are located near the centromere region
of the chromosome. Here are some typical damage
forms:
Break centromere (BCm). BCm in the centromere region
appears when the chromosome breaks into two parts. As
this type of damage occurs often it can be inferred that
the centromere part of the chromosome is the most sensi-
tive to genotoxic substances. Such deformations can lead
to the chromosome form designated the isochromosome
(Figure 4K).
Gap centromere (GCm). GCm represents a narrow chro-
matid breakage in the basal part of the chromatid. It
appears as an unstained part of the chromosome (Fig-
ure 4L).
Single break centromere (SBCm). SBCm is breakage of
the basal part of the chromatid, by the displacement of
the broken longer or shorter chromatid (Figure 4M).
Double break centromere (DBCm). DBCm is the break-
age and displacement of the basal parts of two chromat-
ids (Figure 4N).
Multiple break centromere (MBCm). MBCm arises when
the chromosome breaks in the centromere region and
then breaks into four chromatid fragments. MBCm
occurs very rarely (Figure 4O).
3. Aberrations resulting in changes in the
chromosome number
Polyploidy refers to a numerical change in a whole set
of chromosomes. Organisms in which a particular
chromosome is underrepresented (monosomy) or over-
represented are said to be aneuploidy. Monosomy in
A. cepa is to represent without subtelocentric chromo-
some (2n = 15; 2n = 16 – 1) in the 6th pair (Figure 5).
Euploidy (Polyploidy) A. cepa is to represent with six
sets (6x) and eight sets (8x) to multiply (to increase)
basic chromosome number. Hexaploid (6x) and Octaploid
(8x) chromosomes are examples of euploidy (Figure 6).
 Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics
Figure 2. (A) Diploid metaphase chromosome; and (B) its karyotype from the root cells of onion (Allium cepa L.) containing
2n = 16 with 6 m, 8 sm and 2 st chromosomes (Figure 2b).
4. Chromosome reorganization/reconstruction
In a translocation, a segment from one chromosome is
transferred to a nonhomologous chromosome or to a
new site an the some chromosome. Translocation in
onion A. cepa involves 3th and 5th chromosome. In a
translocation, a segment arm (3p) is transferred to a 5th
chromosome (Figure 7).
Translocation in onion A. cepa is to transfer the
chromosome arm (3p) from 3th chromosome on the 5th
chromosome. Rearrangement novel karyotype is:
2n = 16; t: 3p–; 5p+ (Figure 7).
5. The combined damage types on a particular
chromosome
There are different types of chromatid damage in the
centromere region and in chromosome chromatids. For
example: BCm+GCt (Figure 4K), BCm+GCt (Figure 4P),
BCm+SBCt+GCt (Figure 4R), BCm+ARCt+GCt
(Figure 4S), BCm+DBCt+GCt (Figure 4T), and BCm+
GCt+MBCt (Figure 4U).
29
The number of damaged chromosomes increases with
the level of the genotoxicity of the substance studied
(Table 2, Figure 8A–D). The same holds for the number
of deformations seen on a particular chromosome (Tables
3, 4).
Discussion
The chromosome damage technique has become more
and more important with increasing interest in the
genotoxic effect of pollutants, and following recommen-
dations to use onion chromosome as an indicator of mu-
tagens in the environmental samples. CAs appear in two
forms – the deviations of the metaphase chromosome
number (numerical aberration) and the morphological
deformations (structural aberration) of the chromosomes
themselves.
We have shown several different types of chromosome
and chromatid damage, resulting in numerous changes of
chromosome morphology. We determined 15 types of
structural chromosome damage, one translocation and two
30 P. Firbas and T. Amon
Figure 3. Subtelocentric chromosomes with satellites (arrows);
some chromosomes are damaged.
types of changed chromosome numbers. In the literature
the Allium anaphase–telophase test has been most often
performed without the application of the colchicine (Rank
2003). Genotoxic substances in the cells induce the aber-
ration called c-mitosis, which is the result of damaged
mitotic apparatus, and leads to metaphase chromosomes
being freely distributed in the cell.
Meristem cells of Allium roots treated with the cyto-
static colchicine show a much clearer picture of the
metaphase chromosomes. The observations led us to the
conclusion that there is a positive correlation between
the level of genotoxicity of the substrate and the number
of CAs – including one or several damages on a single
chromosome up to damages on multiple chromosomes
and even changed chromosome number. Our results also
show that substrates with high genotoxic level induce a
greater number of damaged chromosomes as well as
more identical or different damages on the same chromo-
some. Low genotoxic levels induce less-damaged chro-
mosomes as well as fewer damaged sites on a particular
chromosome.
It has proved very convenient to apply colchicine
which arrest dividing cells at metaphase (Al-Sabti and
Kurelec 1985; Al-Sabti 1989; Kumar and Panneerselvam
2007; Ragunathan and Panneerselvam 2007; Palanikumar
et al. 2011; Panneerselvam et al. 2012).
Rainfall and snowfall and petrochemical wastewater
predominantly induced breaks, fragments, ring chromo-
somes and dicentric chromosomes (Al-Sabti 1989).
Al-Sabti and Kurelec (1985) took environmental samples
from the river Sava and made a two phase analysis. In
the first phase he was sampling just the water from the
river. In the second phase he added to the river samples
also the benzene and chloride. Both phases showed
breaks, fragments, ring chromosomes and dicentric
chromosomes, but these anomalies were more
pronounced in the second phase. CA measurement in the
Allium metaphase test is suitable for measuring the
cytogenotoxic potential of chemicals present in waters.
Cytogenetic effects of the food preservative potassium
metabisulphite in Allium cepa root meristem cells
induced CAs: breaks, gaps, multiple breaks and chroma-
tid breaks (Kumar and Panneerselvam 2007). Sodium
azide is a well-known potent mutagen, although it fails
to induce CAs in root cell type such as break, gap,
iso-chromatid break and exchange (Ragunathan and
Panneerselvam 2007). CAs increase with increasing con-
centration of the test chemical and a longer period of
treatment. Curcumin induces CA in A. cepa root tip cells
in an insignificant manner when compared with an
untreated control. Sodium azide alone induces CAs sig-
nificantly with increasing concentrations. The total num-
ber of aberrations was significantly reduced in A. cepa
L. root tip cells pretreated with curcumin. Curcumin has
antimutagenic potential against sodium azide induced
CAs in Allium cepa root meristem cells (Ragunathan and
Panneerselvam 2007). Glycidol is used as a stabilizer in
the manufacture of vinyl polymer-induced CAs in
A. cepa L. root meristem cells. Different concentrations
of glycidol induced cytological abnormalities such as
breaks, gaps, exchange, multiple breaks and chromosome
fragments (Panneerselvam et al. 2012). Increasing
concentrations increased the number of CA.
To date, a variety of plant compounds have been
studied extensively with regard to antimutagenic, anticar-
cinogenic, and antigenotoxic activity. Nevertheless, very
few of them have been studied in terms of clastogenicity.
The clastogenic activity of curcumin (the active compo-
nent of turmeric) and aloin (the active component of
aloe) had significant dose- and time-dependent clasto-
genic effects on the test plant system (Allium cepa L.).
Cytological abnormalities, such as breaks, gaps,
exchange, multiple breaks, and chromosome fragments,
were scored. The frequency of CA increased as the con-
centration increased (Palanikumar et al. 2011).
Various types of structural chromosomal abnormali-
ties can be cytologically divided into chromosome type
and chromatid type. Chromosomal type damage are pro-
duced when breakage and reunion involves the whole
 Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics 31

Figure 4. Chromatid and chromosome damage: (A, B) SBCt; (C) DBCt; (D) IctB; (E) MBCt; (F) CRCt; (G) ARCt; (H) TCs; (I)
DCs; (J) RCs; (K) BCm; (L) GCe; (M) SBCe; (N) DBCe; (O) MBCe; (P) BCm+GCs; (R) BCm+SBCt+GCt; (S) BCm+ARCs+GCt;
(T) BCm+IctB+BCt; (U) BCm+GCt+MBCt.

Figure 5. Monosomy (2n – 1) in the chromosome set of the onion (Allium cepa L.). Subtelocentric chromosome (arrow) is without
homologue couple in chromosome set.

Table 2. Correlation between the genotoxicity level and the number of damaged chromosomes in chromosome set. Samples: envir-
onmental complex mixture as wastewater; river, spring, tarn and lowland lakes, to stain out to deposit positive, and drinking water in
Slovenia; negative control = reverse osmosis tap water filtration; control= 10 ppm : methyl methanesulphonate (MMS).

Level of No. of chromosomes damaged in

Environmental complex mixture genotoxicity (%) metaphase cell
Negative control (reverse osmosis – tap water) 1 ppb MMS 2 1
River, spring, tarn and lake water, 0.1 ppm MMS 3–7 1–4
Lowland lake and river water, effluent wastewater, 1 ppm MMS 5–25 2–7
Effluent urban/communal system wastewater, 10 ppm MMS 25–35 3–10
Effluent urban/communal/industrial and agricultural wastewater, to 30–50 7–16
stain out to deposit

chromosome. These lesions are produced in interphase
(G1 and G0) before the DNA and chromosome material
has been duplicated during the DNA synthesis period.
This will lead to chromosome damage visible at the
next mitosis. Chromatid type damage is produced by
genotoxic chemical agents when cells are exposed in the
S-phase or G2 states of interphase.
Very specific chromatid damage appears in the cen-
tromere region of the chromosome and on its chromatids.
The centromere is a part of the chromosome. The most
32 P. Firbas and T. Amon

Figure 6. Euploidy in the chromosome set of the onion (Allium cepa L.); Basic chromosome number (x=8) is increased from 6
times (six sets, 6x – hexaploid, left image) to 8 times (eight sets, 8x – octaploid, right image). This was likely the cause of
environmental xenobiotic chemicals.

Figure 7. Translocation (t: 3p –; 5p+) in the chromosome set of the onion (Allium cepa L).

common damages are near to the centromere region or Chromatid damages are therefore classified according to
in the centromere region itself. This implies that the cen- their location: damage in the centromere region and
tromere region is most sensitive to genotoxic agents. damage on the other chromosome parts. The scope of
 Caryologia: International Journal of Cytology, Cytosystematics and Cytogenetics 33

Figure 8. Different number chromosome damage in metaphase cells obtained from the meristem root-type cells of onion (Allium
cepa L.): (A) one damaged chromosome; (B) four damaged chromosomes; (C) eight damaged chromosomes; (D) whole chromosome
set is damaged.

Table 3. Different subtypes of damage on a particular chro- Table 4. The most frequent appearances of identical sub-types
mosome. of chromosome damages on a single chromosome.

Number of damages on a single Chromosome damage Chromosome damage Number of damages on a single
chromosome type type chromosome
1 BCm BCm 1
1 DBCt GCt 1–4
1 MBCm SBCt 2
2 IctB, RCs DBCt 2
2 RCs, SBCt MBCt 2–4
2 BCm, RCs CRCt 1
2 CRCt, ARCt ARCt 1
3 BCm, CRCt, ARCt RCs 1
4 BCm, IctC, GCt, MBCt DCs 1
5 BCm, GCt, RCs, IctB, IctB 2
MBCt MBCm 1
34 P. Firbas and T. Amon
damage can include one to eight, sometimes even 12
chromosomes. All the chromosomes in the cell can even
be damaged. The number of damaged chromosomes
increases with the level of the genotoxicity of the sub-
stance studied, and the same holds for the number of
deformations seen on a particular chromosome. Most
agents shown to be carcinogenic and/or mutagenic to
plants and animals have also been shown to be carcino-
genic in man, so further investigations are necessary to
identify contaminants of our ecosystem, and thus reveal
more information on diseases such as cancer (Al-Sabti
1989).
Polyploidization refers to a numerical change in a
whole set of chromosomes. It has already been shown
that the influence of chemical or physical agents on the
eggs immediately after fertilization could also be
responsible for polyploidy induction in fish (Al-Sabti
1991).
Abnormal separation of sister chromatids caused by
their failure to separate (move apart) properly during cell
division. Cells with an extra chromosomes are referred
to as trisomic (2n + 1), and cells which lack a chromo-
some as monosomic (2n – 1). Plant cytogenetics, using
the Allium metaphase test, identifies the same chromo-
some damages as identified in human cytogenetics,
including chromosome and chromatid damages, aneuplo-
dy, euploidy and translocation. These CAs cause clinical
defects on the human body (Schauer 1981).
The cytogenetic effects of maleic hydrazine (MH) in
a human lymphocyte culture result in chromatid gaps,
chromatid breaks, isochromatid gaps and isochromatid
breaks (Gokalp and Kaymak 2002). Genotoxicity studies
of methyl methanesulphonate (MMS) induced in human
lymphocyte culture chromosome damage: chromosome
breaks, chromosome exchanges, chromatid breaks and
chromatid exchanges (Slapšytė and Sukackaitė 2003).
Equally induced, chemical chromosome damage in meri-
steme root cells of onion A. cepa.
The Allium metaphase test is also suitable for the
detection of radiation, since radiation causes similar CAs
as genotoxic substances (Al-Sabti 1989). So these two
tests, for radioactive emissions and genotoxic substances,
can be combined in a single testing session. A triradial
three-arm chromosome configuration arises by the inter-
action of two chromosomes each having an isochroma-
tide breakage. Many types of triradial chromosomes can
be found in mammalian cells. The main reason for this
type of damage is ionizing radiation that induces the for-
mation of dicentric and ring chromosome (IAEA 1986;
Hatayoglu and Orta 2007).
Genotoxic substance research is of great importance
for the protection of the environment because it enables
an insight into the influence of genotoxic substances on
organisms. The goal of the research is to develop a valu-
able tool for the monitoring of the environment. The
Allium metaphase test also shows us the source of the
pollution. With the Allium and related genotoxic tests
one can determine the influence of chemical substances
on a healthy organism. This test also shows an excellent
correlation with similar tests applied on mammals and
other vertebrates. So the results can be extrapolated to
man.
Using colchicine we are able to modify the basic
Allium test on three levels: (i) an Allium general toxicity
test by detecting elongation or inhibition and malforma-
tion of the roots of onion bulbs; (ii) an Allium anaphase-
telophase test including an aberration in the cell division
principle in the late anaphase and early telophase; and
(iii) an Allium metaphase test used for study of the spe-
cific morphology (structure) of metaphase chromatids
and chromosome damage.
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