EnvironmentalMutagenesis

ELSEVIER Mutation Research 334 (1995) 185-195

The improved Allium/Vicia root tip micronucleus assay

for clastogenicity of environmental pollutants
Te-Hsiu Ma a,,, Zhidong Xu a, Chengen Xu a, Heike McConnell a,
Eugenia Valtierra Rabago b, Gemma Adriana Arreola b, Hongen Zhang c
a Department of Biological Sciences, Western Illinois University, Macomb, IL 61455, USA
b Centro de Estudios Academicos sobre Contaminacion Ambienta~ Universidad de Queretaro, QRO, Mexico
c Shandong Academy of Medical Sciences, Shandong, People's Republic of China

Received 17 February 1994; revision received 5 August 1994; accepted 15 August 1994

Abstract

The meristematic mitotic cells of plant roots are appropriate and efficient cytogenetic materials for the detection
of clastogenicity of environmental pollutants, especially for in situ monitoring of water contaminants. Among several
cytological endpoints in these fast dividing cells, such as chromosome/chromatid aberrations, sister-chromatid
exchanges and micronuclei, the most effective and simplest indicator of cytological damage is micronucleus
formation. Although the Allium cepa and Ficia faba root meristem micronucleus assays (Allium/Vicia root MCN)
have been used in clastogenicity studies about 12 times by various authors in the last 25 years, there is no report on
the comparison of the efficiency of these two plant systems and in different cell populations (meristem and F 1) of the
root tip as well as under adequate recovery duration. In order to maximize the efficiency of these bioassays, the
current study was designed to compare the Allium and the Vicia root MCN assays on the basis of chromosome
length, peak sensitivity of the mitotic cells, and the regions of the root tip where the MCN are formed. The total
length of the 2n complement of Allium chromosomes is 14.4/zm and the total length of the 2n complement of Vicia
is 9.32/~m. The peak sensitivity determined by serial fixation at 12-h intervals after 100 R of X-irradiation is 44 h.
The slope of the X-ray dose-response curve of Allium roots derived from the meristematic regions was lower than
that derived from cells in the F 1 region. Higher efficiency was also demonstrated when the MCN frequencies were
scored from the F 1 cells in both Allium and Vicia treated with formaldehyde (FA), mitomycin C (MMC), and maleic
hydrazide (MH). The results indicated that scoring of MCN frequencies from the F 1 cell region of the root tip was
more efficient than scoring from the meristematic region. The X-ray linear regression dose-response curves were
established in both Allium and Vicia cell systems and the coefficients of correlations, slope values were used to
verify the reliability and efficiency of these two plant cell systems. Based on the dose-response slope value of 0.894
for Allium and 0.643 for Vicia, the Allium root MCN was a more efficient test system. The greater sensitivity of the
Allium roots is probably due to the greater total length of the diploid complement and the higher number of

* Corresponding author.

0165-1161/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved

SSDI 0165-1161(94)00077-8
186 T.-H. Ma et aL /Mutation Research 334 (1995) 185-195

metacentric chromosomes. The Allium/Vicia root MCN test system was applied to determine the clastogenicity of
saccharin (SC) and wastewater from Rio Queretaro and the Arena canal in the city of Queretaro, Mexico. The
minimum effective dose (MED) is 10 R for X-rays, 50 mM for FA, 2.2 mM for MMC, 0.01 mM for MH, and 40 ppm
for SC.

Keywords: Allium; Vicia; Micronucleus assay; Bioassay

I. Introduction have a large data base with an extensive list of

The mitotic root meristems of Allium cepa and
Vicia faba have been the pioneer cytogenetic ma-
terials for clastogenicity studies of physical and
chemical agents since the early 1930s. Based upon
the review papers published under the U.S. EPA
Gene-Tox Program (Grant, 1982a; Ma, 1982),
chromosome aberration frequencies in these root
tips were utilized as the indicators of clastogenic-
ity in most studies prior to the 1980s. In recent
years, chromatid aberrations (Cortes and Mateos,
1991), sister-chromatid exchanges (SCE) (Cortes
et al., 1987; Kuglik and Slotova, 1991) and mi-
cronuclei (MCN) (Arora et al., 1969; Degrassi
and Rozzoni, 1982; Ma and Xu, 1986; Wang and
Tang, 1986a,b, 1987; Dash et al., 1988; Panda et
al., 1990; Ruan et al., 1992) have been used more
frequently as parameters of clastogenicity. Root
tips frequently used for cytogenetic studies in the
past five decades were from Allium cepa and
Vicia faba (Grant, 1982b). Both of these systems
chemical agents which have been tested (Grant,
1982a; Ma, 1982). In the course of searching for
an inexpensive, quick and easy bioassay for moni-
toring clastogenicity of water pollutants, the AI-
lium/Vicia micronucleus root tip system (root
MCN) was considered to be most promising and
relevant for in situ monitoring if it could be
proven to be efficient and reliable. The use of the
root MCN as the end point in detecting water
soluble clastogens was first carried out in the
study of Arora et al. (1969) in Vicia roots. The
same study was repeated by Degrassi and Rizzoni
(1982).
As a rule, micronueleus formation is the result
of acentric fragments or laggards being excluded
from the nucleus proper during mitosis. The MCN
are revealed in the subsequent generation in the
interphase or prophase cells (F 1 cells). Since the
MCN are formed in the F 1 interphase cells, effi-
cient scoring should not be done in the meristem-
atic mitotic cells. Thus, in the root MCN system,

I CAp w M I F~
Fig. 1. Photomicrograph of the longitudinal section of Allium root showing the root cap, meristem (M) and daughter (F l) cell
regions.
 T.-H. Ma et al. / Mutation Research 334 (1995) 185-195
a longer recovery time covering two rounds of
mitosis after treatment is required. This is de-
signed to capture the maximum chromosome
damage which was inflicted in the interphases
(G1, S, G2) of the meristematic cells and later
becomes micronuclei in the F 1 cell population
(Ma and Xu, 1986). In general, root development
is initiated at the apex of the root tip by mitotic
divisions in the meristematic region (about 1 mm
in length above the root cap) (Fig. 1) and the F 1
cells (about 1 mm) are moved upward to lengthen
the root structure. A very small portion of the
meristematic cells divide transversely to increase
the girth of the root tip, whereas the majority
divide longitudinally. Based upon this ontoge-
netic scheme, the majority of the MCN should be
in F 1 ceils except, on some rare occasions, when
there is a mitotic delay.
Although no mention was made by any of the
earlier authors that the MCN frequencies were
scored from the cells of the meristematic region,
judging by the photomicrographs and mitotic in-
dices presented in these publications, the MCN
frequencies were probably not scored from F 1
cells. Furthermore, most of the earlier studies
used several recovery periods from 24 up to 120
h. Thus the MCN frequencies obtained in this
way would lose their fidelity and the efficiency of
the test system would be reduced because the
sensitivity of chromosomes to clastogens varies
greatly throughout the mitotic cycle (Scott et al.,
1990). In order to increase the efficiency of the
root MCN tests a rigorous scheme is established
in the present study by scoring MCN frequencies
from the F 1 cell population (the second millime-
ter above the root cap), which represents the cells
of peak sensitivity that were fixed at the end of
the 44-h recovery period. By comparing the rela-
tive MCN frequencies of the meristematic and
the daughter (F 1) cells, and the relative differ-
ences between the control and treated groups,
the relative efficiency of these two schemes could
be evaluated. Comparison has also been made
between Allium and Vicia root tips for their
specific sensitivity to different clastogens. In or-
der to validate their efficiency and reliability,
X-ray dose-response curves in both root systems
have been established. Moreover, a variety of
187
common clastogens as well as industrial wastewa-
ter have also been tested with this system to
determine its efficacy as an environmental moni-
tor.
2. Materials and methods
Three varieties of AUium cepa (onion) were
used in this collaborative study. One was the
Mexican variety which has a small bulb accompa-
nied by a thick stem, which seems to be a hybrid
between the bulb onion and the scallion. The
second variety was an American large yellow bulb
onion of Allium cepa species and the third was
the small white variety generally called pearl
onion. Roots at about 2-3 cm long were sus-
pended in solutions of various agents for treat-
ment, and maintained in tap water for root ger-
mination and recovery after treatment. The de-
veloping roots in all experiments were maintained
in the room at 22 _+ 2°C since temperature is a
crucial factor in the maintenance of the mitotic
cycle in root development (Gonzalez-Fernandes
et al., 1971). X-ray treatment of onion roots was
carried out by exposing the bulb in an upside-
down position so that the root tips (around 20-30)
received roughly the same dose of X-rays.
Two varieties of Vicia (broad bean) were used.
One was Vicia faba var. minor, and the other was
a Chinese variety. The secondary (lateral) roots
of Vicia at about 1-2 cm in length were sus-
pended in solutions of the chemical agents for
treatment. X-irradiation of Vicia roots was car-
ried out by arranging the roots flat against a
plastic tray. Although both the onion and broad
bean roots were intended to be treated at a
distance of 15 cm from the X-ray filament, the
dosages actually received by the root tips may
have varied slightly because they were not situ-
ated exactly at the same level.
A series of preliminary experiments were car-
ried out using the young roots of Allium cepa and
secondary roots of Vicia faba to determine the
frequencies of X-ray-induced micronuclei as an
indicator of efficiency. The X-ray machine
(Standard X-rays Co.) used in this study was
operated at 80 kV, 5 mA, with 1 mm Al filter and
188 T.-H. Ma et al. / M u t a t i o n Research 334 (1995) 185-195

Table 1
Linear dose responses of X-ray-induced micronucleus frequencies in regions of the meristematic (M) cells and the daughter (F 1)
cells of Allium cepa (small white bulb) root tips
Experimental Dosage N u m b e r of samples N u m b e r of cells scored M C N / 1 0 0 0 cells
group (R) M F1 M F1 M FI
Control 0 3 3 2487 2495 0.5 0.5
Treated-1 25 3 3 3535 3471 12.1 19.3
Treated-2 50 3 3 3505 2996 25.7 38.4
Treated-3 75 - 3 _ a 2548 _ a 51.5
Treated-4 100 3 3 2574 3019 42.8 59.1
Treated-5 125 3 3 2977 2925 56.3 95.9

a The root tip cells in the M region were poorly stained and no result was obtained from these groups.

the dose rate was 40 R / m i n . A series of increas-
ing dosages from 10 to 125 R were used to
establish a dose-response curve and an X-ray
baseline for comparison with the clastogenicity of
other agents. Formaldehyde, maleic hydrazide
and M M C (Sigma Chem. Co.) were used as po-
tent clastogens. In addition, saccharin and indus-
trial wastewater were used as common water con-
taminants to determine the efficacy of these root
M C N systems. A series of increasing dosages in
terms of mM or p p m of the chemical agents were
used for treatment in order to obtain the mini-
m u m effective dose (MED). The acute dose of 6
h treatment was followed by 44 h recovery time
and the roots were then fixed for slide prepara-
tion. Roots which received a chronic dose were
treated continuously for 48 h without a recovery
period and then fixed for slide preparation. Con-
trol groups were maintained for each series of
experiments. The root tips were fixed in aceto-al-
cohol (1:3 ratio). Meristematic and F t cell regions
were p r e p a r e d from each root tip separately by
simply cutting the meristem section (first millime-
ter behind the root cap) and the F 1 section (sec-

100

80

CI3

60

Q
O
-7
40

20

I I
0 50 1 O0 1 $0

X-RAY D O S A G E S (R)
Fig. 2. Linear dose-response curves of X-ray-induced micronuclei frequencies in regions of the meristematic (©, M region) cell and
daughter (zx, F 1 region) of Allium.
 T.-H. Ma et al. / Mutation Research 334 (1995) 185-195 189

ond millimeter) as shown in Fig. 1. These two coverglasses. A double staining method combin-
sections were squashed separately in two differ- ing the Feulgen reaction and the aceto-carmine
ent regions on the same slide under two different squash technique was used for the purpose of

Fig. 3. Photomicrograph of cell populations and micronuclei in meristem (M) and daughter (F l) cell regions. Micronuclei are
marked with arrows.
190 T.-H. Ma et al. /Mutation Research 334 (1995) 185-195
accentuating both the nuclear contents and the
contour of the cell wall.
The mitotic indices of both the meristematic
and F 1 cell regions of selected groups of Vicia
and Allium experiments were determined. The
MCN frequencies of these two regions were
scored. The means and standard deviations (SD)
were obtained from the scores of 5-6 slides for
each experimental group. Generally 1000-2000
cells were scored from each of the five or six
slides of an experimental group and the means
were derived from about 5000-12000 cells and
expressed in terms of MCN/1000 cells. Analysis
of variance (ANOVA), Student's t-test, or Dun-
nett's t-statistic were used to determine signifi-
cant differences between treated and control at
the 0.05 level of significance. The MCN frequen-
cies derived from meristematic and F 1 cell re-
gions were computed separately. A treated/con-
trol ( T / C ) ratio was used to compare the effi-
ciency of the MCN scoring from the meristem
and F 1 regions of the root tips. The T / C ratio
exhibits the magnitude (fold increase) of the dif-
ference between the treated and the control MCN
frequencies. A greater T / C ratio denotes a higher
efficiency. Linear regression dose-response curves
were established and the coefficients of correla-
tion, slope, and intercepts of the dose-response
curves were calculated to mark the efficiency of
the system and the efficacy of the system as a
clastogenicity bioassay.
3. Results and discussion
For the purpose of evaluating the relative effi-
ciency of MCN scoring from the F 1 region and
the meristematic cell population, the X-ray dose-
response curves were established for both regions
of the Allium roots. The results are shown in
Table 1 and Fig. 2.
Judging from the slope values (meristematic
region: 0.434; F 1 region: 0.74) of the dose-re-
sponse curve, it is clear that greater efficiency can
be attained when the MCN frequencies are scored
from the F 1 cell region rather than from the
general meristematic region. The general appear-
ance of the cells and MCN in these two regions is
shown in Fig. 3. There is a greater degree of
synchrony of interphase cells in the F 1 region
than in the meristematic region, which facilitates
the MCN scoring. It was known that micronu-
cleus-bearing cells usually do not divide in the F 1
cells and tend to degenerate in the F 2 generation
(Arora, 1961). Scoring MCN from F 1 cells has the
advantage of recording both chromosome breaks
which are generated in the early (G 0 and the late
interphase (G 2) of the parental cells when the
conventional 44-48-h recovery time is used. Arora
and his co-workers (1969) believed that some of
the MCNs were able to divide, according to their
radioisotope thymidine labelling studies. Their
claim contradicted the early findings of Das
(1962). Furthermore, the autoradiographic evi-
dence of labelled MCN should not be used as a
reliable indicator of the MCN division. The occa-
sionally incorporated DNA precursor, [3H]TdR,
in MCN could well be the result of unscheduled
DNA synthesis of the damaged DNA strands.
Another possible misconception for the dividing
MCN could be a mis-interpretation of the com-
mon 'twin MCN' which is the result of separation
of the isochromatid breaks in the parental ceils
which become identical MCN in the F 1 cells.
In most of the earlier publications, micronu-
cleus frequencies were scored from a series of
increasing lengths of recovery periods (24-72 h)
to obtain an average value (Degrassi and Rizzoni,
1982). Others used a 5-day recovery period (Dash
et al., 1987; Panda et al., 1990). Some researchers
score MCN from interphase ceils and others score
MCN together with chromosome abnormalities.
The inconsistent use of one method over the
Table 2
Sensitive peak for X-ray-induced micronuclei in Vicia faba
(Chinese variety) root tips (X-ray dose: 100 R) scored from
the F 1 region
Recovery Number Cells MCN/ _+SD Remarks
duration of scored 1000
(h) samples cells
40 5 5000 35.8 11.6
44 5 5000 57.2 15.1 high peak
48 5 5000 54.7 23.3
64 5 5000 31.7 15.4
72 5 5000 20.0 5.6
 T.-H. Ma et al. ~Mutation Research 334 (1995) 185-195 191

Table 3 Table 4
Linear dose-response of X-ray-induced micronucleus frequen- Linear dose-response curve of X-ray-induced micronucleus
cies in the daughter (F I) cells of the treated meristem of frequencies in the daughter (F 1) cells of the treated root
Allium cepa (large yellow bulb) meristem of Vicia faba (Chinese variety)
Experi- Dosage Number Number MCN/ +SD Experi- Dosage Number Number MCN/ +SD
mental (R) of of cells 1000 mental (R) of samples of cells 1000
groups samples scored cells group scored cells
Control 0 6 7443 1.63 0.92 Control 0 5 5000 0.85 0.01
Treated-1 10 6 8043 4.60 1.97 Treated-1 10 5 5000 1.60 2.07
Treated-2 20 6 8367 13.09 2.45 Treated-2 20 5 5000 3.80 1.30
Treated-3 30 6 7375 22.02 6.52 Treated-3 30 5 5009 3.99 2.34
Treated-4 40 6 7135 35.72 6.36 Treated-4 40 5 5038 10.90 4.00
Treated-5 50 6 7260 43.86 7.39 Treated-5 50 5 5062 21.20 10.18
Treated-6 60 6 6766 49.32 4.00 Treated-6 60 5 5045 41.30 4.60
Treated-7 70 6 6935 61.87 7.02 Treated-7 70 5 5010 40.40 6.40

others has reduced the efficiency of the test sys-
tem. In order to increase the fidelity of the test
system which scores MCN in F 1 ceils, data collec-
tion should be centered at the sensitive peak
stage for the induction of chromosome breaks. In
this study, the peak sensitivity was determined by
using a serial fixation scheme for recovery times
of 40, 44, 48, 64, and 72 h after X-ray treatment.
Then the MCN frequencies for each of the recov-
ery groups were scored from the F 1 cell region.
Results of this experiment indicate that the 44-h
recovery time gives the highest MCN frequency
(Table 2).
This 44-h recovery period agrees with some
earlier studies in Vicia (Arora et al., 1969). Based
on the 24-h mitotic cycle in these roots, the 44-h
recovery time captures the MCN which were in-
duced around the G1 or S stage of the parental
meristematic cells and revealed at the interphase
or prophase in the daughter cells.
After the sensitive peak (44-h recovery time)
and the efficient scoring region (F~) were deter-
mined, two identical series of linear regression
dose-response curves were established in Allium
and Vicia, under a series of increasing dosages
between 10 and 70 R of X-rays. The dose-re-
sponse curves for Allium and Vicia roots are
shown in Tables 3 and 4, and graphically illus-
trated in Fig. 4. The high values of the correla-
tion coefficients (0.968 for Allium and 0.89 for
Vicia) indicate the reliability of the root MCN
assay. The higher slope value in Allium (0.89)
than in Vicia (0.64) is a good indicator for the
greater efficiency of the Allium system.
A separate series of experiments were de-
signed to determine the general efficiency of A1-
lium and Vicia for the induction of MCN with a
single dose of 150 R of X-rays. Allium roots yield
considerably more MCN than Vicia roots when
MCN were scored from the F 1 cells. The differ-
ence in MCN frequencies between these two
plant species is shown in Table 5.
The greater efficiency of induction of MCN in
Allium was most likely due to the greater length
of the chromosomearms and the number of
metacentric chromosome arms in Allium. The

Table 5
Comparison of radiosensitivity between Allium (large yellow bulb) and Vicia (Chinese variety) in X-ray-induced micronuclei scored
from the F t region of root tips (150 R of X-irradiation)
Experimental Number of cells/ Allium root Vicia root Remarks
group number of samples MCN/1000 cells MCN/1000 cells
Mean SD Mean SD
Control 9000/9 0.5 0.3 0.8 0.6
Treated 9000/9 99.6 9.1 70.0 7.9
192 T.-H. Ma et al. /Mutation Research 334 (1995) 185-195

1.46 /zm a n d that of the 10 short acrocentrics

70 i i i i i i i derived from 220 m e a s u r e m e n t s is 0 . 6 4 / z m . T h e
total length of m e t a p h a s e arms in the 16
60 m e t a p h a s e c h r o m o s o m e s (32 arms) is a b o u t 14.4
/zm in A l l i u m in contrast to a total length of 9.32
50 o /zm in the Vicia c o m p l e m e n t of 12 chromosomes.
,d If the acentric f r a g m e n t s c o n t r i b u t e most of the
M C N s in the F~ cells, t h e n roughly, the chance
40 o " " for A l l i u m roots to form M C N should be a b o u t
1.5 times that for Vicia roots. This probability
7.. 3O -
roughly coincides with the M C N frequencies given
Z
in T a b l e 5. I n the practical application of a root
M C N assay for clastogens, A l l i u m roots can pro-
vide a b o u t 30 roots in a b o u t 2 4 - 4 8 h from a
10 d o r m a n t bulb. I n the case of Vicia, it would take
a n average of 5 - 7 days to furnish the same n u m -
I/O /A A
ber of secondary roots from a b e a n seed.
0 10 20 30 40 50 60 70 80 F o r m a l d e h y d e , a w e l l - k n o w n clastogen in
T r a d e s c a n t i a , was used as a testing a g e n t in b o t h
X - R A Y DOSAGES (R) the Vicia a n d the A l l i u m root M C N assays to
Fig. 4. Linear dose-response curves of X-ray-induced mi-
validate these systems. T h e results are shown in
cronucleus frequencies in the daughter (F1) cells of the treated
meristem of Allium (©) and Vicia (zx). T a b l e 6.
A g a i n , the A l l i u m root M C N assay showed a
g r e a t e r sensitivity a n d the overdose effect of the
average l e n g t h of the m e t a p h a s e c h r o m o s o m e chemical was exhibited at a relatively lower dosage
(colchicine a c c u m u l a t e d ) of A l l i u m derived from level in A l l i u m t h a n in the Vicia system. I n the
the m e a s u r e m e n t of 60 c h r o m o s o m e s is 0 . 9 / z m . Vicia system, t h e r e was again a d o s e - r e l a t e d in-
T h e average length of the two long m e t a c e n t r i c c r e m e n t of M C N frequencies especially in the
c h r o m o s o m e s derived from 44 m e a s u r e m e n t s is m e r i s t e m a t i c region. T h e r e was also a steady

Table 6
Formaldehyde-induced micronuc!ei scored from the regions of meristem (M) and daughter (F1) cells of Allium (Mexican variety)
and Vicia (V. faba minor) root tips
Experimental MCN/1000 cells T/C(X) a Mitotic index Remarks
group (dose) M F1 M F1 M F1
Allium
Control 4 1 1 1 10.8 1.3
T-1 (25 mM) 83 53 20.8 53.0 7.2 2.1 positive *
T-2 (50 mM) 22 12 5.5 12.0 5.5 1.4 positive *
T-3 (100 mM) 0 0 - - 2.7 0.2 overdose b
Vicia
Control 7 1 1 1 3.3 1.1
T-1 (25 mM) 15 1 2.1 1.0 16.9 0.9
T-2 (50 mM) 30 11 4.3 11.0 9.6 5.2 positive *
T-3 (100 mM) 89 5 12.7 5.0 14.6 8.6 mitotic delay
a T/C(X), the MCN frequency of the treated group is X times as high as that of the control group.
b Overdose, severe damage indicated by dead cells and pycnotic nuclei.
* Positive response at the 0.05 level of significance based on ANOVA and Dunnett's t statistical analysis.
 T.-H. Ma et al. / Mutation Research 334 (1995) 185-195 193

increase of mitotic indices in both meristematic
and F~ regions. This is generally a sign of mitotic
delay or a metaphase accumulation phenomenon
similar to the colchicine effect. The decline of
MCN frequencies in the F 1 region in the group
treated with 100 mM could also be due to the
mitotic delay. The relatively high efficiency of the
Allium system is substantiated by the T / C ( X )
values at the effective dose levels of formalde-
hyde.
In order to evaluate the efficacy of Allium and
Vicia root MCN assays on known clastogens,
MMC experiments similar to those conducted by
Arora et al. (1969) and Degrassi and Rizzoni
(1981) were repeated under the current recovery
time and MCN scoring procedure. The results
are shown in Table 7.
MMC is a DNA synthesis inhibitor and an
alkylating agent. While the same dosages (2.2 and
4.4 mM) of MMC were used in both the Allium
and the Vicia roots, MMC was highly toxic to the
Allium root tip cells, and unfortunately no result
was obtained from the F 1 cells for comparison.
The lack of MCN in the F 1 cells in Allium roots
could be attributed to the inability of the MMC-
damaged meristematic cells to divide and give
rise to F~ cells. The drastic decline of the mitotic
indices in both meristematic and F 1 regions of the
Allium cells could corroborate this 'lack of F 1
cell-lack of MCN' assumption. In the MMC-
treated Vicia roots, the results demonstrated a
dose-related increase of MCN in both meristem-
atic and F 1 regions, and the increases were more
pronounced in the F 1 cell regions where the mi-
totic indices remained very low as expected. Al-
though the apparent MCN frequencies were
higher in the meristematic region, the fold in-
crease of the treated over the control, T/C(X),
was greater in the F 1 region than in the meris-
tematic region.
The MCN frequencies induced by 2.2 and 4.4
mM MMC in Vicia roots (Table 7) were propor-
tionally lower than those reported by Arora et al.
(1969). These discrepancies could be due to
mainly the MCN scoring procedure. It is obvious
that the control value of the meristematic region
in our experiment is 7/1000 cells which coincides
with the control value of Arora's results but our
control value of the F 1 region is 1/1000 cells.
With the highest MCN frequencies of 200/1000
cells in both our and Arora's results, the T / C ( X )
value shows an 83-fold increase. This is a clear
indication of the high efficiency in using F 1 cell
populations for MCN scoring.
Above all, the actual scoring was much easier
in the more synchronized interphase F 1 cell popu-
lation with very low mitotic indices as compared
with the meristematic cell population (Fig. 3).

Table 7
MMC-induced micronuclei scored from regions of meristem (M) and daughter (F I) cells of Allium (Mexican variety) and Vicia (V.
faba minor) root tips
Experimental M C N / 1 0 0 0 cells T/C(X) a Mitotic index Remarks
group (dose) M F1 M F1 M F1
Allium
Control 4 1 1 1 10.8 1.3
T-1 (2.2 m M ) 95 0 23.4 - 4.0 0 toxic, inhibited
mitosis
T-2 (4.4 mM) 13 0 3.3 - 0.7 0 toxic, inhibited
mitosis
I~cia
Control 7 1 1 1 3.3 1.1
T-1 (2.2 mM) 73 40 10.4 40.0 9.2 0.5 positive *
T-2 (4.4 mM) 202 83 29.0 83.0 8.8 0.4 positive *
a T / C ( X ) , the M C N frequency of the treated group is X times as high as that of the control group.
* Positive response at the 0.05 level of significance based on A N O V A and D u n n e t t ' s t statistical analysis.
194 T.-H. Ma et al. / Mutation Research 334 (1995) 185-195

Table 8
Maleic hydrazide- and saccharin-induced micronuclei scored from the region of daughter (F 1) cells of Allium (large yellow bulb)
root tips
Experimental Dosage Number of Cells MCN/1000 _+SD Remarks
group (mM) samples scored cells
Maleic hydrazide
Control 0 6 14 105 2.60 0.64
Treated-1 0.01 6 13 455 5.28 0.85 positwe *
Treated-2 0.10 6 13 640 12.44 3.33 positwe *
Treated-3 1.00 6 13085 17.25 3.09 positive *

Saccharin
Control 0 5 5625 1.41 0.64
Treated-1 40 5 5775 2.50 0.85
Treated-2 80 5 5895 3.42 0.75 positwe *
Treated-3 120 5 5370 12.05 2.12 positwe *

* Positive response at the 0.05 level of significance based on A N O V A and Dunnett's t statistical analysis.

Furthermore, MCN in the synchronized popula-
tion of the F t ceils can be scored with an auto-
mated 'Tradescantia micronucleus image analysis
system' with modified software for the root MCN
system (Ma et al., 1992).
The Allium root MCN test was used to deter-
mine the clastogenicity of maleic hydrazide (MH),
a well-known clastogen, and saccharin (SC), a
popular water contaminant, under these newly
established experimental procedures by scoring
MCN in the F a cell region only. The results are
given in Table 8.
M H and SC have drastic differences in clasto-
genicity. The M E D of MH is around the 10-100
/xM level while the MED of SC is around 100
mM. This demonstrates that the Allium root
MCN assay was able to detect and differentiate a
1000-fold difference in clastogenicity.
Although both Vicia and Allium root MCN
assays seem to be efficient in detecting clastogens
such as X-rays, FA, MMC, MH and SC, the
ability to detect a low concentration of common
water pollutants is only known through their
chromosome aberration endpoint (Fiskesj6, 1985;
Wang and Tang, 1987; Grant et al., 1989). In this
study, the Vicia root MCN assay was applied to
wastewater in the city of Queretaro, Mexico. The
results are given in Table 9.
Based upon these studies, the root MCN assay
seems to be ineffective in detecting water pollu-
tants. The clastogenicity of wastewater from the
same source was detected with the Tradescantia
micronucleus bioassay (Ruiz et al., 1992) after
several-fold dilutions. Current results do not show
the distinctly high frequencies of MCN as de-
tected by the Tradescantia bioassay. In the Vicia

Table 9
Industrial wastewater-induced micronuclei scored from regions of meristem (M) and daughter (F 1) cells of Vicia (I/.. faba minor)
root tips
Experimental Cells scored Mitotic index (%) MCN/1000 cells Remarks
group M F1 M F1 M F1
Control 2500 2500 9.1 3.5 2.4 2.8
Rio Q R O a 2500 2500 9.0 2.1 6.6 8.3 positive ~
Arena canal b 2500 2500 7.3 3.8 7.8 4.5 positive *

a Rio QRO, the Queretaro river receives industrial water from the Arena canal.

b The Arena canal contains the effluent of industrial wastewater.

* Positive at the 0.05 level of significance based on A N O V A and Dunnett's t statistical analysis.
 T.-H. Ma et al. / Mutation Research 334 (1995) 185-195
root MCN test of the Arena canal wastewater,
there was a sign of mitotic delay. There is no
doubt that these root MCN systems are efficient
in detecting the clastogenicity of chemical and
physical agents. The question on the efficacy of
the root MCN assays in detecting the clastogenic-
ity of water pollutants (especially in situ monitor-
ing) should be addressed, and careful studies are
needed before the root MCN assay in water
pollution monitoring is widely applied.
Acknowledgements
The authors express their deepest appreciation
to Prof. William F. Grant of McGill University
for his assistance in preparing the manuscript and
to Mr. Byeong-Seon Jeong for his assistance in
the statistical analysis and graphic preparations.
References
Arora, O.P., V.C. Shah and S.R.V. Rao (1969) Studies on
micronuclei induced by mitomycin-C in the root cells of
Vicia faba, Exp. Cell Res., 56, 443-448.
Cortes, F.P. and S. Mateos (1991) Premature onset of mitosis
and potentiation of chromosome damage induced by poly-
o-lysine in plant cells: evidence for G 2 repair, Mutation
Res., 247, 147-151.
Cortes, F., P. Escalza and M. Diazrecasens (1987) Factors
affecting the production of SCEs by maleic hydrazide on
root-tip chromosomes of Allium cepa, Mutation Res., 192,
125-130.
Das, N.K. (1962) Synthetic capacities of chromosome frag-
ments correlated with their ability to maintain nucleolar
material, J. Cell Biol., 15, 121-130.
Dash, S., K.K. Panda and B.B. Panda (1988) Biomonitoring of
low levels of mercurial derivatives in water and soil by
Allium micronucleus assay, Mutation Res., 203, 11-21.
Degrassi, F. and M. Rizzoni (1982) Micronucleus test in Vicia
faba root tips to detect mutagen damage in fresh water
pollution, Mutation Res., 97, 19-33.
Fiskesjr, G. (1985) Allium test on river water from Braan and
Saxan before and after closure of a chemical factory,
Ambio, 14, 99-103.
Gonzalez-Fernandez, A., G. Gimenez-Martin and C. de la
Torre (1971) The duration of the interphase periods at
195
different temperatures in root tip cells, CytoBiologie, 3,
367-371.
Grant, W.F. (1982a) Chromosome aberration assays in Al-
lium. A Report of the U.S. Environmental Protection
Agency Gene-Tox Program, Mutation Res., 99, 273-291.
Grant, W.F. (1982b) Plant mutagen assays based upon chro-
mosome mutations, in: E.J. Klekowski Jr. (Ed.), Environ-
mental Mutagenesis, Carcinogenesis and Plant Biology,
Vol. 2, Praeger Press, New York, pp. 1-24
Grant, W.F., H.G. Lee, D.M. Logan and M.F. Salamone
(1989) The use of Tradescantia and Vicia faba bioassays
for the in situ detection of mutagens in an aquatic environ-
ment, Environ. Mol. Mutagen., 14 (Suppl. 15), 75.
Kuglik, P. and J. Slotova (1991) Different effects of pretreat-
ment with tritiated thymidine (tritiated) on radiation-in-
duced sister chromatid exchanges (SCEs) and micronuclei
in Vicia faba root tip, Biol. Plant., 33, 291-297.
Ma, T.H. (1982) Vicia cytogenetic tests for environmental
mutagens. A report of the U.S. Environmental Protection
Agency Gene-Tox Program, Mutation Res., 99, 259-271.
Ma, T.H. and Z. Xu (1986) Validation of a new protocol of
the Allium micronucleus test for clastogens, Environ. Mu-
tagen., 8 (Suppl. 6), 65-66.
Ma, T.H., J. Xu, W. Xia, X. Jong, W. Sun and G. Lin (1992)
Proficiency of the Tradescantia-micronucleus image analy-
sis system for scoring micronucleus frequencies and data
analysis, Mutation Res., 270, 39-44.
Panda, K.K., M. Lenka and B.B. Panda (1990) Monitoring
and assessment of mercury pollution in the vicinity of a
chloroalkali plant, I. Distribution, availability and genotox-
icity of sediment mercury in the Rushikulya Estuary, In-
dia, Sci. Total Environ., 96, 281-296.
Ruan, C., Y. Liang and J. Liu (1992) Application of l/icia faba
root tips micronucleus test in the rapid detection of muta-
genic environmental pollutants, China J. Environ. Sci., 13,
56-58.
Ruiz, E.F., V.M.E. Rabago, S.U. Lecona, A.B. Peres and
T.H. Ma (1992) Tradescantia-Micronucleus (Trad-MCN)
bioassay on clastogenicity of wastewater and in situ moni-
toring, Mutation Res., 270, 45-51.
Scott, D. and H.J. Evans (1967) X-ray induced chromosomal
aberrations in Vicia faba. Changes in response during the
cell cycle, Mutation Res., 4, 579-599.
Wang, Y. and D. Tang (1986a) I/icia faba root tip cell mi-
cronucleus tests on 15 chemicals, China Environ. Sci., 6,
19-23.
Wang, Y. and D. Tang (1986b) Combined micronucleus ef-
fects induced by fluoride and strong mutagens on higher
plant cells, Acta Sci. Circumstant., 6, 240-241.
Wang, Y. and D. Tang (1987) Pollution detected by Vicia faba
root tip cell micronucleus test and comparison with other
index, China Environ. Sci., 7, 45-52.