Optimizing the Utility of Allium cepa L. var. aggregatum (sibuyas Tagalog) for the Allium Test by Elucidating its Mitotic Periodicity and Rhythmicity Under Varying Light Conditions

Abstract

The occurrence of pattern of mitotic activity has long been studied in different plants; in the onion Allium cepa, determination of its mitotic activity has led to its utilization in the Allium test for cytotoxicity and mutagenicity of test substances. In this study, the pattern of mitotic activity of A. cepa var. aggregatum and the effect of light exposure on mitotic activity were determined to test the utility of A. cepa L. var. aggregatum as an alternative to the common onion, A. cepa, for the Allium test. Bulblets of A. cepa var. aggregatum were allowed to root for three days in tap water under three different set-ups: constant light exposure set-up (Light), constant dark set-up (Dark) and 12 hours light-12 hours dark set-up (Light-Dark). The root tips from the bulblets were then excised and subjected to microscopic observation for the mitotic index (MI) every hour after the third day. The results showed no significant difference observed across the three set-ups. However, MI for the Dark and Light set-ups were periodic, showing peaks or maxima of MI falling between 11 AM and 12 PM, whereas that of Light-Dark set-up was rhythmic, having an hourly fluctuation, but also showed maximum between 11 AM and 12 PM. It is recommended that A. cepa var. aggregatum root tips be excised between 11 AM and 12 PM for the Allium test.

Full text

Optimizing the utility of allium cepa L. var. aggregatum (sibuyas Tagalog)
43Science Diliman (January-June 2011) 23:1, 43-51
Optimizing the Utility of Allium cepa L. var. aggregatum
(sibuyas Tagalog) for the Allium Test by Elucidating its Mitotic
Periodicity and Rhythmicity Under Varying Light Conditions
Ambrocio Melvin A. Matias* and Ian Kendrich C. Fontanilla
Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City
Telefax: (63-2) 920-5471
*Corresponding author: Email: kelvin4225@yahoo.com
ABSTRACT
The occurrence of pattern of mitotic activity has long been studied in different plants; in the onion Allium
cepa, determination of its mitotic activity has led to its utilization in the Allium test for cytotoxicity and
mutagenicity of test substances. In this study, the pattern of mitotic activity of A. cepa var. aggregatum
and the effect of light exposure on mitotic activity were determined to test the utility of A. cepa L. var.
aggregatum as an alternative to the common onion, A. cepa, for the Allium test. Bulblets of A. cepa var.
aggregatum were allowed to root for three days in tap water under three different set-ups: constant light
exposure set-up (Light), constant dark set-up (Dark) and 12 hours light-12 hours dark set-up (Light-Dark).
The root tips from the bulblets were then excised and subjected to microscopic observation for the
mitotic index (MI) every hour after the third day. The results showed no significant difference observed
across the three set-ups. However, MI for the Dark and Light set-ups were periodic, showing peaks or
maxima of MI falling between 11 AM and 12 PM, whereas that of Light-Dark set-up was rhythmic, having
an hourly fluctuation, but also showed maximum between 11 AM and 12 PM. It is recommended that A.
cepa var. aggregatum root tips be excised between 11 AM and 12 PM for the Allium test.
Keywords: Allium cepa var. aggregatum, periodicity, rhythmicity, mitotic index, light, Allium test

Matias, AMA and Fontanilla, IKC
44
INTRODUCTION
The Allium test is widely used as an environmental
monitoring tool due to its ease of use and low cost as
well as high correlation of its results with animal models
(Evandri et al. 2000; Fijesko 1979; Quilang et al. 2008;
Rathore et al. 2006; Vidakoviæ et al. 1993; Yi & Meng
2003). The test took its origin from Levan (1938), who
studied the effects of colchicine on mitosis using Allium
cepa. Since then, A. cepa has been used as a test
organism for cytotoxicity and mutagenicity for
environmental monitoring, which involved exposing the
roots of Allium in a test substance, cutting the root and
observing the root cells under the microscope for mitotic
activity and chromosomal aberrations (Fijesko 1985).
In the Allium test, root tips should be cut during optimal
mitotic activity, which is measured in terms of the
mitotic index or MI (the ratio of observed dividing and
non-dividing cells). Jong (1997) suggested that cutting
should be done during midday (12:00-1:00 PM) based
on previous studies that determined the optimal mitotic
activity of A. cepa. However, studies determining
optimal mitotic activity show inconsistent results (Lewis
1901; Kellicot 1904; Solomon & Trent 1941). Lewis
(1901), using a 4-hour interval set up, showed mitotic
maxima at 12 PM and 12 AM, demonstrating
periodicity, which is characterized by the occurrence
of one or two peaks of high mitotic activity. However,
Winter (1929) argued that Lewis’ results were uniform
rather than showing periodicity as shown by the
comparison of the percentage of dividing cells at
different times. In a similar work by Kellicot (1904)
where the onions were planted in soil and a 2-hour
interval was employed, maxima were achieved at 11
AM and 1 PM. Later on, Solomon & Trent (1941)
observed mitotic activity under varying light conditions
at one-hour intervals. The results showed rhythmicity,
or the occurrence of hourly fluctuations in mitotic
activity, under normal light condition. However,
periodicity was evident in continuous light and
continuous dark set-ups.
Effect of light on mitosis is demonstrated in other
systems (Yeoman & Davidson 1971; Nemota &
Furuya 1985). In Chattonella antiqua (red tide
flagellate), light can induce cell division, but it can also
inhibit division by exposing cells that divide under
continuous light (Nemota & Furuya 1985). In callus
cultures, light tends to have inhibitory effect on cell
division (Yeoman & Davidson 1971; Fraser et al. 1967).
This decreased cell division, resulting to decreased
callus formation, might be due to reduction of some
substance by light (Yeoman & Davidson 1971). It is
currently suggested that cell division is affected by
circadian clock since cell cycle genes, i.e. cyclins and
cyclin-dependent-kinase (CDK), are under circadian
control (Moulager et al. 2007). Thus, alteration of
exposure to light might change patterns of mitotic
activity.
Recent works involving the Allium test include those
of Evandri et al. (2000), which showed that water in
polyethylene bottles could induce cytogenetic
aberration; and Quilang et al. (2008), which
demonstrated the effects of polychlorinated biphenyls
(PCB’s) on Allium root cells. Both studies made use
of the common onion variety of A. cepa, though other
studies also employed other varieties of A. cepa and
even other species. For instance, Yi & Meng (2003)
observed increased anaphase aberration and
micronuclei as well as the presence of pycnotic cells in
root tips of A. sativum (garlic). In another modified
Allium test, Vidakovic (1993) used A. cepa var.
aggregatum (A. ascalonicum in the literature) as test
organism for waste drilling fluid, which promoted
cytotoxicity, particularly inhibition of mitotic activity, in
the onion roots. Allium cepa var. aggregatum, also
known as the bunching onion or locally as “sibuyas
tagalog,” has since been synonymized with A.
ascalonicum (Rabinovitch & Currah 2002). Its utility
by Vidakovic (1993) could attest to its suitability,
perhaps even more so than the common onion variety
of A. cepa since A. cepa var. aggregatum were found
to grow faster and in greater numbers than those of
the common onions based on authors’ personal
observations.
Since different species and varieties are being
employed for the Allium test, there is a need to
ascertain the period of optimal mitotic activity for each
type of Allium other than the common onion variety in
order to optimize the time to cut the root tips. This study
therefore aimed to investigate the periodicity and
rhythmicity of mitotic activity in A. cepa var.
aggregatum root tips. Furthermore, since mitotic
Science Diliman (January-June 2011) 23:1, 43-51

Optimizing the utility of allium cepa L. var. aggregatum (sibuyas Tagalog)
45
activity is hypothesized to be affected by light, this study
also investigated the effects of varying durations of
light exposure to the periodicity of mitotic activity in A.
cepa var. aggregatum, by measuring the mitotic index
(MI). Other factors that could affect periodicity of
mitotic activity such as hormone activity and
temperature were not considered in this study.
MATERIALS AND METHODS
Allium cepa var. aggregatum were purchased from
the local market in San Jose, Nueva Ecija. Scaly leaves
were removed until the bulb leaves were exposed. For
each bulb, only one bulblet was used. Three set-ups
were prepared similar to Solomon & Trent (1941) with
modification of light source: 24 hour light exposure
(Light), 12-hour-light-exposure then 12-hour-dark
exposure (Light-Dark) and no light exposure (Dark).
For each set-up, 18 bulblets were prepared as in Quilang
et al. (2008) in which each was placed on top of a 100
mL glass bottle filled with tap water to expose the
bottom end of the bulblet to water. The Light set up
was exposed to continuous light, the Light-Dark set-up
was alternately exposed to light and placed in a dark
locker every 12 hours (i.e. 7 AM and 7 PM,
respectively), and the Dark set-up was placed in a dark
locker during the entire duration of the experiment. The
Light and Light-Dark set-ups were exposed to light by
placing them in front of fluorescent bulbs; light intensity
ranged from 70 to 171.5 Fcandle and was measured
using an ExTech EA31 Easy ViewTM light meter. Only
18 bulblets were prepared for each set up due to the
limited space on the laboratory bench that allowed
uniform exposure of the set-ups to the light apparatus.
The roots were allowed to grow for three days.
On the 4th day, roots were excised every hour for 24
hours starting at 7 PM until 6 PM the next day. For
every hour, 3 bulblets from each set-up were selected
for root excision. Around 3-5 roots were cut from each
bulblet using a pair of dissecting scissors. Since there
were only 18 bulblets for each set up, each bulblet was
sampled for roots for a total of four times to complete
the hourly sampling over a 24 hour period. All 18
bulblets for each set up were randomly arranged prior
to root excision. The roots were placed into 1.5-ml
microfuge tubes containing Farmer’s solution (1 part
acetic acid, 3 parts ethanol) for fixation, after which
they were stored at 40 C prior to use.
The root tips were prepared for examination through
the squash method following El-Shahaby et al. (2003)
with some modifications. Around 1-3 mm of the root
tip was cut and placed on a glass slide, then exposed to
1 N HCl for 1-2 minutes, after which the HCl was
blotted off using tissue paper. The root tip was then
macerated using the cover slip. Aceto-orcein was added
onto the macerated tissue; after 5-8 minutes, the slide
was passed through a flame to heat-fix the stain on the
tissue. A cover slip was placed on top of the tissue,
and the excess aceto-orcein was blotted off using tissue
paper. The slide was subsequently viewed under a 40x
objective. For each root tip sample, 1000 cells were
scored, taking note at which stage the cell was in:
interphase, prophase, metaphase, anaphase or
telophase. Scoring of cells for all the slides began in
the upper left side of the slide and proceeded through a
convention of moving the slides from left to right until
1000 cells were counted.
The mitotic index (MI) of each root tip sample for every
hour (or the number of mitotic cells over 1000) was
determined. For the determination of periodicity and
rhytmicity, the MI for each hour was plotted. One-
way Analysis of Variance (ANOVA) was performed
for every hour, comparing the MI of each set-up. For
each hour, there were 3 bulblets, and root tips were
excised from these bulblets. Each root tip cut from a
bublet for every hour was considered as a replicate,
yielding three replicates for each hour.
To determine whether there was difference in the MI
obtained from different treatments (Light set-up, Dark
set-up and Light-Dark set-up), One-way Analysis of
Variance (ANOVA) was employed using Excel 2007.
Prior to ANOVA, data was transformed using arc sin
(Zar 1999). Two trials were done for this experiment.
RESULTS
For Trial 1, the MI obtained from the Dark set-up across
different time intervals were very similar with those of
the Light-set-up, with the curves of the two set-ups
showing a similar pattern (Figure 1). In the Light set-
Science Diliman (January-June 2011) 23:1, 43-51

Matias, AMA and Fontanilla, IKC
46
up, the maxima MI were observed at 1AM, 12 PM
and 8 PM, with the highest MI value obtained at 12
PM. In the Dark set-up, 11 AM had the highest MI
followed by 12 AM and 8 PM. In both set ups, three
maxima MI were observed: (1) 12 AM to 1 AM; (2) 11
AM to 12 PM; and (3) 8 PM; highest maxima was
obtained at 11 AM to 12 PM. In the Light-Dark set-
up, the maxima MI were at 12 AM, 6 AM and 12 PM;
two of these maxima were within the range of the
maxima MI in the other two set-ups. Unlike the other
two set-ups, however, the MI of Light-Dark set-up
appeared to be fluctuating at an hourly interval
characterized by an increase in one hour followed by a
decrease in the succeeding hour. Furthermore, the MI’s
of Light-Dark set-up were generally lower than in the
other set-ups.
In Trial 2, the Light set-up had maxima MI at 12 AM,
12 PM, and 10 PM with highest maxima at 12 PM;
Dark set-up at 12 AM, 12 PM and 11 PM with highest
at 12 AM; the Light-Dark at 12 AM, 12 PM and 7 PM,
with highest at 12 PM. The Light and Dark set-ups
followed a similar pattern in Trial 1 in terms of the
presence of maxima MI which were distinctly higher
than the other points. In comparison to these two, the
Light-Dark set-up had a fluctuating pattern from 12
AM to 12 PM, but continually decreased at 1 PM to 5
PM and increased at 7 PM then followed a fluctuating
pattern (Figure 2). Based on the plots, Trial 1 and Trial
2 had almost similar results in terms of the patterns.
However, unlike in Trial 1, the MI of Light-Dark set-
up in Trial 2 was relatively high making it similar with
the other two set-ups at 11 AM to 1 PM; hence the
three plots seemed to overlap at this time interval.
In comparing the MI for the three set-ups, a significant
difference based on ANOVA was obtained in Trial 1,
though this was only observed at two time periods, 1
Science Diliman (January-June 2011) 23:1, 43-51
Figure 1. Plot of Mitotic Index for each hour of the day obtained from the first trial showing periodicity for Dark and Light
while rhythmicity for Light-Dark set-up. Error bar corresponds to standard error.

Optimizing the utility of allium cepa L. var. aggregatum (sibuyas Tagalog)
47
AM and 9 PM, (p= 0.063 and p= 0.036, respectively)
(Table 1). No significant difference was obtained from
Trial 2 based on ANOVA (Table 2).
DISCUSSION
In both trials, the maxima fell in the range of 11 AM to
12 PM and 11 PM to 1 AM, which is in agreement
with previous works on the common onion variety of
A. cepa by Kellicot (1904) and Lewis (1901). These
results agree with those of Solomon and Trent (1941);
however, the maxima for the continuous light differed
slightly, which was observed at 7 AM, 11 AM, 3 PM
and 10 PM. The results for the Dark set-up were similar
with those of Solomon & Trent (1941) except that the
frequency of mitosis in the Dark-set-up in this study
was lower than that obtained from the common onion.
The Light-Dark set-up appeared to have lower MI than
the other two set-ups, particularly in Trial 1. In Trial 2,
the Light-Dark set-up appeared to have a similar MI
value with the other two set-ups. This result, however,
differed with that of Solomon and Trent (1941) in which
the day-night set-up (Light-Dark) had the highest MI
of all the three set-ups. In all set-ups, the frequency of
mitosis was lower than that observed for the common
onion variety by Solomon & Trent (1941).
The intensity of light used in the experiment was 70-
171.5 Fcandle for both trials. This intensity is low
compared to the presumed intensity of light from the
sun (720 – 11000 Fcandle), which was used by Solomon
& Trent (1941) for their Light-Dark set-up. The
disparity in terms of the MI for this study and that of
Solomon & Trent (1941) could be attributed to the
different light intensities.
By definition of periodicity by Solomon & Trent (1941),
which is the occurrence of waves or peaks for the
Science Diliman (January-June 2011) 23:1, 43-51
Figure 2. Plot of the Mitotic Index for each hour of the day obtained from the second trial showing periodicity for Dark
and Light while rhythmicity for Light-Dark set-up. Error bar corresponds to standard error.

Matias, AMA and Fontanilla, IKC
48
mitotic activity at different times of the day, the MI for
A. cepa var. aggregatum is periodic in both Dark and
Light set-ups. The maxima for both were distinct and
higher than the other points. On the other hand, the MI
of the Light-Dark set-up appeared to be rhythmic,
defined by the occurrence of hourly fluctuations in
mitosis.
In this study, we tested how light could cause these
variations in mitosis. Using ANOVA, it was found that
there was no significant difference between each
treatment for each hour in Trial 2 (Table 2).
Furthermore, the significant difference observed in Trial
1 did not justify the significance of light in defining the
pattern for the mitotic activity, particularly as the
difference occurred only in two time periods, at 1 AM
and at 9 PM (Table 1). The Dark-set up and Light-set
up had almost similar pattern (periodic pattern). This
suggested that alteration of condition (i.e. from light to
dark) affects mitotic activity, thus showing different
 Tr ia l1
 Darkset‐upLightset‐upLig h t‐Darkset‐up
TimemeanMISDmeanMI SDmeanMI SD
12AM0.0270± 0.00100.0210± 0.00000.0247± 0.0042
1AM0 .01 97 ± 0.00800.0240± 0.00360.0117± 0.0006*
2AM0 .01 90 ± 0.00350.0140± 0.00460.0177± 0.0023
3AM0 .01 73 ± 0.00290.0167± 0.00400.0170± 0.0046
4AM0 .01 63 ± 0.00210.0143± 0.00810.0107± 0.0015
5AM0 .01 50 ± 0.00440.0130± 0.00300.0113± 0.0059
6AM0 .01 53 ± 0.00250.0127± 0.00420.0200± 0.0046
7AM0 .01 80 ± 0.00600.0120± 0.00560.0137± 0.0015
8AM0 .01 53 ± 0.01240.0167± 0.00740.0137± 0.0029
9AM0 .01 53 ± 0.00850.0137± 0.00650.0140± 0.0046
10AM0.0173± 0.00810.0173± 0.00670.0170± 0.0053
11AM 0.0333± 0.01600.0173± 0.00420.0150± 0.0046
12PM 0.0197 ± 0.00350.0310± 0.0130 0.0230± 0.0072
1PM0.0160± 0.00350.0217± 0.00840.0140± 0.0035
2PM0.0133± 0.00720.0157± 0.00400.0080± 0.0044
3PM0.0157± 0.00600.0120± 0.00870.0183± 0.0060
4PM0.0120± 0.00100.0167± 0.00550.0090± 0.0030
5PM0.0163± 0.00420.0107± 0.01070.0167± 0.0102
6PM0.0173± 0.01360.0160± 0.00610.0140± 0.0062
7PM0.0187± 0.00290.0183± 0.00590.0110± 0.0035
8PM0.0250± 0.00780.0257± 0.01340.0150± 0.0044
9PM0.0177± 0.00250.0230± 0.00350.0133± 0.0040*
10PM 0.0217 ± 0.00760.0157± 0.0031 0.0173± 0.0006
11PM 0.0167 ± 0.00640.0143± 0.0035 0.0160± 0.0035

* corresponds to set-up in which there is significant difference (á=0.05)
Table 1
The average Mitotic Index for each hour and set-up obtained from Trial 1 starting from 12 AM to 11 PM.
ANOVA was used to determine the difference between each set-up for each hour.
Values expressed as Mean±SD (Standard Deviation)
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49
patterns. Similarly, the effect of light could have been
due to the previous light/dark regime in which cells were
exposed; exposure to light after cell division could
therefore have prevented further divisions (Nemota &
Furuya 1985).
In addition to light, cell division is also regulated by cyclin
and cyclin-dependent-kinase (CDK) proteins, which
increases activity of CDK and release the transcription
factor for genes of DNA replications, respectively.
Moulager et al. (2007) suggested that cyclins and
CDK’s are under circadian control. These proteins, in
turn, are induced by phytohormones, particularly
cytokinin. Cytokinin is said to regulate cell cycle at both
the G1/S phase and G2/M phase progressions (Zhang
et al. 2004). Cytokinin concentrations also vary across
different times of exposure to light (Nova’kova et al.
2005). The role of cylins, CDK’s and cytokinins in the
rate of cell division should therefore not be ruled out.
 Tri al 2
 Darkset‐upLightset‐upLight‐Darkset‐up 
TimemeanMI SDmeanMISDmeanMI SD
12AM0 . 02 67 ± 0.00210.0203± 0.0081 0. 0 21 0± 0.0087
1AM0 .01 50 ± 0.00350.0137± 0.00 46 0.0 14 3± 0.0045
2AM0 .01 27 ± 0.00210.0137± 0.00 25 0.0 19 0± 0.0062
3AM0 .01 47 ± 0.00830.0127± 0.00 31 0.0 10 7± 0.0055
4AM0 .01 67 ± 0.00290.0157± 0.01 16 0.0 13 0± 0.0053
5AM0 .01 63 ± 0.00570.0050± 0.00 44 0.0 08 7± 0.0042
6AM0 .01 23 ± 0.00400.0137± 0.00 35 0.0 19 0± 0.0090
7AM0 .01 20 ± 0.00720.0113± 0.00 55 0.0 14 7± 0.0015
8AM0 .00 77 ± 0.00380.0140± 0.00 52 0.0 20 7± 0.0083
9AM0 .00 83 ± 0.00290.0117± 0.00 81 0.0 12 7± 0.0064
10AM0 . 01 47 ± 0.00500.0107± 0.0059 0. 0 21 0± 0.0010
11AM 0 . 01 07 ± 0.00550.0163± 0.0081 0. 0 11 0± 0.0069
12PM 0. 02 30 ± 0.00300.0243± 0.0074 0. 0 23 3± 0.0091
1PM0. 01 33± 0.00930.0133± 0. 00 25 0. 017 0± 0.0082
2PM0. 00 77± 0.00320.0100± 0. 00 46 0. 015 0± 0.0046
3PM0. 01 50± 0.00260.0080± 0. 00 66 0. 011 7± 0.0012
4PM0. 01 50± 0.00350.0070± 0. 00 26 0. 008 7± 0.0035
5PM0. 01 27± 0.00510.0133± 0. 01 03 0. 008 0± 0.0044
6PM0. 01 13± 0.00230.0087± 0. 00 15 0. 011 7± 0.0032
7PM0. 01 30± 0.00260.0180± 0. 00 26 0. 021 0± 0.0104
8PM0. 01 10± 0.00350.0170± 0. 00 26 0. 014 7± 0.0049
9PM0. 01 43± 0.00150.0170± 0. 00 46 0. 017 7± 0.0065
10PM 0. 01 27 ± 0.00150.0183± 0.0040 0. 0 17 7± 0.0015
11PM 0. 02 20 ± 0.00850.0090± 0.0060 0. 0 16 7± 0.0025

No significant difference in all groups (á=0.05)
Table 2
The average Mitotic Index for each hour and set-up obtained from Trial 2 starting from 12 AM to 11 PM.
ANOVA was used to determine the difference between each set-up for each hour.
Values expressed as Mean±SD (Standard Deviation)
Science Diliman (January-June 2011) 23:1, 43-51

Matias, AMA and Fontanilla, IKC
50
CONCLUSION
Even though it was not shown that light affects the
pattern of mitotic activity, the periodicity and rhythmicity
of mitosis was still evident in the experiment. In this
study, the duration of exposure to light at 70-171.5
Fcandle illumination did not affect the mitotic activity
in the root tips of A. cepa var. aggregatum. However,
a pattern of mitotic activity was still seen, and these
were periodic for the Light and Dark set-ups and
rhythmic for the Light-Dark set-up. The mechanism
causing these patterns is still not clear but is being
suggested to be controlled by the cyclin and CDK
proteins as well as the plant hormone cytokinin.
These observations have practical applications for the
Allium test, particularly in the usage of A. cepa. var.
aggregatum, a close relative of A. cepa, as model
organism. Root excision is suggested to be done around
11 AM – 1PM where MI is highest; this time period
also falls within the range given by Jong (1997) for
root excision of A. cepa.
ACKNOWLEDGEMENTS
The authors wish to thank the Institute of Biology,
particularly the Genetics Research Laboratory, for the
use of equipment and chemicals, and Ms. Erika Alvero,
Dr. Janet Puzon and Dr. Lilian Ungson for their helpful
comments on the study.
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