ALLIUM CEPA ROOT CHROMOSOMAL ABERRATION ASSAY: AN EFFICIENT TEST FOR EVALUATING GENOTOXICITY FROM DIFFERENT SOURCES OF SOIL

1. A PROJECT ON
ALLIUM CEPA ROOT CHROMOSOMAL
ABERRATION ASSAY: AN EFFICIENT TEST FOR
EVALUATING GENOTOXICITY FROM
DIFFERENT SOURCES OF SOIL
BY,
ARINDAM CHAKRABORTY
B.PHARM, 7TH SEM.
ROLL:20801914016 , SEC: I
UNDER THE GUIDANCE OF
Mr. SOUMYADEEP GHOSH(LECTURER )
DEPT. OF PHARMACEUTICS
BHARAT TECHNOLOGY.

2. INTRODUCTION
 In recent decades, soil has been exposed to a number of pollutants by
various natural and others activities. These contaminants in soil has
potential to pose severe health risk to humans through various routes of
exposure such as direct ingestion, contaminated drinking ground water
and food crops; dermal contact and through food chain. Plant bioassays
are relatively inexpensive; can be easily handled; more sensitive and
simpler than other methods used for detection of genotoxicity of
environmental pollutants. Plant roots are extremely useful in biological
testing because root tips are the first to be exposed to toxicants dispersed
in soil or in water.

3. SOIL POLLUTION IN INDIA
 Industrial sector in India is witnessing rapid growth since the last decade of
twentieth century with reforms in economic laws and with establishment of
special economic zones (SEZ). Such rapid industrial growth has also
increased threat to the environment. It indicates that soil resources are facing
threats from deliberate use of contaminated organics, amendment materials
and irrigation water or from atmospheric depositions, spillage of effluents
etc.
 EFFECTS OF SOIL POLLUTION:
1. Organ damage.
2. Skin disease.
3. Cancer.

4. IMPORTANCE OF CHOOSING Allium cepa AS
A BIOMARKER
 The importance of Allium cepa test contributes knowledge in preventing toxicity
in the environment. Onion (Allium cepa L.) a potential biomarker of genotoxic
studies. Widely used as a bio-indicator of genotoxicity from the different aquatic
environs. This test helps to evaluate mutagens and detecting toxic substances
found in the environment. Plant roots are extremely useful in biological testing
because root tips are the first to be exposed to toxicants dispersed in soil or in
water. Therefore, the root tip chromosomal aberration assays constitute rapid and
sensitive methods for biomonitoring the extent of pollution and to evaluate the
effects of toxic and mutagenic substances in the natural environment.

5. PLANT PROFILE
SYNONYMS: Onion, onion plant, peyaj
BIOLOGICAL SOURCE
Scientific name: - Allium cepa
Family: - Amaryllidaceae
Taste: - Very pungent to mild, even to sweet.
CHEMICAL CONSTITUENCES: Quercetin, fructose, quercetin-3-glucoside,
isorhamnetin-4-glucoside, xylose, galactose, glucose, mannose, organosulfur compounds,
allylsulfides, flavonoids, flavenols, S-alk(en)yl cysteine sulfoxides, cycloalliin, selenium,
thiosulfinates, and sulfur and seleno compounds.
GEOGRAPHICAL SOURCES: In 2014, world production of dried onions was 88.5
million tones, led by China and India producing 25% and 22% of the total, respectively. Not
only that Russia, USA, Bangladesh, Brazil also produces large number of onion.

6. COLLECTION OF SAMPLES
 Study area and collection of samples: The study area in present
investigation are: -
1. Uttarpara.
2. Hasnabad.
3. Uluberia.

7. METHODS
 ALLIUM CEPA ROOT CHROMOSOMALABERRATION ASSAY:
The common onion (Allium cepa) bulbs used for the present study were procured
from the local market. Young onion bulbs of appropriate size were denuded by
removing outer loose scales and scrapped at the bottom to expose root primordial.
The genotoxic potential of soil samples was estimated using Allium cepa root
chromosomal aberration assay by applying two modes of treatment in situ and root
dip.

8.  ROOT DIP TREATMENT:
The soil extracts were prepared by suspending soil in distilled water in ratio of
1:2 (w/v) on shake incubator for continues shaking for 24 hrs.
Filtration
The filtered extract was considered as 100 % and different concentrations of soil
extract were made
The denuded onion bulbs were placed on beaker containing distilled water for
rooting
After 72hrs, the emerged roots of about 0.5 - 1.0 cm length were treated for 3 h
Placing them on treatment beakers containing different concentrations of each
soil extract
Distilled water used as negative control

9.  CYTOLOGICAL INVESTIGATIONS:
After treatment, the bulbs were thoroughly washed, root tips were plucked and fixed in
farmer’s fluid (glacial acetic acid: ethanol=1:3) for 24 hrs
Preserved in 70 % ethanol and stored at lower temperature till further use
3 root tips per plants were cut using forceps at A length of 10 mm and placed into A
petri dish with 2 ml acetic acid and hydrochloric acid solution
Transfer A root to the slide using blade cut off most of the unstained part of the root and
discard it
The roots tips were then heated in water bath then incubate for 5 minutes
Remove the tube from water bath
Cover the root with the aceto carmine stain and incubate the root in the stain for 12
minutes
Remove the hcl from the test tube and rinsed the roots in water about 3 times.

10.  CYTOLOGICAL INVESTIGATIONS:
Cover the root tip by A cover slip then press down formally with one finger for
flattening the sample.
Rinse it
Put one more drop of stain and leave it for 1minutes
Cover the root tip by A cover slip
Examine under the microscope at low power and high power

11. OBSERVATION
CONCENTRAION(%) MEAN ROOT
LENGTH
NUMBER OF
DIVING CELLS
NUMBER OF
ABERRANT CELLS
MITOTIC
INDEX(%)
0 1.447± 1.072 -- -- --
1 0.227±0.024 436 31 40.5
2 0.197±0.005 376 33 34.3
3 0.113±0.657 289 37 25.2
CONCENTRAION(
%
MEAN ROOT
LENGTH
NUMBER OF
DIVING CELLS
NUMBER OF
ABERRANT CELLS
MITOTIC
INDEX(%)
0 8.447±3.072 -- - -
1 7.589±2.756 952 250 70.2
2 7.175±2.156 890 225 66.5
3 6.782±1.883 815 193 62.2
IN FORMALIN
IN WATER
(5000 cells (5 slides) per concentration of each water samples)

12. OBSERVATION
CONCENTRAION(%) MEAN ROOT
LENGTH
NUMBER OF
DIVING CELLS
NUMBER OF
ABERRANT CELLS
MITOTIC
INDEX(%)
25 3.192±1.312 460 28 43.2
50 2.819± 1.118 445 33 41.2
75 2.227 ± 0.912 422 39 38.3
CONCENTRAION(
%
MEAN ROOT
LENGTH
NUMBER OF
DIVING CELLS
NUMBER OF
ABERRANT CELLS
MITOTIC
INDEX(%)
25 3.187± 1.487 650 15 64.5
50 2.728 ± 1.217 635 19 61.6
75 2.867 ± 1.170 610 21 58.9
SAMPLE NO:1 (UTTARPARA)
SAMPLE NO:2 (HASNABAD)
(5000 cells (5 slides) per concentration of each water samples)

13. OBSERVATION
CONCENTRAION(%) MEAN ROOT
LENGTH
NUMBER OF
DIVING CELLS
NUMBER OF
ABERRANT CELLS
MITOTIC
INDEX(%)
25 3.348 ± 1.217 465 26 43.9
50 3.131 ± 1.129 437 31 40.6
75 2.753 ± 1.114 396 37 35.9
SAMPLE NO: 3 (ULUBERIA)
(5000 cells (5 slides) per concentration of each water samples)
Stages of mitotic division in root cells of Allium cepa in low power microscope and High power
microscope

14. OBSERVATIONS

15. OBSERVATION

16. RESULT & DISCUSSION
 Maximum root growth was observed in the control (8.447±3.072) and there were no
morphological deformities found.The roots were whitish in color, unbroken and straight.
 At tested concentrations, 1% was obtained the highest root growth from Water (7.589 ± 2.756)
and (0.227±0.024) in Formalin. It also recorded the highest dividing cells and fewer in a number
of aberrant cells in formalin.
 On the other hand, the 2% concentration attained the second highest root growth from water
(7.175 ± 2.156) and Formalin (0.197±0.005). It results to the second highest number of dividing
cells and fewer numbers of aberrant cells in formalin.
 While the 3% tested concentrations show the poorest root growth from water (6.782 ± 1.883)
and Formalin (0.113±0.657). Indeed, the suppression of mitotic activity constantly caused by
genotoxicity and cytotoxicity. Consequently, mitotic index is also a result of mito depression
during cell division.

17. CONCLUSION
 This study shows the importance of Allium cepa in testing genotoxic substances.
Hence, mitotic index is a marker of increasing and decreasing of cells during
mitosis. The indication of chromosomal and mitotic aberration in the root tip
cells suggests genotoxicity. The present study clearly indicates significant
genotoxic effects in soil. Allium cepa genetic material can be used to evaluate
the effects of potential genotoxic and cytotoxic substances in the environment.

18. REFERENCES
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19. THANK YOU..