`

ESSENC
CE - Interna
ational Jourrnal for Envvironmentaal Rehabilitaation and Conservation
n
Volume V: No. 2 2014 [1
126 – 132] [ISS
SN 0975 - 62722]
[ww
ww.essence-jouurnal.com]

Proceedings of
o National Coonference
“Environmental Conserrvation and Cllean India Proogramme” Deccember 2014, India
I

mium (Crr) in Eleusiine coracaana - Mitootic analysis

Genotooxic Effectts of Chrom

mar1, Shalab
Anil Kum bh Gupta2, G.K.
G Dhingraa1 and Poojaa Kukreti1

Received: October 04, 2014 ~ Acceepted: Decem

mber 11, 20144 ~ Online: December
D 31, 2014

Abstractt Keyy Words: Genotoxicity

G | Eleusine coracana
| Chhromium | Mitotic effects
The purppose of the current
c workk was to assess
the genotoxic effect of Chromiuum (Cr) in one o Intrroduction
of the moost economiically imporrtant crop plant Conntamination of soil in cultivated
c fi
fields by
of Garrhwal, Eleeusine corracana. Tw wo induustrial effluuents loadedd with toxicc heavy
accessionns of E. coracana werre subjected to mettals has em merged as a new thhreat to
detailed root mitoticc studies. Thhe effect of Cr agriiculture. Moost of the effluents andd wastes
on roott growth varied with w different conntain heavy metals in ann amount suufficient
concentraations used. No roots weere availablee in enough to caause toxicitty to cropp plants
10-1 M and 10-2 M duringg the whole (Huutchinson annd Whiteby, 1974, Tem mple and
experimeent. At 10-3M,M the Mitootic Index (M MI) Biseessar, 1981;; Khan et all., 2006). Exxcessive
was extremely low (0.017
( in E11 and 0.008 in accuumulation of o heavy metals
m like nickel,
E2). In thhe course off mitosis various anomallies copper, cobalt, etc. in sooil originatinng from
viz. fraggmentation, clumping, stickiness of mettal mining and proccessing andd other
chromosoomes, rando om groupingg at anaphaase, techhnological human acttivities havve been
chromosoome bridgees at anapphase, and c- repoorted on a number off occasions (Foy et
metaphasse were observed mostly. Most of the t al.,11978; Maliszzewaska et al.,
a 1958).
-3
anomaliees were ob bserved at 10 M Cr Vonn Rosen (19554) groupedd Cr into verry strong
concentraationinboth the accesssions of E. activve heavy metal
m category. The chemical
c
coracanaa. sym
mbol of Chhromium iss Cr. The atomic
For correespondence: num
mber is 24. The relativve atomic mass is
1
Deptt. off Botany, Govt. College
C Rishikessh, (U.K.), Indiaa 51.9996g. Its dennsity is 7.199g/cm3 at 200°C. The
2
Deptt. off Botany, Govt. College
C Karanprrayag, (U.K.), Inndia boilling point annd melting point of Cr iss 2672°C
Email: singhaniya_akr@ @yahoo.com
and 1857°C resspectively. TheT vapor preessure is
-66
10 Pa at 844°C C and it is solublein diluted
126
 Kumar et al. /Vol.
/ V [2] 20114 /126 – 132

hydrochlloric and sullphuric acidd. Cr is present filteer papers) with

w cotton pads ‘sanddwiched’
everywheere and can be found inn three formss: - betw ween them m equidisstantly in various
metal orre, trivalentt chromium m (Cr III)aand treaatments in petri dishes and
a incubateed under
o
hexavalennt chromium m (Cr VI). Chromium is whiite light at 22 C. Ten molar soluutions of
used tommake steel an
nd other alloys, for chrom me C 2O7 rangingg between 100-10M to 10-1M were
K2Cr
plating, and
a as an additive
a to liimit corrosioon. preppared. The control seets were raaised in
Its comppounds are colored hence given the t disttilled waterr and treatedd sets were raised
r in
name chrromium. Hexavalent chrromium occuurs various molar cooncentrations of K2Cr2O7. Only a
naturally, notably
y in w
water-saturatted fixeed amount (about 50 ml) m of distilleed water
conditioons and it is an indicaator of hum man or CrC solutionss was addedd in petri disshes. The
pollutionn. This form
m is relatively soluble and
a variious molarr concentrattions of Cr C were
can moove more readily from fr soil to preppared in Hoaggland’s soluttion
groundwaater. For mitotic studdies, 20 roott tips of conntrol and
Materialls and Meth
hod treaated seedlinggs of E. coraacana were fixed in
The Eleeusine cora acana (Fingger millet) is 3:1 absolute ethanol and glacial aceetic acid
commonlly known as a Mandwaa or Kodu in having a pinch of ferric chlooride. The fiixed root
Garhwal, belongs to th he family Pooaceae. Eleussine tip samples weere stored in 70% ethhanol in
coracanaa, a species native
n to troppical East Afrrica refriigerator. Aftter 24 hours of fixation, the root
is a short stemmed, drry land adapteed, millet withw tips were boiledd in 3% acetoo-carmine andd left for
excellent storage charaacteristics andd an outstandding overrnight. Afterr that these were smeaared and
mineral coontent (Gaur,, 1999). squaashed in 1.5% aceto-carm mine. The following
fo
paraameters weree used for thee analyses:
For the present
p study
y, the seeds were procurred
from twoo local sourcees as followss: a) Numbeer of non-divviding cells,
b) Numbeer of dividingg cells,
Accessioons Placee
c) Numbeer of activelyy dividing cellls, and
E1 Ch
haubattakhal, 1800m asl d) Percentt frequency of
o mitotic anoomalies.

E2 Uttarkashi,
U 12200 m asl Mittotic index was also calculated
c b using
by
following formuula:
nt accessions of E. coracanna.
ble 1. Differen
Tab
Num mber of cellls in mitosis (actively dividing
asl = aboove sea leveel cellls)

The seedds of each accession of o E. coracaana

Number of cells in mitosis (actively
(E1, E2) were first suurface steriliized with 0.11%
diividing cells)
mercuric chloride (HgCl
( 2) sollution for 3-43
Mitootic index = Total numberr of cells (nonn-dividing
minutes and washed d thoroughly and then withw
cells + diviiding cells + actively
distilled water. Thee seeds weere placed on
dividing cells)
double laayered filter papers (3 mm,
m Whattm man
127
 Kumar et al. /Vol.
/ V [2] 20114 /126 – 132

Results and
a Discusssion
Both thee accession ns of E. coracana were
subjectedd to detailed root mitootic studies in
control and
a treated plants.
p The effect
e of Cr on
root g
growth varied
v wiith different
concentraations used. No roots weere availablee in (a) Prophaase (b) Meetaphase
10-1 M and 10-2 M duringg the whole
experimeent. The mean data reelated to noon-
dividing cells (NDCC), dividing cells
c (DC) and
a
actively dividing cellls (ADC) is given in tabble
2.
Mitotic Index (MI)) was also calculated by
formula given in maaterials and methods. The T (c) Stickiness at anaphase
a (d) Stickinesss at anaphase &
metaaphase
mitotic index
i (MI) reflects thee frequency of
cell diviision and is i regarded as important
parameteer for deterrmining thee rate of rooot
growth. AtA 10-3M, th he MI was extremely low
(0.017 inn E1 and 0.0 008 in E2). The graphiical
representtation of mitotic
m indexx in different
concentraations in both the acccessions of E. (e) Stickiness at anaphase
a (f) Bridge at anaphase
coracanaa is shown in n figure 1. Inn the coursee of
mitosis, various
v anommalies were observed. The T
frequencyy distributio
on of differennt anomaliess in
different concentrattions of Crr in both the t
accessionns of E. coraacana is shoown in tablee 3.
Followinng mitotic annomalies weere observedd in
treated pllants: (g) Fragmenttation (h) Random
m grouping at
metaaphase
a) Fragmentatiion (plate 1 g),
g
b) Stickiness of chromoosomes (pllate
1c,d,e),
c) Random gro ouping at anaaphase (plate 1
h),
d) Chromosom me bridges at anaphaase
(plate 1 f), and
a
(i) C-metaphase
e) C-metaphase (plate 1 n, i).
Platte 1 (a-i): M
Microphotograpphs showing different mitootic
a
anomalies in Eleusine
E coracaana

128
 Kumar et al. /V
Vol. V [2] 2014 /12
26 – 132

E1 E
E2
Cr Concentration Parameter
Mean ± SE Range Mean ± SE Rang
ge
NDC 44.10 ± 1.19 32.00
3 - 61.000 44.43 ± 1.13 32.00 - 58.00
Control DC 1.43 ± 0.10 1
1.00 - 3.00 1.47 ± 0.10 1.00 - 3.00
ADC 1.59 ± 0.15 1
1.00 - 4.00 1.87 ± 0.16 1.00 - 4.00
NDC 40.25 ± 1.82 1
18.00 - 65.000 45.38 ± 1.17 35.00 - 60.00
10-10M DC 1.92 ± 0.17 1
1.00 - 4.00 1.50 ± 0.10 1.00 - 3.00
ADC 1.56 ± 0.12 1
1.00 - 3.00 2.00 ± 0.17 1.00 - 4.00
NDC 36.93 ± 1.95 1
15.00 - 62.000 47.10 ± 1.04 35.00 - 60.00
10-9M DC 1.81 ± 0.17 1
1.00 - 4.00 1.06 ± 0.09 0.00 - 2.00
ADC 1.57 ± 0.12 1
1.00 - 3.00 1.65 ± 0.19 0.00 - 4.00
NDC 40.68 ± 1.85 2
22.00 - 64.000 48.30 ± 0.86 37.00 - 60.00
10-8M DC 2.00 ± 0.16 1
1.00 - 4.00 0.88 ± 0.09 0.00 - 2.00
ADC 1.52 ± 0.09 1
1.00 - 3.00 1.31 ± 0.19 0.00 - 4.00
NDC 36.43 ± 1.33 1
19.00 - 56.000 49.95 ± 0.80 37.00 - 60.00
10-7M DC 1.73 ± 0.15 1
1.00 - 4.00 0.71 ± 0.09 0.00 - 2.00
ADC 1.50 ± 0.11 1
1.00 - 3.00 1.00 ± 0.15 0.00 - 3.00
NDC 46.60 ± 1.26 3
35.00 - 66.000 50.65 ± 0.90 37.00 - 65.00
10-6M DC 2.19 ± 0.17 1
1.00 - 4.00 0.76 ± 0.11 0.00 - 2.00
ADC 1.39 ± 0.08 1
1.00 - 2.00 1.04 ± 0.14 0.00 - 3.00
NDC 48.08 ± 1.43 3
32.00 - 66.000 51.10 ± 0.90 37.00 - 65.00
10-5M DC 1.95 ± 0.17 1
1.00 - 4.00 0.76 ± 0.11 0.00 - 2.00
ADC 1.75 ± 0.14 1
1.00 - 4.00 0.77 ± 0.13 0.00 - 3.00
NDC 52.35 ± 1.32 4
40.00 - 70.000 51.60 ± 0.88 40.00 - 65.00
10-4M DC 1.60 ± 0.19 0
0.00 - 4.00 0.73 ± 0.11 0.00 - 2.00
ADC 1.58 ± 0.13 1
1.00 - 3.00 0.55 ± 0.11 0.00 - 2.00
NDC 56.30 ± 1.19 4
41.00 - 70.000 52.70 ± 0.74 45.00 - 65.00
10-3M DC 1.16 ± 0.19 0
0.00 - 4.00 0.82 ± 0.12 0.00 - 2.00
ADC 1.00 ± 0.14 0
0.00 - 3.00 0.42 ± 0.10 0.00 - 2.00
Table 2: Mean data relaated to non-dividinng cells (NDC), div
viding cells
(DC) and actively
a dividing cells (ADC) in n different
Accessions off Eleusine coracanaa.

129
 Kumar et al. /V
Vol. V [2] 2014 /12
26 – 132

Accession Cr
C Concentration a b c d e f g h i j
Control 8.33 4.17
4 4.17 0.00 8.33 0.00 0.00 0.00 29.17 0.00
10-10M 14.29 0
0.00 0.00 0.00 8.33 0.00 0.00 0.00 16.67 0.00
10-9M 0.00 4
4.17 0.00 0.00 8.33 0.00 0.00 0.00 41.67 0.00
10-8M 18.75 4
4.17 12.50 0.00 8.33 0.00 8.33 0.00 20.83 0.00
E1 10-7M 36.36 4
4.17 0.00 0.00 12
2.50 0.00 0.00 0.00 12.50 0.00
10-6M 28.57 0
0.00 12.50 0.00 8.33 0.00 8.33 0.00 12.50 0.00
10-5M 31.25 4
4.17 12.50 0.00 8.33 0.00 8.33 0.00 12.50 0.00
10-4M 21.74 8
8.33 20.83 0.00 4.17 0.00 12.50 0.00 29.17 0.00
10-3M 16.13 299.17 29.17 0.00 0.00 0.00 20.83 0.00 29.17 0.00
Control 0.00 4
4.17 0.00 0.00 8.33 0.00 0.00 0.00 29.17 0.00
10-10M 0.00 0
0.00 0.00 0.00 8.33 0.00 0.00 0.00 33.33 0.00
10-9M 0.00 0
0.00 0.00 0.00 4.17 0.00 0.00 0.00 29.17 0.00
10-8M 0.00 4
4.17 0.00 0.00 8.33 0.00 0.00 0.00 29.17 0.00
E2 10-7M 0.00 8
8.33 20.83 0.00 8.33 0.00 0.00 0.00 25.00 0.00
10-6M 11.76 8
8.33 20.83 0.00 4.17 0.00 0.00 0.00 29.17 0.00
10-5M 16.67 4
4.17 20.83 0.00 8.33 0.00 0.00 0.00 20.83 8.33
10-4M 13.04 122.50 20.83 0.00 0.00 12.50 0.00 8.33 20.83 8.33
10-3M 11.11 122.50 33.33 4.17 0.00 12.50 0.00 8.33 20.83 8.33

Table 3: Mean data related to perceent frequency of different

mitotic abno
ormalities in diffeerent accessions of
o Eleusine
coracana.

a. Clump
ping f. Chroma
atin erosion
b. Restitu
ution Nucleus g. Chroma
atin fragmentattion
c. C- Mettaphase h. Laggard
ds
d. Random Grouping off Chromosomes i. Binuclea
ate condition
e. Late movements
m of ch
hromosomes to
o metaphase pla
ate j. Trinucleeate condition

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Cr Conceentration E1 E2 Thee formation ofo bridges coould be attriibuted to

Con
ntrol 0.034 0.039 chroomosome stickiness annd to chrom mosome
10-110M 0.036 0.041 breaakage and reeunion. The induction
i off lagging
-9
10 M 0.039 0.033 couuld be attribuuted to the failure
fa of thee normal
-8
10 M 0.034 0.026 orgaanization and
a functioon of the spindle
10-7M 0.038 0.019 apparatus. Suchh type of abnnormalities is i due to
-6
10 M 0.028 0.020 the loss of microtubule of spindle fibeers. This
10-5M 0.034 0.015 wass supported by previouss reports off several
-44
10 M 0.028 0.010 authhors (Patil and
a Bhat, 1992;
1 Salamm et al.,
10-33M 0.017 0.008 19993; Chidambaram et al., 2009).
-22
10 M 0.000 0.000
Thee mitotic inddex (MI) refflects the frequency
Table 4
4: Mitotic index
i (MI) in various of cell
c divisionn and is reggarded as im mportant
concentration
ns in differennt accessions
of Eleusine coracana
c paraameter for determiningg the rate of root
growwth. Heavy metal accum mulation in soil and
Mitoticc Index (M
MI) its importancce on thhe morphoological,
0..050 biocchemical annd cytologicaal aspects ofo plants
MI Values

0..040
0..030 havve received more
m attentioon in recent times
t by
0..020
0..010 E1 manny workerrs (Abbasii et al., 1992;
0..000
E2 Premmkumar et al.,
a 2001& 2014;2 Prakassh et al.,
20004).
In E.coracana,, the mitotiic index deecreased
Fig. 1 Graphical reppresentation of mitotic indeex
proggressively with the increase in Cr
(MI) in variouus concentratioons in differennt conncentrations in both the t accessions. In
accessions of Eleusine
E coraccana treaated roots thee mitotic inddex was founnd to be
At 10-3 M Cr con ncentration, most of the
t wer than the control. Att 10-3M, thee mitotic
low
anomaliees like chromosome stickineess, indeex was extrremely low (0.017 in E1 and
clumpingg, chromosome briddges and c- 0.0008 in E2).
metaphasse were observed in both the t
In the
t present studies,
s induuced toxic efffects on
accessionns of Eleusin
ne.
chroomosomes during
d cell division succh as c-
Cr inducced alterationn of nuclearr structure and
a mitoosis, anaphhase bridgess and chrom mosome
inhibitionn of cell diivision in roots
r has beeen stickkiness, are in agreemennt with the findings
observedd by many y authors (Levan,
( 19445; of Levan
L (19455) and Liu et al., (19992). The
Corradi ete al., 1991;; Villalobos--Pietrini et al.,
a preccocious moovement off the chrom mosome
1993). The
T most ap ppropriate reason
r for the
t migght have been causeed by thee early
increase in abnorm malities mayy be due to term
minalization,, stickiness of chromossome or
malfuncttion and inacctivation of number of key k because of the movement of the chrom mosome
enzyme for
f ion uptak ke and spinddle control.
131
 Kumar et al. /Vol.
/ V [2] 20114 /126 – 132

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