Volume 3 (2) ™ April - June 2013 ISSN 2231 – 0304

International Journal of
BIOTECHNOLOGY AND
BIOSCIENCES

Dr. Riyaz Z. Sayyed

Editor-in-Chief
International Journal of Biotechnology and Biosciences
Volume 3 (2) ™ April - June 2013

CONTENTS

Editorial

BIODEGRADABLE PLASTICS FOR CLEAN ENVIRONMENT 71 - 75

Sayyed R Z

Original Paper

BACTERIAL RESISTANCE TO ANTIBIOTICS AND DISINFECTANTS : TODAY AND TOMORROW 76 - 82

Sarode Y N and Zodpe S N

STUDIES ON BACTERIOLOGY AND ANTIBIOGRAM OF PATHOGENS FROM WOUND INFECTIONS 83 - 88

Sirsat S V, Nemade S P, and Musaddiq M

PLANT PARASITIC NEMATODES IN AGRICULTURE AND HORTICULTURE 89 - 91

Kumari R

ISOLATION OF EFFICIENT PHOSPHATE SOLUBILIZING MICRO-ORGANISMS FROM SOIL SAMPLES OF 92 - 95

JALGAON, DISTRICT OF NORTH MAHARASHTRA, INDIA
Chaudhari S and Sayyed R Z

RHAMNOLIPID PRODUCTION BY PSEUDOMONAS AERUGINOSA ISOLATED FROM PETROL PUMP SOIL 96 - 100
Chitanand M P and Jadhav A R

BIODEGRADATIVE POTENTIAL OF BACTERIA FROM CONTAMINATED SOIL WITH REFERENCE TO 101 - 104
CHLORPYRIFOS
Rehman W and Khan S

STUDIES ON PROXIMATE COMPOSITION OF OREOCHROMIS MOSSAMBICUS FROM 105 - 108

TALKHADI LAKE, KHARBAV, BHIWANDI
Dalvie V V

COMPARATIVE ANALYSIS OF MITOTIC ABERRATIONS AND OTHER PARAMETER INDUCED BY 109 - 115
EMS, SA AND NMU IN HORSE GRAM [MACROTYLOMA UNIFLORUM (LAM) VERDCOURT
Kulkarni G B
 CONTENTS

LAND COVER CHANGE MONITORING OF PATHRI RESERVE FOREST 116 - 120

Srivastava N and Srivastava A

STUDY OF CYANOBACTERIA OF THE ACROTHERMAL SPRING AT VAKVALI UNHAVARE, 121 - 126

MAHARASHTRA, INDIA
Pathan A A, Chaudhari G S and Nandan S N

STUDY ON SPECIES DIVERSITY OF NOSTOCEAE IN JALGAON DISTRICT MAHARASHTRA, INDIA 127 - 131
Mahajan S R

BIOLOGICAL ANALYSIS OF WATER (GODAVARI RIVER) AT NASIK, MAHARASHTRA 132- 134

Patil Y S and Thakur H A
 Original Pape Int. J. Biotech Biosci ISSN 2231-0304
INTERNATIONAL
Kulkarni and Pardhe 800K HOUSE I>. LTO.
smc.111Jr Vol 3 (2): pp 109-115 (2013) .

COMPARATIVE ANALYSIS OF MITOTIC ABERRATIONS AND OTHER

PARAMETER INDU.CED BY EMS, SA AND NMU IN HORSE GRAM
[MACROTYLOMA UNIFLORUM (LAM) VERDCOURT]

Kulkarni Ganesh B*
Botany Department, J.E.S. R.G. Bagdia Arts, S.B. Lakhotiya Comm. & R. Bezonji Science Colli
Jalna, Maharashtra 431203 India
E-mail*: dr.kgb98@gmail.com

Abstra�t
The present investigation provide a comparative account of the effect of chemical mutagens of different
concentrations of EMS, SA and NMU on mitotic aberrations, seed germination, seedling survival, plant height
and mitotic index in Horse gram [Macrotyloma uniflorum Lam. (Verdcourt)]. Among all the mutagens, EMS
was found to be more effective for the induction for chromosomal aberrations, seed germination and survival
percentage of seedling than to SA and NMU Mitotic index and· other parameter reduced gradually as the
different concentration of mutagen increased. The cytological study revealed various types of mitotic
· aberrations among them dominant were fragments, stickiness, precocious movement, laggards, bridges, macro­
and micronuclei more frequents.

Key words: EMS, SA, NMU, Horse gram, Mitotic index, Mitotic aberrations.

INTRODUCTION
The legume, members of family Fabaceae produces seeds
that contain 2-3 times proteins than the cereals. A human
nutrition consumption point of view, the Fabaceae stood
second most important family in concern with the content
of protein. Horse gram [M uniflorum, (Naik, 2000)] is a
multipurpose, rich protein crop, cultivated in India,
Pakistan, Bangladesh, Srilanka, Nepal and West Africa. In
India Horse gram is sown late in the rainy season by poor
former, tribal localities, in marginal, drought prone areas
of Maharashtra. Although, it is farmer's as well as
researcher's neglected crop.Making hybridization is a less
attractive and time consuming route for crop improvement.
Mutation breeding has become increasingly popular in
recent times as an effective tool for crop improvement
(Acharya et al., 2006). Mutation breeding supplement
conventional plant breeding as a source of increasing
variability and could confer specific improvement without
significantly altering its acceptable phenotype (Ojomo et
al., 1979). Some legumes crops that have been improved
through mutation breeding are soybeans, Mungbeans, Urad
beans, French beans, peas and Fenugreek (Petropolos,
1973, 2002).
Horse gram, though it is self pollinated, there is a
need to create variation to facilitate genetic improvement
by mutation breeding. Chemical mutagen provides a good

Vol 3 (2) :. April - June 2013 109

 w.ry
IilTERNA$ON.fi.L
eook H(rusE F. r-flr"
scope for selection, as a tool for introducing alterations in
the genotype to enhance the variability of characters.
Cytological analysis with respect to mitotic behavior is one
of the most dependable indexes to estimate the potency of
mutagens.
The main objective of the present experiment was
to investigate the comparative chromosomal aberration
affected by EMS, SA and NMU on mitotic division in root
tip cells ofhorse gram and to explore their possible genetic
consequences and co relation with seed germination,
seedling survival and their potential to induce genetic
variability which may prove asset for cytogenetic and plant
breeders for the improvement of horse gram. The mitotic
variations also induced in root tip cells of horse gmm may
be useful in studying the mode of action of mutagen as
well as inducing the cyto-morphological mutation on later
stage. The successful utilization of sodium azide to
generate genetic variability in plant breeding has been
reported in barley (Kleinhofs and Sander 1975) and other
crops (Avila and Murty, 1983; Micke, 1988; Routaray, e/
a|.,1995).
The present paper deals witheffect of these
mutagen onthe mitotic aberrations, seed germination,
seedling survival, plant height and mitotic index in Horse
gram.
MATERIAL AND METHODS
The dry, healthy and uniform sized seeds of Horse
gram lMacrotyloma uni/lorum (Lam) Verdcourt], were
pre-soaked in distilled water for 6 h, and then treated with
different concentrations of (0.1, 0.2 and0.3%) EMS, (0.05,
0.1 and 0.15%) SA and (0.4, 0.6 and 0.8%) NMU. The
solutions were prepared in sodium phosphate buffer with
7.0 pH for 8 h with constant intermittent shaking. The
treated seeds were washed in running tap water I to 2 h to
remove the residual effect of the mutagen sticking to the
seed coat. One set ofthe seeds was kept untreated to act as
control for comparison. Hundred seeds were used from
each dose and control. In order to find out the effect of
EMS, SA and NMU on seed germination and root tip
collection, 50 seeds were spread over moist Whatman's
filter paper no.l petri plates. The root tips were excised
(2mm) and washed with distilled water, fixed in Glacial
Acetic Acid (GAA): Ethanol (1:3) for 24 h, again washed
with distilled water and stored in 70%o alcohol. For
cytological observations, the fixed roots were hydrolyzed
at 60 'C for 15 min. in lN HCl. After washing the tips
were transferred to %olron alum for 30 min as a mordant
and then transferred to 2%o Aceto Hematoxylene stain for
th. The squashing was done in 45Yo GAA. The slide
showing the uniform spreading was sealed with wax and
110
screened Mitotic study. Mitotic index (MI) were calculated
by
MI = Total number of dividine cells X 100
Total number of cells examined
The other parameter such as seedling survival and
plant height were calculated with the help of field data.
The experimental data were statisti cally analyzed using the
analysis of variance techniques according to Panse and
Sukhatme (1989).
General information of used mutagen:
Ethyl Methane Sulfonate (EMS) C3H8O3SI is a
mutagenic, teratogenic and possibly carcinogenic organic
compound. It produces random mutations by nucleotide
substitution, particularly by guanine alkylation and
forming the abnormal base O-6-ethylguanine. It can induce
mutations at a rate of 5xl0a to 5x10-2 per gene without
substantial killing. During DNA replication, DNA
polymerases that calalyze the process frequently replace
the G:C base pair to an A:T pair. These changes became
harmful to cells and can result in disease.
Sodium Azide [(i.e. Potassium azide and Ammonium azide
NaN3 ] is the inorganic compound which is colourless
azide salt is used for the preparation of other azide
it is highly soluble
compounds. Being an ionic substance,
in water and extremely toxic.Sodium azide is an ionic
solid. Two crystalline forms are known, rhombohedral and
hexagonal. The azide anion is very similar in each, being
centrosymmetric with N-N distances of 1.18 A. The Na*
ion is pent coordinated. Sodium azide is a useful probe
mutagen. Azide inhibits cytochrome oxidase by binding
irreversibly to the heme cofactor in a process similar to the
action of carbon monoxide.
N-Nitroso-N-Methyl Urea tNIvflJ) CzHsN:Oz I is a highly
reliable carcinogen, mutagen, and teratogen. NMU is an
alkylating agent, and exhibits its toxicity by transferring its
methyl group to nucleobases in nucleic acids.
RESULT AND DISCUSSION
The effect of all the chemical mutagens on Mitotic
Index (MI), Chromosomal Aberrations of Horse gram is
represented in Table I and dose dependent reduction was
observed in seed germination, seedling survival and plant
height ware noted in Table 2. Maximum germination was
recorded in control (84.45%), whereas it decreased in
treated populations from 68.44 to 42.38 % in (0.1- 0.3%)
EMS; 61.27 to 48.71 % in (0.05- 0.15%) SA and 78.74 to
56.03 % in (0.4 - 0.8%) NMU. In the present
investigation, the mutagens EMS, SA and NMU show the
decrease in reduction percentage of seed germination, as
increase in concentration. EMS shows more effective for
lnternational Journal of Biotechnology and Biosciences
 COMPAMTIVE ANALYSIS OF MITOTIC ABERMTIONS AND OTHER PARAMETER INDUCED
BY EMS, SA AND
NMU IN HORSE GRAM [MACROTYLOMA UNIFLORUM (LAM) VERDCOURT]

Table 1. Showing frequency of various abnormalities by treated with different concentration

of EMS, SA
and NMU in root tip cells of horse gram lMacrotytoma uniflorum (Lam) Verdcourtl.

Concen- Total Total no. Mitotic Abnormalities (7o) Total%o

Tration no. of of Divi- Index
(%) Bi- Clump Preco Lagg- Chro Re- Stray Frag of
cells ding cells o/o
nucleolat ing cious ards moso orien- Abnorm
Obser Observed Chrom ments
e (macro alities
ved move mal tation o-somes
& micro) ment Bridge
s s
Control 2551 776 30.41 0.00
EMS 2035 476 22.94 0.27 0.23 0.13 0.01 0.09 0.12 0.8s
0.1
0.2 2194 343 15.63 0.34 0.38 0.32 0.02 0.12 0.33 t.57
0.3 2245 2r7 09.66 0.51 0.44 0.55 0.28 0.1 0.31 0.70 2.89
SA 2402 474 t9.73 0.2s 0.34 0.12 0.34 0.09 0.1'1 0.21 t.02 2.54
0.0s
0.1 2581 367 14.21 0.39 0.51 0.34 0.22 0.25 0.35 1.94 3.98
2432 227 9.33 0.46 0.63 0.54 0.39 0.34 0.41 0.24 2.60 5.61
0.15
NMU 2342 568 24.25 0.17 0.41 0.04 0.1 0.28 0.12 0.97 2.09
0-4
2472 465 18.81 0.23 0.82 0.17 0.09 0.13 0.48 0.12 1.21 3.3s
0.6
2406 343 t4.26 0.41 0.39 0.26 0.15 0.22 0.66 0.37 1.67 5. l3
0.8

Table 2. Comparative account of mitotic index, seed germination and seedling survival by EMS,
sA and NMU of horse gram lMacrotyloma aniflorum (Lam) verdcourtl.

Concen- Mitotic Seed S.E. Seedling S.E. Plant s.E.

Tration Index germination (+) survival (+) height (+)
(%) o/
/o (%) (%\ (cm)
Control 30.41 84.45 0.41 91.37 1.03 122.24 0.72
EMS 0.1 22.94 68.44 0.33 81.67 0.77 107.64 0.s4
0.2 t5,63 5s.17 0.09 74.4s 1.67 96.71 0.83
0.3 09.66 42.38 0.76 62.24 0.82 83.55 0.96
sA 0.0s 19.73 61.27 0.89 88.44 t.t2 103.41 1.62
0.1 14.21 54.55 1.06 5.55 0.90
7 84.82 0.43
0.15 9.33 48.71 2.14 67.77 0.66 72.12 0.86
NMU 0.4 24.25 78.74 0.47 84.62 0.76 118.35 0.96
0.6 18.81 62.66 0.81 79.07 0.45 98.03 1.29
0.8 14.26 56.03 0.93 71.12 l.l I 80.78 0.77

Vol 3 (2) .'. April - June 2013

111
 WP
IT'ITEflHATIONAL
B{)OK HOIJSE F. LTD.

Fig 1. Showing the graph of Mitotic Index ("/r') and total o/o of Abnormalities by treated with different
concentration of EMS, SA and NMU in root tip cells of Horse gram lMacrotyloma uniflorum (Lam) Verdcourtl.

s Mitotic lndex % r Abnormalities %

35

30

25

20

15

10

0
:AN
:- > t.,{.
!.1
(ft
<g = g P= 3-
-,o 3*
a :a 2. Z.
r U
-:
r f-l- a a Z-d
/-z
\J
-r-l ra
a
H

Fig 2. Graph Showing the comparison between various parameter viz Mitotic Index (oh),total7o of abnormalities,
Seed germination o/o and Seedling survival 7o of various treated concentration of EMS, SA and NMU on Horse
gram lMacrotyloma uniflorum (Lam) Verdcourtl.

ffi Mitotic lndex % : Seed germination (%)

tu Seedling survival (%) m Plant height (cm)

r.o
C,
f
z

tt2 I nternationa I Journal of Biotech nology and Biosciences

 COMPARATIVE ANALYSIS OF MITOTIC ABERRATIONS AND OTHER PARAMETER INDUCED BY EMS, SA AND
NMU rN HORSE GRAM IMACROTYLOMA UNIFLORUM (LAM) VERDCOURT]
inhibition of seed germination as compare to SA and
NMU.
Similar results were observed by Rank and
Nielsen (1997) in Allium cepa, by Bhat et al. (2007),
Siddiqui et al. (2007) in Trigonella foenumgraecum L.and
also in Vicia faba L. by Agarwal and Ansari (2001).
Srivastava (1979) Mensah and Obadoni (2007)inArachis,
and Goyal and Khan (2010) in Urdbean with all chemical
mutagen considered that reduction in germination
percentage was due to weakening and disturbance of
growth processes resulting in early elimination of
seedlings.
Mitotic Index was observed in percent study
22.94, 15.63, 9.66 in 0. l, 0.2, 0.3 Yo conc. of EMS, 19.73,
14.21, 9.33 in 0.05, 0.1, 0.l5oh conc. of SA and 24.25,
18.81, 14.26 in 0.4, 0.6, 0.8 Yo conc. of NMU respectively.
Similarly total percentage of abnormalities were 0.85, 1.57
and2.89 in EMS, 2.54,3.98,5.61 in SA and 2.09, 3.85 and
5.13 in NMU for similar concentration. There is negative
relationship between mitotic index and percentage of
abnormalities as previously noticed by many workers such
as Banerjee (1992) effect of Tobacco on Allium and Anis
and Wani (1992) in Fenugreek.
The behavior of chromosomes at different stages
was observed normal in control. As a result of EMS, SA
and NMU treatment, multivalent formation at metaphase,
stickiness and precocious movements, clumping of
chromosomes at metaphase, fragments and single and
double bridges at anaphase occurred in different
frequencies (i.e. % of abnormalities ) and increased with
increasing concentrations of mutagen.
As a result of mutagenic treatment of seed with
EMS, SA and NMU, the root tip cells of horse gram
showed varying degrees of mitotic inegularities like
stickiness, fragments, , precocious movements of
chromosomes, bridges with or without fragment which
generally increased with the increasing concentrations of
EMS, SA and NMU. Similar results were noted by Bose
and Saha (1970) in tomato, Roy er al. (1971) in Cucumis
sativus L, Hassan and Ahmad (2000) in Allium and Khan,
et al. (2009) variations in Cichorium.
The clumping of chromosomes was the most
common abnormality observed during the present
investigation. Bivalents were found clumped into a single
group due to stickiness. Similar results have also been
reported by Abbasi and Anis (2002) in Trigonellafoenum-
graecum. Stickiness could be due to de-polymerization of
nucleic acid caused by mutagenic treatment. Stickiness in
chromosomes is a result of partial dissociation and altered
pattern of organization of nucleoprotein.
Vol 3 (2) .'. April - June 2013
Presence of chromosome bridges may be due to
stickiness or formation of dicentric chromosomes caused
by breakage and reunion (Bempong, 1973). The stickiness
of chromosomes makes their separation and free
movement incomplete and thus they remain connected by
bridges in Allium cepa (Kabarity et al., 1974). This may
also be due to the defective formation of the spindle
apparatus. This is justified by Badr (1986) Abraham and
Rajalakshmi (19S9) in V. faba, and Adegoke (198a) in
Allium cepa. Similarly Goyal and Khan (2010) in Urdbean
reported that sodium azide induces chromosomal damages
leading to bridge formation during mitotic division and
hence increased phenotypic aberration.
Fragmentation might have arisen due to stickiness
of chromosomes and consequent failure of separation of
chromatids to poles and was also observed by many
workers (Amer, 1965). Fragments may also be acentric
chromosomes that are formed as a result of inversion.
The mechanism of mitotic index and aberration
brought by the three Mutagens indicates more sensitive to
EMS than SA and NMU. Although these chemicals
showed inhibitory effect on seed germination, plant height
and seedling survival. These mutagens have enough
potential to include genetic variability for quantitative and
qualitative variation which can be favorable exploited by
cytogenetic and plant breeders in improving the genotype
of horse gram.
The survival ofseedling percentage also gradually
decreased with gradual increase in concentration of
mutagen was observed in present investigations. Seedling
was survival percentage were 81.67, 74.45,62.24 for the
conc. of 0.1, 0.2 and 0.3 % of EMS; 88.44, 75.55 and
67 .77 for the concentration of 0.05, 0.1 and 0.15 % of SA
and 84.62, 79.07 and 71.12 for concentration of 0.4, 0.6
and 0.8 % of NMU as against 91.37 in control. Similar
finding also recorded by Bhat et al. (2007), Siddiqui er a/.
(2007), Agarwal and Ansari (2001). Srivastava (1979),
Mensah and Obadoni (2007) in Arachis and Maxim et al.
(2009) Carum carvi L.
Itmay be concluded that EMS, SA and NMU
causes toxic effect on root tip cells and other vegetative
part of plant body of Horse gram, this toxicity induces
different types of genetic and chromosomal variations and
enhances the rate of variations in morphological and yield
characters for selection of better qualities and also
increases the speed ofevolution.
ACKNOWLEDGEMENTS
The author expresses sincerely thanks to the UGC
(WR) for financial assistance, Principal and HOD, Botany
113
 AOOI( HOUSE F. LTD.
tur&,
Department, J.E.S.College, Jalna (M.S.) for providing the Bhat TA Sharma M & Anis M (2007).
laboratory facilities for conducting an experiment. Comparativeanalysis of mitotic aberationinduced by
Diethyl Sulphate (DES) and Sodium Azide (SA) in Vicia
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