Assessment on cytotoxic and mutagenic potency of Gamma rays and EMS in Vigna mungo L. Hepper

Abstract

To determine the mutagen agent potency and deduce an optimum dose, cytological analysis for induced chromosomal aberrations is considered as an accurate index in mutation breeding. Therefore, the present investigation was carried out to estimate the relative frequency and spectrum of meiotic abnormalities at various stages of cell division using Gamma rays, EMS and their combination treatments in M1 generation of black gram (Vigna mungo L. Hepper) varieties Pant U-30 and T-9. The analysis of M1 plants revealed a wide range of induced meiotic abnormalities like univalents, multivalents, laggards, chromatin bridges, micronuclei and chromosome stickiness by different mutagen doses. In addition to these, behavioral changes like precocious separation of chromosomes at metaphase, unequal separation of bivalents at anaphase, disturbed polarity and cytomixis at telophase were observed at low frequency. A linear relationship between the frequency of chromosomal aberrations and the strength of mutagen doses was observed in both the varieties and mutagen types. It was found that the combined treatments induced meiotic abnormalities at a higher frequency as compared to individual treatments of Gamma rays and EMS. Comparative estimation of induced cytological abnormalities suggested higher mutagenic sensitivity of var. Pant U-30 than the var. T-9 towards the treatments used. This also confirmed the relatively broader genetic tolerance level of black gram var. T-9 in inducing a significant amount of viable mutations without much lethality at the mutagen doses recommended in this study. Therefore, it can be concluded that the combine treatments of Gamma rays and EMS (300Gy+0.2%, 200Gy+0.3%) are relatively more effective for induction mutations in var. T-9 at the genotypic tolerable level.

Full text

Artículo original Biotecnología Vegetal Vol. 19, No. 3: 193 - 204, julio - septiembre, 2019
Instituto de Biotecnología de las Plantas. UCLV. MES.
eISSN 2074-8647, RNPS: 2154
Assessment on cytotoxic and mutagenic potency of Gamma rays
and EMS in Vigna mungo L. Hepper
Sonu Goyal1, Mohammad Rafiq Wani2,Rafiul Amin Laskar1*,Aamir Raina1,3, Samiullah
Khan1
1Mutation Breeding Laboratory, Department of Botany, Aligarh Muslim University. Aligarh-202
002. Aligarh. Uttar Pradesh. India.
2Department of Botany, Abdul Ahad Azad Memorial Degree College. Bemina-190 018 Cluster
University. Srinagar. Jammu and Kashmir. India.
3Botany Section, Women’s College, Aligarh Muslim University. Aligarh-202 002. Aligarh. Uttar
Pradesh. India.
*Author for correspondence e-mail: rafihkd@gmail.com
ABSTRACT
To determine the mutagen agent potency and deduce an optimum dose, cytological analysis
for induced chromosomal aberrations is considered as an accurate index in mutation breeding.
Therefore, the present investigation was carried out to estimate the relative frequency and
spectrum of meiotic abnormalities at various stages of cell division using Ga mma rays, EMS and
their combination treatments in M1 generatio n of blac k g ram ( Vigna mungo L. Hepper) varieties
Pant U-30 and T-9. The analysis of M1plants revealed a wide range of induced meiotic
abnormalities like univalents, multivalents, laggards, chromatin bridges, micronuclei and
chromosome stickiness by different mutagen doses. In addition to these, behavioral changes
like precocious separation of chromosomes at metaphase, unequal separation of bivalents at
anaphase, disturbed polarity and cytomixis at telophase were observed at low frequency. A
linear relationship between the frequency of chromosomal aberrations and the strength of
mutagen doses was observed in both the varieties and mutagen types. It was found that the
combined treatments induced meiotic abnormalities at a higher frequency as compared to
individual treatments of Gamma rays and EMS. Comparative estimation of induced cytological
abnormalities suggested higher mutagenic sensitivity of var. Pant U-30 than the var. T-9
towards the treatments used. This also confirmed the relatively broader genetic tolerance
level of black gram var. T-9 in inducing a significant amount of viable mutations without much
lethality at the mutagen doses recommended in this study. Therefore, it can be concluded
that the combine treatments of Gamma rays and EMS (300Gy+0.2%, 200Gy+0.3%) are relatively
more effective for induction mutations in var. T-9 at the genotypic tolerable level.
Keywords: genotypic sensitivity, induced mutagenesis, meiotic aberration frequency, mutation
breeding, pollen mother cells
Evaluación de la potencia citotóxica y mutagénica de los rayos Gamma y
EMS en Vigna mungo L. Hepper
RESUMEN
Para determinar la potencia del agente mutagénico y una dosis óptima, el análisis citológico de
las aberraciones cromosómicas inducidas se considera un índice adecuado en la inducción de
mutaciones. Por lo tanto, la presente investigación se llevó a cabo para estimar la frecuencia
relativa y el espectro de anormalidades meióticas en varias etapas de la división celular
usando rayos Gamma, EMS y sus tratamientos combinados en la generación M1 de variedades
de grano negro (Vigna mungo L. Hepper) Pant U-30 y T-9. El análisis d e las plantas M1 reveló
una amplia gama de anormalidades meióticas inducidas como univalentes, multivalentes,

194 Biotecnología Vegetal Vol. 19, No. 3, 2019
rezagados, puentes de cromatina, micronúcleos y adhesividad cromosómica por diferentes
dosis de los agentes mutagénicos. Además de estos, se observaron cambios como la separación
precoz de los cromosomas en la metafase, la separación desigual de los bivalentes en la
anafase, la polaridad alterada y la citomixis en la telofase a baja frecuencia. Se observó una
relación lineal entre la frecuencia de las aberraciones cromosómicas y la intensidad de las
dosis de agentes mutagénicos, tanto en las variedades como en los tipos de agentes
mutagénicos. Se encontró que los tratamientos combinados inducían anormalidades meióticas
con mayor frecuencia en comparación con los tratamientos individuales de rayos Gamma y
EMS. La estimación comparativa de las anormalidades citológicas inducidas sugirió una mayor
sensibilidad mutagénica de la var. Pant U-30 que T-9 hacia los tratamientos utilizados. Esto
también confirmó el nivel de tolerancia genétic a relativamente más amplio de la var. T-9 en la
inducción de una cantidad significativa de mutaciones viables sin mucha letalidad a las dosis
de los agentes mutagénicos recomendadas en este estudio. Por lo tanto, se puede concluir
que los tratamientos combinados de rayos Gamma y EMS (300Gy + 0.2%, 200Gy + 0.3%) son
relativamente más efectivos para la inducción de mutaciones en la var. T-9 en el nivel de
tolerancia genotípica.
Palabras clave: células madre de polen, frecuencia de aberración meiótica, inducción de
mutaciones, mutagénesis inducida, sensibilidad genotípica
INTRODUCTION
Black gram (Vigna mungo L. Hepper), also
known as urdbean, believed to have originated
in India is under cultivation from ancient times
(Purseglove, 1974). It is an important rainfed
short duration legume crop of India and
considered as a major source of dietary
protein (Abraham et al., 2013). It is a lso g rown
in Pakistan, Srilanka, Bangladesh, countries
of South East Asia, Africa and America. In
India, during 2015-16, urdbean was grown
over an area of 3.62 million hectares with the
production and productivity of 1.94 million
tones and 537 kg ha-1, respectively. It is
grown in various agro-ecological conditions
and cropping systems with diverse agricultural
practices both in rainy (Kharif) and post-rainy
(Rabi) seasons (Gupta et al., 2001; Singh and
Ahlawat, 2005). Rainy season records higher
yields than post rainy due to conducive
environment viz., high day temperature, less
moisture stress, adequate solar radiation etc.
for crop growth and yield. In coastal
ecosystems, it is grown in rice fallows with
re sidual moisture after the harvest of paddy.
Urdbean is generally eaten as an
accompanying dish with cereal preparations
like rice (Oryza sativa L.) and chapatti
(Triticum aestivum L.). It is also consumed
in the form of ‘dal’ (whole or split, husked/
un-husked) or perched. Urdbean is very rich
in protein containing about 24% in its seed.
Analysis of amino acid profile shows that seed
protein of urdbean has a low concentration
of sulphur containing amino acid such as
methionine and cysteine (Srivastava and Ali,
2004). Comparatively higher lysine content
makes it an excellent complement to cereals in
terms of balanced human nutrition (Tsou et al.,
1979). Gr een pods are dire ct ly co nsumed as a
vegetable by the bulk populace. It is also
prescribed as medicine both externally and
internally in paralysis, rheumatism and certain
diseases of the human nervous system (Singh,
1982) besides being recommended as
medicinal diet to cure flatulence due to its
easier digestibility. It also provides nutritious
green fodder and feeds to the livestock.
The increasing demands for food supply due
to the exponential rate of population growth
necessitate the acceleration of agricultural
output globally. Grain legumes are an essential
part of dietary requirements, especially in
South Asian and African countries, due to
their tremendous nutritional value and wide
environmental adaptability. Therefore,
induction of genetic variation in high potential
grain legumes like black gram is necessary in
the present unstable agro-climatic condition
for sustained availability of improved varieties
with high yield and wide adaptability to
farmers (Goyal et al., 2019).
Mutation breeding using individual and
combined treatments of various physical and
chemical mutagens has been employed
successfully in genetic improvement of
several grain legumes (Laskar, 2018; Laskar
et al., 2019).

Induced mutations may be resorted to
developing superior genotypes due to their
direct or cumulative effect on genetic
background (Baisakh et al., 2011). Mutagenic
concentration is directly proportional to both
rates of mutation and biological damages
(Khursheed et al., 2015). Therefore, it is
imperative to optimize the mutagen doses to
be used in the mutation breeding experiment
for obtaining the maximum rate of mutation
with reasonable germination and lesser growth
inhibitions. Kozgar et al. (2014)
comprehensively documented the important
cytogenetic tests for genotoxicity, genetic
variation, cytotoxicity and mutagenic potency
in induced mutagenesis experiments. Mutagen
act directly on the genetic material of the
species which in turn alters specific gene
expressions and meiosis is directly linked with
inheritance of traits through gametes in the
subsequent generations (Khursheed et al.,
2015). Therefore, the assessment on induced
meiotic abnormalities has been considered to
be the most reliable indices for accurate
estimation of mutagenic potency and genetic
sensitivity in applied field of induced
mutagenesis (Khan et al., 2015). In higher
plants, chromosomal aberrations induced by
mutagens have been extensively reported by
different authors (Datta and Biswas, 1985;
Kaul and Murthy, 1985; Reddy, 1990; Zeerak,
1991; Ahmad, 1993; Saeed, 1993; Sinha and
Gandhi, 1994; Kumar and Dubey, 1998; Kumar
and Singh, 2003; Kumar and Gupta, 2007;
Khan et al., 2015).
Although, cytological studies on major grain
legumes are considerable, however detailed
reports on urdbean meiotic abnormalities is
still limited for access and thus expected to
generate new insights for urdbean
improvement using induced mutagenesis. With
consideration of available facts and findings
on urdbean mutagenesis, the present
investigation was carried out to estimate the
extent of chromosomal aberrations induced
at various stages of meiotic division in M1
generation using Gamma rays and EMS alone
as well as in combination.
MATERIALS AND METHODS
Plant material
Two varieties of black gram (V. mungo)
namely T-9 and Pant U-30 were used in the
present study. Seeds of both varieties were
obtained from G. B. Pant University of
Agriculture and Technology, Pantnagar,
Uttaranchal, India. Both varieties are well
adapted to agro-climatic conditions of Uttar
Pradesh including the site of study viz.,
Aligarh.
Mutagenic treatments, field seed culture and
cytological analysis
Dry seeds of each variety with a moisture
content of 12% were irradiated with 100, 200,
300 and 400 Gy doses of Gamma rays from
60CO source at the National Botanical
Research Institute, Lucknow, Uttar Pradesh,
India. For chemical treatments, healthy
seeds of uniform size of each variety were
presoaked for 9 hours in distilled water and
treated with 0.1, 0.2, 0.3 and 0.4% of EMS
(ethylmethane sulphonate–a monofunctional
alkylating agent, manufactured by Sissco
Research Laboratories Pvt. Ltd., Mumbai,
India) for 6 hours with intermittent shaking
at room temperature (25±1 ºC). The solution
of EMS was prepared in phosphate buffer
pH 7. After treatment, the seeds were
thoroughly washed in running tap water to
remove the excess mutagen from the seed
surface.
To understand the relative effect of Gamma
rays and EMS, combination treatments were
also employed following the procedure
adopted by Khursheed et al. (2015). For
combination treatments, dry seeds of each
variety were first irradiated with Gamma rays
at 200 and 300 Gy doses and then treated
with 0.2 and 0.3% EMS (i.e. 200 Gy+0.2%
EMS, 300 Gy+0.2% EMS, 200 Gy+0.3% EMS
and 300 Gy+0.3% EMS). The procedure
adopted was similar to that for individual
treatment.
Three replications of 100-seeds each were
sown for every treatment and control in each
variety in a randomized complete block design
(RCBD) at the Agriculture Farm, Aligarh Muslim
University, Aligarh, India. The spacing was
maintained at 30 cm (seed to seed in a row)
and 60 cm (between the rows) in the field.
The experiment was conducted during summer
(Zaid) season of 2008. Recommended
agronomic practices were employed for the
preparation of field, sowing and subsequent
management of the population.
Biotecnología Vegetal Vol. 19, No. 3, 2019 195

196 Biotecnología Vegetal Vol. 19, No. 3, 2019
The meiotic analysis was conducted on 30
randomly selected plants from each treatment
and control. For meiotic studies, young flower
buds were fixed in Carnoy’s fluid (1 part glacial
acetic acid: 3 parts chloroform: 6 parts ethyl
alcohol) for 24 hours. Ferric chloride was added
to the fixative to get the better staining. After
24 hours of fixation, flower buds were
transferred to 70% alcohol. Anthers were
smeared in 1% acetocarmine solution and pollen
mother cells were examined for their behavior
at various stages of microsporogenesis under
the microscope. The observations were made
in a binocular microscope with a magnification
of 10X × 100X with oil immersion lens and
photographs were taken using MOTIC BA210
fitted with an inbuilt Nikon Camera. The
frequencies of meiotic abnormalities were
estimated by using the following formula:
Frequency of meiotic abnormalities
(%)=(Number of abnormal Pollen Mother Cells)/
(Total Pollen Mother Cells observed) X100
RESULTS AND DISCUSSION
In the present study, a broad spectrum of
meiotic abnormalities was induced by Gamma
rays, EMS and their combination treatments
in M1 generation of urdbean (Table 1, Table
2). The spectrum of meiotic abnormalities
observed in various mutagenic treatments
included univalents, multivalents, laggards,
chromatin bridges, micronuclei and
chromosome stickiness. In addition, precocious
separation of chromosomes at metaphase,
unequal separation of bivalents at anaphase,
disturbed polarity and cytomixis at telophase
were observed at low frequency (Figure 1,
Figure 2). Meiosis is genetically the most
significant activity of an organism consisting
of highly coordinated physiological,
biochemical, cytogenetic and phenotypic
events which lead to gene recombination,
chromosome reduction and gamete formation.
Although these changes are continuous,
mutation of any of the genes disrupts meiosis
and culminates in gametes sterility and other
abnormalities. Hence, the study of the meiotic
behavior of mutagenized population forms a
reliable index for estimating the genotype
specific potency of mutagens (Khan et al.,
2015).
Almost all the mutagenic treatments induced
a high degree of stickiness among the
bivalents which adversely affected the
normal disjunction. Stickiness has been
reported to be the result of partial
dissociation of nucleoproteins and alteration
in the pattern of organization of
chromosomes by Evans (1962), while Rao
and Laxmi (1980) attributed it to be due to
the disturbances of cytochemically balanced
reactions by the mutagens. In addition,
Gaulden (1987) postulated that stickiness
may result from defective functioning of one
or two types of specific non-histone proteins
involved in chromosome organization which
are necessary for chromatid separation and
segregation. The altered functioning of
these proteins leading to stickiness is caused
by mutations in the structural genes coding
for them (hereditary stickiness) or by the
action of mutagens (induced stickiness).
Chromosome stickiness has been reported
to decrease the pollen fertility in some crop
species (Rao et al., 1990; Pagliarini et al.,
2000). The occurrence of univalents
indicates non-homology between certain
chromosomes in the compliment. The
mutagenic treatments induce structural
changes in some of the chromosomes which
restrict the pairing and hence the formation
of univalents. Reduction in chromosome
pairing has also been attributed to mutations
in the genes governing homologous
chromosome pairing and/or chromosomal
structural changes (Gottschalk and
Villalobes-Pietrini, 1965; Reddy et al., 1991;
Singh, 1992). At metaphase-I, some of the
univalents disjuncted early. Such
chromosomal divergences in the form of
precocious movements are pointed towards
the structural differentiation of the
homologous pair. Stickiness of chromosomes
at metaphase-I adversely affected the
normal disjunctions of chromosomes at
anaphase-I, which resulted in the formation
of laggards and unequal separation of
chromosomes at anaphase stage. The
laggards observed, in the present study,
have also been reported earlier and may be
the result of delayed terminalization,
stickiness of chromosome or the failure of
chromosomal movement (Jayabalan and Rao,
1987).
Such abnormalities lead to micronuclei
formation (Zeerak and Zargar, 1998) and
non functional spores. The frequency of

Biotecnología Vegetal Vol. 19, No. 3, 2019 197
Figure 1. Pollen Mother Cells showing meiotic abnormalities in the treated
population of urdbean. (A) Diakinesis with 11 bivalents; (B) Metaphase-I
with 11 bivalents; (C) Metaphase-I with univalents; (D) Metaphase-I
showing stickiness of chromosomes; (E) Metaphase-I showing stickiness
of chromosomes; (F) Metaphase-I with 3I+6II+1III+1IV; (G) Metaphase-I
showing precocious movement of chromosomes; (H)Anaphase-I showing
movement of chromosomes towards the poles; (I) Anaphase-I showing
chromosomes stickiness with difficulty to segregate. Bars = 10 µm.
micronuclei was less at telophase-II as
compared to the frequency of univalents
and laggards, indicating that some
chromosomes were included in the main
nucleus. This seems to be the normal
behavior of many species (Koduru and Rao,
1981). Unequal separation of chromosomes
would lead to the production of aneuploid
gametes (Zeerak, 1992). Chromosome
bridges were frequently observed at
anaphase-I. Chromosome breakage and
reunion of broken ends could lead to the
formation of bridges (Ignacimuthu and
Babu, 1989). Anaphase bridges were also
recorded by Suganthi and Reddy (1992)
in Triticum aestivum, Zeerak (1992) in
Lycopersicon esculentum L., Fatma and
Khan (2009) in Vicia faba and Khan and
Tyagi (2009) in Glycine max L.
Multivalent formation in mutagenic treated
plants has been attributed to many breaks
induced by mutagens which may lead to
reciprocal translocation and resulting in
multivalent formation. Such type of multivalent
formation has also been reported in brinjal
(Solanum melongena L.) (Satyanarayana and
Subhash, 1982), urd and mung beans ( Vigna
radiata L. Wilczek) (Ignacimuthu and Babu,
1989), rice (Oryza sativa L.) (Radhadevi and
Reddi, 1997) and chilli (Capsicum frutescens)
(Dhamayanthi and Reddy, 2000). In the
present study, among individual mutagenic
treatments, Gamma rays produced a higher
number of multivalents. Gamma rays and EMS
treatments have been reported to induce the
formation of multivalents in V. radiata
(Ignacimuthu and Sakthivel, 1989) and Lens
culinaris Medik (Re ddy and Annadurai, 1992).

198 Biotecnología Vegetal Vol. 19, No. 3, 2019
Figure 2. Pollen Mother Cells showing meiotic abnormalities in the treated
population of urdbean. (A) Anaphase-I (Control); (B) Anaphase-I with lagging
chromosome; (C) Anaphase-I showing chromatin bridge; (D) Anaphase-I
showing unequal separation of chromosomes; (E) Telophase-I with lagging
chromosomes; (F) Telophase-II (Control); (G) Telophase-II showing
disturbed polarity; (H) Telophase-II showing eliminated chromosomes forming
micronucleus; (I) Telophase-I showing cytomixis. Bars = 10 µm
Disturbed polarity at telophase-II could be
due to alteration in the gene controlling
biochemical pathways of the substance that
determine the position of spindle poles.
Similar reports were made in wheat (Triticum
aestivum L.) (Suarez and Bullrich, 1990),
barley (Hordeum vulgare) (Kumar and Singh,
2003) and faba bean (Vicia faba L.) (Ansari
and A li, 2009). Cytomixis generally refers to
the migration of chromatin from one cell to
the other through cytoplasmic connections.
Although cytoplasmic connections are very
common in angiosperms, the movement of
nuclear material through them is rare. In
the present study, although no visual
transfer of chromosome through cytoplasmic
connections could be detected, the
formation of cytoplasmic channels between
the two cells suggests that screening of
mutagen treated population for varied ploidy
level and its utilization in plant breeding can
yield interesting results. Cytomixis may have
serious genetic consequences by causing
deviations in chromosome number and may
represent an additional mechanism for the
origin of aneuploidy and polyploidy (Sarvella,
1958; Siddiqui et al., 1979). Cytomixis has
been detected at higher frequency in
genetically imbalanced species such as
hybrids as well as apomictic and polyploid
species (Gottschalk, 1970; Yen et al., 1993).
The abnormalities induced were found to be
dose-dependent where a linear correlation
established between increasing strength of
mutagen treatments and increasing
frequency of meiotic aberrations (Figure 3).
Although the types of chromosomal
abnormalities were more or less common in
both the varieties, yet the frequency of

Biotecnología Vegetal Vol. 19, No. 3, 2019 199
such aberrations was comparatively more
in var. Pant U-30 than the var. T-9 (Table
3, Figure 4) indicating that it is more
sensitivity towards the mutagens. Combined
treatments were most effective for induction
of meiotic aberrations, whereas Gamma rays
individually induced more aberrations than
EMS (Table 3, Figure 4). These results
support the general hypothesis that physical
mutagens produce more cytological
abnormalities than chemical ones (Kozgar,
Figure 3. Dose dependent linear relationship between chromosomal
aberrations frequency and mutagen treatment concentrations in urdbean
(Vigna mungo L. Hepper).
2014). However, EMS was earlier found to
be more effective in inducing meiotic
irregularities than Gamma rays individually
as well as in combination with Gamma-ray
treatment (Dhamayanthi and Reddy, 2000).
Dose dependent increase in meiotic
abnormalities has also been reported by
Ignacimuthu and Babu (1989) in urdbean (V.
mungo) and mung beans (V. radiata) and
Dhamayanthi and Reddy (2000) in chilli
(Capsicum annuum L.).
Figure 4. Relative total frequency (%) of meiotic abnormalities induced in
urdbean (Vigna mungo L. Hepper). A: Comparison between Gamma rays, EMS
and their combination; B: Comparison between urdbean varieties Pant U-30
and T-9.

200 Biotecnología Vegetal Vol. 19, No. 3, 2019
The scarcity of available genetic variations is
among the prime constraints in black gram
breeding, thus induced mutagenesis is one of
the most preferred complimentary technique
for generation of accessible mutation(s) in
the black gram genotype(s). In mutation
breeding, the selection of suitable mutagens
agents and their doses are prerequisite for
subsequent generation of mutagenised
population(s) and genotypic improvement of
any crop. In the present study, meiotic
analysis of induced chromosomal aberrations
facilitated the process of mutagen potency
assessment in two widely cultivated black
gram genotypes. The wide range of induced
chromosomal aberrations at different
treatment doses and the aberrations
frequencies estimated in the study showed
higher mutation rates with lower lethality can
be obtained by employing physical (Gamma
rays) and chemical (EMS) mutagens agents
together at lower doses. The meiotic
aberrations resulted directly from the
interactions between applied mutagen doses
and black gram chromosomes. Therefore, it
provides authentic information about the
genotypic tolerable limits at which useful
mutations can be induced by employing
suitable Gamma rays and EMS treatments in
black gram. Moreover, it implies that the
mutagenic populations generated in both T-9
and Pant U-30 varieties using Gamma rays
and EMS alone as well as in combination are
useful source for isolation of early putative
mutants through mutation breeding in
subsequent generations.
Table 1. Frequency and spectrum of meiotic abnormalities in various mutagenic treatments in
M1 generation of black gram ( Vigna mungo L. Hepper) var. T-9.

Table 2. Frequency and spectrum of meiotic abnormalities in various mutagenic treatments in
M1 generation of black gram ( Vigna mungo L. Hepper) var. Pant U-30.
In the present study, broad array of
chromosomal aberrations have been induced
at various stages of meiotic division in M1
generation using Gamma rays and EMS alone
as well as in combination. The combined
mutagen agent doses resulted in higher
frequency of abnormalities as compared to
the individual doses of Gamma rays and EMS
in both the varieties Pant U-40 and T-9.
However Pant U-40 reflected higher mutagenic
sensitivity than the var. T-9. Differential
sensitivity of the varieties, observed in the
present study, suggested that mutagenic
potency completely depends on the genetic
makeup of the target species and cannot be
generalize for all biological systems. Hence, prior
selection of varieties, mutagen agent doses and
durations are the key to achieve a desirable
spectrum of mutation. Comparative estimation
of induced cytological abnormalities suggested
higher mutagenic sensitivity of var. Pant U-30
than the var. T-9 towards the treatments
used. This reflects a relatively broader genetic
tolerance level of black gram var. T-9.
Biotecnología Vegetal Vol. 19, No. 3, 2019 201

202 Biotecnología Vegetal Vol. 19, No. 3, 2019
Table 3. Total (pooled) frequency of meiotic abnormalities induced by Gamma rays, EMS and
their combinations in M1 generation of black gram ( Vigna mungo L. Hepper).
CONCLUSIONS
Combined treatments of Gamma rays and EMS
are most effective for induction of meiotic
aberrations in black gram (Vigna mungo L.
Hepper) varieties Pant U-30 and T-9, whereas
individually Gamma rays induce more
aberrations. The higher frequency and wide
spectrum of aberrations induced in
combination treatments suggests physical and
chemical mutagens when employed together
at predetermined tolerable doses could
increase the rate of mutations in black gram
genotypes. Also, the meiotic anomalies
observed in the study suggests associated
alteration of gene(s) expression in the
mutagenised populations that may govern the
trait(s) of the interest of breeder which could
get stabilized in advanced generations.
ACKNOWLEDGMENTS
The authors are thankful to the University
Grants Commission (UGC) New Delhi, India,
for providing financial assistance and to the
Chairman, Department of Botany, AMU,
Aligarh, India, for infrastructure facilities.
Conflict of interest
The authors declare that there is no conflict
of interest regarding the publication of this
article.
Conflict of interest
The authors declare that they have no conflict
of interest.
REFERENCES
Abraham B, Vanaja M, Reddy PRR, Sivaraj
N, Sunil N, Kamala V, Varaprasad KS (2013)
Identification of stable and high yielding
genotypes in black gram (Vigna mungo L.
Hepper) germplasm. Ind J Genet 73(3):
264-269
Ahmad S (1993) Meiotic studies in two
cultivars of Cicer arietinum L. after Gamma
irradiation. Cytologia 58: 61-65
Ansari MYK, Ali Z (2009) Cytomorphological
variabilities induced by aniline in Vicia faba L.
J Cytol Genet 11: 57-64
Baisakh B, Das TR, Nayak BK (2011) Efficacy
of physical and chemical mutagenic treatments
for micro-mutants in urd bean. J Food
Legumes 24(2): 106-109
Datta AK, Biswas AK (1985) A EMS induced
bushy mutant of Nigella sativa L. with
desynaptic behavior of chromosomes.
Cytologia 50: 535-543
Dhamayanthi KPM, Reddy VRK (2000)

Biotecnología Vegetal Vol. 19, No. 3, 2019 203
Cytogenetic effects of Gamma rays and
ethylmethane sulphonate in chilli pepper
(Capsicum annuum L.). Cytologia 65: 129-
133
Evans HJ (1962) Chromosome aberrations
induced by ionizing radiations. Int Rev Cytol
13: 221-308
Fatma S, Khan S (2009) Diethyl sulphate induced
meiotic abnormalities in Vicia faba L. J Phytol
Res 22(2): 195-198
Gaulden ME (1987) Hypothesis: some mutagens
directly alter specific chromosomal proteins (DNA
topoisomerase II and peripheral proteins) to
produce chromosome stickiness which causes
chromosome aberration. Mutagenesis 2: 357-395
Gottschalk W (1970) Chromosome and nucleus
during microsporogenesis of Pisum sativum.
Nucleus 13: 1-9
Gottschalk W, Villalobes-Pietrini RV (1965) The
influence of mutant genes on chiasmata formation
in Pisum sativum. Cytologia 30: 88-97
Go yal S, Wani MR, L aska r RA, Raina A, Ruhul A,
Khan S (2019) Induction of morphological
mutations and mutant phenotyping in black
gram (Vigna mungo L. Hepper) using Gamma
rays and EMS. Vegetos 32(4): 464-472; doi:
10.1007/s42535-019-00057-w
Gupta S, Gupt a SR, Di ksh it H K, S ing h RA (20 01)
Variability and its characterization in Indian
collection of black gram (Vigna mungo (L.)
Heppe r). Plant Genet Re sour Ne ws 127: 2
Ignacimuthu S, Sakthivel N (1989) Induced
chromosomal abnormality and pollen sterility in
Vigna radiata (L.) Wilczek cv PS 16. Cytologia
54: 109-114
Ignacimuthu S, Babu CR (1989) Induced
chromosomal abnormality and pollen sterility in
wild and cultivated urd and mungbean. Cytologia
54: 159-167
Jayabalan N, Rao GR (1987) Gamma radiation
induced cytological abnormalities in
Lycopersicon esculentum Mill. var. Pusa Ruby.
Cytologia 52: 1-4
Kaul MLH, Murthy TGK (1985) Mutant genes
affecting higher plant meiosis. Theor Appl Genet
70: 449-466
Khan H, Laskar RA, Khan S ( 2015) Comparative
estimation of induced cytotoxicity and
mutagenic potency of EMS and SA using Vicia
faba L. as a biological system. In: Krishnan
S, Rodrigues BF (eds). Advances in Plant
Science and Biotechnology, pp. 172-186. Goa
University, India; ISBN: 978-81-908791-4-9
Khan MH, Tyagi SD (2009) Cytological effe cts
of different mutagens in soybean (Glycine max
(L.) Merrill). Front Agric China 3(4): 397-401
Khursheed S, Laskar RA, Raina A, Amin R, Khan
S (2015) Comparative analysis of cytological
abnormalities induced in Vicia faba L. genotypes
using physical and chemical mutagenesis.
Chromosome Science 18: 47-51
Koduru PRK, Rao MK (1981) Cytogenetics of
synaptic mutants in higher plants. Theor Appl
Genet 59: 197-214
Kozgar MI, H ussain S, Wani MR, K han S (201 4)
The role of cytological aberrations in crop
improvement through induced mutagenesis. In:
Ahmad P, Wani M, Azooz M, Phan Tran LS (eds).
Improvement of crops in the era of climatic
changes, pp. 283-296. Springer, New York
Kumar G, Gupta P (2007) Mutagenic efficiency
of lower doses of Gamma rays in black cumin
(Nigella sativa L.). Cytologia 72(4): 435-440
Kumar G, Singh V (2003) Comparative analysis
of meiotic abnormalities induced by Gamma rays
and EMS in barley. J Indian Bot Soc 82: 19-22
Kumar S, Dubey DK (1 998) Effect of Gamma
rays, EMS and dES on meiosis in Latyrus
sativus. J Cytol Genet 33: 139-147
Laskar RA (2018) Studies on the induction of
mutations for improvement of yield and yield
attributing traits in Lentil (Lens culinaris
Medik.). PhD Thesis, Aligarh Muslim University,
Aligarh, India
Laskar RA, Khan K, Deb CR, Tomlekova N, Wani
MR, Raina A, Amin R (2019) Lentil (Lens
culinaris Medik.) Diversity, Cytogenetics and
Breeding. In: Al-Khayri J, Jain S, Johnson D
(eds). Advances in Plant Breeding Strategies:
Legumes, pp. 319-369. Springer, Cham; doi:
10.1007/978-3-030-23400-3_9

204 Biotecnología Vegetal Vol. 19, No. 3, 2019
Pagliarini MS, De Freitas PM, Batista LAR
(2000) Chromosome stickiness in meiosis of a
Brazilian Paspalun accession. Cytologia 65:
289-294
Purseglove JW (1974) Tropical crops:
Dicotyledons. Wiley, London
Radhadevi K, Reddi TVV (1997) Cytogenetics
of interchange heterozygotes in scented rice
by recurrent treatments with Gamma rays. J
Cytol Genet 32: 129-133
Rao NB, Laxmi N (1980) Gamma ray induced
meiotic abnormalities in Capsicum annuum L.
Caryologia 33: 509-518
Rao PN, Ranganadham P, Nirmala A (1990)
Behaviour of a sticky desynaptic mutant in
pearl millet. Genetics 81: 221-227
Reddy VRK (1990) Cytology of some
morphological mutants in Triticale. Acta
Botanica Indica 18: 146-149
Reddy VRK, Annadurai M (1992) Cytological
effects of different mutagens in lentil (Lens
culinaris Medik). Cytologia 57: 213-216
Reddy VRK, Revathi R, Nalini R (1991) Effect
of physical and chemical mutagens on
meiotic behaviour in barley and wheat. J
Indian Bot Soc 70: 113-118
Saeed A (1993) Meiotic studies in two
cultivars of Cicer arietinum L. after Gamma
irradiation. Cytologia 58: 61-65
Sarvella P (1958) Cytomixis and the loss of
chromosomes in meiotic and somatic cells
of Gossypium. Cytologia 23: 14-24
Satyanarayana B, Subhash K (1982) Studies
on EMS induced mutations in egg plant.
Geobios 9: 137-141
Siddiqui NH, Khan R, Rao GR (1979) A case
of cytomixis in Solanum nigrum L. Complex.
Curr Sci 48(3): 118-119
Singh DP (1982) Genetics and breeding of
black gram and green gram. Res Bull GB Pant
Univ Agric Technol 109: 13–14
Singh DP, Ahlawat PS (2005) Green gram
(Vigna radiata) and black gram (V. mungo)
improvement in India: past, present and
fu ture p rospec ts. Indian J A gric Sci 75: 243-
250
Singh RN (1992) Chromosomal abnormalities
and fertility in induced autotetraploid
Helianthus annuus in C1 and C2 generations.
Cytologia 57: 277-281
Sinha A, Gandhi I (1994) Mitotic abnormalities
caused by Gamma rays in some Vicia species. J
Mendel 1(3-4): 183-184
Srivastava RP, Ali M (2004) Nutritional Quality
of Common Pulses. Indian Institute of Pulses
Research, Kanpur, India
Suarez E, Bullrich L (1990) Meiotic spindle
disturbances in a commercial bread wheat
cultivar. Cytologia 55: 79-86
Suganthi CP, Reddy VRK (1992) Effects of
Gamma rays and EMS on meiosis in some
cereals. J Cytol Genet 27: 103-114
Tsou CS, Hsu MS, Tan ST, Park HG (1979) The
protein quality of mungbean and its
improvement. Acta Horticulturae 93: 279-289;
doi: 10.17660/ActaHortic.1979.93.26
Yen C, Yang JL, Sun GL (1993) Intermeiocyte
connections and cytomixis in intergeneric hybrid
of Roegeneria ciliares (Trin) Nevski with
Psathyrostachys huashanica K eng. C yt ol og ia 58:
187-193
Zeerak NA (1991) Cytogenetical effect of
Gamma rays and ethylmethane sulphonate in
brinjal (Solanum melongena L.). Cytologia 56:
639-643
Zeerak NA (1992) Cytogenetical effects of
Gamma rays and ethylmethane sulphonate
in tomato (Lycopersicon esculentum var.
cerasiforme). Phytomorphology 42(1-2):
81-86
Zeerak NA, Zargar GH (1998) Cytological
studies in brinjal (Solanum melongena va r.
local long kashmiri). In: Manna GK, Roy SC
(eds). Perspective in Cytology and Genetics,
pp. 369-374.AICCG Publications, Kalyani,
India
Recibido: 26-06-2019
Aceptado: 21-07-2019
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