77Journal of Oilseed Brassica, 7 (1) January, 2016
Determination of LD
50
of ethyl methanesulfonate (EMS) for induction of
mutations in rapeseed-mustard
Prashant Yadav, HS Meena*, PD Meena, Arun Kumar, Riteka Gupta, S Jambhulkar
1
,
Reema Rani and Dhiraj Singh
ICAR-Directorate of Rapeseed-Mustard Research, Bharatpur-321303, Rajasthan, India
1
Bhabha Atomic Research Centre, Trombay, Mumbai-400 085, Maharashtra, India
*Corresponding author: singh_hari2006@yahoo.co.in
(Received: 22 Nov 2015; Revised: 22 Dec 2015; Accepted: 27 Dec 2015)
Abstract
Mutation breeding is an effective way to enrich genetic variability in crop plants. There are two basic means,
physical and chemical mutagens for inducing mutations. Among chemical mutagens, the alkylating agent,
ethyl methanesulfonate (EMS) is the most commonly used mutagen in plants as it causes a high frequency of
nucleotide substitutions i.e. point mutations. Hence, an optimum dose is highly desired to produce the high
frequency of mutations with minimum killing of treated individuals. Therefore, the present investigation was under-
taken to determine the LD
50
of EMS and effect of different dosages of EMS on seed germination of two
Indian mustard varieties (viz. RH-749 and NRCHB-101) and one of its important wild relative
Sinapis alba. Results revealed the significant effects of EMS dosages and treatment periods on seed germination.
The EMS doses (LD
50
) at 0.42%, 0.73% and 0.3% for duration of 12 h were found to be optimum for Indian
mustard varieties (RH-749, NRCDR-101) and S. alba respectively. The LD
50
of EMS for Brassica juncea
was higher than the S. alba and it also varied for two varieties of B. juncea. This information would be highly
useful for initiating mutation breeding programme in rapeseed-mustard crops.
Key words: Brassica, EMS, LD
50
, mutagenesis, Sinapis alba
Journal of Oilseed Brassica, 7 (1) : 77-82, January 2016
Introduction
Rapeseed-mustard crops, particularly the Indian
mustard has a highly significant role in Indian
agriculture. It is second important oilseed crop at
national level contributing nearly 1/3
rd
of the edible
oil pool of the country (Pratap et al., 2014). Considering
the population growth rate, increased per capita
edible oil consumption and mounting imports of
vegetable oils, rapeseed-mustard crops signifies a
positive trend and offers enormous scope for yield
enhancement on long term basis. In spite of achieving
impressive productivity gains through development
of many improved cultivars; still there is compelling
need to further increase and stabilize the productivity
of this crop (Meena et al., 2015). To enhance the
production of any crop, a good variability should be
present in the primary gene pool (Kumar et al.,
2013a; Kumar et al., 2015). Genetic variation plays
a critical role in developing well-adapted improved
cultivars. Since, there is limited genetic variability in
primary gene pool of Brassica juncea; the various
tools to generate new genetic variability shall be
employed. However, cross breeding has restricted
usages due to limited genetic variability in nature
(Sestili et al., 2010). Mutation breeding might be
the effective alternate to augment genetic variation,
particularly for traits having low level of genetic
variation (Szarejko and Forster, 2007). Numerous
reports on successful mutation breeding are available
in various oilseed crops (Bacelis, 2001; Spasibionek,
2006; Ferrie et al., 2008; Parry et al., 2009).
Induced mutations have been used mainly to generate
variation that could rarely be found in germplasm
collections. Mutagenesis have been employed to
improve a large number of desirable characters like
earliness, dwarfness, biotic and abiotic stress
resistance, seed yield and oil quality (Schnurbush et
al., 2000; Parry et al., 2009). Many physical and
78 Journal of Oilseed Brassica, 7 (1) January, 2016
chemical mutagens are available to induce
mutations in crop plants. The nature of alteration in
genetic makeup of crop plant depends on the
specific mode of action of a particular mutagen
(Feldmann et al., 1994; Meinke et al., 1998).
Several rearrangements in DNA fragments may
occur based on dose level and treatment time of
various mutagens that may result in production of a
range of mutants. The information on right dose of
a particular mutagen for specific crop, species or
genotype is highly important to induce mutants with
desirable frequency. Ethyl Methane Sulphonate
(EMS) is a chemical mutagen, produces random
mutations in plant genome and is reported to be the
most effective and powerful mutagen (Minocha &
Arnason., 1962; Hajra 1979) that typically produces
only point mutations (Okagaki et al., 1991). In Brassica
crops, Fowler and Stefansson (1975) obtained many
morphological variations using EMS in B. napus.
Similarly, Khalatkar and Indurkar (1991) reported
various mutations for “00” genotypes in B. juncea
and early flowering mutant in B. napus have been
reported by Landge and Khalatkar (1995). Usually,
mutagen treatments reduce seed germination, growth
rate, vigour and fertility. There is substantial killing
of plants during different stages of development, thus
considerably reduces the survival of resulting plants.
The dose required for high mutagenic efficiency
depends on properties of the mutagenic agent and
material treated. Hence, an overdose might kill too
many treated individuals and an underdose will
produce fewer mutations. The optimum dose will
produce the high frequency of mutations and cause
minimum killing. Many workers felt that a dose near
to LD
50
should be the optimum which varies with
crop species and mutagen used (Singh, 2000).
Therefore, the information on suitable dose of a
particular mutagen for a specific crop, various
species and different cultivars of a same species is
highly useful. In Rapeseed-mustard crops, there are
limited information on optimum dose of chemical
mutagens and the reports on species wise and cultivar
differences are not available. Therefore, keeping the
above points in view the present investigation was
conducted to determine the LD
50
of EMS for
Brassica species and to determine the effect of ploidy
levels (diploid and tetraploid) of the species and
different varieties of the same species on optimum
dose of chemical mutagen (EMS).
Materials and Methods
The present study was conducted at ICAR-
Directorate of Rapeseed-Mustard Research,
Bharatpur (India) during 2014-15. The experimental
material consisted of two different species of Brassica
having two different ploidy levels viz., Sinapis alba
(Diploid, 2n=2x=24, SS) and B. juncea
(Tetraploid, 2n=4x=36, AABB) and two varieties of
the same species B. juncea (Indian Mustard) viz.
RH-749 & NRCDR-101. Ethyl methanesulfonate
(EMS) was used as chemical mutagen for induction
of mutation. The study was conducted under
controlled conditions in laboratory with three
replications. Hundred seeds of each genotype for
each replication were first presoaked in distilled
water for 2 h and then treated overnight with 8
different concentrations of EMS (0.1%, 0.25%,
0.5%, 0.75%, 1.0%, 1.25%, 1.5% and 2%) for 12 h
at 24
º
C with constant shaking at 100 rpm. The over-
night treated seeds were washed 3-4 times under
running tap water to remove residual EMS from the
seeds. The seeds were placed on petri dishes on
distilled water soaked whatman paper disks. The
seeds for control were treated with distilled water
only. Petri dishes were placed on tissue culture racks
for seed germination. The room temperature was
maintained at 25 ± 2
º
C. Distilled water was
constantly applied twice a day with 8 h differences
to maintain moisture in the paper. All sprouted seeds
were considered as germinated either the resulting
seedlings were normal or abnormal. Observations
were recorded on 3
rd
and 6
th
days after treatment
(DAT), the germinated seeds were counted from
each petri plate. Percent seed germination was
calculard using average of the three replications and
data was analyzed for determining LD
50
.
Results and Discussion
Mean germination percentage of S. alba and B.
Juncea (Var. RH-749 & NRCDR-101) genotypes
at 8 different doses of EMS is presented in Table 1.
The germination was recorded on 3rd and 6th DAT
and the LD
50
was calculated on the basis of
percent germination on 3rd day. The seedlings
having normal root and shoots were considered for
79Journal of Oilseed Brassica, 7 (1) January, 2016
Table 1: Effect of different doses of EMS on germination of Indian mustard varieties (RH-749 &
NRCHB-101) and S. alba
Dose of EMS C** 0.10% 0.25% 0.50% 0.75% 1% 1.25% 1.50% 2%
DAT* 33636363636363636
RH-749 91 82 94 72 85 33 62 6 18 2 10 0 0 0 0 0 0
NRCHB-101 98 92 96 89 95 88 94 40 76 7 58 0 6 0 0 0 0
S. alba 99 84 93 33 68 25 57 4 37 5 28 1 9 0 0 0 0
*Days after treatment; **Control
Fig 1: Decrease in germination percentage with increasing concentrations of EMS in B. juncea (RH-749 &
NRCHB-101) and S.alba
calculating the LD
50
as most of the seedlings from
delayed germinated seeds were abnormal. The
results indicated that the average germination was
decreased with increasing in the concentration of
mutagen (EMS). The percent germination was
reduced from 91, 98 and 99 to 2, 7 and 5 percent
with 1% of EMS for genotypes RH-749, NRCHB-
101 and S. alba, respectively. At the same time,
almost all the genotypes showed zero (0.00%)
germination at the concentration of 1.25% and higher
doses of EMS. This revealed that the doses of EMS
higher than 1% are highly lethal irrespective of
genotype and species. The germination percentage
on 6
th
day after treatment (DAT) was found higher
than the germination percentage on 3
rd
DAT
indicating the delay in seed germination due to
effect of mutagen treatment (Fig 3). Most of the
seedlings emerged from delayed germinated seeds
were abnormal which could not developed into
normal seedlings. This showed the effect of
mutagenic treatment on seed germination and its
lethality. Delayed germination was observed with
most of the EMS dosage (Fig 3).
80 Journal of Oilseed Brassica, 7 (1) January, 2016
Fig 2: Differences in LD50 of EMS in B. juncea (RH-749& NRCHB-101) and S. alba
Fig 3: Effect of EMS on delayed germination of Indian mustard varieties (RH-749 & NRCHB-101) and
S. alba
81Journal of Oilseed Brassica, 7 (1) January, 2016
Induced mutagenesis is a tool to create new
variability in any crop with a higher frequency than
the spontaneous mutations (Chopra, 2005). For
artificially induced mutations either with physical or
chemical mutagens, LD
50
has been considered as
the best dose for high frequency of mutations
(Anbarasan et al., 2013). In the present investigation
the LD50 was calculated on the basis of seed
germination at different doses of EMS. The LD
50
of two B. juncea varieties RH-749 and NRCHB-
101 was found at 0.42% and 0.73% of EMS
dosages respectively (fig. 1). Similarly, Khatri et al.,
(2005) reported the optimum concentration of EMS
in B. juncea cv. S-9 ranging from 0.7% to 1% with
a treatment period of 3-4 hours. The variation in the
LD
50
of EMS for the two varieties of the same
species was found different, suggesting that it may
vary from one genotype to another (fig. 2). It might
be due to the differences in their genetic constitution
and their parentage. Similarly, the LD
50
for the S.
alba was found nearly 0.30% which was lower than
the LD
50
of both the genotypes of B. juncea (fig.
2). It shows that the LD
50
for B. juncea is higher
than the S. alba. The provable reason for the higher
LD
50
for B. juncea genotypes compared to S. alba
might be due to the differences in their ploidy level
as the tetraploid genome of B. juncea may have
higher buffering capacity towards mutagenic effects
as compared to diploid genome of S. alba. Emrani
et al. (2011) reported 0.8% EMS concentration
optimum (LD
50
) for B. napus which is again higher
in comparison to our results with B. juncea
genotypes. However, they treated the seeds for 6
hours in EMS in contrast to the present study (12
hours) and may be the longer treatment period
requires lesser dose of EMS, besides the genotypic
difference this may also be a reason. Furthermore,
these two species are amphidiloid (tetraploid) in
nature but the number of chromosomes in B. napus
(2n=38, AACC) are higher than the B. juncea
(2n=36, AABB) and have one different genome.
Kumar and coworkers (2013b) reported 0.6% dose
of EMS as LD
50
with 8 hours of treatment period in
Helianthus annuus. Similarly, Talebi and coworkers
(2012) reported the LD
50
of EMS in Malaysian rice
as 0.5% with 12 hour of treatment period. The
information generated in the present investigation
may be highly useful for initiating mutation breeding
programmes in rapeseed- mustard crops as the LD
50
of a particular mutagen for a particular genotype
may not be the same for all the genotypes of the same
species or for different species of the same genus.
Acknowledgements
Authors are grateful to the Board of research in
Nuclear Sciences (BRNS), Government of India for
providing financial assistance in the form of BRNS
project entitled “Induced mutagenesis for isolation
of Alternaria blight resistant mutant in Brassica
juncea”
bearing sanction No. 35/14/44/2014-
BRNS and Director, ICAR-DRMR for providing
laboratory facilities.
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