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BIOINFOLET 4p) : 181- 187,2007
EFFECTIVITY AND EFFICIENCY OF GAMMA RAYS, SODIUM AZIDE AND
ETHYL METHANESULPHONATE IN LINSEED.
Rupesh S. Badere'and Aruind D: Choudhary
Department of Botany, RTM Nagpur University Campus, Amravati Road,
Nagpur 440033. rsbadere @ rediffmail.com
" Department of Botany, Histop coltege, Civit Lines, Nagpur 440001.
ABSTRACT
The present paper reports about the effectivity and efficiency of gamma rays,
sodium azide and ethylmethanesulphonate in Linum usitatissimum. Gamma rays
were found to be the most effective mutagen amongst the three followed by so-
dium azide and ethyl methanesulphonate. Similarly, the sodium azide was found
to be the most efficient mutagen followed by ethyl meth4nesulphonate and gamma
rays. On the basis of our findings we propose sodium azide to be the preferable
mutagen for induction of mutations in linseed due to its high efficiency coupled
with fairly good effectivity.
Key words : Linseed, Linum, induced mutagenesis, effectivity, efficiency
lndia is blessed with multitude of rich simpler infrastructural needs and its
oleaginous genetic plant wealth. Oilseed crops applicability. Mutations have been instrumental
comprise of groundnut, rapeseed, mustard, in crop improvement since they were proved to
soybean, safflower, sesame, niger, castor and bb inducible. Since then, mutations are being
linseed. lnspite of being world's fourth largest employed directly or indirectly for improving
oil economy; the imports of edible oil has risen various traits of plants. More than one thousand
manifold during last decade in lndia. This clearly five hundred direct mutants have been released
indicates the need to increase oil production. as varieties in the last century. ln addition to
Linseed is grown in lndia since ancient times this about sevcn hundred mutants have been
for its oil. lt shares 5.1% of area covered under used in crosses to breed improved varieties
oilseeds and has ils 2To share in terms of (Laguda,2004).Althoughmutagensbringabout
productionamongstthem(Rao,1991).Attempts changes in nucleotide sequence of DNA, the
for linseed improvement have been made mode of action of each mutagen is distinct.
earlier through various approaches including Moreever, a mutagen may effectively bring
induced mutiations (Sinha et al. 1981, Wang et. about mutations, but the accompanying
al., 2000). However, still there remains a wide undesirable effects like lethality or sterility may
scope of improvement in linseed in almost all decrease its efficiency. Thus, in orderto exploit
aspects including yield, oil content and oil inducedmutagenesisforcropimprovement,the
ffi$Til": ;ff *:'i:'J":3:;:iciencv of a
While genetic engineering is becoming
a popular tool for crop improvement; induced As mentioned earlier, induced mutations
mutations still carries its relevance in terms of have been helpful in linseed improvement.
182
However, studies about the effectivity and
efficiency of mutagens have not been repoded.
Hence, the present study was carried out to
assess the effectivity and efficiency of gamma
rays, sodium azide and ethyl
methanesulphonate in linseed (Linum
usitatissimum L.).
Seeds of two cultivars of linseed (Linum
usitatissimum L.), NL-97 and RLC-6 were
treated with gamma rays (GR), Sodium azide
(SA) and Ethyl methanesulphonate (EMS). GR
treatment was given using 60Co as radiation
source (USlC, Nagpur University Campus,
Nagpur). One hundred twenty-five seeds of
each variety were irradiated with the dose of
200,400,600 and 800 Gy (100R = 1Gy). ln
case of chemical mutagens, one hundred seeds
were treated per concentration of mutagen. ln
this case dry 3h and 6h presoaked seeds in
distilled water (PSW) were treated with mutagen
solution for 1Bh at 25 + 10C with continuous
shaking. Then the seeds were thoroughly
washed and post-soaked for t h in distilled
water. SA (E Merck, Germany) was used at the
concentration of 0.005, 0.010 and O.02Oy. (wl
v), while EMS (Sigma Chemicat Company,
USA) was used at the concentration of 0.05,
0.10 and 0.20% (w/v). The dry and water
soaked seeds served as control for all the
mutagenic treatments. The mutagen treated
seeds were sown in plots along with the control
seeds. ln M, generation, pollen sterility was
used as a measure of lethality induced by
mutagens. The pollen sterility was determined
on the basis of stainability and shape of pollen
grains. Five buds each from twenty{ive plants
per dose/concentration were taken and the
pollens from these buds were stained wilh 2o/"
acetocarmine. Unstained and irregular pollens
were counted as sterile. The percentage of
sterile pollens was calculated for each dose/
concentration. The M1 population was
harvested at maturity and seeds were collected
from each ptant individually.
Seeds obtained from M, plants were
sown to raise M, generation in the next season.
BIOINFOLET
Due to limited space, large population could not
be grown. About 25 seeds were sown per row.
The population so raised was screened for
various morphological traits. This helped in
identifying different variants. The M, population
was screened for various morphological
mutants such as tall, dwarf, highly branched,
white flower, variegated seed, stunted & sterile,
early flowering, high yielding and frequency of
mutants was calculated in terms of percentage
on M, plant basis. The mutagenic effectivity and
efficiency were calculated by the following
formulae (Kharkwal, 1 998):
Mutagenic effectiveness = Mf/tc or Mf/Gy
Mutagenic efficiency = Mf/S
Where, Mf - mutation frequency.
t - period of treatment in case of
chemical mutagen.
c - concentration of mutagen in
case of chemical mutagen.
Gy - dose of gamma radiation.
S - %pollensterility.
ln the present study pollen sterility was
selected as a measure of lethality. The mutagen
treatment increased the pollen sterility in allthe
cases. GR affected both the varieties equally
at lower doses. At higher doses, however, var.
RLC-6 seemed to be more prone to the lethality
of GR. The effect of the chemical mutagens
differed in affecting the poilen fertility. SA
affected pollen fertility equally in both the
varieties except at the concentration of 0.01%.
On the other hand, lethality of EMS has been
very well demonstrated in RLC-6 than NL-97.
Presoaking grossly increased the deleterious
effect of mutagen as evident from increased
pollen sterility. Moreover, the sterility was more
pronounced in var. RLC- 6 treated with SA
(Table 1). The induction of polten steritity is
common a outcome of mutagen treatment due
to factors like chromosomal irregularities
(Konzak et. al., 1961, Bora et. a!., 1961),
chromosomal aberrations (Ehrenberg et al.
1961), and gene mutation and cryptic
deficiencies (Bender and Gaul 1966, Sato and
Gaul, 1967)"
Various morphological mutants were
Vol.4 (3),2007
isolated in the present investigation. These were
induced by all the mutagens used. However,
no mutant could be detected in var. RLC-6
treated with 800Gy of GR and in 3h pSW set of
var. NL-97 treated with 0.2% EMS. The
frequency of mutants varied with variety,
mutagen, dose/concentration and treatment. lt
was more in NL-97 as compared to RLC-6 in
case of GR. ln contrast, the chemical mutagens
induced the mutants with higher frequency in
RLC - 6 than in NL - 97. presoaking modified
the efffect of mutagen in the persent study. ln
case of SA, presoaking seemed to increase the
mutation frequency in NL - 97, while it
decreased the mutation frequency in RLC -6.
However, the mutation frequency remained
almost equal in both the varieties treated with
EMS (Table 1).
The detection of a mutant in the treated
population depends upon various factors and
hence, mutation frequency varies with the
treatment. According io Auerbach (1967) all
mutations may not necessarily bring about
observable change. Similarly, presence or
absence of a particular mutant in a mutagen
treated population depends on the availability
of mutagenic loci to the mutagen (Reddy,
et. al., 1994). During chemical mutagen
treatment seeds become metabolically active.
At this time various mutually exclusive factors,
the one which favour mutation induction and
the other which repair the mutagenic lesions,
are active inside the cell. The presoaking results
in leaching of protective compounds (Kamara,
et . al., 1960), oxygen enrichment (Latteral,
1961) and increase in permeability of cell
membrane (Walles, 1967). All these factors act
towards increasing the mutation frequency.
Similarly, due to presoaking, seeds gain
capacity to repair the damage caused by
mutagen to DNA (Cerda-Olmedo and Hanawalt,
1967, Strauss, et. a\.,1968), thus resulting in
the decrease in mutation frequency. Hence, the
frequency of mutation is a resultant of these
two opposing factors acting simultaneously.
Effectivity is the measure of mutation
183
frequency per unit dose/concentration. The
effectivity was found to be dependent on
genotype and the dose/concentration of
mutagen. Differential effectivity of GR on the
Iwo varieties was found at the dose of 200 Gy;
whereas at rest of the doses, effectivity of GR
was more or less similar in both the varieties.
The effectivity of SA was more pronounced in
RLC-6 as compared to NL-97. ln case of NL-
97 the effectivity of SAdecreased with increase
in concentration of mutagen. presoaking mosily
enhanced the effectivity of SA in NL-97. lt was,
however, decreased in case of RLC-6. Unlike
SA, the effectiveness of EMS was almost similar
in both the varieties in dry and presoaked sets
of EMS (Table 1). ln pooted data of both the
varieties, GR seemed to be most effective at
the dose of 200 Gy. Whereas, amongst
chemical mutagens, SA was most effective at
moderate concentration and EMS displayed its
highest effectivity at lowest concentration. The
effectivity of SA and EMS decreased due to
presoaking (Table 1). However, when mutagen
was pooled over dose/concentration and
varieties, SA was found to be most effective in
3h PSW set (Fig. 1). As far as mutagens are
concerned, SA proved to be most effective
mutagen in present investigation (Fig. 2).
The efficiency of a mutagen gives an idea
about the undesirable effects associated with
its mutation induction ability. ln the present study
GR demonstrated highest eff iciency at the dose
of 400 Gy in both the varieties. Whereas,
amongst chemical mutagens, SA was found to
be more efficient than EMS. Similarly, the
efficieny of SA was found to be more in RLC-6
as compared to NL-97. The presoaking of seeds
prior to treatment modified the efficiency of SA
in both the varieties. The efficiency of SA
increased due to presoaking in NL-97, whereas
it decreased in RLC-6. On the other hand EMS
was more efficient in inducing mutations in NL-
97 than RLC-6. However, its efficiency was
decreased due to presoaking in both the
varieties (Table 1). ln pooled data of both the
varieties, efficiency of GR was found to be most
at 400 Gy of dcse. Amongst chemical
184
mutagens, SA was most efficient at the
concentration of 0.01% in linseed; whereas
EMS was most efficient at lowest concentration
of 0.05%. The efficiency of EMS decreased with
increase in concentration. Presoaking lowered
the efficiency of both the mutagens in linseed
(Table 1). Various treatments of both the
chemical mutagens displayed ihe similar
pattern of efficiency in linseed' The efficiency
of SA and EMS was highest in dry seeds and
least in 6h PSW seeds (Fig. 1)' Amongst
mutagens, GR proved to be the most efficient
mutagen (Fig. 2).
The decreased efficiency is indicative of
increased sterility with respect to mutation
frequency. The presoaking makes cell
permeable and hence increases the entry of
mutagen into the cell (Walles, 1967). The
increased mutagen concentration induces
sterility at faster rate than mutation frequency
(Konzak et. a\.,1965). Hence, the decreased
efficiency might be due to induction of sterility
at faster rates than the mutations.
The different mutagens used in present
study showed different effectivity and efficiency.
These differences might be due to the different
sets of conditions produced by a mutagen, to
which cerlain genes respond in a unique way
(Shukla, 1972), specificities to a direct reaction
between mutagens and DNA(Auerbach, 1976)
BIOINFOLET
and mode of action of chemical and physical
mutagens (Jain and Raut, 1966, Alexander and
Lett, 1967). ln present investigation, SA was
found to be the most effective mutagen in
linseed. lt was followed by EMS and GR in
terms of effectivity. However, GR proved to be
the most efficient mutagen in linseed varieties.
The next efficient mutagen was SAfollowed by
EMS. The effectivity of SA, EMS and GR in
linseed are in agreement with the report of
Khalatkat et. al., (1979) in Hordeum vulgare.
However, the most efficient mutagen in H.
vulgare was found to be SA (Khalatkar, et. al.,
1979). Similarly, NMU (an alkylating agent like
EMS) was found to be 2-5 times efficient than
GR in Lens culinaris var. -36 (Dixit and Dubey
19BO). ln a recent finding Gaikwad and
Kothekar (2004) reported EMS to be more
effective and efficient mutagen as compared to
SA in Lens culinaris var. L- 4611 and L- 4639.
ln the present study intervarietal differences in
effectivity and efficiency of a mutagen were also
seen. Such responses have been reported by
Kothekar (1989) in mothbean and Kharkwal
(1998) in chickpea.
Thus, this study proves SA to be a
preferable mutagen for mutagenization of
linseed due to its highest effectivity alongwith
fairly high efficiency.
Fig. 1 Mutagen pooled over dose/concentration and varieties.
Tradrnatrtr
D
! 0'6
lit 0.4
60b
:: I
Yol. 4 (3),2007
Table 1 : Effectivity and Efficiency of various mutagens in induction of mutation in lhe var.
NL-97 and RLC-6 of linseed.
185
NL.97 RLC.6 Treatment pooled
over varieties
Treatment % T" Effect- Effic-
letha- mutat- iveness iency
lity ions
% 7o Effect- Effic- Effectiv- Efiicie_
letha- muta- iveness iency eness ncy
lity tions
200Gy 22.62 6.44
400Gy 19.76 22.80
600Gy 38.18 14.41
B00Gy 35.58 38.73
0.32
0.06
0.02
0.05
Gamma rays
4.29 21.43 5.00 0.03
1 .15 20.26 21.0s 0.05
0.38 51.90 5.00 0.08
1 .09 57.73 0.00 0.00
0.18 0.26
0.06 1.95
0.05 0.24
0.05 1.09
0.23
1.04
0.10
0.00
Dry set
0.0050/. 12.75 6.78 75.33
0.01% 9.97 12.48 69.33
0.02% 15.75 5.77 16.03
3h PSW
0.005% 12.25 20.67 229.67
0.01% 7.67 19.95 110.83
0.02o/o 6.55 7.87 21.86
6h PSW
0.005% 7.27 18.41 204.56
0.01% 12.47 6.91 38.39
a.o2% 30.81 14.35 39.86
122.78
129.36
40.60
0.16 151.89
0.51 118.06
0.29 15.82
0.33 141.00 1.43
0.95 75.64 0.75
0.35 33.96 0.41
SA
0.53 12.24
1.25 14.01
0.37 17.23
1.69 42.20
2.6 43.92
1 .20 12.33
2.53 21.20
0.55 21.43
0.47 29.14
15.32170.22
34.09 189.39
23.46 65.17
6.67 74.11
22.55125.28
3.52 9.78
6.97 77.44
20.32't12.89
10.10 28.06
1.25
2.43
1.36
0.89
1.83
0.87
0.93
1.56
0.75
Dry set
0.05% 15.04
0.1% 8.82
0,22% 16.23
3h PSW
0.05% 37.25
0.1% 18.34
0.22% 23.49
6h PSW
0.05% 22.10
0.1Y" 27.50
0.2"/o 33.75
14.83 16.48
9.10 5.06
25.59 7.11
19.56 21.73
1.08 0.6
0.00 0
2.10 2.33
7.95 4.42
6.38 1.77
EMS
0.99 31.58
1.03 28.52
1.58 33.08
0.53 20.07
0.06 54.67
0 57.80
0.10 31 .18
0.29 40.00
0.19 45.69
22.30 24.78 0.71 20.63 o.B5
15.75 8.75 0.55 6.91 0.79
9.41 2.61 0.28 4.86 0.73
10.27 11.41 0.51 16.57 0.52
8.18 4.54 0.15 2.30 0..t1
s.69 1.58 0.10 1.58 0.10
0.08 2.59 0.09
4.25 4.94 0.27
0.08 1.38 0.13
2.56 2.84
9.84 5.45
3.53 0.98
186
Fig. 2 Mutagen pooled over treatment and varieties.
BIOINFOLET
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I
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