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Research in Plant Biology, 1(4):23-32, 2011 ISSN : 2231-5101
www.resplantbiol.com
Regular Article
Assessment of chemical mutagenic effects in mutation
breeding programme for M1 generation of Carnation
(Dianthus caryophyllus)
Rajib Roychowdhury and Jagatpati Tah*
Cytogenetics and Plant Breeding Section, Botany Department (UGC-CAS),
The University of Burdwan, Burdwan-713104, West Bengal, India
*Corresponding author e-mail: jptahbu@gmail.com
Mutation breeding is one of the most reliable technique in improving crop plants.
Mutagens are used to bring the variability in floricultural crop, like Dianthus. Among
chemical mutagens, the efficiency and effectiveness are necessary to study the effective
dose that can brings the broad spectrum of variability. In present study, three different
concentrations (0.1, 0.4 and 0.7%) of colchicine (Col), ethyl methane sulphonate (EMS)
and sodium azide (SA) were used to treated Dianthus seeds to assess seed germination
behaviour, lethality, pollen sterility and mutagenic effectiveness. It was noted that
increase in the dose of EMS and SA, germination percentage and survivability were
decreased; whereas colchicine doses were proportional to increase germination
percentage at seedling stage, but they were not survived till maturity. Higher lethality
over control (32.89) was shown by 0.7% EMS. Pollen sterility also increased with
increasing mutagenic doses. The maximum pollen sterility (61.1%) was observed under
0.7% colchicine. So, the effect of chemical mutagenesis on seedling and pollen sterility
with EMS (especially 0.7%) treatment is much more beneficial as compared to colchicine
and SA. 0.4% colchicine is effective for other agronomical characters. The highest
mutagenic frequency (13.953) was observed at 0.4% Col and lowest one (4.464) at 0.1%
Col. The mutagenic effectiveness was maximum (86.42%) at 0.1% EMS and minimum
(13.824%) in 0.7% Col. The highest mutagenic efficiency (6.977) was recorded in 0.4% Col
and lowest (0.995) in 0.7% SA on the basis of survivability. The effectiveness of the three
chemicals on Dianthus is ranked as EMS>Col>SA.
Key Words: Chemical mutagens, Dianthus caryophyllus, effectiveness and efficiency,
mutation, mutation breeding.
Mutation breeding has been widely
used for the improvement of plant
characters in various crops. It is a powerful
and effective tool in the hands of plant
breeders especially for autogamous crops
having narrow genetic base (Micke, 1988).
The prime strategy in mutation breeding
has been to upgrade the well-adapted plant
varieties by altering one or two major
agronomic metrical traits which limit their
productivity or enhance their quality.
Dianthus caryophyllus L., commonly known
as Carnation, belongs to the angiospermic
family Caryophyllaceae, is an important
Rajib Roychowdhury and Jagatpati Tah / Research in Plant Biology, 1(4):23-32, 2011
24
floricultural crop all over the world and
ranks just next to Rose in popularity (Laurie
et al., 1968; Staby et al., 1978). This genus is
important by having pharmacological
properties, aromatic things and polymer-
phism in morphology, genetics and
hybridization (Facciola, 1990; Hughes, 1993;
McGeorge and Hammett, 2002; Su Yeons,
2002; Lee et al., 2005). In this modern era, an
agronomic demand of high yielding
cultivar of this crop was noticed. One way
of creating variability in such a self-
pollinated crop is attempting crosses
between two genotypes complementing the
characters of each other but, due to
autogamous nature of this crop,
hybridization at appropriate time is a
difficult process. The only alternative left
with breeders to create variability is
mutation breeding. This method can be
used as a potential source of creating
variability (Novak and Brunner, 1992).
Mutation can produce the development of
Dianthus cultivars with more desirable
floral characteristics and higher
productivity (Roychowdhury and Tah,
2011; Roychowdhury, 2011). It is a tool and
being used to study the nature and function
of genes which are the building blocks and
basis of plant growth and development,
thereby producing raw materials for genetic
improvement of economic crops (Adamu et
al., 2007). Mutation induction offers
significant increase in crop production
(Kharkwal and Shu, 2009) and the
possibility of inducing desired attributes
that either cannot be found in nature or
have been lost during evolution. Treatment
with mutagens alters genes or breaks
chromosomes. Gene mutations occur
naturally as errors in DNA replication. Most
of these errors are repaired but some may
pass to the next cell division to become
established in the plant offspring as
spontaneous mutations. Gene mutations
without phenotypic expressions are usually
not recognized. Consequently, genetic
variation appears rather limited and
breeders have to resort to mutation
induction (Novak and Brunner, 1992).
Mutagenic agents have been used to induce
useful phenotypic variations in plants for
more than seventy decades (Anitha Vasline
et al. 2005). During the past 70 years, more
than 2543 mutant cultivars from 175 plant
species including ornamentals, cereals,
oilseeds, pulses, vegetables, fruits and fibers
have been officially released in 50 countries
all over the world (Maluszynski et al., 2000;
Chopra, 2005). Chemical mutagenesis (the
non-GMO approach) is a simple approach
to create mutation in plants for their
improvement of potential agronomic traits.
In any mutation breeding programme,
selection of an effective and efficient
mutagen is very essential to produce high
frequency of desirable mutation. Many
chemical mutagens have been employed for
obtaining useful mutants in various crop
species (Singh and Singh, 2001). However
the various workers emphasizes that
artificial induction of mutation by
colchicine (Col), ethyl methane sulphonate
(EMS) and sodium azide (SA) provides tool
to overcome the limitations of variability in
plants especially Carnation and induces
specific improvement without disturbing
their better attributes (Mensah and
Obadoni, 2007; Islam, 2010; Roychowdhury
and Tah, 2011). It might be considered that,
these chemical induced growth
abnormalities were mainly due to cell death
and suppression of mitosis at different
exposures. Colchicine is a chromosome
doubling agent that possesses anti-
microtubular action. EMS is a common
alkylating agent, whereas sodium azide is
responsible for creating point mutation in
DNA level. However, these chemicals have
also proved their worth as mutagens to
Rajib Roychowdhury and Jagatpati Tah / Research in Plant Biology, 1(4):23-32, 2011
25
induce genetic variability. Thus, they
become important tool to enhance
agronomic traits of crop plants. The role of
mutation breeding in increasing the genetic
variability for desired traits in various crop
plants have been proved beyond doubt by a
number of scientists (Tah, 2006; Adamu and
Aliyu, 2007; Khan and Goyal, 2009; Kozgar
et al., 2011; Mostafa, 2011). Several factors
such as properties of mutagens, duration of
treatment, pH, pre and post treatment,
temperature and oxygen concentrations etc.
influence the effect of mutagens. The dose
of a mutagen applied is an important
consideration in any mutagenesis
programme. Generally, it was observed that
higher the concentrations of the mutagen
greater the biological damage. To enhance
the mutagenic effectiveness and efficiency
of sodium azide and especially the
metabolite, more knowledge about the
effect of time, pH value, temperature, seed
soaking and various concentrations are
required (Khan et al., 2009). Carnation offers
many opportunities exploitation of
mutations, recombination and of increasing
genetic variability in quantitatively
inherited agronomic characters. Induced
mutations are also useful when it is desired
to improve easily identifiable characters.
The present studies have provided evidence
on the induction of genetic variability
connected with germination behaviour and
metrical traits in Dianthus crop. Thus,
induced genetic variability can effectively
be exploited for evolving mutant strains
possessing desirable attributes and to assess
the mutagenic effectiveness and efficiency.
Materials and Methods
The genotype used for mutagenic
treatment was Carnation (Dianthus
caryophyllus L.), a promising and leading
Dianthus variety of which dry (10%
moisture) and healthy seeds were obtained
from Globe Nursery, Kolkata. It is suitable
to grow in Burdwan agro-climatic
conditions under timely and late sown
condition. Three different concentrations
(0.1, 0.4 and 0.7 % as w/v) of three
chemicals viz. colchicine (Col), ethyl
methane sulphonate (EMS) and sodium
azide (SA) were freshly prepared using
phosphate buffer (pH 7.0) for conducting
the mutagenic treatments (Roychowdhury,
2011; Roychowdhury and Tah, 2011) . For
each chemical treatment, 300 healthy seeds
were taken and were at first surface
sterilized by 0.01% (w/v) mercuric chloride
(HgCl2) for 5 minutes and thoroughly
washed thrice with single distilled water for
10 minutes in each and then presoaked with
double distilled water for 10 hours to
initiate metabolic activities. After pre-
soaking the seeds were blotted, dry and
then placed in freshly prepared solutions of
aforesaid three mutagens with their three
different concentrations. The seeds were
kept in the mutagenic solution for 6 h at
room temperature 28±2°C with intermittent
shaking for providing uniform treatment to
the dipped seeds. An equal number of same
genotypes were soaked in distilled water
which served as control. To avoid
dissociation of chemicals, the acidity of the
solutions was controlled by using buffer
solution. After the treatment time is over,
the seeds were thoroughly washed in
running tap water for three hours to remove
the chemical present in them and then
blotted dry. For laboratory experimentation,
treated seeds were then sown in absorbent
cotton-wet petridish for recording the
germination behaviour like germination
percentage, survival after germination and
maturation and lethality over control
(LOC). The germination percentage per
treatment with three replicates was counted
and recorded on 21st day after seed sowing.
Percent inhibition or stimulation over
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26
control (lethality over control, LOC) were
calculated as [Control-Treated/Control] X
100. Pollen fertility as well as sterility was
tested for each treatment by using 2.0%
(w/v) freshly prepared Aceto-carmine
solution and examined under the low
power (15x) of compound light microscope
(Olympus). Dark stained and normal sized
pollen grains were considered as fertile and
those of irregular shaped and sized with
light or no stain were considered as sterile.
The number of plants survived till maturity,
i.e., at the time of flowering phase, were
scored from each treatment and recorded as
per cent survival and compared with the
control. The germinated seeds were finally
transferred to experimental plots.
In field experimentation, seeds of
ten treatments of Col, EMS and SA as well
as untreated (control) sown during winter
(2009-2010) for raising M1 generation was
done in three different plots having 4 m
length and 25 cm apart by adopting 25 x 20
cm spacing following Randomized Block
Design (RBD) layout with three replications
for each genotypes/treatments at Crop
Research Farm, Botany Department (UGC-
CAS), The University of Burdwan,
Burdwan. This experimental site is situated
at 23.530 N, 22.560 S latitude and 83.250 E, 860
W longitude and 86 meter above the mean
sea level (msl). In M1 generation the
observations on germination, flowering,
seedling survival and other characters were
noted. The all normal recommended
cultural practices and plant protection
measures were followed timely to raise
good crop stand. Uniform agronomical
measures were provided for this M1 crop in
the field experimentation. The data were
recorded on five randomly selected plants
from each replication for some agro-
economic traits studied viz. days to Seed
germination, shoot height (cm) at 21 days
after planting, number of leaves/plant,
stem diameter (cm), leaf area (cm2), fresh
weight (g) of vegetative growth, dry weight
(g) of vegetative growth, days to flowering,
number of flower/plant, flower longevity
(days),seeds/inflorescence, 1000 seed
weight (g). Mutagenic effectiveness and
efficiency were assessed following the
methods of Nilan et al (1965) and Sisikala
and Kamala (1988).
Results and Discussion
The data on seed germination
parameters and pollen fertility in first
mutant (M1) generation for colchicine (Col),
EMS and sodium azide (SA) treatments in
Dianthus caryophyllus are given in table 1. It
was evidenced from table 1 that with
increase in the mutagenic concentration or
dose, the percentage germination had gone
down except in Col, where it gears up;
however, the effects of the chemicals
differed considerably from each other. It
was also recorded by Nandanwar and
Khamankar (1996) and Sing et al. (1997) in
mungbean and Mehetre et al. (1994) in
soybean. In Carnation, as compared to the
control (76%), the germination percentage
was lower in EMS and SA treatments. It
was noted that 67.67, 64.67 and 51% on 0.1,
0.4 and 0.7% of EMS; 69.67, 62.3 and 52.67%
on 0.1, 0.4 and 0.7% of SA, respectively. In
0.1, 0.4 and 0.7% of Col., it was 77.3, 80.3
and 84.3%, respectively, i.e., all values were
higher than the control set. Similar results
were also reported for EMS in soybean
(Pedavai and Dhanavel, 2004) and in
mungbean (Singh and Kole, 2005).
Similarly, the survival rate during
germination period of the treated seeds
reduced with increased dose of mutagens in
M1 generation, except in colchicine, where it
was fully opposite. The lowest laboratory
germination of 51% with lowest survival
seedling (153 out of 300) was recorded in
0.7% EMS. Survival at flowering stage or at
Rajib Roychowdhury and Jagatpati Tah / Research in Plant Biology, 1(4):23-32, 2011
27
maturity due to different mutagenic doses
was ranged 38.9-71.3% in colchicine, 41.7-
69.6% in EMS and 46.4-78.9% in sodium
azide, whereas control at 91.67, that means
compared to control, treatments were still
less. We see that, colchicine treatment firstly
gears up the germinability, but they were
not survived no longer till the maturity. A
reduction in germination and plant survival
in M1 generation of Dianthus due to
mutagenic treatments has also been
reported by Mahna et al. (1989) in Vigna
mungo and Afsar et al. (1980) in Rice. They
observed that, in general, an increase in SA
concentration resulted in decrease the
germination; the plant survival was also
decreased with the increase mutagenic
dose, which is in accordance with the
present findings.
Mutagens are known to induce
lethality at the seedling stage in M1
generation (Table 1). It was revealed from
the observation that colchicine have the
negative value of lethality over control
(LOC) when compared to the control set
(0.00) indicating that low lethality rate (i.e.
higher survival rate) at the seedling stage.
LOC value of both EMS and SA were
positive higher value than that of control
indicating their higher rate of lethality.
Higher LOC (32.89) was recorded in 0.7%
EMS, where 0.7% SA showed second higher
LOC value (30.7). The behavior in terms of
lethality of Col, EMS and SA at highest
concentrations was noted.
The above results could be
attributed to the effect of mutagens on the
meristematic tissues of the seeds. These
may be due to physiological and acute
chromosomal damage (Singh et al., 1997;
Nilan et al. 1976), delay in the onset of
mitosis (Yadav, 1987), chromosomal
aberrations induced enzyme activity such
as catalase and lipase and hormonal activity
resulted in reduced germination
(Ananthaswamy et al., 1971) and
survivability. The probable reason for
reduction in germination might be due to
the disturbed base pair relationship due to
the molecular action of applied mutagens.
Disturbance in the formation of enzymes
involved in the germination process may be
one of the physiological effects caused by
Col, EMS and SA leading to decrease in
germination. Reduced growth due to higher
doses was also explained differently by
different workers. It may be attributed to
one or more of the following reasons (i) the
increase in growth promoters, (ii) the
sudden increase in metabolic status of seeds
at certain levels of dose, (iii) the increase in
destruction of growth inhibitors, (iv) drop
in the auxin level or inhibition of auxin
synthesis and (v) decline of assimilation
mechanism. Taking these as preliminary
consideration.
In the present investigation, the
pollen sterility among all the mutagenic
treatments show gradual increase with
respect to the increase in concentrations,
whereas pollen fertility gradually decreases.
Pollen sterility ranged from 28.7 to 61.1 for
Col, 30.4 -58.3 for EMS and 21.1 – 53.6 for
SA (Table 1). The maximum pollen sterility
(61.1%) was observed under the treatment
0.7% Col. The dose treatment of Col and
EMS was found to be more effective to
produce maximum pollen sterility as
compared to SA. The relative sensitivity of
Dianthus cultivars to various mutagenic
treatments was assessed by studying the
biological damage' induced in M1, in terms
of seed germination, pollen sterility and
fertility. In the present study, reduction in
seed germination and pollen fertility was
concentration dose dependent and linear.
Promoting effects of low doses of Col, EMS
and SA on biological parameters have been
earlier reported by Dubey (1988). In most
cases, meiotic abnormalities are responsible
Rajib Roychowdhury and Jagatpati Tah / Research in Plant Biology, 1(4):23-32, 2011
28
for pollen sterility (Mathusamy and
Jayabalan, 2002; Khan and Wani, 2005). In
addition to chromosomal aberrations, some
genetic and physiological changes might
have caused pollen sterility.
Table 1: Effect of colchicine, EMS and sodium azide on seed germination and pollen fertility in
M1 generation of Dianthus caryophyllus
Mutagen C (%) Total
seed
soaked
Survival
seedling
Germination
percentage
(%)
Lethality
over
control
(%)
Survival at
flowering
(%)
Pollen
fertility
(%)
Pollen
sterility
(%)
Control --------- 300 228 76 0.00 91.67 (209) -- --
COL 0.1 300 232 77.3 -1.75 84.48 (196) 71.3 28.7
COL 0.4 300 241 80.3 -5.7 75.52 (182) 59.6 40.4
COL 0.7 300 253 84.3 -10.96 68.77 (174) 38.9 61.1
EMS 0.1 300 203 67.67 10.96 79.31 (161) 69.6 30.4
EMS 0.4 300 194 64.67 14.91 73.2 (142) 56.3 43.7
EMS 0.7 300 153 51 32.89 51.63 (79) 41.7 58.3
SA 0.1 300 209 69.67 8.33 79.43 (166) 78.9 21.1
SA 0.4 300 187 62.3 17.98 64.7 (121) 61.3 38.7
SA 0.7 300 158 52.67 30.7 46.2 (73) 46.4 53.6
COL = Colchicine, EMS = Ethyl methane sulphonate, SA = Sodium azide, C = Concentration.
Different responses of various
agronomical characters, which are very
much important in any crop improvement
programme, by applied doses of three
mutagens are represented in table 2. It
reveals differences among character values
that fluctuate treatment to treatment. It was
observed that under all three treatments,
most of the characters were decreased with
increasing concentration. For all the
characters studied, 0.4% colchicine
treatment gave the better value than that of
control. Effect of 0.1% EMS was more or
less non-effective, indicating nearest values
to the control set. Increase concentration of
SA negatively affect to the characters.
Present study reveals that wide
spectrum of viable mutants were observed
in the mutagenic treatments (Table 3).
Mutagenic frequency, effectiveness and
efficiency were computed based on
survival, lethality, sterility and injury in M1
generation. The treatment at 0.4% Col
produced a higher frequency of mutation
(13.953), in terms of viable mutants and
percentage lethality, followed by 0.7% EMS
(13.542). The lowest mutation frequency
(4.464) was observed in 0.1% Col. The
highest concentrations were more effective
in inducing higher mutation frequencies in
EMA and SA; whereas moderate dose of
Col (0.4%) produces its higher value. The
mutagenic effectiveness was the maximum
at 0.1% EMS (86.42%) and minimum at
0.7% Col (13.824). The aim is to derive more
efficiency with an optimum mutagenic
dose. For obtaining high efficiency, the
mutagenic effect should overcome other
effects in the cells such as chromosomal
aberrations and toxic effects. According to
Rajib Roychowdhury and Jagatpati Tah / Research in Plant Biology, 1(4):23-32, 2011
29
Konzak et al. (1965), the greater efficiency
of low dose of mutagens appeared in
relation to the fact that lethality and injury
increase with increase in dose at faster rate
than mutations. The maximum mutagenic
efficiency was observed at 0.4% Col (6.977)
and lowest in 0.7% SA (0.995). The
effectiveness of these three mutagens in
Dianthus caryophyllus can be ranked as
EMS>Col>SA.
Table 2: Effect of Colchicine (COL), Ethyl Methane Sulphonate (EMS) and Sodium Azide (SA) on seed
germination, flower characters and some agronomic characters of Dianthus caryophyllus in M1 generation
CH. C. DS SH NL SD LA FW DW DF NF FL SI SW
Control - 5±0.33 24.33±1.9 56.9±1.9 0.22±0.2 3.35±0.4 36.83±1.6 3.75±0.5 27.33±1.6 37.00±1.4 45.04±1.6 26.8±1.3 1.59±0.1
COL 0.1 5±0.14 19.63±2.6 72.4±1.8 0.29±0.1 4.34±0.6 34.60±1.5 3.04±0.3 25.31±1.9 31.00±1.7 44.20±2.3 23.1±1.6 1.60±0.4
0.4 3±0.07 26.47±2.2 82.8±2.1 0.51±0.2 4.66±0.5 45.38±2.3 4.19±0.7 26.67±2.2 41.33±1.3 53.18±2.7 27.4±1.8 1.67±0.3
0.7 4±0.03 23.20±2.25 84.3±2.1 0.34±0.2 3.35±0.3 39.61±2.1 3.13±0.7 24.33±1.4 38.23±1.7 47.52±1.6 29.6±1.3 1.35±0.3
EMS 0.1 5±0.72 20.53±2.0 42.7±2.5 0.31±0.1 4.50±0.4 36.39±1.9 3.13±0.8 25.00±1.8 34.67±1.6 42.34±1.9 26.1±1.4 1.45±0.4
0.4 6±0.48 22.53±2.3 36.2±2.2 0.26±0.3 3.92±0.6 33.23±1.7 2.87±0.4 25.82±2.1 36.12±1.3 38.40±1.4 24.4±1.9 1.63±0.2
0.7 8±0.09 21.20±2.5 52.5±1.8 0.30±0.2 3.65±0.8 30.04±1.9 2.64±0.3 25.83±1.5 39.03±2.1 33.28±2.1 19.2±1.5 1.61±0.4
SA 0.1 4±0.97 21.60±1.8 64.6±2.7 0.21±0.1 3.76±0.5 31.65±2.4 3.01±0.4 25.00±1.4 42.67±2.5 42.09±2.4 24.3±1.8 1.52±0.2
0.4 6±0.12 18.27±2.1 51.4±2.4 0.19±0.2 3.35±0.4 29.42±2.1 2.70±0.7 26.03±2.2 31.90±1.8 39.51±1.4 21.7±1.5 1.49±0.3
0.7 6±0.93 15.30±2.4 41.7±1.9 0.18±0.3 3.10±0.3 26.25±1.5 2.96±0.4 27.21±1.9 33.52±1.5 36.39±1.7 17.8±1.8 1.54±0.3
CH. = Chemical, C. = Concentration (%), DS = Days to Seed germination, SH = Shoot height (cm) at 21 days after planting, NL
= Number of leaves/plant, SD =Stem diameter (cm), LA = Leaf area (cm2), FW = Fresh weight (g) of vegetative growth, DW =
Dry weight (g) of vegetative growth, DF = Days to flowering or Maturity time, NF = Number of flower/plant, FL = Flower
longevity (days), SI = Seeds/inflorescence, SW = 1000 seed weight (g), ± = Standard error.
Table 3: Mutagenic frequency, effectiveness and efficiency of colchicine, EMS and sodium azide on Dianthus
Mutagen Concen
-tration
(C)
Total
Plant
Studied
(P)
Viable
Mutants
Observed
(V)
Percentage
Lethality
(L)
Mutation
Frequency %
(M=V/Px100)
Mutagenic
Effectiveness
(M/C)
Mutagenic
Efficiency
(M/L)
COL 0.1% 112 5 4 4.464 44.64 1.116
0.4% 86 12 2 13.953 34.883 6.977
0.7% 93 9 7 9.677 13.824 1.382
EMS 0.1% 81 7 3 8.642 86.42 2.88
0.4% 79 9 5 11.392 28.48 2.278
0.7% 96 13 8 13.542 19.346 1.673
SA 0.1% 77 6 3 7.792 77.92 2.597
0.4% 103 9 8 8.738 21.845 1.092
0.7% 85 11 13 12.941 18.487 0.995
Rajib Roychowdhury and Jagatpati Tah / Research in Plant Biology, 1(4):23-32, 2011
30
Conclusion
It is advocated that the effect of chemical
mutagenesis on seedling and pollen
sterility with EMS (especially 0.7%)
treatment is much more beneficial as
compared to Colchicine and SA. 0.4% Col.
is effective for other agronomical characters
and as per effectiveness, the rank is
EMS>Col>SA. Hence, these chemical
mutagens could be used as a means of
improving the genetic background of
Dianthus cultivar with desirable alleles for
further improvement in seed yield and its
component major traits.
Acknowledgements
We thank to Prof. Alok
Bhattacharya, Head of the Department of
Botany (UGC-CAS) at The University of
Burdwan for providing necessary facilities.
We want to acknowledge Dr. Abhijit
Bandyopadhyay and Dr. Tinkari Dalal in
this department for their several sorts of
help. We also thank the anonymous
reviewers for critical suggestions and
useful inputs.
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