Plant Breed. Biotech.

2020 (September) 8(3):252~264 Online ISSN: 2287-9366

https://doi.org/10.9787/PBB.2020.8.3.252 Print ISSN: 2287-9358
RESEARCH ARTICLE

Inducing Potential Mutants in Bread Wheat Using Different

Doses of Certain Physical and Chemical Mutagens

Ghada M.Sh.M. Abaza1†, Hassan A. Awaad2†, Zakaria M. Attia1, Khalid S. Abdel-lateif3, Mohamed A. Gomaa2,
Safy M.Sh.M. Abaza4, Elsayed Mansour2*
1
Plant Research Department, Nuclear Research Center, Atomic Energy Authority Inchas, Inchas 13759, Egypt
2
Agronomy Department, Faculty of Agriculture, Zagazig University, Zagazig 44511, Egypt
3
Genetics Department, Faculty of Agriculture, Menoufia University, Shebin 32511, Egypt
4
Department of Genetics and Agricultural Genetic Engineering, Faculty of Agriculture, Zagazig University, Zagazig 44511, Egypt

ABSTRACT Mutation is an effective strategy not only for creating novel variation into crop genome but also for direct releasing
adapted and high-yielding genotypes. The current work explores inducing genetic variability in bread wheat using physical and
chemical mutagens. Three wheat cultivars were treated by three mutagens; gamma irradiation (five doses; 250, 300, 350, 400 and 450
Gray); laser ray (three treatments; 1, 1.5, and 2 hour exposure) and EMS (three concentrations; 0.2, 0.3 and 0.4%). Besides, a
combination of physical (laser) and chemical (EMS) mutagens using middle range of each treatment (1.5 hour laser and 0.3% EMS) was
attempted to be applied. The treated seeds were sown in the first season and 4050 M1 plants were harvested. The harvested seeds were
sown in the second season, and 78750 M2 plants were obtained. The selection was performed in second season (M2) based on
morpho-physiological and yield traits; flag leaf area, flag leaf chlorophyll content, plant height, spike length, grain yield per plant and
its components. Based on evaluated traits fourteen mutants were selected to be evaluated in the third generation (M3). The results
indicated that the used mutagens had direct impact and significantly improved agronomic traits in derivative mutants compared to their
parent cultivars. Moreover, the maximum increment in yield related traits were obtained by 0.4% EMS, 1 and 2 hour-laser, 350-Gy, 1.5
hour × 0.3% EMS and 250-Gy. The obtained results highlighted the importance of these doses of applied mutagens to induce useful
genetic variability in bread wheat for improving grain yield and contributing traits.
Keywords Mutation breeding, Genetic variation, Gamma ray, Laser beams, Ethyl methanesulfonate

INTRODUCTION to current and expected future high population growth rate,

the gap between wheat production and consumption would
Wheat (Triticum aestivum ssp) is one of the most im- increase, adding further challenges to the limited food
portant cereal crops worldwide (Shewry 2009). Its total resources in this developing country. Furthermore, current
cultivation area in 2018 was 214.29 million hectares with climate change, particularly in the Mediterranean region,
global production of 734.05 million tons (FAOSTAT, obstructs efforts to increase the yield, and these changes are
http://www.fao.org/statistics/en/). Egypt was involved in expected to increase (García‐Ruiz et al. 2011; Cook et al.
these statistics with cultivation area 1.32 million hectares 2016; Mansour et al. 2017; 2018). This situation highlights
produced 8.8 million tons. Nonetheless, Egypt is amongst the importance of wheat breeding to increase current pro-
the top wheat importers, with around 10.16 million tons per duction.
year (FAOSTAT, http://www.fao.org/statistics/en/). Due Genetic variability presents the principle of plant breed-

Received June 15, 2020; Revised July 11, 2020; Accepted July 15, 2020; Published September 1, 2020
*Corresponding author Elsayed Mansour, sayed_mansour_84@yahoo.es, Tel: +20-552245274, Fax: +20-55221688
†
These authors contributed equally.

Copyright ⓒ 2020 by the Korean Society of Breeding Science

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0)
which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Using Physical and Chemical Mutagens for Inducing Promising Mutants in Bread Wheat ∙ 253

ing. It could be created through hybridization or mutations (NMU) hydroxylamine and sodium azide (Wu et al. 2005;
followed by selection (Addisu and Shumet 2015). In com- Talebi et al. 2012; Luz et al. 2016). The chemical mutagens
parison to mutation, improving through hybridization is la- result frequently point to mutation changing single nucleo-
borious, time-consuming, with low genetic variation (Hanafiah tide pairs (Siddiq et al. 1968; Rakszegi et al. 2010; Kurowska
et al. 2010). Mutation is considered as a forward genetic et al. 2012). EMS is very effective and most popular chemical
approach, which provides novel phenotypes that can be ex- mutagen (van Harten 1998). It produces haphazard point
ploited in classical breeding programs (Parry et al. 2009). mutation in plant material by nucleotide substitution. EMS
As such, mutation can be seen as a powerful strategy for reacts with guanine or thymine through the addition an
direct release of improved varieties (Dhaliwal et al. 2015). ethyl group. It induces high frequency of nucleotide sub-
Essentially, up to date, mutation breeding has been used for stitutions with majority of changes about 70 to 99% in the
developing 3288 mutant varieties in different crops, which obtained mutants are GC to AT base pair transitions (Sega
are registered in the database of the International Atomic 1984; Till et al. 2007). Accordingly, these mutations gene-
Energy Agency (FAO/IAEA, http://mvgs.iaea.org). Most rate allelic versions of genes causing phenotypic variations
mutant cultivars have been developed in ornamentals, cereals, (Dhaliwal et al. 2015). Additionally, the combination be-
legumes and oil crops. Regarding bread wheat, 237 mutant tween physical and chemical mutagens can be implemented
varieties were realized; 195 using physical mutagens, 40 by irradiating seeds first by a physical mutagen followed by
using chemical mutagens and 2 varieties using their com- treating with a chemical mutagen in a solution (van Harten
binations. Furthermore, the released mutant varieties for 1998). This approach is interesting for enhancing mutation
major crops have been cultivated over large areas, in- spectrum and frequency (Suprasanna et al. 2015). Mutation
creasing food production and thus contributing to food se- breeding was performed previously in bread wheat by
curity (Suprasanna et al. 2015). various researchers using physical and chemical mutagens
The used physical mutagens in plants comprise non- in different doses. Rakszegi et al. (2010) used 0.6 and 0.9%
ionizing (e.g. UV) or ionizing (e.g. X and gamma rays, EMS to mutagenize bread wheat (cv. Cadenza) and they
alpha and beta rays) radiation. The physical mutation fre- obtained 3750 M6 mutant lines displayed wide diversity in
quently causes double-strand DNA breaks and produces morphological and agronomic traits. Likewise, Mishra et
large deletion in DNA and thereby causes visual effects on al. (2016) utilized 0.2% EMS solution to develop mutant
the chromosome structure. Gamma rays is the most wide- lines for amylose variation in bread wheat. Moreover,
spread physical mutagen among plant breeders due to its Singh and Datta (2010) utilized gamma radiation (10 to 100
convenience as well as its capability of penetrating deeper Gy) for improving yield attributes in bread wheat. In addi-
in the tissues (Suprasanna et al. 2015). It induces nucleo- tion, Xiong et al. (2018) used gamma radiation (250 Gy) for
tide substitutions and small deletions of 2-16 bp, in addition improving genetic diversity in bread wheat.
mutation frequency is expected to be one mutation per 6.2 The aims of this study were to induce genetic variability
Mb (Sato et al. 2006). Furthermore, laser beams were de- in bread wheat using physical and chemical mutagens and
monstrated that are able to induce chromosome mutation evaluate the impact of using different doses on major ag-
by Berns et al. (1969). It was found that longer irradiation ronomic traits in three commercial bread wheat cultivars.
using laser beams damages the genetics of plant cells but Additionally, to identify the most potential mutants in terms
lower doses cause bio-stimulation effects (Dudin 1990; of improving agronomic traits, compared to parent cultivars.
Rybinski 2001; Chen et al. 2005). Its stimulation mechanism
is due to synergism between polarized monochromatic laser
beams and plant photoreceptors (Koper et al. 1996). On the MATERIALS AND METHODS
other hand, the chemical mutagens contain ethyl methane-
sulfonate (EMS), hydrogen fluoride (HF), methyl methane- Mutagenesis and obtaining mutant seeds
sulfonate sulphonate (MMS), N-Nitroso-N-methylurea Seeds of three bread wheat cultivars (Sids-12, Sakha-94,
254 ∙ Plant Breed. Biotech. 2020 (September) 8(3):252~264

and Gemmiza-9) were obtained from Wheat Research 250, 300 and 350 Gy were selected for both Sids-12 and
Section, Field Crops Research Institute, Egyptian Agri- Gemmiza-9 cultivars, while Sakha-94 was irradiated with
cultural Research Center, Egypt (Supplementary Table 350, 400 and 450 Gy. For the laser beams treatment, seeds
S1). The used cultivars in this study were selected based on were irradiated by laser ray with 1, 1.5, and 2 hour ex-
their high-yielding production, whether they are used as posure. The helium-neon laser (He-Ne) at the wavelength
commercial cultivars and also have different pedigrees. of 632.8 nm and power density of 1MW-2 was applied as
Two hundred seeds of each cultivar were treated separately the source of radiation. For the EMS treatment, seeds were
by three mutagens: two physical mutagens (gamma ray and soaked at 0.2, 0.3 and 0.4% (v/v) EMS concentration for 8
laser beams) and one chemical mutagen (EMS). All these hours. For the combination treatment was used only for
treatments were carried out at the Nuclear Research Center, parent cultivar Sids-12, seeds were treated first by laser for
the Egyptian Atomic Energy Authority, Egypt. Gamma 1.5 hours and then followed by soaking in 0.3% EMS
60
irradiation at a cobalt 60 ( Co) was used to irradiate seeds. solution as middle range of each treatments. After each
A preliminary experiment of gamma irradiation sensitivity treatment, seeds were washed in distilled water at ambient
test was conducted using Petri-dishes with nine doses; 0, temperature and then were air-dried. The applied doses of
150, 200, 250, 300, 350, 400, 450 and 500 Gray (Fig. 1, used mutagens in the three parent cultivars were presented
Table 1). Based on reduction percent in seedling epicotyl in Fig. 2.
length, according to Konzak and Mikaelson (1995), three
gamma-ray doses were selected for each cultivar. Doses

Fig. 1. Epicotyl length (cm) of seedling of the three wheat cultivar; Sids-12 (Sd), Sakha 94 (Sh-94) and Gemmiza-9 (Gm-9)
following irradiation of its seeds with 9 doses of gamma rays (0, 150, 200, 250, 300, 350, 400, 450 and 500 Gy).
 Using Physical and Chemical Mutagens for Inducing Promising Mutants in Bread Wheat ∙ 255

Table 1. Mean values of epicotyl length of seedling for the three wheat cultivars as affected by different gamma-rays
doses and percentage of reduction as compared to the parent cultivar (0 dose).
Sids-12 Sakha-94 Gemmeiza-9
Dose (Gy) Epicotyl length Reduction Epicotyl length Reduction Epicotyl length Reduction
(cm.) (%) (cm.) (%) (cm.) (%)
Parent cultivar (0) 4.38 - 5.12 - 4.18 -
150 2.44 44.29 4.78 6.64 3.74 10.52
200 3.01 31.27 4.43 13.47 4.18 0
250 2.72 37.90 4.23 17.38 2.42 42.10
300 2.42 44.74 3.73 27.14 1.99 52.39
350 2.16 50.68 3.15 38.47 1.36 67.46
400 0.95 78.31 2.16 57.81 1.15 72.48
450 1.32 69.86 1.75 65.82 0.74 82.29
500 1.04 76.25 1.11 78.32 0.81 80.62
LSD0.05 0.738 1.067 0.932

Fig. 2. Schematic diagram showing different mutagen treatments applied in three parental cultivars and selected ones from
derivative mutants.

experimental site is mostly loamy sand (77% sand) (Sup-

Field experimental site plementary Table S2). In addition, meteorological data, i.e.
Three field experiments were performed in the experi- averages of minimum and maximum air temperatures and
mental farm of the Nuclear Research Center, the Egyptian total rainfall are presented in Supplementary Table S3.
Atomic Energy Authority, Egypt (30º 24’ N and 31º 35’ E), Sowing was on the 21st of November of each season,
for three winter growing seasons between 2011 and 2014. representing the optimal period for wheat cultivation in
The soil physical and chemical characteristics of this Egypt. Ammonium nitrate (33% N), Calcium Superphos-
256 ∙ Plant Breed. Biotech. 2020 (September) 8(3):252~264

phate (15.5% P2O5) and Potassium Sulphate (48% K2O) spike (NFSS), number of sterile spikelets/spike (NSSS),
fertilizers were applied at the recommended rates of 240 kg number of spikes/plant (NSP), grain number per spike (GNS),
N/ha, 70 kg P2O5/ha and 60 kg K2O/ha, respectively. Other 1000-grain weight (TGW, g) and grain yield/plant (GYP,
agronomic practices including irrigation, pest, disease and g) were also estimated.
weed control were followed as recommended for the wheat
production in the study region. Statistical analysis
The analysis of variance (ANOVA) was applied for all
Field trials and generation advance investigated traits. In addition, least significant difference
In the first season (2011-2012), seeds were immediately (LSD) values were performed at the 5% probability level
sown after treatment in three separate experiments; one for (P ＜ 0.05). The statistical analyses were made using SAS
each parent cultivar and its treatments. The experimental software (version 9.2, SAS Institute, Cary, NC, USA).
design was randomized complete block design in three
replications. Individual seeds were sown in plots consisting
of five rows 3-m long with 30 cm apart, and seeds were RESULTS
spaced with 10 cm on the row. Seeds harvested from 4050
M1 plants were sown in the second season (2012-2013) in Analysis of variance
randomized complete block design with three replications, Analysis of variance for studied traits is presented in
and 78750 M2 plants were obtained. Selection was per- Table 2. The results suggested that the used mutagens had
formed on M2 individual plants at harvesting based on a direct impact on all evaluated traits with statistically
morpho-physiological and yield traits; flag leaf area, flag significant variations. This clearly depicts genetic vari-
leaf chlorophyll content, plant height, spike length, grain ability among the obtained families for all investigated
yield per plant and its components. Fourteen promising traits as the mean values are significantly different from
mutants were selected: five from Sids-12, four from Sakha- those of the parent cultivars. This finding confirms the
94, and five from Gemmiza-9. The seeds of these selected efficiency of tested mutagens and possibility for inducing
families were sown in the third season (2013-2014; M3 more variations for morpho-physiological and yield traits.
generation) to be evaluated and compared to the parent
cultivars. The selected mutants were not derived from all Mean performance
used mutagens as shown in Fig. 2. Three separate experi- Flag leaf area significantly increased by used mutagens,
2
ments were performed for the three parent cultivars and with values varying between 19.26 and 56.9 cm (values of
2
their derivative mutants using randomized complete block three parents were 18.8, 22.5 and 43.7 cm ). Respecting
design in three replications, to evaluate the promising four- Sids-12, the highest values were assigned to mutants de-
teen M3 mutants. rived from 1.5 hour-laser × 0.3-EMS and 0.4-EMS fol-
lowed by 250-Gy, 350-Gy and 2 hour-laser, which were
Agronomic measurements significantly higher than their parent cultivar (Fig. 3A).
Fifteen plants were chosen from each replication to mea- Likewise, in Sakha-94, the mutants derived from 350-Gy
sure morpho-physiological and yield traits. Flag leaf area and 2 hour-laser followed by 0.4-EMS exhibited larger flag
2
(FLA, cm ) was recorded on main stems at the time of 100% leaf area than their parent cultivar. For Gemmiza-9, the
heading as flag leaf length × flag leaf width × 0.72. Flag leaf highest values were obtained using 300-Gy, 1 hour-laser
chlorophyll content (FLCC) was measured at anthesis and 250-Gy followed by 350-Gy. As illustrated in Fig. 3B,
using Chlorophyll Meter SPAD-502Plus. Plant height (PH) flag leaf chlorophyll content responded differently to the
was measured at maturity as the distance (cm) from the treatments, with values ranging between 50.84 and 64.9
base of the plant to the tip of the spike, excluding awn. In SPAD reading (values of three parents were 46.3, 51.4 and
addition, spike length (SL, cm), number of fertile spikelets/ 54.7). Apart from 2 hour-laser in Sids-12 and 350-Gy in
 Using Physical and Chemical Mutagens for Inducing Promising Mutants in Bread Wheat ∙ 257

Table 2. Analysis of variance (mean squares) of the investigated traits for fourteen M3 mutant families and parent cultivars.
Source of
d.f. FLA FLCC PH SL NFSS NSSS NSP NGS TGW GYP
variance
Sids-12
Treatments 5 77.65** 51.39** 90.41** 7.865** 10.73** 4.560** 2.557** 1.326** 13.42** 149.07**
Residual 10 2.014 1.269 0.783 0.053 0.066 0.003 0.092 0.026 0.390 0.220
Total 17 24.152 16.03 27.32 2.350 3.215 1.343 0.819 0.406 4.600 44.005
Sakha-94
Treatments 4 51.49** 58.93** 21.53** 3.442** 10.16** 0.337* 26.21** 0.144** 16.47** 99.56**
Residual 8 1.006 0.775 1.249 0.083 0.113 0.062 0.140 0.009 0.630 0.799
Total 14 15.46 17.46 7.713 1.038 2.984 0.132 7.572 0.047 5.096 29.29
Gemmiza-9
NS
Treatments 5 216.09** 3.774** 56.55** 4.677** 3.497** 3.547** 4.478 0.264** 23.44** 172.31**
Residual 10 5.528 0.280 4.490 0.042 0.266 0.083 0.485 0.003 0.350 2.739
Total 17 67.41 1.64 19.47 1.405 1.272 1.094 1.760 0.080 7.151 52.67
NS
, *, ** not significant, significant at the 0.05 and 0.01 probability levels, respectively. d.f.: degrees of freedom, FLA:
flag leaf area, FLCC: flag leaf chlorophyll content, PH: plant height, SL: spike length, NFSS: number of fertile
spikelets/spike, NSSS: number of sterile spikelets/spike, NSP: number of spikes/plant, GNS: grain number per spike,
TGW: 1000-grain weight and GYP is grain yield/plant.

Gemmiza-9, all mutants showed chlorophyll content higher and the parent had 11.5 cm) (Fig. 3D). Similarly, the num-
than those of parent cultivars. For Sids-12, the mutagens ber of fertile spikelets/spike was significantly affected by
250-Gy had the highest value followed by 0.4-EMS, 350- the treatment. The highest number of fertile spikelets was
Gy and 1.5 hour-laser × 0.3-EMS, which were significantly obtained in Sids-12 by 0.4-EMS (22.7 spikelets, while the
higher than their parent cultivar. On the other hand, for parent had 17.3 spikelets) followed by 350-Gy (21.2 spikelets).
Sakha-94, the highest values were obtained by 350-Gy, In addition, all used mutagens in Sakha-94 displayed high
0.4-EMS and 2 hour-laser. Meanwhile, in Gemmiza-9 the number of spikelets compared to the parent cultivar. For
mutagens 300-Gy and 1 hour-laser exhibited the highest Gemmiza-9, the mutagens 300-Gy, 0.3-EMS and 1 hour-
flag leaf chlorophyll content. laser exhibited the highest number of fertile spikelets (22.3,
Furthermore, plant height differed significantly in the 21.6 and 21.6 spikelets respectively compared to the parent
three cultivars under the influence of the different mutagens. that had 19.4 spikelets) (Fig. 3E). On the other hand, apart
The shortest plant height was assigned to 350-Gy, 250-Gy from 250-Gy in Sids-12, the number of sterile spikelets/
and 2 hour-laser in Sids-12 (73.8 cm), which was signifi- spike in Sids-12 decreased significantly by the used muta-
cantly shorter than the parent cultivar (76.3 cm). Further- gens compared to parent cultivars (Fig. 3F). Similarly, in
more, the shortest plant height in Sakha-94 was displayed Sakha-94; 350-Gy, 400-Gy and 2 hour-laser significantly
by 0.4-EMS. In contrast, 2 hour-laser exhibited tallest plant decreased number of sterile spikelets/spike as well as
height with no significant difference from the parent cul- 250-Gy, 300-Gy, 0.3-EMS and 1 hour-laser in Gimmeza-9.
tivar Sakha-94 (86.7 cm). In addition, 300-Gy and 0.3-EMS Moreover, our results demonstrate significant differ-
in Gemmiza-9 exhibited plant height shorter than parent ences among the mutants in terms of the number of spikes/
cultivars (Fig. 3C). In general, spike length increased sig- plant, as it increased significantly than the parent cultivars
nificantly by different mutagens compared to parent culti- in Sids-12 and Sakha-94. However, there are no significant
vars. The tallest spike length was assigned for 0.4-EMS in differences for Gemmiza-9 (Fig. 4A, Supplementary Fig.
Sids-12 (18.5 cm and the parent had 14.2 cm) as well as in S1). In Sids-12 and Sakha-94, the mutants derived from
Sakha-94 (14.2 cm and the parent had 11.7 cm), while 0.4-EMS and 2 hour-laser displayed the highest number of
300-Gy recorded the highest value in Gemmiza-9 (14.6 cm spikes. Otherwise, number of grains/spike changed signifi-
258 ∙ Plant Breed. Biotech. 2020 (September) 8(3):252~264

Fig. 3. Mean performance of M3 mutant families and parent cultivars for Flag leaf area (A), Flag leaf chlorophyll content
(B), Plant height (C), spike length (D), number of fertile spikelets/spike (E) and number of sterile spikelets/spike
(F). The bars on the top of the columns represent the SE and different letters on the column differ significantly
by LSD (P ＜ 0.05).

cantly by the used mutagens in Sids-12 and Gemmiza-9, 12 (78.7 and 77.9 g in the same order) followed 0.4-EMS
while it was not affected in Sakha-94. The highest number (75.0 g), compared to the parent cultivar (60.9 g). In addi-
of grains was displayed by 0.4-EMS in Sids-12 (90 grains) tion, the highest grain weight in Sakha-94 was displayed by
followed by 250-Gy, 350-Gy and 2 hour-laser, being sig- 350-Gy (65.9 g) followed 2 hour-laser (62.9 g), compared
nificantly higher than the parent cultivar (68.7 grains). In to the parent cultivar (46.6 g) (Fig. 4C). In Gemmiza-9, the
Gemmiza-9, the mutants derived from 350-Gy followed highest grain weight was assigned for 350-Gy (50.1 g)
250-Gy presented the highest number of grains (80 and followed by 250-Gy (48 g), as compared to the parent
76.6 respectively) compared to the parent cultivar (40.9 cultivar (42.3). Finally, we found significant differences in
grains) (Fig. 4B). grain yield/plant between the obtained mutants and the
In the same context, 1000-grain weight also significantly parent cultivars. Grain yield was increased by all mutagens
differed in its response to the used mutagens. The highest in Sids-12 and Sakha-94, but in Gemmiza-9 it was in-
grain weight was assigned to 350-Gy and 250-Gy in Sids- creased only by 250-Gy, 350-Gy and 1 hour-laser. The high-
 Using Physical and Chemical Mutagens for Inducing Promising Mutants in Bread Wheat ∙ 259

Fig. 4. Mean performance of M3 mutant families and parent cultivars for number of spikes/plant (A), grain number/spike
(B), 1000-grain weight (C) and grain yield/plant (D). The bars on the top of the columns represent the SE and
different letters on the column differ significantly by LSD (P ＜ 0.05).

est grain yield was given in Gemmiza-9 by 350-Gy (42.2 g) respect to plant height, the maximum reduction was as-
followed 250-Gy (35.3 g) compared to the parent cultivar signed for 0.4-EMS in Sakha-94 followed by 350-Gy in
(23.7 g). On the other hand, in Sids-12 the highest grain Sids-12 and 300-Gy in Gimmeza-9. Moreover, the maxi-
yield was assigned to 0.4-EMS (33.9 g) followed by 2 mum increase in spike length and number of fertile spikelets/
hour-laser (31.9 g), relative to the parent cultivar (14 g). spike was obtained by 0.4-EMS in Sids-12 and Sakha-94,
Also, in Sakha-94 the highest value was presented by 2 while in Gemmiza-9 the maximum increase was recorded
hour-laser (33.8 g) followed by 0.4-EMS (25 g) and 350- by 300-Gy. In addition, the maximum reduction in the
Gy (23.7 g) compared to the parent cultivar (18.3 g) (Fig. number of sterile spikelets/spike was observed in Gemmiza-
4D). 9 by 0.3-EMS and in Sakha-94 by 350-Gy. Furthermore,
The increments in M3 derivative mutants compared to the highest increase in number of spikes/plant was pre-
their parent cultivars in evaluated traits were calculated and sented by 0.4-EMS and 2 hour-laser in Sids-12 as well as by
expressed as a percentage (Table 3). The results indicated 2 hour-laser in Sakha-94 while in Gimmeza-9 was dis-
that the highest increase in flag leaf area was obtained by played by 1 hour-laser and 250-Gy. Likewise, the largest
1.5 hour × 0.3% EMS in Sids-12 while in Sakha-94 was increase in grain number per spike was shown by 0.4-EMS
produced by 350-Gy and 2 hour-laser as well as in Gemmiza- in Sids-12, while by 2 hour-laser in Sakha-94 and by
9 was recorded by 300-Gy. Moreover, the largest increase 350-Gy in Gemmiza-9. Similarly, it was observed the most
in flag leaf chlorophyll content in Sids-12 was assigned for increase in 1000-grain weight in all mutants was recorded
250-Gy. Meanwhile, 350-Gy, 2 hour-laser and 0.4-EMS by 350-Gy. Additionally, for grain yield/plant, the highest
possessed the highest increase in flag leaf chlorophyll increase was assigned for 0.4-EMS in Sids-12, whereas in
content in Sakha-94 as well as 300-Gy in Gimmeza-9. With Sakha-94 was displayed by 2 hour-laser and in Gemmiza-9
260 ∙ Plant Breed. Biotech. 2020 (September) 8(3):252~264

Table 3. Increasing or reduction of the evaluated traits expressed as the percentage compared to parent cultivar for M3
wheat mutants.
FLA FLCC PH SL NFSS NSSS NSP NGS TGW GYP
Sids-12
‒3.17d
cd
250-Gy 9.52 18.61a 2.61d 18.18c - 21.51c 25.40b 27.96a 88.38e
‒4.09
c c e b b c b a c
350-Gy 10.45 7.15 16.10 22.33 - 24.11 24.15 29.28 110.48
‒3.04e ‒2.74c
d
7.42c
d
51.30a 22.43c 16.21c
b
2 hr 8.02 9.64 - 128.16
0.4-EMS 26.21b 8.85b 1.11a 30.44a 30.87a - 53.66a 31.02a 23.15b 142.09a
‒0.07
a d b d d b d d d
1.5 hr × 0.3 30.30 5.56 3.88 9.81 - 35.46 18.61 13.19 102.05
ANOVA ** ** ** ** ** ** ** ** **
Sakha-94
‒3.13c ‒31.18d
a
350-Gy 44.66 21.13a 7.98c 18.35d 6.83c 0.04c 41.39a 29.40c
‒1.63 ‒15.21
c b b d b b c b d d
400-Gy 2.41 9.71 0.86 25.92 4.62 1.31 25.46 13.18
2 hr 44.71a 21.06a 0.74a 13.99b 23.56c ‒19.01c 37.61a 2.32a 34.96b 84.17a
0.4-EMS 15.14b 21.26a ‒5.89d 21.80a 27.20a ‒2.66a 15.34b 1.82ab 30.17c 36.11b
ANOVA ** * ** ** ** ** ** * ** **
Gemmiza-9
250-Gy 49.31
c
1.95c ‒0.69c 22.74b 4.37c ‒30.57b 3.70a 87.15b 13.39b 48.62b
300-Gy 65.54a 4.46a ‒2.71d 26.91a 14.45a ‒30.00b ‒3.70c 61.29d 1.58c 2.19d
350-Gy 36.74d ‒0.84d 1.58a 20.14c 3.91c 4.57a 0.00b 95.50a 18.26a 78.06a
1 hr 58.14b 4.28a 0.54b 19.70c 11.27b ‒60.29c 3.70a 68.62c ‒3.61d 17.66c
‒0.48 ‒72.57 ‒3.11
e b c d b d c c b d
0.3-EMS 6.35 2.82 4.34 11.06 66.18 13.39 1.94
ANOVA ** ** ** ** ** ** ** ** ** **
*, ** significant at 0.05 and 0.01 probability levels,respectively. Means followed by different letters differ significantly
by LSD (P ＜ 0.05). FLA: flag leaf area, FLCC: flag leaf chlorophyll content, PH: plant height, SL: spike length, NFSS:
number of fertile spikelets/spike, NSSS: number of sterile spikelets/spike, NSP: number of spikes/plant, GNS: grain
number per spike, TGW: 1000-grain weight and GYP: grain yield/plant.

was recorded by 350-Gy. yielding genotypes (Dhaliwal et al. 2015). Mutation

breeding has been used for developing many mutant vari-
eties in different crops mainly in cereals, legumes and oil
DISCUSSION crops and actually have been released to farmers (FAO/
IAEA, http://mvgs.iaea.org). These released varieties are
Genetic variability is considered as the cornerstone in cultivated over large areas and increasing food production
wheat breeding and improving agronomic traits (DePauw and thus contributing to food security. Moreover, realized
and O’Brien 2016). It could be generated through hy- mutants in important trait as phenology, quality and grain
bridization as well as mutation followed by selection yield can be used without legislative restrictions and social
(Addisu and Shumet 2015). Creating genetic variability opposition facing the genetic modification approach (Parry
through hybridization is time-consuming and laborious, et al. 2009).
nevertheless with low genetic variation (Hanafiah et al. It is valuable to use different mutagens for achieving
2010). Otherwise, mutation is powerful approach to pro- desirable mutation in bread wheat. Because, even higher
vide novel genetic variation into crop genome that can be rates of mutagenesis may be achieved in bread wheat, it is
exploited in breeding programs to improve agronomic more tolerant for mutation than diploid species (Sidhu et al.
traits and mitigate negative impacts associated with en- 2015). One of the main limiting factors of mutation rate in
vironmental constraints. Furthermore, mutation is an ef- bread wheat is the fertility of the mutagenized plants (Parry
fective method for direct releasing adapted and high- et al. 2009; Rakszegi et al. 2010). Therefore, in this study
 Using Physical and Chemical Mutagens for Inducing Promising Mutants in Bread Wheat ∙ 261

different chemical and physical mutagens and their com- gens reduced plant height which is desirable in modern
bination were used to generate different mutagenized popu- high yielding cultivars. In particular, 350-Gy, 250-Gy and
lations. 2 hour-laser in Sids-12 as well as 0.4%-EMS, 350-Gy and
Various physical and chemical mutagens have been 400-Gy in Sakha-94 and 300-Gy in Gemmiza-12. Besides,
demonstrated with significant effects on chromosome struc- the combination between physical and chemical mutagens
ture and thereby phenotypic variations in plant materials (middle range of laser ray and EMS; 1.5 hour laser and
(Maluszynski 2001; Kodym and Afza 2003; Suprasanna et 0.3% EMS) was also attempted to be applied for enhancing
al. 2015). In the current study, gamma ray and laser beams mutation spectrum and frequency. This approach is inter-
as physical mutagens as well as ethyl methanesulfonate esting for promoting mutation spectrum and frequency
(EMS) as chemical mutagens were used to enrich the (Suprasanna et al. 2015). Remarkably, this treatment dis-
genetic diversity in bread wheat. The useful mutations are played high traits compared to each mutagen separately
obtained when appropriate doses are applied. Therefore, to (1.5 hour laser or 0.3% EMS). Consequently, these findings
identify appropriate doses of gamma irradiation, a prelimi- highlight the importance of the selected doses to induce
nary experiment was conducted with nine doses. Geno- useful genetic variability in bread wheat materials for im-
typic differences for response to gamma-ray have been proving grain yield and contributing traits.
observed. Accordingly, three doses; 250, 300 and 350 Gy Moreover, the used parent cultivars are commercial
were selected for two cultivars (Sids-12 and Gemmiza-9), cultivars appear in Egyptian recommended list and were
while the third cultivar (Sakha-94) was irradiated with 350, selected for this study based on their high-yielding pro-
400 and 450 Gy. Gamma irradiation has advantage of its duction and also have different pedigrees. Accordingly,
ability of penetrating deeper in plant tissues and inducing they showed different responses to used mutagens. High
nucleotide substitutions with small deletions of 2-16 bp yield was displayed by Sids-12 followed by Sakha-94 and
(Sato et al. 2006; Suprasanna et al. 2015). Besides, lower Gemmiza-9 as presented in Table 3. Fourteen potential M3
doses of laser beams (1, 1.5 and 2 hours) were applied to mutants with improved agronomic traits compared to pa-
cause bio-stimulation (Chen et al. 2005). The stimulation rent cultivars were preliminary identified. Therefore, these
mechanism of laser beams resulted from synergism be- derivative mutants need to be evaluated in next generations
tween polarized monochromatic and plant photoreceptors in multi-environments trials to validate their performance
(Koper et al. 1996). Furthermore, ethyl methanesulfonate and adaptability. Accordingly, these mutants could be ex-
(EMS) was applied with three concentrations (0.2, 0.3 and ploited in wheat breeding programs for enriching the gene-
0.4%). It is very effective chemical mutagen causes point tic diversity in bread wheat.
mutation with altering single nucleotide pairs generating Previously, phenotypic mutations were induced in bread
phenotypic variations (van Harten 1998; Rakszegi et al. wheat using physical and chemical mutagens and displayed
2010; Kurowska et al. 2012). Generally, the applied doses significant improvement in agronomic traits compared to
in the three mutagens caused significant variations in all parent cultivars. In this context, Singh and Datta (2010)
evaluated traits (Table 2). Which indicates to direct impact utilized gamma radiation with doses 10 to 100 Gy for im-
of these doses on studied traits with statistically significant proving yield related traits in bread wheat. They docu-
variations and clearly induced genetic variability. Moreover, mented that gamma irradiation increased grain yield due to
these doses exhibited considerable improvement in morpho- increasing of spikes number and grains number per spike as
physiological and yield traits compared to their parent well as improving plant vigor and flag leaf area. Moreover,
cultivars which revealed to proper doses have been applied. Rakszegi et al. (2010) used 0.6 and 0.9% EMS to muta-
Obviously, the mutagens doses exhibited different effect genize bread wheat (cv. Cadenza). Their results showed
on the evaluated agronomic traits. The highest stimulation useful genetic diversity in evaluated morphological and
in yield traits was recorded by 0.4% EMS, 1 and 2 hour- agronomic traits as plant height, spike length, number of
laser, 350-Gy and 250-Gy (Figs. 3, 4). Alongside, these muta- spikes per plant, presence of awns and grain color com-
262 ∙ Plant Breed. Biotech. 2020 (September) 8(3):252~264

pared to parent cultivar. In addition, Albokari (2014) laser, 350-Gy, 1.5 hour × 0.3% EMS and 250-Gy. More-
applied gamma radiation in doses 150 and 200 Gy in wheat. over, fourteen promising mutants were detected with im-
The results revealed to selected 11 M3 putative mutants proved morpho-physiological and yield traits. According-
displayed reduced plant height and early heading as well as ly, these mutants could be exploited in wheat breeding
improved grain yield compared to parent cultivar. Like- programs for enriching the genetic diversity in bread
wise, Mishra et al. (2016) utilized 0.2% EMS solution to wheat.
develop mutant lines for amylose variation in bread wheat.
They obtained 101 EMS-mutant lines showed desirable
variation in amylose content. Moreover, Guo et al. (2017) ACKNOWLEDGEMENTS
used 0.5, 1.0 and 1.5% EMS for inducing mutation in bread
wheat (cv. Jing 411). Twenty potential and stable M3 The authors thank the cooperatives of Wheat Research
mutants were selected base on their improved performance Section, Field Crops Research Institute, Agricultural
in days to heading, plant height, number of tillers, spike Research Center and Nuclear Research Center, Atomic
length and 1000-grain weight. Furthermore, Kenzhebayeva Energy Authority, Inchas, Egypt.
et al. (2017) used gamma ray in doses 100 and 200 Gy for
inducing useful genetic variation in bread wheat. The
results proposed that 200-Gy exhibited protein content CONFLICT OF INTEREST
more that the parent genotype (cv. Almaken) by approxi-
mately 11% as well as grain iron and zinc concentrations The authors declare that they have no conflict of interest.
higher than the parent genotype by 3-times. Moreover,
some mutant lines displayed larger 1000-grain weight,
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