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.
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Key words: Mutation breeding, Genetic variation, Gamma ray, Laser beams, Ethyl methanesulfonate
INTRODUCTION
Wheat (
Triticum aestivum ssp) is one of the most im-portant cereal crops worldwide (
Shewry 2009). Its total cultivation area in 2018 was 214.29 million hectares with global production of 734.05 million tons (FAOSTAT,
http://www.fao.org/statistics/en/). Egypt was involved in these statistics with cultivation area 1.32 million hectares produced 8.8 million tons. Nonetheless, Egypt is amongst the top wheat importers, with around 10.16 million tons per year (FAOSTAT,
http://www.fao.org/statistics/en/). Due to current and expected future high population growth rate, the gap between wheat production and consumption would increase, adding further challenges to the limited food resources in this developing country. Furthermore, current climate change, particularly in the Mediterranean region, obstructs efforts to increase the yield, and these changes are expected to increase (
García‐Ruiz et al. 2011;
Cook et al. 2016;
Mansour et al. 2017;
2018). This situation highlights the importance of wheat breeding to increase current pro-duction.
Genetic variability presents the principle of plant breed-ing. It could be created through hybridization or mutations followed by selection (
Addisu and Shumet 2015). In com-parison to mutation, improving through hybridization is la-borious, time-consuming, with low genetic variation (
Hanafiah et al. 2010). Mutation is considered as a forward genetic approach, which provides novel phenotypes that can be ex-ploited in classical breeding programs (
Parry et al. 2009). As such, mutation can be seen as a powerful strategy for direct release of improved varieties (
Dhaliwal et al. 2015). Essentially, up to date, mutation breeding has been used for developing 3288 mutant varieties in different crops, which are registered in the database of the International Atomic Energy Agency (FAO/IAEA,
http://mvgs.iaea.org). Most mutant cultivars have been developed in ornamentals, cereals, legumes and oil crops. Regarding bread wheat, 237 mutant varieties were realized; 195 using physical mutagens, 40 using chemical mutagens and 2 varieties using their com-binations. Furthermore, the released mutant varieties for major crops have been cultivated over large areas, in-creasing food production and thus contributing to food se-curity (
Suprasanna et al. 2015).
The used physical mutagens in plants comprise non-ionizing (
e.g. UV) or ionizing (
e.g. X and gamma rays, alpha and beta rays) radiation. The physical mutation fre-quently causes double-strand DNA breaks and produces large deletion in DNA and thereby causes visual effects on the chromosome structure. Gamma rays is the most wide-spread physical mutagen among plant breeders due to its convenience as well as its capability of penetrating deeper in the tissues (
Suprasanna et al. 2015). It induces nucleo-tide substitutions and small deletions of 2-16 bp, in addition mutation frequency is expected to be one mutation per 6.2 Mb (
Sato et al. 2006). Furthermore, laser beams were de-monstrated that are able to induce chromosome mutation by
Berns et al. (1969). It was found that longer irradiation using laser beams damages the genetics of plant cells but lower doses cause bio-stimulation effects (
Dudin 1990;
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 other hand, the chemical mutagens contain ethyl methane-sulfonate (EMS), hydrogen fluoride (HF), methyl methanesulfonate sulphonate (MMS), N-Nitroso-N-methylurea (NMU) hydroxylamine and sodium azide (
Wu et al. 2005;
Talebi et al. 2012;
Luz et al. 2016). The chemical mutagens result frequently point to mutation changing single nucleo-tide pairs (
Siddiq et al. 1968;
Rakszegi et al. 2010;
Kurowska et al. 2012). EMS is very effective and most popular chemical mutagen (
van Harten 1998). It produces haphazard point mutation in plant material by nucleotide substitution. EMS reacts with guanine or thymine through the addition an ethyl group. It induces high frequency of nucleotide sub-stitutions with majority of changes about 70 to 99% in the obtained mutants are GC to AT base pair transitions (
Sega 1984;
Till et al. 2007). Accordingly, these mutations gene-rate allelic versions of genes causing phenotypic variations (
Dhaliwal et al. 2015). Additionally, the combination be-tween physical and chemical mutagens can be implemented by irradiating seeds first by a physical mutagen followed by treating with a chemical mutagen in a solution (
van Harten 1998). This approach is interesting for enhancing mutation spectrum and frequency (
Suprasanna et al. 2015). Mutation breeding was performed previously in bread wheat by various researchers using physical and chemical mutagens in different doses.
Rakszegi et al. (2010) used 0.6 and 0.9% EMS to mutagenize bread wheat (cv. Cadenza) and they obtained 3750 M6 mutant lines displayed wide diversity in morphological and agronomic traits. Likewise,
Mishra et al. (2016) utilized 0.2% EMS solution to develop mutant lines for amylose variation in bread wheat. Moreover,
Singh and Datta (2010) utilized gamma radiation (10 to 100 Gy) for improving yield attributes in bread wheat. In addi-tion,
Xiong et al. (2018) used gamma radiation (250 Gy) for improving genetic diversity in bread wheat.
The aims of this study were to induce genetic variability in bread wheat using physical and chemical mutagens and evaluate the impact of using different doses on major ag-ronomic traits in three commercial bread wheat cultivars. Additionally, to identify the most potential mutants in terms of improving agronomic traits, compared to parent cultivars.
MATERIALS AND METHODS
Mutagenesis and obtaining mutant seeds
Seeds of three bread wheat cultivars (Sids-12, Sakha-94, and Gemmiza-9) were obtained from Wheat Research Section, Field Crops Research Institute, Egyptian Agri-cultural Research Center, Egypt (
Supplementary Table S1). The used cultivars in this study were selected based on their high-yielding production, whether they are used as commercial cultivars and also have different pedigrees. Two hundred seeds of each cultivar were treated separately by three mutagens: two physical mutagens (gamma ray and laser beams) and one chemical mutagen (EMS). All these treatments were carried out at the Nuclear Research Center, the Egyptian Atomic Energy Authority, Egypt. Gamma irradiation at a cobalt 60 (
60Co) was used to irradiate seeds. A preliminary experiment of gamma irradiation sensitivity test was conducted using Petri-dishes with nine doses; 0, 150, 200, 250, 300, 350, 400, 450 and 500 Gray (
Fig. 1,
Table 1). Based on reduction percent in seedling epicotyl length, according to
Konzak and Mikaelson (1995), three gamma-ray doses were selected for each cultivar. Doses 250, 300 and 350 Gy were selected for both Sids-12 and Gemmiza-9 cultivars, while Sakha-94 was irradiated with 350, 400 and 450 Gy. For the laser beams treatment, seeds were irradiated by laser ray with 1, 1.5, and 2 hour ex-posure. The helium-neon laser (He-Ne) at the wavelength of 632.8 nm and power density of 1MW-2 was applied as the source of radiation. For the EMS treatment, seeds were soaked at 0.2, 0.3 and 0.4% (v/v) EMS concentration for 8 hours. For the combination treatment was used only for parent cultivar Sids-12, seeds were treated first by laser for 1.5 hours and then followed by soaking in 0.3% EMS solution as middle range of each treatments. After each treatment, seeds were washed in distilled water at ambient temperature and then were air-dried. The applied doses of used mutagens in the three parent cultivars were presented in
Fig. 2.
Three field experiments were performed in the experi-mental farm of the Nuclear Research Center, the Egyptian Atomic Energy Authority, Egypt (30º 24’ N and 31º 35’ E), for three winter growing seasons between 2011 and 2014. The soil physical and chemical characteristics of this experimental site is mostly loamy sand (77% sand) (Sup-plementary Table S2). In addition, meteorological data, i.e. averages of minimum and maximum air temperatures and total rainfall are presented in
Supplementary Table S3. Sowing was on the 21
st of November of each season, representing the optimal period for wheat cultivation in Egypt. Ammonium nitrate (33% N), Calcium Superphos-phate (15.5% P
2O
5) and Potassium Sulphate (48% K
2O) fertilizers were applied at the recommended rates of 240 kg N/ha, 70 kg P
2O
5/ha and 60 kg K
2O/ha, respectively. Other agronomic practices including irrigation, pest, disease and weed control were followed as recommended for the wheat production in the study region.
In the first season (2011-2012), seeds were immediately sown after treatment in three separate experiments; one for each parent cultivar and its treatments. The experimental 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 spaced with 10 cm on the row. Seeds harvested from 4050 M
1 plants were sown in the second season (2012-2013) in randomized complete block design with three replications, and 78750 M
2 plants were obtained. Selection was per-formed on M
2 individual plants at harvesting 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. Fourteen promising mutants were selected: five from Sids-12, four from Sakha-94, and five from Gemmiza-9. The seeds of these selected families were sown in the third season (2013-2014; M
3 generation) to be evaluated and compared to the parent cultivars. The selected mutants were not derived from all used mutagens as shown in
Fig. 2. Three separate experi-ments were performed for the three parent cultivars and their derivative mutants using randomized complete block design in three replications, to evaluate the promising four-teen M
3 mutants.
Fifteen plants were chosen from each replication to mea-sure morpho-physiological and yield traits. Flag leaf area (FLA, cm2) was recorded on main stems at the time of 100% heading as flag leaf length × flag leaf width × 0.72. Flag leaf chlorophyll content (FLCC) was measured at anthesis using Chlorophyll Meter SPAD-502Plus. Plant height (PH) was measured at maturity as the distance (cm) from the base of the plant to the tip of the spike, excluding awn. In addition, spike length (SL, cm), number of fertile spikelets/ spike (NFSS), number of sterile spikelets/spike (NSSS), number of spikes/plant (NSP), grain number per spike (GNS), 1000-grain weight (TGW, g) and grain yield/plant (GYP, g) were also estimated.
Statistical analysis
The analysis of variance (ANOVA) was applied for all investigated traits. In addition, least significant difference (LSD) values were performed at the 5% probability level (P < 0.05). The statistical analyses were made using SAS software (version 9.2, SAS Institute, Cary, NC, USA).
RESULTS
Analysis of variance
Analysis of variance for studied traits is presented in
Table 2. The results suggested that the used mutagens had a direct impact on all evaluated traits with statistically significant variations. This clearly depicts genetic vari-ability among the obtained families for all investigated traits as the mean values are significantly different from those of the parent cultivars. This finding confirms the efficiency of tested mutagens and possibility for inducing more variations for morpho-physiological and yield traits.
Flag leaf area significantly increased by used mutagens, with values varying between 19.26 and 56.9 cm
2 (values of three parents were 18.8, 22.5 and 43.7 cm
2). Respecting Sids-12, the highest values were assigned to mutants de-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 significantly higher than their parent cultivar (
Fig. 3A). Likewise, in Sakha-94, the mutants derived from 350-Gy and 2 hour-laser followed by 0.4-EMS exhibited larger flag leaf area than their parent cultivar. For Gemmiza-9, the highest values were obtained using 300-Gy, 1 hour-laser and 250-Gy followed by 350-Gy. As illustrated in
Fig. 3B, flag leaf chlorophyll content responded differently to the treatments, with values ranging between 50.84 and 64.9 SPAD reading (values of three parents were 46.3, 51.4 and 54.7). Apart from 2 hour-laser in Sids-12 and 350-Gy in Gemmiza-9, all mutants showed chlorophyll content higher than those of parent cultivars. For Sids-12, the mutagens 250-Gy had the highest value followed by 0.4-EMS, 350-Gy and 1.5 hour-laser × 0.3-EMS, which were significantly higher than their parent cultivar. On the other hand, for Sakha-94, the highest values were obtained by 350-Gy, 0.4-EMS and 2 hour-laser. Meanwhile, in Gemmiza-9 the mutagens 300-Gy and 1 hour-laser exhibited the highest flag leaf chlorophyll content.
Furthermore, plant height differed significantly in the three cultivars under the influence of the different mutagens. The shortest plant height was assigned to 350-Gy, 250-Gy and 2 hour-laser in Sids-12 (73.8 cm), which was signifi-cantly shorter than the parent cultivar (76.3 cm). Further-more, the shortest plant height in Sakha-94 was displayed by 0.4-EMS. In contrast, 2 hour-laser exhibited tallest plant height with no significant difference from the parent cul-tivar Sakha-94 (86.7 cm). In addition, 300-Gy and 0.3-EMS in Gemmiza-9 exhibited plant height shorter than parent cultivars (
Fig. 3C). In general, spike length increased sig-nificantly by different mutagens compared to parent culti-vars. The tallest spike length was assigned for 0.4-EMS in Sids-12 (18.5 cm and the parent had 14.2 cm) as well as in Sakha-94 (14.2 cm and the parent had 11.7 cm), while 300-Gy recorded the highest value in Gemmiza-9 (14.6 cm and the parent had 11.5 cm) (
Fig. 3D). Similarly, the num-ber of fertile spikelets/spike was significantly affected by the treatment. The highest number of fertile spikelets was obtained in Sids-12 by 0.4-EMS (22.7 spikelets, while the parent had 17.3 spikelets) followed by 350-Gy (21.2 spikelets). In addition, all used mutagens in Sakha-94 displayed high number of spikelets compared to the parent cultivar. For Gemmiza-9, the mutagens 300-Gy, 0.3-EMS and 1 hour-laser exhibited the highest number of fertile spikelets (22.3, 21.6 and 21.6 spikelets respectively compared to the parent that had 19.4 spikelets) (
Fig. 3E). On the other hand, apart from 250-Gy in Sids-12, the number of sterile spikelets/ spike in Sids-12 decreased significantly by the used muta-gens compared to parent cultivars (
Fig. 3F). Similarly, in Sakha-94; 350-Gy, 400-Gy and 2 hour-laser significantly decreased number of sterile spikelets/spike as well as 250-Gy, 300-Gy, 0.3-EMS and 1 hour-laser in Gimmeza-9.
Moreover, our results demonstrate significant differ-ences among the mutants in terms of the number of spikes/ plant, as it increased significantly than the parent cultivars in Sids-12 and Sakha-94. However, there are no significant differences for Gemmiza-9 (
Fig. 4A,
Supplementary Fig. S1). In Sids-12 and Sakha-94, the mutants derived from 0.4-EMS and 2 hour-laser displayed the highest number of spikes. Otherwise, number of grains/spike changed signifi-cantly by the used mutagens in Sids-12 and Gemmiza-9, while it was not affected in Sakha-94. The highest number of grains was displayed by 0.4-EMS in Sids-12 (90 grains) followed by 250-Gy, 350-Gy and 2 hour-laser, being sig-nificantly higher than the parent cultivar (68.7 grains). In Gemmiza-9, the mutants derived from 350-Gy followed 250-Gy presented the highest number of grains (80 and 76.6 respectively) compared to the parent cultivar (40.9 grains) (
Fig. 4B).
In the same context, 1000-grain weight also significantly differed in its response to the used mutagens. The highest grain weight was assigned to 350-Gy and 250-Gy in Sids-12 (78.7 and 77.9 g in the same order) followed 0.4-EMS (75.0 g), compared to the parent cultivar (60.9 g). In addi-tion, the highest grain weight in Sakha-94 was displayed by 350-Gy (65.9 g) followed 2 hour-laser (62.9 g), compared to the parent cultivar (46.6 g) (
Fig. 4C). In Gemmiza-9, the highest grain weight was assigned for 350-Gy (50.1 g) followed by 250-Gy (48 g), as compared to the parent cultivar (42.3). Finally, we found significant differences in grain yield/plant between the obtained mutants and the parent cultivars. Grain yield was increased by all mutagens in Sids-12 and Sakha-94, but in Gemmiza-9 it was in-creased only by 250-Gy, 350-Gy and 1 hour-laser. The highest grain yield was given in Gemmiza-9 by 350-Gy (42.2 g) followed 250-Gy (35.3 g) compared to the parent cultivar (23.7 g). On the other hand, in Sids-12 the highest grain yield was assigned to 0.4-EMS (33.9 g) followed by 2 hour-laser (31.9 g), relative to the parent cultivar (14 g). Also, in Sakha-94 the highest value was presented by 2 hour-laser (33.8 g) followed by 0.4-EMS (25 g) and 350-Gy (23.7 g) compared to the parent cultivar (18.3 g) (
Fig. 4D).
The increments in M
3 derivative mutants compared to their parent cultivars in evaluated traits were calculated and expressed as a percentage (
Table 3). The results indicated that the highest increase in flag leaf area was obtained by 1.5 hour × 0.3% EMS in Sids-12 while in Sakha-94 was produced by 350-Gy and 2 hour-laser as well as in Gemmiza 9 was recorded by 300-Gy. Moreover, the largest increase in flag leaf chlorophyll content in Sids-12 was assigned for 250-Gy. Meanwhile, 350-Gy, 2 hour-laser and 0.4-EMS possessed the highest increase in flag leaf chlorophyll content in Sakha-94 as well as 300-Gy in Gimmeza-9. With respect to plant height, the maximum reduction was as-signed for 0.4-EMS in Sakha-94 followed by 350-Gy in Sids-12 and 300-Gy in Gimmeza-9. Moreover, the maxi-mum increase in spike length and number of fertile spikelets/ spike was obtained by 0.4-EMS in Sids-12 and Sakha-94, while in Gemmiza-9 the maximum increase was recorded by 300-Gy. In addition, the maximum reduction in the number of sterile spikelets/spike was observed in Gemmiza 9 by 0.3-EMS and in Sakha-94 by 350-Gy. Furthermore, the highest increase in number of spikes/plant was pre-sented by 0.4-EMS and 2 hour-laser in Sids-12 as well as by 2 hour-laser in Sakha-94 while in Gimmeza-9 was dis-played by 1 hour-laser and 250-Gy. Likewise, the largest increase in grain number per spike was shown by 0.4-EMS in Sids-12, while by 2 hour-laser in Sakha-94 and by 350-Gy in Gemmiza-9. Similarly, it was observed the most increase in 1000-grain weight in all mutants was recorded by 350-Gy. Additionally, for grain yield/plant, the highest increase was assigned for 0.4-EMS in Sids-12, whereas in Sakha-94 was displayed by 2 hour-laser and in Gemmiza-9 was recorded by 350-Gy.
DISCUSSION
Genetic variability is considered as the cornerstone in wheat breeding and improving agronomic traits (DePauw and O’Brien 2016). It could be generated through hy-bridization as well as mutation followed by selection (
Addisu and Shumet 2015). Creating genetic variability through hybridization is time-consuming and laborious, nevertheless with low genetic variation (
Hanafiah et al. 2010). Otherwise, mutation is powerful approach to pro-vide novel genetic variation into crop genome that can be exploited in breeding programs to improve agronomic traits and mitigate negative impacts associated with en-vironmental constraints. Furthermore, mutation is an ef-fective method for direct releasing adapted and high-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 crops and actually have been released to farmers (FAO/ IAEA,
http://mvgs.iaea.org). These released varieties are cultivated over large areas and increasing food production and thus contributing to food security. Moreover, realized mutants in important trait as phenology, quality and grain yield can be used without legislative restrictions and social opposition facing the genetic modification approach (
Parry et al. 2009).
It is valuable to use different mutagens for achieving desirable mutation in bread wheat. Because, even higher rates of mutagenesis may be achieved in bread wheat, it is more tolerant for mutation than diploid species (
Sidhu et al. 2015). One of the main limiting factors of mutation rate in bread wheat is the fertility of the mutagenized plants (
Parry et al. 2009;
Rakszegi et al. 2010). Therefore, in this study different chemical and physical mutagens and their com-bination were used to generate different mutagenized populations.
Various physical and chemical mutagens have been demonstrated with significant effects on chromosome structure and thereby phenotypic variations in plant materials (
Maluszynski 2001;
Kodym and Afza 2003;
Suprasanna et al. 2015). In the current study, gamma ray and laser beams as physical mutagens as well as ethyl methanesulfonate (EMS) as chemical mutagens were used to enrich the genetic diversity in bread wheat. The useful mutations are obtained when appropriate doses are applied. Therefore, to identify appropriate doses of gamma irradiation, a prelimi-nary experiment was conducted with nine doses. Geno-typic differences for response to gamma-ray have been observed. Accordingly, three doses; 250, 300 and 350 Gy were selected for two cultivars (Sids-12 and Gemmiza-9), while the third cultivar (Sakha-94) was irradiated with 350, 400 and 450 Gy. Gamma irradiation has advantage of its ability of penetrating deeper in plant tissues and inducing nucleotide substitutions with small deletions of 2-16 bp (
Sato et al. 2006;
Suprasanna et al. 2015). Besides, lower doses of laser beams (1, 1.5 and 2 hours) were applied to cause bio-stimulation (
Chen et al. 2005). The stimulation mechanism of laser beams resulted from synergism be-tween polarized monochromatic and plant photoreceptors (
Koper et al. 1996). Furthermore, ethyl methanesulfonate (EMS) was applied with three concentrations (0.2, 0.3 and 0.4%). It is very effective chemical mutagen causes point mutation with altering single nucleotide pairs generating phenotypic variations (
van Harten 1998;
Rakszegi et al. 2010;
Kurowska et al. 2012). Generally, the applied doses in the three mutagens caused significant variations in all evaluated traits (
Table 2). Which indicates to direct impact of these doses on studied traits with statistically significant variations and clearly induced genetic variability. Moreover, these doses exhibited considerable improvement in morpho physiological and yield traits compared to their parent cultivars which revealed to proper doses have been applied. Obviously, the mutagens doses exhibited different effect on the evaluated agronomic traits. The highest stimulation in yield traits was recorded by 0.4% EMS, 1 and 2 hour-laser, 350-Gy and 250-Gy (
Figs. 3,
4). Alongside, these mutagens reduced plant height which is desirable in modern high yielding cultivars. In particular, 350-Gy, 250-Gy and 2 hour-laser in Sids-12 as well as 0.4%-EMS, 350-Gy and 400-Gy in Sakha-94 and 300-Gy in Gemmiza-12. Besides, the combination between physical and chemical mutagens (middle range of laser ray and EMS; 1.5 hour laser and 0.3% EMS) was also attempted to be applied for enhancing mutation spectrum and frequency. This approach is inter-esting for promoting mutation spectrum and frequency (
Suprasanna et al. 2015). Remarkably, this treatment dis-played high traits compared to each mutagen separately (1.5 hour laser or 0.3% EMS). Consequently, these findings highlight the importance of the selected doses to induce useful genetic variability in bread wheat materials for im-proving grain yield and contributing traits.
Moreover, the used parent cultivars are commercial cultivars appear in Egyptian recommended list and were selected for this study based on their high-yielding pro-duction and also have different pedigrees. Accordingly, they showed different responses to used mutagens. High yield was displayed by Sids-12 followed by Sakha-94 and Gemmiza-9 as presented in
Table 3. Fourteen potential M
3 mutants with improved agronomic traits compared to pa-rent cultivars were preliminary identified. Therefore, these derivative mutants need to be evaluated in next generations in multi-environments trials to validate their performance and adaptability. Accordingly, these mutants could be ex-ploited in wheat breeding programs for enriching the gene-tic diversity in bread wheat.
Previously, phenotypic mutations were induced in bread wheat using physical and chemical mutagens and displayed significant improvement in agronomic traits compared to parent cultivars. In this context,
Singh and Datta (2010) utilized gamma radiation with doses 10 to 100 Gy for im-proving yield related traits in bread wheat. They docu-mented that gamma irradiation increased grain yield due to increasing of spikes number and grains number per spike as well as improving plant vigor and flag leaf area. Moreover,
Rakszegi et al. (2010) used 0.6 and 0.9% EMS to muta-genize bread wheat (cv. Cadenza). Their results showed useful genetic diversity in evaluated morphological and agronomic traits as plant height, spike length, number of spikes per plant, presence of awns and grain color com-pared to parent cultivar. In addition,
Albokari (2014) applied gamma radiation in doses 150 and 200 Gy in wheat. The results revealed to selected 11 M
3 putative mutants displayed reduced plant height and early heading as well as improved grain yield compared to parent cultivar. Like-wise,
Mishra et al. (2016) utilized 0.2% EMS solution to 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) used 0.5, 1.0 and 1.5% EMS for inducing mutation in bread wheat (cv. Jing 411). Twenty potential and stable M
3 mutants were selected base on their improved performance in days to heading, plant height, number of tillers, spike length and 1000-grain weight. Furthermore,
Kenzhebayeva 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 more that the parent genotype (cv. Almaken) by approxi-mately 11% as well as grain iron and zinc concentrations higher than the parent genotype by 3-times. Moreover, some mutant lines displayed larger 1000-grain weight, grain area, length and width about 40% than the parent genotype. Additionally,
Hussain et al. (2018) used 0.4 to 0.9% EMS to induce mutations in bread wheat (cv. NN-Gandum-1) for improving resistance to leaf and yellow rust. They obtained 3634 M
2 plants from each of the M1 plant. Out of these, 0.91% and 0.55% had complete resist-ance to leaf and yellow rust, respectively. Besides,
Xiong et al. (2018) used gamma radiation in dose of 250 Gy for improving genetic diversity in bread wheat. The induced wheat mutant lines showed reduction in plant height and increasing in 1000-grain weight.
In conclusions, Mutation is a valuable tool to induce genetic variation that could be used in wheat breeding. In the current study certain physical mutagens (gamma ray and laser beams) and chemical mutagens (ethyl methane-sulfonate (EMS)) were applied for improving morpho-physiological and yield traits. The used mutagens caused significant variations in all evaluated traits in M3 lines indicating their efficiency for inducing further useful gene-tic variability in other plant materials for improving ag-ronomic traits in bread wheat. The highest yield and con-tributing traits were obtained by 0.4% EMS, 1 and 2 hour-laser, 350-Gy, 1.5 hour × 0.3% EMS and 250-Gy. More-over, fourteen promising mutants were detected with im-proved morpho-physiological and yield traits. According-ly, these mutants could be exploited in wheat breeding programs for enriching the genetic diversity in bread wheat.
Supplemental Materials
ACKNOWLEDGEMENTS
The authors thank the cooperatives of Wheat Research Section, Field Crops Research Institute, Agricultural Research Center and Nuclear Research Center, Atomic Energy Authority, Inchas, Egypt.
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CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
Fig. 1Epicotyl 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).
Fig. 2Schematic diagram showing different mutagen treatments applied in three parental cultivars and selected ones from derivative mutants.
Fig. 3Mean 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).
Fig. 4Mean 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).
Table 1Mean 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).
Table 1
|
Dose (Gy) |
Sids-12 |
|
Sakha-94 |
|
Gemmeiza-9 |
|
|
|
|
Epicotyl length (cm.) |
Reduction (%) |
|
Epicotyl length (cm.) |
Reduction (%) |
|
Epicotyl length (cm.) |
Reduction (%) |
|
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 |
|
Table 2Analysis of variance (mean squares) of the investigated traits for fourteen M3 mutant families and parent cultivars.
Table 2
|
Source of variance |
d.f. |
FLA |
FLCC |
PH |
SL |
NFSS |
NSSS |
NSP |
NGS |
TGW |
GYP |
|
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 |
|
Treatments |
5 |
216.09** |
3.774** |
56.55** |
4.677** |
3.497** |
3.547** |
4.478NS
|
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 |
Table 3Increasing or reduction of the evaluated traits expressed as the percentage compared to parent cultivar for M3 wheat mutants.
Table 3
|
FLA |
FLCC |
PH |
SL |
NFSS |
NSSS |
NSP |
NGS |
TGW |
GYP |
|
Sids-12 |
|
250-Gy |
9.52cd
|
18.61a
|
‒3.17d
|
2.61d
|
18.18c
|
|
21.51c
|
25.40b
|
27.96a
|
88.38e
|
|
350-Gy |
10.45c
|
7.15c
|
‒4.09e
|
16.10b
|
22.33b
|
|
24.11c
|
24.15b
|
29.28a
|
110.48c
|
|
2 hr |
8.02d
|
‒3.04e
|
‒2.74c
|
7.42c
|
9.64d
|
|
51.30a
|
22.43c
|
16.21c
|
128.16b
|
|
0.4-EMS |
26.21b
|
8.85b
|
1.11a
|
30.44a
|
30.87a
|
|
53.66a
|
31.02a
|
23.15b
|
142.09a
|
|
1.5 hr × 0.3 |
30.30a
|
5.56d
|
‒0.07b
|
3.88d
|
9.81d
|
|
35.46b
|
18.61d
|
13.19d
|
102.05d
|
|
ANOVA |
** |
** |
** |
** |
** |
|
** |
** |
** |
** |
|
Sakha-94 |
|
350-Gy |
44.66a
|
21.13a
|
‒3.13c
|
7.98c
|
18.35d
|
‒31.18d
|
6.83c
|
0.04c
|
41.39a
|
29.40c
|
|
400-Gy |
2.41c
|
9.71b
|
‒1.63b
|
0.86d
|
25.92b
|
‒15.21b
|
4.62c
|
1.31b
|
25.46d
|
13.18d
|
|
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 |
49.31c
|
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
|
|
0.3-EMS |
6.35e
|
2.82b
|
‒0.48c
|
4.34d
|
11.06b
|
‒72.57d
|
‒3.11c
|
66.18c
|
13.39b
|
1.94d
|
|
ANOVA |
** |
** |
** |
** |
** |
** |
** |
** |
** |
** |
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