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The Effect of Single and Combined Use of Gamma Radiation and Ethylmethane Sulfonate on Early Growth Parameters in Sorghum

Success in inducing genetic variation through mutagenic agents is dependent on the source and dose of application. The objective of this study was to determine the optimum doses of a single and combined use of gamma radiation and ethylmethane sulfonate (EMS) for effective mutation breeding in sorghum. The study involved two concurrent experiments as follows: in experiment I, the seeds of four sorghum genotypes (‘Parbhani Moti’, ‘Parbhani Shakti’, ‘ICSV 15013′, and ‘Macia’) were treated using gamma radiation (0, 300, 400, 500 and 600 Gy), EMS (0, 0.5 and 1.0%), and gamma radiation followed by EMS (0 and 300 Gy and 0.1% EMS; 400 Gy and 0.05% EMS). In experiment II, the seeds of two genotypes (‘Macia’ and ‘Red sorghum’) were treated with seven doses of gamma radiation only (0, 100, 200, 300, 400, 500 and 600 Gy). Overall, the combined applied doses of gamma radiation and EMS are not recommended due to poor seedling emergence and seedling survival rate below LD50. The best dosage of gamma radiation for genotypes Red sorghum, Parbhani Moti, Macia, ICSV 15013 and Parbhani Shakti ranged between 392 and 419 Gy, 311 and 354 Gy, 256 and 355 Gy, 273 and 304 Gy, and 266 and 297 Gy, respectively. The EMS optimum dosage ranges for genotypes Parbhani Shakti, ICSV 15013, Parbhani Moti and Macia were between 0.41% and 0.60%, 0.48% and 0.58%, 0.46% and 0.51%, and 0.36% and 0.45%, respectively. The above dose rates are useful to induce genetic variation in the tested sorghum genotypes for greater mutation events in sorghum breeding programs.


Introduction
Use of climate-smart crop cultivars is a key mitigation strategy against the unpredictable impacts of climate change associated with threats such as drought and heat stress, flooding, soil erosion, and salinization [1]. Sorghum (Sorghum bicolor [L.] Moench) is one of the key crops adapted to grow under arid and semiarid and harsh conditions where other crops fail to survive under similar environmental conditions. This makes sorghum a crop of choice in sub-Saharan Africa (SSA), Asia and similar agro-ecologies. However, yields in sorghum are low in SSA, with a mean grain yield level of less than 1 t/ha far below the potential grain yield of the crop, which reaches up to more than 4 t/ha [2]. Therefore, Table 1. Mean-square values and significance tests for emergence percentage (%E), seedling survival percentage (%SS) and shoot length (SLT) of four sorghum genotypes evaluated with variable doses of gamma radiation, EMS concentrations and combined treatments in Experiment I.

Effect of Single Doses of EMS
Significant interactions (p < 0.01) were observed between genotype and EMS concentrations on seedling emergence percentage and survival rate (Table 1). This suggests that the optimum EMS dose in sorghum mutation breeding is significantly influenced by the genotype. The means of seedling emergence percentage, survival percentage and shoot length showed a significant reduction with increased EMS concentration ( Table 2). The lowest seedling emergence value, 4.6%, was recorded in genotype Parbhani Moti at 1% EMS, showing significant differences when compared with the 17.6% and 11.0% recorded in genotypes Parbhani Shakti and ICSV 15013, respectively. The lowest seedling survival rate value, 2.4%, was recorded in genotype Macia at 1% EMS, showing non-significant differences when compared with genotypes Parbhani Shakti, ICSV 15013 and Parbhani Moti with values of 6.4%, 4.8% and 3.0%, respectively. The LD 50 s for seedling emergence for genotypes Parbhani Shakti, ICSV15013, Parbhani Moti and Macia were 0.60%, 0.58%, 0.46% and 0.45%, respectively ( Figure 2). The LD 50 s for seedling survival rate for genotypes Parbhani Moti, ICSV 15013, Parbhani Shakti, and Macia were 0.51%, 0.48%, 0.41% and 0.36% EMS, respectively. The GR 50 s for shoot length for genotype ICSV 15013, Parbhani Shakti, Parbhani Moti, and Macia were 1.35%, 0.90%, 0.87% and 0.67% EMS, respectively ( Figure 2).

Effect of Combined Doses of Gamma Radiation and EMS
Highly significant interactions (p < 0.01) were observed between genotype and combined use of gamma radiation doses and EMS concentrations on seedling emergence percentage and survival percentage ( Table 1). The mean of seedling emergence percentage, survival percentage and shoot length showed a significant reduction with increased combined dosage ( Table 2). Seedling emergence percentages of genotypes Parbhani Moti, Macia, Parbhani Shakti and ICSV 15013 at dose rates of 300 Gy and 0.1% EMS were 27.6%, 19.7%, 16.8% and 14.3%, respectively ( Figure 3). The lowest seedling emergence value, 1%, was recorded in genotype Macia at dose rates of 400 Gy and 0.05% EMS ( Table 2). Genotype Macia could not survive a dose rate of 400 Gy and 0.05% EMS, compared with Parbhani Moti, ICSV 15013 and Parbhani Shakti, which survived with 1.4%, 1.0% and 0.6%, respectively. Genotype Parbhani Moti showed the lowest mean shoot length value of 10.3 cm, recorded at dose rates of 400 Gy and 0.05% EMS, showing non-significant differences when compared with genotypes ICSV 15013  Figure 3). emergence percentages of genotypes Parbhani Moti, Macia, Parbhani Shakti and ICSV 15013 at dose rates of 300 Gy and 0.1% EMS were 27.6%, 19.7%, 16.8% and 14.3%, respectively ( Figure 3). The lowest seedling emergence value, 1%, was recorded in genotype Macia at dose rates of 400 Gy and 0.05% EMS ( Table 2). Genotype Macia could not survive a dose rate of 400 Gy and 0.05% EMS, compared with Parbhani Moti, ICSV 15013 and Parbhani Shakti, which survived with 1.4%, 1.0% and 0.6%, respectively. Genotype Parbhani Moti showed the lowest mean shoot length value of 10.3 cm, recorded at dose rates of 400 Gy and 0.05% EMS, showing non-significant differences when compared with genotypes ICSV 15013 and Parbhani Shakti, with 12.8 and 12.4 cm, respectively. The growth reduction of shoot length for genotypes Parbhani Shakti, ICSV 15013, Parbhani Moti and Macia at the dose rate of 300 Gy and 0.1% EMS were 64.6%, 57.3%, 53.1% and 44.9% of control, respectively ( Figure 3).

Experiment II
Significant interactions (p < 0.01) were observed between genotype and gamma radiation dosage on germination percentage, seedling emergence percentage, seedling survival percentage, number of panicles per m 2 , number of productive panicles per m 2 , panicle length, plant height and seed viability (Table 3). Table 4 summarizes the means of traits studied. The germination percentage for genotype Macia was significantly reduced by doses ≥300 Gy while there was a non-significant reduction in the genotype Red sorghum ( Table 4). The lowest seedling emergence was recorded in genotype Macia (0.7%) at a dose of 600 Gy showing significant difference compared to genotype Red sorghum (13.9%). The seedling survival rate and shoot length demonstrated a similar trend as emergence percentage. Genotype Macia could not survive at 600 Gy as compared with Red sorghum, which survived the dose with 9.2% ( Table 4). The mean of shoot length was significantly decreased by doses of ≥400 Gy in both genotypes. The LD50s for germination percentage for genotypes Red sorghum and Macia were 6158 and 1092 Gy, respectively (

Experiment II
Significant interactions (p < 0.01) were observed between genotype and gamma radiation dosage on germination percentage, seedling emergence percentage, seedling survival percentage, number of panicles per m 2 , number of productive panicles per m 2 , panicle length, plant height and seed viability ( Table 3). Table 4 summarizes the means of traits studied. The germination percentage for genotype Macia was significantly reduced by doses ≥300 Gy while there was a non-significant reduction in the genotype Red sorghum ( Table 4). The lowest seedling emergence was recorded in genotype Macia (0.7%) at a dose of 600 Gy showing significant difference compared to genotype Red sorghum (13.9%). The seedling survival rate and shoot length demonstrated a similar trend as emergence percentage. Genotype Macia could not survive at 600 Gy as compared with Red sorghum, which survived the dose with 9.2% ( Table 4). The mean of shoot length was significantly decreased by doses of ≥400 Gy in both genotypes. The LD 50 s for germination percentage for genotypes Red sorghum and Macia were 6158 and 1092 Gy, respectively (Figure 4). The LD 50 s for seedling emergence for the genotypes Red sorghum and Macia were 419 and 355 Gy, respectively (Figure 4). The LD 50 s for seedling survival rate for genotypes Red sorghum and Macia were 392 and 288 Gy, respectively. The GR 50 s for shoot length for genotypes Red sorghum and Macia were 686 and 658 Gy, respectively (Figure 4).  The number of panicles m −2 was significantly reduced by doses ≥400 and 600 Gy in Macia and Red sorghum, respectively ( Table 4). The number of panicles m −2 in Red sorghum was significantly increased at 100 Gy and decreased at 600 Gy, revealing different responses of genotypes to gamma radiation doses. Plant height of the genotype Red sorghum was significantly reduced by doses ≥100 Gy whereas genotype Macia was reduced by doses ≥400 Gy. Seed viability was significantly decreased by doses ≥200 and 500 Gy for genotype Macia and Red sorghum, respectively. In general, the result showed that genotype Red sorghum demonstrated stronger tolerance to the tested gamma radiation doses, suggesting that high doses are needed to induce mutagenesis.  The number of panicles m −2 was significantly reduced by doses ≥400 and 600 Gy in Macia and Red sorghum, respectively ( Table 4). The number of panicles m −2 in Red sorghum was significantly increased at 100 Gy and decreased at 600 Gy, revealing different responses of genotypes to gamma radiation doses. Plant height of the genotype Red sorghum was significantly reduced by doses ≥100 Gy whereas genotype Macia was reduced by doses ≥400 Gy. Seed viability was significantly decreased by doses ≥200 and 500 Gy for genotype Macia and Red sorghum, respectively. In general, the result showed that genotype Red sorghum demonstrated stronger tolerance to the tested gamma radiation doses, suggesting that high doses are needed to induce mutagenesis.

Effect of Single Doses of Gamma Radiation
The present study found highly significant interactions between genotypes and gamma radiation doses on assessed traits, viz., germination percentage, seedling emergence percentage, seedling survival percentage, number of panicles per m 2 , number of productive panicles per m 2 , panicle length and seed viability (Tables 1 and 3). The optimum dose rates calculated on the basis of seedling emergence and survival rates for genotypes Red sorghum, Parbhani Moti, Macia, ICSV 15013 and Parbhani Shakti ranged between 392 and 419 Gy, 311 and 354 Gy, 256 and 355 Gy, 273 and 304 Gy, and 266 and 297 Gy, respectively (Figures 1 and 4). Moreover, results showed that GR50 for shoot length ranged between 436 and 686 Gy, respectively (Figures 1 and 4). However, these dose rates will mean lower seedling emergence, poor seedling survival rate and weak plants. High dose rates produce low plant population and poor plant growth in the M 2 and M 3 generations, in turn affecting the identification and selection of useful mutants (Tables 2 and 4). Golubinova and Gecheff [35] applied gamma radiation dose rates ranging between 100 to 400 Gy on Sudan grass and found that the LD 50 values estimated on the basis of seedling survival rate were more suitable for large scale mutagenesis than using data on germination and sterility. The authors stressed the importance of using the optimum dose rate which obtains good and healthy plant populations to be grown to maturity. Mudibu et al. [34] suggested that a high mutagen dose rate leads to an increased proportion of changes such as chromosomal aberrations, lethality, injury, and sterility. The present study found that low gamma radiation dose rates (≤200 Gy) showed no significant effect on plant growth except for the germination rate and the number of panicles per m 2 (Table 4). Meanwhile, a high dose reduced seedling emergence and survival rates, the number of panicles and the number of productive panicles per m 2 , plant height and seed viability below the target of 50% (Table 4). Similar findings were reported in chili pepper [36] and African nightshade [37]. The differences observed in biological traits suggest that the identified optimum doses of single gamma radiation may produce useful mutants, as reported by Tadele [38] and Manova and Gruszka [39].

Effect of Single Doses of EMS
Highly significant interactions were found between genotypes and EMS concentration on seedling emergence and survival rates ( Table 1). The LD 50 s calculated on the basis of seedling emergence and survival rate for genotypes Parbhani Shakti, ICSV 15013, Parbhani Moti and Macia was between 0.41% and 0.60%, 0.48% and 0.58%, 0.46% and 0.51%, and 0.36% and 0.45%, respectively ( Figure 2). The GR 50 s for shoot length for genotypes ICSV 15013, Parbhani Shakti, Parbhani Moti, and Macia were 1.35%, 0.90%, 0.87% and 0.67% EMS, respectively ( Figure 2). GR 50 may be associated with undesirable effects on growth promoters and various chromosomal aberrations which affect emergence, seedling survival rate and seed set [40]. Therefore, the GR 50 s determined were not suitable for large scale mutagenesis. Mani [17] applied an EMS concentration of 0.3% for 8 h in three sorghum varieties and found germination rates between 63% and 80% and a survival rate between 66% and 82%. This result suggested the need to increase the concentrations to obtain LD 50 s for each genotype. In the present study, the mean seedling emergence, survival rates and seedling height had significant reductions with increased EMS concentrations ( Table 2). This result corroborates the findings in rice [41], soybean [34] and wheat [42] which reported that seedling emergence, survival and growth were influenced by genetic variations and EMS concentrations.

Effect of Combined Doses of Gamma Radiation and EMS
Significant interactions were found between genotypes and dose rates for the combined use of gamma radiation followed by EMS concentrations on seedling emergence and survival rates (Table 1). However, results were unable to determine the optimum dose rates for combined applications of gamma radiation and EMS for large scale mutagenesis on the test genotypes. The test dose rates were high and produced undesirable effects, including reduction of seedling emergence and survival rates. The mean of all traits assessed was drastically decreased with the test doses (Table 2). Results showed that, with a combined treatment of 300 Gy followed by 0.1% EMS concentration, seedling emergence percentages of genotypes Parbhani Moti, Macia, Parbhani Shakti and ICSV 15013 were 27.6%, 19.7%, 16.8% and 14.3%, respectively (Figure 3). At the same dose rate, the seedling survival rates of genotypes Parbhani Shakti, Parbhani Moti, Macia, and ICSV 15013 were 16.2%, 15.9%, 3.8% and 3.5%, respectively. These results can be explained by the optimum dose rates obtained in a single use of gamma radiation and EMS on the test genotypes shown above. This result suggests the need to apply lower gamma radiation doses and EMS concentrations to obtain desired level of seedling emergence and survival rates and in turn to promote healthy plant growth for effective selection.

Study Site and Plant Material
The study involved two concurrent experiments carried out under field tests at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, India, (17 • 30 32.9" N 78 • 16 50.8" E) and Mannheim Crop Research Station, Tsumeb, Namibia (19 • 10 06.7" S 17 • 45 54.1" E). The study used seeds of five sorghum genotypes, viz., Parbhani Moti (SPV 1411), Parbhani Shakti (ICSR 14001), ICSV 15013, Macia (SDS 3220) and Red sorghum. The genotypes were selected for their unique traits; e.g., Macia is one of the popular cultivars in the Southern African region. It is a high-yield semi-dwarf and early-maturity cultivar [43]. Meanwhile, the genotype Red sorghum is a late-maturity cultivar widely cultivated in Namibia [44], and the genotypes Parbhani Shakti, Parbhani Moti and ICSV15013 have unique traits including high contents of grain Fe and Zn and are currently being tested for wider adoption and drought tolerance.

Experiment I
This study examined the effect of a single or combined use of gamma radiation and EMS. Four sorghum genotypes ('Parbhani Moti', 'Parbhani Shakti', 'ICSV 15013 , and 'Macia') were subjected to mutagenesis using five doses of gamma radiation (0, 300, 400, 500 and 600 Gy), three EMS doses (0, 0.5 and 1.0%) and subsequent use of gamma radiation followed by EMS (0 and 300 Gy and 0.1% EMS; 400 Gy and 0.05% EMS). Batches of 1000 dry seeds per treatment were irradiated at Bhabha Atomic Research Centre, Nuclear Agriculture and Biotechnology, Mumbai, India. The minimum gamma radiation dose (300 Gy) was selected following favorable responses as previously reported by Bretaudeau [23], Human et al. [45] and Mizuno et al. [24]. EMS dose rates were selected following the germination rate and seedling responses reported by Jiao et al. [46], Mani [17] and Xin et al. [26]. Prior to EMS treatment, 100 seeds per treatment were placed in mesh bags replicated five times for pre-soaking under running tap water for six hours at room temperatures, according to the procedure described by Mba et al. [47] and Ndou et al. [48]. Seeds were removed to dry off excess water and placed in respective prepared EMS solutions for five hours. The seed was rinsed under running tap water for three hours. Treated seeds were sown in 10-m-long single-row plots using a randomized complete block design (RCBD), with five replications in the experimental field of ICRISAT, Patancheru, India. Inter-row spacing of 75 cm and intra-row spacing of 10 cm was used for planting. To ensure adequate soil moisture, supplemental irrigation was provided. Compound synthetic fertilizer as a source of nitrogen (N), phosphorus (P) and potassium (K) was used at the rate of 30 kg N ha −1 , 45 kg P 2 O 5 ha −1 and 30 kg K 2 O ha −1 , in that order. The fertilizer was homogeneously broadcasted to the plots before planting.

Experiment II
This experiment examined the effect of gamma radiation from germination to maturity. The experiment involved two genotypes ('Macia' and 'Red sorghum'), which were treated with seven doses of gamma radiation (0, 100, 200, 300, 400, 500 and 600 Gy). The doses of 100 and 200 Gy were included following the result of Experiment I. Batches of 1000 dry seeds per treatment were irradiated at the Joint FAO/IAEA laboratories in Seibersdorf, Austria. Gamma-irradiated seeds were sown in 10-m-long single-row plots laid in an RCBD with three replications in the field at Mannheim Crop Research Station, Namibia. All standard agronomic practices were followed as described above. Urea (46% N) was applied at the rate of 30 kg N ha −1 after a 50% emergence.

Data Collection
The following traits were assessed following the methods described by FAO/IAEA [49], Horn and Shimelis [50] and Mba et al. [10]. Germination percentage was recorded by counting sprouted seeds per treatment after 3 days using the moist paper towel germination method under room temperature as described by Sako et al. [51]. Emergence percentage was recorded by counting the number of seedlings raised above ground from sowing to seven days after sowing. Seedling survival percentage was recorded by counting the number of surviving seedlings at 21 days after 50% emergence. The shoot length was recorded from measuring five randomly selected plants per plot from the base to the tip of the top leaf, and the average was expressed in cm 21 days after 50% emergence. The total number of panicles and productive panicles per plot was counted at harvest and expressed in the number of panicles m −2 . Panicle length was recorded from five randomly selected panicles per plot by measuring from the base to the tip of panicles and expressed in cm. The plant height was measured from five randomly selected plants from the base of the plant to the tip of the panicle and expressed in cm. Seed viability was recorded using a germination test as described above.

Data Analysis
Data on germination percentage, emergence percentage, seedling survival percentage, shoot length, number of panicles m −2 , number of productive panicles m −2 , panicle length, plant height and seed viability were subjected to analysis of variance (ANOVA) using Genstat (18th edition) statistical software [52]. Mean comparisons were conducted using Fisher's least significant difference procedure when significant differences were detected in the ANOVA. The mean lethal dose (LD 50 ) and mean growth reduction (GR 50 ) were estimated through the simple linear regression model by fitting the straight-line equation y = mx + c; where y is the response variable, x is the independent variable, m and c represent the slope and constant, respectively.

Conclusions
This study determined doses of gamma radiation and EMS singly or in combination in sorghum based on early growth parameters. Combined doses of gamma radiation and EMS produced poor seedling emergence and seedling survival rate below the target of LD 50 . Therefore, combined doses were not recommended for large scale mutation induction in sorghum. The optimum gamma radiation dose rates aiming at LD 50 for seedling emergence and survival rate for genotypes Red sorghum, Parbhani Moti, Macia, ICSV 15013 and Parbhani Shakti ranged between 392 and 419 Gy, 311 and 354 Gy, 256 and 355 Gy, 273 and 304 Gy, and 266 and 297 Gy, respectively. The optimum EMS dose rates aiming at LD50 for seedling emergence and survival rate for genotypes Parbhani Shakti, ICSV 15013, Parbhani Moti and Macia were between 0.41% and 0.60%, 0.48% and 0.58%, 0.46% and 0.51%, and 0.36% and 0.45%, respectively. These dose rates may induce genetic variation in the tested sorghum genotypes for genetic enhancement in sorghum breeding programs.