Improving desirable agronomic traits of M2 lines on fourteen Ethiopian Sesame (Sesamum indicum L.) genotypes using Ethyl Methane Sulphonate (EMS)

Sesame is an important oilseed crop cultivated in Ethiopia as a cash crop for small holder farmers. However, low yield is one of the main constraints of its cultivation. Boosting and sustaining production of sesame is thus timely to achieve the global oil demand. This study was, therefore, aimed at identifying mutant genotypes targeted to produce better agronomic traits of M2 lines on fourteen Ethiopian sesame genotypes through seed treatment with chemical mutagens. EMS was used as a chemical mutagen to treat the fourteen sesame genotypes. Quantitative and qualitative data were recorded and analyzed using analysis of variance with GenStat 16 software. Post-ANOVA mean comparisons were made using Duncan’s Multiple Range Test (p≤ 0.01). Statistically significant phenotypic changes were observed in both quantitative and qualitative agronomic traits of the M2 lines. All mutant genotypes generated by EMS treatment showed a highly significant variation for the measured quantitative traits, except for the traits LBL and LTL. On the other hand, EMS-treated genotypes showed a significant change for the qualitative traits, except for PGT, BP, SSCS, LC, LH and LA traits. Mutated Baha Necho, Setit 3, and Zeri Tesfay showed the most promising changes in desirable agronomic traits. To the best of our knowledge, this study represents the first report on the treatment of sesame seeds with EMS to generate desirable agronomic traits in Ethiopian sesame genotypes. These findings would deliver an insight into the genetic characteristics and variability of important sesame agronomic traits. Besides, the findings set up a foundation for future genomic studies in sesame agronomic traits, which would serve as genetic resources for sesame improvement.


Introduction
Sesame (Sesamum indicum L., 2n = 26) is one of the ancient oilseed crops widely cultivated, mainly in the arid and semi-arid regions of Africa, Asia and South America, as a source of targeting key functional agronomic traits for the Ethiopian sesame genotypes is still demanding.Therefore, it is vital to develop new mutants using EMS, which can accelerate the breeding program and improve sesame genotypes.The main objective of this study was to use EMS to induce desirable agronomic traits in 14 Ethiopian sesame genotypes, with the aim of accelerating breeding programs and improving the crop's productivity.

Collection of seeds and sterilization
Sesame seeds of fourteen genotypes comprising four landraces, an introduced variety from Israel and nine varieties released from different research centers in Ethiopia, which were acquired from Humera Agricultural research center (HuARC), Tigrai, Ethiopia, were used in this study (Table 1).Disease-free, dry and normal shaped seeds were used for the experiment.The seeds were soaked in 3% teepol detergent solution for 5 min after being washed with running tap water for 20 min and rinsed with distilled water.70% ethanol was used to disinfect the seeds for 45 sec at room temperature and rinsed with sterile distilled water three times.Sulfuric acid and glacial acetic acid were used to treat the seeds and soaked in sterile distilled water for 16 hrs [25,27].

Treatment with EMS
Different concentrations of EMS, i.e. 0.0, 0.25%, 0.5%, 0.75%, and 1% were studied to find the optimum concentration [29,30].Seeds of 14 sesame genotypes were pre-soaked in cold tap water at 4˚C for 24 hrs and then soaked in 0.5% EMS solution (HiMedia Laboratories, Pvt. Ltd., Mumbai-400086, India) with So ¨renson phosphate (Sigma-aldrich, Munich, Germany) as a buffer adjusted to pH = 3 with H 3 PO 4 (Sigma-aldrich, Munich, Germany) for 4 hrs with constant shaking at 20 rpm at 18-24˚C.EMS is a mutagenic chemical, and it is important to reduce the harmful effects of EMS on the environment and human health, provided it is properly stored, used, disposed of, and transported.The procedures developed in the work of Weldemichael et al. [25] were used in this study.After washing, both the treated and control seeds were planted in well prepared beds in Kebabo, Humera and Sheraro to get M1 lines.Untreated seeds of all genotypes have been used as control for comparison throughout the experiment.Besides, no data were taken from M1, which was rather advanced into M2 (second generation mutant) lines from which all the quantitative and qualitative data were used for analyses in this study.Finally, the M2 plants derived from the M1 seeds were being considered as mutants.

Field evaluation and recorded data of M2 lines
The experiment was laid down in a randomized complete block design (RCBD) having three replications.Each genotype of the M2 lines was randomly assigned and sown in rows, in a plot area of 2.8m by 5m with 1m between plots and 1.5m between blocks keeping inter-and intrarow spacing of 0.4m and 0.1 m, respectively.Each experimental plot was treated equally as per Table 1.List of 14 sesame genotypes with their pedigree, altitude, average gain yield, oil content and days to maturity.

Released by
Baha *m.a.s.l: meters above sea level ARC: Agricultural Research Center, HU; Haramaya University Source: [28] https://doi.org/10.1371/journal.pone.0287246.t001 the agronomic recommendations for the crop in the growing area.During the growth period, all required production practices like weed management and fertilization were carried out as recommended.Data were collected from fifteen plants of each M2 sesame lines at 75% maturity.Both quantitative and qualitative data (all in cm) were recorded properly from all the treated and control genotypes.Recorded quantitative data include: ground distance to first branch (GDFB), internode length (IL), plant height (PH), length of basal leaf (LBL), length of top leaf (LTL), width of basal leaf (WBL), width of top leaf (WTL), length of marginal leaf (LML), width of marginal leaf (WML), petiole length of basal leaf (PLBL), petiole length at middle (mid-level/mid-height) leaf (PLML), petiole length of top leaf (PLTL).Likewise, recorded qualitative data were: main stem color (MSC), stem hairiness (SH), stem branch (SB), plant growth type (PGT), branching pattern (BP), stem shape in cross section (SSCS), leaf color (LC), leaf hairiness (LH), leaf arrangement (LA), leaf shape (LS), basal leaf profile (BLP), basal leaf margin (BLM), lobe incision of basal leaf, and leaf angle to main stem (LAMS) (Table 2).

Data analyses
The collected data for the different quantitative and qualitative traits were measured and subjected to analysis of variance (ANOVA) using GenStat 16 Software [32].Post-ANOVA mean comparisons were carried out using Duncan's Multiple Range Test at a significance level of p � 0.01 [33].

Effects of EMS on quantitative agronomic traits of M2 lines
Results of ANOVA clearly indicated highly significant effects among the genotypes, concentrations of EMS and their interaction effect on the quantitative data including GDFB, IL, LTL, WBL, WTL, LML, WML, and PLTL (p � 0.01; Table 3).On the other hand, the remaining Sources: [31] https://doi.org/10.1371/journal.pone.0287246.t002 quantitative leaf traits including LBL, PH, PLBL, and PLML showed non-significant interaction effect among the genotypes and concentrations of EMS (Table 3).
Similarly, the control genotypes showed higher PLTL values as compared to the treated ones, the highest mean PLTL values being recorded from the control genotypes Setit 1, Hirhir, and Humera 1 (14.67,14.33 and 14.00 cm, respectively) while the lowest being recorded from the treated Humera 1 (3.67 cm) genotype.

Effects of EMS on qualitative agronomic traits of M2 lines
In this study, the treatment of seeds with EMS was found to be instrumental to bring major changes in multiple agronomic traits of the M2 lines of the tested Ethiopian sesame genotypes.The genotypes, EMS concentrations, and their interaction effects showed significant changes on the fourteen qualitative agronomic traits, namely main stem color, stem hairiness, stem branch, leaf shape, basal leaf profile, basal leaf margin, lobe incision of basal leaf, and leaf angle to main stem.However, the application of EMS did not result in any significant changes in plant growth type, branching pattern, stem shape in cross section, leaf color, leaf hairiness, or leaf arrangement in any of the genotypes tested (Table 5).
2. Stem hairiness.The treatment of seeds with EMS caused changes in the stem hairiness from weak or sparse to medium in the genotypes ADI, Baha Necho, Baha Zeyit, and Borkena.In addition, significant change was observed in stem hairiness of Bounji, Hirhir, Humera 1, Gondar 1, Setit 1 and Zeri Tesfay genotypes from glabrous to weak/sparce.On the other hand, the application of EMS did not change the steam hairiness of the genotypes ACC44, Setit 2, and Setit 3. 3. Stem branch.The treatment of seeds with EMS significantly changed the stem branch in ADI, Baha Necho, Bounji, Gumero, Hirhir and Setit genotypes from opposite to mixed.On the other hand, no change was observed in the genotypes ACC44, Baha Zeyit, Borkena, Gondar 1 and Humera 1 genotypes when treated with EMS.
4. Leaf shape.The treatment of seeds with EMS brought changes in the shapes of the leaves in two genotypes, namely Hirhir and Setit 1, from lanceolate to ovate.On the other hand, all the other genotypes showed no change when treated with EMS.
6. Basal leaf profile.EMS treatment brought about significant changes in basal leaf profile in ACC44, ADI, Bounji, Hirhir, Setit 1, Setit 2, and Setit 3 genotypes from flat to reverse cup shaped.The remaining genotypes showed no change when treated with the chemical mutagen.
7. Basal leaf margin.The treatment of EMS caused changes in basal leaf margin in ACC44, Baha Necho, Baha Zeyit and Bounji genotypes from entire to serate.The remaining genotypes, however, did not show any change when treated with the chemical.

8.
Leaf angle to main stem.Treatment of EMS brought significant changes in the leaf angle to main stem in five genotypes.Changes from dropping to acute were observed in ACC44, Setit 2 and Setit 3; from dropping to horizontal in Borkena; and from horizontal to acute in Humera 1 genotypes.

Discussion
Sesame (Sesamum indicum L.) is an important oilseed crop used for food, feed, medicinal and industrial applications.Inherently, low genetic yield potential and susceptibility to biotic and abiotic stresses contribute to low productivity in sesame.Mutation breeding has been one of the breeding strategies used to minimize yield reducing factors in sesame [34].As a result, more than 147 sesame mutants associated with various agronomic traits, such as leaf, capsule, male sterility, flower, disease resistance, and maturity, have been reported globally [35][36][37].For example, in Egypt, Cairo white 8 and Senai white 48 mutants were developed using induced mutations and released for their nonbranching habits and white seed coat color [35].
In addition, various mutant sesame varieties with increased yield, high oil content and resistance to diseases such as phytophthora blight were developed and released in India, South Korea, and Sri Lanka [34,38,39].In Ethiopia, however, yield remained very low due to a diverse set of factors, including shattering capsules, determinate growth habit, branching, reduced plant height, diseases, insect pests, drought, waterlogging, salinity, and lodging.So far, no study has been conducted to improve the aforementioned desirable agronomic traits of Ethiopian sesame genotypes through mutation breeding.Therefore, improvement of sesame using mutation breeding would play a vital role to identify superior genotypes with desirable oil and yield, shattering resistance capsules, improved branching habit, determinate growth, and better number of capsules per plant.On the other hand, limitations and drawbacks of mutation breeding as a means of genetic improvement in sesame plants including low frequency, pleiotropic effects, undesirable side effects, lethal mutations, and difficult selection process would be taken in to considerations.In this study, desirable qualitative and quantitative plant height related traits were investigated in 14 sesame genotypes using induced mutation with EMS.As a result, the lowest plant height was recorded from Setit 1 (56.0 cm) and Setit 2 (52.0) treated with EMS.These results were in line to those of Zhang et al. [11] where plant height of mutant genotypes was reduced from 170 to 110 cm.Moreover, the results of this study were consistent with previous studies that have shown a significant reduction in plant height of mutated dw607 (dwf1) using EMS mutagenesis [16,38].According to their findings, the plant height was reduced by more than 40% (from 176.00 to 118.25 cm) as compared to the wild type, Yuzhi 11.This finding demonstrated that yield of dwarf varieties derived from dw607 significantly increased under suitable management conditions.These findings suggest that mutation breeding is crucial for producing sesame genotypes with desirable traits, such as determinate growth habit, shattering resistant capsules, synchronous flowering, and homogeneous maturity in a short time.To the best of our knowledge, all the sesame genotypes used in this study had indeterminate growth habits, thus causing non-uniform ripening of capsules that make mechanical harvesting difficult and result in seed loss at harvest.These results agree with the findings of other studies, in which induced mutation improved desirable agronomic traits such as determinate growth in sesame [40].According to these data, the maximum plant height acceptable for all harvest was 150 cm and lower plants were more preferable [20].This finding has important implications for developing sesame genotypes having lower plant height.Besides, these findings suggested that higher plant height usually shows less fruiting density, is associated with shy branching, sensitive to lodging, undesirable for high yield and unsuitable for mechanical harvesting, urging more investigation for the development of dwarf sesame genotypes.As a result, reduction of plant height after seed treatment with sodium azide at low pH value has been reported [41][42][43].The combination of these findings hence supports the conceptual premise that plant height decides the plant architecture and contributes a significant role in the yield of sesame [44].In addition, dropping plant height as a means of enlightening lodging resistance is very significant for sesame breeders.Thus, early flowering, increased yield, and reduced plant height are imperative targets for the genetic improvement of sesame.
With regard to internode length, several gammas irradiated mutant lines have been described in St. Augustine grass and in bermudagrass [45][46][47].The dwarfing trait and short internode length have also been reported in the mutant dw607 [16].In the present study, short internode length was observed on ACC44, ADI and Hirhir genotypes treated with EMS.The findings of this study validate the findings of the previous works in identifying the role of chemical mutagens to improve genetic diversity in higher plants [48,49].Besides, the results of this study agree with the previous studies, which have been developed to facilitate the identification, isolation and cloning of genes used in designing crops with improved quality and yield traits in many crops such as barley, cotton, rice, peanuts, wheat, and beans [50].These findings suggest that the observed variations in sesame genotypes are due to the induced mutations.

Conclusion
The study analyzed the effect of EMS on improving desirable agronomic traits in M2 lines of fourteen Ethiopian sesame (Sesamum indicum L.) genotypes.This study showed significant changes in the majority of the qualitative and quantitative agronomic traits for the first time in Ethiopian sesame genotypes using EMS.EMS is a powerful chemical mutagen that can potentially produce desirable agronomic traits.Improving the determinate growth habit of sesame, reducing the plant height and inter node length would lead to synchronous maturity and facilitate mechanical harvesting, reducing yield losses at harvest.Further investigations to develop mutant sesame genotypes with other desirable traits such as larger seed size, improved pod shattering resistance, determinate growth habit, more uniform and shorter maturation period, modified plant architecture and size, earliness, resistance to diseases as well as higher oil content and modified fatty acid composition are strongly recommended.Moreover, the genetic variability induced in sesame plants after mutagenesis should be determined by marker assisted selection.

Table 4 .
(Continued) GDFB: ground distance to first branch; IL: internodes length; LBL: length of basal leaf; WBL: width of basal leaf; LML: length of marginal leaf; WML: width of marginal leaf; LTL: length of top leaf; WTL: width of top leaf; PLBL: petiole length of basal leaf; PLML: petiole length at middle (mid-level/mid-height) leaf; PLTL: petiole length of top leaf.Means followed by different letters indicate significant differences at P�0.01, i.e., means with different letters in the column are significant, while means with the same letter(s) in the column are non-significant. https://doi.org/10.1371/journal.pone.0287246.t004