This investigation used three Egyptian lentil cultivars namely, Giza 9, Giza 29, and Giza 51. The previous three cultivars were performed from leguminous Crops Research Department, Field Crops Research Institute, Agricultural Research Center (ARC), Giza, Egypt. These varieties were known to be widely famous as genetic material highly tolerant for biotic and abiotic stresses. Therefore, the genetic improvement may give super new variety traits.
Accordingly, the EMS was used in this investigation to cause the process of mutagenesis. Highly positive and significant limit of genetic improvement was the right decision after reviewing a large number of papers included a lot of mutagenesis as it was of great importance, feasibility, and effectiveness in this regard.
One thousand pure seeds of the three lentil cultivars were placed in a 500-ml flask for each genotype and ultrapure water was added to about 5 cm level above the seeds (~ 100 ml). Seeds were soaked overnight at room temperature for 20 h. Subsequently, the water was decanted and 50 ml of 0.1%, 0.2%, and 0.3% concentrations of EMS (v/v) in water was added. Seeds were incubated for 12 at room temperature followed by decantation of the EMS and rinsing with 100 ml of ultrapure water (5 times, 4 min each) and 200 ml of ultrapure water (4 times, 15 min each). Seeds were then rinsing under running tap water for 4 h planting in petri dishes and when planting stage started immediately transfer to pots.
Preparing of ethyl methane sulfonate (EMS) stocks
1) The first dose 0.1%: 1 g EMS was dissolved in 1000 ml distilled water.
2) The second dose 0.2%: 2 g EMS was dissolved in 1000 ml distilled water.
3) The third dose 0.3%: 3 g EMS was dissolved in 1000 ml distilled water.
After mutagenesis, each genotype was grown under the four treatment conditions, the control besides the three doses of EMS (0.1, 0.2, and 0.3%) in half of November 2017 in pots experiment. Each treatment was replicated 5 times. Each replicate consisted of 10 pots to produce the first mutagenic generation (M1). The seeds of the three lentil varieties for the four treatments were grown in half of November 2018 in pots. Each treatment was replicated 5 times where each replicate consisted of 10 pots to get and produce the second mutagenic generation (M2) in 2018. All germination, morphological, and growth traits were calculated for M2 generation in 2018.
The following traits in M2 generation were recorded.
1- Germination percentage was calculated by counting only normal seedlings 8 days after planting according to ISTA (1985).
2- Seedling fresh weight (g) was determined by weighting normal seedlings according to Krishnasamy and Seshu (1990).
3- Seedling dry weight (g) was determined using 10 normal seedlings dried in a hot-air oven at 110 °C for 17 h to obtain the seedling dry weight according to Krishnasamy and Seshu (1990).
4- Shoot length
5- Root length
6- Seedling length (cm) was measured as an average of ten normal seedlings 8 days after sowing.
7- Germination energy percentage was calculated according to the method of Ruan et al. (2002).
8 and 9- Seedling vigor indices 1 and 2 were measured according to Krishnasamy and Seshu (1990).
10:- Electrical conductivity
Note: seedling vigor index 1 and seedling vigor index 2 were performed according to the method of Abdul-Baki et al. (1973) and electrical conductivity was conducted according to the method of Thomas (1960) and Matthews and Bradnock (1967) and modified methods by ISTA (2006) and Matthews and Powell (1981), respectively.
All data were statistically analyzed as a factorial experiment design as the technique of analysis of variance (ANOVA) as described by Gomez and Gomez (1984) and SAS program version (1985).
Improvement or positive and negative value percentage of induced mutagenesis (M2 generation) in lentil (Lens culinaris L.) seedling is as follows: it was assessment by yield under stress (the value of trait for EMS treatment)—(the value of trait under control)/(the value of trait under control) × 100.
Correlation coefficients: simple phenotypic correlation coefficients among all studied traits for the control and the three EMS treatments were estimated using the formula suggested by Miller et al. (1958).
Molecular and biochemical studies
2,2′-azino-di-(3-ethyl-benzothiazole-6-sulfonic acid) (ABTS), hydrogen peroxide of analytical grade (30% w/v), 1-chloro-2, 4-dinitrobenzene (CDNB), and reduced glutathione (GSH) are products of Sigma-Aldrich Co. The buffers and other chemical reagents used in this study were of laboratory grade.
Protein determination: protein concentration was measured by the Bio-Rad assay using bovine serum albumin as a standard (Bradford 1976). Measurements were done on the shimadzu UV spectrophotometer at 595 nm.
SDS-protein electrophoresis: sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to study the protein banding patterns of the three lentil genotypes under the control and treatments. Protein fractionation was performed according to the method of Laemmli (1970) as modified by Studier (1973).
Preparation of crude homogenate: fresh weight sample (1 g) was frozen, then homogenized in 10 ml of 50 mM cold Tris-HCl buffer, pH 7 containing 2 mM β-mercaptoethanol. The homogenates were centrifuged at 10,000 rpm for 20 min and the supernatant was used as a crude material for enzymatic assays.
Peroxidase (POX): peroxidase activity was determined according to the method of Childs and Bardsley (1975), with slight modifications using ABTS as a reducing substrate, in a reaction mixture (1 ml) containing 2 mM H2O2, 0.36 mM ABTS, and 100 mM sodium phosphate buffer (pH 6) and peroxidase concentration which gave linear response for of 3 min. The change in absorbance at 414 nm was followed at 1 min intervals. One unit of POX activity was defined as the amount of enzyme that oxidized 1 mmol ABTS per minute at 25 °C under the assay conditions.
Glutathione S-transferase (GST): the GST activity was determined spectrophotometrically with aromatic substrate (CDNB) by monitoring the change at 340 nm due to thioether formation at 25 °C as described by Habig et al. (1974). The assay mixture contained in a total volume of 1 ml, 0.1 M potassium phosphate buffer, pH 6.5, and 1 mM (CDNB) in ethanol 1 mM GSH and the enzyme solution. One unit of glutathione S-transferase activity is defined as the amount of enzyme which catalyzes the formation of 1.0 μmole of thioether per minute at 25 °C under the assay conditions.
Native-polyacrylamide gel electrophoresis (Native-PAGE) was conducted according to Stegemann et al. (1985) to identify isozyme variations between controls and EMS treatments of three lentil genotypes. Two isozymes systems, POX and PPO, were analyzed. After electrophoresis, gels were stained according to their enzyme systems with the appropriate substrate and chemical solutions and then incubated at room temperature in dark for complete staining. For POX activity, 0.125 g of benzidine-dihydrochloride HCl as a substrate, 2 ml glacial acetic acid and was completed with distilled water up to 50 ml. The gel was placed into this solution and 5 drops of hydrogen peroxide were added and incubated at room temperature until bands appeared. The gel was incubated at room temperature until bands appear (Brown 1978). For PPO activity, 15 mg cathecol and 50 mg sulfanilic acid, as substrates, were dissolved in 100 ml of 0.1 M sodium phosphate buffer, pH 6.8. The gel was placed into this solution and incubated at 30 °C for 30 min until bands appeared (Manchenko 2002).