Effect of Salinity Stress on Osmoprotectant (Glycine Betaine and Prline) of Two Maize (Zea mays L.). Varieties Cultivated in Sokoto Metropholis

Plant Material and Stress Treatment

Two maize cultivars (Oba super 2 16-11-kd-155-159 and sammaz-37) were used in this study. The maize cultivars were obtained from Sokoto Agricultural Development Project (SADP), Sokoto State, Nigeria. seed were directly sown into sandy soil (85.9 % sand, pH 6.34) the soil initially contained 0.64 % carbon, 0.060 kg nitrate-nitrogen, 0.3 9 kg sodium, 0.82 kg potassium, 5.8 kg cation exchange capacity, 0.45 kg calcium and 0.50 kg magnesium. Germination was carried out in the Botanical Garden of the Department of Biological Sciences, Usmanu Danfodiyo University, Sokoto. Salinity irrigation water treatments were applied as sodium chloride (NaCl) solution at the following levels (0.00, 50, 100 and 150 mM). Plants were irrigated daily with different NaCl concentration. Each treatment was replicated three times. Roots and mature leaves were harvested after 21 days of salt treatment and samples were immediately taken for analysis.

Hydrogen Peroxide (H2O2)

Hydrogen peroxide content was estimated according to the method of [13]. Exactly 0.5 g fresh tissues of roots and matured leaves were homogenized in 5 ml of 0.1% (w/v) trichloroacetic acid (TCA) in an ice bath. The homogenate was centrifuged at 3000 rpm for 15 min. Then 0.5 ml of the supernatant was made up to 1 ml with 10 mm potassium Phosphate buffer (pH 7.0) to which 1 ml of 1M KI was added. The absorbance was measured at 390 nm against blank in three replicated sample. The blank sample was prepared by 0.4 ml of distilled water and 0.4 ml of working reagent. And 0.4 ml of working reagent was prepared as a standard test.

Determination of Lipid Peroxidation Marker

Malondialdehyde (MDA): MDA was determined using the method of [14]. Exactly 0.5 g each of fresh roots and matured leaves were homogenized in 1 % metaphosphoric acid and then incubated at room temperature for 30 min. 1 ml of 10 % trichloroacetic acid (TCA) was added and centrifuged at 2000 rpm for 15 min and the supernatant was used for the assay. 1 ml of the supernatant was mixed with 1 ml of 5 % thiobarbituric acid (TBA) in the test tubes, followed by addition of equal volume of 40% trichloroacetic acid. The mixture was placed in boiling water for 30 min at 95 0C. The samples were allowed to cool at room temperature and then the absorbance were measured at 532 nm in three replicated sample. The blank sample was prepared by 1.5 ml of distilled water and 1.5 ml of working reagent. And 1.5 ml of working reagent was prepared as a standard test.

Determination Of Osmoprotectants

Glycine Betaine (GB): The glycine betaine level was determined according to the method of [15]. A 0.5 g of the fresh tissues (root and mature leaves) was homogenized for extract preparation with 5 ml of toluene-water mixture (0.05% toluene) and then filtered. After filtration 0.5 ml of extract was mixed with 1 ml of 2 N HCl solution and then 0.1 ml of potassium tri-iodide solution (containing 7.5 g iodine and 10 g potassium iodide in 100 ml of 1 N HCl) was added and shaken in an ice-cold water bath for 30 minute. Then 10 ml of 1, 2-dichloroethane was poured into the test tube and shake gently. Absorbance was measured at 365 nm against blank in three replicated sample. The blank sample was prepared by 0.1 ml of distilled water and 0.1 ml of working reagent. And 0.1 ml of working reagent was prepared as a standard test.

Proline: The proline content was estimated according the method of [16]. Exactly 0.5 g each of roots and matured leaves tissues of maize was homogenized in 5 ml 3% aqueous sulfosalicylic acid in an ice bath. The homogenate was centrifuged at 3000 rpm for 15 min at 4 oC and then filtered. 2ml of the filtrate was added to 2 ml acid ninhydrin and 2 ml of glacial acetic acid in a test tube. The mixture was boiled for 1 hour and then later the test tube was placed in an-ice cold water for the termination of the reaction. 4 ml of toluene was added to the reaction mixture and stirred for 30 sec. The toluene layer was separated and warmed at 37°C and the absorbance was measured at 520 nm against blank sample in three replicated. The blank sample was prepared by 2.0 ml of distilled water and 2.0 ml of working reagent. And 2.0 ml of working reagent was prepared as a standard test.

Hydrogen Peroxide Levels in Salt-Stressed Maize Cultivars

Increase in salinity stress resulted a significant increase in hydrogen peroxide (H2O2) content in roots at 50 mM NaCl and young leaves at 50 and 100 mM NaCl of Oba super 2. In sammaz 37 variety significant increase in hydrogen peroxide content was observed only at 50 mM NaCl in young and matured leaves (Figure 1).

Malondialdehyde (MDA) Levels of Maize Seedlings Under Salinity Stress

MDA increased significantly (p<0.05) in root, mature leaves and young leaves (Figure 2). The effect of salt stress was observed at highest concentration (100 and 150 mM NaCl) in roots, matured and young leaves of both the varieties investigated.

Glycine Betaine Content of Maize Seedling under Salinity Stress

Figure 3 shows that salt stress resulted in no significant increase in GB level in root and mature leaves of Oba super 2 while in the young leaves there is slight decrease in GB level. Sammaz 37 variety shows no significant increase in GB by increasing salt concentration in all the tissues examined. Proline Levels in Salt Stress Maize Cultivars Levels of proline in root of oba super 2 variety decreased at all salt regimes when compared with control, but in young leaves proline levels decrease significantly only at 150 mM NaCl. While in the mature leaves increase in salinity resulted to a decrease in proline level at all salt regime when compared to the control Figure 4. In roots of sammaz 37 the decrease in proline levels was observed by increasing salt concentration. In young leaves Proline level shows significant decreased (p˂0.05) only at 150 mM NaCl whereas in the mature leaves proline levels increased significantly at 50, 100 and 150 mM NaCl.

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Figure 1: Hydrogen Peroxide levels (µg/g) of two varieties of maize exposed to the same level of different concentrations of NaCl for 21 days. Values are expressed as mean ± SEM of three replicates. Significant difference between the means was determined using Duncan’s test at (p < 0.05), mean in the column followed by the same letter (s) are not significantly difference at 5% level.

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Figure 2: Malondialdehyde levels (µg/g) of two varieties of maize exposed to the same level of different concentrations of NaCl for 21 days. Values are expressed as mean ± SEM of three replicates. Significant difference between the means was determined using Duncan’s test at (p < 0.05), mean in the column followed by the same letter (s) are not significantly difference at 5% level.

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Figure 3: Glycine Betaine levels (µg/g) of two varieties of maize exposed to the same level of different concentrations of NaCl for 21 days. Values are expressed as mean ± SEM of three replicates. Significant difference between the means was determined using Duncan’s test at (p < 0.05), mean in the column followed by the same letter (s) are not significantly difference at 5% level.

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Figure 4: Proline levels (µg/g) of two varieties of maize exposed to the same level of different concentrations of NaCl for 21 days. Values are expressed as mean ± SEM of three replicates. Significant difference between the means was determined using Duncan’s test at (p < 0.05), mean in the column followed by the same letter (s) are not significantly difference at 5% level.