Effect of Calcium Salts on Salinity Stress on Morphology …
307
3 Methodology
3.1 Experiment Designing and General Morphological
Seeds were grown with Hoagland media (Hoagland and Arnon 1950). The 15-dayold seedlings were treated with 200 mM of NaCl solutions with the three different
calcium salts CaCl 2 , CaSO 4 and CaCO 3 in 5 mM, 10 mM and 15 mM concentration,
respectively. Rice seedlings from each concentration were randomly selected after
7 days for the measurements of shoot length, biochemical estimations and estimations
of antioxidant activities.
3.2 Estimations of Chlorophyll Content
Chlorophyll content was measured by using the method of Arnon (1949) at 645 nm
and 663 nm and calculated using the following equation: Chlorophyll a: 12.7(A 663 )–
2.69(A 645 ) and Chlorophyll b: 22.9(A 645 )–4.68(A 663 ).
3.3 Estimations of Proline Content
Proline content was analysed by acid ninhydrin according to the modified procedure
of Bates et al. (1973). Reacted compound was extracted by toluene and measured at
520 nm.
3.4 Estimations of Carbohydrates
Reducing sugars were extracted with hot 80% ethanol and then estimated by using
dinitrosalicylic acid reagent calorimetrically at 540 nm wavelength and starch was
extracted with perchloric acid and then estimated with ‘Anthrone reagent’ (Miller
1972).
3.5 Estimations of Lipid Peroxidation Rate
The level of lipid peroxidation is expressed as µg/g malondialdehyde content
extracted with thiobarbituric acid–trichloroacetic acid (Agarwal 2007).
307
3 Methodology
3.1 Experiment Designing and General Morphological
Seeds were grown with Hoagland media (Hoagland and Arnon 1950). The 15-dayold seedlings were treated with 200 mM of NaCl solutions with the three different
calcium salts CaCl 2 , CaSO 4 and CaCO 3 in 5 mM, 10 mM and 15 mM concentration,
respectively. Rice seedlings from each concentration were randomly selected after
7 days for the measurements of shoot length, biochemical estimations and estimations
of antioxidant activities.
3.2 Estimations of Chlorophyll Content
Chlorophyll content was measured by using the method of Arnon (1949) at 645 nm
and 663 nm and calculated using the following equation: Chlorophyll a: 12.7(A 663 )–
2.69(A 645 ) and Chlorophyll b: 22.9(A 645 )–4.68(A 663 ).
3.3 Estimations of Proline Content
Proline content was analysed by acid ninhydrin according to the modified procedure
of Bates et al. (1973). Reacted compound was extracted by toluene and measured at
520 nm.
3.4 Estimations of Carbohydrates
Reducing sugars were extracted with hot 80% ethanol and then estimated by using
dinitrosalicylic acid reagent calorimetrically at 540 nm wavelength and starch was
extracted with perchloric acid and then estimated with ‘Anthrone reagent’ (Miller
1972).
3.5 Estimations of Lipid Peroxidation Rate
The level of lipid peroxidation is expressed as µg/g malondialdehyde content
extracted with thiobarbituric acid–trichloroacetic acid (Agarwal 2007).