Investigating physiological and biochemical

3.3.2.3 Chlorophyll a,b
During the experiment, before dealing amount of proline, chlorophyll ‘a’ and 
chlorophyll ‘b’ contents were measured in the laboratory. Photosynthetic pigments 
(chlorophyll a and b) were measured in fresh leaf samples, a week before the harvest. 
One plant per replicate was used for chlorophyll determination. Prior to extraction, 
fresh leaf samples were cleaned with deionized water to remove any surface 
contamination. Leaf samples (0.5 g) were homogenized with acetone (80% v/v), 
filtered and make up to a final volume of 5 mL. Then the solution was centrifuged at 
3000 rpm for 10 minutes. Pigment concentrations were calculated from the 
absorbance of extract at 663 and 645 nm using the formula (Ashraf et al., 1994; 
Arnon, 1975) given below: 
Chlorophyll a (mg/g FW) = [12.7× (A663) - 2.69× (A645)] × 0.5 
Chlorophyll b (mg/g FW) = [22.9× (A645) - 4.69× (A663)] × 0.5 
Chlorophyll a+b (mg/g FW) = [20.2× (A645)- 8.02× (A663)]×0.5 
3.3.2.4 Relative water contents (RWC)
Relative water contents were measured as described by Yamasaki and Dillenburg 
(1999). Leaves were collected from mid section of plant in order to minimize age 
effect. For each treatment, 10 leaves were taken. To obtain their fresh weight (fr wt), 
the leaves were weighed just after removal from the stem. In order to determine turgid 
weight (t wt), the leaves were kept in Double Distilled water (DDW) inside a closed 
perti dish for four hours. After gentle wiping the water from the leaf surface with 
tissue paper, the leaves were weighed. To determine dry weight (dry wt), the leaf 
samples were dried at 80 ºC for 48 h. Values for fr wt, t wt and dry wt were used to 
calculate leaf RWC using the equation below: 
 
100 

---

98 
3.3.2.5 Electrolyte leakage (EL)
EL is used to assess membrane permeability as described by Lutts et al. (1995). 
Samples were washed three times with DDW to remove surface contamination. 
Young leaf discs from each sample were taken. Leaf discs were placed in a closed vial 
containing 10 mL of DDW and incubated on rotator shaker for 24 h subsequently the 
electrical conductivity of solution (EC1) was determined. Samples were then 
autoclaved at 120 ºC for 20 min and last electrical conductivity (EC2) was noted after 
cooling the solution at room temperature. The electrolyte leakage was calculated as: