Page 274 - e-Book

Root length and number of leaves of plant were. Leaf area was calculated in square

2
centimeters (cm ) by the method of Jose et al. (2000). Fresh weight of the whole plant was
weighed using an electronic weighing balance (Danwer DW500NANO). For dry weight, the

whole plant was oven dried for 48 hours at 72ºC and was measured in grams by the method
of Kacar (1972).

Chlorophyll Content
0.5 g of leaf sample was extracted in about 7 ml of DMSO which was then kept at 65°C

in an incubator. After extraction, sample tube was taken off from the incubator and sample
tube was further filled up to 10 ml with DMSO. Absorbance of both blank (pure DMSO) and

sample were taken at 645 nm and 663 nm. Hiscox and Israelstam (1979) suggested the use of

Arnon’s (1949) equations:
-1
Chl a (g l ) = 0.0127 A663 – 0.00269 A645;
−1
Chl b (g l ) = 0.0229 A645 – 0.00468 A663;
−1
Total Chl (g l ) = 0.0202 A645 + 0.00802 A663.

Phenolic Content

0.5 g of leaves crushed in a pestle and mortar in 80% ethanol and centrifuged at 10,000

rpm for 20 minutes. Supernatant was saved. The supernatant was dehydrated and dried matter
is fluxed in 5 ml distilled water. About In 0.2 ml of solution, water is added to make the

volume 3 ml. Then 0.5 ml of Folin-Ciocalteau and 2 ml of 20% Na2CO3 was put and mixed.
Absorbance of sample plants and standard was taken at 650 nm. The concentration of phenols

was calculated from the standard curve by Malick and Singh (1980) method.

Flavonoids
About 0.1 g of leaf sample was added to the 1 ml of 80% methanol to prepare the sample

solution and then centrifuged for 20 minutes at 10,000 rpm. About 1.25 ml of deionised water
and 75µl of a 5% sodium nitrite was added to the 250µl supernatant of the sample. After five

minutes, 150µl of 10% AlCl3.H2O solution was added and filtered for six minutes. About

500µl of 1 molar NaOH and 275µl of distilled H2O were added to the above prepared sample
and the concentration of sample and standard were taken at 510 nm to calculate the content of

total flavonoids by the method of Jia et al. (1999).

264