Page 261 - e-Book

as control-uninfected and infected-treated plants were watered with nutrient solution once a

week till the harvesting period (30days).

BIOCHEMICAL STUDIES
Sample preparation for biochemical estimation :(Allam et al.,1980)

The plants were depotted and cleaned with tap water to remove sand particles, then

rinsed with distilled water and blotted with filter paper thirty days following inoculation. To
minimize water loss, the plants were split into root and shoot sections and weighed as

promptly as feasible for evaluation. To get a consistent weight, the roots and shoots were
separated and dried in a hot air oven for a week. Separately, these dry samples were ground

to a 60 mesh powder. Total sugar content of various samples was calculated using the process
of Seifter et al. (1950), starch estimation by Jayaraman, 1981, and -Amylase activity

(Bernsfeld, 1995). Chlorophyll concentrations were assessed using the Arnon (1949) method.

Estimation of Enzymes
Kun and Abood were the first to use Tripheny Tetrazolium Chloride (TTC) as an

artificial electron acceptor (1949). TTC was used to determine the activity of the various
enzymes studied in the current research. The intensity of the lowered red colourded formazon

(which indicates hydrogen liberation from the substrate and absorption of by colourless TTC
was reduced to red coloured formazon). Indicative of the enzyme activity was matched with

the artificially reduced formation standard as follows. The formazon was made by reacting

100 mg of TTC with 5 ml of 10% NaOH. The formazon was dried and weighed, and a known
concentration of 1 to 25 microgram was prepared by diluting it in toluene. These were then

put through a spectrophotometer to create a standard curve. In enzyme research, add a known

amount of buffered extract to the known amount of buffered extract (Buffer beings specific
for each enzyme study). 1.0 ml of newly manufactured 0.01 percent TTC and 1 ml of the

particular substrate were mixed together and incubated at 40°C for 30 minutes. The red
coloured formazon that emerged as a result of reduced TTC (an indicator of enzyme activity)

was extracted with 6ml toluene and the colour intensity was measured at Spectrophotometer.
Preparation of Enzyme Powder

Fresh root and shoot systems were separated and ground in a cold Butanol, Benzene,

Acetone combination (in a 2:1:1 ratio) before being kept in the fridge overnight. The extract
was then dried at room temperature before being ground into a fine powder. This enzyme

powder was used to perform a variety of enzymatic tasks. Kannan's (1968 b) approach was
used to estimate the enzyme's activity.

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