Genomics and Applied Biology 2015, Vol. 6, No. 4, 1-6
3
Table 2 calculated pressure from stress treatment and soil
moisture content from regression equation
Soil Moisture Content (%)
Pressure (bar)
ASM (%)
33.46
-
0.69
100.00
31.32
-
2.46
93.60
30.19
-
3.39
90.23
29.18
-
4.22
87.21
28.14
-
5.08
84.10
27.09
-
5.94
80.96
26.12
-
6.74
78.06
25.29
-
7.43
75.58
24.91
-
7.74
74.45
24.32
-
8.22
72.68
23.78
-
8.67
71.07
23.40
-
8.98
69.93
23.11
-
9.22
69.07
22.86
-
9.43
68.32
21.28
-
10.73
63.60
20.83
-
11.10
62.25
19.51
-
12.19
58.31
19.30
-
12.36
57.68
18.63
-
12.91
55.68
18.11
-
13.34
54.12
17.81
-
13.59
53.23
17.52
-
13.83
52.36
17.10
-
14.01
50.11
16.72
-
14.47
49.01
16.00
-
15.08
47.82
1.1 Estimation procedure:
1.1.1 Proline
Proline content of the leaf sample was estimated by the
method of Bates et al. (1973) and expressed as µg g
-1
of
fresh weight.
A fresh leaf sample of 0.5g was macerated with 10mL
of aqueous sulphosalicylic acid (3%) using a pestle and
mortar. The extract was centrifuged at 4000 rpm for 10
minutes. The supernatant solution of 2 ml was taken in
a test tube and to this 2 mL of acid ninhydrin and 2 mL
of glacial acetic acid was added. The solution was kept
in water bath for one hour at 100°C and it was cooled
under tap water. After cooling, the solution was
transferred into a separating funnel and 4 mL of toluene
was added. The funnel was uniformly shacked for 30
seconds. Two different layers were formed. The
colorless bottom layer was discarded and the upper
pink color layer was collected. The Optical Density
was recorded at 520 nm against blank as toluene.
1.1.2 Acid ninhydrin
(2.5g of ninhydrin was taken and mixed with 60 ml of
glacial acetic acid and 40 ml of 6 M orthophosphoric
acid. The solution was stirred well and slightly warmed
in hot water bath until the content dissolved.)
1.2 Polyphenol Oxidase (PPO)
The Poly Phenol Oxidase (PPO) activities of the leaf
sample was estimated at all the stages of the crop by the
method of Bray and Thrope (1954) and expressed as
unit
-1
min
-1
mg of protein
-1
.
The leaf sample of 0.5g was macerated with 10 ml of
sodium phosphate buffer (0.1M, pH 7.0) using a pestle
and mortar. The extract was centrifuged at 10000 rpm
at 4°C for 20 minutes. The supernatant solution of 0.5
mL was taken in a test tube and 2 ml of sodium
phosphate buffer (125µmoles, pH 6.8) , 0.5 mL of
pyrogallol solution (50 µmoles) was added and kept in
water bath for 5 minutes at 25 to 30°C or at room
temperature and 0.5 ml of H
2
SO
4
was added. The
Optical Density was recorded at 420 nm against blank.
2 Result
2.1 Proline content
The proline content showed an increasing trend from
5
th
MAP to 7
th
MAP (Table 3). Between the treatments,
M
1
(control) had higher proline content of than M
2
(stress) at 7
th
MAP. Analyzing the effect of sub-plot
treatments, S
1
recorded an increased proline
accumulation. This treatment was followed by S
2
and
S
3
. The treatment, S
12
showed a lesser proline
accumulation at 7
th
MAP in main and ratoon crop.
The interaction effects of M at S and S at M revealed
significant differences at all the stages of growth. The
treatment M
2
S
1
recorded the highest proline content
followed by M
2
S
2
, M
2
S
3
and M
2
S
4
at 7
th
MAP. The
treatment M
2
S
10
, M
2
S
10
, M
2
S
11
and M
2
S
12
found to
accumulate the proline at significantly lower level
than the other treatments at 7
th
MAP in main and
ratoon crop.
2.2 PPO enzyme activity
Polyphenol oxidase activity steadily increased in 7
th
MAP at main and ratoon crop (Table 3). Main plot
treatments differed significantly at 5
th
and 7
th
MAP
growth stages. Significantly higher enzymatic activity
