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MATERIALS AND METHODS
MATERIALS AND METHODS
Fenugreek (Trigonella foenum-graecum L.) has autogamous white flowers, ovate
trifoliate leaves, branched stems, straight or slightly-curved pods, golden yellow seeds
and nodule bearing roots. Certified seeds of fenugreek (cultivar RMT-1) were
procured from Jawaharlal Nehru Krishi Vishwa Vidhyalaya (JNKVV), Jabalpur,
Madhya Pradesh, India.
Experiments were conducted during 2011-2012 and 2012-2013 under following set
up:
Laboratory experiments:
For laboratory experiments uniform and healthy seeds (ten seeds per petriplate) were
screened and surface sterilized using 0.01% mercuric chloride solution followed by
thorough washing with running water and finally with distilled water and placed in
petriplates lined with Whatman’s filter paper number 1 in two layers for germination.
Seeds were maintained at different concentrations of three different salts of potassium
i.e., 0.001 M, 0.01 M and 0.1 M of K2O, KCl and KNO₃ using distilled water as
control. Seedlings were put to analysis/ estimations seven days after sowing.
Germination percentage was recorded three days after sowing. Shoot/ Root length,
fresh/ dry weight, other growth parameters and activity of different enzymes were
worked out in seedlings (7 DAS old).
Pot experiments:
Separate pots were maintained for each treatment and each pot used was having
0.1281 m2 area. Nitrogen (urea) and phosphate (single super phosphate) were applied
at the rate of 60 kg ha-1 and 40 kg ha-1 respectively. Three salts of potassium i.e. K2O,
MATERIALS AND METHODS
KCl and KNO₃ were applied in three varying doses i.e., 20 kg ha-1, 40 kg ha-1 and 60
kg ha-1 respectively and control was maintained without supplementation of
potassium. Different growth, physiological and biochemical parameters were analysed
after twenty five (vegetative phase) and forty days (just before flowering) of seed
sowing.
Leaf Area:
Leaf area was measured following the gravimetric method (Shahane and Mungikar
1984 and Mungikar 1986). Leaf shape was drawn on a paper and the paper was cut
subsequently, and these paper leaves were weighed and area of the leaves was
calculated comparing it with the standard preweighed paper of known area.
Chlorophyll contents:
Chlorophyll contents in leaves were estimated following Arnon’s method (1949). 100
mg fresh leaf sample was homogenized in 80% acetone (80 ml acetone + 20 ml
distilled water to make up 100 ml) followed by centrifugation at 3000g for five
minutes; the volume was then made up to 5 ml with 80% acetone. Thereafter, optical
density was recorded at 480, 645 and 663 nm with spectrophotometer. Chlorophylls
were calculated using the formulae given hereunder:
Chlorophyll a (mg/gm/fr wt) = (0.0127 x A663) - (0.00269 x A645).
Chlorophyll b (mg/gm/fr wt) = (0.0229 x A645) - (0.00468 x A663).
Total Chlorophylls (mg/gm/fr wt) = (0.0202 x A645) + (0.00802 x A663).
Carotenoids (mg/gm/fr wt) were calculated following Kirk and Allen’s (1965) formula
and expressed in milli gram per gram fresh weight.
A480 + (0.114 x A663) - (0.638 x A 645).
MATERIALS AND METHODS
Nitrate reductase activity:
Nitrate reductase activity (in vivo) was determined following Srivastava (1974) as
adopted by Sharma and Agarwal (2002). 300 mg fresh plant sample was cut into small
pieces and 5 ml of reaction mixture [0.1M phosphate buffer maintained at 7.5 pH and
200 mM KNO3 and 0.5% n-propanol (v/v)] was added to it and subsequently
incubated in dark for 2 hours at 300C. After incubation 0.8 ml aliquote was taken and
0.4 ml of each 1% sulphanilamide (prepared in 3N HCl) and 0.2% 1-nephthylethylene
diamine dihydrochloride were added to it followed by thorough mixing and left for 20
minutes to develop colour. Optical density was recorded at 540 nm. Nitrate reductase
activity was calculated using standard curve of nitrite and expressed as µM NO2
produced h-1 gm-1 of the fresh weight.
Aminotransferase activity:
Aminotransferase activity was determined following method given by Reitman and
Frankel (1957). 100 mg sample was homogenized using pestle and mortor in 1.5 ml
chilled sodium-potassium buffer (pH 7). After centrifugation for 15 minutes at 4000g
the supernatant was collected. 0.05 ml of supernatant (enzyme extract) was added to
0.1 ml buffer substrate (for both alanine aminotransferase and aspartate
aminotransferase), mixed and incubated for 30 minutes (for alanine aminotransferase)
and 1 hour (for aspartate aminotransferase ) at 370 C in water bath. Thereafter, 0.1 ml
DNPH (prepared in 1N HCl) was added and resultant was allowed to stand for 20
minutes. Finally l ml sodium hydroxide was added and mixed. After five minutes
optical density was recorded at 540 nm. For blank water and for standard pyruvate was
MATERIALS AND METHODS
taken with 0.1 ml buffer substrate and 0.1 ml DNPH (2, 4-dinitrophenyl hydrazine),
making up the volume as in the case of test.
Ascorbic acid Content:
Ascorbic acid was determined following Omayl et al. (1979). 1 gm sample is
crushed in 5ml of 10% TCA followed by centrifugation at 3500g for twenty minutes.
Re-extracted and the volume was made up to 10ml after that 0.5 ml aliqote was taken
followed by addition of 1ml DTC reagent (prepared by 3gm DNPH+ 0.4gm thiourea
and 0.05gm CuSO4 in 100 ml of 9N H2SO4). Subsequently it was incubated for three
hours at 370C in water bath after that 0.750 ml of ice cold 65% H2SO4 was added and
left for thirty minutes and read at 520 nm in spectrophotometer. Ascorbic acid content
was calculated using standard curve of ascorbic acid and expressed as mg gm-1 fresh
weight.
Enzyme extraction:
Fresh plant material (100 mg) was homogenized using prechilled mortar and pestle in
2.0 ml extraction mixture (pH 7.5) containing 0.1 mM EDTA and 0.1% PVP.
Homogenate was centrifuged at 12,000 g for 15 minutes and supernatant was used for
enzyme assay. Entire process was carried out at around 4oC.
Superoxide dismutase (SOD) activity:
SOD (EC 1.15.1.1) was assayed following Dhindsa et al. (1981). The activity was
measured by the ability of enzyme extract to inhibit the photochemical reduction of
NBT. The assay mixture consisted of 1.5 ml reaction buffer (pH 7.8), 0.2 ml of
200mM methionine, 0.1 ml enzyme extract with equal amount of 1M Na2CO3,
2.25mM NBT (nitroblue tetrazolium chloride) solution, 3mM EDTA, 60µM riboflavin
MATERIALS AND METHODS
and 1.0 ml of double distilled water. Test tubes were incubated for 15 minutes under
light of 15 W inflorescent lamp. Blank A having components of the reaction mixture
along with enzyme extract were placed in dark. Blank B containing all the constituents
of reaction mixture except enzyme was placed in light along with the samples.
Reaction was terminated by switching off the light and samples were removed and
wrapped with black cloth. Blank A containing enzyme extract and maintained in dark
developed no colour. Absorbance of samples and blank B was recorded at 560 nm
against blank A. The difference in % reduction in colour between blank B and sample
was calculated. Reduction (50%) in the colour was considered as one unit of enzyme
activity and is expressed as enzyme unit (EU) mg-1 protein h-1.
Catalase (CAT) activity
CAT (H2O2; oxidoreductase; EC 1.11.1.6) activity was estimated following the
method given by Aebi (1984). Assay mixture contained 0.750 ml of 50mM potassium
phosphate buffer (pH 7) and 0.1 ml enzyme extract. Reaction was initiated by adding
0.1 ml of 100 mM H2O2. Disappearance of H2O2 was monitored by measuring a
decrease in absorbance at 240 nm for 2 minutes. Activity was calculated by using
Extinction Coefficient (ε) 0.036 mM-1 cm-1 and expressed in EU mg-1 protein h-1.
Guaiacol peroxidase:
Guaiacol peroxidase activity (EC 1.11.11.11) activity was estimated following Chance
and Maehly (1955). Assay mixture contained 1.0 ml of 100mM phosphate buffer (pH
7) and 0.05 ml of 10mM Guaiacol and 0.1 ml enzyme extract. Reaction was initiated
by adding 0.1 ml of 100mM H2O2 and guaiacol peroxidase activity was determined by
following the disappearance of H2O2 by observing a decrease in absorbance at 470 nm
MATERIALS AND METHODS
for 2 minutes. Activity was calculated using Extinction Coefficient (ε) 26.6 mM-1 cm-1
and expressed as (EU) mg-1 protein h-1.
Protein estimation:
Lowry et al. (1951) method was followed for protein estimation. 0.5 ml supernatant
(obtained after homogenization of samples) and was mixed with 20% TCA (tri-chloro
acetic acid) followed by keeping for 30 minutes and centrifugation at 5000 g for 15
minutes. Residue is dissolved in 1 ml NaOH (0.1N) and 0.1 ml of alkaline supernatant
was mixed with 1 ml of freshly prepared reagent C (50 ml of reagent A+1 ml of
reagent B; where reagent A is 20% Na2CO3 dissolved in 0.1N NaOH and reagent B is
0.5% CuSO4.5H2O in 1% sodium potassium tartarate) and left for 15 minutes at room
temperature. 1 ml Folin-Ciocalteau’s reagent (1N) was added to it and kept in dark for
30 minutes. Optical density was read at 660 nm and amount of protein determined
using a standard curve of Bovine Serum Albumin (BSA).
Total nitrogen:
Nitrogen estimation was done following micro-Kzeldahl’s method as suggested by
Jackson (1973) and modified by Iswaran and Marwaha (1980). 1g dried plant sample
was taken in a 100 ml Kzeldahl digestion flask and moistened with 5 ml distilled water
followed by the addition of concentrated H2SO4 (15 ml). Few drops of 1N KMnO4 are
added till pink colour appears. Thereafter, catalyst mixture (3gm K2SO4 + 0.3 gm
FeSO4.5H2O+ 0.15 gm CuSO4.5H2O) was added to it. The sample was then digested
at low flame for half an hour till the colour of mixture turns from brown to yellowish
green. Subsequently, flasks were cooled and contents were transferred to 100 ml
MATERIALS AND METHODS
volumetric flasks and the volumes made up to 100 ml with distilled water and
ammonia in the digested solution was then estimated using micro-Kzeldahl’s method.
Potassium, Sodium and Calcium:
The analysis was done flame photometrically as adopted by Tomar and Agarwal
(2013). 0.5 gm dried plant material was taken in a conical flask and digested in triacid
mixture (H2SO4+HNO3+HClO4 in 9:3:1 ratio). The colorless digested material was
filtered through Whatman filter paper No.1 into 100 ml volumetric flask making up
the total volume to 25 ml. 5 ml aliquot was then taken and made up to 25 ml with
distilled water and was read directly on digital flame photometer, employing Na, K
and Ca filters separately and percentage of Na, K and Ca was determined using
standard curves.
Phenols:
Phenols were estimated following the method of Malik and Singh (1980). 0.5 gm
powdered plant sample was homogenized in 80% ethanol (ten times volume).
Homogenate is centrifuged at 10,000 g for twenty minutes. Supernatant was saved and
residue was re-extracted in 80% ethanol (five times volume). Supernatant was
evaporated to dryness and dissolved in 5.0 ml distilled water. 0.1 ml of aliquot was
taken out and volume was made up to 2.0 ml with distilled water. Folin-Ciocalteau’s
reagent (0.5 ml, 1N) was then added and after three minutes 20% Na2CO3 (2.0 ml)
was added followed by keeping in boiling water bath for one minute. Phenols react
with phosphomolybdic acid in Folin-Ciocalteau’s reagent in alkaline medium and
produce a blue coloured complex. Absorbance was recorded at 650 nm against blank
MATERIALS AND METHODS
using spectrophotometer. Concentration of phenols was expressed in mg gm-1 dry
weight, equivalent to catechol.
Tannins:
Tannins were estimated according to method of Swain and Hills (1959). Powdered
plant sample (0.05 gm) was taken in test tubes and 7.5 ml distilled water was added to
it followed by keeping in boiling water bath. After 30 minutes it was cooled to room
temperature and centrifuged at 2000 g for 20 minutes. Supernatant was collected and
final volume made up to 10 ml adding distilled water. 0.1 ml of supernatant was mixed
with 1.9 ml of distilled water making it 2.0 ml. Folin-Denis reagent (1.0 ml) and 35%
Na2CO3 (2.0 ml) were added to it and kept in dark for 45 minutes at room temperature.
Absorbance was measured at 700 nm. Tannin content of samples was expressed as mg
gm-1 dry weight tannic acid equivalents and computation was done using standard
curve.
Phytic acid:
Phytic acid was estimated following method of Wilcox et al. (2000). Phytic acid is
extracted in 0.4mM HCl. 100 mg powdered sample was mixed thoroughly with
0.4mM HCl (1.0 ml) followed by centrifugation at 10,000 g for 20 minutes. 0.1 ml
supernatant was made up to 1 ml using distilled water. 1 ml colorimetric reagent (3M
H2SO4, 2.5% ammonium molybdate and 10% ascorbic acid mixed in equal volume
with two volume of distilled water) was added to it and kept at room temperature for
one hour. Optical density was measured against blank at 650 nm with
spectrophotometer. Concentration of phytic acid was calculated using K2HPO4 as
standard and expressed in mg gm-1 dry weight.
MATERIALS AND METHODS
Free amino acids:
Free amino acids were estimated following the method outlined by Sadasivam and
Manickam (2004). Plant sample (0.5gm) was ground in pestle and mortor adding 80%
ethanol (5ml) and centrifuged at 2000 g for 20 minutes and supernatant was collected.
Extraction was repeated once again using the residue and supernatant were pooled
together. Extract so prepared was used for the quantitative estimation of free amino
acids. Extract (0.1ml) was made up to 2 ml with distilled water and to this ninhydrin
(1ml) and later equal volume of diluent (equal volume of water and n-propanol) was
added to it. After 20 minutes, optical density was recorded at 570 nm against a blank
prepared taking 80% ethanol (0.1 ml) in place of the extract. Computation was done
using standards.
Ninhydrin solution was prepared by dissolving 160 mg of stannous chloride in 100 ml
0.2 M citrate buffer (pH 5), which was added to ninhydrin (4 gm) dissolved in 100 ml
methyl cellosolve (2 methoxy- ethanol) and kept for incubation for 20 minutes.
Free proline:
Free proline estimation was done following Bates et al. (1973). Extraction was done in
3% sulphosalicylic acid followed by centrifugation at 3000g for ten minutes. Extract
was subsequently reacted with acid ninhydrin (prepared by adding 2.5 gm ninhydrin to
60 ml glacial acetic acid mixed with 40 ml 6 M phosphoric acid) followed by the
addition of equal amount of distilled water and keeping the resultant at 1000C for one
hour in oven. The reaction was terminated at cooling temperature. Thereafter, free
proline was separated using toluene in a separating funnel and optical density was
MATERIALS AND METHODS
recorded at 520 nm in spectrophotometer using toluene as blank and calculations were
done employing proline standards and expressed in µM gm-1 dry weight.
Total free sugars:
Anthrone method was employed for the determination of free sugars based on Fong et
al. (1953) and Jain and Guruprasad (1989). Plant sample was boiled in 80% ethyl
alcohol, followed by centrifugation at 5000 g for ten minutes. The residue was again
crushed in 80% ethyl alcohol and then centrifuged. The supernatant was pooled
together. The alcoholic extract (1ml) so prepared was evaporated to 0.2 ml in water
bath and the volume was made up to 1 ml using distilled water. 1N HCl (1ml) was
added and the tubes were kept in boiling water bath for 40 minutes for hydrolysis. 4
ml of 0.2% anthrone (prepared in chilled concentrated H2SO4) was then put to 0.5 ml
of hydrolysed extract and the tubes were subsequently kept in boiling water bath for
10 minutes. Later, it was brought to room temperature and optical density was
recorded at 620 nm. Glucose standards were used for the computation of total free
sugars.
Starch:
Starch was determined using anthrone method (Sadasivam and Manickam, 2004) as
followed by Pandey (2000). Residue (pellet) of the sample from which free sugars
were extracted was dried in water bath, subsequently 2 ml distilled water was added
followed by keeping in water bath for 15 minutes. Thereafter, cooled and 2 ml 9.2N
Perchloric acid was added and stirred for 15 minutes, followed by centrifugation at
5000 g for ten minutes. Supernatant was collected in 50 ml volumetric flask. 2 ml of
4.6N Perchloric acid was added to the residue and stirred for 15 minutes then
MATERIALS AND METHODS
centrifuged. Finally supernatants were combined and volume made up to 10 ml with
distilled water. 0.1 ml aliquot was taken and volume made up to 1 ml with distilled
water. 4 ml anthrone reagent was added to it and the tubes were then kept in a boiling
water bath for 10 minutes followed by cooling on ice. Optical density was recorded at
630 nm after bringing it to room temperature. Computation was done using standard
curve.
Chloride:
One gram dry sample was boiled in distilled water (100 ml) for 30 minutes. After
cooling the extract was filtered and was titrated against 0.1N AgNO3 solution till a
permanent brick red precipitate persists (Eaton et al. 2005). Potassium chromate (5%)
(K2CrO4) is used as indicator.