MATERIALS AND METHODS
Experiments were conducted during the year 2010-2013 to study the effects of
micronutrients (Zn, Cu and Fe) disorder in wheat (Triticum aestivum L.) plants on their
growth (shoot length, fresh and dry matter production), biochemical responses
(chlorophyll a, b, and total chlorophyll and protein content) and some antioxidative
enzymes activity (peroxidase and catalase), reproductive yield and grain quality (grain
protein content and grain Zn, Cu and Fe contents). Standard methods were used to
conduct the experimental works.
Pot culture, Sand culture as well as field observation experiments were conducted
to find out the objectives of the work. In the field observation, five different locations
Lucknow (Jankipuram) L1, Barabanki (Murliganj) L2, Khalilabad (Gorayabhar) L3,
Lucknow (Mohanlalganj) L4, Lucknow (Bakshitalab) L5 of Uttar Pradesh state (India)
were selected for the observation and data collection under field conditions. The soils of
Lucknow district were collected for pot experiments to find out the objectives of work.
The details of materials and methods adopted for the experimental work are described in
this chapter.
3.1 Cleaning of glasswares
The glasswares used for the analytical work was routinely cleaned first with
detergent, and then by boiling 6N HCL, followed by thorough washing with running tap
water. If required, chromic acid was also used especially for the pipettes, burettes and
spectrophotometer cuvettes. The acid cleaned glass wares were rinsed thrice with GDW
and dried in oven before use.
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3.2 Collection of soil
Soil samples from Lucknow district in Uttar Pradesh state (India) were collected
from unpolluted area, surface soil (0–25 cm) was collected in bulk from an area of 20×20
m2 having level surface and soil of uniform texture. Before collection, it was ensured that
the selected area had received no manure or fertilizer and does not have any industrial
contamination, or any major road intersections that may subsequently contribute to
automobile pollution. After scraping top 1cm soil layer to remove surface vegetation, the
areas were dug with a shovel up to 25 cm depth. The collected soil was allowed to dry for a
day. The large soil aggregates were broken into smaller pieces. The soil was thoroughly
mixed, filled in alkathene lined gunny bags and transported to the laboratory. Soil of
different study locations in Uttar Pradesh state (India) were also collected before the
sowing of crop.
3.3 Preparation of soil
The bulk soil samples collected from the uncontaminated area of Lucknow and
different study locations in Uttar Pradesh state (India) were air dried on alkathene sheet
in shade for 23 days. After air drying, the soil clods were broken with clean wooden
mallet and made free from the plant remains. The soil was thoroughly pulverized and
stored in alkathene lined jute gunny bags. Composite soil were analysed for important
physio-chemical properties such as texture, pH, electrical conductivity, bulk density,
organic matter contents, CaCO3 content and some available nutrient elements (Zn,
Cu, and Fe) before the experiment.
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Map showing five different study area in Uttar Pradesh State (India)
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Table 3.1 Physico-chemical properties of soil, used for the experiments.
Soil Properties
Field locations observed
Lucknow
Barabanki
Khalilabad
MohanlalGanj
Bakshitalab
Clay loam
Clay loam
Sandy loam
Sandy loam
Bulk density (g/cc)
Sandy
loam
1.50±0.05
1.78±0.01
1.87±10.02
1.70±0.01
1.40±0.03
pH
7.90±0.30
6.20±0.10
6.10±0.20
7.84±0.10
7.20±0.40
E.C (dS m-1)
0.31±0.01
0.52±0.02
0.49±0.04
0.46±0.04
0.37±0.01
CaCO3 (%)
1.97±0.10
0.98±0.09
1.08±0.10
1.77±0.20
1.97±0.10
O.M. (%)
0.83±0.10
2.64±0.1
2.18±0.10
1.48±0.10
0.38±0.10
Zn (mg kg-1)
0.48±0.02
0.75±0.03
0.62±0.01
0.69±0.01
0.52±0.04
Cu (mg kg-1)
0.25±0.03
0.38±0.06
0.32±0.01
0.28±0.02
0.30±0.01
Fe (mg kg-1)
2.38±0.01
4.80±0.04
3.95±0.08
2.89±0.03
2.62±0.01
Texture
± - S.E. value (n=3).
3.4 Soil Analysis
Texture
The soil sample (100 g) was sieved through a set of sieves of varying pore size
like 10, 22, 44, 60, 85 and 120 mm. on a plastic sheet. After sieving the soil retained on
every sieve was collected. The different fraction of the sieved sample was weighed,
separately. The proportional composition of various soil separates on weight basis was
determined to find the textural class of the soil sample. The soil separates from 10 and 22
mm. sieves constituent the sand fraction of soil, Similarly separates from 44 and 60 mm.
sieves constitute the silt fraction and that of 85 and 120 mm. sieves together with sieved
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soil constitute the clay fraction of soil. Depending upon the proportionate percentage
composition of sand, silt and clay, the texture of soil sample was determined on
comparing the percentage fractions with the histograms of soil texture.
pH
10 g soil sample was taken in a clean and dry test tube. To this was added a pinch
of barium sulphate and 25 ml fresh distilled water (free of CO2). It was shaken vigorously
for a minute and then intermittently for 5 minutes. The test tubes were left as such for 1
hour to allow the soil solids to settle down. A small amount of clear soil supernatant was
decanted in a watch glass. Its pH was tested by pH meter. Then suspension take in
contact of electrode of pH meter and then the reading of pH-meter was taken.
Organic matter
The organic matter content was determined in the soil by the method of Walkley
and Black (1934). To 0.5 g of soil sample taken in a 250 ml conical flask was added
10 ml of 1N K2Cr2O7 solution and mixed by swirling the flask. This was followed by
addition of 20 ml concentrated H2SO4. The contents were mixed by gentle rotation for 1
min to ensure complete contact of the reagent with the soil and the mixture was allowed
to stand for 30 min. This was followed by sequential addition of 70 ml glass distilled
water, 10 ml 85 % (w/v) H3PO4, 0.02 g NaF and 1 ml diphenylamine indicator. The
mixture was titrated against 0.5N ferrous ammonium sulphate and the change in colour
from bluish black to brilliant green was taken as the end point. A standard blank without
soil was run in the same way. The organic matter content in the soil samples was
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expressed as in percentage (%).The organic matter content of the soil was determined as
percent organic matter calculated by the formula given below:
Organic matter % = 10 (1-T/S) × 1.34
Where, T = Titrate value of the soil sample
S = Titrate value of the blank
Calcium carbonate
The calcium carbonate content was determined in soil by the method of Piper
(1942). Five g of air dried soil was taken in a 250 ml conical flask. To this was added 100
ml of 0.5 N HCl and the contents were mixed thoroughly. After standing for an hour, 20
ml aliquot was drawn from the supernatant and back titrated against the pre-standardized
0.5 N NaOH using bromo-thymol blue as indicator. The change in colour from orange to
sky blue was taken as the end point. The CaCO3 content in the soil samples was
expressed as in percentage (%).
Electrical conductivity
1:2.5 soil suspension was prepared by taking 20 g soil in 100 ml aerated distilled
water and shaking it mechanically for 2 hours. The conductivity of the soil suspension
was measured on conductivity meter, a century analysis kit.
Bulk density
A pre-weighted 50 cc glass bottle was filled with dried soil and the bottle was
tapped 15 to 20 times. The weight of the soil was determined by subtracting the weight of
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the empty bottle. The volume of the water occupied in the sample boule was measured
and bulk density was calculated as follows:
Bulk density =
𝑊2 − 𝑊1
𝑔 𝑐𝑐 −1
𝑉
Where:
W1 = g weight of empty bottle (50 cc)
W2 = g weight of bottle +soil
V = volume of water filling the bottle
DTPA extractable trace metals
Trace metals was estimated in soil by the method of (Lindsay and Norvel, 1978).
Weighed 20 g of air dried soil in a 125 ml Erlenmeyer flask. Add 40 ml of DTPA
extracting solution. Covered each flask with stretchable parafilm and secure up rights on
a reciprocating shaker. Shake at a speed of about 176 cycles/minutes for 2 hours. It was
filtered through Whatman filter paper number 42. Filterated was analyzed by atomic
absorption spectrophotometer (Perkin-Elmer-250).
Iron (Fe)
100 g soil was transferred to a 100 ml conical flask. To it was added 50 ml
ammonium acetate pH 4.8 in there fractions. The flask was gently shaken and contents
were transferred to a Buchner funnel fitted with a vacuum pump for rapid filtration.
Whatman filter paper No. 42 was used for filtration. 40 ml of the filtrate was taken in a
100 ml conical flask and evaporated to dryness on a hot plate. The solution was
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evaporated to dryness and digested using 5 ml aqua regia and 1 ml perchloric acid. The
digest was made to 10 ml with distilled water. Total 10 ml aliquot was transferred to in a
50 ml test tube for estimation of Fe by the method of Humphries (1956). To this, 10 ml
sodium acetate pH 4.8 was added followed by 10 ml of freshly prepared mixed reagent.
The mixed reagent was prepared by mixing 0.25% orthophenanthroline, 10%
hydroxylamine, hydrochloride and ammonium acetate pH 4.6 solution in a ratio of
(2:1:2). The test tube was heated on a boiling water bath for half an hour and then cooled.
The final volume was made to 25 ml. The optical density was measured in a Systronics
Colorimeter using blue green filter.
3.5 Pot culture
For conducting the clay pot culture experiments to study the effect of
micronutrients (Zn, Cu and iron) disorder on growth, biochemical parameters,
reproductive yield and grain quality of wheat plants, the soil was taken from
Mohanlalganj, Lucknow. Before collection of soil, it was ensured that the selected sites
had not received any manure and fertilizer for last 5 years. The surface soil upto 35 to 50
cm was collected in bulk from an area having soil of uniform texture. The experiment
was conducted in earthen pots (size 25x30 cm), filled with soil. Pot culture experiments
were performed on ten healthy seeds of wheat (Triticum aestivum L.) Ver. H D 2851 and
H D 2858. For pot culture experiment various doses of Zn and Cu were amended in soil
to study the effect of Zn and Cu on growth, biochemical constituents, reproductive yield
and grain quality of wheat plants. In experiment 4.1A five doses of Cu (control, 1.0, 5.0,
10.0 and 25.0 mg Kg-1) were amended in soil and in 4.1B five doses of Zn (control, 5.0,
15.0, 25.0 and 50.0 mg Kg-1) were amended in soil. In experiment 4.2 studies were
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conducted on the effect of Cu and Zn interaction in soil on various levels of Cu and Zn.
In 4.2A experiment soil amended with a high dose of Cu 50 mg Kg-1alone and mixed
with various levels of and Zn (0.0, 25.0, 50.0 and 100 mg Kg-1) and In 4.2B experiment
a high dose of Zn 100 mg Kg-1 alone and mixed with various levels of Cu ( 0.0, 5.0, 25.0
and 50.0 mg Kg-1) in soil.
3.6 Field experiment
The studies were carried out on the agricultural fields of five different locations of
Uttar Pradesh (India), where wheat was grown. Randomized sampling system was used
for this study. In this five agricultural fields occupied with different variety of wheat
plant were selected to determine the effect of various soil conditions on growth (length
and biomass), reproductive yield and grain quality (protein, Zn, Cu and Fe contents) of
wheat. All the observations regarding growth and reproductive yield of crop were taken
time to time and at the time of harvesting 1x1 m2 areas were harvested first for data
collection on growth, reproductive yield and grains quality of wheat plants.
3.7 Sand culture technique
To study the effect of micronutrients disorder on growth, biochemical parameters,
reproductive yield and grain quality of wheat plants, plants were grown in refined sand at
an ambient temperature during the course of study (minimum 50 C to maximum 300 C) in
a glass house. In sand culture experiment the technique for growing plants in sand was
essentially the same as developed by Hewitt (1952) and adapted for under Indian
conditions according to Agarwala and Sharma (1961). White silica sand used for culture
studies were obtained from sand stone beds of Shankargarh, Allahabad, Uttar Pradesh.
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The sand was sieved and the fraction retained between 20 and 60 mesh sieves,
particle size 0.25 to 0.84 mm, was used to carry out the experiments. The sand was first
water washed and then purified against macro and micronutrients by treatment with hot
hydrochloric acid (HCl). The water washing of the sand freed it from clay, silt and other
lighter particles that were removed by upward displacement with water. The water
washed sand was treated with a mixture of hot 17 % hydrochloric acid and 1 % oxalic
acid (Hewitt, 1952). After acid treatment sand was washed thoroughly with water. Before
starting experiment the acid washed sand was leached with 4 mM solution of calcium
nitrate purified by phosphate adsorption (Hewitt, 1952), to bring down its pH to near
neutral pH (around 6.5). The pots used for growing the plants were of high quality
polyethylene and had a central drainage hole at the bottom. This was covered with an
inverted watch glass with a thin felt of glass wool underneath its rim. This retained the
sand in the pots while allowing free drainage of nutrients and aeration of roots
Nutrient solution
The basal nutrient solution was prepared by the method given by Hewitt (1966).
Macro and micronutrients solution were prepared by using AR (analytical regent) grade
chemical regents. Calcium nitrate was prepared by the method of Hewitt (1966) Iron
ethylene diamine tetra acetate (Fe-EDTA) was prepared by the method of Jacobson
(1951). First of all the stock solution of micronutrients including potassium nitrate,
magnesium sulphate and sodium hydrogen phosphate were prepared. For making basal
nutrient solution, Fe-EDTA and stock macronutrients were diluted 1000 times by adding
distilled water.
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Table 3.2 Composition of Macro-Micronutrients in the basal nutrient solution prepared
as follows:
Macronutrients
Form in which supplied
Level of supply (m. equiv./L)
Potassium(K)
KNO3
4
Calcium (Ca)
Ca(NO3)2
8
Nitrogen (N)
Nitrate of Ca&K
12
Phosphorus(P)
NaH2PO4.H2O
4
Magnesium(Mg)
MgSO4.&H2O
4
Sulphur(S)
Sulphate of magnesium
4
Sodium(Na)
NaH2PO4.2H2O
4
Micronutrients
Form in which supplied
Concentration (ppm)
Iron(Fe)
Fe-EDTA
5.600
Manganese(Mg)
MnSO4.H2O
0.550
Boron(B)
H3BO3
0.370
Copper(Cu)
CuSO4.5H2O
0.064
Zinc(Zn)
ZnSO4.2H2O
0.065
Molybdenum(Mo)
Na2Mo4.2H2O
0.020
Chloride(Cl)
NaCl
3.550
Cobalt(Co)
CoSO4.7H2O
0.006
Nickel(Ni)
NiSO4.7H2O
0.006
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3.8 Plant analysis
Growth observations
The plants were sampled for determination of fresh and dry weight at three stages
of growth, development stage, after clear appearance of visible symptoms and on
reproductive growth stages. For determination of fresh weight plant samples were
collected, thoroughly washed with running tap water and rinsed two or three times with
distilled water to remove surface contamination. After rinsing the plant samples were
gently blotted to wipe out the absorbed water and separated into different plant parts and
weighed quickly on balance to avoid excessive loss of water by evaporation. The dry
weight of plants was determined after drying these fresh samples in pre heated oven at
700 C for 48 hours. The oven dried material was transferred to desiccators and weighed
after cooling at room temperature. The dried plant material was also utilized for
elemental analysis after wet digestion. Among the growth parameters, plant height and
number of tillers were also measured at 15, 30 and 45 DAT. Dry matter production per
plant was recorded at the time of harvesting. Plant height was measured from the base of
the plant to the growing tip by holding the plant vertically. The mean plant height was
expressed in centimeter (cm). Total number of tillers was counted in randomly selected
three plants at 15, 30 and 45 DAT and average was worked out to obtain number of
branches per plant.
Reproductive yield
The main component of reproductive yield in wheat plant is number of
inflorescence, length of inflorescence, number of grain per inflorescence and grain yield.
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Number and length of inflorescence and number of grain per inflorescence, was
calculated from three selected plants in each treatment. 100 grain weight of wheat was
calculated by harvesting mature, inflorescence and at this stage, 100 grains were selected
randomly from each treatment and their weight was recorded in gram (g).
Tissue concentration of metals
Oven dried plant material samples (1g) were digested in concentrated HNO3: HCl
V/V (3:1) till a clear solution was obtained, know final volume was made in volumetric
flask by adding double distilled water and estimated by using atomic absorption
spectrophotometer (AAS Parkin Elmer Lambda 40) at 228.8nm detection. Metal
concentrations were calculated by the formula given below:
Metal concentration (µg g-1dw) = XV / W
Where:
X= reading in ppm on AAS.
V= Final volume (ml).
W= Dry weight of the plant material (g).
3.9 Biomolecules
Pigments
Chlorophyll contents were measured by the method of Lichtenthaler (1987). In
this method 100 mg of fresh leaf tissue in 10 ml acetone (80%) was homogenized by
using glass pestle-mortal along with a pinch of calcium carbonate (CaCO3) to avoid
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oxidation. Thereafter, the samples were centrifuged at 5000 rpm for 10 minutes. In one
test tube 80% acetone solution was also taken for the reading of blank. The supernatant
was observed in spectrophotometer (AAs Perkin Elmer Lambda 40) at the wavelength
645, 652 and 663nm for estimation of chlorophyll a, chlorophyll b and total chlorophyll,
respectively. The units of all biomolecules were expressed in mg/g fresh weight of tissue.
Carotenoids content were calculated on leaf fresh weight basis according to formula
given by Duxbury and Yentsch (1956) and the results were expressed on fresh weight
basis in mg g-1.
Pigments content were calculated as follows:
Chlorophyll a (mg g-1fr. wt.) = [12.7(A663)-2.69(A645)] x V/1000xWl (g).
Chlorophyll b (mg g-1fr. wt.) = [22.9(A645)-4.68(A663)] x V/1000Xw
Total Chlorophyll (mg g-1fr. wt.) = [20.2(A645)-8.02(A663)] x V/1000xW
Where:
A663 and A645 = Absorbance at these wavelength
V
= Final extract volume (ml)
W
= Weight of sample (g)
Carotenoid (mg g-1fw) = [7.6(A480)-1.49(A510)] x V/1000xW
Where:
A480 and A510 = Absorbance at these wavelength
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V
= Final extract volume (ml)
W
= Weight of sample (g)
Protein content
Protein content was estimated by the method of Lowry et al. (1951). Four
reagents (A-D) were used in this process.
A.
Sodium potassium tratarte (2%)
B.
Copper sulphate (1%)
C.
Sodium carbonate (2%) in 0.1N NaOH
D.
Folin and ciocalteu’s phenol reagent diluted in 1:2 ratios.
About 500 mg of test plants were crushed in 5ml of 10% trichloro acetic acid and
centrifuged at 10000 rpm for 10 minutes. After decanting the supernatant, pellets were
washed with 5ml of 1N NaOH twice, again centrifuged in 5ml of 1N NaOH and final
supernatant was collected. Reagent A (50 ml) and B (1ml) were added to reagent C to
make 100ml. 5ml of above (A+B+C) solution was added to final supernatant (0.5ml) and
kept for 10-15minutes at 300 C. Reagent D (0.5ml) was finally added and thoroughly
mixed. After 45 minutes, the absorbance was recorded at 750 nm. Bovine serum albumin
(sigma) was used as standard.
Protein content calculated as follows:
Δ O.D. = O.D. Sample – O.D. blank
Protein = Δ O.D. x 5 x 158.43 μg/g fresh weight tissue.
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Catalase activity
Catalase activity was assayed following to the method prescribed by Euller and
Josephson (1927) with some recent modifications for the estimation of catalase activity
by Bisht (1978). 5% extract of plant tissue was prepared. The catalase activity was
assayed by taking two test tubes for each sample, one for catalase activity and the other
for its blank. In each test tube 2ml citrate phosphate buffer (pH 7.0) was added then 2ml
0.5% H2O2, 2ml D.W., 2ml tissue extract was added in a series and incubated for 10
minutes. Thereafter, the reaction was stopped by adding 2ml 4N H2SO4 and thus was
done in the case of sample showing catalase activity and in the case of blank, H2SO4 was
added before addition of H2O2, finally titrated against 0.01N KMnO4.
Catalase activity was calculated as followes:
Blank – Sample × 40
Activity was measured in terms of ml H2O2 hydrolyzed g-1 fresh weight.
Peroxidase activity
Peroxidase activity was estimated by using the method of Luck (1963). For the
estimation, 2.5% extract was prepared. In test tube, 2ml citrate phosphate buffer (pH 6.0)
was added, to this 1ml 0.5% H2O2 and further 1ml extract was added to the reaction
mixture and then incubated for 10minutes. To stop the reaction 2ml of 4NH2SO4 was
added. In the case of blank H2SO4 was added before using 1ml extract to the reaction
mixture thus no reaction takes place in the mixture. Then, the mixture was centrifuged at
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40 C at 500 rpm for 10 minutes and optical density was measured with the help of
spectrophotometer at 485 nm.
Peroxidase activity was expressed in terms of ml H2O2 hydrolyzed per gm fresh
weight of tissue, calculated as follows:
Δ O.D. = Sample – Blank × 4
Activity was measured in terms of ml H2O2 hydrolyzed g-1 fresh weight.
3.10 Test plants
Wheat (Triticum aestivum L.) belongs to family poaceae. Wheat is the most
important food crop of the world and it is extensively cultivated in most parts of the
world. In India wheat is grown as Rabi crop. It is sown from early October to late
November. Wheat used as a main source of food all over the world. It is the staple food
of India and meets the major dietary requirements. Wheat occupies first position by
covering 68 % of the total cropped area in India; wheat is adapted to a variety of climate.
Wheat is an annual herb with 0.6-1.5m high culms, which are differentiated into nodes
and internodes. The roots formed in the seedling stage are ephemeral and are soon
replaced by adventitious fibrous roots. Grain is a dry, one-seeded, indehiscent fruit,
known as caryopsis. The endosperm makes up to about 82% of the grain by weight and is
delimited by aleurone layer which is rich in proteins and minerals.
3.11 Statistical analysis
The significant difference between the tested and the control sample was analyzed by
taking least significant difference (LSD). * Value significant at P<0.05 and ** value
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significant at P<0.01 levels. The significant values of all the parameter were mean of 3
measurements each. The following statistical formulas were used for the analysis of the
data obtained in present studies.
(I)
Mean or X
X=∑X/n
(II)
Standard Error (SE)
SE=SD/√n
(III)
Standard Deviation (SD)
SD=(X1-X)/n
Where:
X1=individual values
X=arithmetic mean of individual
values
n= number of observations
(IV)
LSD value
LSD= tn-2 √2*MSwithin/n = tn-2*q’
Where:
MS = Mean square
q = Studentized range
n = Treatment
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