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Materials and Methods 31

MATERIALS AND METHODS

This chapter deals with the description of material used for the study,

experimentations performed and methodologies employed for the determination of various

traits during the course of investigation.

3.1 Plant Material

Chickpea (Cicer arietinum L.) genotypes were chosen as the experimental model for

the present study. Healthy and authentic seeds of 21 C. arietinum genotypes denominated by

accession numbers namely 1C269810, 1C269831, 1C269843, IC269848, IC269814,

IC269819, IC269862, IC269837, IC269205, IC269875, IC269055, IC269853, IC269803,

IC269867, IC269850, IC269834, IC269812, IC269870, IC269794, IC269817 and IC269845

were obtained from National Bureau of Plant Genetic Resources (NBPGR), Indian

Agricultural Research Institute (IARI), New Delhi, India (Table 1).

3.2 Botanical Description

The scientific classification of C. arietinum is summarized as follows: Kingdom –

Plantae, Division – Magnoliophyta, Class – Magnoliopsida, Order – Fabales, Family –

Fabaceae, Sub-family – Papilionaceae, Genus – Cicer, Species – arietinum.

Cicer arietinum is an annual cool-season grain legume, commonly known as

bengalgram, gram, chana, kadle etc. It is an erect, 12-50(-100) cm tall, herbaceous plant,

which branches from the base. The plant is mostly covered with glandular or non-glandular

hairs but some genotypes do not possess hair. There are two types of chickpea, depending on

seed color, shape, and size. Kabuli type, the seeds of this type are large, round or ram head,

and cream-colored. The plants are medium to tall (up to one m) in height, with large leaflets

and white flowers, and contain no anthocyanin pigmentation. The second type is Desi type,

which has small seeds, angular in shape. The seed colour varies from cream, black, brown,

yellow to green. There are 2-3 ovules per pod but on an average 1-2 seeds per pod are

produced. The plants are short, more prostrate with small leaflets and purplish flowers, and

contain anthocyanin pigmentation. Chickpea plants have a strong taproot system with 3 or 4

rows of lateral roots. Chickpea roots bear Rhizobium nodules and play a vital role in

sustaining long-term productivity of soil through biological nitrogen fixation. Chickpea

leaves are petiolate, compound, and uniimparipinnate (pseudoimparipinnate). The solitary


flowers are borne in an axillary raceme. Chickpea flowers are complete and bisexual, and

have papilionaceous corolla. It is a highly self-pollinated specie because of its cleistogamous

flower.

3.3 Habitat and Cultivation

Chickpea, being a cool-season plant, grows best if daytime temperatures are between

21 and 29°C and night temperatures are between 18 and 21°C. It is a relatively drought-

tolerant crop. The long taproot allows the plant to use water to a greater depth than other

pulse crops. In the absence of disease, chickpea performs best when there is 8 to 12 inches of

rainfall during the growing season, and when it is cropped on soils that are well drained.

Chickpea (Cicer arietinum L.) is cultivated mainly in Algeria, Ethiopia, Iran, India Mexico,

Morocco, Myanmar, Pakistan, Spain, Syria, Tanzania, Tunisia and Turkey. It is grown in

tropical, sub-tropical and temperate regions. Kabuli type is grown in temperate regions, while

the desi type chickpea is grown in the semi-arid tropics. In India, it is extensively cultivated

as a winter crop, since it thrives well under receding soil-moisture condition. In India, mid

October to mid November is the ideal period for sowing chickpea. It is grown on different

types of soil ranging from sandy to sandy loams to deep black cotton soils. The best soils for

chickpea growth are deep loams or silty clay loams devoid of soluble salts.

3.4 Seed zinc analysis

3.4.1 Sample preparation

Seeds were ground to a fine powder with mixer grinder and sieved to <1 mm. Five ml

of concentrated nitric acid was added to 250 mg of powder sample and kept for overnight.

The next day, 5 ml of triacid mixture of concentrated nitric, perchloric and sulphuric acids in

40:4:1 ratio was added to it and then digested at high temperature (up to 205ºC) in microwave

digester. After digestion, the sample was allowed to cool and the volume was made up to 25

ml, using deionized water, and further dilutions were made if the concentration of the

solution was too high. These dilutions were used for zinc estimation.

3.4.2 Estimation

Zinc content was estimated in the sample by using atomic absorption

spectrophotometer (AAS ZEEnit 65, fitted with graphite tube of Wall type, Analytikjena,

Germany).


Standard curve preparation: a stock solution of 1000 ppm zinc was prepared by using

ZnSO4. From this stock solution 20,40,60,80 and 100 ppm concentrations were prepared and

further diluted with deionized water and resultant solutions were fed to AAS and their

absorbance were recorded. A standard graph was constructed by plotting concentration on X-

axis and absorbance on Y-axis; this standard curve was further used for estimating zinc

content in the sample. The seed zinc content was calculated using the following formula and

was expressed in µg per seed.

Average ppm × volume of digested sample × vol. made up

106

× weight of the sample × Aliquot taken

Table 2. Seed Zn content (µg/seed) of 21 chickpea genotypes (with code and accession

numbers) used in the present study.

Code no. Accession no. Seed Zn content Code no. Accession no. Seed Zn content

G1 1C269810 4.067 G12 IC269853 3.84

G2 1C269831 5.523 G13 IC269803 3.953

G3 1C269843 5.73 G14 IC269867 4.077

G4 IC269848 5.523 G15 IC269850 4.007

G5 IC269814 4.77 G16 IC269834 3.827

G6 IC269819 5.203 G17 IC269812 4.813

G7 IC269862 5.237 G18 IC269870 5.167

G8 IC269837 5.157 G19 IC269794 5.01

G9 IC269205 6.547 G20 IC269817 5.333

G10 IC269875 4.757 G21 IC269845 4.813

G11 IC269055 5.627

Values are the means of three replicates.

Zinc =


3.5 Experimental Set-Up

To accomplish the objectives set-out in the current work, the following five major

experiments were carried out according to standardized protocols. Experimental procedures

followed during each experiment are detailed below.

3.5.1 Experiment 1

The aim of this experiment was to screen zinc (Zn) accumulation variability in C.

arietinum genotypes under Zn-deficient (0.01 mg ZnSO4 L-1

nutrient solution) condition.

Under sand culture and using a randomized block design, the experimental set-up consisted

of 21 genotypes, 3 replications and 2 treatments, using 126 plastic pots (7.5 cm x 10 cm)

filled with 500 g of autoclaved acid-washed sand (Hewitt, 1966).

Ten equal-sized seeds of each of the selected genotypes (Table 1) were sterilized with

0.1% mercuric chloride for 5 min and then rinsed vigorously with deionized water and

germinated in the dark on fine sand moistened with deionized water. After one week, 5

uniformly germinated seeds were sown at the same depth (1.5 cm) in sand-filled plastic pots,

supplied with modified Hoagland’s nutrient solution (Peterson, 1969). The pots were lined

with polyethylene plastic film to avoid contamination. Two Zn concentrations (0.01 and 0.5

mg ZnSO4/L nutrient solution) were selected to provide the deficient and optimal Zn levels

respectively. The nutrient solution was prepared from the analytical grade chemicals. The pH

of the nutrient solution was adjusted to 5.6 ± 0.2 immediately prior to use, with 0.1 M KOH,

and iron (Fe) was added in chelate form rather than as an inorganic iron in order to prevent

precipitation. There were three replicates per treatment, each replicate consisting of three

plants (nine plants/ treatment). A 200 ml of the nutrient solution was applied to the root zone

every two days under laboratory conditions. Temperature was controlled using heaters and

fans, keeping day temperature in the range of 20-30ºC and night temperature above 15ºC,

whereas light intensity was maintained at 1500 lux for 12 h a day, with 16–60% relative

humidity. The pots were kept on benches in a randomized block design. Plants were thinned

after a week’s interval to keep one plant per pot. Harvesting was done after 30 days of

seedling transplant (early growth stage). Roots and shoots were separated, washed with

deionized water, and oven-dried at 70ºC for 48 h, before estimating the biomass (g/plant) by

weighing the dried material. For estimation of Zn concentration, dried root and shoot samples

were prepared and analyzed on atomic absorption spectrophotometer (AAS ZEEnit65),


following the methods described in section 3.4.1 and 3.4.2. Plants were analyzed for their Zn-

accumulating capacity and relative shoot growth based on shoots dry matter production.

Table 3. Experimental design ´Exp. 1´.

No. of genotypes

selected

Form of zinc Treatment

(mg/ L nutrient solution)

Replicates

21 ZnSO4.7H2O Zn0.01 Zn0.5 3

0.01 0.5

3.5.2 Experiment 2

Experiment 2 was conducted on the basis of the findings of Experiment 1. The aim of

this experiment was to evaluate Zn-accumulation, Zn-efficiency, dry matter production, and

yield under low (0.01 mg ZnSO4/L nutrient solution) and adequate (0.5 mg ZnSO4/L nutrient

solution) Zn conditions.

During the Rabi season (October, 2011- February, 2012) at Jamia Hamdard, New

Delhi, in sand culture under green-house conditions, three and four genotypes exhibiting

respectively high and low Zn-accumulating capacity were selected for evaluation of their

performance based on vegetative growth and grain yield was investigated in these genotypes.

The average day/night temperatures were 33/20 ± 2°C, with relative humidity in the range of

60-70%, during the cropping cycle. The genotypes were laid in randomized block design with

three replications each. Ten uniform-sized grains were surface- sterilized and germinated on

moistened sand, following the procedure described above. After one week, 5 uniformly

germinated seedlings were transferred to pots filled with acid-washed autoclaved sand

supplied with a modified Hoagland’s nutrient solution (Peterson, 1969). The first sampling

was done 40 days after transplant (DAT) (pre-flowering stage) and two plants were

maintained in each pot till 120 DAT (harvesting stage). Dry shoot-root weight and ratio, Zn-

concentration and -efficiency, and the C, N, S and protein percentage in leaves were

estimated. Yield and its attributes were calculated at harvest. The genotypes were ranked with

respect to dry matter production, Zn content, grain yield (g/plant) and zinc efficiency (ZE).

Zn uptake by root, relative Zn transport (%) to shoot and zinc accumulation by these

genotypes were determined.


Table 4. Experimental design for ´Exp. 2´.

Type of Genotypes Form of zinc

Treatment

(mg/ L nutrient solution)

Replicates

Zn0.01 Zn0.5

Higher zinc accumulating

(G5, G8, G20)

ZnSO4.7H2O 0.01 0.5 3

Lower zinc accumulating

(G2, G14, G18, G19)

ZnSO4.7H2O 0.01 0.5 3

3.5.3 Experiment 3

Experiment 3 was conducted on the basis of the findings of Experiments 2.

Considering C. arietinum genotypes exhibiting the highest Zn-accumulation (HZnG) and the

lowest Zn-accumulation (LZnG) under Zn-fertilization regimes, the Experiment 3 consisted

of the following two sub-aims: (a) to evaluate the effect of zinc fertilization on growth and

yield attributes of higher (HZnG) and lower (LZnG) zinc-accumulating genotypes and to

optimize the dose of zinc fertilizer. (b) to assess nitrogen-utilization efficiency (N-use

efficiency) by assaying enzymes involved in nitrogen metabolism (NR, NiR, GS, GOGAT

and GDH), the modulation of Zn-dependent enzymes (super oxide dismutase and carbonic

anhydrase) and various physiological and biochemical parameters.

To achieve the above aim, ten healthy uniform-sized seeds of two genotypes of

chickpea selected for the study were surface-sterilized with 0.1% mercuric chloride for 5 min

and then vigorously rinsed with deionized water before germination. Seeds were germinated

in non-contaminated sand moistened with deionized water in the dark. After one week, five

most vigorous and equally developed seedlings were transferred to 23-cm-diameter soil-filled

earthen pots lined with polythene bags (to avoid contamination). Entire experiment was

conducted under naturally illuminated field condition with relative humidity of 70-76%. Zinc

at a concentration of 0, 2.5, 5 and 10 mg/kg was added to the soil as ZnSO4.7H2O and

thoroughly mixed. Recommended basal doses of N, P, K and S were applied and mixed

thoroughly in soil in order to get 25 kg N , 20 kg P, 30 kg K and 20 kg S /ha. The sources of

N, P, K, and S were urea, single super phosphate, muriate of potash (KCl) and gypsum


respectively. Plants (5 per pot) were watered every alternate day with double deionized water

(DDW). Watering schedule was adjusted throughout the experimental duration in order to

avert leaching. The treatments were arranged in a randomized design with three replicates

and five plants per pot. Growth was visually assessed at every 5 weeks until harvest in

February. The necessary after-care operations, such as thinning, hand weeding, inter

cultivation and plant protection measures, were carried out as and when required to maintain

good and healthy seed crop. The crop was sprayed with Dithane M-45 @ 2.5 mg per litre at

35, 45, 75 days after sowing to control infestation of Aschoyta blight and Fusarium wilt.

Endosulfan 35 EC @ 1.5 ml per litre was spayed at 35, 50, 70 and 90 days after sowing

(DAS) for control of pod borer and other sucking pests. After 40 DAS (pre-flowering stage),

first sampling was done and four plants were maintained in each pot up to 60 DAS. After 60

days of sowing (flowering stage), second sampling was done and three healthy plants of

uniform size were maintained in each pot until third sampling at 90 DAS (post-flowering

stage). Data on plant growth, biomass (dry weights of shoot and root), root nodulation (total

number of nodules/root system), number of branches, number of leaves per plant were

recorded at 40, 60 and 90 DAS. Various biochemical parameters were also determined at

different growth stages, i.e. pre-flowering, flowering and post-flowering stages. Nitrogen

metabolizing and Zn-dependent enzymes were assayed at different stages of plants i.e., 40, 60

and 90 DAS. At termination, 120 days after sowing (harvesting stage), all yield related

parameters were determined.

Table 5. Experimental design for ´Exp. 3´.

Type of Genotypes Treatment

(mg/ ZnSO4 kg-1

soil)

Replicates

Zn0 Zn2.5 Zn5 Zn10

Highest zinc accumulating

(G8)

0 2.5 5 10 3

Lowest zinc accumulating

(G14)

0 2.5 5 10 3


Table 6. Some physico-chemical properties of the soil used in pot-culture experiments.

Soil characteristics Values

Texture Loamy Sand (83.6% sand, 6.8% silt and 9.6% clay)

pH 7.1

Conductivity E.C. 0.23 ds m-1

Organic C 0.28%

Dry bulk density 1.39 Mg m-3

Available N 128 kg ha-1

Available P 14 kg ha-1

Available K 158 kg ha-1

DTPA-extractable zinc 0.78

Table 7. Weather condition for the experimental site (2011-2012).

Jan Feb Mar Apr May Jun July Aug Sep Oct Nov Dec

T. max 21 24 33 36 40 41 35 34 34 35 29 24

T.min 8 10 17 21 27 29 27 26 25 19 12 9

R.average 25 22 18 7 8 65 211 173 150 31 1 5

(R = Rainfall, mm; T = Temperature, °C)

3.5.4 Experiment 4

Experiment ‘4’ was conducted to determine the effect of nitrogen (N) levels on zinc

distribution, and growth and yield of chickpea genotypes differing in their accumulation and

efficiency for zinc.

To evaluate N on Zn-partitioning and plant growth at constant Zn level, the two C.

arietinum genotypes differing in Zn-accumulation were grown in pots filled with acid washed

sand at constant zinc level with varying levels of nitrogen (0, 12.5, 25 and 50 mg/kg of sand)


as urea. Recommended basal doses of P, K, S, and Zn were applied and mixed thoroughly in

sand in order to get 20 kg P, 30 kg K, 20 kg S and 10 kg Zn /ha. The sources of K, P, S and

Zn were muriate of potash (KCl), single super phosphate, gypsum and ZnSO4 respectively.

Ten uniform-sized seeds were surface-sterilized and germinated on moistened sand following

the procedure described above. After one week, 5 uniformly germinated seedlings were sown

in sand-filled pots. The necessary aftercare operations and plant-protection measures were

carried out as and when required to maintain a good and healthy crop. The crop was also

given with protective irrigation, depending upon the water requirement. The first sampling

was done at 90 DAS and two plants per pot were maintained up to harvesting stage (120

DAS). At 90 DAS, number of root nodules, leghaemoglobin (LHb) content, leaf N content,

leaf Zn concentration and number of root nodules were estimated. Grain yield determination

was performed at the harvest (120 DAS).

3.5.5 Experiment 5

Experiment ‘5’ was carried out to evaluate phosphorus regimes on Zn-partitioning

and plant growth at constant Zn level.

On the perspective of P regime influence evaluation on Zn-partitioning and plant

growth at constant Zn level, the two C. arietinum genotypes differing in zinc accumulating

capacity were grown in clay pot filled with acid-washed sand (Hewitt, 1966) at constant level

of zinc and varying amount of phosphorus (0, 13.5 and 27.0 mg/kg of sand) as single super

phosphate. Recommended basal doses of N, K, S, and Zn were applied and mixed thoroughly

in sand in order to get 25 kg N, 30 kg K, 20 kg S and 10 kg Zn /ha. The sources of K, N, S

and Zn were muriate of potash (KCl), urea, gypsum and ZnSO4 respectively. The

experimental design was block randomized with three replicates and three treatments.

Moisture content was maintained at an optimum level (12%) by weighing method throughout

the experiment with deionized water. Plants were maintained up to 90 DAS and all measures

were taken for healthy plant growth as described above. The shoot and root dry matter

production, and the estimation of Zn and P were performed.

3.6 Chemicals used

All the chemicals used in this study were of GR or AR quality. The major and minor

salts, buffer components, etc. were procured from E. MERCK and SRL. Most of the

biochemicals used were purchased from Sigma Aldarich (St. Louis Mo, USA). Before use, all


glass and plastic ware was carefully cleaned, using the procedures described by Norvell and

Welch (1993).

3.7 Parameters

The following parameters were studied at different sampling times.

Growth characteristics

o Plant height

o Number of branches per plant

o Number of leaves per plant

o Leaf area index

o Plant biomass

o Shoot: root ratio

o Relative shoot growth

o Number of root nodules

Physiological and biochemical characteristics

o Zn uptake

o Relative Zn transport

o Zinc accumulation

o Zinc efficiency

o Shoot zinc -and phosphorous -uptake

o Analysis for elements (C, N, S) and protein concentration

o Photosynthetic pigments

o Soluble protein content

o In vivo nitrate reductase activity


o In vitro nitrate reductase activity

o In vitro nitrite reductase activity

o In vitro gluatamine synthetase activity

o In vitro glutamate synthase activity

o In vitro glutamate dehydrogenase activity

o Superoxide dismutase activity

o Carbonic anhydrase activity

o Leghaemoglobin content

o Nitrate content

o Nitrogen, zinc and phosphorus estimations

Yield and its attributes

o Number of pods per plant

o Number of seeds per pod

o Grain yield per plant

o Hundred seed weight

3.8 Methods

3.8.1 Growth characteristics

3.8.1.1 Plant height

The plant height was measured from the base to the tip of the plant in each treatment

in the three randomly tagged plants. The plant height was measured with the help of metre

scale and the average was computed and expressed in centimeters.

3.8.1.2 Number of branches

The number of branches per plant on three randomly tagged plants were counted

manually and the average was computed.


3.8.1.3 Number of leaves per plant

The leaves of three randomly tagged plants in each treatment were counted manually

at 40, 60 and 90 days after sowing and average was computed.

3.8.1.4 Leaf area index (LAI)

Three plants were randomly selected from each pot-treatment. LAI was determined

using the LI-COR model 3100 LI Area Meter. The leaf area index was calculated by dividing

the leaf area per plant by area occupied by the plant.

Leaf area / plant (cm2)

LAI =

Area / plant (cm2)

3.8.1.5 Plant biomass measurement

Plants were carefully uprooted from the pot area and roots of plant samples were

rinsed several times with deionized water and immersed in 20 mM Na2-EDTA (disodium

ethylenediaminetetraacetate) for 20 min to remove Zn adhering to the root surface (Yang et

al., 2006). Plants were then briefly rinsed in three lots of deionized water and gently blotted.

Roots and shoots were separated and dried at 70ºC for 48 h in Precision™ mechanical

convection oven (GCA Corporation). After getting constant weight, the dried plant parts were

cooled in the ambient conditions for two hours. Weighing of plant parts was done with

Mettler™ balance (type H6), which is accurate up to four decimal places. Values were

expressed in gram per plant (g/plant).

3.8.1.6 Shoot: root ratio

The shoot: root ratio was computed by dividing the shoot dry weight by root dry

weight.

3.8.1.7 Relative shoot growth

Relative shoot growth was calculated as the ratio of shoot dry matter at deficient Zn

level (Zn0.01 = 0.01 mg ZnSO4/L nutrient solution) to that at sufficient Zn level (Zn0.5 = 0.5

mg ZnSO4/L nutrient solution) and expressed as percentage.


3.8.1.8 Number of root nodules

Three randomly selected plants were carefully uprooted from each pot with root

system intact. The roots were washed in running tap water and nodules were detached

carefully with forceps and number of nodules per plant was counted as average.

3.8.2 Physiological and biochemical characteristics

3.8.2.1 Root Zn uptake

Zn uptake per gram root dry matter (µg/g root DW) was calculated by dividing the

plant Zn content (µg/plant) with root dry weight (g/plant).

3.8.2.2 Relative Zn transport

Relative Zn transport (%) was determined by dividing the shoot Zn content (µg/plant)

with the total plant Zn content (µg/plant).

3.8.2.3 Zinc accumulation

The amount of Zn accumulated by plant from the soil solution was estimated by

subtracting seed Zn from the total plant Zn. All values were expressed as µg per plant.

3.8.2.4 Zinc efficiency (ZE)

The term ZE is defined as the ability of a genotype to grow well under Zn-deficient

conditions (Graham, 1984). The efficiency of crop genotypes is basically concerned with the

uptake, transport and utilization of zinc for the production of dry matter and grain bulk. The

efficient genotypes are able to utilize zinc more effectively than the other genotypes and are

better yielding under low soil Zn conditions (Blair, 1993; Graham and Rengel, 1993).

Moreover, ZE is linked to the differences in the ability of the genotype to maintain an optimal

activity of the important zinc regulating enzymes, viz., copper zinc super oxide dismutase

(Cu/zinc SOD) and carbonic anhydrase (CA). Also, activity of a large number of enzymes

which have zinc as an integral component of their structure (zinc enzymes), has been

correlated with zinc availability to plants. Differences in internal utilization or mobility of

zinc have been shown to be involved in the expression of zinc efficiency. Therefore, plant

zinc efficiency is an important parameter. Hence, it is relevant to investigate whether there is

variation in ZE for grain yield among chickpea genotypes differing in their zinc accumulation


ability. ZE was computed at 40 DAS among seven selected chickpea genotypes (G2, G5, G8,

G14, G18, G19 and G20) by using the following formula (Khan et al, 2000):

3.8.2.5 Shoot zinc -and phosphorus -uptake

The uptake of zinc (Zn) and phosphorus (P) were determined by multiplying shoot

dry matter (g/plant) with the concentration of each element separately. Zinc uptake was

expressed as µg per plant and phosphorus uptake was expressed as mg per plant.

3.8.2.6 Analysis for elements (C, N, S) and protein concentration

Leaf samples were collected from each pot and wiped free of any adhering dust. The

samples were first washed in running tap water for 1 min followed by 1 min in distilled

water. The samples were oven-dried at 70oC for 48 hrs and ground to fine powder with

mortar-pestle. Sample-powder were weighed and prepared in low weight aluminium boats

and tungsten was used as combustion agent. Five standard samples were run before leaf

samples were subjected to analysis, where small amount of sulphalinic acid was used instead

of leaf material. The samples were subjected to carbon (C), nitrogen (N), Sulphur (S), C/N

ratio and Protien analysis using Elementar (CHNS Analyser, Vario EL III, Germany).

Nitrogen values were converted into protein values by multiplying by a factor of 6.25. All

values were expressed as percentage (%).

3.8.2.7 Photosynthetic pigments

Fresh leaves were collected from Cicer arietinum L. plants at the three developmental

stages: pre-flowering, flowering and post-flowering stages, i.e. 40, 60 and 90 DAS

respectively, to investigate variation in pigments such as Chlorophyll ‘a’, Chlorophyll ‘b’ and

Total chlorophyll contents.

3.8.2.7.1 Pigment extraction procedure

The Hiscox and Israelstam (1979) method was used to estimate the chlorophyll

content in the samples. The method involves the estimation of plant pigments without

Zn efficiency of chickpea genotype = Grain yield at Zn0.01

Grain yield at Zn0.5

× 100


maceration. Leaves were kept on a moist filter paper in an icebox, washed with cold distilled

water and then chopped. 100 mg of the chopped leaf material was taken in vials in triplicates

containing 7 ml of dimethyl sulfoxide (DMSO). The vials were then kept in an oven at 65°C

for 1 hr for complete leaching of the pigments. Thereafter, the volume of DMSO was made

up to 10 ml. The chlorophyll content was then measured immediately. The absorbance of

DMSO containing pigments was recorded at 645 and 663 nm using a Beckman

spectrophotometer (model DU 640, Fullerton, USA).

3.8.2.7.2 Pigment estimation procedure

Values of optical densities (ODs) were used to compute the chlorophyll a, chlorophyll

b, and total chlorophyll contents, using the following formulae given by Maclachalam and

Zalik (1963) for chlorophyll a, Duxbury and Yentach (1956) for chlorophyll b, and Arnon

(1949) for total chlorophyll

Where,

D = Distance travelled by the light path

W = Weight of the leaf material taken

V = Volume of the extract

OD = Optical density

3.8.2.8 Soluble protein content

The total soluble protein content of different sample was estimated following the

method of Bradford (1976).

3.8.2.8.1 Reagents used: i) Extraction buffer: 0.1 M phosphate buffer (pH 7.0) was used as

extraction buffer; ii) 10% (w/v) TCA; iii) 0.1 N NaOH; and iv) Bradford’s reagent.


Bradford’s reagent preparation: In a dark/covered volumetric flask, 50 ml of 90% ethanol

was mixed with 100 ml of orthophosphoric acid (85%). To the above, 100 mg of Coomassie

Brilliant Blue (G) dye was added; volume was made up to 1 L using double distilled water

and stirred well on a magnetic stirrer. Subsequently, the solution was filtered through

Whatman filter paper No.1 and stored in dark condition. The final concentrations of

components in the reagent were 0.01% Coomassie brilliant blue G-250 (w/v), 4.75% ethanol

(w/v) and 8.5% O-phosphoric acid (w/v).

3.8.2.8.2 Soluble protein extraction

Chopped fresh leaf tissues (0.5 gm) were homogenized in 1.5 ml of 0.1 M phosphate

buffer (extraction buffer) at 4°C with the help of a pre-cooled mortar and pestle and kept in

ice during the process of homogenization. The homogenate was transferred to a 2 ml

eppendorf and centrifuged at 5000 rpm for 20 min at 4°C. An equal amount of chilled 10%

TCA was added to 0.5 ml of the supernatant, which was again centrifuged at 3300 rpm for 30

min. The supernatant was discarded and the pellet left was washed with acetone. It was then

dissolved in 1 ml of 0.1 N NaOH.

3.8.2.8.3 Soluble protein estimation

To 0.2 ml aliquot, 1 ml of Bradford’s reagent was added and vortexed. The tubes were

kept for 10 min for optimal color development. The absorbance was then recorded at 595 nm

on a Beckman spectrophotometer (DU 640, Fullerton, USA). The soluble protein

concentrations were quantified with the help of a standard curve prepared from the standard

of Bovine Albumin Serum (BSA) from Sigma, USA. The protein content was expressed in

mg g-1

F.W.

3.8.2.9 In vivo nitrate reductase activity

Nitrate reductase (NR) activity was estimated by the intact tissue assay method of

Jaworski (1971), which is based on the reduction of nitrate to nitrite.

3.8.2.9.1 Enzyme extraction

Leaf material (250 mg) was suspended in screw capped vial containing 2.5 ml each of

phosphate buffer (0.1 M, pH 7.5), potassium nitrate solution (0.2 M) and iso-propanol (5%),

and 2 drops of chloramphenicol (0.5%). After sealing, the vials were incubated at 30° C in

the dark for about 2 h.


3.8.2.9.2 Enzyme assay

NR activity in the medium was determined by taking 0.4 ml of incubated solution and

0.3 ml each of sulphanilamide (1% in 3N HCl) and NEDD (0.02%). After 20 min, the

solution was diluted with 4 ml of distilled water to make the final volume up to 5 ml and its

optical density measured at 540 nm. A standard curve was plotted using varying

concentrations of potassium nitrate and used for calculation.

3.8.2.10 In vitro nitrate reductase activity


Extraction of NR was done by the method of Hageman and Flesher (1960). One gram

of leaves was homogenized using mortar and pestle in 4 ml of grinding medium (pH 7.5)

containing tris (hydroxymethyl) aminomethane (0.1 M), cystein (0.01 M), ethylene diamine

tetra acitic acid (EDTA, 0.0003 M). The homogenate was centrifuged at 20, 000 g for 15

minutes. The supernatant was assayed for NR activity.


NR activity was measured by modification of the method described by Evans and

Nason (1953). The assay mixture contained 1 ml of phosphate buffer (0.1 M, pH 7.0), 0.2 ml

of potassium nitrate (0.1 M), 0.5 ml of diphosphopyridine nucleotide (DPNH, 1.36 × 10-3

M),

0.2 ml of enzyme extract and 0.1 ml of distilled water. The assay was initiated by addition of

first, DPNH and immediately thereafter the enzyme extract. The mixture was incubated at

27°C for 15 minutes and the reaction was stopped by adding sulphanilamide (1 % in 1.5 N

HCl). 1 ml of NEDD (0.02 %) was added and the contents mixed by inverting the tubes. The

colour was allowed to develop for 5 minutes before centrifuging at 1,500 g for 10 minutes to

remove the turbidity. The absorbance was determined by reading each sample against its own

blank (complete except DPNH) at 540 nm.

3.8.2.11 In vitro nitrite reductase activity


For nitrite reductase (NiR) enzyme extraction, crude homogenates were prepared

according to Gupta and Beevers (1984), using one gram of sample in 5 ml of extraction

buffer consisting of phosphate buffer (100 mM, pH 8.8), EDTA (5 mM) and cysteine-HCl (1


mM). The homogenates were fractionated with saturated ammonium sulphate. The material

precipitated between 40 and 75% saturated ammonium sulphate was pelleted by

centrifugation at 10,000 g for 25 minutes and re-suspended in 0.5 ml of phosphate buffer (50

mM, pH 7.5) containing 10% glycerol.


NiR activity in extracts was assayed by the method of Ida and Morita (1973). The

assay mixture contained in a final volume of 2 ml Tris - HCl buffer (100 mol, pH 7.5),

sodium nitrate (3.0 µmol), methyl viologen (2 µmol) and 20-120 milli units of enzyme in

open test tubes. The reaction was started by adding 0.3 ml of freshly prepared solution

containing 24 µmol of sodium dithionite in sodium bicarbonate (0.2 M) to the assay mixture,

which was kept in an ice bath. After reaction had proceeded for 20 min at 30°C, it was

terminated by shaking the test tube vigorously until the blue color of reduced methyl viologen

completely disappeared. Then, 0.1 ml of this aliquot was withdrawn with 5.9 ml of water,

followed by addition of 1 ml each of sulphanilamide (1% in 3 N HCl) and NEDD (0.02%).

Absorbance of the diazo-color was read at 540 nm after standing for 20 minutes. Non-

enzymatic loss of nitrite was determined with a control assay mixture without enzyme.

Enzyme-dependent disappearance of the substrate was estimated by subtracting the amount

of nitrate, which disappeared in the control assay. A standard curve was plotted using sodium

nitrate for calculations.

3.8.2.12 In vitro assays of gluatamine synthetase (GS), glutamate synthase (GOGAT)

and glutamate dehydrogenase (GDH) enzymes


Extracts used for GS, GOGAT and GDH assays were prepared by grinding leaf

sample (1 g) in 4 ml of grinding medium prepared according to Cooper and Beevers (1969).

The grinding medium consisted of tricine buffer (165 mM), sucrose (0.4 M), potassium

chloride (10 mM), magnesium chloride (10 mM), EDTA (10 mM) and ß-mercaptoethanol (10

mM). The homogenate was centrifuged for 7 min at 500 g to remove unbroken cells and cell

fragments. The supernatant solution was then centrifuged at 10,000 g for 15 min yielding a

crude mitochondrial pellet and supernatant. The supernatant solution was centrifuged at 1,

00,000 g for 30 min and the resulting clear solution was used for GS and GOGAT assays.


The high speed centrifugation was necessary to remove NADH oxidase which otherwise

interfered with the GOGAT and GDH assay

3.8.2.12.2 In vitro glutamine synthetase assay

For determination of GS activity, method of Lea et al. (1990) was followed. 0.5 ml of

the synthetase assay mixture contained glutamate (50 mM), hydroxylamine hydrochloride (5

mM), magnesium sulphate (50 mM) and ATP (20 mM) in tris-HCl (100 mM, pH 7.8). The

pH values of glutamate and hydroxylamine solution were carefully adjusted to pH 7.8. The

ATP solution was made fresh before each assay and pH adjusted to just above 7.0.The

reaction was started by the addition of 0.2 ml of enzymes extract and incubated at 30°C. The

reaction was terminated by the addition of 0.7 ml of FeCl3.6H2O in 0.02 N HCl. Control

tubes were incubated comprising the enzyme extract and all the reagents except ATP. For

precipitation of protein, the tubes were centrifuged at 10,000 g for 5 min. The absorbance of

the supernatant was determined at 540 nm. Standard curve was constructed using L-glutamic

acid γ-monohydroxamate to obtain an accurate quantification of GS activity.

3.8.2.12.3 In vitro glutamate synthase assay

The GOGAT activity was determined by the method of Mohanty and Fletcher (1980).

The reaction mixture contained in a final volume of 3 ml tris - HCl buffer (75 µmol, pH 7.5),

α-ketoglutarate (10 µmol), L-glutamine (15 µmol), NADH (0.3 µmol) and enzyme

preparation (0.5 ml). The reaction was initiated by adding NADH and the rate was measured

at 340 nm at 25ºC for 5min. The control lacked glutamine, NADH and α-ketoglutarate.

Enzyme activity was calculated using the extinction coefficient of NADH at 340 nm i.e. 6.3 x

103 litres mol

-1 cm

-1.

3.8.2.12.4 In vitro glutamate dehydrogenase assay

The estimation of GDH activity was done using the method of Mohanty and Fletcher

(1980). The reaction mixture contained in a final volume of 3 ml phosphate buffer (75 µmol,

pH 7.5), α-ketoglutarate (10 µmol), ammonium chloride (300 µmol), NADH (0.3 µmol) and

suspension medium containing enzyme (0.2 ml). The reaction was initiated by adding

NADH, and the oxidation of NADH was followed at 340 nm and at 25°C for 5 minutes. The

blank consisted of the complete reaction mixture without ammonium chloride. Enzyme

activity was calculated using the extinction coefficient of NADH at 340 nm i.e. 6.3 × 103

litres mol-1

cm-1.


3.8.2.13 Superoxide dismutase activity

The method given by Dhindsa et al. (1981) was followed with a slight modification

for the estimation of superoxide dismutase (SOD) activity.

3.8.2.13.1 Reagents used: i) 1 M Sodium bicarbonate solution (NaHCO3); ii) 200 mM

Methionine solution; iii) 2.25 mM Nitroblue tetrazolium (NBT) solution; iv) 3 mM EDTA;

v) 60 M Riboflavin; vi) Extraction and reaction buffers.

Extraction buffer preparation: The extraction buffer was prepared using 0.1 M phosphate

buffer (pH = 7.5), 0.05% (v/v) Triton X-100, 1% (w/v) polyvinylpyrrolidone (PVP) and 3

mM EDTA.

Reaction buffer preparation: The reaction buffer contained 0.1 M phosphate buffer (pH 7.5)

and 1% polyvinylpyrrolidone (PVP).

3.8.2.13.2 Extraction Procedure

Fresh plant tissues (0.05 g) were homogenized in 2.0 ml of extraction mixture with

the help of mortar and pestle. The process was carried out under cold condition (4oC). The

mortar and pestle was kept in ice during the course of homogenization. The homogenate was

transferred to centrifuge tubes and centrifuged at 10,000 rpm for 10 min at 4oC.


SOD activity in the supernatant was assayed by its ability to inhibit the photochemical

reduction. The assay mixture, consisting of 0.5 ml of enzyme extract, 1.5 ml reaction buffer,

0.2 ml of methionine, 0.1 ml NaHCO3, 0.1 ml NBT solution, 0.1 ml EDTA, 0.1 ml riboflavin

(immediately after keeping in light) tubes, which were incubated under the light of 15 W

influorescent lamp for 10 min at 25/28°C. Blank A containing all the above substances of the

reaction mixture, along with the enzyme extract was placed in the dark. Blank B containing

all the above substances of reaction mixture except enzyme was placed in light along with the

sample. The reaction was terminated by switching off the light and the tubes were covered

with a black cloth. The non-irradiated reaction mixture containing enzyme extract did not

develop light blue color. Absorbance of samples along with blank B was read at 560 nm

against the blank A. The difference of % reduction in the color between blank B and the

sample was then calculated. 50% reduction in the color was considered as one unit of enzyme

activity and the activity was expressed in Enzyme Unit (EU) mg-1

protein h-1

.


3.8.2.14 Carbonic anhydrase activity

Activity of CA in leaf was estimated according to the method described by Makino et

al. (1992). Sampled leaves were homogenized in 10 mL of buffer containing 50mM HEPES-

NaOH (pH 7.5), 10mM DTT, 0.5mM EDTA and 10% (v/v) glycerol.Triton X-100 was added

to the homogenate to a final concentration of 0.1% (v/v). The homogenate was centrifuged

(15,000g, 10 min), and the supernatant was used for the determination of CA. Activity of CA

was determined in Wilbur-Anderson unit following time-dependent reduction in pH from

8.25 to 7.45 at 0-4°C. The Unit [U] of enzyme activity was calculated according to the

formula U = 10 (T – T0)/T0, where T and T0 represent the time required to change the pH

from 8.25 to 7.45, with and without the extract of crude enzyme, respectively. The enzyme

activity was presented as U per mg protein.

3.8.2.15 Leghaemoglobin content

Like most legumes, chickpea has the potential to fix atmospheric nitrogen through

symbiotic relationship with soil microorganisms. The nitrogen is converted by the Rhizobia

on the root nodules of the plant to an available form by leghaemoglobin, and making the

plant an efficient source of nitrogen. Thus to find out capacity of this plant to fix atmospheric

nitrogen, the leghaemoglobin content was determined from fresh root nodules. The

leghaemoglobin content of fresh, bold and pink nodules was determined by the method of

Wilson and Reisenauer (1963) with Drabkin’s solution (Wilson and Reisenauer 1963).

3.8.2.15.1 Extraction and estimation

Nodules were separated from roots, the level of leghaemoglobin was immediately

analysed. Nodules (500 mg) were homogenized in aliquots of Drabkin’s reagent (10 ml) and

leghaemoglobin was quantified spectrophotometrically at A540, as described by Wilson and

Reisenauer (1963). Bovin haemoglobin was used as a standard, and values are expressed as

milligrams per gram of nodule mass (fresh weight) (mg/g nodules F.W).

SOD activity (EU mg-1

protein h-1

) =

% Reduction in color between blank and sample x Dilution factor x 60

Incubation time x mg protein in sample


3.8.2.16 Nitrate content

Nitrate content of leaves was estimated using the method given by Grover et al.

(1978).

3.8.2.16.1 Reagents used: i) 0.1 N NaOH; ii) 0.01 M Hydrazine sulfate; iii) 10% Acetone;

iv) 1% Sulphanilamide; v) 0.02% N-1 Naphthyl ethylene diamine dihydrochloride (NEDD);

and vi) Copper sulphate (CuSO4)- Zinc sulphate (ZnSO4) catalyst.

3.8.2.16.2 Procedure

Fresh leaves (0.5 g) were taken in conical flask; 50 mg charcoal and 10 ml distilled

water were added to it and boiled for 4-5 mins. After filteration, the volume was made up to

50 ml by adding DDW. 1 ml of this aliquot was taken and 0.5 ml CuSO4 solution, 0.25 ml

hydrazine sulfate, 0.25 ml of 0.1 N NaOH were added to it. The vials were kept in water bath

incubator for 10 mins at 330C and then transferred to ice and 0.5 ml chilled acetone and 1.0

ml sulphanilamide and NEDD [N-(1-napthyl) ethylene diamine dihydrochloride] were added.

The volume was increased to 6 ml by adding 1.5 ml DDW and the vials were kept for 20

mins for colour development. Optical density was measured at 540 nm on Beckman DU 640

spectrophotometer. The nitrate content was expressed as n mole g-1

F.W. The concentration

of nitrate was determined against the standard curve prepared by using KNO3 (potassium

nitrate) solution.

3.8.2.17 Nitrogen, zinc and phosphorus estimations

The N content (% dry weight) was analyzed by packing the known weight of plant

sample-powder in low weight aluminum boats with the help of Elementar system (CHNS

Analyzer, Vario EL.), and expressed as %. For estimation of Zn concentration, dried shoot

samples were ground to fine powder, and then digested at high temperature (up to 205ºC) in

triacid following the methods described above. Zn concentration was analysed by atomic

absorption spectrophotometer (AAS ZEEnit65) and concentration of P was determined by

using the Vanadomolybdate yellow colour method (Jackson, 1973) in acid-digested extract

solution. The content of Zn was expressed as mg kg–1

DW and P was expressed as g kg–1

DW

in plant samples.

3.8.3 Yield and its attributes

All the plants were allowed to grow to maturity. The pods were shelled manually and

the seeds were separated and weighed after harvesting (120 DAS). The number of pods per


plant, number of seeds per pod, number of seeds per plant and the weight of 100 seeds were

recorded. Subsequently, the grain yield per plant (g/plant) was determined. Procedures

adopted are described below.

3.8.3.1 Number of pods per plant

The total pods per plant were counted in each treatment from three randomly tagged

plants and average was worked out and expressed as number of pods per plant.

3.8.3.2 Number of seeds per pod

The total number of seeds from three randomly selected pods from tagged plants were

manually removed and counted. The mean seeds per pod were calculated.

3.8.3.3 Grain yield (g) per plant

Seeds were cleaned before being weighed for grain yield per plant (g/plant). The

number of seeds obtained from three random pods were weighed carefully with the help of

electronic balance and expressed as grain yield per plant in grams.

3.8.3.4 Hundred seed weight (g)

One hundred seeds in three replications from each treatment were counted and their

weight was recorded by using electronic balance as per ISTA procedure (Anonymous, 1999).

The average weight was expressed in grams.

3.9 Statistical Analysis

Each experiment was performed three times and all the determinations were obtained

from three replicates (N= 3). Statistical analysis of the data was performed, using the Graph

Pad Prism software program (Graph Pad Prism 5, 2003 Analytical Software). Results are

expressed as mean ± standard error. The values were submitted for analysis of variance

(ANOVA) for each factor (dose and genotypes) and their interaction. Tukey test was applied

to detect the statistical significance of differences (P ≤ 0.05) between means.

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