36

3. MATERIALS AND METHODS

3.1. Study material

Brassica juncea L. var. RLC1 was chosen as the study material for the present

investigation. Certified seeds of B. juncea L. var. RLC1 were procured from Punjab

Agricultural University, Ludhiana, India.

3.1.1. Classification, occurrence and uses of B. juncea

3.1.1.1. Classification

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Capparales

Family : Brassicaceae

Genus : Brassica

Species : juncea

3.1.1.2. Occurrence and uses

B. juncea, is a herbaceous plant. Also known as Indian mustard, it is cultivated widely

in India, China, Nepal, Bangladesh and other Asian countries. Its leaves and seeds are

used as vegetable, salad and condiment in various cuisines all over the world. Its seeds

contain about 25% of oil which is used for cooking (Panda, 1999). B. juncea has

medicinal value due to the presence of glycosides, flavonoids, sterols and phenolic

compounds (Kumar et al., 2011). B. juncea is suitable for phytoremediation purposes

because of high biomass, short life span and heavy metal tolerance. It has been widely

studied for its potential to accumulate heavy metals like Cu, Cd, Pb, Cr, Ni etc.

(Ariyakanon and Winaipanich, 2006; Lim et al., 2004; Jagtap et al., 2013). In addition,

B. juncea is also a potential biofuel crop (Blackshaw et al., 2011).

37

3.2. Raising of B. juncea plants

3.2.1. Treatment of seeds

3.2.1.1. Sterilization of seeds

Sterilization of the seeds was performed by rinsing them for 15 min with 0.4% solution

of sodium hypochlorite. It was followed by the repeated washings with distilled water.

3.2.1.2. Presoaking treatment of B. juncea seeds with 24-epibrassinolide (24-

EpiBR)

24-EpiBR used in the present study was purchased from Sigma Aldrich Pvt. Ltd., New

Delhi, India. A mother stock solution (106 nM) of 24-EpiBR was prepared in high

performance liquid chromatography (HPLC) grade methanol and was stored in a deep

refrigerator at -20oC. On the basis of field applications and earlier studies, 0, 100, 1 and

0.01 nM concentrations of 24-EpiBR were finalized for the present investigation.

Different working stock solutions of 24-EpiBR were prepared from the mother stock

solution by serial dilutions. The seeds were given the 8 h pre-soaking treatments of the

above mentioned concentrations of 24-EpiBR.

3.2.2. Treatments of Cu

The Cu salt used as the source of Cu(II) was CuSO4.5H2O. The concentrations of Cu(II)

used in the present investigation were determined on the basis of IC50 (concentration at

which 50% growth was inhibited) for B. juncea seedlings. 0, 0.25, 0.50 and 0.75 mM

Cu(II) treatments were selected to give Cu(II) treatments in growth medium of B.

juncea plants for further experimentation. The above mentioned concentrations of

Cu(II) and 24-EpiBR were applied alone and in binary combinations. Different

combinations of Cu(II) and 24-EpiBR were used, which are as below:

1. Control

2. 0.25 mM Cu(II)

3. 0.50 mM Cu(II)

4. 0.75 mM Cu(II)

5. 100 nM 24-EpiBR

38

6. 1 nM 24-EpiBR

7. 0.01 nM 24-EpiBR

8. 0.25 mM Cu(II) + 100 nM 24-EpiBR

9. 0.50 mM Cu(II) + 100 nM 24-EpiBR

10. 0.75 mM Cu(II) + 100 nM 24-EpiBR

11. 0.25 mM Cu(II) + 1 nM 24-EpiBR

12. 0.50 mM Cu(II) + 1 nM 24-EpiBR

13. 0.75 mM Cu(II) + 1 nM 24-EpiBR

14. 0.25 mM Cu(II) + 0.01 nM 24-EpiBR

15. 0.50 mM Cu(II) + 0.01 nM 24-EpiBR

16. 0.75 mM Cu(II) + 0.01 nM 24-EpiBR

3.2.3. Raising of B. juncea plants in field

Fig. 3.1. Field prepared according to randomized block design

39

An open area (approx. size: 8 m x 6 m) receiving plenty of sunlight all over the day,

was selected. It was ploughed with a tractor. All weeds were manually removed. Field

was levelled properly. Field was prepared according to randomized block design (Fig.

3.1). Three replicative blocks were prepared for each of the 16 concentrations. So, total

48 blocks of size 36" x 36" were prepared separated by gaps of enough size making it

easy to move between the blocks for working in the field. From initial experiments with

water holding capacity, each block of size 36" x 36" x 6" required 45 L of water to

reach soil saturation. CuSO4.5H2O treatments at 0, 0.25, 0.50 and 0.75 mM Cu(II)

concentrations were given in these blocks for each treatment. Seeds pre-soaked with 24-

EpiBR were then sown in the field with proper spacing. Plants were grown under

natural environmental conditions. Normal agronomic practices were followed. Plants

were harvested randomly from each treatment for analysis after 30 (vegetative stage)

and 60 (reproductive stage) days of sowing. After 90 days (fully mature plants), all the

plants were harvested (Fig. 3.2).

Fig. 3.2. Scheme of raising of B. juncea plants in field

40

3.2.4. Raising of B. juncea seedlings in seed germinator

Petri dishes were autoclaved and were lined with Whatman No. 1 filter paper. The Petri

dishes were labelled in triplicates for the treatments: Control, 0.50 mM Cu(II), 100 nM

24-EpiBR and 0.50 mM Cu(II) + 100 nM 24-EpiBR. The Petri dishes were applied with

distilled water or 0.50 mM Cu solution as per the labelling. Sterilized seeds of B. juncea

given pre-soaking treatment with distilled water or 100 nM 24-EpiBR were sown in the

respective labelled Petri dishes with proper spacing. Seedlings were raised under

controlled conditions in a seed germinator with a photoperiod of 16 h; temperature, 25 ±

0.5oC and light intensity, 175 µmol m

-2 s

-1. 7-day old seedlings were harvested for the

analysis of various physiological and biochemical parameters.

3.3. Analysis of B. juncea plants

Harvested seedlings and plants were processed for physiological and biochemical

analysis. Physiological studies included study of growth parameters, gas exchange

parameters, Cu(II) uptake, seed microsculpture, cell death and stomatal behaviour.

Biochemical analysis included studies on carbohydrate metabolism, lipid metabolism,

lipid peroxidation, plant growth regulators (PGRs), polyphenols, elements and cell

peroxidation (Fig. 3.3).

Fig. 3.3. Analysis of B. juncea plants

41

3.3.1. Studies done on the 30, 60 and 90-day old B. juncea plants raised in the field:

3.3.1.1. Physiological studies

3.3.1.1.1. Growth parameters

Growth parameters were measured for 30, 60 and 90-day old B. juncea plants. It

included the measurement of shoot and root lengths and fresh weights and calculation

of number of leaves per five plants.

3.3.1.1.2. Gas exchange parameters

Gas exchange parameters like stomatal conductance, intercellular CO2 concentration,

photosynthetic rate and transpiration rate were measured by using infra red gas analyzer

(IRGA), LI-COR 6400XT.

Principle

LI-COR 6400XT is an open photosynthetic gas exchange system. Plant leaf is enclosed

in a transparent chamber. The CO2 fixation rate of the enclosed leaf is estimated by

measuring the alteration in the concentration of CO2 of the air flowing through the

chamber.

Procedure

The measurements were done in the morning time from 9.00 to 11.00 a.m. Different

conditions for operating the instrument included 1000 µmol m-2

s-1

photon flux density,

80-90% of air relative humidity, 25oC of air temperature and 400 µmol mol

-1 of CO2

concentration.

3.3.1.1.3. Cu(II) uptake

Cu(II) uptake by the plants was measured by atomic absorption spectrophotometer

(Shimadzu 6200).

Principle

Atomic absorption spectrophotometer is used to find out concentrations of metals in

solutions. The sample solution is aspirated in argon or acetylene powered flame. In the

flame the molecules in the sample solution get dissociated into atoms. Thermal

excitation of these atoms occurs in the flame but they remain in the ground state. A

42

hollow cathode lamp produces a monochromatic light, which passes through the top of

the flame. The atoms absorb this light. Beer’s law is followed here, as the absorption is

directly proportional to the concentration of metal atoms in the flame and path length in

the flame.

Reagents

Concentrated (Conc.) nitric acid

Conc. perchloric acid

CuSO4.5H2O

Procedure

The plant material was digested by employing the method of Khan et al. (2012) with

some modifications. The plant material was dried in an oven at 80oC for 24 h. It was

crushed into powder form. 0.5 g of the powdered plant samples was taken into digestion

beakers. To it 5 ml of conc. nitric acid was added and the mixture was heated on a hot

plate on low heat until the disappearance of nitric acid fumes. After it 2.5 ml of conc.

perchloric acid was added and heated first gently and then vigorously. When the

contents of the beakers were reduced to 1-2 ml, they were removed from the hot plate

and cooled. The contents of the beakers were transferred to some bigger glassware and

the volume was made to 50 ml with double distilled water. The resulting solution was

then filtered through Whatman No. 1 filter paper. Standard solutions of CuSO4.5H2O

were prepared and were aspirated through atomic absorption spectrophotometer to form

a calibration curve. After it individual plant samples were aspirated and their readings

were noted. Double distilled water was aspirated after each test sample for the cleaning

purpose.

3.3.1.1.4. Study of seed microsculpture

Seed microsculpture was studied using Zeiss environmental scanning electron

microscope, EVO LS10.

Principle

Environmental scanning electron microscope gives electron micrographs of wet and

uncoated biological samples. A beam of electrons is generated, which travels through

43

vacuum and is focussed by electromagnetic lenses to the specimen and scans the surface

of the specimen. The electron beam when interacts with the specimen, then

backscattered, secondary and augor electrons; light photons and x-rays are generated,

which are collected by different detectors in specimen chamber and image is produced

on the monitor of a computer.

Procedure

Seeds of B. juncea plants were mounted on Al adhesive tape applied on the surface of

specimen stubs. These were then given gold coating. Images of seed microsculpture

were produced by environmental scanning electron microscope under low vacuum,

extended pressure varying from 90-120 Pa and magnification, 1.00 KX.

3.3.1.2. Biochemical analysis

3.3.1.2.1. Carbohydrate metabolism

3.3.1.2.1.1. Spectrophotometric analysis

Carbohydrates such as total sugars, reducing sugars, glucose, fructose and sucrose were

measured with the help of spectrophotometer thermo electron corporation, Genesys

10UV.

Principle

A spectrophotometer performs qualitative and quantitative analysis of a sample solution

by measuring the amount of light absorbed by it. It is based upon Beer-Lambert law,

which states that there is a linear correlation between the light absorbed by a sample

solution and its concentration. It is written as: A = log (Io/I) = εlc, where, A is

absorbance, Io is intensity of light incident upon sample cell, I is intensity of light

leaving sample cell, ε is molar extinction coefficient, l is path length and c is the

concentration of sample solution.

Preparation of carbohydrates extract

Reagents

Ethanol (70% and 80%)

Saturated solution of basic lead acetate

Sodium oxalate crystals

44

Procedure

For the extraction of carbohydrates, method of Singh and Luthra (1988) was used. 5 g

of B. juncea leaves was taken in a 100 ml conical flask. Free sugars were extracted with

80% ethanol (2 times) and then with 70% ethanol (4 times) by keeping flask in boiling

water bath. Extracts were pooled together and the ethanol was removed at 40oC by

using rotary vacuum evaporator. The remaining aqueous syrup was transferred to 100

ml conical flask and 98 ml distilled water was added to make the volume up to 100 ml.

To precipitate out proteins, 1 ml solution of basic lead acetate was added. Further

distilled water was added to make the final volume up to 100 ml. Filtration of the

contents was carried out through Whatman No. 40 filter paper. Sodium oxalate crystals

were used to remove the excess lead ions by precipitation and the precipitates were

removed by filtration. So, a clear protein free test extract was obtained which was then

used to determine sugars.

Estimation of various carbohydrates

3.3.1.2.1.1.1. Total sugars

Reagents

Sulphuric acid (95%)

Phenol (5%)

Procedure

Total sugars were estimated using method of Dubois et al. (1956). 1 ml of test extract

was taken in a test tube, followed by the addition of 5% phenol and then 5 ml of 95%

sulphuric acid. After 10 min, tubes were held under running water for some time to

bring them to room temperature. After 20 min, absorbance was taken at 490 nm against

blank. The concentration of total sugars was determined from the standard curve

prepared using glucose standards.

3.3.1.2.1.1.2. Reducing sugars

Reagents

Reagent A: Anhydrous sodium carbonate (2.5 g), potassium sodium tartarate (2.5 g),

sodium bicarbonate (2 g) and anhydrous sodium sulphate (20 g) were dissolved in

distilled water and the final volume was made up to 100 ml.

45

Reagent B: CuSO4.5H2O (0.6 g) was dissolved in 4 ml distilled water. 1 drop of conc.

sulphuric acid was added.

Reagent C: It was prepared freshly by mixing reagent A and B in the ratio 25:1.

Reagent D: 2.8 g of ammonium molybdate was dissolved in 50 ml distilled water and

2.3 ml of conc. sulphuric acid was added. Separately, 0.33 g of sodium arsenate as

sodium arsenate was dissolved in 2.8 ml water and this solution was added to the

ammonium molybdate solution, drop wise. The resulting solution was incubated at 37oC

for 24 h and was stored in an amber coloured bottle.

Procedure

These were determined using the method of Nelson (1944). 1 ml of test extracts was

taken in test tubes, to them 1 ml of reagent C was added. The tubes were heated in a

boiling water bath for 20 min. The tubes were cooled to room temperature. 1 ml of

reagent D was added and was mixed thoroughly. The volume was made up to 10 ml.

The intensity of blue colour developed was measured at 520 nm against reagent blank.

The concentration of reducing sugars was determined from standard curve prepared

using glucose standards.

3.3.1.2.1.1.3. Glucose

Reagents

Reagent A: To 25 ml of 0.1 M potassium phosphate buffer (pH 7.0), 50 mg of glucose

oxidase was added followed by the addition of 2.5 mg of peroxidase.

Reagent B: 30 mg of o-dianisidine was added to 5 ml of methanol. The solution was

filtered and was stored in an amber coloured bottle in a refrigerator.

Reagent C: It was prepared by the mixing of 6 ml of reagent A, 3 ml of reagent B and

51 ml of 45% of glycerol.

Procedure

Glucose was determined by employing the method of Gascon and Lampen (1968).

Took 1 ml of the test extract in a test tube. To it 1 ml of solution C was added and the

contents were incubated for 20 min at 30oC. After it 2 ml of 2 N hydrogen chloride was

46

added. The intensity of pink colour developed was taken at 540 nm. The amount of

glucose was estimated from the standard curve of glucose.

3.3.1.2.1.1.4. Free fructose

The content of free fructose was estimated by taking the difference in the contents of

total reducing sugars and the glucose.

3.3.1.2.1.1.5. Sucrose

Reagents

Potassium hydroxide (6%)

Resorcinol solution (0.1% of resorcinol solution in glacial acetic acid)

Hydrogen chloride (30%)

Procedure

Sucrose was estimated by using the method of Roe (1934) with little modifications in it.

1 ml of test extract was taken in a test tube. To it was added, 1 ml of 6% potassium

hydroxide. The tubes containing the mixture were heated in a boiling water bath for 20

min for the destruction of free fructose. The tubes were cooled to room temperature.

After it 1 ml of 0.1% resorcinol solution and 3 ml of 30% hydrogen chloride were

added to the test tubes. At 80oC, incubation of tubes was done for 10 min. Absorbance

was measured at 490 nm. The amount of sucrose was measured from the standard curve

of glucose.

3.3.1.2.1.2. Ion chromatograph analysis

Other carbohydrates such as sorbitol, mannitol, cellobiose, arabinose and xylose were

analysed through Metrohm ion chromatograph, Orion-960

Principle

Ion chromatography separates the polar molecules or ions on the basis of their affinity

to ion exchanger. Its subtypes are anion exchange chromatography and cation exchange

chromatography. Anion exchange chromatography involves the attraction of negatively

charged molecules to a solid support having positive charge. Cation exchange

47

chromatography involves the attraction of positively charged molecules to a solid

support having negative charge.

Procedure

The plant material was dried in oven for 24 h at 80oC. It was then crushed to powder

form. 5 g of this powdered plant sample was dissolved in 10 ml of double distilled

water. The mixture was then incubated at room temperature for 12 h. It was first filtered

through Whatman No. 1 filter paper, then through nylon filter membrane of pore size

0.22 microns. The filtrate was analysed through ion chromatograph. The injection

volume was 20 µL. 100 mM sodium hydroxide was used as the eluent. Flow rate was 1

ml/min. Column temperature was set at 33oC and pressure was 11.9 MPa.

3.3.1.2.2. Lipid peroxidation

Lipid peroxidation was measured in terms of malondialdehyde (MDA) content. Lipid

peroxidation was estimated by using spectrophotometer thermo electron corporation,

Genesys 10 UV.

Principle

Reactive oxygen species abstracts electrons from unsaturated fatty acids and lipid

radicals are formed. Molecular oxygen generates lipid peroxy radicals by reacting with

lipid radicals and a chain reaction is started resulting in the formation of lipid peroxides.

MDA is produced in these reactions which forms an adduct with thiobarbituric acid

which absorbs at 532 nm.

Reagents

0.1% trichloroacetic acid

0.5% thiobarbituric acid in 20% trichloroacetic acid

Procedure

Method of Heath and Packer (1968) was employed to measure lipid peroxidation.

Homogenisation of 1 g of plant sample of 30, 60 and 90-day old B. juncea was done in

3 ml of 0.1% solution of trichloroacetic acid. After homogenisation, samples were

centrifuged for 5 min at 10,000 rpm. The resulting supernatant was collected. 3 ml of

48

0.5% solution of thiobarbituric acid was added to the supernatant. The mixture was

heated in a water bath for 30 min at 95oC. It was cooled immediately. Absorbance was

taken at 532 and 600 nm.

Calculation

Here, A is absorbance at 532 nm - absorbance at 600 nm and extinction coefficient is

155 mM-1

cm-1

3.3.1.2.3. Lipid metabolism

The contents of lipids were measured by using spectrophotometer thermo electron

corporation, Genesys 10 UV.

Preparation of lipid extract

Reagents

Methanol and chloroform mixture (2:1)

Chloroform

Procedure

Method used by Kates (1970) was used for the extraction of lipids. 12.5 g of B. juncea

leaves were blended with 60 ml of methanol-chloroform (2:1, v/v). The resulting

homogenate was filtered and the filter residue was reblended with a mixture of 60 ml

methanol-chloroform (2:1, v/v) and 16 ml of water. The homogenate was filtered. Filter

residue was washed with 30 ml of methanol-chloroform (2:1, v/v). Filtrates were

combined and were taken in a separatory funnel. To the mixture was added, 50 ml of

chloroform and 58 ml of water. Phases were allowed to separate. Chloroform layer was

withdrawn and was dried with the help of a rotary vacuum evaporator. The residual

lipids were immediately dissolved in 50 ml of chloroform.

49

Estimation of various lipids

3.3.1.2.3.1. Phospholipids

Reagents

10% of magnesium nitrate prepared in 95% of ethanol

Hydrogen chloride (0.5 N)

Reagent A: It was prepared by mixing ascorbic acid (10% w/v) and ammonium

molybdate (0.42 w/v in 1 N sulphuric acid).

Procedure

First organic phosphorous was converted to inorganic form by using the method of

Ames and Dubin (1960). 1 ml of test extract was taken in a test tube and chloroform

was evaporated. To each test tube, 0.18 ml of magnesium nitrate. The contents were

heated on a low flame until they became dry and then on a strong flame until the

disappearance of brown fumes. The tubes were cooled. 1.8 ml of hydrogen chloride was

added to each test tube. The tubes were capped with water ampules and heated in a

boiling water bath for 15 min. This process converted the organic phosphorous into

inorganic form. Then to estimate the phosphorous, the tubes were cooled. To the 1.8 ml

of sample solution obtained above 4.2 ml of reagent A was added and incubated at 45oC

for 20 min in a water bath. The tubes were cooled and the absorbance was taken at 820

nm against a blank. The concentration of phosphorous in the samples was calculated

from a standard curve of potassium dihydrogen orthophosphate.

3.3.1.2.3.2. Glycolipids

Reagents

Phenol (5%)

Conc. sulphuric acid (36 N)

Procedure

These were determined with the method of Dubois et al. (1956). 0.5 ml of the lipid

sample in chloroform was taken in a test tube. The chloroform was evaporated. 2 ml of

50

distilled water was added and was shaken vigorously. 1 ml of 5% phenol was added and

was mixed by shaking. 5 ml of conc. sulphuric acid was added and the tubes were kept

at room temperature for 30 min. The absorbance was taken at 490 nm against a blank.

The concentration of glucose was estimated from the standard curve of glucose.

3.3.1.2.3.3. α-tocopherol

Reagents

0.07% α-α' bipyridyl solution in ethanol

Ferric chloride (0.2%)

Procedure

Estimation of α-tocopherol was done using the method of Gaunt and Barlow (1971). 1

ml of the lipid sample was taken in a test tube. To it 3-5 ml of 0.07% α-α' bipyridyl

solution was added. The tube was shaken thoroughly. After it, addition of 0.50 ml of

ferric chloride was done. Exactly 2 min after it, absorbance was taken at 520 nm. The

amount of α-tocopherol in the sample was calculated from the standard curve prepared

by using α-tocopherol in chloroform.

3.3.1.2.3.4. Total sterols

Reagents

Chloroform

Acetic anhydride

Conc. sulphuric acid

Procedure

Method of Sperry and Webb (1950) was used.0.5 ml of lipid sample was taken in a test

tube. To it 5 ml of chloroform and 1 ml of acetic anhydride were added and mixed

thoroughly. To this mixture, 0.1 ml of conc. sulphuric acid was added. Absorbance was

taken at 625 nm against blank. Concentration of total sterols was measured from a

standard curve of ergosterol.

51

3.3.1.2.3.5. Esterified sterols

Reagents

1% solution of digitonin in 80% ethanol

Petroleum ether (40oC-60

oC)

Procedure

1 ml of lipid sample was taken a test tube and to it, 0.25 ml of 1% of digitonin was

added and mixed thoroughly. The mixture was evaporated by heating at 60oC. After it 3

ml of petroleum ether was added to the test tubes. The tubes were covered and heated

in a water bath at 60oC such that half of the solvent got evaporated. The test tubes were

cooled and the esterified sterols were then measured using the method of Sperry and

Webb (1950), the same method used for the estimation of total sterols.

3.3.1.2.4. PGRs

The endogenous content of plant growth regulators in the plant samples was analysed

through Agilent 6410 Triple-Quad liquid chromatography mass spectrophotometer

(LCMS).

Principle

LCMS combines the physical separation potential of HPLC with mass analysis potential

of mass spectrophotometer. In it the molecules in the test samples are converted into

ions or charged molecules which are separated on the basis of their mass to charge ratio

in an analyzer by electromagnetic fields.

Reagents

80% methanol (HPLC grade)

0.5% solution of formic acid in Milli-Q water

Absolute methanol (HPLC grade)

Sample preparation

0.5 g of the plant leaves were homogenised in 5 ml of 80% of methanol. The resulting

extract was centrifuged and the supernatant was filtered through nylon filter membrane

52

of 0.22 microns pore size. The resulting filtrate was used to measure the endogenous

content plant hormones including polyamines (putrescine, spermidine, spermine and

cadaverine); jasmonic acid; abscisic acid; auxins (indole-3-acetic acid, indole-3-butyric

acid and phenylacetic acid) and salicylic acid using LCMS.

LCMS analysis

The endogenous content of the plant hormones was estimated using the method of

Banerjee and Kulkarni (2011). Injection volume of the samples was 2 µL. 0.5% solution

of formic acid was the mobile phase A and methanol was the mobile phase B. Column

temperature was set at 40oC. Flow rate was 200 µL min

-1. Run time in the positive

mode was 16 min and in the negative mode was 6 min.

3.3.1.2.5. Polyphenols

Polyphenols were estimated through Shimadzu ultra performance liquid

chromatography (UPLC) Naxera system (Shimadzu, USA) coupled with photodiode

array detector.

Principle

UPLC separates components in a mixture for their identification and quantification. It

consists of a stationary phase with particles smaller than 2 µm, to give better sensitivity,

resolution and speed than HPLC (Srivastava et al., 2010). As the particle size of column

packing reduces, efficiency and thereby resolution enhances. It uses high pressures

ranging from 8000 to 15000 PSI. Liquid solvent containing sample is passed at high

pressure through a column containing solid adsorbent material. Individual components

in the sample are separated on the basis of their differential interaction with the

adsorbent material, which results in their variable flow rates.

Reagents

80% methanol (HPLC grade)

Absolute methanol (HPLC grade)

0.01% solution of acetic acid in Milli-Q water

53

Sample preparation

Homogenisation of 0.50 g of plant sample was done in 4 ml of 80% methanol. The

homogenised samples were then centrifuged at 13,000 rpm for 20 min. Supernatant was

collected and filtered through 0.22 microns pore sized nylon filter membrane. The

resulting filtrate was analysed through UPLC to determine the presence of polyphenols

such as chlorogenic acid, gallic acid, caffeic acid, catechin, kaempferol, ellagic acid,

epicatechin, coumaric acid, quercetin, umbelliferone and rutin.

UPLC analysis

5 µL of the filtrate obtained above was injected into UPLC system. C-18 column (150 ×

4.6 mm) with 5 µM pore size carried out the chromatographic process at room

temperature. Flow rate was 1 ml min-1 and λ was 280 nm. 0.01% solution of acetic acid

was solvent A and methanol was solvent B. The software provided with the UPLC

system determined the peaks. Calibration curves were formed by plotting

concentrations against peak areas. The compounds were detected on the basis of their

spectral similarity and retention time.

3.3.1.2.6. Elements

3.3.1.2.6.1. N and S

N and S contents were estimated though elemental analyser elementar, Vario EL III.

Principle

It is based on catalytic tube combustion of the dry test sample. Gases formed during the

process are separated by their adsorption on different columns which are ultimately

detected by thermal conductivity detector.

Sample analysis

The plant samples were dried in oven for 24 h at 80oC. The dried samples were then

crushed to powder form. 3 mg of this powdered plant sample was then analysed through

elemental analyser.

3.3.1.2.6.2. Na and K

Na and K contents were estimated through flame photometer (Systronics 128)

54

Principle

In a flame photometer due to thermal dissociation of compounds of alkali and alkaline

earth metals in a flame, some atoms are produced which are excited to higher levels of

energy. On returning to the ground state, these atoms emit radiations with wavelengths

lying in the visible region of the spectrum. Each element emits radiations of a specific

wavelength. Na and K emit radiations at a wavelength of 589 and 766 nm and give

yellow and violet colour in a flame, respectively.

3.3.1.2.6.2.1. Na

Reagents

Sodium chloride

Procedure

Preparation of samples for Na was done as for the determination of Cu(II) (Section:

3.3.1.1.3). The standard solutions of Na were prepared by dissolving sodium chloride in

distilled water. A calibration curve was prepared by running these standards through

flame photometer. After that the plant samples were aspirated through the instrument

and their readings were noted. Double distilled water was aspirated through the

instrument after every test sample.

3.3.1.2.6.2.2. K

Reagents

Potassium chloride

Procedure

Samples for K were prepared as for Cu(II) (Section: 3.3.1.1.3). The standard solutions

of K were prepared by dissolving potassium chloride in distilled water. A calibration

curve was prepared by running these standards through flame photometer. After it the

plant samples were aspirated through the instrument and their readings were noted.

Double distilled water was aspirated through the instrument after every test sample.

55

3.3.1.2.6.3. Ca and Mg

Titration method was used to estimate Ca and Mg content in the plant samples.

Principle

In titration, titrant is slowly added to a known volume of unknown concentration until

the reaction approaches neutralisation, which is visible in the form of colour change.

3.3.1.2.6.3.1. Ca

Reagents

Ethylenediaminetetraacetic acid (EDTA) solution: 0.93 g of EDTA was dissolved in 1 L

of double distilled water.

1 M sodium hydroxide

Murexide as indicator: 0.1 g of murexide (ammonium purpurate) and 50 g of sodium

chloride were crushed in a mortar and the mixture was stored in an amber coloured

bottle.

Procedure

Samples for the determination of Ca were prepared as used for Cu(II) (Section:

3.3.1.1.3). Titration method of Allen et al. (1976) was followed to determine Ca

content. 5 ml of test sample was taken in a titration flask. 5 ml of 1 M solution of

sodium hydroxide was added to it and the volume was raised to 100 ml with double

distilled water. After it, 0.1 of murexide was added. The mixture was shaken

thoroughly. Titration with EDTA solution was carried out until the appearance of bluish

end point. Blank determination was carried out by taking 5 ml of double distilled water

in place of the test sample and after that the above procedure was followed for titration.

The blank reading was subtracted from the sample values. The amount of Ca in the test

samples was calculated by using the following formula:

Here, A = 0.1 mg Ca/1 ml EDTA solution

56

3.3.1.2.6.3.2. Mg

Reagents

EDTA solution: 0.93 g of EDTA was dissolved in 1 L of double distilled water.

Indicator solution: 0.25 g of erichrome black T was dissolved in 50 ml of industrial

spirit.

Triethanolamine

Buffer solution: 67.5 g of ammonium chloride was dissolved in double distilled water.

570 ml of 0.88 ammonia solution was added to it and the volume was made up to 1 L

with double distilled water.

Procedure

Preparation of samples for Mg was done as for the determination of Cu(II) (Section:

3.3.1.1.3). Titration method of Allen et al. (1976) was followed to determine Mg

content. 5 ml of the test sample was taken in a titration flask. 7.5 ml of buffer solution, 1

ml of triethanolamine and 5 drops of indicator solution were added. The volume was

raised to 100 ml with double distilled water. The resulting solution was shaken

thoroughly. Titration with EDTA solution was carried out until the colour changed from

red to clear blue. Blank determination was carried out by taking 5 ml of double distilled

water in place of the test sample and after that the above procedure was followed for

titration. The blank reading was subtracted from the sample values. The Ca + Mg

content in the test samples was calculated by using the following formula:

Here, A = 0.1 mg Mg/1 ml EDTA solution

The Ca results were subtracted from the Ca + Mg results to determine the content of

Mg.

57

3.3.2. Studies done on the 7-day old B. juncea seedlings raised in the Petri dishes in

seed germinator:

3.3.2.1. Study of cell peroxidation and cell death

Cell peroxidaiton and cell death was studied in the roots of 7-day old B. juncea

seedlings using Nikon laser scanning confocal microscope, A1R with resonant scanner

made by Nikon corporation, Japan.

Principle

Confocal microscope is used to generate three dimensional images of non-biological

and biological specimens. It involves sequential illumination in contrast to simultaneous

illumination in the conventional microscopes. At a time, one volume element of

specimen is focussed as a spot by illumination from a laser source. The specimen emits

fluorescence. Through a detector pinhole this fluorescence is directed to a

photomultiplier. The point is displayed as a pixel on the screen of a computer. For

producing the entire image, a mirror moves the light point line by line over the entire

specimen and simultaneous image is generated on a monitor (Singh and Gopinathan,

1998).

Cu metal stress leads to the generation of peroxides. To study the extent of peroxides

generated plant sample is labelled with the fluorescent d , -dichlorofluorescin

diacetate. Peroxides oxidize this dye and produce green fluorescence, which is finally

detected using confocal microscope. Excessive Cu metal stress also results in cell death.

To check cell viability, plant samples are labelled with propidium iodide. Propidium

iodide penetrates through the damaged plasma membranes of dead cells and intercalates

itself in the double helicle structure of DNA and produces red fluorescence, due to

which nuclei appear as red spots in the image produced by the confocal microscope.

Reagents

2 , 7 -dichlorofluorescin diacetate (10 µM)

Propidium iodide (25 µM)

58

Procedure

The method of Ortega-Villasante et al. (2005) was used with slight modifications. A

portion of roots of 7-day old B. juncea seedlings, about 0.5 cm, from the root tips was

cut and was labelled for 15 min with a solution of 0.5 ml of 2 , 7 -dichlorofluorescin

diacetate (10 µM) and 0.5 ml of propidium iodide (25 µM). Root samples were

observed with the help of a confocal microscope.

3.3.2.2. Stomatal studies

Stomata in B. juncea leaves were studied using Zeiss environmental scanning electron

microscope, EVO LS10.

Procedure

Leaves of the 7-day old B. juncea seedlings were cut from petioles and were mounted

on Al adhesive tape applied on the surface of specimen stubs. To study stomata, leaves

were observed with the help of environmental scanning electron microscope under low

vacuum, extended pressure varying from 90-120 Pa and magnification, 1.50 KX.

3.4. Statistical Analysis

The data was analysed statistically using self coding software. Mean and standard

deviation (SD) were calculated and the data was presented as mean ± SD.

Where, is an observation, is the sample mean and n is the number of observations.

Two way analysis of variance (ANOVA): In order to test the null hypothesis (Ho), that

no two sample means were different from each other, two way ANOVA was used. The

alternative (H1) hypothesis tested that at least two sample means are significantly

different from each other at a given P level.

Honestly significant difference (HSD): To determine whether different means were

statistically different from each other or not, HSD was calculated, using Turkey’s

multiple comparison test.

59

Multiple linear regression: In multiple regression analysis, instead of a single

independent variable, effect of two or more independent variables on the value of

dependent variable, is estimated.

Where, is dependent variable; are 1st and 2

nd independent variables,

respectively; are partial regression coefficients and a = Y intercept.

Multiple regression with interaction: The type of effect induced by the interaction

between Cu(II) and 24-EpiBR on a parameter was determined by applying multiple

regression with interaction on the data.

β-regression: It gave relative effects of independent variables: X1 [Cu(II)] and X2 (24-

EpiBR) on the dependent variable, Y (any parameter), irrespective of their units. A

higher absolute value of β-regression implies that the variable has more relative effect

on the dependent variable. Table 1. gives the interaction in terms of β-regression

coefficients (Rupinder et al. 2009).

Table 3.1. Interaction in terms of β-regression coefficients (modified after

Rupinder et al., 2009).

Interaction Variables

X1 X2 X1 X2

β-regression coefficients

β1 β2 β3

Synergistic + + +

Synergistic - - -

Antagonistic + + -

Antagonistic - - +

Mixed: X1 antagonistic to X2, but X2 synergistic to X1 + - +

Mixed: X1 synergistic to X2, but X2 antagonistic to X1 + - -

Additive +/- +/- 0

60

Multiple correlation: To find out correlation between three sets of variables (Y, X1 and

X2) we determined the multiple correlation coefficient (r).

Where, r is multiple correlation coefficient, is dependent variable; are 1st and

2nd

independent variables, respectively; are regression coefficients and n is

sample size.

% variability explained: Coefficient of determination (r2) is the variability explained by

the correlation of dependent variable as a function of independent variables and is

represented as percent.