Materials and Methods

MATERIALS AND METHODS

1. PLANT MATERIAL

Centella asiatica (L.) Urb. is a common plant belonging to the family

Umbelliferae (Apiaceae). Healthy Plants were collected from the paddy fields

in Malappuram and Kozhikode Districts for the present study. Multiplication

of the plants were done by planting the cuttings of runner collected from the

healthy plants grown in garden pots filled with garden soil, sand and dried

powdered cow dung mixed in the ratio 2:1:1. The pots with plants were

watered regularly and allowed to establish and maintained in the net /poly

house of the Department of Botany, University of Calicut, throughout the

course of the investigation.

2. NUTRIENT CULTURE STUDIES

Cuttings of plants growing in garden pots were collected from healthy

plants and rooting was done by planting in trays filled with soil. When the

cuttings develop roots from the nodes the plants were carefully removed from

the soil, which was used for the nutrient culture studies.

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2. 1 Preparation of Nutrient Solution

The modified Hoagland's solution after Epstein (1972) as described by

Taiz and Zeiger (2002) was prepared and used for the present study.

Composition of the modified Hoagland‟s solution is shown in Table 1.

The stock solution of each nutrient was prepared separately and

appropriate volumes were mixed to make up nutrient solution of final volume

and concentration. The pH of the final solution was adjusted to 6.8 using

0.1 N HCl or NaOH.

2.2 Preparation of Mercuric Chloride and Triadimefon Solutions

One molar solution of mercuric chloride (HgCl2) was prepared as the

stock solution. Trial experiment was conducted by planting rooted propagules

of C.asiatica plants in nutrient solution containing different concentrations of

mercuric chloride such as 5, 10, 15 and 20M in order to determine the

optimum concentration in which the experimental plants survived and

exhibited about 50 percent growth retardation. Out of the various

concentrations tried 15M was found to be optimum for the present study.

Appropriate volumes of HgCl2 solution were mixed with required volume of

nutrient stock solution and final volume was made up to 1000ml with 15M

HgCl2.

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 Triadimefon stock solution was prepared by dissolving 1gm of

triadimefon in one litre of Hoagland‟s solution. Rooted propagules of

Centella asiatica were planted in nutrient solution to which different

concentration of triadimefon was added. From the trials, plants grown in

15mgl-1 triadimefon solution were selected for further experiments. In order to

determine the antagonistic effect of triadimefon, the selected concentrations

of both HgCl2 and triadimefon solution were mixed together and experimental

plants were also allowed to grow in it. So a combination of mercuric chloride

and triadimefon solution was prepared by adding 15 mg of traidimefon to

1000 ml of 15 µM HgCl2 solution prepared in Hoagland nutrient medium and

the present investigation includes the following sets:

1. Control plants growing in Hoagland's medium (Represented as C),

2. Plants grown in 15M HgCl2 solution (Represented as Hg),

3. Plants grown in 15mgl-1 triadimefon solution and (Represented as Tri),

and

4. Plants grown in a combination of 15M HgCl2 + 15mgl-1 triadimefon

solution (Represented as Hg + Tri).

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 Table 1: Composition of a modified Hoagland nutrient solution employed in the present study.

Concentration Concentration Volume of stock Final

Molecular
Compound of stock of stock solution per litre concentration
weight
solution solution of final solution of element
g mol-1 mM g l-1 ml M ppm
Macronutrients
KNO3 101.10 1,000 101.10 6.0 16,000 224
Ca(NO3). 4H2O 236.16 1,000 236.16 4.0 6,000 235
NH4H2PO4 115.08 1,000 115.08 2.0 4,000 160
MgSO4. 7H2O 246.48 1,000 246.49 1.0 2,000 62
1,000 32
1,000 24

Micronutrient
KCl 74.55 25.0 1.864 50.0 1.77
H3BO3 61.83 12.5 0.773 25.0 0.27
MnSO4. H2O 169.01 1.0 0.169 2.0 0.11
ZnSO4. 7H2O 287.54 1.0 0.288 2.0 2.0 0.13
CuSO4.5H2O 249.68 0.25 0.062 0.5 0.03
H2MoO4 (85% 161.97 0.25 0.040 0.5 0.05
MoO3)
NaFeEDTA 558.50 53.7 30.00 0.3-1.0 16.1-53-7 1.00-
(10% Fe) 3.00
Adopted from Taiz and Zeiger ( 2002)

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 Good quality of plastic containers with size 20155cm were selected

to take nutrient solution. Slightly large sized plastic net trays of size 30205

cm having holes of 55 mm were placed above the plastic container. The

roots of the propagules were inserted through the holes of the tray in to the

nutrient solution taken in the container. Plastic twine of diameter with 1mm

was tied interweavingly length-wise and breadth-wise on the trays forming a

mesh to provide mechanical support to the propagules. The trays were placed

in such a way that only the roots become in touch with the nutrient solution.

Ten trays for each treatment were taken and 1000 ml of Hoagland‟s solution

was added to one tray, which served as the control. Three trays each were

filled separately with 1000 l of nutrient + HgCl2, nutrient + triadimefon and

combination of both HgCl2 and triadimefon. Additional nutrient solution was

added to replenish the nutrient medium at an interval of 4 days as and when

required.

3. SAMPLING

Random sampling was followed by collecting a minimum of six plants

from the trays of each treatment and control at intervals of 4 days from the

day of treatment up to 24th day there by constituting seven samples of each

treatment. The collected plants were thoroughly washed in tap water, blotted

to remove water adhered on it and separated into root, runner and leaves. All

these samples were used separately for various studies.

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4. MORPHOLOGICAL STUDIES

Growth of the treated as well as control plants was assessed in terms of

root length, runner (stem) length, petiolar length, leaf area, tolerance index

percentage, stomatal index and total biomass. The fresh weight and dry

weight of individual plant parts like roots, runner and leaves were also

determined.

The length of root, runner and petiole was measured using a graduated

scale and was expressed in centimetres. The runner length was measured from

the basal part of the runner to the extreme tip of the plant. In C. asiatica the

root system appear as a tuft of “fibrous like” and so the length of the longest

root in each tuft is taken in to consideration for measuring the length of the

roots.

The leaf area of the treated and control plants were measured using

graph paper method and was expressed in cm2. All these data were taken

from randomly selected six plants each and the mean values were calculated.

4.1 Fresh Weight

For fresh weight determination the plants were carefully removed from

the nutrient solution, washed thoroughly in water and blotted to dryness. The

plant parts such as root, runner, and leaves were separated. Fresh weight of

individual plant parts was determined using a Shimadzu electronic balance.

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4.2 Tolerance Index Percentage

Tolerance index (TI) percentage was calculated according to the

method of Turner (1994).

Observed value in solution with metal

TI%  X 100
Observed value in solution without metal

For calculating Tolerance Index root length, runner length and leaf area

were taken as parameters.

4.3 Studies on Stomata

To study stomatal density, number of stomata on abaxial and adaxial

sides of the leaf were counted under a light microscope, preparing

impressions using a colourless nail-polish. Stomatal index was calculated

according to the following method of Meidner and Mansfield (1968).

Number of stomata per unit area

Stomatal index   100
Number of stomata per unit area  number of epidermal cells per unit area

4.4 Biomass Determination

Total biomass content of the control and experimental plants were

calculated on per plant basis. For biomass determination each plant from

treated and control were selected and the root, stem (runner) and leaf tissues

were taken separately for dry weight determination. The separated root, stem

and leaf tissues were oven dried initially at 1000C for one hour and then at

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600C till the weight becomes constant. Total biomass per plant was calculated

from the sum of the dry weight of root, stem (runner) and leaf tissues.

5. ANATOMICAL / HISTOCHEMICAL STUDIES

Free-hand sections of the root, stem and leaf tissues of control and

treated plants of C. asiatica samples on 4th, 12th, and 24th days were taken and

stained with safranin and semi permanent slides were prepared. Sections were

observed using Nikon microscope (Model Nikon ECLIPSE E400) and

photomicrographs were taken using Nikon Digital Camera (Model D x M

1200F) and image analyser.

6. PHYSIOLOGICAL/ BIOCHEMICAL STUDIES

6.1 Chlorophyll

Chlorophyll estimation of leaves of treated and control plants was done

according to the method of Arnon (1949). Two hundred milligram of fresh

leaf tissues of each sample were homogenized using chilled acetone in a pre-

chilled clean glass mortar and pestle. The homogenate was centrifuged for 10

minutes and the supernatant was collected. The residue was again extracted

with 80% acetone and centrifuged. The supernatant was pooled together and

the extraction process was repeated until the residue become colourless.

Volume of the combined supernatant was noted. The absorbance of the

solution was measured at 645 nm and 663 nm against the solvent (80%

acetone) as blank.

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 The amount of chlorophyll present in the extract was calculated in mg

chlorophyll per gram tissue according to the following equation (Arnon,

1949).

V
mg chlorophyll a/g tissue = 12.7 (A663) – 2.69 (A645)
1000  w

V
mg chlorophyll b/g tissue = 22.9 (A645) – 4.68 (A663)
1000  w

mg total

V
chlorophyll /g tissue = 20.2 (A645) + 8.02 (A663)
1000  w

Where A = Absorbance at specific wave lengths

V = Final volume of chlorophyll extract in 80% acetone

and W = Fresh weight of tissue extracted

6.2 Protein

Protein content of individual plant parts sampled at each interval was

estimated using Folin – Ciocalteau reagent according to the method of Lowry

et al. (1951).

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6.2.1 Extraction

Five Hundred mg of fresh tissues of treated as well as control plant

parts, such as root, runner and leaves were homogenized in 5ml of 50 mM

Phosphate buffer (pH 7.5) containing 100 mM 2-mercapto ethanol using pre-

chilled glass mortar and pestle. Volume of the homogenate was measured.

One millilitre of the homogenate in duplicate was taken in clean dry test tubes

for the estimation of total protein. The remaining part of the homogenate was

transferred to centrifuge tubes and centrifuged for 10 minutes. The

supernatant was collected and used for the estimation of soluble proteins. One

millilitre each of the homogenate as well as supernatant was taken in

duplicate and equal volume of 10% (w/v) trichloroaceticacid was added and

kept in a refrigerator for one hour for flocculation. The protein precipitated

was collected by centrifugation for 5 minutes, the supernatant was decanted

off. The residue was washed twice with cold 2% trichloroaceticacid (w/v)

followed by washing with anhydrous acetone and then by 80% (v/v) acetone.

6.2.2 Estimation

The pellet obtained after centrifugation was digested in 5 ml of 0.1 N

sodium hydroxide by heating in a bath of boiling water for 5 minutes. The

suspension was clarified by centrifugation and supernatant was collected.

Suitable aliquots were taken in triplicate and the volume was made up to 1 ml

with double distilled water. To the aliquots 5ml of alkaline copper reagent

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was added and shaken well. After 10 minutes 0.5 ml of IN Folin- Ciocalteau

reagent was added and shaken well immediately. The tubes were kept for 30

minutes for colour development. The optical density of the solution was read

at 700 nm using a GENESIS 20 Spectrophotometer. Bovine Serum Albumin

fraction V powder was used as standard.

6.3 PAGE Studies of Proteins

SDS polyacrylamide gel electrophoresis was carried out according to

the method of Gaal et al. (1980). Five hundred milligrams of the root, stem

and leaf tissues of the plants of control as well as the treatments were

homogenized using a chilled mortar and pestle in 50mM phosphate buffer in

the presence of polyvinyl polypyrrolidone as phenolic binder and 10% sodium

dodecyl sulphate. The 10% homogenate was centrifuged at 16,000Xg for 20

minutes using a Plastocraft model ROTA R4 Rv/Fm refrigerated centrifuge at

40C and the supernatant was collected.

6.3.1 Preparation of Gels

The resolving gel was prepared by mixing 3.3ml of

acrylamide/bissacrylamide (30% T and 2.67 % C), 5ml of 1.0M resolving gel

buffer (pH 8.8), 50l of 10% ammonium persulphate, 50l of 10% SDS and

5.0l TEMED. The mixture was made upto 10ml with deionized water.

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 The stacking gel was prepared by mixing 0.99ml of acrylamide/

bisacrylamide (30% T and 2.67% C), 3ml of 0.5M resolving gel buffer (pH –

6.8), 30l of 10% ammonium persulphate, 30l of 10% SDS and 5l

TEMED. The mixture was made upto 6ml with deionized water.

6.3.2 Gel Casting

The gel was cast in a Genei Mini vertical gel casting unit. The glass

plates, the comb and the spacers of the casting unit were wiped clean with

alcohol using tissue paper. The glass plates were then wiped with acetone.

The dried glass plates were clamped on the casting unit with the spacers

placed in between them. The resolving gel was poured into the casting unit

and the top was layered with a small volume of deionized water to avoid

contact with air. After completion of polymerization, the water was removed

with strips of filter paper. Then the comb was placed and the stacking gel was

poured carefully. The gel was topped with deionized water. After

polymerization, the comb was removed carefully and the well were cleaned

thoroughly.

6.3.3 Electrophoresis

Fourty millilitre of the extract containing 20% sucrose was added to

each well. Bromophenol blue was used as the tracking dye. Medium range

molecular weight Marker (Genei) was loaded in one of the wells.

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Electrophoresis was carried out using the electrophoretic reservoir buffer,

Tris-glycine, pH-8.4. Initially the gels were maintained at a voltage of 80v.

Once the stacking has taken place, the voltage was raised to 120v and was

maintained there till the electrophoretic run was completed. At the end of the

run, the gel was carefully removed and was stained with 0.2% (w/v)

coomassie brilliant blue R 250 in methanol acetic acid mixture. After 3 hours

of staining, the gels were destained in methanol acetic acid mixture and stored

in 7% (v/v) acetic acid.

The gels were analysed in a Broad Geldoc and molecular weight of the

bands was determined using the software, Quantity-One.

6.4 Total Free Amino Acids

Total free amino acids of plant parts such as root, runner and leaves of

treated and control plants of C. asiatica were estimated according to the

method of Lee and Takahashi (1966).

6.4.1 Extraction

One gram of the fresh tissue was homogenized in 80% (v/v) alcohol

using a clean glass mortar and pestle. The homogenate was transferred to a

round-bottomed flask fitted with vertical condenser and refluxed on a boiling

water bath for 2 hours. Then the suspension was centrifuged and the

supernatant was collected. The residue was re-extracted with 80% alcohol and

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after each centrifugation the supernatant was combined with the original

supernatant. The combined supernatant was then evaporated to dryness over

a boiling water bath, eluted using a known quantity of 10% iso-propanol. This

extract was used for the determination of total free amino acids using

ninhydrin.

6.4.1.1 Preparation of ninhydrin – citrate – glycerol reagent

One ml of 1% (w/v) ninhydrin solution in 0.5M citrate buffer (pH 5.5)

was mixed thoroughly with 2.4 ml of glycerol and 0.4 ml of 0.5 M citrate

buffer (pH5.5)

6.4.2 Estimation

To 0.2 ml of the sample, 3.8 ml of ninhydrin – citrate – glycerol

mixture was added. After shaking well, the mixture was heated in a boiling

water bath for 12 minutes and cooled to room temperature, by keeping in tap

water. Within one hour the optical density of the resultant solution was

measured at 570nm using a GENESIS 20 Spectrophotometer. The reagent

blank was prepared by mixing 0.2 ml water and 3.8 ml ninhydrin – citrate –

glycerol. Glycine was used as the standard.

6.5 Proline

Proline content in the plant parts was estimated according to the

method of Bates et al. (1973).

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6.5.1 Extraction

One gram fresh tissue of plant parts such as root, runner and leaves

each of the experimental and control plants was homogenised in 10 ml of 3%

(w/v) aqueous sulfosalicylic acid using a clean glass mortar and pestle. The

homogenate was transferred to centrifuge tubes and centrifuged for 10

minutes, and the supernatant was collected and estimation of proline was done

using acid ninhydrin.

6.5.1.1 Preparation of acid ninhydrin

Acid Ninhydrin was prepared by dissolving 1.25 gm of Ninhydrin in a

mixture of 30 ml of glacial acetic acid and 20 ml of 6 M ortho

phosphoricacid.

6.5.2 Estimation

From the supernatant 2 ml was taken in test tubes in triplicate and

equal volumes of glacial acetic acid and acid ninhydrin were added to it. The

tubes were then heated in a bath of boiling water for one hour and then the

reaction was terminated by placing the tubes in an ice bath. For colour

development 4 ml of toluene was added to the reaction mixture and stirred

well for 20-30 seconds. Then the toluene layer was separated carefully and

brought to room temperature. The colour intensity of the solution was

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measured at a wave length of 520 nm using PHOTOCHEM colorimeter. L-

Proline was used as the standard.

6.6 Phenolics

Total phenolics was estimated using Folin-Denis Reagent (Folin and

Denis, 1915). Tannic acid was used as the standard.

6.6.1 Extraction

One gram of fresh tissue of treated and control plant parts was

homogenized in 10 ml of 80% (v/v) alcohol using a clean glass mortar and

pestle. The homogenate was quantitatively transferred to a round bottomed

flask fitted with vertical condenser, and refluxed for two hours over a boiling

water bath. The suspension was then cooled, centrifuged for 10 minutes and

the supernatant was collected. The residue was re-extracted with 80% alcohol

and refluxed for one hour. The supernatant was collected, and combined with

the original. The total volume of the supernatant was noted.

6.6.1.1 Preparation of Folin-Denis reagent

To 750 ml of distilled water, 100 g sodium tungstate, 20 g

phosphomolybdic acid and 50 ml ortho phosphoric acid were added. This

mixture was refluxed for 2 hrs, cooled and diluted to 1 litre. Normality of the

reagent was determined by titrating it against an alkali NaOH with

phenolphthalein as an indicator.

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6.6.2 Estimation

Aliquots of 2ml in triplicate were pipetted out and equal volume of

Folin – Denis reagent was added. The contents were thoroughly mixed and

after 3 minutes 2 ml of IN sodium carbonate was added. This mixture was

kept for one hour, after thorough mixing, for colour development. The optical

density of the resultant solution was measured at 700 nm using a GENESIS -

20 Spectrophotometer.

6.7 Peroxidase Assay

Peroxidase activity of plant parts such as root, runner and leaf was

measured according to the method of Abeles and Biles (1991).

6.7.1 Extraction of Enzymes

From the randomised tissue samples1.0 g each of root, stem (runner)

and leaf of control as well as treatments, were weighed and a 10%

homogenate was prepared by grinding in 50 mM Tris-HCl buffer (pH 7.5)

and 50 mg of polyvinyl poly pyrrolidone using a pre-chilled mortar and

pestle. The extract was filtered through two layers of muslin cloth and was

made up to 10 ml. The filtrate was transferred to centrifuge tubes and

centrifuged in a cold centrifuge at 15,000 rpm for 15 minutes at 0 oC using

Plastocraft cold centrifuge model (ROTA R4 RV/FM). The supernatant was

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transferred to a clean test tube and stored in ice bath. One part was used for

enzyme assay and the rest for protein estimation.

6.7.2 Enzyme Assay

Assay system consisted of 1.5ml of 100 mM phosphate buffer, 0.3ml

of 10 mM guaiacol, 0.3ml enzyme extract and 0.6ml distilled water except

hydrogen peroxide. The hydrogen peroxide 10 mM (0.3ml) was added to

initiate the enzyme activity. Immediately after the addition of hydrogen

peroxide the mixture was transferred to a Spectrophotometer cuvette and the

activity was measured at 470 nm by direct measurement of optical density

using a Shimadzu UV-visible Spectrophotometer UV- (1601) for 3 minutes at

intervals of 30 seconds. The blank was prepared by adding 0.3ml water to the

reaction mixture instead of enzyme extract. The optical density was

determined by subtracting the initial absorbance from the optical density at

180th second, and the unit activity was calculated on per gram tissue fresh

weight basis. The unit activity of peroxidase enzyme was expressed as the

change in absorbance per minute per gram fresh weight of tissue.

The rate of formation of guaiacol dehydrogenation product is a

measure of the peroxidase activity. The enzyme activity was expressed in

terms of specific activity, which is the activity per mg protein.

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7. BIOACCUMULATION STUDIES

Bioaccumulation studies were conducted by estimating mercury

content present in the tissue components such as root, stem (runner) and leaf

samples. Mercury content retained in the residual solution also was analysed.

Different plant parts – root, stem and leaf tissues were sampled at each

interval upto 24 days of growth in the culture medium artificially

contaminated with mercury, a combination of mercury + triadimefon and

triadimefon along with control. Accumulation of Hg in the samples collected

at each interval like 4, 8, 12, 16, 20 and 24 days were recorded.

Mercury content was analyzed using Atomic Absorption

spectrophotometer (AAS). Samples were prepared according to the method of

Allan (1969). Oven dried plant materials were used for the analysis. Known

weight (500mg) of each sample was wet digested by refluxing in 10ml of

concentrated nitric acid and perchloric acid mixed in the ratio of 10:4 until the

solution became colourless in Kjeldahl‟s flasks and made upto 50ml. Atomic

Absorption Spectrophotometer (PERKIN ELMER A Analyst 300) available

at Cashew Export and Promotion Council (CEPC), Kollam, Kerala, was used

for estimating mercury in all the samples.

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8. STATISTICAL ANALYSIS

All experiments were repeated a minimum of six times and the mean

values are given in tables and figures. Standard deviation and standard error

were also calculated. The values in tables were mean value  standard error.

Test of significance was done using Fisher‟s „t‟ test.

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