Introduction. and Materials

7/31/2019 Introduction. and Materials

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MATERIALS AND METHODS

Arbuscular Mycorrhizal (AM) fungi is one of the most important microbes of soil that

form symbiotic associations with most of the terrestrial plants on the earth. These fungi are

chiefly responsible for Phosphorus (P) uptake. Arbuscular mycorrhizal fungi are of considerable

interest because of their archaic existence, ability to form symbiotic associations with 85% of

plant taxa and their potential use as a biofertilizer to increase yield of crop and tree species.

Hence, an attempt was to study the effect of AM fungi on the growth and yield of Abelmoscus

esculentus.

SOIL SAMPLE COLLECTION

The soil samples used for the isolation of Arbuscular mycorrhizal fungi were collected

from Soukku, Banana, Sugarcane and Coconut field at Nagercoil, Tirunelveli, Tenkasi and

Kanyakumari. Four samples were collected from each sampling site and mixed to form a

composite sample. The collected soil samples were stored at 4C for two months for spore

development.

ISOLATION AND QUANTIFICATION OF SPORES

The soil samples were analyzed for AM fungal spores, following a wet sieving and

decanting procedure (Gerdemann, 1995). 250gm of soil was mixed in 1000ml of water and

allowed the heavier particles to settle down for few seconds. The liquid was poured through

coarse sieve (750 mm, 450 mm, 250 mm and 45 mm) to remove large pieces of organic matter.

The filtrate was mixed with some more quantity water, well shaked and allowed heavier particles

to settle for few more seconds. The suspension was passed through a sieve fine enough to filter

the desired spores. The materials retained on the sieve were washed to ensure that all colloidal

material pass through the sieve. Small amounts of remaining debris were transferred to a

petridish and examined under a dissecting microscope. (Leyva et al., 2008)

CARRIER BASED INOCULUM PRODUCTION


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The isolated spores were used to establish pot cultures, since, soil inoculums are

considered to be more rapidly infective than spore inoculums. AM fungi are propagated in pot

cultures by placing the mycorrhizal inoculums with sterilized soil. Onion (Allium cepa) was used

as a host plant to propagate the AM fungal spores for two months. The presence and abundance

of AM fungi propagates are constantly monitored. At the end of two months, soil was stopped

watering and the cultures were allowed to dry slowly. The mycorrhizal inoculum was harvested

and stored, later used it for plant inoculation. For quality control of mycorrhizal inoculum,

evaluation of the proportion of root length colonized by AM fungi (percentage of mycorrhizal

colonization) isolation and quantification of spores per gram of soil was done (Corkidia et al.,

2008).

ROOT SAMPLE COLLECTION

For mycorrhizal assay, root was collected carefully, by excavating from soil, assuring

sufficient terminal feeder roots attached to lower order roots.

CLEARING AND STAINING SPECIMENS

The roots were cut into small pieces (0.5 cm -1.0 cm) and heated/boiled in beaker

containing 10% potassium hydroxide (KOH) solution for 20 minutes to 1 hour till it become soft

in a well-ventilated exhaust hood. Added KOH solution clears the host cytoplasm and nuclei,

which readily allows stain penetration. Pour off the KOH solution and rinse the root sample in a

beaker with at least three complete changes of water or until no brown color appears in the rinsed

water. Do not agitate the root vigorously. Cover the root in the beaker with alkaline H2O2

(NaoH-3ml; H2O2 (10%)30 ml; water-567 ml) and keep it room temperature for 10 to 20 min

or until roots are bleached. Rinse the root in beaker thoroughly using at least threecomplete

changes of tap water to remove H2O2 Cover the root in the beaker with 1% HCL and soak for 3

to 4 min and then pour of the solution. Do not rinse after this soak because the specimens must

be acidified for proper staining. Cover the root in the beaker with 0.01% trypanblue lactic acid

staining solution and warm it again in beaker for 10 minutes. (Lactic acid solution; lactic acid-

875 ml, glycerol-63 ml; tap water-63 ml; trypanblue-0.1 g). Remove excess of stain and cover


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the root with above solution without trypanblue for destaining and mycorrhizal assay can be

done (Khadge., 1992). Mycorrhizal infection can now quantify in the samples.

SOIL ANALYSIS

The four different soil samples were used for the pot experiment to analyze the effect of

AM on the physiological and biochemical changes on plant They were collected from Nagercoil,

Tirunelveli, Sivakasi, Thenkasi and the samples were labeled asS1, S2, S3 and S4 respectively.

NITROGEN, PHOSPHOURS AND POTASSIUM ANALYSIS OF SOIL SAMPLES

The pH nutrient analysis of soil samples (S1.S2,S3 and S4) such as Nitrogen, phosphorus

and potassium were analyzed in Regional Research Institute at Kovilpatti.

DESIGNING OF POT EXPERIMENT

To assess the effect of AM fungi the pot experiment was selected. Four different types of

soil samples were taken in pots and the seeds were sown in the sterilized soil. An AM fungus

was inoculated as the soil containing AMF (soil inoculums) used as an inoculum. The plants

were analyzed for various parameters after 45 days of germination.

EFFECT OF ARBUSCULAR MYCORRHIZAL FUNGI ON MAIZE PLANT ON FOUR

DIFFERENTSOILS

The effect of AM fungi on the physiological and biochemical changes on plant grown in

different soil samples were analyzed after forty five days of seedling.

DAY OF EMERGENCE

After seedling the day of emergence was noted for all the plants grown in different soil.

ROOT INFECTION

The percentage of root infection was calculated according to Kaushik et al ., 2008.


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MEASUREMENT OF ROOT LENGTH

To measure the root length of plant, the plants were uprooted carefully from soil, washed

with water and its length was measured. An average value of the root was taken in to

consideration and expressed in centimeter (cm).

MEASUREMENT OF SHOOT LENGTH

To measure the shoot length of plant, the plants were uprooted carefully from soil,

washed with water and its length was measured. An average value of the shoot was taken in to

consideration and expressed in centimeter (cm).

EFFECT OF AM ON FRESH WEIGHT AND DRY WEIGHT OF PLANT

The plant parts are collected and he fresh weight of the plants was calculated. After

drying the plants in oven, dry weight of the plants was calculated and expressed in gram (g).

EFFECT OF AM ON CHLOROPHYLL CONTENT OF PLANT

0.5 gm of control and AM treated leaf material were ground separately in 80% acetone

and centrifuged at 5000 rpm for 5 minutes. Extraction with 80% acetone was repeated till the

pellet becomes non-green color. The supernatants were polled after each centrifugation and the

combined supernatant was used for the estimation of chlorophyll. Absorbance was read at 645

nm and 662 nm.

Thimmaiah. S.K., (1999) formulae were used for establishing the content of Chl a, Chl b

and total Chlorophyll.

Chl a (mg/g) =10.65 xA662 -2.35 x A645

Chl b (mg/g) =17.51 xA645 - 3.96 x A662


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Total chlorophyll (mg/g) =6.69 x A662 -16.26 x A645

ESTIMATION OF CAROTENOIDS

The carotenoids present in the acetone extract were quantified by measuring absorbance

at 470 nm and amount of carotenoids were calculated by the following formula of 1000 x A470-

2.27x Ca80.2 x Cb Carotenoids 229

ESTIMATION OF ANTHOCYANIN

The estimation of anthocyanin content was determined by Thimmaiah. S.K., (1999). 100

mg fresh leaves were taken and ground with 10ml of ethanol and filtered through Whatman No 1filter paper. One ml of extract along with Methanolic - Hcl was added to 1 ml of peroxide

reagent and kept in dark for 15 minutes and the absorbance was read at 525 nm. Anthocyanin

content was determined by O.D value (A525) / gram of leaf tissue.

ESTIMATION OF PROLINE

Free proline from plant tissues may be selectively extracted in aqueous Sulphosalicylic

acid and its concentration was measured using Ninhydrin method Thimmaiah. S.K., (1999). 200

mg of leaf sample was taken and ground with 10 ml Sulphosalicylic acid and filtered with what

man 1 filter paper. 2ml of the extract along with 2ml acid Ninhydrin and 2 ml of glacial acetic

acid was taken, mixed well and kept in boiling water bath (100C) for one hour. It was cooled in

an ice for 5 minutes and added with 4ml of toluene. The tubes were agitated vigorously. The

upper pink chromophore layer was separated and the absorbance was read at 520 nm. The

amount of proline was estimated using proline as the standard

ESTIMATION OF PHENOL

The phenolic content was estimated by Folin Ciocalteu method (Thimmaiah. S.K.,

(1999).100 mg fresh leaves were taken and ground with 10 ml of ethanol and filtered through

what man no 1 filter paper. One ml of extract was added with 2 ml of 20% sodium carbonate. It


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was shaken well and kept in boiling water bath for 1 minute and cooled. The blue solution

obtained was diluted to 25 ml with water and the absorbance was read at 650 nm. The amount of

phenol was estimated using Catachol as the standard.

ESTIMATION OF PROTEIN BY LOWREY ET AL METHOD (1951) (Thinmmaiah.S.

K.,1999)

Reagents

1. Alkaline Copper mixture,2. 0.1N NaOH3. 10% TCA,4. Folins Ciocalteu Reagent.5. Procedure

100 mg of leaf sample of both control and treated plants were taken and homogenized

separately with 10 ml distilled water with the help of mortar and pestle. The extract was

centrifuged at 3000 rpm for 5 minutes. The supernatant was taken and the pellet was

discarded. 1 ml of ice cold TCA was added with the supernatant and kept it in ice for 10

minutes. The supernatant was centrifuged at 50000 rpm for 10 minutes. The pellet was taken

and dissolved in 1 ml of 0.1 N NaOH. It was used as test solution. From this test solution 0.5

ml was taken and make up to 1 ml with distilled water. Add solution was mixed thoroughly

and boiled for 10 minutes and kept in dark for half an hour. The absorbance was noted at 650

nm. Blank was also prepared with 0.5 ml of distilled water, 0.5 ml of folin reagent and add

5.5 ml of alkaline copper reagent. The amount of protein content was calculated from the

standard graph of protein constructed with bovine serum albumin (BSA) as marker protein.

(An aliquot of 100 mg BSA showed 0.197 absorbance of 650 nm)

CARBOHYDRATE ESTIMATION BY ANTHRONE METHOD (Thimmaiah. S. K., 1999)

The carbohydrate content can be measured by hydrolyzing the polysaccharides into

simple sugars using dilute hydrochloric acid and estimating the resultant monosaccharides. In


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hot acidic medium glucose was dehydrated to hydroxymethyl furfural. This compound

formed with Anthrone a green colored product with absorption maximum at 630 nm.

Reagents

1.

2.5 N-HCL,2. Anthrone reagent,3. Standard glucose

Procedure

Weighed 100 mg of each samples (control and AM treated) in to sampling tubes. It was

hydrolyzed by keeping it in a boiling water bath for three hours with 5 ml of 2.5 N-HCL and

cooled to room temperature. Then neutralized with sodium carbonate until effervescence cease.

The volume was made up to 100 ml and centrifuged. Standard was also prepared by taking

0,0.2,0.4,0.6,0.8,1 ml of the working standard. The volume was made to 1 ml in all the tubes

including sample tubes with distilled water. Then added 4 ml of anthrone reagent and heated for

8 minutes in a boiling water bath. Cooled rapidly and read the green to dark green colors at 630

nm. The standard graph was drawn and the amount of carbohydrate present in the sample tubes

was calculated. Calculation

Amount of carbohydrate present in 100 mg of sample = mg of glucose / volume of

Test sample x100

ESTMATION OF STARCH BY ANTHRONE METHOD (Thimmaiah. S. K., 1999) Reagents

1. Anthrone: Dissolve 200 mg anthrone in 100 ml of icecold 95% sulphuric acid.2. 80% ethanol3. 52% Perchloric acid4. Standard glucose: stock-100 mg in 100 ml water.5. Working standard- 10 ml of stock diluted to 100 ml with

water (100 mg/ml)Method


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0.1 to 0.5 gm of the sample was homogenized in hot 80% ethanol to remove sugars. It

was centrifuged and retained the residue. The residue washed repeatedly with hot 80% ethanol

till the washing did not gave color with Anthrone reagent. The residue was dried over a water

bath. To the residue, 5 ml of water and 6.5 ml of 52% Perchloric acid were added and extracted

at 0 c for 20 minutes. Centrifuged and save the supernatant. The extraction was repeated using

fresh Perchloric acid. Centrifuge and pool the supernatant and make up to 100 ml. Pipette out 0.1

or 0.2 ml of the supernatant and make up to the volume to 1 ml with water. The standards were

prepared by taking 0.2, 0.4, 0.6, 0.8 and 1.0 ml of the working standard and make up the volume

to 1 ml in each tube with water. 4 ml of Anthrone reagent was added to each tube and heated for

8 minutes in a boiling water bath. Cooled rapidly and read the intensity of green to dark green

color at 630 nm.

Calculation

The glucose content in the sample was found using the standard graph and multiply the

value by a factor 0.9 to arrive at starch content.

ESTIMATION OF NITROGEN BY MICRO-KJELDHAL METHOD Reagents

1.Conc.H2SO4

2.Mercuric oxide

3.Potassium sulphate

4.Potassium hydroxide- Sodium thiosulphate solution:

Dissolve 600g of NaOH and 50 g of Na 2SO3.5H2O in water and make up

to 1 liter.

5.0.2N standard HCL or H 2SO4

6.4% Boric acid solution:

Dissolve 4g of H3BO3 in warm water and diluted 100 ml distilled water.

7.Mixed indicator solution:


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Mix 2 parts of 0.2% methyl red in ethanol with 1 part of 0.2% methylene blue in

ethanol.

Methods

40-100 mg of finely powdered homogenous sample was taken into 30 ml digestion

flask.2g K2SO4 and 90 mg of HGO, 2ml of Conc.H2SO4 Were added and mixed well. Boiling

chips/glass beads were added and digested the sample over digestion rack. Cooled and add

minimum quantity of water along the sides of the flask to dissolve solids and transferred

quantitatively to the distillation apparatus with successive rinsing with water. A 100ml conical

flask containing 5ml of boric acid solution with a few drops of mixed indicator in such a way

that the tip of the condenser dipped inside the solution.10 ml sodium hydroxide sodium

thiosulphate solution to the digest in the apparatus through the funnel and rinse with water.

Distilled and collected the ammonia in boric acid. The color change from violet to green was an

indication of ammonium absorbed. Rinsed the tip of the condenser with water and titrated the

distilled sample against the standard HCl or H2SO4 until the appearance of original violet color

as the end point. Run a blank digested similarly with an equal volume of water after washing the

distillation apparatus by back suction with excess of water. Calculation

%Nitrogen= (ml HCl in sample)-(ml HCl in blank) x Normality of

acid x 14.01 x 100

Weight of the sample

EFFECT OF AM ON Ca AND Fe CONTENT

The control and AM treated leaf sample were digested with concentrated nitric acid and

30% hydrogen peroxide and Ca, Fe content were determined by using Atomic Absorption

Spectrophotometer (AAS) .

SDS PAGE Reagents

Separating gel (12%) 10 ml:

Distilled H2O = 3.3 ml

30% acryl amide, bis acryl amide = 4 ml


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1.5 M Tris (pH 8.8) = 2.5 ml

10% SDS = 0.1ml

10% APS = 0.1 ml

TEMED = 0.004 ml

Stacking gel (5%)3 ml


30% acryl amide, bis acryl amide = 0.5ml

1 M Tris (pH 6.8) = 0.38 ml

10% SDS = 0.03ml

10% APS = 0.03 ml

TEMED = 0.003 ml

Sample loLoading buffer (10 ml)

Tris pH (6.8) = 2.5 ml

10% SDS = 4 ml

100% Glycerol = 2ml

-mercapto ethanol = 0.8 ml

Bromophenol blue (0.1%) = 0.3 ml


Tank buffer

Glycine = 36g

Tris = 7.5g

APS 10%:

Dissolve 300mg of solid APS in 5ml of Distilled H2O.

Staining solution: (200 ml):


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Coomasive brilliant blue = 0.3 g

Methanol (or) ethanol = 80 ml

Glacial acetic acid = 20 ml

Distilled H2O = 100ml

Stock Acryl amide

Mix 29.2 g Acryl amide, 0.8 g bisacrylamide and final volume is made upto 100ml.

1.5 M Tris- HCL (pH 8.8)

Dissolve 18.15 g of Tris in 50 ml of Dis. H2O. Adjust the pH to 8.8 with HCL; make

the volume to 100 ml.

10% SDS

Dissolve 18.15 g of Tris in 50 ml of Dis. H2O and make the final volume equal to 10 ml.

Procedure

Thoroughly clean the glass plates and spacer then assemble them proper by hold the

assembly together bulldog clips clamp in an upright position white petroleum gelly or 2% agar

(meltated in a boiling water bath) is then applied around the edges of the spacers.

To hold them in place and seal the chamber between the glass plates.Prepare the

sufficient volume of separating gel mixture. Mix gently and carefully pour the gel solution in the

chamber between the glass plates.

Layered distilled water on the top of the gel and leave to set for 30 to60 minutes.

Prepare the sufficient volume of stocking gel mixture. Remove the water from the top of

the gel and wash with a little stocking solution. Pour the stacking gel mixture, place the comb in

the stacking gel and allow the gel to set (30-60 minutes).

After the stacking gel has polymerized remove the comb without distorting shape of well

carefully install the gel after removing the clips, agar etc in the electrophoresis apparatus.

Fill it with electrode buffer and remove and any trapped, air bubbles at thebottom of gel.


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Connect the cathode of the top and turn on the DC power briefly to check the electrical

circuit. Plate can be kept cooled using a suitable facility. Heat generator during the run is

dissipated and does not affect the gel and resolution. Prepare samples for electrophoresis,

following suitable extraction procedure.

Adjust the protein concentration in each sample using the 5-strength sample buffer and

water in such a way that the same account of proteins (50-200 g) in a volume (25-50l) not

greater than the sample well. Sample solutions in a boiling water bath for 2-3 minutes to ensure

complete interaction between proteins and SDS. Cool the sample solutions and carefully inject it

into a sample well through an electrode buffer. Marking the position of wells on the glass plate

with a marker pen and the presences of chromo phenol blue in the sample buffer facilitates easy

loading of the samples. Similarly load the a few wells with standard marker proteins in the

sample buffer.

Turn on the circuit to 10-15 mA for initial 10-15 minutes until the samples travel through

the stacking gel. The stacking gel helps concentration of the samples. Then continue the run at 30

mA until the Bromo phenol blue reaches the bottom of the gel (about 3 hours).

The gel may be run at a high current (60-70 mA) for short period (1 hour) with proper

cooling. After the run is complete carefully remove the gel from between the plates &immerse in

staining solution (3 hour)&uniform shaking. Transfer the gel to suitable container with at least

200-300 ml ofdestaining solution and shake gently.

Destaining process should be stopped at appropriate stage the visualize as many bands as

possible. Gel as can be photographed stored in polythene bags or dried in vacuum for permanent

record.

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Introduction. and Materials