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 4ºC 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

For quality control of mycorrhizal inoculum. Cover the root in the beaker with 0. AM fungi are propagated in pot cultures by placing the mycorrhizal inoculums with sterilized soil.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.The isolated spores were used to establish pot cultures.01% trypanblue lactic acid staining solution and warm it again in beaker for 10 minutes. later used it for plant inoculation. soil inoculums are considered to be more rapidly infective than spore inoculums. since. glycerol-63 ml. trypanblue-0. tap water-63 ml. assuring sufficient terminal feeder roots attached to lower order roots. soil was stopped watering and the cultures were allowed to dry slowly. The mycorrhizal inoculum was harvested and stored. Added KOH solution clears the host cytoplasm and nuclei. 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.. 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. (Lactic acid solution. Do not rinse after this soak because the specimens must be acidified for proper staining. lactic acid875 ml. water-567 ml) and keep it room temperature for 10 to 20 min or until roots are bleached. Onion (Allium cepa) was used as a host plant to propagate the AM fungal spores for two months. Do not agitate the root vigorously. evaluation of the proportion of root length colonized by AM fungi (percentage of mycorrhizal colonization) isolation and quantification of spore’s per gram of soil was done (Corkidia et al. ROOT SAMPLE COLLECTION For mycorrhizal assay. root was collected carefully. The presence and abundance of AM fungi propagates are constantly monitored. Cover the root in the beaker with alkaline H2O2 (NaoH-3ml. by excavating from soil. 2008). CLEARING AND STAINING SPECIMENS The roots were cut into small pieces (0.5 cm -1. At the end of two months. which readily allows stain penetration. Remove excess of stain and cover .1 g). H2O2 (10%) –30 ml.

Four different types of soil samples were taken in pots and the seeds were sown in the sterilized soil.. DAY OF EMERGENCE After seedling the day of emergence was noted for all the plants grown in different soil. 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. NITROGEN. Thenkasi and the samples were labeled asS1. 1992). S2.S2. ROOT INFECTION The percentage of root infection was calculated according to Kaushik et al . 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. . Sivakasi. PHOSPHOURS AND POTASSIUM ANALYSIS OF SOIL SAMPLES The pH nutrient analysis of soil samples (S1.S3 and S4) such as Nitrogen. 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. 2008..the root with above solution without trypanblue for destaining and mycorrhizal assay can be done (Khadge. S3 and S4 respectively. The plants were analyzed for various parameters after 45 days of germination. Mycorrhizal infection can now quantify in the samples. An AM fungus was inoculated as the soil containing AMF (soil inoculums) used as an inoculum.

the plants were uprooted carefully from soil. Chl a (mg/g) =10. An average value of the shoot was taken in to consideration and expressed in centimeter (cm). (1999) formulae were used for establishing the content of Chl a.35 x A645 Chl b (mg/g) =17. washed with water and its length was measured. dry weight of the plants was calculated and expressed in gram (g).. Absorbance was read at 645 nm and 662 nm.96 x A662 . The supernatants were polled after each centrifugation and the combined supernatant was used for the estimation of chlorophyll.MEASUREMENT OF ROOT LENGTH To measure the root length of plant. EFFECT OF AM ON CHLOROPHYLL CONTENT OF PLANT 0. washed with water and its length was measured. MEASUREMENT OF SHOOT LENGTH To measure the shoot length of plant. Thimmaiah.65 xA662 -2.51 xA645 . Chl b and total Chlorophyll.3. S.5 gm of control and AM treated leaf material were ground separately in 80% acetone and centrifuged at 5000 rpm for 5 minutes.K. An average value of the root was taken in to consideration and expressed in centimeter (cm). After drying the plants in oven. the plants were uprooted carefully from soil. 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. Extraction with 80% acetone was repeated till the pellet becomes non-green color.

The tubes were agitated vigorously.Hcl was added to 1 ml of peroxide reagent and kept in dark for 15 minutes and the absorbance was read at 525 nm. It was cooled in an ice for 5 minutes and added with 4ml of toluene.. mixed well and kept in boiling water bath (100°C) for one hour.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. (1999). Anthocyanin content was determined by O.K. S.K. 100 mg fresh leaves were taken and ground with 10ml of ethanol and filtered through Whatman No 1 filter paper.27x Ca –80. It . The amount of proline was estimated using proline as the standard ESTIMATION OF PHENOL The phenolic content was estimated by Folin – Ciocalteu method (Thimmaiah. (1999). One ml of extract along with Methanolic . 2ml of the extract along with 2ml acid Ninhydrin and 2 ml of glacial acetic acid was taken.Total chlorophyll (mg/g) =6.26 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 A4702.69 x A662 -16.. (1999).D value (A525) / gram of leaf tissue. S.2 x Cb Carotenoids 229 ESTIMATION OF ANTHOCYANIN The estimation of anthocyanin content was determined by Thimmaiah. The upper pink chromophore layer was separated and the absorbance was read at 520 nm. 200 mg of leaf sample was taken and ground with 10 ml Sulphosalicylic acid and filtered with what man 1 filter paper. S. 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.K.

S. It was used as test solution. The supernatant was taken and the pellet was discarded.1N NaOH 3.197 absorbance of 650 nm) CARBOHYDRATE ESTIMATION BY ANTHRONE METHOD (Thimmaiah. 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.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. From this test solution 0. Alkaline Copper mixture.5 ml of folin reagent and add 5.1999) Reagents 1. K. The extract was centrifuged at 3000 rpm for 5 minutes. 0.5 ml of alkaline copper reagent.5 ml of distilled water.5 ml was taken and make up to 1 ml with distilled water. 4. 0. The absorbance was noted at 650 nm. The amount of protein content was calculated from the standard graph of protein constructed with bovine serum albumin (BSA) as marker protein. 1999) The carbohydrate content can be measured by hydrolyzing the polysaccharides into simple sugars using dilute hydrochloric acid and estimating the resultant monosaccharides. K. Folins Ciocalteu Reagent. The supernatant was centrifuged at 50000 rpm for 10 minutes. 5. The pellet was taken and dissolved in 1 ml of 0. 10% TCA. The amount of phenol was estimated using Catachol as the standard.. In . ESTIMATION OF PROTEIN BY LOWREY ET AL METHOD (1951) (Thinmmaiah. Add solution was mixed thoroughly and boiled for 10 minutes and kept in dark for half an hour..S. 1 ml of ice cold TCA was added with the supernatant and kept it in ice for 10 minutes. Blank was also prepared with 0. (An aliquot of 100 mg BSA showed 0. 2.1 N NaOH.

Reagents 1. Working standard. Standard was also prepared by taking 0.6.0. K. 2.1 ml of the working standard. Cooled rapidly and read the green to dark green colors at 630 nm. 1999) Reagents 1. Standard glucose: stock-100 mg in 100 ml water. 5.8.4. 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. 80% ethanol 3. S. 52% Perchloric acid 4. Then neutralized with sodium carbonate until effervescence cease.10 ml of stock diluted to 100 ml with water (100 mg/ml)Method . Standard glucose Procedure Weighed 100 mg of each samples (control and AM treated) in to sampling tubes. Anthrone reagent. The volume was made to 1 ml in all the tubes including sample tubes with distilled water. This compound formed with Anthrone a green colored product with absorption maximum at 630 nm.0.0. It was hydrolyzed by keeping it in a boiling water bath for three hours with 5 ml of 2.5 N-HCL.hot acidic medium glucose was dehydrated to hydroxymethyl furfural. Anthrone: Dissolve 200 mg anthrone in 100 ml of ice –cold 95% sulphuric acid. Then added 4 ml of anthrone reagent and heated for 8 minutes in a boiling water bath.0. 3. 2.2. 2..5 N-HCL and cooled to room temperature. The volume was made up to 100 ml and centrifuged. The standard graph was drawn and the amount of carbohydrate present in the sample tubes was calculated.

6. 7.5 ml of 52% Perchloric acid were added and extracted at 0 c for 20 minutes.Potassium hydroxide.Conc.Potassium sulphate 4. 4 ml of Anthrone reagent was added to each tube and heated for 8 minutes in a boiling water bath.2 ml of the supernatant and make up to the volume to 1 ml with water. The extraction was repeated using fresh Perchloric acid. ESTIMATION OF NITROGEN BY MICRO-KJELDHAL METHOD Reagents 1. It was centrifuged and retained the residue.9 to arrive at starch content.1 to 0.2N standard HCL or H 2SO4 6. 0.4. The residue washed repeatedly with hot 80% ethanol till the washing did not gave color with Anthrone reagent. 5 ml of water and 6.0.5H2O in water and make up to 1 liter.Mixed indicator solution: . Calculation The glucose content in the sample was found using the standard graph and multiply the value by a factor 0.0.Mercuric oxide 3.1 or 0. 0. Centrifuged and save the supernatant.H2SO4 2. Pipette out 0. Cooled rapidly and read the intensity of green to dark green color at 630 nm.4% Boric acid solution: Dissolve 4g of H3BO3 in warm water and diluted 100 ml distilled water.2. Centrifuge and pool the supernatant and make up to 100 ml.0 ml of the working standard and make up the volume to 1 ml in each tube with water. 5.8 and 1.Sodium thiosulphate solution: Dissolve 600g of NaOH and 50 g of Na 2SO3.5 gm of the sample was homogenized in hot 80% ethanol to remove sugars. The residue was dried over a water bath. 0. The standards were prepared by taking 0. To the residue.

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.H2SO4 Were added and mixed well.Mix 2 parts of 0. Calculation %Nitrogen= (ml HCl in sample)-(ml HCl in blank) x Normality of acid x 14. The color change from violet to green was an indication of ammonium absorbed.3 ml 30% acryl amide. 2ml of Conc.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. Run a blank digested similarly with an equal volume of water after washing the distillation apparatus by back suction with excess of water. Methods 40-100 mg of finely powdered homogenous sample was taken into 30 ml digestion flask.2% methylene blue in ethanol.2g K2SO4 and 90 mg of HGO. Boiling chips/glass beads were added and digested the sample over digestion rack.2% methyl red in ethanol with 1 part of 0. Distilled and collected the ammonia in boric acid. 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. SDS PAGE Reagents Separating gel (12%) 10 ml: Distilled H2O = 3. 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. bis acryl amide = 4 ml . Fe content were determined by using Atomic Absorption Spectrophotometer (AAS) .10 ml sodium hydroxide –sodium thiosulphate solution to the digest in the apparatus through the funnel and rinse with water.

1 ml 30% acryl amide.4 ml Tank buffer Glycine = 36g Tris = 7.8) = 2. bis acryl amide = 0.8) = 2.1ml = 0.5g APS 10%: Dissolve 300mg of solid APS in 5ml of Distilled H2O.8) = 0.004 ml Stacking gel (5%)3 ml Distilled H2O = 2.5 ml 10% SDS 10% APS = 0.3 ml Distilled H2O = 0.1 ml TEMED = 0.5 ml 10% SDS = 4 ml 100% Glycerol = 2ml в-mercapto ethanol = 0.1%) = 0.5 M Tris (pH 8.5ml 1 M Tris (pH 6.03ml = 0.1.003 ml Sample loLoading buffer (10 ml) Tris pH (6.38 ml 10% SDS 10% APS = 0.8 ml Bromophenol blue (0. Staining solution: (200 ml): .03 ml TEMED = 0.

Mix gently and carefully pour the gel solution in the chamber between the glass plates. Adjust the pH to 8.2 g Acryl amide. 1. Pour the stacking gel mixture. H2O.8 g bisacrylamide and final volume is made upto 100ml.15 g of Tris in 50 ml of Dis.3 g Methanol (or) ethanol = 80 ml Glacial acetic acid Distilled H2O = 20 ml = 100ml Stock Acryl amide Mix 29. air bubbles at thebottom of gel.8 with HCL. place the comb in the stacking gel and allow the gel to set (30-60 minutes). agar etc in the electrophoresis apparatus. Remove the water from the top of the gel and wash with a little stocking solution.5 M Tris. Fill it with electrode buffer and remove and any trapped.Prepare the sufficient volume of separating gel mixture. H2O and make the final volume equal to 10 ml. To hold them in place and seal the chamber between the glass plates. 10% SDS Dissolve 18. 0. Layered distilled water on the top of the gel and leave to set for 30 to60 minutes. make the volume to 100 ml.15 g of Tris in 50 ml of Dis. 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.8) Dissolve 18.HCL (pH 8. . Prepare the sufficient volume of stocking gel mixture.Coomasive brilliant blue = 0. After the stacking gel has polymerized remove the comb without distorting shape of well carefully install the gel after removing the clips.

Gel as can be photographed stored in polythene bags or dried in vacuum for permanent record. Turn on the circuit to 10-15 mA for initial 10-15 minutes until the samples travel through the stacking gel. . following suitable extraction procedure. After the run is complete carefully remove the gel from between the plates &immerse in staining solution (3 hour)&uniform shaking. The gel may be run at a high current (60-70 mA) for short period (1 hour) with proper cooling. Plate can be kept cooled using a suitable facility. Then continue the run at 30 mA until the Bromo phenol blue reaches the bottom of the gel (about 3 hours). Sample solutions in a boiling water bath for 2-3 minutes to ensure complete interaction between proteins and SDS. Similarly load the a few wells with standard marker proteins in the sample buffer. Transfer the gel to suitable container with at least 200-300 ml ofdestaining solution and shake gently. 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 µg) in a volume (25-50µl) not greater than the sample well. Heat generator during the run is dissipated and does not affect the gel and resolution. Prepare samples for electrophoresis. 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.Connect the cathode of the top and turn on the DC power briefly to check the electrical circuit. Destaining process should be stopped at appropriate stage the visualize as many bands as possible. The stacking gel helps concentration of the samples.

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