Methods

2.1 STUDY OF ECOLOGICAL DIVERSITY

40 transects (4m X 10m) was used to sample terrestrial as well as epiphytic species. The minimum horizontal distance between two transects was greater than 100m (Ding et al 2015). Each transect was further divided into 2m X 5m quadrates. Species that were rooted within the quadrate were included in the analysis. We followed Sandord’s definition of an ‘individual’; a group of rhizomes and leaves belonging to one species, which forms a clearly delimited stand (Zotz and Schultz 2008). Vascular epiphytic species and number of individuals was recorded for each tree in the field using binoculars, sample pole and single rope climbing (Perry 1978). Most epiphytic fern species could be identified and counted from the ground since most of the canopies were easily visible. In cases where binoculars failed to provide proper resolution, 7 trees (16.6%) were climbed. Most of these climbed trees were large, having dbh (diameter at breast height) greater than 40cm and mainly occur in the central part of the islands. Voucher specimens of the identified species, rhizosphere type specimens and all lectotypes are stored in the herbarium collection at Bangabasi Evening College.

2.2 STUDY OF MORPHO-ANATOMICAL CHARACTERS

Stomatal density was calculated using the method proposed by (Friedrich 1973) where the fronds of the collected ferns were cut into 1cm2 pieces and the suitable epidermal layer was peeled for studying the stomata. The peeled portion devoid of mesophyll cells containing chlorophyll were placed under the high power objective (40X) of a Weissman binocular microscope and observed. Suitable fields were identified, photographed and counted. Stomatal frequency was calculated using the following formula:

{(Total no of stomata/cm2 area) ÷ (total leaf area)} X 100.

Since stomatal frequency is often considered to be an absolute measure, stomatal index (Salisbury 1928) was also calculated from the data:

Stomatal index = {(number of stomata present / unit area of leaf) ÷ ∑(number of stomata + number of epidermal cells)} X 100.

2.3 CHEMICAL AND BIOCHEMICAL ANALYSIS OF SOIL AND PLANT SPECIMENS

2.3.1 Chemical Analysis of Soil

2.3.1.1 Soil pH:

Apparatus

• pH Meter — Direct reading type conforming to IS : 2711-1979*, with glass electrode and a calomal reference electrode or any other suitable electrode.
• Balance ( Analytical ) — sensitive to O'OOl g.
• Three 100-ml Glass Beakers — with covering glasses and stirring rods.
• Two 500ml Volumetric Flask,
• Wash Bottle — containing distilled water.
• Mortar with Rubber Covered Pestle,

Buffer Solutions

• Buffer Solution pH 4'0 ( at25°C ) — Dissolve 5-106 g of potassium hydrogen phthalate in distilled water and dilute to 500 ml with distilled water.
• Buffer Solution pH 9.2 ( at 25°C ) —Dissolve 9'54 g of sodium tetraborate ( borax ) in distilled water and dilute to 500 ml.
• Soil Specimen — The soil sample received from the field shall be prepared in accordance with IS : 2720 ( Part 1 )-1983f. All aggregations of particles shall be broken down so that, if the samples were sieved on a 425-micron IS Sieve, discrete particles would be retained. The sample, after having been broken up, shall be thoroughly mixed and then sub- divided either by quartering or by riffling until a representative sub-sample is obtained.

Procedure

30 g of the soil from the sample, prepared as in 2,3, shall be taken in a 100-ml beaker. 75 ml of distilled water ( see Note ) shall be added to it. The suspension shall be stirred for a few seconds. The beaker shall then be covered with a cover glass and allowed to stand for one hour, with occasional stirring. It shall be again stirred well immediately before testing.

The pH meter shall be calibrated by means of the standard buffer solutions following the procedure recommended by the manufacturer. The electrodes shall be washed with distilled water dried with the help of an ordinary filter paper and then immersed in the soil suspension. Two or three readings of the pH of the soil suspension shall be made with brief stirring in between each reading. The reading should agree within ± 0*05 pH units (the pH readings of the soil suspension should reach a constant value in about one minute. No readings should be taken until the pH meter has reached equilibrium). The electrodes shall be removed from the suspension immediately and washed with distilled water. The calibration of the pH meter shall be again checked with one of the standard buffer solutions. If the instrument is out of adjustment by more than 0*05 pH units, it shall be set to the correct adjustment till consistent readings are obtained (when not in use, the electrodes shall be left standing in a beaker of distilled water).

2.3.1.2 Soil Alkalinity:

Alkalinity of water is the capacity of that water to accept protons. It may be defined as the quantitative capacity of an aqueous medium to react with hydrogen ions to pH 8-3 (phenolphthalein alkalinity) and then to pH 3-7 (total alkalinity or methyl orange alkalinity). The equation in its simplest form is as follows:

COp+ H+ = HCO; - (pH 83)

From pH 8*3 to 3-7, the following reaction may occur:

hco; + H + « H 2 C0 3

Sample Preparation — The sample aliquot used for analysis should be either free from turbidity or should be allowed to settle prior to analysis.

Apparatus

• pH Meter
• Burette — 50-ml capacity.
• Magnetic Stirrer Assembly
• Reagents
• Distilled Water— Distilled water used should have pH not less than 6-0. If the water has pH less than 6-0, it shall be freshly boiled for 15 minutes and cooled to room temperature. Deionized water may be used provided that it has a conductance of less than 2 jiS/cm and a pH more than 6 0. Sulphuric Acid— Dilute 5*6 ml of concentrated sulphuric acid ( relative density 1-84) to one litre
• with distilled water. Standard Solution of Sulphuric Acid — 002 N. Phenolphthalein Indicator — Dissolve 0-5 g of phenolphthalein in 100 ml, 1:1 (v/v), alcohol water mixture. Mixed Indicator Solution — Dissolve 0*02 g methyl red and 0-01 g bromocresol green in 100 ml, 95 percent, ethyl or isopropyl alcohol.

2.3.1.3 Total Organic Matter:

This procedure is used for the routine estimation of soil organic matter by the loss of weight in a sample heated at a temperature high enough to burn organic matter but not so high as to sdecompose carbonates.

Summary of Methods

A sample of soil is dried at 105° C to remove moisture. The sample is weighed, heated at 360° C for 2 hours and weighed again after the temperature drops below 150° C.

Safety

Care should be exercised in handling hot samples. Be sure to cool the oven to 150° C before removing the samples from the oven. Use a good pair of tongs and grasp the sample firmly.

Interferences

Any material that losses moisture below 360° C is a potential source of error. Therefore, soil moisture must be removed before the base weight of the sample is taken. Also, ignited samples must not be allowed to re-absorb moisture from the air before they are weighed.

Gypsum loses water of hydration gradually. Soils containing gypsum should be heated initially at 150° C instead of 105° C. Some hydrated clays may also lose water below 360° C. It is important that the results of this method be calibrated against organic carbon, preferably using a carbon analyser, on soils from the area for which the test will be used.

Apparatus and Materials

• Oven, or muffle furnace capable of being heated to 400° C and controlled to within ± 10° C.
• Beakers, 20 ml
• Crucible rack, stainless steel
• Balance accurate to ± 0.001 g in a draft free, low humidity environment
• Soil scoop calibrated to hold 5 g of light-colored silt loam soil
• Drying oven, 105° C

Reagents

An advantage of this method is that no reagents are required.

Methods

• Place a 5 g scoop of soil into a tared 20-ml beaker
• Dry for 2 hours or longer at 105° C
• Record weight to ± 0.001 g
• Bring oven to 360° C. Samples must then remain at 360° C for two hours.
• Cool to < 150° C
• Weigh to ± 0.001 g, in a draft-free environment

Calculations

Calculate percent weight loss-on- ignition (LOI) LOI= (wt. at 105°C) – (wt. at 360° C) x 100 Wt. at 105° C

Estimate % organic matter. Organic matter is estimated from LOI using regression analysis. Select soils covering the range in organic matter expected in the area serviced by the lab. Determine % organic matter using a carbon analyzer or by the Walkley-Black procedure for organic carbon. Regress OM on LOI.

Reporting: Data are reported as % LOI or as estimated % O.M.

2.3.1.4 SOIL SALINITY:

The main methods of measuring total water-soluble salts in a soil sample are the (1) weight method and (2) conductivity method. The data obtained from the weight method are reliable, but the operation is tedious and time-consuming. The conductivity method is simple.

Weight Method

This method is based on a water extract from a soil sample. The extract is evaporated to dryness and then dried at 105–110 °C to constant weight. The total dried residue contains both water-soluble salts and water-soluble organic matter. H2O2 is used to remove the organic matter in the residue. What remains are the total water-soluble salts from the soil.

Instruments and Reagents

Instruments: evaporating dish, water bath, dryer, electrothermal drying oven, analytical balance.

Reagents: 15 % H2O2 and 2 % Na2CO3.

Method

Draw 50.0 ml of solution from a soil sample of known weight (w), place in an evaporating dish and weigh (w 0). Evaporate to dryness in a water bath and then dry in an electrothermal drying oven at 105–110 °C for 4 h. Remove from the oven and place in a dryer for 30 min, then weigh using an analytical balance. Return sample to the electrothermal drying oven for 2 more hours, cool down and reweigh. Repeat these steps until a constant weight (w 1) is obtained; the weight difference between the two times should not be more than 1 mg. Calculate the weight of the dried residue. Add 15 % H2O2 in drops to wet the residue. Evaporate to dryness in a water bath. Repeat this treatment until the entire residue turns white. Dry the white residue to constant weight (w 2) according to the method described above. Calculate the content of the total water-soluble salts in the soil.

Calculation of Total Water-Soluble Salts

Total dried residue=(w1−w0)/w×100 % where w is the weight of the soil sample (g) that the drawn extract is equivalent to.

2.3.1.5 Soil Available Phosphorus:

Plant available phosphorus (P) is extracted from the soil with 0.03 N NH4F in 0.025 N HCl (Bray P1 extract). This extractant primarily measures P adsorbed by Al compounds. The Al is complexed by F- ions, liberating P. Lesser amounts of Fe-, MN-, and Ca-P may be extracted, along with water-soluble P. Extracted P is reacted with ammonium molybdate to form a blue phosphomolybdate compound in the presence of a reducing agent.The concentration of P is determined colorimetrically or by UV – Vis spectrophotometer at 645nm. Potassium is extracted simultaneously with P and analyzed separately.

Reagents

• Stock P-A solution (1.25 N HCl, 1.5 N NH4F): Add 54 ml of 48% HF to 700 ml of deionized water. Neutralize to pH 7.0 with NH4OH. Add 108 ml of concentrated HCl (11.6 N) and dilute to 1 liter.
• Dilute P-A solution (0.025 N HCl, 0.03 N NH4F): Dilute 20 ml of stock P-A solution to 1 liter with deionized water.
• P-B solution (0.87 N HCl, 0.38% ammonium molybdate, 0.5%H3BO3): Dissolve 3.8 g ammonium molybdate, (NH4) 6Mo7O24⋅4H2O, in 300 ml of deionized water at about 60°C. Cool. Dissolve 5.0 g boric acid, H3BO3, in 500 ml of deionized water, and add 75 ml concentrated HCl (11.6 N). Then, add the molybdate solution and dilute to 1 liter with deionized water.
• P-C powder: Thoroughly mix and grind to a fine powder 2.5 g of 1-amino-2- napthol-4 sulfonic acid, 5.0 g sodium sulfite (Na2SO3), and 146 g of sodium metabisulfite (Na2S2O5).
• P-C solution: Dissolve 8 g of dry P-C powder in 50 ml of warm deionized water. Let stand overnight, if possible. A fresh reagent should be prepared every three weeks. (Upon standing, some material may crystallize out, but this is still satisfactory.)
• Standard P solution (1000 ppm P, 500 ppm P)
• Working standards (0, 1.0, 2.5, 5, 10, 20, 40 ppm P, prepares with same matrix as the samples.)

Methods

• Place a 1.5 g scoop of soil into a 50-ml Erlenmeyer flask.
• Add 15 ml of P-A solution with Automatic Brewer Pipette.
• Shake the suspension on oscillating shaker for 5 minutes.
• Filter through filter paper into a 15- ml funnel tube.
• Pipette a 3.0-ml aliquot of filtrate with constant suction pipette apparatus and transfer to a 10- ml colorimeter tube.
• Add 3.0 ml of P-B solution with the same pipette apparatus and mix well.
• Add 3 drops of P-C solution, and mix immediately.
• Read color after 15 min., but before two hr., with a UV-Vis spectrophotometer.
• UV – Vis spectrophotometer should be set at 645 nm.
• Calibrate the instrument to read directly in ppm P in soil using working standards. These standard preparations are treated in the same manner as the soil extracts. (color development is complete in 15 minutes. and standards should be read within two hours.).

Calculations

In lieu of direct calibration of the colorimeter scale, calculate extractable P, ppm P in soil = ppm P in solution x 15 ml/1.5 g = ppm P in solution x 10.

2.3.1.6 Estimation of other parameters:

Acidity, Total fluoride content, organic carbon, lead and hexavalent chromium was estimated using methods described as standards in APHA 23rd Edition - 2310B; APHA 23rd Edition, 2520 B: 2017; IS 2720 (Part XXII): 1972; EPA 3050B - 1996 and APHA 23rd Ed - 3500-Cr -B: 2017 respectively.

2.3.2 Biochemical Analysis

2.3.2.1 Determination of Total Phenolics:

Extraction of the plant material:

The dried powdered leaves and rhizomes (100 g) were extracted by maceration with 1000 mL 70% ethanol for 3 d at room temperature with occasional shaking. The extract was filtered and the marc was re-extracted by the same process until plant materials were exhausted.

Total Phenol Estimation:

The collected filtrates were pooled and evaporated to dryness under reduced pressure to yield the dry extracts (yield w/w: 8.69 %) and was stored at 4 °C until used. The total phenolic content of the leaf and rhizome extracts was determined by using Folin-Ciocalteu reagent following a slightly modified method of Ainsworth. Gallic acid was used as a reference standard for plotting calibration curve. A volume of 0.5 mL of the plant extract (100 µg/mL) was mixed with 2 mL of the Folin-Ciocalteu reagent (diluted 1:10 with de-ionized water) and were neutralized with 4 mL of sodium carbonate solution (7.5%, w/v). The reaction mixture was incubated at room temperature for 30 min with intermittent shaking for colour development. The absorbance of the resulting blue colour was measured at 765 nm using double beam UV-VIS spectrophotometer (UV Analyst-CT 8200).

The total phenolic contents were determined from the linear equation of a standard curve prepared with gallic acid. The content of total phenolic compounds expressed as mg/g gallic acid equivalent (GAE) of dry extract.

2.3.2.2 Physiological analysis

Analysis of Transpirational rate and stomatal density

24. Measurement of the rate of transpiration using potometer

A simple experiment is described below to demonstrate how the rate of transpiration of common plants found in Hong Kong can be determined with the use of potometer. Moreover, students can also be given the opportunity to estimate the stomatal density of leaves from different plants to evaluate the relationship between different transpiration rates and this morphological feature.

Estimation of transpiration rate using a photometer

Procedure

1. Use a sharp razor blade to cut a leafy shoot under water.
2. Insert the leafy shoot through the hole of the stopper provided with the potometer.
3. Fill the potometer with water and fit the stopper holding the leafy shoot to the apparatus.
4. Use vaseline to seal all the connections of the apparatus.
5. Trap an air bubble in the capillary tube by the following procedures:
   • dip the end of the capillary tube into a beaker of water,
   • close the tap of the reservoir,
   • take away the beaker of water and allow the plant to transpire for a while, and
   • re-immerse the capillary tube into the beaker of water again.
6. Wait for about 5 minutes for the plant to equilibrate.
7. Estimate the rate of transpiration by measuring the distance moved by the air bubble per unit time. Take another measurement, and average the two readings.
8. Find out the total leaf surface area of the experimental plant using a graph paper.
9. Transpiration rate can be expressed in terms of water transpired per unit time per unit area of leaf surface.
10. If time permits, estimate the rate under different environmental conditions, e.g. under direct incandescent light illumination, under low light condition, in rapid air movement (provided by a fan) or in still air.

B. Estimation of stomatal density on leaf surface

Procedure

1. Use a needle or a pair of fine forceps to make a shallow cut on the lower surface of the leaf.
2. Use a pair of fine forceps to peel off the lower epidermis.
3. Mount the epidermis in a drop of water on a slide.
4. Using 100x magnification, move the slide to find a portion which occupies the entire field of view, count the number of stomata within the area occupied.
5. Repeat step (4) 3-5 times to obtain a good average value.
6. Repeat steps (1) to (5) for the upper epidermis of the same leaf.
7. Given a copper wire of known diameter, estimate the area covered by the microscope field of view at 100x magnification.
8. Calculate the stomatal density of the upper and lower epidermis of the plant.