Assignment

Group 1:

Name: Roll numbers

Aqsa Mukhtar B-BOT-17-01

Najma Parveen B-BOT-17-03

Ayesha Mehboob B-BOT-17-05

Amina Murtaza B-BOT-17-07

Aimon Sikandar B-BOT-17-09

Samia Manzoor B-BOT-17-11

Farah Khan B-BOT-17-15

Subject: Plant Physiolog

Assignment Topic: General Lab Techniques and Skills of Plant Physiology.

The Women University, Multan

Context:-

1. Measurement of water potential

2. Measurement of Osmotic Potential by freezing point depression

3. Measurement of water content in plants

4. To determine turgor pressure of plant tissue

5. Photosynthesis (rate of photosynthesis oxygen and Co2 measurement)

6. Chlorophyll a & b estimation

7. Spectrophotometric estimation of carotenoids

8. Column chromatography Technique for carotenoids estimation

9. Spectrophotometer for absorption of plant pigment

10. Nutrient analysis: Micronutrients and Macronutrients

11. To conduct pots experiment under different stresses (salinity, heavy metal
and drought)
1. Measurement of water potential

Chardakov'smethod:

The Chardokov method provides a quick means to determine plant tissue water potentials. This
method depends on the change in density in a solution that occurs after a tissue has been
immersed in it. The solution gains or losses water depending on the water potential of the
tissue. If the density of a solution does not change (no net movement of water) then this
solution has the same water potential as the tissues that were incubated in it. It is assumed that
solute movement between tissue and solution is negligible. Density changes can be observed by
watching whether a drop of the original solution floats or sinks in the test solution after tissue
incubation. Alternately, for a more accurate measurement of changes in the solution density, a
refractometer can be used.
1. Dispense 10 mL of water or a sucrose solution (0.1 - 0.8 molal) into each of nine
appropriately-labeled test containers
(note: sorbitol, mannitol or polyethylene glycol can be used in place of sucrose).
2. Use a cork borer to prepare at least 27 uniform tissue samples. Cut them to the same length
(ca. 4.0 cm) with a razor blade and be sure not to include any fragments of the skin. Work
quickly to minimize evaporation and keep the tissue wrapped in a moist towel.
3. Put two or preferably three tissue section in each solution (water or sucrose). If necessary,
add more of the appropriate solution to completely submerge the cores but the final volume in
each tube must be the same.

4. Incubate the tissue section for at least 1.5 h, preferably longer.  Periodically swirl the
containers.  Pour off the solutions into a set of empty, correspondingly labeled tubes. Mix the
tubes thoroughly with a vortex mixer.
5. Record the temperature of the solutions.
6. Using a Pasteur pipet, remove a small amount of water dyed with methylene blue (to dye the
sucrose solution, dip a dry probe into methylene blue powder and then mix).
7. Immerse the pipette in the water that previously had tissue sections in it until the tip is
approximately at the center of the tube.

8. Slowly release a drop of the methylene blue solution from the pipette and note whether the
drop of the dye sinks, disperses, or floats to the surface in this solution and subjectively
estimate whether it does so rapidly or slowly.  Do this gently.
9. Record your results and repeat this procedure for each of the sucrose solutions. Be sure to
use a different pipet for each dye stock.
2. Osmotic potential measurement by freezing point depression:-
   Osmotic potential (Ψs) represents the effect of solutes on the energy state of water. Osmotic
potential is related to other properties of the solution such as vapor pressure, boiling point, and
freezing point.

Freezing point depression is generally determined on tissue sap. Sap samples are prepared by
freezing the tissue to rupture cells and then squeezing the frozen material by hand through
cheesecloth or with a hydraulic press. Alternately, the tissue can be homogenized (without
water) in a blender or with a mortar and pestle. First, calibrate the thermometer by placing 3
mL of water in the sample test tube. Immerse this unit in a beaker (600 mL) containing salt and
ice. The ice bath should be between -5 and -10°C. Suspend a thermometer in the sample
making sure that it doesn't touch the bottom or sides of the tube. Stir the sample continuously
and watch the decrease in temperature. When the temperature reaches 0°C, record the
temperature every 10 seconds. Continue readings as the sap cools. Note the temperature
closely. Periodically tap the thermometer gently to induce freezing. When freezing occurs there
will be a rapid increase in temperature. Note this temperature which is the apparent freezing
point (Δf') and continue to record temperatures until they become fairly constant. [In some
cases, the sample may freeze without releasing a detectable heat of fusion. If this occurs,
discard the sample and begin again.  You can often determine that this has occurred if there is a
plateau in the cooling rate of the sap.] The freezing point of the water should be zero. If the Δf’
of the water is not 0°C, then correct the Δf' of the sap sample accordingly.

3. To measure Relative water content in plants:

Relative water content (RWC) is probably the most appropriate measure of plant water status
in terms of the physiological consequence of cellular water deficit. Water potential as an
estimate of the energy status of plant water is useful in dealing with water transport in the soil-
plant-atmosphere continuum.

Normal values of RWC range between 98% in fully turgid transpiring leaves to about 30-40% in
severely desiccated and dying leaves, depending on plant species. In most crop species the
typical leaf RWC at around initial wilting is about 60% to 70%, with exceptions.

Usually, between 4 to 6 samples (replications) are taken from a single treatment or genotype.
Each sample represents a different plant, if possible. Top-most fully expanded leaves are
sampled, unless the interest is in profiling leaves on the plant. In large broad-leaves (sunflower,
cotton, etc) leaf discs are cut from the leaves, to obtain a total of about 5-10 cm2/sample.
Sample size does not have to be the same for all samples. Avoid large veins. Leaf discs should
be large enough (around 1.5 cm in diameter) so as to reduce the area of cut leaf
surface/sample. Alternatively, a sharp cork borer may be used, cutting the leaf over a piece of
dense rubber or a large rubber stopper. It is important that sampling will proceed quickly. In
smaller composite leaves (groundnuts, alfalfa, clover, chickpeas) several leaflets make up a fast
and convenient sample. In cereals, a sample may constitute of a mid-leaf section of about 5-10
cm2 cut with scissors. With larger leaves such as maize or sorghum a section measuring, say,
about 1×7 cm can be cut with scissors from the area between the mid-vein and the edge. Each
sample is placed in a pre-weighed airtight (possibly also oven proof) vial. Cereal leaf sample
should be placed in a vial slightly longer than the sample, with its basal part to the bottom. Vials
should be immediately placed in a picnic cooler (around 100C-150C) but not frozen on ice. In
the Lab vials are weighed to obtain leaf sample weight (W), after which the sample is
immediately hydrated to full turgidity for 3-4h under normal room light and temperature. Some
prefer to hydrate samples on the lower shelf of a lab refrigerator (about 100C). Leaf discs and
small leaflets are usually hydrated by floating on de-ionized water in a closes petri dish. Cereal
leaf samples receive water into the vial to a level of 1-2 cm after which the vial is capped.

4. To determine Turgor pressure of plant tissue

Turgor pressure is the force within the cell that pushes the plasma membrane against the cell
wall. It is also called hydrostatic pressure, and defined as the pressure measured by a fluid,
measured at a certain point within itself when at equilibrium.

Procedure
1. Get 2 pieces of tissue of approximately the size of potato
2. Get two weigh boats Label one “A” for salt water and the other “B” for distilled water.
3. Fill the weigh boat labeled “A” about half full with salt water.
4. Fill the weigh boat labeled “B” about half full with distilled water.

5. Obtain the mass of the first piece of tissue sample using the triple beam balance. Record the
mass.
6. Now place the first piece of tissue sample in the weigh boat “A” (the salt water weigh boat).
7. Obtain the mass of the second piece of tissue sample using the triple beam balance. Record
the mass.
8. Now place the second piece of tissue sample in the weigh boat “B” (the distilled water weigh
boat).
9. Allow the tissue sample to soak for 15-20 minutes.
10. Once the tissue sample have soaked for the required time, remove the tissue sample in the
weigh boat “A”.
11. Use the paper towel to gently dry off excess water from the tissue sample.
12. Use the balance to obtain the mass. Record the mass of the tissue sample.
13. Determine if this piece of potato is stiff or not. Record it.
14. Now, remove the tissue sample in the weigh boat “B”.
15. Use the paper towel to gently dry off excess water from the tissue sample.

16. Use the balance to obtain the mass. Record the mass.

17. Determine if this piece of potato is stiff or not. Record your observation.

Tissue sample stiff or not will help in determining of turgor pressure

5. Photosynthesis (rate of photosynthesis, oxygen and Co2 measurement):

Photosynthesis is the process by which green plants and microbes manufacture food. They
need carbon dioxide (CO2) in the air, which enters the plant through leaves, and water (H2O)
from the soil, which roots absorb. Light captured by chlorophyll in the leaves combines water
and carbon dioxide to produce glucose (C6H12O6) and oxygen (O2).

The equation for photosynthesis is:

6CO2 + 6H2O + Sunlight ----------> C6H12O6 + 6O2

The glucose undergoes various reactions with other minerals to produce sugars,

Measuring Photosynthesis

Since photosynthesis is a chemical reaction, its levels are monitored by the rate of occurrence.
Changes in the levels of its inputs and outputs are used to calculate the photosynthetic rate.
Thus, there are various methods to measure photosynthesis:

Uptake of CO2 by plants: Since CO2 is needed for photosynthesis, measuring how much of it is
taken up by the plants gives us information on how much of photosynthesis is happening.

Release of O2: The amount of O2 produced during photosynthesis can be measured.

Dry matter content: Dry matter is comprised of all the solids in a plant minus the water
content. These solids are all produced as a result of photosynthesis. So, dry matter can be used
to measure this process.

Carbohydrate production: This is an indirect way of measuring photosynthesis by its products.

Parts of a plant can be harvested, dried and weighed at intervals. The difference in weights
gives the increase in carbohydrates (sugars) due to photosynthesis.

Measure light-dependent photosynthesis using Hill’s reaction: In the first step of

photosynthesis, oxygen is produced by chloroplasts by splitting water molecules using energy
from light. Using dichlorophenolindophenol (DCPIP) as the terminal electron acceptor of oxygen
atoms, Hill’s reaction measures the light-dependent phases of photosynthesis.

Chlorophyll fluorescence: When chlorophyll absorbs light, its molecules are raised to an
“excited state”. It returns to its normal state by releasing the energy. Part of this is used to
power photosynthesis; another portion is emitted as radiation called fluorescence radiation.
Since fluorescence radiation is complementary to photosynthesis it is used to measure it in
higher plants as well as algae and bacteria.

Commercial Instruments

Gas exchange is used most often as the way to measure photosynthesis, and there are a few
different techniques. CO2 measurement uses infrared light, while O2 measurement requires
electrochemical sensors.

Infrared Gas Analyzer: CO2 absorbs infrared light. When infrared light is directed at a plant or
leaf in a closed space or chamber, there is less CO2 because the plants have used it in
photosynthesis. So, there is more infrared light left unabsorbed. (Clarify this please. I
understand this to mean that as CO2 levels decrease, less infrared light is absorbed. Is this
correct? The incoming and outgoing CO2 from the leaf chamber is measured by infrared
spectroscopy with an infrared gas analyzer. The difference gives us the amount of CO2, from
which the rate of photosynthesis can be calculated.

Electrochemical Gas Sensor: O2 doesn’t absorb infrared light, so it is measured by

electrochemical sensors. The gas passes through a membrane to which electrodes are
connected. O2 gets changed to water after receiving electrons, in the form of hydrogen ions, (I
added this - is it correct? The electrons have to come from somewhere, and you can’t turn
oxygen into water without hydrogen.) From the electrode, a process which is measured as an
electric current. The amount of current used is proportional to the amount of O2 present from
which the rate of photosynthesis can be calculated.

Oxygen measurements:-

Procedure:--

To begin the experiment, take a few twigs of the aquatic plant and insert the cut ends of their
stems into the tube of the funnel like so. Quickly but carefully immerse the funnel into the
bottom of the beaker containing water. Now, fill the test tube to the brim with water. Hold the
mouth of the tube with your thumb and invert the tube into the beaker and let it sit over the
tube of the funnel. The setup is now complete. Add a pinch of Sodium or potassium
bicarbonate into the beaker to kick start the photosynthesis process. Place the setup in
sunlight. A few minutes after exposure to sunlight, tiny air bubbles evolved from the cut ends of
the stems of the aquatic plant. These air bubbles travel up through the tube of the funnel and
eventually collect in the upper end of the test tube. As time passes, more air bubbles collect in
the test tube. Leave the setup in sunlight until the test tube is completely filled with the gas.
Now, carefully remove the test tube from the beaker while covering the mouth of the tube with
your thumb. Remove the thumb and immediately introduce a glowing splinter into the test
tube you’ll notice that the glowing splinter reignites with a pop sound. This test confirms that
the gas produced by the aquatic plants and collected in the test tube is indeed oxygen.

Carbon dioxide Measurements:-

Put a Hydrilla about 10 cm long into conical flask containing distilled water. Insert the oxygen
sensor into the conical flask and seal the conical flask. Place a bench lamp at 0.25 m from the
Hydrilla. Place a water tank between Hydrilla and banch lamp. Turn on banch lamp and allow
Hydrilla to equilibrate for five minutes. After 5 minutes record the measurements with the
sensor.

6. Chlorophyll-a & b estimation

Introduction

Chlorophyll a is found in all green plants including algae. For this reason it can be used to
estimate the quantity of algae present in a water body. Chlorophyll a constitutes approximately
1 to 2% of the dry weight of planktonic algae. Level of chlorophyll a indicates the quality of a
water body with respect to its fertilization.

Extraction of chlorophyll a & b

Before the chlorophyll a & b can be determined it must be extracted from the bulk of the water
sample as it would normally be too dilute to measure directly. This should be done by
concentrating the sample by centrifugation or filtration and then extracting the chlorophyll
pigment with acetone as given in the Standard Analytical Procedure (SAP) for chlorophyll a & b

Chromatography

Samples may be ground either by hand, using glass mortars and pestles and the addition of
acid-washed quartz sand, or mechanically, using a high-speed blender. In either case it must be
insured that the chlorophyll is extracted completely from the tissue, (i.e., the tissue is devoid of
green color)

Sample extracts, now in centrifuge tubes, are centrifuged for twenty minutes at high speed
(approximately 500 x gravity). The supernatant solution is decanted into a 10 mL graduated
cylinder and the volume brought to 10 mL with 80% aqueous acetone.
At this point, the supernatant may be stored at -10°C.

Dilution is required if the initial reading is out of the linear range of instrument detection. The
supernatant is diluted by adding 80% aqueous acetone, as necessary, to give a reading in the
range of 0.2 to 0.8 absorbance units at wavelengths of 645 nm and 663 nm. A solution which
has an absorbance of 1.00 should, when diluted in half, have an absorbance of 0.50. Final
volume of the diluted sample extract will be recorded. The 80% aqueous acetone is used as the
blank to zero the instrument initially and after every wavelength resetting. Zero checks, and any
deviations from zero, must be recorded. All samples being analyzed will be read at 645 nm,
then read again after resetting the wavelength to 663 nm. Dilutions must be accurately made
and recorded in order to calculate the chlorophyll concentration in the original tissue sample.
Calculations for chlorophyll concentrations are made after the absorbances are read at both
wavelengths.

7. Spectrophotometeic estimation of carotenoids

The spectrophotometer was used to measure TCC and the HPLC was used to measure TCC and
trans-BC. Carotenoids samples were extracted. Five g of homogenized sample were added to
50-mL centrifuge tubes with 10 mL of acetone. After 10 min, 10 mL of petroleum ether were
added and mixed using an ultra-turrax for 30 seconds. The samples were then centrifuged at
3000 rpm for 10 min at 10 °C. The upper (organic) phase was collected separately and the
extraction was repeated two additional times with 5 mL of acetone and 5 mL of petroleum
ether. Ten mL of NaCl 0.1 M solution were added to the organic extract and centrifuged at 3000
rpm for 7 min at 10 °C. The lower (aqueous) phase was discarded and the washing process was
repeated twice. Because of the wide range of carotenoid concentration, final extraction
volumes of 30, 35, and 40 mL were adjusted for low, middle, and high TCC samples. An in-house
quality control sample of freeze-dried orange fleshed sweet potato was stored at -80 °C and
used to assess between-run and within-run variations. From the obtained extracts, TCC was
measured in the μ Quant spectrophotometer at an absorbance of 450 nm using an absorption
coefficient (A1%1cm) of 2592 for all-trans-β-carotene in petroleum ether.

8. Column chromatography Technique for carotenoids estimation

Two grams of clean, dry sample leaves were weighed out, chopped with a razor blade and
placed into a mortar. Four mL of cold reagent grade ethanol (95%), a pinch of clean silica sand
to facilitate grinding, and 100 mg of CaCl2 to neutralize acids and prevent magnesium loss from
the chlorophyll were added. The leaf material was homogenized with a pestle.

The column was equilibrated with 6 mL of 28.5% ethanol (three parts reagent grade ethanol:
seven parts distilled water). The solution was drawn down to the top of the column. Two mL of
the leaf extract were placed in the reservoir and then allowed to pass through the column until
the extract solution had been drawn down to the top of the column. The column was rinsed
with 6 mL of ethanol solution, which was again drawn down to the top of the column. The
tube's reservoir was filled with 81% ethanol (8.5 parts reagent grade ethanol: 1.5 parts distilled
water) and connected to a solvent reservoir containing the same ethanol solution. This was
accomplished by inserting an 18-gauge needle, attached to plastic tubing, through a 00-rubber-
stopper and inserting the stopper into the syringe. An Erlenmeyer flask acted as a solvent
reservoir. Two mL fractions were collected until the green pigments had eluted from the
column, (about 15 to 20 tubes, depending upon the column). The reservoir was then
disconnected and the ethanol solution was brought to the top of the resin.
The column was washed with 95% ethanol, until all of the yellow pigment (carotene) had been
collected.

9. Spectrophotometer for absorption of plant pigment

Using a Beckman DU-650 spectrophotometer, absorption spectra (350 to 750 nm) were
determined for each of the tubes. Tubes containing the highest concentrations of each of the
first three peaks were diluted with the 81% ethanol solution to allow comparison of their
spectra to that of the carotenes on a single chromatogram.

Use a stock solution to prepare a series of 4 dilutions of a dietary supplement that contains
chlorophyll among other compounds (stock chlorophyll derived compounds at 0.05mg/ml).
Each dilution in the series will differ from the previous dilution by 1/2, so the dilution ratio for
each tube is 1/2, 1/4, and 1/8. Calculate the concentrations of the chlorophyll derivative that
the tubes (in micrograms/ml) will contain after the dilutions have been prepared, and record
them.

Label four 13mm test tubes with the dilution ratios: 1/2, 1/4, 1/8, 1/16. With a 5.0mL
serological pipette add 4.0mL distilled water to each tube. Using a clean pipette, add 4.0mL of
stock solution to the tube labeled 1/2 and mix by inversion (not vortexing). Use square of
Parafilm to cover test tube opening. Keeping thumb on Parafilm, invert test tube twice. Use a
clean pipette to add 4.0mL of 1/2 dilution to the tube labeled 1/4; mix. Use a clean pipette to
add 4.0mL of 1/4 dilution to the tube labeled 1/8; mix. Use a clean pipette to add 4.0mL of 1/8
dilution to the tube labeled 1/16; mix.

Determination of Absorbance versus Concentration

You will now use all four dilutions (stock, 1/2, 1/4, 1/8, 1/16) to demonstrate the relationship
between absorbance and concentration. Do not read the stock solution, as this will give you an
absorbance value that is too high. Set the spectrophotometer to the wavelength that gave
maximal absorption for chlorophyll a. Zero the instrument using water as the blank. Read the
absorbance of the four concentrations and record them.

10. Nutrient analysis

Introduction:

The role of plant nutrients in crop production is well established. There are 16 essential plant
nutrients. These are carbon (C), hydrogen (H), oxygen (O), nitrogen (N), phosphorus (P),
potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), Sulphur (S), zinc (Zn), manganese (Mn),
copper (Cu), boron (B), molybdenum (Mo) and chlorine (Cl). These nutrient elements have to be
available to the crops in quantities as required for a yield target. Any limiting or deficient
nutrient (or nutrients) will limit crop growth. The nutrient elements enter the plant in ionic
form from the soil solution. Ion transport to the root surface may take place through ion
diffusion and bulk transport (mass flow). The interpretation of plant analysis data is usually
based on the total concentrations of nutrients in the dry matter of leaves or other suitable plant
parts compared with standard values of “critical nutrient concentrations” (“critical values”).

Macronutrients

Nitrogen:
Total N in plants is estimated by the Kjeldahl method. In plants, N is present in protein form,
and digestion of the sample with H2SO4 containing digestion mixture (10 parts potassium
sulphate and 1 part copper sulphate) is required for estimation.

Procedure:

1. Weigh 1 g of soil sample. Place in a Kjeldahl flask. 2. Add 0.7 g of copper sulphate, 1.5 g of
K2SO4 and 30 ml of H2SO4. 3. Heat gently until frothing ceases. If necessary, add a small
amount of paraffin or glass beads to reduce frothing. 4. Boil briskly until the solution is clear
and then continue digestion for at least 30 minutes. 5. Remove the flask from the heater and
cool, add 50 ml of water, and transfer to a distilling flask. 6. Place accurately 20–25 ml of
standard acid (0.1M HCl or 0.05M H2SO4) in the receiving conical flask so that there will be an
excess of at least 5 ml of the acid. Add 2–3 drops of methyl red indicator. Add enough water to
cover the end of the condenser outlet tubes. 7. Run tap-water through the condenser. 8. Add
30 ml of 35-percent NaOH in the distilling flask in such a way that the contents do not mix. 9.
Heat the contents to distil the ammonia for about 30–40 minutes. 10. Remove the receiving
flask and rinse the outlet tube into the receiving flask with a small amount of distilled water. 11.
Titrate excess acid in the distillate with 0.1M NaOH. 12. Determine blank on reagents using the
same quantity of standard acid in a receiving conical flask.
Phosphorus:
The P content of the plant sample is converted to orthophosphates by digestion with an acid
mixture (di-acid or tri-acid). The digested sample is used for P estimation. When
orthophosphates are made to react with moly date and vanadate, a yellow-colored
vanadomolybdophosphoric heteropoly complex is formed. The intensity of the yellow color is
directly proportional to the concentration of P present in the sample, which can be read on the
spectrophotometer.

Procedure:

Preparation of the standard curve: Put 0, 1, 2, 3, 4, 5 and 10 ml of standard solution (50 μg P/ml) in 50-
ml volumetric flasks. Add 10 ml of vanadomolybdate reagent to each flask and make up the volume. The
P contents in these flasks are 0, 1, 2, 3, 4, 5 and 10 μg P/ml, respectively. The standard curve is prepared
by measuring these concentrations on a spectrophotometer (420 nm) and recording the corresponding
absorbances. 2. Take 1 g of plant sample and digest as per the wet digestion method, and make the volu
me up to 100 ml. 3. Put 5 ml of digest in a 50-ml volumetric flask, and add 10 ml of vanadomolybdate
reagent. 4. Make up the volume with distilled water, and shake thoroughly. Keep for 30 minutes. 5. A
yellow color develops, which is stable for days and is read at 420 nm on spectrophotometer. 6. For the
observed absorbance, determine the P content from the standard curve.

Potassium:
Potassium estimation can be done on a flame photometer, an AAS or by the volumetric sodium
tetra phenyl boron method. In a soil/plant analysis laboratory, the use of an AAS is very
common and a large number of elements are estimated using this equipment.
Procedure:
Set up the AAS and standardize. The relevant parameters for K estimation on an AAS are: lamp
current = 6 m A°; wavelength = 766.5 nm; linear range = 0.4–1.5 μg/ml; slit width = 0.5
nm; integration time = 2 seconds; flame = air acetylene. 2. Preparation of the standard
curve: Prepare the standard curve using 0, 5, 10, 15 and 20 μg K/ml. The curve will show a
linear relationship between the concentration of K and absorbance on a specific wavelength as
read from the AAS. Acid-digest 1 g of plant sample and make up to 100 ml. Keep the sample for
estimation in the range 5–10 mg K/kg (5–10 μg K/ml) by further diluting as appropriate. 4.
Prepare a blank in the same way without adding plant digested material. 5. Take an aliquot of 5
ml for estimation and make up to 100 ml. Atomize on the calibrated AAS, on which the
standard curve has also been prepared. 6. Record the absorbance against each sample. 7. From
the standard curve, note the concentration of K for the particular absorbance observed for the
sample.
Sulphur:
As dry ashing leads to volatilization loss of S present in the organic combination, and the wet
oxidation based on tri-acid mixture includes H2SO4, these two methods cannot be used for S
determination in plant samples. Therefore, di-acid (HNO3–HClO4) digestion is used.
Procedure:
Preparation of the standard curve: Same as for S estimation in soil. 2. Digest 1 g of plant
sample in di-acid and make the volume up to 100 ml. 3.Transfer 10 ml of the aliquot to a 100-ml
volumetric flask. 4. Add 1 g of sieved BaCl2 and shake for 1 minute. 5. Add 1 ml of gum acacia
acetic – acid solution, make the volume up to the mark and shake for 1 minute. 6. Run a blank
in an identical manner. 7. Measure the turbidity 25–30 minutes after the precipitation at 440
nm. 8. Read the S content in the sample from the standard curve against the similar absorbance
as noted for the sample.
Calcium:
Procedure:
1. Take 1 g of prepared plant sample. Digest in di-acid, and make the volume up to 100 ml. 2.
Dilute the sample solution to 10–20 times depending on expected content of Ca, which can be
estimated from the standard curve prepared for the purpose. 3. Set up and calibrate the AAS
using the relevant parameters: lamp current = 10 m A0; wavelength = 422.7 nm; linear
range = 1–4 μg/ml; slit width = 0.5 nm; integration time = 2 seconds; flame = nitrous oxide
acetylene. 4. After setting the AAS, atomize the standard solutions of different concentrations
of Ca and record the absorbance for the respective concentrations of Ca. Plot the concentration
of Ca on the x-axis and the corresponding absorbance on the y-axis in order to prepare the
standard curve. 5. Put 5 ml of the sample solution in a 100-ml volumetric flask and make up the
volume, atomize, and observe the absorbance. Note the corresponding concentration for the
absorbance recorded that represents the content of Ca in the sample solution.
Micronutrients:
Procedure:
The estimation of Zn, Mn, Cu, Fe, B and Mo can be done in the same manner as the estimations
of K, Ca and Mg (above) using an AAS. The preparation of the standard curve for each of these
metal elements is different. In brief, they are:
Zn: 1 g of pure zinc dissolved in 20 ml of HCl (1:1) and diluted to 1 000 ml gives 1 mg/ml Zn
stock solution. Working solutions can be obtained by diluting the stock solution from 100 to 1
000 times to obtain 10 μg to 1 μg/ml.
Fe: 1 g of pure iron wire is dissolved in 30 ml of HCl (1:1) and diluted to 1 000 ml to obtain 1
mg/ml of standard Fe. By diluting further, a working solution of a different concentration is
obtained.
Mn and Cu: Standard Mn and Cu solutions are prepared by dissolving 1 g each of pure Mn and
Cu metal in 30 ml of HCl (1:1) and making the volume up to 1 000 ml. It will give 1 mg/ml Mn
and Cu solutions. Further dilutions can be done to obtain working solutions.
B: Dissolve 8.819 g of Na2B4O7.10H2O in warm water. Dilute to 1 liter to obtain 1 mg/ml B
stock solution. Dilute further for working solutions and preparation of standard curve.
Mo: Dissolve 0.15 g of MoO3 in 100 ml of 0.1M NaOH. Dilute to 1 liter to obtain 1 000 μg/ml
or 1 mg/ml of Mo stock solution. Dilute further for working solutions and preparation of
standard curve.

11. To conduct pots experiment under different stress conditions.

Pots experiments, as a complement to field measurements, allow the investigation of plants
under controlled conditions without distracting effects of heterogeneous environmental
factors.
Pot preparation:
Plants are grown in artificial environment in various containers with various media and placed
under growing conditions which can range from growth chambers to the laboratory window sill.
The pot size has huge effect on plant growth. The plastic pots should be 1 ft. in height and 30cm
in diameter. The pots should have a hole or perforated plates. Hole is covered with Muslin
cloth.
Filling of pots:
The pots are filled with sand, soil and peat moss. Sand should be water washed. Seeds should
be sown after equal distances and covered with sand. No wash is required for soil. Soil should
be garden soil or clay. Soil mixture should be well prepared. By adding water, soil will become
saturated after one or two days. Soil should be soft for sowing seeds.
How to apply different stresses:
Salt is added as stress source in the form of solution in sand pots. Different sets of pots are
made. First row of pots is controlled. Second row of pots is under salinity stress. For solution
preparation, in 100ml water calculated salt is added.
Heavy metals are used as stress source in soil pots. Heavy metals in soil are measured and
mixed. Water supply is limited so that leaching does not occur in the uncontrolled rows of pots.
After sowing seeds, when plants become healthy, in case of drought stress, controlled row of
pots are supplied with water. Uncontrolled rows of pots have no water supply. Duration of no
water supply is used as a source of stress.

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