To Investigate Water Potential of Plant Tissue by Dye

Method (Falling Drop Method or Chardakov’s Method)

V. S. Chardakov, a Russian scientist devised this method in

1948.

Principle:

Water always moves from lower solute concentration to higher solute

concentration OR water moves from higher water potential to lower (more
negative) water potential.
Procedure:
1- Prepare 500 ml of 0.5 M solution of sucrose by dissolving 85.5g sucrose
(342) in distilled water.

2- Dilute this stock solution so as to prepare 10 ml of each solution ranging from

0.15 to 0.5 M (0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5). Take these solutions in 8
separate test tubes, placed in a row, label this series of test tubes as control
series.
3- Place another set of 8 smaller test tubes in a row behind the control series.
Transfer 4 ml each of the solutions of the control series into these test tubes and
label them as test series. Stopper the tubes.

4- Take leaves and cut eight rectangular pieces of uniform size from these leaves.
The pieces of leaf tissue should be small.
5- Transfer one piece of leaf to each solution of the test series, immediately after
cutting. The piece should be completely immersed in the solution.
6- Add a drop or two of methylene blue solution (0.2% W/V in water) to each
solution of the control series and shake so that the solution is tinted blue, wait for
30 minutes.
7- After 30 minutes, draw a small amount of control solution from the first tube
of the test with a dropper.
8- Immerse the end of dropper in the corresponding solution of the test series of
the same grade. Gently release a drop of the blue test solution. Hold a white
paper behind the tube and watch, if drop rise, falls OR stands still and diffuse
out.

Observations
> If the drop falls in any molar concentration, the test solution has
decreased in its density over the initial value. In other words water has
diffused out of the leaf tissue into the test solution to make the solution less
concentrated or less dense.

> Rising of the drop in any concentration of the series shows that the
solution from which the drop has been taken has lesser density than the
test solution into which it has been released. The increase in density of the
test solution is caused by diffusion of water from the solution into the leaf
tissue.
> The diffusing out of drop in any concentration of the test series
shows that there is no gain or loss of water in the test solution. Hence this
solution is isotonic with the leaf tissue.
The molarity of this solution is therefore equal to the concentration of leaf tissue.
The osmotic potential of the solution in which there is no rise or fall of the drop
is therefore, equal to water potential of the leaf.
9- Record the observation in the following table. Indicate the movement of the
drop by words “up” “down” OR “null”. The values of the osmotic potential in
the table are calculated at 20 C0.

Table: Results of water potential determination of the leaf tissues.

Molarites of Direction of
Test series tube sucrose Osmotic potential drop
no. solution movement
1 0.15 -4
2 0.20 - 5.3
3 0.25 - 6.7
4 0.30 - 8.1
 5 0.35 - 9.6
6 0.40 - 11.1
7 0.45 - 12.7
8 0.50 -14.3

Precautions:
1- Use separate dropper for each grade.

2- Take care that the tube of test series is not disturbed while releasing the drop
into the solution contained in it.

3- Release the drop gently in the middle of the test

solution. Hypotonic:

A solution having a lower osmotic pressure than an adjacent solution or

a solution under comparison. There is net outflow of solvent molecules from a
hypotonic medium into a more concentrated solution separated by a permeable
membrane.

Hypertonic:
Having higher osmotic pressure than an adjacent solution under
comparison. If a hypertonic solution is placed within the confines of permeable
membrane and surrounded by a solution of lower osmotic pressure, then there
is a net influence of solvent molecules, due to diffusion, until the solute
concentration become equal.

Isotonic:
Having same osmotic pressure as the solution under comparison. No net
migration of concentration from one to the other.
 Determination of Leaf Gas Exchange by Infra-Red
Gas Analyzer (IRGA)
Reflects the efficiency of leaves to exchange gasses (H2O vapors and CO2) and
photosynthesis. Determined by Infra-Red Gas Analyzer (IRGA).
Principle:
Works on the principal of absorption of infra-red rays by water and carbon
dioxide. Very efficient and accurate equipment available in many model.
Parameters determined by IRGA
 IRGA can determine more than 40 gas exchange parameters
 The most important ones are
– Transpiration or Evaporation Rate (E)
– Net CO2 Assimilation Rate (A)
– Stomatal Conductance (gs)
– Intracellular CO2 Concentration (Ci)
– Ci/Ca Ratio
– Water Use Efficiency (WUE)