Ph. D.

AGRICULTURE
Stress Crop Production (2+1)

PRACTICAL-2
Determination of water potential of
plant tissues

Dr. P. K. Chovatia
Associate Professor
Dept. of Agronomy
Ex. 2 Determination of water potential of plant tissues
Chardakov Method for Determining Water Potential
Background information:
 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 looses 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.
Protocol:
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 from
the potato. 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 potato cores 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 cores 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 (Table 1)
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.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 (Table 2) and repeat this procedure for each of
the sucrose solutions. Be sure to use a different pipet for each dye
stock.
Table 1: Temperature Data – Temperature of the solutions in
which the potato cores were incubated
Temperature (C) Temperature (K)
Table 2: Response of drops (float, sink, hover) when placed in
solutions in which potato cores have been incubated
[Sucrose](molality) Drop Response
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Analysis & Conclusions:

Determine the approximate sucrose concentration for which

there is no net change in density after tissue incubation (i.e.,
drop disperses, the Ψwtissue = Ψw solution).
Measuring Water Potential by the Gravimetric Technique
Background:

This technique for measuring water potential is similar in

theory to the Chardakov method and shares the advantage of being
simple to perform and doesn't require expensive equipment.
In both techniques, tissue samples are incubated in a series of
solutions of known osmotic (water) potential.
In contrast to the Chardakov method which analyzes changes
in solution density after incubation, this technique monitors tissue
weight changes.
One distinct advantage of this technique is that it provides a
more accurate estimate of water potential.
In this method, tissue samples are weighed before and after
incubation in a series of solutions of known osmotic (water)
potential.
Then, the percent change in weight of the tissue is plotted versus
solution concentration (or osmotic potential).
The water potential of the tissue is considered to be equal the
osmotic potential of the incubating solution at which there is no
change in tissue weight (i.e., where the curve intercepts the x-axis).
A "kink" may be observed in the graph below the x-axis.
This is due to incipient plasmolysis that occurs at low solution water
potential.
Water potential values determined by this method may be
slightly more negative than those obtained by the Chardokov
method. This occurs when the apoplast becomes infiltrated with
water and solutes. This increases the tissue weight and may lead to
small errors.
Protocol:

1.Dispense 10 mL of water or sucrose (0.1 - 0.8 molal) into each of

nine appropriately-labeled 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
from the potato. Cut them to the same length with a razor blade (ca.
4.0 cm). 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. Weigh two or preferably three cores, record your data in Table 2
and then place the cores in one of the test solutions. Repeat for all
solutions. Weigh the cores to the nearest 0.01 g.
4.If necessary, add more of the appropriate sucrose solution to
completely submerge the cores – but, the final volume in each tube
must be the same.
5. Incubate the cores for 1.5 - 2.0 hours. Periodically swirl.

6. After 1.5 - 2.0 hours, record the temperature of the solutions

(Table 1). Then remove the tissues, gently blot on paper towels and
reweigh. Record your data in Table 2. Examine the cores as you
weigh them. Describe their relative turgor (stiff/limp).
Data:
1.Complete Table 2. Use the following equation to calculate the percent
change in weight for each tissue by the following equation: % change =
(final - initial)/initial x 100
2.Plot % change in weight vs. sucrose concentration (molality). Draw the
best fit line for your data.
3.From the graph, determine the concentration of the sucrose solution in
which there was no net weight gain (i.e., % change = 0). At this point, the
water potential of the solution equals the water potential of the potato
cores. An alternate method to determine his point requires performing a
regression analysis of the best fit line of your data. The equation for this
line is in the form, Y = mx + b. Substitute in this equation, Y = O, and
then solve for X (the point at which the line crosses the X axis and equals
the sucrose concentration in which there is no net change in weight of the
cores = water potential of the cores). Which method do you think will be
more accurate?
4.Calculate the osmotic (= water) potential of this solution
Table 1: Temperature Data – Temperature of the solutions in which
the potato cores were incubated
Temperature (C) Temperature (K)
Table 2: Change in weight of potato cores incubated in sucrose solutions
[Sucrose] Initial Weight Final Weight Change in % Change in
(molality) (g) (g) Weight (final - Weight
initial) (g)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Analysis & Conclusions:

1.Determine the approximate sucrose concentration for which there

is no net change in density after tissue incubation (i.e., drop
disperses, the Ψwtissue = Ψw solution).
 Determination of water potential of plant tissues
The water potential of a plant tissue can be determined by the
following principle. If the tissue shows no net gain or loss of water
when immersed in a solution of known molarity, its water potential
is equal to that of the external solution.
Samples of the tissue are allowed to come to equilibrium in a
range of solutions of different concentrations. When the tissue
shows neither an increase nor a decrease in mass or length, the water
potential of the potato tissue is the same as that of the external
solution.
A.By length
Procedure
1. Use 1M sucrose solution and distilled water to prepare a series of
10 cm3 sucrose solutions in boiling tubes of different
concentrations: 1M, 0.8M, 0.6M, 0.4M 0.2M and 0.0M. Label the
boiling tubes.
2. Use Cut them all to the same length of 5 cm. It is important to
work quickly to avoipotential a cork borer to obtain cylinders of
potato tissue with the same diameter. of the tissued loss of water
through evaporation as this would lower the water.
3. Immerse two cylinders of potato tissue in each tube and cover the
tubes with sealing film.
4. Leave the set up for one hour.
5. Remove the cylinders from each tube. Measure the length of the
cylinders, and calculate the percentage change in length using the
formula below:
 final length – initial length
% change in length = x 100
initial length

6. Find the mean percentage change in length of the cylinders at each

concentration.
7. Plot a graph of the mean percentage change in length (vertical
axis) against the concentration of sucrose solution (horizontal axis).
8. From the graph, determine the concentration of sucrose solution
which causes no change in length of the tissue.
9. The water potential of the potato tissue can be expressed in terms
of the molarity of sucrose solution that causes no change in length of
the tissue.
B. By weight
Procedure
1. Repeat steps (1) and (2) in method A.
2. Slice up each cylinder into six discs of approximately equal
thickness. Place each group of discs on a separate piece of filter
paper to blot dry the water on the surfaces.
3. Weigh each group of discs and record the results.
4. Put the groups of discs in each of the labelled tubes. Cover the
tubes with sealing film.
5. Leave the set up for one hour.
6. Remove the discs from each tube. Blot off any surplus fluid
quickly and gently with filter paper and re-weigh them. Record the
new weight of each group of discs.
7. Calculate the percentage change in weight using the formula
below:
 final weight – initial weight
% change in weight = x 100
initial weight weight

8. Plot a graph of the percentage change in weight (vertical axis)

against the concentration of sucrose solution (horizontal axis).
9. From the graph determine the concentration of sucrose solution
which causes no change in weight of the tissue.
10. The water potential of the potato tissue can be expressed in terms
of the molarity of the sucrose solution that causes no change in
weight of the tissue.