Plant Physiology (Biology 327)  - Dr. Stephen G. Saupe;  College of St. Benedict/ St. John's University;  Biology Department; Collegeville, MN  56321; (320) 363 - 2782; (320) 363 - 3202, fax;    ssaupe@csbsju.edu

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.

Question: What is the water potential of potato tissue?

Hypothesis: Water potentials (Ψw) will be negative and should range from -0.1 to -1.0 MPa (Bland and Tanner, 1985). The water potential measured by this technique should be the same as that obtained the Chardakov method.

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 (see the Chardakov Lab for equations)
 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] (molality) Initial Weight (g) Final Weight (g) Change in Weight (final - initial) (g) % Change in Weight 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Analysis & Conclusions:
What is the water potential calculated by this method? Does it correspond to the value obtained by the Chardakov technique? Which is more accurate? Explain. Do the cores show various degrees of turgor?  Explain.

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Last updated:  01/07/2009     � Copyright  by SG Saupe