Spring.wmf (18300 bytes) 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

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.

Question: What is the water potential of potato tissue?

Hypothesis: Water potentials (Ψw) of the potato tuber cells will be negative and should range from -0.1 to -1.0 MPa (Bland and Tanner, 1985; Barcelo et al, 1994).

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. 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 (Table 2) and repeat this procedure for each of the sucrose solutions. Be sure to use a different pipet for each dye stock.

Data:

  1. Complete Tables 1 and 2.
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:

  1. Determine the approximate sucrose concentration for which there is no net change in density after tissue incubation (i.e., drop disperses, the Ψw tissue = Ψw solution).
  2. Calculate Ψs (= Ψw) in an open system such as a solution in a beaker):
    3.

    Ψs = -miRT                where

    m = molality (moles/1000 g)
    i = ionization constant (1.0 for sucrose)
    R = gas constant (0.00831 liter MPa mol-1 K-1 )
    T = Temperature in degrees K (C + 273 = K)
    note: the units are MPa

    NOTE:  the following is the preferred SI form for the equation. The answer will be in Jm-3 which are equivalent to pressure (Pa). Divide by 106 to convert to MPa.

    Ψs = -miRT                            where

    M = molality (1 molal = 1 x 103 mol m-3 H2O)
    i = ionization constant (1 for sucrose)
    R = gas constant (8.31 JK-1 mol-1 )
    T = temperature (K)

  3. What is the water potential of the potato tuber cells? How does this value correlate to the expected values? Is this technique accurate? Explain.
     
  4. We incubated the cores for about 1.5 hours.  How can we determine if this is an adequate time?
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Last updated:  01/07/2009     � Copyright  by SG Saupe