PURPOSE:  To measure freezing resistance in winter acclimated woody plants by determining the temperature at which freezing occurs and the temperature at which tissue death occurs.

MATERIALS: Twig samples, refrigerator, thermocouples with probes and switchbox, dissecting needle, masking tape, erhlenmeyer flasks with cork stoppers, centrifuge or other small test tubes, wedging material (cotton, washer or cardboard in step E), 60% ethanol, dry ice, insulated box or food chest, graph paper.

INTRODUCTION:  Native trees and shrubs in northern United States and Canada are often subjected to winter temperatures of -30^oC or lower.  Their vigorous renewed growth in spring is a clear indication that the living tissues are not damaged by these temperatures.  One may ask, then, at what temperature is the living tissue injured to the point that death results?  In this activity you will determine this temperature experimentally using a thermocouple as you gradually cool a twig to -30^oC or more in a dry ice/ethanol bath.

The freezing of water is an exothermic (heat releasing) process which produces 80 cal/g.  You will measure the increase in temperature that occurs when water in the xylem tissue and intercellular water (water outside or between the cells) freezes.  In winter-acclimated plants, usually no tissue injury occurs with this exotherm.  As cooling continues, a second exotherm occurs as intracellular (inside the cells) ice formation occurs.  You may observe a slight increase in temperature, or a time interval in which no decrease in temperature occurs.  It is this exotherm that marks the point when tissue death occurs.

PREDICTIONS: Use what you know about plant physiology and winter temperatures to make predictions.

1.  At what temperature do you think intercellular water will freeze in local trees and shrubs?  At what temperature do you expect tissue death to occur as a result of the formation of intracellular ice?

PROCEDURE:

A.  Cut a branch from a tree or shrub outdoors in winter.  The branch should have twigs about 3-4 mm in diameter and at least 5 cm long.  Use more than one species for comparison.  Place the cut ends of the branches in water and allow to thaw for 24 hours in a refrigerator before exposing them to room temperatures.  A sudden change in temperature can be more detrimental to living tissue than a long exposure to low temperatures.

B.  Cut a twig sample that will fit into a test tube (see step E).  Puncture the bark with a dissecting needle at an acute angle from the axis of the stem.  The needle should not penetrate deeper than the vascular cambium located just below the bark.

C.  Insert the probe of a thermocouple into the puncture hole and hold it in place with masking tape.

D.  Use tape to mount a second thermocouple probe 2-3 mm above the bark on the opposite side of the twig.  In this way the twig temperature and the air temperature can be monitored simultaneously.  The difference between the two will indicate when heat energy is released due to the freezing of water inside the twig.

E. Place a ring of cardboard, a rubber washer, or a wad of cotton over the lower end of the stem sample.  Wedge the wired stem sample into the bottom of a standard centrifuge tube or small test tube so that it is not touching the sides of the tube.

F. Place the centrifuge tube in an erhlenmeyer flask containing 60% ethanol.  The ethanol should come within 1 cm of the top of the tube.  Be careful not to get ethanol into the centrifuge tube with the sample.  If this happens you must start over with a new twig.

G. Stopper the flask and place it in an insulated box or food chest containing dry ice.

H.  Monitor both stem temperature (Ts) and air temperature (Ta) sensors, taking readings at one minute intervals as the temperature decreases.  Cooling should be controlled so that the rate does not exceed 1^oC/min by moving the flask closer to or farther from the dry ice.  Continue to record temperatures until there is no further cooling.

I.  Calculate the difference between the stem and air temperature ( _ T). Construct a graph with air temperature on the horizontal axis, and on the vertical axis.  Label the exotherm(s) with an arrow.

CONCLUSIONS:

2. At what temperature did the first exotherm occur for the species you tested?  How close was this to your prediction?

3.  What was the lowest temperature you were able to get using the dry ice bath?  Did you get a second exotherm?  What was your evidence?  How close was that temperature to your prediction?

4.  How does freezing resistance in the different species tested compare?  Which species showed the greatest resistance?  Which species showed the least?

DISCUSSION:

5. Using a reference book, determine the geographic distribution of each species tested.  Is there a correlation between northern range and freezing resistance?

6. Why was the thermocouple probe inserted into the vascular cambium and not into the xylem tissue below?

7. What chemical or physiological characteristics of living tissue may allow plants to resist freezing?

8. What was the lowest recorded air temperature (^oC) for your area during this winter season?  What was the lowest temperature ever recorded for your area?  Has winter kill occurred among trees in your area?  If so, describe the evidence.

9. What other kind of organisms could you test for freezing resistance using this method? 

Name ______________________________

PLANT FREEZING RESISTANCE DATA SHEET