PURPOSE:  To investigate the effects of exposure to cold temperatures on seed germination.

MATERIALS:  Wild plant seeds stored indoors since autumn, 6 petri dishes for each species tested, filter paper, distilled water, forceps, scissors, tape or glue, refrigerator at 1-5^oC.

INTRODUCTION:  The seeds of most native temperate zone plants will not germinate when they reach maturity in the autumn even though the weather may be favorable for growth.  They are in an inactive, dormant state.  Breaking seed dormancy takes time, and may require exposure to specific environmental conditions before germination.  One common requirement is exposure to a chilling period.  The artificial exposure of seeds to a period of low temperature in order to stimulate germination is called stratification.

In this laboratory exercise you will conduct a stratification experiment.  You will determine if the length of the chilling period affects the rate and speed of germination in seeds of several temperate zone plant species.  Which seeds will germinate best with and without cold exposure?  If cold exposure is necessary, how long must the chilling period be to get maximum seed germination?

PREDICTION: Use what you know about how wild seeds germinate in nature and the winter weather in your region to make a prediction.

1.  What chilling period is necessary for maximum germination rates among seeds of plants in your area:  Days?  Weeks?  Months?  Explain your ideas.

PROCEDURE:

A.  Collect seed samples from a variety of wild plants that are common to your area.  They must be mature seeds from the past growing season that have not been exposed to the low temperatures of autumn or winter. 

B. Prepare a series of 6 petri dishes for each species you plan to test by lining them with two circles of filter paper.  Cut out the labels indicating the length of the chilling period (0, 2, 4, 6, 8, and 10 weeks) and glue or tape them to the covers of the dishes.

C.  Count out 25 seeds on to the dry filter paper in each series of labeled petri dishes.  Dish 0 will serve as the control group, containing unstratified seeds (seeds that have not been exposed to low temperatures).  The other five groups of experimental seeds will be stratified for periods of 2-10 weeks by placing the dishes in the refrigerator.  All dishes should be kept in dry storage at room temperature until needed. 

D.  Starting on a Friday, take the 10-week dish from each series and add distilled water to saturate the filter paper.  (A thin film of water should cover the bottom of the dish.) Place the 10-week dishes in a refrigerator where the temperature is maintained at 1-5^oC.

E. After two weeks take the 8-week dish from each series, add distilled water and place in the refrigerator.

F. At two week intervals repeat this procedure for the 6, 4, and 2 week dishes.  Dish 0 should NOT be subjected to chilling. 

G.  On the Friday when the chilling periods are complete, take the dishes from the refrigerator.  Add distilled water to Dish O until there is a thin film in the bottom.  Add distilled water to the refrigerated dishes if necessary. Place all the dishes in a convenient location at room temperature. 

H.  Starting on Monday, examine the seeds daily for evidence of germination for two weeks, or until no more seeds germinate.  (For the purpose of this investigation, germination has occurred when any part of the embryo, usually the hypocotyl, becomes visible.)  Count and discard the germinated seeds, recording the data on the dish labels.  Make sure that the filter paper does not dry out by adding more distilled water as needed.

I.  Calculate the total number of germinated and ungerminated seeds and the percent germination on each dish label.

J.  Record your data on the class data sheet.  In the last column, record the day when germination first occurred for each species.

K. Construct a line graph from the class data, plotting percent germination on the vertical axis and chilling period in weeks on the horizontal axis.  Plot each species tested by the class on the same graph.

CONCLUSIONS:

2. Did most of the species require a short chilling period (2-4 weeks) or a long chilling period (6-10 weeks) for maximum germination? 

3. Did any of the species you tested germinate well without exposure to cold temperature?  If so, which one(s)?

4. Is there a difference in the time required to begin germination as the stratification period increases? 

DISCUSSION:

5. Think about how you set up this experiment.  List three minimum environmental conditions necessary for any seed germination.  Considering the results of this experiment, what other requirement must be met for maximum germination in most northern plants?

6. What do you think is the survival advantage to northern plant species of having seeds that require a chilling before germination?

7. Some species may have had poor germination in all the dishes.  Give at least two possible explanations for such results.

8. What results would you expect if arctic plant seeds had been cold-stratified in this experiment?  Explain your ideas.

9. What results would you expect if tropical plant seeds had been cold-stratified in this experiment?  Explain your ideas.

10. What results would you expect if seeds of a crop plant like corn or wheat had been cold-stratified in this experiment?  Explain your ideas.

GOING FURTHER:

Another type of seed dormancy results from mechanical resistance of the seed coat to embryo enlargement.  Crack or scarify (mechanically abrade) the very hard seed coats of 25 Okra seeds, being careful not to damage the embryos.  Place 25 treated and 25 untreated seeds in separate moist dishes at room temperature and make daily observations for 7-10 days.

Exposure to dry rather than moist chilling may have an influence on the length of the cold period necessary to overcome dormancy.  This can be investigated by chilling two sets of seed samples for 10 weeks, one moist and one dry.  After chilling, maintain both sets of seeds at room temperature under moist conditions, and compare percent germination.