Plants & Human Affairs (BIOL106)  -  Stephen G. Saupe, Ph.D.; Biology Department, College of St. Benedict/St. John's University, Collegeville, MN 56321; ssaupe@csbsju.edu; http://www.employees.csbsju.edu/ssaupe

Tetrazolium Test for Seed Viability

    Germination percentage is a good measure of seed quality.  One disadvantage of conventional germination tests is that they require a waiting period of two weeks or more. The tetrazolium test is a quick chemical test that can be conducted in a short period of time with minimal equipment. 

    In this test, a seed is incubated in a dilute (0.1%) solution of 2,3,5-triphenyltetrazolium chloride.  Initially the tetrazolium solution is colorless but changes to red when it comes into contact with hydrogen (reduction) derived from enzymes in the respiration process.  Embryos showing active respiration are considered "viable" and turn red.  The darker the color the greater the respiratory activity in the seed.  Light pink color indicates an seed with reduced viability when compared to a seed that stains dark red.

    There is some evidence that microwaving a seed will improve its ability to germinate (Coburn & Airhart, 1988).  This effect may be the result of increased permeability due to the heating.  Microwave frequency is not as important as the final temperature of the seed with 75 C being reported to be optimal.  In this experiment we will use the tetrazolium test to monitor the effect of microwaves on maize seed viability.

Exercise 1: Tetrazolium Testing of Maize:

Question:  What percentage of maize grains will show staining with tetrazolium?  Where will the grains stain red?

Hypotheses:  Corn grains will show high degrees of tetrazolium staining.   The embryos will stain red.

Protocol:

1. Obtain about 50 maize grains that were soaked overnight.
2. Boil approximately 25 in water for 2 minutes (boiled control)
3. Cut the grains in half and discard one half.  Place
4. Place the one half of seed in a petri dish containing 0.1% tetrazolium.  Place the cut side down and allow to incubate for about 30 minutes.  When working with the tetrazolium, wear gloves and use a forceps to handle the grains.
5. Examine the embryo for tetrazolium staining.  Daw an embryo that has been stained in Fig 1.  Record your staining data in Table 1.
6. Complete table 1 by gathering data from the remainder of the class and then calculate the percent viability (= # grains stained/total x 100)

Results:

Analysis/Questions:

1. What do you conclude about your hypothesis that maize grains will show high degree of viability?
   
   
   
    
2. Why did we boil some of the maize grains? 
   
    
3. Explain the effect of boiling on tetrazolium staining.
   
    
4. Which part(s) of the embryo stained with tetrazolium?  Explain why you did or did not expect that.

Exercise 2:  Comparing Tetrazolium Results with Conventional Germination Results

Question:  Is tetrazolium viability a good predictor for the germination percentage of maize?

Hypothesis:  Based on the tetrazolium results, the germination percentage of corn will be ______________.

Protocol:

1. Prepare a "rag doll" germinator according to the directions provided in class.
2. Count the number of grains that germinated in a few days and record it in table 2.  Complete the table.

Results:

Statistical Test:
    It is likely that we will need to perform a statistical test to determine if our germination rate and tetrazolium viability  are significantly different.  To do this we will use a Chi square 2 x 2 contingency table test.  We will set it up using the model below.  The statistical test is available through the Concepts of Biology web site and we will discuss it in class.

Analysis/Questions:

1. How do the tetrazolium results compare with those from the conventional technique?  Is tetrazolium staining a good predictor for seed germination in maize?

Exercise 3:  The Effect of Microwaves on Maize Seed Viability

Question:  Will the viability of maize grains increase when treated with short duration of microwaves?

Hypothesis:  Unhydrated maize grains that are microwaved for short periods will show greater viability than those not microwaved.  Hydrated maize grains will quickly loose their viability when microwaved

Protocol:

1. Lots of grains were placed in a flask on the rotating plate of an Amana microwave and microwaved at full power for either 10, 30, 60 and 90 seconds.
2. The grains were removed and soaked overnight
3. Using the tetrazolium test described above, test the viability of these treatments.

Results:

Analysis/Questions:

1. Plot % viability vs. treatment time (sec)
    
2. Describe the effect of microwaving on maize grain viability.  How do the results compare to our hypothesis?  Explain.

Last updated:  01/07/2005 / � Copyright  by SG Saupe / URL:http://www.employees.csbsju.edu/ssaupe/index.html