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

Testing for Seed Viability 

Objectives:  The purpose of this lab experience is to provide an opportunity to:

  1. measure seed viability by the tetrazolium and germination tests

Introduction
     G
ermination 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 water-soluble and colorless.   The tetrazolium is reduced by respiratory enzymes (dehydrogenases) to triphenylformazon which is insoluble and red.  Thus, if respiratory activity is present, which is presumed to be a sign of metabolism, the seeds will turn red and are considered "viable."  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:

 

 

 

 

 

 

Fig 1.  Sketch of tetrazolium stained seed

 

Table 1:  Tetrazolium staining of maize grains
Treatment   # grains in treatment # grains stained % viability
Fresh Individual      
Class      
Boiled Individual      
Class      

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:

Table 2.  Germination of maize grains
Grains total  
Grains germinate  
% Germination  

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.

  Not viable Viable
Tetrazolium result    
Germination result    


 

Null hypothesis (Ho):  
x2 =  
p =  

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 5, 10, 15, 30, or 60 seconds.
  2. The grains were removed and soaked overnight
  3. Using the tetrazolium test described above, test the viability of these treatments.

Results:

Table 3.  Tetrazolium staining of microwaved maize grains
Treatment (sec) # grains in treatment # grains stained % viability
0 (data from Ex 1)      
5      
10      
15      
30      
60      

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