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FRSA
SEED ANALYSIS FACT SHEET: TZ
TZ test for seed viability
What is the TZ test?
The TZ test estimates seed viability. Prepared seeds
are soaked in a solution of water with 2,3,5 triphenyl tetrazolium chloride.
The abbreviation TTC is used for the chemical while the abbreviation TZ is
used for the solution and the test in general. In the living tissues
of the seeds, active enzymes (dehydrogenases) that produce free H+ ions convert
the TTC chemical to formazan, an insoluble red dye that stains the living
tissues red. The reaction takes from a few hours to one or two days,
depending on the type of seed and other factors.
What information does a TZ test provide?
TZ results are expressed as percent viability. The TZ
test becomes a dormancy test when it is done on the ungerminated seeds left
after a standard germination test or as a separate test along side the germination
test. A TZ test alone cannot measure dormancy.
TZ tests can also provide vigor information when the reaction
variables are tightly controlled and the analyst uses check samples (samples
of known high vigor). The nature and extent of mechanical damage, thermal
damage, aging, embryo maturity and insect and fungal pathogens can also be
detected with a TZ test.
What are the limitations of the TZ test?
The TZ test is generally more labor intensive than the germination
test. So even though the test can be done in 1-3 days, the preparation
and evaluation often involves careful dissection skills and examination under
a microscope. Viral infections and chemical damage are nearly impossible
to detect with a TZ test. Some kinds of mechanical damage can be overlooked
if they are not on the surfaces being examined by the analyst. Test
artifacts can confuse evaluation for seeds that are difficult to manipulate.
Who would want information from a TZ test?
· Seed producers who need ballpark information regarding
seed viability without the weeks of waiting needed for some germination tests
· Dealers who need a quick means of double checking viability
information on a label
· Anyone who needs dormancy information
· Analysts who want a backup for germination results
or a means of checking the efficacy of various dormancy breaking treatments.
How is the TZ test done?
There are five steps in a TZ test. Some steps may be
omitted depending on the species of the seed. Analyst experience and
preference may also affect the choice of method.
1. preconditioning: Most seeds need to be imbibed with
water before they are cut or prepared for the staining solution. Seeds are
placed on moistened germination media (blotters or towels) overnight or for
several hours.
2. preparation: The imbibed seeds are then pierced or cut to
expose the embryo to the staining solution. Some seeds require removal
of covering structures like the fruit coat or the seed coat. Other
species can simply be placed directly into the stain without any preparation.
3. staining: The prepared seeds are placed into a TZ solution
that has an appropriate TZ concentration for the species being tested and
the preparation method chosen. Generally this is a one percent solution
for whole or pierced seeds and a one-tenth percent solution for seeds that
have been cut to expose the embryo. The time and temperature for staining
are other variables that are species specific.
4. preparation for evaluation: Again, this is species
specific. Some dissection may be needed after staining to more clearly
see the embryo structures. Lactic acid or glycerol may be used to “clear”
seeds with dark seed coats if dissection is too difficult or time consuming.
These clearing solutions lessen the chance of artifacts from the preparation
method (2).
5. evaluation: the analyst must know the seed structures
and be able to evaluate staining patterns of the essential structures.
Some parts of the seed naturally do not stain. For example, seeds in
the grass family have a non-staining endosperm. The stain color itself
also has to be evaluated. A bluish purple color may indicate frost
damage, while an orange red may indicate that the stain is improperly buffered.
Staining may be uneven, due to the rate of solution uptake or the uneven
metabolic activity in the different parts of the seed. The soundness
and turgidity of the tissues are also examined. The many considerations
for evaluation are discussed in handbooks and numerous technical papers in
many scientific journals.
How commonly is the TZ test used?
State laws differ on the legal use of the tetrazolium test.
Colorado Seed Act Rules and Regulations refer in Part 11 to the use of the
AOSA Rules for Testing Seed. The AOSA Rules refer to the use of the
TZ test for determination of the viability of ungerminated seeds at the end
of a germination test. The TZ test is not allowed to be used as a substitute
for germination test information intended for a label.
TZ testing resources:
International Seed Testing Association. 1999. Biochemical test
for viability. Seed Sci.Technol. 27, (supplement): 201-244.
PETERS, J. (ed.) 2000. Tetrazolium Testing Handbook.
Contribution No. 29 to the Handbook on Seed Testing revised 2000. AOSA.
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