Agriculture Reference
In-Depth Information
GEnErAL ProCEdurES
Detailed procedures for performing the tetrazolium test are outlined in handbooks prepared by both AOSA
and ISTA. These handbooks cover the details of tetrazolium techniques for seeds of various kinds of agri-
cultural, vegetable, lower, and tree (and shrub) seeds. Analysts in laboratories that test many species should
have a copy of these handbooks.
This chapter will describe the basic principles of the test and enable the analyst to adapt these tech-
niques and achieve successful results with a wide range of species. This requires a good understanding of
seed structures, and the relationship between seed morphology and the seedling structures that develop
from the embryo. Analysts with this understanding and the necessary skills, along with proper training and
experience, should have no trouble in performing tetrazolium tests. Otherwise, success with tetrazolium
testing would be dificult. A tetrazolium test consists of the following basic steps: (1) preparation of dry
seed before moistening, (2) moistening, which hydrates the seeds to activate the respiratory enzymes and
softens the tissues for cutting and piercing, (3) additional preparation for staining such as cutting or piercing
to allow TZ solution into the internal tissues, (4) staining with TZ solution for various periods of time, (5)
preparation for evaluation (e.g., use of lactic acid), and (6) interpretation of staining patterns. Each of these
steps is described in detail below.
The methods and procedures in the ISTA Tetrazolium Testing Handbook were used extensively in
the preparation of this chapter. This has been done with permission from the International Seed Testing
Association. A new AOSA Tetrazolium Testing Handbook is also now available, complete with similar
information and extensive illustrations. This handbook is especially useful in that it contains detailed testing
methods for many families, genera and species, as well as detailed descriptions and illustrations needed for
interpreting test results.
PrEPArATIon of dry SEEd bEforE MoISTEnInG
The seed coats of many species are so hard and impermeable that some kind of mechanical abrasion of the
seed coat is necessary before moisture can penetrate into the interior tissues of the seed. The TZ test should
be performed on pure seed (of the species in question) free from weed or other crop seeds. As speciied
by the AOSA or ISTA rules, two to four replicates of 100 seeds should be randomly selected for each test.
MoISTEnInG
Seeds need to be moistened with water to soften the seed coat and activate the enzymatic systems.
Respiration rate increases as a result of the dehydrogenase activity in imbibed seeds. This step is necessary
for the TZ reaction with the viable tissues resulting in the red staining. It also facilitates the cutting, pierc-
ing, or removal of the seed coat or other structures during preparation for staining. Moistening should be
done over an extended period (usually overnight) for large-seeded legumes to avoid imbibitional injury to
excessively dry, brittle or aged tissues. Although an experienced analyst can usually recognize injury due
to imbibition, it makes accurate interpretation of viability more dificult. Hydration should be done on or
between moist paper towels; however, many kinds of seeds such as tall fescue and ryegrass can be hydrated
by soaking directly in water without affecting interpretation of viability. Care should be taken to avoid
excessive water, especially during initial imbibition. Fully imbibed seeds are more easily sectioned, and
interpretation of staining patterns more reliable when a clean-cut surface of the embryo is exposed. Staining
also proceeds more rapidly and uniformly in imbibed seeds.
Moistening is usually most compatible with the normal laboratory routine if it is done overnight;
however, some species such as orchardgrass need only several minutes to a few hours of hydration. Large
seeds may be placed in moist rolled towels and small seeds on top of or between moist blotters or ilter
paper. Two to four replicates of 100 seeds each should be used for each test. The temperature at which the
