Agriculture Reference
In-Depth Information
seeds are moistened does not need to be highly precise. However, germination processes are initiated during
this period, and at higher temperatures, radicle protrusion may occur in fast-germinating species such as
sorghum. Such radicle protrusion is a useful supplementary indicator of seed viability.
If it is necessary to conduct a tetrazolium test during a single working day, imbibition may be acceler-
ated by soaking the seeds in a container of warm water or in a growth chamber at 30-35°C for 3-4 hours.
This is particularly useful for cereals and grass species. Large-seeded legumes such as soybeans which are
dry and brittle at low moisture contents should be allowed to imbibe more slowly by gradually regulating
the moisture content of the media. This avoids the introduction of imbibitional injury (artifacts of the prepa-
ration process) and makes it easier to detect pre-existing mechanical injury.
When absolutely necessary, the test may be conducted without pre-moistening if the tissues of the
seed are soft enough to cut or pierce (e.g. orchardgrass). However, it is dificult to section dry, brittle seeds
without breaking the embryo, which may obscure structures, lessening the accuracy of the viability deter-
mination. In emergency cases, however, even a lower degree of test precision may be preferable to having
no knowledge of the seed's viability.
When the seeds are fully hydrated, they may be ready for staining. This is conveniently done the fol-
lowing morning if the seeds have been moistened overnight.
Some seeds, particularly small-seeded legumes, do not require pre-moistening and may be placed
directly in tetrazolium solution without danger of cracking the seed. To accelerate enzymatic activity, a
0.03% solution of hydrogen peroxide may be used. For seeds with deep dormancy, including many native
grasses, better staining can be achieved if seeds are moistened in a solution of gibberellic acid (400-500
ppm) instead of just water.
PrEPArATIon for STAInInG
While many species can be soaked in TZ solution directly after moistening, most species require additional
preparation so the tetrazolium solution will enter the seed and be absorbed by the embryo. This is necessary
because the seed coats of many seeds are impermeable to the large molecules of tetrazolium, even though
water is readily imbibed. This preparation generally consists of cutting, piercing, or puncturing the seed
or removing the seed coat or other structures, which allows the entry and absorption of the TZ solution
throughout the seed. In some cases, it involves elimination of slippery or waxy substances that interfere
with water entry. This procedure requires a good knowledge of both the internal and external anatomy of
the seed and is very exacting in its requirements. Otherwise, additional injury may occur which can make
interpretation dificult.
Several procedures have been developed for preparation of seeds for stain-
ing. Depending on the need, these methods include (1) bisecting longitudinally or
transversely with a razor blade, (2) puncturing the seed coat with a sharp needle, (3)
making seed coat incisions with a razor blade or scalpel, (4) removing the seed coat
as illustrated, and (5) excising the embryo. All of these methods may be successful,
although they differ somewhat in degree of dificulty, time required, preference of
the analyst and the species being tested.
For species not described in the tetrazolium testing handbooks, a little experimentation with various
techniques will suggest practical procedures based on seed coat permeability and size and structure of the
seed, especially the internal morphology. Such experimentation is also suggested for analysts without a
handbook, but with general experience in tetrazolium testing. Permeability of the seed coat to tetrazolium
may be determined by placing imbibed seeds of good viability in tetrazolium solution. If the embryos
remain unstained after several hours, the seed coats are impermeable to tetrazolium and must be opened in
some way. If the size, structure and location of the embryo is not known, these features can be determined
by bisecting a few seeds. Proper sectioning procedures can be determined after the internal seed structure is
known. In general, large-seeded grasses and seeds of cereals are sectioned longitudinally so that with one
