Agriculture Reference
In-Depth Information
q. Successive slices of the nutritive tissue until the outline of the intact embryo becomes clearly
visible [
Asparagus
(Liliaceae);
Aucuba
(Cornaceae);
Iris
and
Belamcanda
(Iridiaceae); most
Smilaceae and Ulmaceae].
r. Longitudinal edges of the seed, including a thin section of the nutritive tissue and/or of the coty-
ledons [
Cannabis
(Moraceae);
Fraxinus
and
Syringa
(Oleraceae);
Romneya
(Papaveraceae)].
s. Pericarp edges (1-2 mm) on three sides of the seed, leaving both fruits attached [
Acer
(Acearaceae)].
t. A fragment of the seed coat from a non-destructive location [
Humulus, Maclura
and
Morus
(Moraceae)].
u. The embryo by pieces. Keep the pieces together as a unit [
Carya
and
Pterocarya
(Juglandaceae)].
v. The intact embryonic axis and the scutellum by thrusting the narrow tip of a blade or a lance
needle through the seed coat and into the endosperm immediately above the germ. The basal
end of the seed is split while the embryo is being lifted out [
Hordeum, Secale
and
Triticum
(Poaceae)].
w. One cotyledon. Stain the cotyledon with the attached embryo [
Arachis
(Fabaceae) and most
Fagaceae].
STAInInG WITH Tz SoLuTIon
Following preparation for staining, seeds are placed in a dilute solution of 2,3,5-triphenyl tetrazolium chlo-
ride (or bromide, in some countries). When this salt enters a living cell, it is reduced by respiratory enzymes
(dehydrogenases) to form an insoluble red compound (formazan), staining the cell red. The tetrazolium test
is thus a method of visualizing the presence of dehydrogenase enzymes which are active only in living cells.
Since dehydrogenase is not active in dead tissues, these remain unstained.
A number of other tetrazolium salts are available with higher and lower molecular weights, faster and
slower staining times and with various colors of red, blue, or violet. Some of these salts are preferred for
staining microscopic preparations. However, none appear to have a practical advantage over tetrazolium
chloride or tetrazolium bromide for seeds.
Staining will proceed under a wide range of tetrazolium concentrations and temperatures. Staining
time is shortened as temperatures are increased to 35°C and as tetrazolium concentrations are increased to
1.0%. Generally, concentrations of 0.5 to 1.0% are most satisfactory for legume, grass, vegetable, lower,
tree, and other seeds that are not bisected through the embryo, while concentrations of 0.1 to 0.5% are more
satisfactory for seeds that are bisected through the embryo. Temperatures between 25 and 35°C are usually
most practical for seed testing laboratories since these temperatures may be attained in seed germinators
and staining is rapid enough for most purposes. The staining time required depends on species and tem-
perature and can be modiied for convenience to the laboratory schedule by adjusting the temperature of
incubation. Furthermore, the staining period may also be interrupted by transferring the test to refrigerated
conditions to accommodate the convenience of the laboratory schedule.
Large-seeded species are placed in tetrazolium solution for a speciied period in darkness or subdued
light. Small-seeded species may be placed on or between ilter paper or paper towels or on watch glassware
to allow absorption of the TZ solution. Staining time varies from 1 to 24 hours or more in seeds with high
levels of dormancy depending on the kind of seed, method of preparation, deterioration, concentration of
testing solution, and temperature. Appropriate staining times are given for each kind and method in the
AOSA and ISTA TZ handbooks. These times should not be considered absolute and the test may be termi-
nated when staining is adequate to recognize viable, weak and dead tissues. It is important to not overstain
the seeds since staining patterns may be obscured and interpretations dificult to make. More experienced
analysts tend to make interpretations at an earlier stage of staining when the physical condition of the tis-
sues is more obvious. In some situations, reasonably good estimates of viability are achieved by merely
soaking seeds in water and examining the physical condition of their seed structures without staining in
