THE TOPOGRAPHICAL TETRAZOLIUM METHOD FOR DETER-
MINING THE GERMINATING CAPACITY OF SEEDS -
Received July 14, 1948
In 1942 the author published the so-called tetrazolium method for de-
termination of the germinability of seeds (3).
the reduction in germinability of the seeds due to a gradual dying-off of
the embryo (2).
The topographical spread of necrosis of embryo tissues
can be demonstrated by means of 2,3,5-triphenyl-tetrazolium-chloride.
This compound is colorless but is reduced (hydrated) into the stable but
nondiffusible red formazan by living cells (3).
tetrazolium is a definite indication of its viability because necrotic cells
remain uncolored. By means of extensive experiments conducted over a
period of years, the procedure has been perfected to demonstrate which
parts of the embryo are at least sufficiently viable to make germination
The recent publication of R. H. PORTER, MARY DURRELL, and H. J.
(1) concerning the topographical tetrazolium method does not,
however, present the method correctly in relation to several essential points.
In order to correct these errors, the present author submits a short descrip-
tion of his method (3).
Use of the tetrazolium method merely involves preparation of the em-
bryo of various species of seeds in such a way that all those parts which
are decisive in the estimation of germinability be made visible.
cereals the anterior face of the embryo must be visible.
carp is impermeable and opaque in most cereals the embryo must be re-
moved from the kernel with a lance-like scalpel or dissecting needle.
cept in maize, a longitudinal section through the center of the embryo is
not adequate because the lateral root primordia remain invisible.
in oats is the pericarp sufficiently transparent to obviate excision of the
The embryo of maize, however, possesses a simple root primor-
dium, and the lateral root primordia (secondary radicles) in the meso-
cotyl are uniformly distributed.
In this instance a longitudinal section
provides the best preparation (5).
The presoaking of the seeds in water
is necessary only to facilitate the removal of the embryo.
germination is neither necessary nor significant for the test because even
non-after-ripened cereals can reduce tetrazolium
described below, the cross-sectioned grains of oats are placed in tetrazolium
solution without pre-soaking.
Only in the case of maize is the staining of
the scutellum important in relation to germinability.
This method is based upon
Coloration of a cell by
Since the peri-
According to the method
This response of
the scutellum in other species of cereals is of no significance.
zolium procedure, in contrast to the germination tests, practically elimi-
nates experimental errors.
Data from two tests of 100 kernels each
treated in this manner have been found to provide the same accuracy as
direct germination experiments carried out as four tests of 100 grains each.
The reason for these results is that the topographic tetrazolium method
practically eliminates systematic errors because the tests are independent
of personal procedure; in the usual germination tests this individual error
amounts to more than half of the total error according to RODEWALD (7).
Only the sampling error remains and this will maximally be 50 per cent.
higher with 200 kernels than with 400 kernels and therefore will not reach
the amount of the total error of germination tests.
processing of several thousand tests has shown that in tetrazolium tests
with 200 kernels each, the errors always were lower than in germination
tests with 400 kernels.
For small cereals and maize the following procedure is most advan-
tageously employed. A 1 per cent. aqueous solution of 2,3,5-triphenyl-
tetrazolium-chloride is used.
This reagent is light-sensitive.
it, exposure both to direct sunlight and prolonged exposure to diffused
sunlight must be avoided.
The solution when preserved in the dark re-
mains unchanged for several months.
six and seven in order to be properly reactive.
extended investigations, a 1 per cent. solution at this pH-value proved most
favorable, because it produced best reaction in all cases.
and barley the desired number of grains is soaked in water from six to 18
hours which is done most conveniently during the night.
then excised by means of a sharp-bladed dissecting needle as follows: the
needle is thrust through the pericarp and testa below the embryo; the
scutellum is severed and the embryo removed.
scutellum) must remain undamaged and free from starchy endosperm and
Embryos extracted in this manner are placed in
It is advisable to use small porcelain blocks having a
round cavity about 30 mm. in diameter.
to insure the continuous submersion of embryos in the solution.
longed contact with the air must absolutely be avoided.
The reaction reaches completion after about seven to eight hours at
room temperature in the dark.
The response can then be accurately de-
Only those embryos are germinable in which the plumule and
adjacent tissue-bearing root primordia are stained.
portion of these tissues constitutes a positive test.
indicated by hatched areas in figure 1.
Id) (whose embryos possess an epiblast) the borderline between stem and
root structures parallels the epiblast.
base of the upper root primordia; in barley (fig. lc) this line is recogniz-
able as a distinct constriction between shoot and root structures.
In fact, statistical
It should have a pH value between
-According to the writer's
For wheat, rye,
The embryo is
The embryo (except the
Special care must be exercised
Coloration -of a small
The critical regions are
In wheat (fig. la) and oats (fig.
In rye (fig. lb) this line lies at the
LAKON: GERMINATING CAPACITIES OF SEEDS
An inexperienced operator may at first consider the excision of em-
bryos both difficult and tedious.
The procedure is in reality extremely
simple and can be learned in a very short time, often within a few hours.
An experienced operator can prepare 200 embryos in about half anhour.
Embryos which are decayed and soft cannot be extracted.
Oats have a thin and transparent seed coat and hence excision of em-
bryos is not necessary.
The grain is evenly cut across by means of small
nippers or scissors whereupon the two halves of the grain drop out of the
The dehulled halves of the grain which carry the embryo maythen
be placed in the tetrazolium solution without pre-soaking.
to place them in small glass dishes about 40 mm. in diameter.
practice, the determination can be made in about 24 hours at room tem-
In some cases, however, it is advisable to extend the observa-
Excised embryos of cereals, about x 13.
Those embryo parts which at least must show coloration after treatment with
tetrazolium in order for the kernel to be germinable are shown by hatch marks.
tion to 48 hours because with oats some individual kernels may show a
faint coloring, which may cause doubt about the reaction.
minability in these cases are the same as for other species of grains, namely
coloration of shoot and root primordia without reference to the scutellum.
The hatched portion of figure ld indicates that part of the oat embryo
which must at least be stained if the grain is to be classed asgerminable.
In the case of maize (5) the grains to be tested are soaked in water
for several hours, preferably over night.
pel, the kernels are cutlongitudinally so that the embryo is bisectedmedian-
ally throughout its entire length.
One half of each grain is put into a
9 cm. petri dish and covered with tetrazolium solution until the grains are
barely submerged therein.
Contact of the tissue surfaces with the airmust
be avoided under all circumstances.
after three to four hours.
Those grains aregerminable. in which the em-
bryo is stained eithercompletely or at least in the region of the shoot, in-
cluding initials of secondary radicles and the scutellum.
a. Wheat; b. Rye;
Then by means of a sharp scal-
The determination will be possible
the corn scutellum, in contrast to other cereals, is significant and at least
half of its area (the medial zone) should show coloration (fig. 2).
tissue connecting the germ and scutellum should be stained in its entirety.
The minimum area requisite as a test of germinability is shown as the
hatched portion of figure 2.
In especially urgent cases involving need for haste one may utilize tepid
water for pre-soaking, and seed-parts submersed in the tetrazolium solu-
tion can be placed in an incubator at 300 C.
may be shortened to a single day.
In this manner the test
Longitudinal median section through a kernel of corn.
Pr, procambial strand; C, coleoptile; P1, plumule; B, base of plumule; A, transition
from plumule to radiele; R. radicle; SW,initials of secondary radieles.
of the embryo which at least must show coloration after treatment with tetrazolium in
order for the kernel to be germinable is shaded by hatch marks.
About x 8.
The staining must be completed within 24 hours at the most.
longer periods, microorganisms may appear and obscure the reaction.
difficulty may, however, be overcome by transferring the seeds or embryos
into a 1/1000 solution of mercuric chloride.
read at a later time.
Bacteria which multiply rapidly, especially in oat and maize tests even
within the normal. reaction time in tetrazolium, may cloud the picture in
case of severe infection of the medium.
stained by tetrazolium, may erroneously simulate staining of the cereal
embryos or of the cut surface of the maize kernels.
In this case the test may be
Living bacteria, which are also
In such cases the
LAKON: GERMINATING CAPACITIES OF SEEDS
tetrazolium solution itself is stained red and this abnormal discoloration
of the reagent enjoins caution.
In such instances seed parts appear im-
properly stained, differing distinctly from normal coloration.
dosperm, for instance may be covered with minute red spots.
ored deposit can, however, be removed by gentle abrasion of the surface
with a dissecting needle or scalpel and the correct test of underlying tissue
can then be accurately determined.
In comparison with direct germination tests, the above tetrazolium pro-
cedure has the following advantages:
1. No great amount of space or large and complex apparatus is re-
Tests can be conducted in a limited space with simple instruments
such as porcelain blocks, petri dishes and small glass dishes, dissecting
needles, scalpels, scissors or nippers.
2. Execution is rapid even for large scale tests, small cereals requiring
no more than 48 hours, and maize no more than one day at the most.
especially urgent cases the. time may be considerably reduced by pre-
soaking in tepid water and incubation at 300 C as described.
3. The method provides reliable and exact results.
impediments to germination as in freshly harvested non-after-ripened
cereals which are not immediately germinable but which possess inherent
germinability of the grain (a condition which gave rise to the term "ger-
minating potency"2 or "Keimpotenz" (6) in 1918), the eventual germina-
tion can be determined with certainty.
DEPARTMENT OF SEED RESEARCH AND SEED TESTING STATION
The tests themselves are simple and
Even in the case of
1. PORTER, R. H., DURELL, MARY, and RoMM, H. J.
phenyl-tetrazolium-chloride as a measure of seed germinability.
Plant Physiol. 22: 149-159.
2. LAKON, GEORG.
Das Schwinden der Keimfiihigkeit der Samen, insbe-
sondere der Getreidefriichte.
Topographischer Nachweis der Keimfiihigkeit der Ge-
treidefriichte durch Tetrazoliumsalze.
Die topographische Selenmethode, emn neues Verfahren
zur Feststellung der Keimfiihigkeit der Getreidefriichte ohne.
Proc. Intern. Seed Testing Assoc. 12: 1-8.
Topographischer Nachweis der Keimfiihigkeit von Mais
Ber. Deutsch. Bot. Ges. 60: 434-444.
The use of 2,3,5-tri-
Ber. Deutsch. Bot. Ges. 57: 191-
Ber. Deutsch. Bot. Ges.