6

Seed Viability
and Viability
Testing
Although the concept of seed viability is well known, there may sometimes be dis-
agreement and confusion as to its precise meaning. To most seed technologists and most
people in the seed industry, viability means that a seed is capable of germinating and
producing a "normal" seedling. Therefore, it is used synonymously with germination capacity.
In this sense, a given seed is either viable or nonviable, depending on its ability to germinate
and produce a normal seedling; thus, only seed lots representing populations of seeds may
exhibit levels of viability.
In another sense, viability denotes the degree to which a seed is alive, metabolically
active, and possesses enzymes capable of catalyzing metabolic reactions needed for
germination and seedling growth. In this context, a given seed may contain both live and dead
tissues, and mayor may not be capable of germination. This meanil1g deals with tissue
viability as well as viability of the entire seed.
In either context, seed viability is probably highest at the time of physiological maturity,
though its environment on the parent plant may not permit germination. After physiological
maturity, the viability of seeds gradually declines. Their longevity depends on the
environmental conditions to which they are exposed.
Numerous tests exist for determining seed viability; these are discussed on the following
pages.

GERMINATION TEST

The germination test is most commonly used to determine seed viability. It has become
so universally accepted that seed germination and viability are probably considered one and
the same by most people. Regardless of its acceptance, the germination test is still an estimate
and has certain limitations as a universal estimate of seed quality. However, if these
limitations are recognized, the germination test is a useful viability index.

L. O. Copeland et al., Principles of Seed Science and Technology

© Kluwer Academic Publishers 2001
Seed Viability 125
Germination Terminology

Why test seeds for germination? The intuitive answer to this question is "Because we
need to know how the seeds will perform in the field." However, there are more reasons than
this. For example, we know that a seed lot consists of a population of seeds, each with its
own distinct capability to produce a plant. A germination test is an analytical procedure to
evaluate seed germination under standardized, favorable conditions that are seldom, if ever,
encountered in the field. Consequently, germination test results often overestimate field
performance of a seed lot. It is now increasingly recognized that seed vigor tests can more
accurately reflect the ability of a seed to perform in the field (see Chapter 8). However, the
use of favorable conditions leads to a maximum germination percentage and this result can be
consistently reproduced. This permits different analysts from different laboratories to obtain
similar germination percentages for the same seed lot. This allows verification of germination
results on seed labels, which permits shipment of seed lots of known value to move through
interstate and global commerce. A germination test allows a seed producer to determine and
compare the quality of a seed lot before it is planted. This information can be used to
determine the planting value of a seed lot and its storage potential to satisfY labeling laws and
provide for standardized marketing of seed.
To the seed analyst, germination has a definite and standardized meaning, which must
lead to uniform interpretations made by analysts in different laboratories. In the Rules for
Testing Seeds of the Association of Official Seed Analysts, seed germination is described as
"the emergence and development from the seed embryo of those essential structures which.
for the kind of seed in question, are indicative of the ability to produce a normal plant under
favorable conditions" (AOSA 2000). The seed analyst looks for the emergence of an
embryonic plant that must consist of a complete root and shoot axis that has the capacity of
normal growth under favorable conditions.
Seed testing associations (AOSA and ISTA-see Chapter 15) have developed sets of
standardized conditions (specifications) for seed germination of different species. These
conditions are published in their official rules, which specifY the optimum germination
conditions for hundreds of kinds of agricultural, vegetable, flower, and tree seeds. The rules
provide optimum germination conditions for each kind of seed. The rules also provide
additional measures that may aid in germination, particularly of dormant seeds. An excerpt of
the rules of the Association of Official Seed Analysts (AOSA) is given in Table 6,1.

Rules for Germination Testing Procedures

The AOSA Rules for Testing Seeds are divided into four groupings: agricultural,
vegetable and herb, flower, and tree and shrub seeds. Each kind of seed is listed according to
its recognized scientific nomenclature as well as common name. Following the name of the
seed is the substrate (germination media) recommended [e.g., petri dishes (P), blotters (B),
sand or soil (S), paper towels (T), or kimpac (C)]. Substrata should (1) be non-toxic to
germinating seedlings, (2) be free of molds, microorganisms, and their spores, and (3) provide
adequate aeration and moisture for germination. Timothy seed should be germinated on any
substratum suspected of being toxic to seedlings since its roots will not develop normally in
the presence of toxic substances.
 N
0\
-

Table 6.1. Some Methods for Testing for Germination.

First Final Specific

count count requirements and Fresh and
Kind of seed Substrata Temperature ·C days days photo numbers dormant seed
Eragrostis trichodes P 20-30 5 14 Light; KN0 3 Pre chill at 5° or 10°C
sand love grass for 6 weeks
Erodium cieutarium B,T 20-30 3 14 Clip seeds
alfileria
Fagopyrum eseulentum B,T 20-30 3 6
buckwheat
Festuea arundinacea P 15-25; 20-30 5 14 Light and KN0 3 Prechill at 5° or 10°C
tall fescue optional for 5 days and extend
test to 21 days
Festuea capillata P 10-25 10 28 KN0 3
hair fescue
Festuea elatior P 15-25; 20-30 5 14 Light and KN0 3
meadow fescue optional
Festuea ovina P 15-25 7 21
sheep fescue
alternate method P 20-30 7 28 Light
Festuea ovina var duriuseula P 15-25 7 21 Light and KN0 3
hard fescue optional
Data from AOSA {1978}.

V:l
~
iSS
~
~
~.
Seed Viability 127
Recommended temperatures (in degrees Celsius) for conducting germination tests are
followed by the fIrst and fmal evaluation times (counts). Temperature recommendations are
based on research that has shown optimum temperatures for each species of seed. The
Association of Official Seed Analysts permits only a 1°C variation from the recommended
temperature. One number in the temperature column indicates a constant temperature
throughout the testing period. Two numbers separated by a dash indicate an alternating
temperature during a 24-hour period-the fIrst temperature maintained for 16 hours followed
by the second temperature for 8 hours. Two temperature recommendations separated by a
semicolon are considered alternate recommendations. In such cases, both temperatures are
permissible and a laboratory may use the temperature that is most convenient. The time of the
fIrst count is considered to be approximate and a deviation of one to three days is permitted.
At this time, the analyst counts and discards germinated seedlings and inspects the test for
potential problems such as lack of moisture or pathogen infestation. The fmal count must be
on the day specifIed since it provides sufficient time for even weak seedlings to germinate.
Thus, the test should be terminated by the fmal count unless dormancy is suspected.
Seeds of certain species require special treatment for maximum germination or breaking
dormancy. Such requirements are listed in the two columns, "Special requirements" (Table
6.1) and include germination-promoting environments such as light and the addition of KN03.
Light is required for the germination of most grasses, many tree and shrub seeds, and some
vegetable seeds. The Association of Official Seed Analysts recommends that light be evenly
distributed with an intensity range of 810 to 1620 lux (75 to 150 foot-candles). For most seed
germination tests, seeds should be subjected to light for only a part of the test period. Eight
hours of light per day is usually sufficient. Potassium nitrate solution (0.2%) is used to
promote germination of some dormant seeds.
Special treatments for overcoming seed dormancy are listed under "Fresh and dormant
seed." In most instances, directions for prechilling are provided (Le., placing the seed on a
moist substratum at low temperatures-generally 10°C-for a specifIed period). Then the
seed is transferred to optimum temperature conditions for the duration of the test. Although
prechilling treatment (stratifIcation) prolongs a germination test substantially, it is essential to
break the dormancy of many species.
Although moisture and aeration are not specifIed in the germination methods, enough
moisture should be provided so that the seeds can imbibe the water before it is evaporated
from the substratum. Too much water creates an anaerobic environment in which essential
oxygen is not available to seeds. A formula determining optimum moisture for seeds
germinating in sand is provided in the "Rules for Testing Seeds" (AOSA 2000). Regardless of
the kind of seed being tested, all germination tests should be examined daily to ensure that the
moisture content of the substratum is optimal. The Association of Official Seed Analysts
further recommends that germination tests be conducted in germinators or germinator boxes
that maintain a relative humidity of 95% or higher to minimize moisture loss from the
germination substrata.
The rules for germination testing specify only environmental conditions and dormancy-
breaking procedures that are of proven effectiveness in promoting germination and lead to
standardized interpretations in routine seed testing. Too frequently, they do not reflect new
knowledge that is available through scientifIc research. For example, it is widely known that
many growth regulators are effective in breaking dormancy, yielding a more accurate
estimate of viability; yet only one inorganic chemical (potassium nitrate) is recognized by the
128 Seed Viability

AOSA "Rules for Testing Seed" (AOSA 2000) as an aid in performing germination tests. Of
the plant hormones, ethylene and gibberellic acid are approved (e.g., green needlegrass) for
helping to break dormancy. Ethylene is used for sunflower.

TETRAZOLIUM TEST

The tetrazolium test is widely recognized as an accurate means of estimating seed

viability. This method was developed at Hohenheim University in Germany in the early 1940s
by Professor Georg Lakon (1928) (see Figure 6.1) who had been trying to distinguish
between live and dead seeds by exposing them to selenium salts. He then tried tetrazolium
salts and found them more effective. Today, the test is used throughout the world as a highly
regarded method of estimating seed viability and is a routine test in many seed testing
laboratories. It is often referred to as a "quick test," since it can be completed in only a few
hours (compared to regular germination tests that require as long as two months for some
species). Tetrazolium test results can be extremely valuable for providing labeling
information for immediate shipment of
seed lots without waiting for completion
of germination tests. It is also a valuable
research technique for estimating seed
viability and determining reasons for
poor germination.

Principle

The tetrazolium test distinguishes

between viable and dead tissues of the
embryo on the basis of their relative
respiration rate in the hydrated state.
Although many enzymes are active
during respiration, the test utilizes the
activity of dehydrogenase enzymes as
an index to the respiration rate and seed
viability. Dehydrogenase enzymes react
with substrates and release hydrogen
ions to the oxidized, colorless,
tetrazolium salt solution, which is
changed into red formazan as it is
reduced by hydrogen ions (Figure 6.2).
Seed viability is interpreted according to
the topographical staining pattern of the
embryo and the intensity of the
coloration.

Figure 6.1. Professor George Lakon (Courtesy

of Prof A. M Steiner).
Seed Viability 129

N - N - CsHs

CSH5 - C
/ + 2 e + 2H+
) C6H5 - C \
\
N = N - CSH5

2,3,5-triphenyl tetrazolium formazan

chloride
(colorless) (red)

Figure 6.2. The chemical reaction that changes the colorless tetrazolium solution into jormazan.

Procedure

Seeds are first imbibed on a wet substratum to allow complete hydration of all tissues.
For many species, the tetrazolium solution can be added to the intact seed. Other seeds must
be prepared by cutting and puncturing in various ways (Figure 6.3) to permit access of the
tetrazolium solution to all parts of the seed. After hydration, the seeds are placed in a
tetrazolium salt solution and held in a warming oven at about 35°C for complete coloration.
Two hours is usually adequate for seeds that are bisected through the embryo. but others
require longer periods of staining. If seeds are held too long in contact with the tetrazolium
solution, they tend to become overstained, making interpretation difficult. Handbooks of
instructions for performing tetrazolium tests and interpretation instructions have been pub
lished by the Association of Official Seed Analysts (AOSA 2000) and International Seed
Testing Association (1985).

Evaluation

Although the living tissues of seeds stain red, interpreting these results to estimate
viability requires considerable skill and experience. Sound embryo tissues absorb tetrazolium
slowly and tend to develop a lighter color than embryos that are bruised, aged, frozen, or
disturbed in other ways. The experienced analyst learns to distinguish between seeds with the
capacity to produce normal seedlings and those that stain abnormally (Figure 6.4).
The tetrazolium test is often called the topographical tetrazolium test because the
pattern, or topography of staining is an important aspect of its interpretation. Many seeds are
neither completely dead nor completely alive. The staining pattern reveals the live and dead
areas of the embryo and enables the analyst to determine if seeds have the capacity to produce
normal seedlings. The cell division areas of the embryo are most critical during germination,
and if they are unstained or abnormally stained, a seed's germination potential is weakened.
The analyst must be familiar with crucial cell division areas of the embryo and learn to
interpret their staining pattern in terms of seed germinability.
130 Seed Viability

Small-seeded grasses

A B c
I

--4f--~- 4t lor
I>--
./
~
~
./ (side view}

Large-seeded grasses

-
.
-€?-.
.~-
. -
..
_ - .....::.---
... -
(top view)

- -

(side view)

Figure 6.3. Preparation procedures for small- and large-seeded grasses for the tetrazolium test.
Small seeded species (above) with a rather soft embryo and endosperm, such as orchardgrass, are
prepared by piercing with a needle near the embryo to allow access by the 1Z solution. Others are
prepared by cutting across or near the embryo with a razor blade. Larger seeded species (below)
are cut (after presoaking) to expose a cross-section ofthe embryo and endosperm (Slightly mod?fied
from AOSA 2000).

Other factors must be carefully observed when interpreting a tetrazolium test. Among
these are flaccid tissues (lack of adequate turgor) and critically located fractures, bruises, and
insect cavities. Any of these factors, when present in a vital position, may cause an otherwise
sound seed to be nongerminable.

Advantages and Disadvantages

The rapidity of the test is its most obvious advantage and may justify its use when speed
is important. Another advantage is its usefulness for dormant as well as for nondormant
seeds, although its nondetection of dormancy (other than hard seed) is sometimes cited as a
disadvantage. Actually, when used in combination with a germination test, it can be useful for
Seed Viability 131

2 3

5 6

7 8 9

No. I GERMINABLE. Seed completely stained.

Nos.2-4 GERMINABLE. Minor unstained areas on cotyledons.
No.5 GERMINABLE. Minor unstained area on upper portion of radiclehypocotyl
axis.
No.6 GERMINABLE. Extreme tip of radicle unstained; minor unstained spots on
cotyledons.
No.7 NON-GERMINABLE. More than extreme tip of radicle unstained.
No.8 NON-GERMINABLE. Unstained area at juncture of cotyledons and radicle-
hypocotyl axis.
No.9 NON-GERMINABLE. Unstained area near point of attachment of cotyledons
and radicle-hypocotyl axis over location where plumule develops.
No. 10 NON-GERMINABLE. Radicle-hypocotyl axis bisected by unstained area.
No. 11 NON-GERMINABLE. Unstained areas on radicle-hypocotyl axis and at point
of attachment of cotyledons to axis.
No.12 NON-GERMINABLE. More than one-half of cotyledonary tissue unstained.
No. 13 NON-GERMINABLE. Radicle-hypocotyl axis unstained.
No. 14 NON-GERMINABLE. Seed stained off color. grayish-red, orange-red or glassy
or transparent red color.
No. 15 NON-GERMINABLE. Seed completely unstained.

Figure 6.4. Tetrazolium staining patterns and their interpretation for clover seeds. Black areas
represent stained, living tissue; white areas represent unstained, dead tissue. Pairs representy both
sides of the seed (From AOSA 1970).
132 Seed Viability

testing dormant seed. The germination test tells the percentage of immediate germination; the
tetrazolium test tells the percentage of live seed; and the difference between the tetrazolium
and germination tests represents the percentage of dormant seed.
Perhaps the greatest disadvantage of the tetrazolium test is its difficulty and the
experience required to interpret it correctly. Another disadvantage has been the lack of
acceptance of tetrazolium test results by seed law enforcement agencies, the seed trade, and
certification agencies; however, this prejudice is gradually disappearing, as tetrazolium
testing procedures become standardized and more analysts are trained to use them.

OTHER BIOCHEMICAL TESTS FOR SEED VIABILITY

The tetrazolium method has largely supplanted other biochemical viability tests for
routine testing of seed quality. An excellent review of rapid biochemical viability tests that
have been attempted (with varying success) has been prepared by Gadd (1950) of the
Swedish seed testing station. The following information was taken largely from his review.

Vital Coloring Methods

The principle of the vital coloring methods is the differential coloration of live versus
dead tissues when exposed to certain dyes. An early method used sulfuric acid. Subsequently,
indigo carmine and other aniline dyes were used. This dye stains dead tissue blue, but it is
incapable of penetrating live tissue, which remains unstained. Gadd states that it is
particularly useful for determining viability of tree seeds.

Enzyme Activity Methods

These methods measure enzyme activity of imbibed seeds as an indication of their

viability. Some enzymes that have been measured are the hydrolyzing enzymes, which are
capable of splitting high-molecular organic compounds of proteins, starch, and fats into less
complicated soluble substances. Examples of such enzymes are lipase, diastase and amylase.
Another group of enzymes are the desmolases, which may be divided into two groups, the
oxidases (catalase and peroxidase), or oxidizing agents, and the dehydrogenases. They are
directly involved in the respiration process and, as a group, are closely correlated with seed
viability.
Oxidase Method i-Catalase. Some investigators have used the catalase content of
seeds as an estimate of seed viability (Leggatt 1929), while others have doubted its usefulness
because of its lack of absolute correlation with seed viability. Catalase activity varies over the
course of germination, and the catalase content of immature seeds has been reported to be
higher than that of ripe seeds. Although some workers have reported good correlations
between viability and catalase content, others have not. The probability of errors in the
detailed and complex method of measuring catalase activity makes it an unreliable measure.
Oxidase Method 2-Peroxidase. The peroxidase content appears to be more closely
correlated with viability than the catalase content. McHargue (1920) used a technique
involving guaiacol which, in the presence of H20 2, is transformed to blue tetraguaiaco-
quinone. By pretreating the seed sample with H20 2 and then grinding the seed, followed by
colorimetric determinations, he achieved results that closely agreed with germination tests.
Brucher (1948) used a similar method without the disadvantage of grinding the seed. He
Seed Viability 133
soaked the seeds for 12 hours in a mixture of guaiacol and benzidine in 10% dilutions of
saturated alcohol solutions and then treated them with the reagent. Unlike McHargue's
technique, this method permitted evaluation of each individual seed, and close correlation was
found between its results and those of regular germination tests. Brucher considered embryos
that were slightly stained or well stained to be capable of germinating. The disadvantage of
these methods is that the color disappears rapidly.
Other Oxidases. Gadd reported that other oxidases generally disappeared gradually
from mature seeds and were unsuitable for viability tests. Phenolase has been tested but with
unreliable results.
Dehydrogenase Activity Tests. These chemical tests are based on the color changes of
certain substances, depending on whether they are in an oxidized or a reduced state. The
tetrazolium test, described earlier, is the best example of a successful dehydrogenase activity
test. Another method is based on the change of methylene blue to colorless methylene white;
however, the seed must be ground and the determination made in a vacuum since exposure to
air reoxidizes the methylene blue. This process is also complicated by the presence of
microorganisms that cause a similar color change. Another test utilizes the dehydrogenase
reduction of dinitrobenzene to a red nitrophenylhydroxylamine compound in the presence of
ammonia. Gadd reported that the color reaction was rapid, and at 40°C takes place in one to
two hours; however, the color quickly disappears and the substance itself is poisonous.
The Selenite Method. The selenite test was a biochemical test of the 1930s based on
the reduction of colorless selenium salts to red elementary selenium by dehydrogenase activity
of living cells. Gadd states that the selenite principle was used as early as 1900 with bacteria
cultures; its potential in seed viability testing was discussed by Hasegawa (1935) and was
popularized by Eidmann (1938) in the 1930s. Eidmann worked primarily with tree seeds,
which he prepared by halving the embryo, piercing the seed coat or excising the embryo. The
usual method, according to Gadd, is to soak the seeds for about 24 to 48 hours at 20 to 30°C,
at which time they are washed in water and the color reaction determined. As in the
tetrazolium test, both the distribution and intensity of coloration are important in interpreting
seed viability. Gadd suggested the following classification of selenite results: (1) completely
and intensively red colored embryos (germinable), (2) slightly colored to intensively colored
embryos with pale spots (germinable), and (3) very slightly colored to entirely uncolored
embryos and those with larger uncolored spots amounting to more than one-third of the
surface (nongerminable). Gadd and Kjaer (1940) reported results of testing cereals with a
combination indigo-carmine-biselenite method in which viability estimates were improved. A
distinct disadvantage of the selenite method is that a poisonous gas slowly develops, making it
dangerous for large-scale testing.

OTHER VIABILITY TESTS

Free Fatty Acid Tests

The breakdown of fats during seed germination was discussed in Chapter 5. Similar
degradative reactions may occur in seeds as deterioration progresses, especially under high
moisture levels, high temperatures, and microorganism infestation, resulting in the formation
of free fatty acids. Consequently, under such conditions, the level of free fatty acids has been
134 Seed Viability
suggested as an index of viability. At best, the test provides only a broad quantitative estimate
of the general level of viability of a seed lot. It has never attained status as a recognized
viability test although it is reported to be useful for cotton seed.

Hydrogen Peroxide (H202) Test

In early seed germination tests, particularly those with tree seeds, hydrogen peroxide
was used as a seed treatment to minimize the effects of fungi. It was soon recognized,
however, that the compound stimulated germination. The reason(s) for this stimulation
remain unknown, although it has been suggested that the hydrogen peroxide degrades to water
and one-half molecule of oxygen, which enhances the immediate oxygen environment
surrounding the seeds and thus stimulates germination. The test is conducted by cutting the
seed coat at the radicle end allowing a 1% solution of hydrogen peroxide to permeate the
interior of the seed. This treatment results in more rapid root protrusion compared to the
standard germination test.
There are a number of other seed viability tests that focus on the integrity of the seed
coat which can have an influence on imbibition damage, seed leakage, and susceptibility to
invasion by pathogens. These include ferric chloride, indoxyl acetate, fast green, and sodium
hypochlorite tests.

Ferric Chloride Test

Mechanically injured legume seeds tum black when placed in a solution of ferric
chloride (Hardin 1980). This is a very fast and useful test that provides a rapid estimate of
the percentage of abnormal seedlings expected from a crop. The seed is placed in a 20%
solution of FeCl2 for 15 minutes, at which time all black staining seeds are separated.
Because of the rapidity of this test, a seed conditioner can examine seeds immediately after
conditioning and make adjustments to equipment as needed to reduce mechanical damage.

Indoxyl Acetate Test

Any damage to seed coats, particularly in soybeans and other large~seeded legumes, is
an indication of mechanical abuse during harvesting and conditioning and serves as an entry
site for pathogen infestation. The indoxyl acetate test (French et a1. 1962) is a rapid
laboratory test that reveals seed coat damage in soybeans and other light colored legume
seeds. Seeds are soaked for 10 seconds in a 0.1 % solution of indoxyl acetate prepared in 95%
ammonia for 10 seconds and allowed to air dry. Lesions in the seed coats that are difficult to
detect become visible because they turn purplish~green against the yellow or white seed
cotyledon background.

Fast Green Test

The fast green test reveals physical fractures in the seed coat of light-colored seeds such
as corn. Seeds are soaked in a 0.1 % fast green solution for only 15-30 seconds. During this
period, the fast green penetrates any area of the seed coat which has been fractured and stains
the endosperm green. After the soak period, the seeds are washed and the fractures then
become apparent in the seed coat.
Seed Viability 135
Sodium Hypochlorite Test

The sodium hypochlorite test is used to identifY soybean seeds with seed coat damage.
Seeds are immersed in a dilute solution of sodium hypochlorite for 10 minutes. Seeds with
seed coat cracks rapidly absorb the sodium hypochlorite and swell to three times their original
size. This enables identification of seeds with seed coat cracks from those without seed coat
deformations.
Other seed viability tests focus on seed membrane and embryo integrity such as the
conductivity, excised embryo, and x-ray tests.

Conductivity Test

Seed producers have long dreamed of a simple method for determining seed viability by
merely subjecting them to an electrical current and noting the different responses of live and
dead seeds. According to Gadd, this concept has been used with varying degrees of success
beginning with Waller in 1901 who showed that live and dead seeds gave different 'blaze
currents" that could be measured with a galvanometer. A more reliable electrical method was
suggested by Fick and Hibbard (1925) who found that after soaking a seed sample in water
for a few hours under controlled temperature conditions, the conductivity of the solution
reflected the general level of viability of the seed sample.
Conductivity tests are based on the premise that as seed deterioration progresses, the cell
membranes become less rigid and more water-permeable, allowing the cell contents to escape
into solution with the water and increasing its electrical conductivity. In the past, the
conductivity test suffered from the disadvantage of being a bulk test where 50-100 seeds were
soaked simultaneously and a mean conductivity value obtained for all seeds. However, a
commercial instrument (Figure 6.5), has been developed and possesses the capability of
monitoring individual seed conductivity values. The instrument provides a rapid indication of
seed viability for seed lots (McDonald and Wilson 1979) and brings a sophistication to the
concept of conductivity tests by improving the value and reliability of test results. However,
there is some doubt about the reliability of test results in the medium quality range.

Excised Emhryo Tests

The excised embryo test (Figure 6.6) provides a unique way of assessing seed viability
that can greatly reduce the time required for viability estimates of dormant seed. It is
particularly useful for seeds of woody species for which the time for viability estimates 9~n
be dramatically reduced. Most of the original research on this technique was performed \ly
Flemion (1936, 1938, 1941, 1948) at the Boyce Thompson Institute. She observed that if
such embryos of dormant seeds were carefully removed without injury and placed on a moist
blotter or filter paper under favorable conditions, they would readily tum green and begin to
grow. When embryo dormancy occurred, growth was slow, although much more rapid than
in the intact seed. Where extreme embryo dormancy existed, no growth occurred. Even in the
latter case, dormancy was more easily overcome when the embryos were excised than when in
the intact seed.
136 Seed Viability

Figure 6.5. This machine has the capability of simultaneously measuring the conductivity of
leachate from 100 individual seeds sequentially (Courtesy of Wavefront, Inc., Ann Arbor, MI).

The excised embryo method is especially valuable with seeds of trees and shrubs where
dormancy is a major problem in evaluating seed viability, and germination sometimes takes
as long as six months. Laboratories that test considerable quantities of tree and shrub seeqs
routinely conduct excised embryo tests; however, a standard germination test may be
performed for comparative results.
One disadvantage of the excised embryo test is the high degree of skill and time required
to prepare the embryos for the test. Considerable caution must be taken to avoid injuring the
embryo during removal. Another disadvantage is that the test does not reveal damage to the
embryo other than in the root-shoot axis that might prevent normal germination of the intact
seed

X-Ray Tests

Although the x-ray test is not a viability test, it does provide information that can help
assess viability. It can reveal morphological deficiencies that indicate the structural potential
for viability. However, by using different metallic salts with different absorption capacity into
live and dead tissue, it can be used to distinguish viable from nonviable seeds.
The most beneficial use of x-rays in seed testing is to obtain a quick indication of
abnormal morphology or mechanical damage that might impair germination capacity.
Perhaps most x-ray work has been performed on seeds of sugar beets and tree seeds. In both
cases, it is particularly useful, since it reveals the inner seed structure within the hard seed
coat and shows developmental deficiencies (Figure 6.7) as well as mechanical fractures. The
Association of Official Seed Analysts has developed a handbook for x-ray testing of seeds
(AOSA 1979).
Seed Viability 137

Figure 6.6. Excised embryo test (Courtesy ofAOSA, Public Service Kit).

Questions

1. What do you consider to be the most useful seed viability test and why?
2. Do you believe the germination test will ever be replaced as the most common seed
viability test?
3. Does the seed analyst's concept of germination differ from that ofthe layperson? How?
4. What is the principle of the tetrazolium test?
5. Do any of the other biochemical viability tests have use in routine seed analysis?
6. List the advantages of other viability tests, including those for free fatty acid, indoxyl
acetate, conductivity, excised embryo, and x-ray tests.
7. List the substrate, temperature, and fmal count days recommended for germination of
tall fescue seeds according to the AOSA "Rules for Testing Seeds."

General References

Association of Official Seed Analysts. 1970. Tetrazolium testing handbook for agricultural
seeds. Association of Official Seed Analysrs Handbook No. 29, ed. D. F. Grabe.
Association of Official Seed Analysts. 2000. Tetrazolium testing handbook. AOSA
Publication.
Association of Official Seed Analysts. 2000. Rules for Testing Seeds. Proceedings of the
Association of Official Seed Analysts 60(2):39.
138 Seed Viability

Normal Empty

Damage-(mechanical) Damage-( deterioration)

Figure 6.7. Radiograph of longleaf pine seed showing normal embryo development, empty seed,
damaged (mechanical) and damaged (deterioration) (From "Radiographic Analysis ofAgricultural
and Forest Tree Seed, " AOSA Handbook No. 31).

Association of Official Seed Analysts. 1979. Radiographic AnalYSis of Agricultural and

Forest Tree Seed: Handbook No. 31, by E. Belcher and J. Bozzo.
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Seed Viability 139

--::-_-:-:-.1941. Further studies on the rapid determination of the germinative capacity of seeds.
Contributions from Boyce Thompson Institute 11 :455-464.
___ .1948. Reliability of the excised embryo method as a rapid test for determining the
germinative capacity of dormant seeds. Contributions from Boyce Thompson Institute 15:
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of cracked seed coats of white beans and other light colored legume seeds. Proceedings of the
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