Seed Viability Testing

What is seed viability?

The viability of the seed accession is a measure of how many seeds are alive and could
develop into plants which will reproduce themselves, given the appropriate conditions.

Why do we test seed viability?

It is important to know that the seeds that are stored in a gene bank will grow to produce
plants. Therefore they must have a high viability at the start and during storage. The viability
of seeds at the start of storage will also determine, within the environmental conditions, the
storage life of the accession.

When should viability be determined?

Viability will need to be determined at the start of storage and at regular intervals during
storage to predict the correct time for regeneration of the accession. The viability test takes
from a few days to weeks or even months to give an accurate result. If possible the results
should be available before the seeds are packaged and placed in the gene bank so that poor
quality seeds can be identified and regenerated before storage. Where the viability cannot be
determined before storage, the seeds should be placed into long-term storage to ensure their
safety whilst awaiting the results of the test.

How should viability be determined?

The most accurate test of viability is the germination test and this will be described here. The
germination test is made under controlled conditions to find out how many seeds will
germinate and produce normal seedlings which could develop into normal reproductively
mature plants. The IBPGR Advisory Committee on Seed Storage recommends that for the
initial germination test of species where a reasonable germination technique is available, a
minimum of two replicates using 200 seeds (100 seeds per replicate) is acceptable, providing
that germination is above 90%. If not, a further 200 seeds should be tested as before and the
overall result for seed viability taken as the mean of the two tests. Other biochemical tests are
available to test viability. These have the advantage of being quicker, but are not as accurate
and require considerable skill and practice in their implementation and interpretation. These
are not recommended by the IBPGR Advisory Committee on Seed Storage for general use as
tests for seed viability.

Germination test is the best indication of the potential of a seed lot to emerge under field
conditions. However, it takes from days to weeks and in some cases even months to
complete. Tetrazolium test, commonly known as the TZ test for seed viability have,
therefore, been developed to furnish quick estimates of seeds' germinability. The test is very
useful in processing, handling, storing and marketing. Large quantities of seed in a short
time, testing dormant seed lotS, vigour rating of the seed lots, ~upplernenting germination
test results and diagnosing the cause of seed deterioration.

The accuracy of the tetrazolium test requires:

1. A sound knowledge oi"the seed and seedling structures,
2. An understanding of the mechanisms of the reactions leading to the staining of the seed.
3. Ability to interpret the staining pattern by critically examining the seed topography and by
keeping other visible seed qualities such as tissue turbidity, abnormal or missing structures
etc., in view and,
4. Experience gained through keen interest and natural skill. Essential Seed Structures for TZ
Test Evaluation Seeds can be grouped into monocots, dicots and polycots on the basis or the
presence of one, two or more than two cotyledons. Embryo structures or structure primordial
arc. Relatively similar for embryos within each groups though theym11Y differ in shape. size
and differentiation/development of embryo structures. The critical features of the seeds of
these groups me described below.
Monocotyledons: Seeds of Gramineae family are a classical example of this class. The most
critical features are the growing root and shoot tips and the scutellum. 1be root region
comprise mainly of the radicle which may be theonly root primordia in some cases e.g. small
seeded grases or it may have severalseminal root primordia besides the radicle c.g. wheat,
maize. barley etc. The radicleties below the mesocotyl region covered under a sheath
coleorhiza. Theshootregion or the plumuleconsists oftheshoot meristem along with one or
more leaf primordial near the base, enclosed within the coleoptilc. Mesocotyl
seperatesplumulefrom the point of attachment to tbe scutel1um.

Dicotyledons: In the dicot seeds with a low degree of differentiation the embryo is embedded
in nutritive tissues and consists of a radicle and two cotyledons. In the seeds with a higher
degree of differentiation. Embryooccupies a larger portion of the seed. The radicle is well
recognizable part of the embryo while the shoot meristem with the primary leaf primordia
generally lies compressed between thetwo cotyledons.
Polycotyledons: Not very common. This type of seed is found in Pinaceae and other conifers.
The embryo is not very well differentiated except for a radicle and more than two cotyledons.
Living nutritive storage tissues encircle the embryo while the shoot meristem is encircled by
the base of the cotyledons.

Equipments required for conducting TZ test

(1) Staining dished e.g. watch glasses for small seeds or petridishes,
(2)Beakers of 100 to 250 ml capacity for larger seeds,
(3)Single edge razor blade,
(4)Needles,
(5) Forceps,
(6) Magnifying devices,
(7) Medicine dropper,
(8) Dispensing bottle,
(9) Germinating, filter paper and paper towel
(10) Oven or incubator.

Preparation of the staining solution

The chemical used for this test is a cream or light yellow coloured water soluble
powder called 2,3,5,- triphenyl tetrazolium chloride. Several concentrations of tetrazolium
solution ranging from 0.1 to 100%are used with comparable results. Generally the 1.0%
solution is used for seeds that are not bisected through the embryo, is bisected. Other low
concentrations much as 0.2% and 0.5% are sometimes used instead of the 0.1% solution. To
prepare a 1.0%solution, one gram of tetrazolium salt is dissolved in the distilled or tap water
to make 100 m!.The pH of the solution should be around 7.0for the proper staining to occur.
Solutions of pH4 or lower will not stain even viable embryos and solution of pH more than 8
will result in too intense staining. If the pH of the water is not in the neutral range, the
tetrazolium salt should be dissolved in phosphate buffer solution. The buffer solution is
prepared as follows. Solution A-Dissolve 9.078 g of Potassium dehydrogenate phosphate
(KH2PO4) in water to make 1000 m!. Solution B-Dissolve 11.876 g of Disodium hydrogen
phosphate (Na2I-IPO4.2H20)to make 1000 ml. Take, 400 ml of solution A and (1OOml of
solution 13and mix them together. In one litter of the buffer solution prepared as above,
dissolve 10g of the tetrazolium salt. This gives a 1.0% tetrazolium solution of pH 7.0 Store
the solution in a dark or amber coloured bottle to prevent the reaction 'which occurs in the
presence of light. The solutions can be stored [or several months in a refrigerator.

Preparation of the clearing solution

A 1clctophenol solution can be used as a clearing agent, if the pigment within the
seed coat or .lemma prevents clear vision after staining. 13yusing the clearing agent the
embryo is visible through the seed coat or lemma. It consist of Lactic acid: 20 parts, Phenol:
20 parts. Glycerine: 40 parL<a;nd Water: 20 p~lrts.,

Temperature
Temperature influences the staining reaction. Staining will take place twice as fast at
30 as at 20oe and twice as fast at 40 as at 30°c. Temperature between 20oe and 40oe have no
effect on accuracy of tetrazolium test but staining proceeds faster at the higher temperature.
The tests can be performed satisfactorily at room temperature.

Working Sample
At least 100 seeds should be tested in replicates of 50 or less. The seeds should be
randomly selected from the pure seed component arid counted in replicates before
conditioning. In order to ensure a contact of the tetrazolium solution with the embryo. Some
conditioning and preparatory steps may be essential. These depend on the type of the seed
permeability and thickness of the seed coat location of the embryo etc.

Preconditioning
It is done to allow complete hydration of all the tissues.
- prevent damage to cotyledons and embryo axes while cutting seeds
- initiate and activate the germination process and to
- have proper penetration of tetrazolium solution.
Some kinds of seeds can directly be placed in water while others must be moistened slowly
moistening is generally practiced for large seeded legumes or for the seed samples which are
dry brittle or are aged to avoid tissue damage due to rapid intake of water. 'Ihis can be done
by keeping the seed on top of or in between the moist blotter or paper towels respectively.
Small seeded crops or seed with a permeable coat can be moistened by direct soaking
without any adverse effects. The moistening time can be considerably reduced by putting the
seeds at a slightly higher temperature (within 40°C) during imbibitions.

Preparation for Staining

In addition to moistening most kinds of seed require some preparatory steps before
staining to
- assure the adequate penetration of the staining solution into the seed.
- accelerate the rate of staining.
- facilitate the evaluation.
Thus depending upon the kind of the seed time available degree of accuracy desired and the
experience of the analyst. one of the following methods can be adopted:
(a) No moistening or preparation required (small seeded legumes with soft coats).
(b) Slow moistening without any preparation (large seeded legumes with soft coats).
(c) Piercing puncturing or cutting of the seed coat (small seeded grasses).
(d) Cutting the seeds longitudinally through the midsection of the embryo and through part of
the endosperm leaving the two halves attached at the base or sliting the seed completely,
keeping only one half for the staining, discard the other.
(e) Cutting the seeds longitudinally slightly off-centre to avoid cutting into the embryo.
(f) Removing the seed coat (with forceps/needles/razor blades etc.) with minimum injury to
the tissues. Sometimes, a thin membrane adheres to the cotyledons even after removal of the
seed coat this may be removed by a sliding motion after an additional 30 min. of soaking
(dicots with hard seed coats).

Staining
Seeds can be stained in watch glasses, petridishes or beakers. Sufficient solution should be
used to cover the seeds and to allow its absorption. A'>a rule of thumb, a concentrated (1.0%)
solution can be used for legumes, cotton and grasses that are not bisected through the embryo
and a dilute (0.25% or 0.50%) solution for grasses and cereals that are bisected thr6ugh the
embryo. Suggested preparation method for staining, concentration of the solution, staining
time at 35° C. Care should be taken not to over stain seeds as this causes degeneration,
making interpretations difficult and uncertain. In general, seeds are placed in the solution and
held at 35° C for complete colouration. After a period in the solution, the length of which
varies according to the condition of the seed, species, temperature and concentration of the
solution. The seed is removed from the tetrazolium solution, rinsed 2-3 times in water and
then evaluated according to the staining pattern. During evaluation, the seed should be left in
a little water to prevent these from drying. If seeds are not to be evaluated' immediately, the
seeds in a little water should be kept in the refrigerator. Whenever required, 2-3 drops of
lactophenol clearing solution can be added to 100 seeds of small seeded grasses, after
completely removing the tetrazolium solution. Small seeds are examined under a stereo
microscope, while, large seeds may be examined with magnifying lenses only. Seed coats of
legumes must usually be removed before examination.
PREPARE THE SEEDS FOR THE TEST

1. Take a random sample of seeds from the accession.

2. Count out 200 seeds for each test.

3. Divide these seeds into at least two replicates. More replicates of fewer seeds in each can
be used to fit into the equipment at your gene bank.

4. In order to prevent damage to the seeds from the rapid uptake of water (imbibitions
damage), seeds with a low moisture content must imbibe water slowly. Any seeds known to
be prone to imbibitions damage should be placed in a humid atmosphere to equilibrate as a
pre-treatment. This is especially important for many species of legumes.

Top of paper method

1. Cut the paper to the size and shape of the dishes.

Equipment
Petri dishes or other containers
Filter paper, paper toweling or rice straw paper
Water
Forceps
Incubators
Permanent markers

2. Place a layer of paper in each dish. If the paper is too thin use a double layer.

3. Label the top and bottom of each dish with the accession number, number of the replicate
and date of the test.

4. Moisten the paper with water.

.

5. Arrange the seeds in a regular equidistant pattern on the surface of the paper.

6. Add more water if required and replace lid.

Between paper method

1. Cut the paper to a convenient size to hold one replicate of the seeds when spaced at regular
intervals. If the paper is too thin use a double layer.

2. Label each sheet on the outside of the paper at one end with the accession number,
replicate of the test and the date of the start of the test.

3. Moisten the paper with water.

Equipment
Filter paper, paper towelling or rice straw paper
Plastic bags
Water
Forceps
Incubators
Permanent marker

4. Arrange the seeds at regular intervals on the paper, leaving at least two centimeters clear
from the edges all round.

.5. Cover the seeds with another sheet of paper and fold in the edges to prevent the seeds
from falling out.
6. Roll the paper loosely towards the end with the label.

7. Place the rolled papers inside ventilated plastic bags or boxes.

8. Put these in an upright position in wire baskets or plastic boxes.

9. Keep the paper moist with water.

Germination in sand

1. Pack clean sterile sand into pots or trays with drainage holes in the bottom.

2. Water the sand until it is moist. Do not use excess water.

Equipment
Sand
Labels
Permanent markers
Pots or trays
Tools for planting
Water
Forceps
Incubators

3. Make holes in a regular equidistant pattern at about the same depth as the size of the seeds
for each replicate of the seeds. Ideally, the distance between holes should be at least three to
five times the seed diameter.

4. Prepare a label with the accession number, date of sowing and replicate of the test and
place in each pot or tray.
5. Fill seeds from each replicate into the holes and cover with sand.

6. Water the sand again to cover the seeds, but do not make it too wet.

CARRY OUT THE GERMINATION TEST

1. Place the prepared germination tests under the light and temperature conditions which
were determined in Step 1.

2. Keep the substrate moist during the test with water, but do not over-water.

3. Run the test for a sufficient period to determine whether the seeds have germinated, or are
dead or dormant. A fixed interval can be used based on previous experience, but the time
taken to germinate will vary among species and accessions of the same species.
 COUNT THE GERMINATED SEEDLINGS

Germination using absorbent paper

1. Seeds sown on top of paper can be counted either through the transparent cover or after
removing the cover of the container. For those between paper, unroll the paper carefully to
avoid tearing the paper or damaging the roots of the young seedlings.

2. Count and record the number of normal seedlings in each replicate.

3. Count and record the number of abnormal seedlings in each replicate. Examples of the
types of abnormal seedlings found in different species have been described by ISTA (1976b).

4. Once a seed has been counted as germinated, the resulting seedling can be discarded.

5. If the seeds are dormant, treat with the appropriate technique to stimulate germination as
advised in Vol. II of Ellis, Hong and Roberts (1985) and continue the test to allow those to
germinate.

Germination in sand

1. In this case the number of seedlings emerged from the sand is usually counted because
early root growth cannot be seen without disrupting the test.

2. Count and record the number of normal seedlings emerged in each replicate.

3. Count and record the number of abnormal seedlings emerged in each replicate.

4. If any fungal or insect infestation is seen during the germination test, sterilize the sand
before using again.

INTERPRET THE RESULTS

1. For each accession, consider the total number germinated in each replicate.

2. Check that the results of the different replicates are compatible by using tolerance tables
(see chapter 14 Ellis, Hong and Roberts, 1985).

3. If the results are compatible, calculate the mean percentage viability of that accession from
the results of all replicates.

4. If the results are not compatible consult the curator for advice on how to proceed.
DECIDE WHETHER TO REPEAT THE TEST

1. Consider the mean percentage viability of the accession.

2. If it is above 90%, accept the test as valid and use this value as the true viability.

3. If the result is 90% or below, repeat the test using a further 200 seeds following the same
procedures.

4. Calculate the mean percentage viability from the results of the two tests and use this as the
overall test result.

ENTER THE DATA INTO THE FILES

1. If the standard test for that species in your gene bank has been followed exactly, enter the
percentage viability into the inventory data file for each accession.

2. If a standard test has not been used, either enter a reference to the full germination data or
state the conditions which vary from the standard test.

Accelerated Aging (AA) Test

Equipment and Supplies

Balance: Analytical balance capable of weighing to 1.0 mg.

Inner aging chamber: A plastic box (11.0 x 11.0 x 3.5 cm) with a lid into which is placed a
plastic or wire tray with a 10.0 x 10.0 x 0.3 cm wire mesh screen (mesh 14 x 18). These trays
can be purchased commercially.

Seed moisture tins: Temperatures ranging from 103-130 C as described for each species in
the International Rules for Seed Testing (ISTA, 1999).

Bottle-top Dispensette (Brinkman): Range from 0-100 ml, for dispensing 40 ml water from a
standard screw-neck bottle or a 50 ml graduated cylinder.

Water: Deionized or distilled

Outer aging chamber: Water-jacketed incubator capable of maintaining a constant

temperature range from 40-45 C +/- 0.3 C (alternative equipment described below).

Alternative outer aging chambers: This may include incubators that have a heating element
immersed in water at the base of the chamber. These chambers frequently do not have precise
temperature control and require the use of auxiliary control to maintain uniform temperature
(YSI model 71, Yellow Springs Instrument Inc., Yellow Springs, OH, USA). When using
these chambers, water may collect inside the top of the outer chamber due to condensation
and drip on the lids of the inner chamber (boxes). If water accumulates on the lid of the inner
chambers, condensation can occur inside the inner chamber (under the lid) and water will
drop onto the seed that will raise seed moisture levels during the aging period. This can
reduce germination and cause excess mold growth. Precautions must be taken to shield the
inner chambers to prevent water droplets from accumulating on the lids during the aging
period. Do not use dry incubators or ovens as outer chambers.

Germination test facilities: See Rules for Seed Testing (AOSA, 1999) or International Rules

for Seed Testing (ISTA, 1999). (The brand name, model numbers and suppliers for outer and
inner aging chambers and other equipment needed for the AA test can be acquired from the
chairperson of the AOSA vigor test committee.)

Procedure

1. The inner chamber AA boxes and screen trays should be thoroughly washed in a 15%
sodium hypochlorite (clorox) solution and dried after each use to prevent fungal
contamination.

2. Place 40 ml (+/- 1 ml) of distilled or demonized water in each inner aging chamber and
insert a dry screen tray, being certain not to splash water onto the screen surface.

3. Determine the inital seed moisture of the sample to be tested using the method appropriate
for the species (ISTA, 1999) and if >14.0% (fresh weight basis), dry the seed to 10 - 14%
before testing.

4. A minimum of 200 seeds, determined on a weight basis (Table 11.2), are placed on the

surface of the screen tray (approximately one layer deep). More than one inner chamber
should be used to obtain the quantity of seeds needed for larger seeded species (Table 11.2).
Preferably, seeds to be aged should be untreated. However, if seeds of the crop species are
primarily marketed with fungicide treatment, treated seeds may be used.

5. The lid is secured (not sealed) on each inner chamber. The inner chambers should be
placed on a shelf, transported and placed in the outer aging chamber at the same time. Allow
an air space of approximately 2.5 cm between inner chambers on each shelf in the outer
chamber to assure temperature uniformity.

6. Precisely monitor the temperature of the outer aging chamber and maintain temperature
at +/- 0.3 C of desired temperature (Table 11.2) during the aging period. Record the time
when inner chambers are placed in the outer chamber.

7. The outer chambers should not be opened during the aging period specified in Table 11.2.
8. Seed in the inner chambers should be removed from the outer chamber at the exact hour
(+/- 15 min) specified (Table 11.2) and planted for standard germination within one hour
after removal.

9. The conditions for the standard germination test are those outlined in the AOSA Rules for
Testing Seeds (AOSA, 1999) or ISTA International Rules for Seed Testing (ISTA, 1999).

10. Include a control sample with each AA test. At the conclusion of the aging period, but
prior to planting for standard germination, remove a small sample of seed (10-20 seeds) from
the inner chamber of the control sample and weigh immediately to test for seed moisture
(fresh weight basis) using the oven method (ISTA, 1999). If seed moistures are lower or
higher than those shown in Table 11.2, the test results may not be accurate and the sample
should be re-tested.

11. When AA tests are initiated for many seed lots on the same day, samples should be
grouped at approximately one hour intervals between two outer chambers to allow adequate
time for samples to be planted immediately after the aging period.

Interpretation of AA Results

The AA test does not provide an absolute vigor or field emergence score, but simply records
germination (percentage normal seedlings) after a period of stress under conditions of high
temperature and seed moisture. When the AA results are compared to the standard
germination results of the same seed lot prior to aging, the AA germination will be either
similar to standard germination (high vigor seed) or less than standard germination (medium
to low vigor seed). One use of these results can be to rank seed lots by vigor and decisions
can be made regarding the storability or planting of each seed lot.
 REFERENCES

Abdul-Baki, A.A., and J.E. Baker. 1973. Are cellular organelles or membranes related to
vigor loss in seeds? Seed [Link].1(1):89-125.

Ader, F. 1958. Vergleichende Untersuchungen uber die Brauchbarkeit des

Tetrazoliumverfahrens in der Routineprufung bei Getreide. (Comparative investigations
concerning the usefulness of the tetrazolium method in the routine testing in cereals).
Saatgut-Wirtsch. 10:45-48, 78-80.

Ader, F. 1962. Die Bestimmung der TetrazoliumTriebkraft bei Getreide. (Determination of

tetrazolium vigor in cereals). Saatgut-Wirtsch. 14(12):338-340.
Babele, G.S. and A.K. Kandya. 1986. Use of TTC for rapid testing of the viability of
Lagerstroemia parviflora (Roxb.) seeds. J. Trop. For. 2(3):226-231.

Baird, P. D., M. M. MacMasters, and C. E. Rist. 1950. Studies on a rapid test for the viability
of corn for industrial use. Cereal Chem. 27:508-513.

Baker, L. C. 1959. Value and limitations of quick viability seed testing. Seed men’s Dig.
10(2):52.
Book, Principles of seed technology, by G. N. Kulkarni.

AOSA. 2009. Seed Vigor Testing Handbook. Contribution No. 32 to the Handbook on Seed
Testing.

Elias, S. G., and L. O. Copeland. 1997. Evaluation of seed vigor tests for canola. Seed
Technol. 19(1) 78-87.

Kim, S. H., Z. R. Choe, J. H. Kang, L. O. Copeland, and S. G. Elias. 1994. Multiple seed
vigor indices to predict field emergence and performance of barley. Seed Sci. &Technol.,
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