Lab 5: Seed Viability and Testing
Objectives addition to the name and cultivar of the seed,
1. Learn which tests are used to assess seed
viability.
2. Compare several seed viability tests,
including visual assessment, seed floating,
tetrazolium chloride staining, and
germination.
3. Compare the germination of common bean
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and mung bean.
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Introduction
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Annuals, biennials, agronomic crops,
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many herbaceous perennials and woody plants
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are propagated directly from seed. Field and
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vegetable crops are often planted directly in the
field. Seeds of bedding plants (annual flowers
and vegetables) and perennials are usually
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planted in flats in greenhouses or enclosed
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structures. For growers to be efficient and
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successful, they must know answers to several
questions about the seed they plant. Are there
any dormancy factors? If dormancy is present,
how is it overcome? Is the seed viable? What
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is the percentage that should germinate? Is
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there debris or other foreign material in with
the seed? Is the seed contaminated with other
undesirable seed? How old is the seed?
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These questions are dealt with by those who
collect, clean, and sell seed. Seed companies
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test samples of all seeds they sell in order to
determine germination percentage and vigor
of the seeds. Seed companies also recommend
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to growers and consumers the optimum
conditions for growing the seed.
Seed testing
Seed testing is required because both state
and federal laws require labeling of seed. In
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data such as germination percentage, purity,
amount of weed seed, and amount of inert
material may be required.
Several tests have been developed that
determine viability and vigor. We will be using
some of these tests on various bean seeds.
Direct Germination Test
This is the standard test for seeds of annual,
biennial, and herbaceous perennial seeds.
Seeds are placed in an environment suitable
for germination, and the final percentage of
seeds that germinate is calculated. Suitable
containers include Petri dishes for small
seeds, plastic boxes, or small flats. The seed
may be germinated on blotter paper, specially
manufactered paper towels, perlite, or sand. A
representative sample of the seed lot must be
obtained, then reduced in size to produce the
Working Sample—the seeds which are actually
tested. Replicate tests, usually of 100 seeds
each, are always done. Direct germination tests
last from several days to several weeks.
Triphenyltetrazolium test (TZ or TTC test)
Seeds are soaked in triphenyltetrazolium
chloride, which reacts with enzymes in living
tissue and produces a red color within hours.
In the TZ test, dehydrogenase enzymes in
seeds (or any living tissue) catalyze a reaction
with triphenyltetrazolium chloride that
produces hydrogen ions. When hydrogen
ions accumulate in the oxidized, colorless
tetrazolium salt solution, the solution is
chemically reduced and changes to a red or
pink color.
The interpretation of the TZ test is precise
and differs according to different species. The
 TZ test is often referred to as a topographical
test, because interpretation depends not only
on the presence of a red color, but also on the
pattern (topography) of staining. The most
important areas to stain are those that contain
meristems, the radicle tip and the shoot
apical meristem. Interpretation is carefully
standardized as described in Rules of Seed
Testing.
The TZ test is also useful because it is
quick and suitable for determining viability
of dormant seeds that require lengthy pre-
germination treatments and do not germinate
readily using the direct germination test.
Float test
The float test can often easily distinguish
between seeds with and without embryos,
because seeds with embryos are heavier and
will sink in water. Saline solutions are required
to provide buoyancy for heavy seeds. This test
must be carefully monitored, and, as with the
cut test, the float test is not a true viability test.
Reading Material
HKDG, 7th: pages 172 - 183.
HKDG, 8th: pages 175 - 184.

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Excised embryo test

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Seeds of woody plants are often dormant

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and require lengthy pre-germination

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procedures to germinate. An excised embryo
Materials

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test is useful if the dormancy is located in the
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seed coats. If the embryo is excised from the
Plant Material
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seed coats and placed in suitable conditions, it
may germinate or give a partial response (e.g. ❏❏ Broad (Fava) Bean, (Vicia faba cv.
greening of cotyledons, radicle growth, etc.) Windsor) seeds
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indicating viability. Nonviable embryos turn ❏❏ Snap Bean (Phaseolus vulgaris cv. Yellow
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brown and deteriorate. Since seed coats often Wax Rocdor) seeds
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contain compounds that inhibit germination, it ❏❏ Mung Bean (Vigna radiata) seeds
is absolutely necessary to remove all seed coats
for reliable test results. Materials
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❏❏ Large (12 ounce) clear plastic cups

Cut test
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The cut test indicates whether the embryo ❏❏ Singled-edged razor blades
is present, not whether the embryo is viable. ❏❏ Germination boxes
As such, it is not a true viability test and is of ❏❏ 2,3,5-triphenyltetrazolium chloride
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limited use. The cut test may be used when no (TTC), 0.5% solution
other test is available. The seed is cut open to
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reveal the presence or absence of an embryo. ❏❏ Plastic Petri dish

❏❏ NaDCC stock solution, 50g/L
X-rays ❏❏ Paper towels
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X-rays may be used to determine the

❏❏ Kimwipe tissues
presence of embryos rather than cutting seeds
open. ❏❏ Ziplock bags
❏❏ Tape

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 Methods — Floating Seed “Viability” Test

❏❏ 1. Each student will be given 2 broad bean (Vicia faba) seeds. Wipe the seeds dry and mark
both sides of your two seeds with your initials using a black Sharpie pen.
❏❏ 2. Students will pool their seeds to make three groups.
❏❏ 3. Place the pooled seeds into a large (12 ounce) clear plastic cup and add water so all
seeds have an opportunity to float without being crowded.
❏❏ 4. Record the number of seeds that float and the number that sink.
❏❏ 5. Use a black Sharpie pen to mark “F” on both sides of seeds that float.

Methods — Cut Seed “Viability” Test

❏❏ 1. Each student will use their two seeds from the floating test to check for viability.

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❏❏ 2. Using a single-edged razor blade, carefully cut each seed in half longitudinally.

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❏❏ 3. Examine each cut seed and record the number of seeds in which you visibly see an

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embryo.

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Look for the embryo, which will look
something like this (see area circled). You
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may have cut the embryo in half when

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opening the seed, so you might see part of

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the embryo in each half of the seed.

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Methods — Tetrazolium Viability Test

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❏❏ 1. After visually examining each seed, lay them in a germination box with the inside
surface facing up.
❏❏ 2. Add TTC (2,3,5-triphenyltetrazolium chloride, 0.5% solution) to the box to just cover
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the seeds.
❏❏ 3. Set aside for 30 minutes to 1 hour to allow the color to develop.
❏❏ 4. Record the number of seeds in which the embryonic tissue stained red.

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 Methods — Germination Test

❏❏ 1. Each student will receive 30 snap bean (Phaseolus vulgaris cv. Rocdor) seeds and
10 mung bean (Vigna radiata) seeds that have been treated with 10% NaDCC stock
solution for 15-30 minutes.
❏❏ 2. Divide the snap bean seeds into three groups of 10.
❏❏ 3. Lay one group of 10 snap bean seeds in a single row about 1 inch from the edge of a dry
paper towel. Fold the paper towel over the seeds twice. Moisten the area of the paper
towel holding the seeds. Fold the paper towel one to two more times, folding in the
edges along the way. Fold the rolled up paper towel in half and moisten slightly under
running tap water. Place the folded paper towel in a Ziplock bag that is left slightly
opened. Label the bag with your name and date.
❏❏ 4. Sow the second group of 10 snap bean seeds in a small Ziplock bag containing moist
vermiculite and shake the bag well. Keep the Ziplock back slightly opened. Label the

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bag with your name and date.

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❏❏ 5. Place the remaining 10 snap bean seeds in a Petri dish lined with moistened Kimwipe

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tissue paper. Place the lid on the Petri dish, tape it closed, and label it with your name

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and date.

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❏❏ 6. Place the 10 mung bean seeds in a Petri dish lined with moistened Kimwipe tissue
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paper. Place the lid on the Petri dish, tape it closed, and label it with your name and date.
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❏❏ 7. Take all four experiments home with you and monitor them daily. Keep them in a warm
location (putting them above a refrigerator works well; just don’t forget about them!)
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❏❏ 8. Record the number of seeds germinated each day. The emergence of a radicle from the
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seed will be the definition of “germinated” for these seeds.

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 Observations and Results — Viability Tests

❏❏ 1. Express your data for each species in each of the viability tests as percent viability:
number of viable seeds
×100%
total number of seeds
❏❏ 2. Did the different tests result in different percent viabilities? Why?
❏❏ 3. Is each of these tests a viability test? Explain.

Observations and Results — Germination Tests

❏❏ 1. Express your data for each of the germination tests as percent germination and a
germination rate (i.e., seeds germinated per day).

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number of germinated seeds
Germination percentage =

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×100%
total number of seeds

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❏❏ 2. Were there differences in the germination percent or rate for the folded towel and Petri

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dish tests? Speculate on the reasons for any differences.
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❏❏ 3. How do the germination percentage and rate results for these two tests compare to the
germination values in vermiculite?
❏❏ 4. How did the germination of mung beans in the Petri dish compare to the germination of
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bush beans?
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