Tetrazolium

Testing

Annette Miller, USDA/ARS

National Center for Genetic Resources Preservation

prepared 2004, updated 2014

• What is the Tetrazolium Test ?

• Categories of TZ tests done in Laboratories

• Legal, regulatory, and labeling issues. State

law case studies.

• Related issues: hard seeds, relative dormancy,

pure seed

• Ways to increase confidence in the TZ test

results.

What is the tetrazolium test?

• Chemical test used to determine seed viability.

• Measure of respiration activity.

Tetrazolium test: Detects signs of

life or metabolic activity in seeds

Germination test: Evaluates

seedling growth and development

Limitation #1: By itself, the tetrazolium test does not measure the capacity for normal cell division, growth speed, or dormancy.

How does the tetrazolium test

measure viability?

For all practical purposes, respiration cannot be measured in dry seeds.

Limitation # 2: TZ test must be done on hydrated seeds.

Hydration initiates respiration.

Enzyme activity (dehydrogenases) involved in respiration reduces the chemical:

2,3,5 triphenyl to

tetrazolium chloride (colorless in solution) (insoluble red compound)

formazan red

METHOD

• preconditioning

• preparation and staining

• evaluation

Preconditioning

Chalaza Hilum

scarify hard

seeds

imbibe on moist

media

for repeatability:

• obtain representative

sample

• determine “pure seed”

by AOSA Rules

(blower necessary for

species with blowing

points)

• randomly draw seeds

for testing

Preparation and Staining:

equipment and materials

proper disposal of

hazardous waste

implements

microscope

incubator

2,3,5, triphenyl

tetrazolium

chloride (TTC)

buffer compounds:

KH2PO4

Na2HPO4

Seed preparation

longitudinal cut

lateral cut

pierce edge slice

Seed staining

concentration technique

0.1% embryo bisected

1% whole seed

edge slice

lateral clip, cotyledon tips

pierce

Staining temperatures: 25-35oC

warmer = faster

Technical preparation problems

bad cut

Chlorophyllous embryos: cut

dry, place in TZ , stain with

cold temps in the dark .

mucilaginous seed coats (cut dry, some spp.

place cut face down on solution-soaked medium)

Rigid fruits

Deeply dormant: use hormones and

longer prep time including prechill.

Evaluation

• The analyst examines stain patterns of a

seed’s essential structures

• Each seed designated “viable” or

“non-viable”

• Seeds are also examined for signs of

mechanical damage, pathogens, and decay

• Consult TZ handbook for evaluation

guidelines

Evaluation issues:

• Embryo size

• Stain pattern and color

• Tissue texture

• Relation to germination

and field performance

Evaluation

• Uneven staining seen even with

standardized time/temperature.

• Banding patterns: normal ? abnormal ?

• Damage or differential TZ uptake?

Artifacts caused by

preparation technique

Difficult embryo extraction:

results in scrapes and bruises.

Tiny seeds hard to manipulate.

Right side has no conducting

tissue included in the cut of

the radicle/hypocotyl.

Radicle/hypocotyl area not

stained. Seed is viable.

Piercing artifact appears white.

Improper chopping motion or

dull blade causes white layer of

unstained tissue on the cut

surface.

Nutritive tissue

+ + + +

- - +

-

grasses

non-living nutritive tissue:

endosperm does not stain columbine

living nutritive tissue:

endosperm must stain

penstemon

living nutritive tissue: endosperm must stain

(dead embryo)

“Loaf of bread” cuts Spherical seeds with linear or curled embryos:

Sometimes several slices are needed to examine all parts of

the embryo. Extra time is needed.

examples: iris, asparagus

Iris

Apiaceae evaluation

?

differential embryo development

false negatives

Check embryo texture. If turgid and

unstained, retest with GA3 pretreatment.

Flaccid embryos are

nonviable

4 categories of TZ tests

• Paired TZ and germination tests for kinds

specifically mentioned in the special

procedures section (4.8) of the Rules

• Ungerminated seed left over at the end of a

germination test

• Stand alone TZ test

• Vigor test

1 test:

Germ + TZ

at end of test

DORMANT BY TZ

2 tests paired:

Germ

TZ

Stand alone TZ

GERMINATION

VIABILITY ONLY

VIABILITY

1 test:

Germination

= Dormancy broken

with special procedures

during germination

= Dormancy

determined by TZ

Category 1: Ungerminated seeds left over at the end

of a germination test

Can’t ignore them:

1. test for viability

AOSA Rules:

section 6.2f definition of Dormant

section 6.9m (EE, TZ, clip, GA3, Cut test, hard seeds)

Table 6 entries with footnote “c”

Germination Table 6: specific requirements and notes; fresh and

dormant.

Amelanchier alnifolia .. TZ may also be used: see sec. 6.9 m (2)

Atriplex canescens... TZ: see sec. 6.9 m (2)

Balsamorhiza sagittata ... TZ may also be used: see sec. 6.9 m (2)

Fraxinus americana ... TZ may also be used: see sec. 6.9 m (2)

Magnolia grandiflora... …or use TZ: see sec. 6.9 m (2)

Malus spp. (same for TZ may also be used: see sec. 6.9 m (2)

Prunus, Pyrus) ...

6.9m (2) is listed with every entry that mentions TZ and only

refers to ungerminated seed at the end of a germination test.

There is no reference to a stand-alone TZ test in table 6.

Category 2: Paired TZ and germination tests done

concurrently for kinds specifically

mentioned in AOSA Rules:

AOSA Rules Section 6.8

• green needlegrass (Nassella viridula)

• firecracker penstemon (Penstemon eatonii)

• needle-and-thread (Hesperstipa comata)

1 test:

Germ + TZ

at end of test

DORMANT BY TZ

2 tests paired:

Germ

TZ

Stand alone TZ

GERMINATION

VIABILITY ONLY

1 test:

Germination

VIABILITY

Category 3: Stand-alone test

THE STAND-ALONE TZ TEST DOES NOT DETERMINE

DORMANCY

VIABILITY ONLY

1 test:

Germ + TZ

at end of test

DORMANT BY TZ

2 tests paired:

Germ

TZ

Stand alone TZ

GERMINATION

VIABILITY ONLY

1 test:

Germination

VIABILITY

Category 3: Stand-alone test

Uses: • Research

• In-house information

• Viability estimate not used for a label

(cannot use for interstate commerce)

• Supplemental label information

• Intrastate commerce label information

in NE, SD, OR, ID, and UT (for

selected species only)

Category 3: Stand-alone test

• AOSA Section 8 lists references for conducting

tetrazolium tests but there are no specific

directions for stand-alone tests.

• Reporting: see section 15 k. and l.

• Recommendations for testing seed in the AOSA

TZ handbook were not intended for tests done for

labeling purposes.

• Tolerances: Section 14.7, Tables 14 l, m, and n

Category 3: Stand-alone test Can I label with a stand-alone TZ test?

NO for: • Interstate commerce: governed by the Federal Seed Act. There is no provision in the Colorado Seed Act, the FSA or RUSSL for listing “viability” instead of germination plus dormant. YES for:

• Intrastate commerce in NE, SD, OR, ID, and UT for selected species only. Some of these laws have more ambiguous wording than others.

• Supplemental information.

Clarification and standardization is needed.

STATE SEED LAW LANGUAGE: NE 81-2,147.02. Container; labeling requirements. (Excerpts regarding viability)

2) For agricultural seeds except for grass seed mixtures as provided in

subdivision (5) of this section:

(i) For each of the following named grasses the percentage of

germination exclusive of dormant seed, the percentage of dormant seed if

present, or the percentage of viability as indicated by a tetrazolium (TZ)

test and the calendar month and year the test was completed to determine such

percentages. Following the percentage of germination, exclusive of dormant

seed and the percentage of dormant seed, if present, the total germination and

dormant seed may be stated if desired. Also, for each of the following named

grasses when extreme dormancy is encountered, the result of a tetrazolium

(TZ) test may be shown in lieu of the percentage of germination to indicate

the potential viability of the seed:

Bluestem: Big Andropogon gerardii

Little Schizachyrium scoparium

Sand Andropogon hallii

Yellow Bothriochioa ischaemum

Dropseed, sand Sporobolus cryptandrus

Buffalograss Buchloe dactyloides

Grama: Sideoats Bouteloua curtipendula

Blue Bouteloua gracilis

Indiangrass Sorghastrum nutans

Lovegrass, sand Eragrostis trichodes

Needlegrass, green Stipa viridula

Prairie sandreed Calamovilfa longifolia

Ricegrass, Indian Oryzopsis hymenoides

Wheatgrass, western Elymus smithii

Switchgrass Panicum virgatum

STATE SEED LAW LANGUAGE: NE Title 25 Chapter 7 Nebraska Seed Law Regulations

003. Label Requirements. (Excerpts) 003.01. Label forms for agricultural, vegetable, and flower seed.

003.01A. The form of label for agricultural seeds, including lawn and turf seeds, shall show the

necessary information as stated in the Nebraska Seed Law, such as:

Germination %

Hard Seed % (for legumes and may be shown if present)

Dormant Seed % (may be shown if present)

Total Germ & Dormant Seed % (may be shown if present)

Total Germ & Hard Seed % (for legumes and may be shown if present)

TZ Viability % (used only for grasses listed in section 81-2,147.02(2)(i))

Date tested or date of expiration (for lawn and turf seed)

008 . Adopt Rules for Testing Seeds

The official methods and tolerances adopted by the Association of Official

Seed Analysts …"Rules for Testing Seeds," …for the methods of computing

tolerances of ….tetrazolium (TZ) test….

Section 5.5 of the Rules for Testing Seeds shall be utilized when computing

tolerances for percentages of ….tetrazolium (TZ).

STATE SEED LAW

LANGUAGE: SD

South Dakota

38-12A-2 excerpts: (section about Label requirements)

(10) Tetrazolium tests may be substituted for germination tests for the

kinds and time prescribed by the secretary in accordance with § 38-12A-20;

ARTICLE 12:36

12:36:04:05. Seeds for which TZ test may be used. For each of the

following grasses, the result of a tetrazolium test may be shown in lieu of the

percentage of germination to indicate the potential viability of the seed:

(1) Needlegrass, green, Stipa viridula;

(2) Wheatgrass, western, Agrophyron smithii.

For all other seeds, the result of a tetrazolium test may be shown along

with the percentage of germination.

STATE SEED LAW LANGUAGE: ID TITLE 22 AGRICULTURE AND HORTICULTURE CHAPTER 4 PURE SEED LAW 22-415.

LABEL REQUIREMENTS—AGRICULTURAL, VEGETABLE, FLOWER, TREE AND SHRUB SEEDS. …

(1) For agricultural seeds and mixtures:…

(h) Germination for each named agricultural seed:

(i) Percentage of germination, exclusive of hard seed;

(ii) Percentage of hard seed, if present;

(iii) The calendar month and year the test was completed to determine the percentages;

(iv) A tetrazolium test is deemed sufficient to meet germination labeling requirements

if the species is included in the director’s published list. …

Idaho Administrative Code IDAPA 02.06.01

300. Viability by TZ%

A TZ (tetrazolium) test may be used in lieu of germination for the following

species. The label shall read “viability by TZ%”

01 Bitterbrush

02 Saltbush

03 Sagebrush

04 Indian ricegrass

05 Winterfat

STATE SEED LAW LANGUAGE: UT

UTAH STATE SEED LAW

R68-8-16. Labeling of Chemical Tests for Viablity (Tetrazolium):

The results of tetrazolium tests performed in accordance with the

current ‘Rules for Testing Seeds’ of the Association of Official Seed

Analysts shall be recognized for labeling purposes.

UTAH STATE SEED ACT 14-16

No explicit mention of TZ testing or how the information is to be stated

on the label. No mention of % dormant in labeling requirements.

Quote from Steve Burningham (Seed Control official for UT):

“The viability percentage is reported in place of germination, with TZ

following the percentage. (85% TZ)” There are no restrictions on

which species may be labeled this way.

STATE SEED LAW LANGUAGE: OR 633.511 Definitions for ORS 633.511 to 633.750

(10) "Percentage of germination" means the percentage of pure

seed of a lot or sample that produces satisfactory sprouts before

the close of a standard germination test as prescribed pursuant

to ORS 633.580.

633.520 (1). Label requirements

(a) the percentage of germination. If germination data is based

on tests other than sprouting, that shall be so stated on the label.

___________________________________________________

Germination = “…satisfactory sprouts”

TZ = “viability” or “dormant” when germ subtracted

TZ SHOULD NEVER BE REPRESENTED AS GERMINATION

TZ DATA: STATE AS “% VIABLE” OR “% DORMANT”

ISTA Rules: All stand-alone TZ tests are done on 400

seeds. Tolerance tables have been

adopted.

Canadian Rules: - TZ tests may be done for supplemental

information or concurrently with germ.

- Not to be used for primary or sole

viability information.

Category 4: TZ as vigor test

Within the viable category, seeds are placed

into indexed categories (e.g. high/medium/low vigor)

Possible for large-seeded species where some

unstained areas are permissable. (documented for

soybeans)

Cannot use for small-seeded species with “completely

stained” criterion for viability.

Will not pick up markedly shortened roots seen in a

germination test. (documented for lettuce:

N. Vivrette, Ransom Seed Lab.)

Related issues:

• Hard Seed

• Relative Dormancy

• Time issues

• Pure Seed

Hard Seeds:

Lupine example: hard seeds not always good

“Up to 75% of the hard seed has been found to be

dead on individual lots.”

-Alvarez and Vivrette 1997

One can incubate clipped seeds or do a TZ. In this

case, TZ is faster.

Issues regarding TZ testing of

Native Seeds

What should the seller put on the label?

Research: N. Vivrette, Ransom Seed Laboratory

Phacelia tanacetifolia (all tests 15oC)

germination dormant by TZ

water light 32

water dark 79

KNO3 light (AOSA) 58 33

KNO3 dark 82

PCH water light 21

PCH water dark (ISTA) 59 32

GA3 91

How much of this lot is “dormant”?

If a state law or the FSA has no provision for “dormant” how

is this communicated to the buyer?

Is it important to know how a viability test was done?

Relative dormancy

We won’t always know how to break

dormancy:

Can we avoid TZ ambiguities

by breaking dormancy? No.

-ecotypic variation within native species

-harvest year and maturity

-species with little germination data

(Can’t do a thermogradient plate analysis of

every sample that comes in the lab.)

Time issues:

• Purity required before doing TZ.

• Analyst proficiency with the species to be tested

affects time spent doing the test.

• Seed quality affects the evaluation time.

• TZ vs. Germination:

More “hands-on” time for the analyst.

Less calendar time for the customer.

Pure seed issues: Differences in “pure seed” determination

directly affect viability results.

• Collection and conditioning methods different

• AOSA pure seed definitions:

consistency a problem e.g.

Artemisia, Krascheninnikovia,

Encelia

• Time: finding pure seed to do the test can

take as much time as a purity. This adds cost

to the test. (e.g. bluestems)

pure, pure, pure, inert

The TZ test is one of

several imperfect tools to

measure viability.

Ways to increase confidence in

the TZ test

• test a larger sample

• use a check sample of known high quality

• invest in analyst training and encourage

consultation and collaboration with other analysts

referees and research

regional groups

workshops

• follow standardized purity rules for

determining pure seed

Summary

• The tetrazolium test measures

viability.

• The tetrazolium test only

measures dormancy when it is

combined with a germination test.

Acknowledgements:

Arkansas Valley Seed Solutions Richard Agnew, RST

Colorado State Seed Lab Marilyn Milhous, RST, CSA

Ransom Seed Lab Susan Alvarez, RST

Aleta Meyr, RST

Dr. Nancy Vivrette, RST

USDA/ARS NCGRP Dr. Loren Wiesner

Laurie Thoma