Upload
others
View
14
Download
0
Embed Size (px)
Citation preview
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