ISTA Tetrazolium CommitteeChair Stefanie Krämer
Relationship betweenTetrazolium
and Germination tests
Monday, 13 June 2010 Agenda Point 11:15-11:45
Stefanie Krämer, Chair ISTA Tetrazolium Committee
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The Germination test is the emergence and development of the seedling to a stage where the aspect of its essential stuctures indicates whether or not it is able to develop further into a satisfactory plant under favourable conditions in the field.
Stefanie Krämer, Chair ISTA Tetrazolium Committee
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Classification in different categories:
Normal seedlings: Intact seedlingsslightly defektssecondary infections
Hard seeds: seeds remain dry and hard
Fresh ungerminated seeds: imbibed seeds, where the germination process is blocked
Abnormal seedlings: corresponding on the species, explained in chapter 5 and in the GER Handbook
Dead seeds: seeds absorb water, are usually soft and discolored, show no sign of seedling development
GERMINATION:
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The Topographical Tetrazolium Test is a biochemical test where seeds show by their biochemical activity the potential to produce normal seedlings.
Stefanie Krämer, Chair ISTA Tetrazolium Committee
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Tetrazolium Test:
Viable seeds:
seeds, which are completely stained
seeds where the unstained, flaccid and/or necrotic tissue does not exceed the permitted maximum area. This areas are prescribed under point 7 in the Tetrazolium workingsheets , in column 7 in chapter sixof table 6A and also in column 7 of the table 1 in the Tetrazolium handbook (so called: small damages).
Classification in different categories:
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Non Viable seeds:(so called large damages)
seeds where the unstained, flaccid and/or necrotic tissue does exceedthe permitted maximum area. seeds with damage at any other essential area (eg. connecting area of
cotyledons)
completely unstained seeds
empty seeds
rotten seeds
seeds with insects
seeds with deformed embryos
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GER: TEZ:
Normal seedlings Viable seeds
Hard seeds Viable seeds or Non Viable seeds
Fresh seeds Viable seeds or Non Viable seeds
Abnormal seedlings Non Viable seeds
Dead seeds Non Viable seeds
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Normal seedling (GER) Viable seed (TEZ)
Primary root is intact, or acceptable defects not affecting the conducting tissues.
Up to ⅓ radicle, measured from the radicle tip is unstained
The hypocotyl is intact or shows accectable defects
(included above)
Cotyledons are intact or show acceptable defects as necrotic spots, healed cracks, 50%of the tissue functining normally, three cotyledons
Up to ⅓ distal area of cotyledons is unstained up to ½ of cotyledons if superficial
The terminal bud and surrounding tissue is intact (usually not visible)
No unstained, necrotic or flaccid spots at the terminal bud and surrounding tissue
Comparison for Medicago sativa
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normal fresh hard abnormal dead
A 65 0 6 2 27
B 68 0 3 2 27
C 65 0 6 3 26
D 66 0 11 2 21
Result
%
66 0 7 2 25
Germination of Medicago
Method: TP, 5 days prechilling 20°C/ 7days
The 7 % hard seeds were tested in TEZ: all seeds were viable
Possible germination 73%
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Tetrzolium Test of Medicago
Method: soaking in water,18h staining 30°C, 1% TEZ concentration.
The percentage of hard seeds included in the percentage of viable seeds.
Viable Non viable
A 75 25
B 80 20
C 74 26
D 65 35
Result % 74 26
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Evaluation scheme Tetrazolium Test of Medicago:
Total %
(A +B)
A: soaked seeds
B: hard seeds
Viable seeds
1 Totally stained 52 23 51 29 56 18 48 17 207 87 52 22 742 Small Cotyledon damage : not more than ½ of distal end is unstained,
if superficial or not more than ⅓ of distal end is unstained, if
pervading3 Small radicle damage: not more than ⅓ unstained, measured from
radicle tip4 Small cotyledon and radicle damage: 2and 3 in a combination
Non viable seeds
5 Large cotyledon damage: more than ½ of distal end, if superficial, is
unstained or more than ⅓ of distal end, if pervading, is unstained,
or an unstained area at leaf base
4 1 1 4 9 1 2 0 2
6 Large radicle damage: more than ⅓, measured from radicle tip is
unstained
1 1 2 1
7 Large cotyledon and radicle damage: l ine 5 and 6 in any combination 3 2 4 4 4 4 6 2 17 12 4 3 7
8 Totally unstained 11 1 11 10 2 17 2 49 5 12 1 139 Other deficiencies eg. rotten 3 5 3 11 3 3
A B A B
%
A B A B A B A B
Date Replication (4x100 seeds) Total
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Germination: 66% normal seedlings , 7% hard seeds, 2% abnormal, 25% dead
Tetrazolium Test: 74% viable, 26 % non viable (2% large cotyledon damages1% large radicle damage7% large cotyledon and radicle damages
13% unstained3% rotten)
Medicago sativa
Many of the dead seeds in GER show in the TEZ partially stained areas
The differenz in the number of hard seeds (7% in GER – 26% in TEZ) may be
because of the prechilling treatment.
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primary root defect Root damage
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Connecting area of cotyledons defect (the terminal bud and surrounding tissue) and primary root defect
Connecting area of cotyledons defect (the terminal bud and surrounding tissue) and root damage
Small superficial cotyledon damage
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primary root defect
primary root defect
root damage
root damage
root damage
Small cotyledon damage,
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Cotyledon defect Cotyledon damage
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Cotyledon and primary root defect Cotyledon and root damage
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Seedling completely deformed
Seedling completely deformed
Seed completely deformed
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Seedling completely deformed
Seedling completely deformed
Seed completely deformed
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Dead seed
Dead seed
Non viable seed
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Dead seeds
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Non viable seeds
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Non viable seeds
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Normal seedling Viable seed(Maximum unstained area)
Primary root may be replaced by secondary roots
2/3 radicle, meassured from radicle tip
Cotyledons are not broken, deformed, decayed, necrotic and more than 50% of the original tissue is functioning normally
½ of distal area of cotyledon
Pisum sativum
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Pisum sativum
Germination: 15% normal, 23% abnormal, 62% dead
Tetrazolium: 14% viable, 86% non viable (34% broken roots10% large cotyledon damages42% unstained)
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Broken radicles
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No root developed Large radicle damage
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Dead seed Non viable seed
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Terminal bud defected, root
short - stumped
Large radicle damage, large
cotyledon damge
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Shoot deformed, fungi
infection on the cotyledonCotyledon with flaccid areas
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Non viable seeds
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Vicia faba
Germination: 57% normal, 21% abnormal, 22% dead
Tetrazolium test: 63% viable, 37% non viable(1% root damages1%deformed
35% broken roots)
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Normal seedling Viable seed(Maximum unstained area)
Primary root may be replaced by secondary roots
2/3 radicle, meassured from radicle tip
Cotyledons are not broken, deformed, decayed, necrotic and more than 50% of the original tissue is functioning normally
½ of distal area of cotyledon
Vicia faba
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No root developed Broken radicle
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Stumped root
Stumped root
Radicle damage
Radicle damage
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Radicle damage
No root developed
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Non viable seeds
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Triticum aestivum
Normal seedling Viable seed
Seedling completely normal developed
Embryo completely stained
At least one seminal root At least one root is stained
Mesocotyl is intact or shows acceptable defects
No damages at the shoot area
Coleoptyle is intact or shows acceptable defects
No damages at the shoot area
Primary leaf is intact or shows acceptable defects
No damages at the shoot area
Up to ⅓ of extremities of scutellum is allowed to be unstained
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Triticum aestivum:
Germination: 87% normal, 5% abnormal, 8% dead
Tetrazolium test: 88% viable, 12% non viable
(1% root damages5% shoot and root damages1% Scutellum damage4 % unstained1% broken seeds)
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Normal seedling
Viable seed
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Non viable seed because of the shoot damage
Coleoptyle defekted
Root area, there is still at least one root viable
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Coleoptyle defekted
Coleoptyle defekted
Non viable seed because of the shoot damage
Root area, there is still at least one root viable
Damage on the epiblast
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Primary infections –only in several cases visible during the Tetrazolium Test
Primary infections
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Primary infections
Germination Tetrazolium
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Sprouted seed
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Dead seed Unstained (non viable) seed
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Non viable seeds – nearly all essential structures are only partially stained (mechanical damages)
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Non viable seeds – nearly all essential structures are unstained or only partially stained
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Zea mays
Germination: 77% normal, 12% abnormal, 11 % dead
Tetrazolium Test: 79% viable, 21% non viable (20% damages on root and/or shoot
1% unstained)
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Normal seedling Viable seed(Maximum unstained area)
Primary root may be replaced by secondary roots
Primary root
In total 1/3 of distal area of margin of the scutellum
Zea mays
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Viable seed
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Viable seed, small scutellum damage
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Large Scutellum damage and shoot damage
only little root growth
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Leaf damage
Primary leaf missing
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Shoot damage and parts of the radicle unstained
No shoot developed, small root
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Shoot damageNo shoot developed
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Dead seedNon viable seed
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Large Scuttelum damageSeedling underdeveloped and infected
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Coleptyle damage, seedling small Scuttelum damage, shoot damage
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Non viable seeds
Large Scutellum and shoot damage
Large Scutellum damage
Large root and shoot damage
Embryo deformed, scutellum on the wrong side
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Non viable seeds, fungi infections
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Viable seedNormal seedlings
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Conclusion:
In many cases there is a rather good correlation between the results of GER and TEZ.
Differences can appear in cases of:DormancyHardseednessMycrobiologcical infections of the seedsChemical treatments
Low results in GER can be checked with TEZ because physical damages ( eg. broken seeds, heat damages) and physiological specifics (eg. dormancy, immature seeds) are rather good representable.
The use of evaluation schemes raises the reproducibility of GER and TEZ. ( But as smaller and more undifferentiated seeds are, as more difficult is the classifaction into different catergories of non viable seeds.)