relationship between tetrazolium and germination tests · relationship between tetrazolium and...

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.


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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.


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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


Seed completely deformed


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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


Tetrazolium test: 63% viable, 37% non viable(1% root damages1%deformed

35% broken roots)


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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


Primary leaf is intact or shows acceptable defects


Up to ⅓ of extremities of scutellum is allowed to be unstained


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Triticum aestivum:


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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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.)

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