14.

TETRAZOLIUMTEST FOR SEED

VIABILITYAND VIGOUR
V.N. PATIL &MALAVlKADADLANI
Germination test is the best indication of the potential of a seed lot to emerge
under field conditions. However, it takes from days to weeks and in some cases
even months to complete. Tetrazolium test, commonly known as the TZ test for
seed viability have, therefore,. been developed to furnish quick estimates of seeds'
germinability. The test is very useful in processing, handling, storing and marketing.
large quantities of seed in a short time, testing dormant seed lotS, vigour rating of
the seed lots, ~upplernentinggermination test results and diagnosing the cause of
seed deterioration.
Machanism of the Reaction
The TZ test isreliable and widely utilized. the method was pioneered by the .
Germanscientist Lakon during the mid nineteens (1939-1958), who recognised
thatall living tissues, which respire, are capable of reducing a colourless chemical
2,3,5 triphenyl tetrazolium chloride or bromide) into a red coloured compound
formazenby H transfer reactions catalysed by the enzyme debydrogenases
. ~
qN-N-C6H5 .+ . I N-N-G HS
C H C
I
2.H '+2(;. 1/ ~
6 5 \ + DEHYDROGENASls C6HSG +HCL-
N=N-~HS
~
\N=N-c. H .
. c.~. . G5
NADP
. NADPH NAD
2,3,,5 TRIPHENYL. NADP
TETRAz.OLIUMCHLORIOE
(COLOURLESS}
FO~A!AN
( RED)
Formazan being non-diffusable, stains the living tissues red. Thus, the living
parts of a viable seed should be stained red when incubated in the solution of this
chemical.
The immense significance and practi~l utility of this test was realised by the
See9scientists and technologists the world over and it was suitably modified and
improvedto assess the seed quality in terms of its viability.
209
Since the tissues within a seed could be at different states of viabilitythey
would be stained differently. Moore (1973) described the use of TZ stainingmore
efficiently on the basis of the topographic pattern of the seed.
The accuracy of the tetrazolium test requires:
1.
A sound knowledge oi"the seed and seedling structures,
2.
An understanding of the machanisms of the reactions leadingto the
staining of the seed.
3.
Ability to interpret the staining pattern by critically examining theseed
topography and by keeping other visible seed qualities such as tissue
turbidity, abnormal or missing structures etc., in view and,
4.
Experience gained through keen interest and natural skill.
Essential Seed Structures for TZ Test Evaluation
Seeds can be grouped into monocots, dicots and polycots on the basis orthe
presence of one, two or more than two cotyledons. Embryo structures or structure
primordia arc. relatively similar for embryos within each groups though theym11Y
differ in shape. size and differentiation/development of embryo structures. The
critical features of the seeds of these groups me described below.
Monocotyledons: Seeds of Gramineae family are a classicalexampleOflhis
class. Tbe most critical features are the growing root and shoot tips and the
scutellum. 1be root region comprise..<;mainly of the radicle which may be theonly
root primordia in some cases e.g. small seeded grases or it may have several seminal
root primordia besides the radicle c.g. wheat, maize. barley etc. The radicleties
below the mesocotyl region covered under a sheath coleorhiza. Theshootregion
or the plumuleconsists oftheshoot meristem along with one or more leaf primordia
near the base, enclosed within the coleoptilc. Mesocotyl seperatesplumulefrom
the point of attachment to tbe scutel1um. The main embryo structures of maize. 11
typical monocot seed are shown in Figure 14.1.
Dicotyledons: In the dicot seeds with a low degree of differentiationthe
embryo is embedded in nutritive tissues and consists of a radicle and two
cotyledons. In the seeds with a higher degree of differentiation. embryooccupies
210
a larger portion of the seed. The radicle is well recognizable part of the embryo
while the shoot meristem with the primary leaf primordia generally lies compressed
between thetwo cotyledons. The structure of a bean seed - a typical dicot seedwith
well developed embryo is given in Figure 14.2.
Polycotyledons: Not very common. This type of seed is found in Pinaceae and
other conifers. The embryo is not very well differentiated except for a radicle and
more than two cotyledons. Living nutritive storage tissues encircle the embryo
while the shoot meristem is encircled by the base of the cotyledons.
Equipments required for conducting TZ test
(1) Staining dished e.g. watch glasses for small seeds or pctridishes,. (2)
Beakers of 100to 250 ml capacity for larger seeds, (3)Single edge razor blade, (4)
Needles, (5) Forceps, (6) Magnifying devices, (7) Medicinedropper, (8) Dispens-
ing bottle, (9) Germinating bl(~tters,filter paper and paper towel.;;,and (10) Oven
or incubator.
Preparationof the staining solution
The chemical used for this test is a cream or light yellow coloured water soluble
powder called 2,3,5,- triphenyl tctrazolium chloride.
Several concentrations of tetrazolium solution ranging from 0.1 to 100%are
used with comparable results. Generally the 1.0%solution is used for seeds that
are not bisected through the embryo, is bisected. Other lowconcentrations much
as 0.2% and 0.5% are sometimes used instead of the 0.1% solution. To prepare a
1.0%solution, one gramof tetrazoliumsalt is dissolved in the distilled or tap water
tomake 100m!. The pHofthe solutionshould be around 7.0for the proper staining
tooccur. Solutions of pH4 or lower will notstain evenviable embryos andsolution
of pH more than 8 will result in too intense staining.
If the pH of the water is not in the neutral range, the tetrazolium salt should be
dissolved in phosphate buffer solution. The buffer solution is prepared as follows:
Solution A-Dissolve 9.078 g of Potassiumdihydrogen phosphate (KH2PO4)
in water to make 1000 m!.
Solution B-Dissolve 11.876 g of Disodium hydrogen phosphate
(Na2I-IPO4.2H20)to make 1000 ml.
211
Take,400 ml of solution A and (JOOml of solution 13and mix them together.
In one litre ofthe buffer solution prepared as above, dissolve 10g of the tetrazolium
salt. This gives a 1.0% tetrazolium solution of pH 7.0
Store the solution in a dark or amber coloured bottle to prevent the reaction
'which aL<;o occurs in the presence of light. The solutions can be stored [or several
months in a refrigerator.
Preparation of the clearing solution
A 1clctophenol solution can be used as a clearing agent, if the pigment within
the seed coat or .lemma prevents clear vision after staining. 13yusing the clearing
. ,
agent the embryo is visible through the seed coat or lemma. It consisL<;of: Lactic
acid: 20 parts, Phenol: 20 parts. Glycerine: 40 parL<; and Water: 20 p~lrts.,
'Temperature
Temperature influences the staining reaction. Staining will take place twice as
fast at 30 as at 20oe and twice as fast at 40 as at 30c. Temperature between 20oe
and 40oe have no effect on accuracy oftetrazolium test. but staining proceeds faster
at the higher temperature. The tests can be performed satisract(~rily at room
temperature.
Working Sample
At least 100 seeds should be tested in replicates of 50 or less. The seeds should
be randomly selected from the pure seed component arid counted in replicates
before conditioning.
In order to ensure a contact of the tetrazolium solution with the embryo. some
conditioning and preparatory steps may be essentiaL These depend on the type of
the seed. permeability and thickness of the seed coat. location of the embryo etc.
Preconditioning
It is done to.
allow complete hydration of all the tissues.
- prevent damage to cotyledons and embryo axes while cutting seeds
,212
- initiate and activate the germination process. and to
- have proper penetration of tetrazoliumsolution.
Some kinds of seeds can directly be placed in wClter.' while others must be
moistenedslowly~Slowmoistening isgenerally practiced for largeseeded legumes
or for the seed samples which are dry. brittle or are aged to avoid tissue damage
due to rapid intake of water. 'Ihis can be done by keeping the seed on top of or in
b~tweenthe moist blotter or paper towels respectively.
Small seeded crops or seed with a permeable coat can be moistened by direct
soaking without any adverse effects. The moistening time can be considerably
reduced by putting the seeds at a slightly higher temperature (within 40C) during.
imbibition. .
Irepanltion for Staining
In addition to moistening. most kinds of seed require some preparatory steps
before staining. to
- assure the adequate penetration of the stainingsolution into the seed.
- accelerate the rate of staining.
- facilitate the evaluation.
Thus. depending upon the kindof the seed. time available. degree of accuracy
desired and the experience of the analyst. one of the following methods can be
adopted:
(a) No moistening or preparation required (s~all seeded legumes with soft
coats).
(b) Slow moistening without any preparation (large seeded legumes with soft
coats).
(c) Piercing. puncturing or cutting'of the seed coat (small seeded grasses).
(d) Cutting the seeds longitudinally through the midsection of the embryo and
through part of the endosperm. leaving the two halves attached at the base or sliting
213
the seed completely, keeping only one half for the staining, discard the other.
(e) Cutting the seeds longitudinally slightly off-centre to avoid cutting into the
embryo.
(f) Removing the seed coat (with forceps/needles/razor blades etc.) with
minimum injury to the tissues. Sometimes, a thin membrane adheres to the
cotyledons even after removal of the seed coat, this may be removed by a
sliding motion after an additional 30 min. of soaking (dicots with hard seed
coats).
Staining
Seeds can be stained in watch glasses,petridishes or beakers. Sufficient solu-
tion should be used to cover the seeds and to allow its absorption. A'>a rule of
thumb, a concentrated (1.0%) solution can be used for legumes, cotton and grasses
that are not bisected through the emrbyo and a dilute (0.25% or 0.50%) solution
for grasses and cereals that are bisected thr6ugh the embryo.
Suggested preparation method for staining, cOncentration of the solution,
staining time at 35 C, and evaluation for a number of important Indian crops are
given in Table 14.1(for further details, refer to Moore, 1995). Careshould be taken
not to overstain seeds as this causes degeneration, making interpretations difficult
and uncertain.
In general, seeds are placed in the solution and held at 35 C for complete
colouration..After a period in the solution, the length of which varies according to
the condition of the seed, species, temperature and concentration of the solution: .
the seed is removed from the tetrazolium solution, rinsed 2-3 times in water and
. thenevaluated according tothe stainingpattern. Duringevaluation, theseedshould
be left in a little water to prevent these fromdrying. If seeds are not tobe evaluated'
immediately, theseeds in a little water should be kept in the refrigerator. Whenever
required, 2-3 drops of lactophenol clearing solution can be added to 100seeds of
small seeded grasses, after completely removing the tetrazolium solution. Small
seeds are examined under a stereo microscope, while,largeseeds may be examined
with magnifying lenses only. Seed coats of legumes must usually be removed
before examination.
214
Tissue Characteristics
(i) Sound tissues
Staining proceeds gradually and uniformly from the exposed surafaces inward.
Changes in the intensity of colour are gradual without distinct boundaries. The
colour intensity is lesser but lustrous in sound tissues than the bruised, damaged or
aged tissues. A gradual reduction in colour intensit y from the s~r[acc to the interior
of the seed indicates slow absorption and firm, sound unstained tissues indicate the
lack of penetration of the tetrazolium solution.
(ii) Weak ,'iable tissues
Such tissues tend to stain greyish red or brighter red than normal. '[be apical
tips of leaves. colcoptiles and radicles tend to reveal earlier evidence of deteriora-
tion than do othcr areas. Weak and aged tissue.<;also lose the turgid ity of the sound
tissues. they appear l1accid and develop mottled stain.
(Hi) Weak non-viable tissues
Tissue colour is often mottl~d, it may be purplish. brownish or greyish red aJ;ld
its intensity may very from abnormal dark to pale pink. Cut surfaces may appear
off white. while the inner tissues may appear dark red. 1be unstained de<Jdtissues
look l1acid, liquid-logged, blurred, chalky white and lack-lustre.
A definite boundary docs not necessarily occur between the non- viable,
abnormallystained. stained and unstained tissues. Hoth types of tissues arc there-
fore.considered dead, regardless of colour differences.
Seed structure and the seed as a whole must be considered while evaluating
stainingpattern. Seed may be considered non- germinable even when there is only
onefractureor one small broken or missing spot inavital positionsuch as the point
of attachment of roots and cotyledons. Seed is considered dead when unstained
areasincludemesocotyl andseminal roots ingrasses and tipsof colcorhiza inwheat
andrye. lor areas of vital importance in interpreting the staining pattern in monocot
arc the plumule tips, the portion where embryo is attached to the scutellum and
seminal root region. In legumes. injuries caused to the growing organs must be
noted more carefully that the injuries of the other organs. Unstained radicle tips in
legumes may not allow the radicle to grow. Improper staining in areas such as
215
raidcle and hypocotyl development, cotyledons and in the plumble region indicate
a non-germinable or abnormal seed.
The tetrazolium percentage is the percentage of normal germinable seeds to
be expect~ when the seed lot is germinated under very favourable conditions.
Properly conducted tetrazolium an.dgermination test results are generally very
close.
Interpretation of TZ-test results
The Seed T~chnology Laboratory. Mississippi State University developed
detailed staining patterns of some of the major crop species for interpreting the 12
test r~su1ts (Fig. 14.3-14.10). Most of the other crops can be evaluated taking one
or the other ofthese staining patterns as the guide. However. for some crops it may
be necessary tDfirst standardisc the staining. 'Thestandardization of the TZmethods
for ragi (Eleusine coracane and khesari (Lathyrus sativlls) have been reported
(Agarwal and Kaur, 1975; Agrawal and Ncgi. 1977). If properly conducted, TZ
test's result is in close agreement with the germination percentage (Agrawal et aI.,
1973). However. discrepancies between the two results might arise due to several
factors such as (Agrawal and Dadlani. 1987) :
(iii)
(iv)
'(vi)
(vii)
(i) Sample difference
(ii) . Improper germination testing
ImproperTZ testing
Seed dormancy
(v)
Presence of hard seeds
Presence of seed-borne organisms
Chcmia11 injury (such as fumigation injury and excess-treat-
ment with mercurial fungicides etc. which may not inhibit the
TZ staining but could affect normal germination).
For the convenience of the seed analysts. viable seed characters are codedby
the Tetrazolium Committee of the ISTA (1985).
216
Terms used
Stained: Refers to viable (functional) tissues as established by staining
characteristics and/or tissue turgidity.
Necrosis: Refers to non-viable (non~functional) tissues as established by
abnormal staining characteristics and/or lack of tissue turgidity~
Evaluation codes for staining patterns of viable seeds of different species (for
column 5 of Table 6.1)
(i) Embryo
A.
Completely stained.
R",
Completely stained except for the type of necrosis listed
below:
Root initial area
6.
Primary root initial or surrounding area provided that at least two root
initials are stained and are adjacent to a functional plumule.
Cotyledons
7.
Superficial
8. Distal 1/4
217
Radicle
1.
Distal tip
2. Distal1!3
3. Distall/2
4. Distal 2/3
5. Entire
rl I I
1
J I
15.
Isolated superficial necroses, other than at junction with cmbry
and at centre of back of outer thickness or both c.otyledonary 10
j I
I
I
II
Scutellum
16.
Necrosis involving no more than 1/3 of upper and lower ends.
17. Distal 1/4
Coleoptile
18.
Distal 1/2
(ii) Nutritive stomge tissue
D.
Completely stained.
E.
Stained except for small surface necroses that are not
tact with embryo cavity.
Advantage of TZ test
(a) Quick and fairly acc:uratc
(b) Can also determine the viability of a dormant seed lot
short timc.
(c) Seeds are not damaged (in dicOls) and can be germim
218
I>isadvantages ofTZ test
(a) Distinction between normal and abnormal seedlings difficult.
(b)
Cannot differentiate between dormant and non-dormant seeds.
(c) Correct evaluation is possible only after prolonged experience
(d) Microorganisms harmful for seedling emergence remain un-
detected.
Tetrazoliumtest for vigour assessment
After differentiating viable seeds from the non-viable ones, the viable seed
groupcanbe reclassified intoseveral vigourgroups on the basis of intensityof stain
and staining pattern of different seed parts. The simplest classification is to divide
viable seeds lot having larger number of sound seeds will be considered more
vigorous.
A better estimate of vigour can be obtained by colorimetric determination of
formazan. Kittock and Law (1968) described a method of estimating seed vigour
on the basis of colour intensity of the stained embryo or seed. The method is
describedbelow:
1.
Prepare tetrazolium solution as described earlier.
2.
Put 100seeds in a petridish lined with moist filter paper and keep at 25 :j:
1C overnight.
3.
Excise the embryonic axes andadd 1ml oftetrazolium solution to 25 axes
in quadruplicate.
4. Incubate in dark at 30C for 4 hours. The incubation time varies with the
crop species. However, for maintaining unifromity, duration should be
kept constant for a crop under study.
5. Drain out the.excess solution and wash in distilled water.
219
_/
,
6.
Soak the axes in 10 ml of methyl cellosolve fm 4 to 6 hours with
occassional stirring till the extraction of red coloured formazan is com-
plete i.e. the axesbecomecolourless.
7.
Decant the extract and read the intensity of the colour at 480 nm. in a
spectronic 20 colourimcter using methyl cellosolve as theblank.
Higher the intensity of the extracted formazan, greater is the vigour.
220
221
Table 14.1
Tetrazolium testing guide fo,. some important crop
species (adapted fmm Moore, 1985)
Crop Moistening Preparatory Staining Staining
period(h)
method after
time(h)
evaluation
moistening
(refer to
the text)
2 3 4 5
eel-eats
Barley
6-18
(a) Cut the seed
3-24 B6
longitudinally
through the mid-
section of the
embryonic axis
Maize 18 -do- 3.24
B5, B1 7
Oat 6-18 - do 2-24 B6
Paddy
18 - do - 6-24 B4
Pearl Millel 6-18 -do- 6-24 B4
Rag!
6-18 -do- 6-24 B4
or cut laterally
Sorghum
6-18
(a)
6-24
B4,817
Wheal 6-18 -do- 3-24
B6, B17 -"
I)ulses
Chick pea
18
(b) Cut through
6-24
B4, Bll, BI8
the seed coat
near midsection
Cowpea
18* - do --entire 18-24
B3, B13, B18
seed length
Green Gram 18* -do- 18-24
B3, B13, 818
Lalhyrus
18'" - do - & 6-24 B3, B13
(c) Severe a
distal end of
cotyledon
Lentil 18*
(b)
6-24
83,B13,818
Pea 18-24 -dO- 6-24
84,813,818
Pigeon pea
18
(b) & (C)
6-24
83,B13
Soybean
18*
(b) or no seed
6-24
83,813,818
coat preparation
needed
Oilseeds
Castor 18
(b) & (c)
6-24
A,D
Groundnut 18*
(b)
6-24
B2,B8,B18
Linseed 18* Treat with alum to 6-24
B2,B9
remove seed coat
sIiperiness & (b)
1 2 3 4 5
Mustmd 18
(d) Puncture the
18-24 81J316
seed coat, cut as
in (b) including
1/2 of circumference.
Safflower 18-24
(e) Cut longitudi-
6-24 I32.1314
nally through the
midsection of
distal end.
Sesame 18 -do- 6-24
131,138
Sunflower 6-18 - do - 6-24 132.1314
Fih,'e Cl'OpS
Cotton 18':' - do 6.24
133,137,1310
.lute 18 -do- 24-48
At:
Sunnhemp
18
(f) Cut through the
18-24 133.I3J3
coat near midsection
of entire cotyledon
length & (c)
FOJ'age
Alfa alfa ()-18 -do- 6-24
m,BJ I
Berseel1l 18 -do- 6-24 133.1313
(Trifolium)
Guinea grass
6-18
(g) Cut through the
6-24 134
midsection of distal
end and expose the
embryonic axis.
Vegetahles
Amaranrhus 18
(b)
18-24 132,139
Beet root 18 Puncture the seed 24-48 B2.B9
coat and remove the
cap covering embryo.
Bitter gourd
6-18
Remove the wings & (g)
6-24 81,1312
Brinjal
18 Puncture and ()-24
A,C
(h) Cut laterally
or longitudinally
through the entire
depth.
Broad bean 24*
(b)&(c)
16-24
134,1313.1318
Carrot 18
(e)
16.48
A,C
Chillies 18
(h)
6-24
A,C
Cucumber 6-18 Remove the 6-24 82.812
Wings & (g)
Dolichos 18
(f)
6-24
133,813,818
bean
Lettuce 6-18
(e)
6-24
82,817
Luffa 6-18 Remove the 16-24 82,816
wings & (d)
Onion 6-18*
(h)
6-24
A,D
222
2 3 4 5
Pumpkin 6-18 Remove the 6-24 B2,B12
wings & (g)
Radish lR (d) 6-24 132,816
Roundgourd 6-18 Remove seedcoat 6-24 B2,1312
sliperiness by
drying & (g)
Tomato 18 Puncture & (h) 1.8-24 132,B14
Flowel's
Aster 6-1f: (e) 6-24 B2,B14
Begonia 18 (e) 18-24 A
Canna If: Remove hard seed- 24-48 A,D
edness&(e)
Coleus 18 Treat with alum to lR-24 B2,811
remove seed coat
sliperiness&(b)
Dahlia 6-18 (e) 6-24 82,1314
Gladiolus 18 Severe through the 24-48 A,C
distal end of the seed
Rose 18 Severe wings & (e) 24-48
Others
Citrus 18 Remove seedcoat 6-24 82,1312
sliperiness by
drying. Remove or
punture the inner coat.
Co!Tea 18 (g) 24-48 A,C
Pine 18 Cut off centre 24-48 A,D
through the coat &
nutritive tissues to
expose the embryo
outlinc.
Rubber 18 -do- 2at40C -
Tobacco 24 (b) 24-48 A,C;
Note:
'" =Slow moistcnIng recommended.
Proparatory methods as indicatcd in co!. 3 are as follows:
a = Cut the seed longitudinally through the midsection of the embryonic axis.
b = Cut through the seed coal near midsection.
c = Severe a distal end of the cotyledon.
d = Puncture the seed coat, cut as in (b) i.ncluding1/2 of circumference,
223
e = Cut longitudinally through the midsection of distal end.
f = Cut through the seed coat near midsection of entire cotyledon length.
g = Cut as in (d) and expose the embryonic axis by spreading the cut surface..'i
slighLlyapart.
h = Cut of seed laterally full depth from the centre outward between the
radicle and the cotyledons.
224
i
I\)
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01
14.1
pericaTp
aleurone layer
. endosperm
scutellum
coleoptile
leaf primordia
shoot meristem
mesocotyl
adventitous root primordia
rad.icle
coleorhiza
shoot meristem
cotyledon attachement
primary leaf primordia
radic1e
micropyle
hilurn
~
0)
strophiole
cotyledon
seed coat
14.2
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