Experiment: 3 Seed Viability Tests
Seed viability refers to the “state of being aliveness”, metabolically active and possesses enzymes capable of catalyzing metabolic reactions needed for germination and seedling growth. To most seed technologist, viability means that a seed is capable of germination and producing a normal seedling. Mainly the following tests are employed in different plant species:
1. Standard
germination test;
2. Enzyme
activity test;
3. Ferric
chloride test;
4. Electrical
conductivity test;
5. Excised
embryo test;
6. X-ray
test;
7. Tetrazolium
test etc.
Tetrazolium
(Tz) Test:- Seed viability is reliably estimated by the
standard germination test but the relatively longer period of time (7-35 days)
required for the completion of germination test. This has necessitated the
search for a quick and effective test to predict seed viability. Hence the
tetrazolium test (Tz) has been developed by German scientist Lakon in 1942.
This biochemical test is quick, reliable, comprehensive and highly correlated
with the following objective: To
make a quick estimate of the viability of seed samples.
Principle:-
All
living cells of the seed which respire can reduce a colourless solution of
tetrazolium chloride or bromide salt into a red colored compound called
formazan. The reduction of the chemical takes place in the seed by the action
of a group of enzymes known as dehydrogenases. Within the seed tissue, it
interferes with the reduction of living cells and accepts H+ from
the dehydrogenases. By hydrogenation of the tetrazolium salt, a red,
stable and non-diffusible substance, triphenyl formazan is produced in living
cells. The reaction is as follows:
Since the reaction takes
place within the respiring (living) cells and the formazan is non-diffusible.
Equipments:-
I.
Cutting
and piercing devices: single edged blades, dissecting needles
and forceps.
II.
Conditioning/
moistening media: paper towels, blotter or filter papers
III.
Glasswares
for staining: petri dishes, beakers of 250 ml
capacity, watch glasses
IV.
Magnifying
devices: magnifying lens, stereoscope microscope
V.
Others:
fine scissors, dispensing bottle, medicine dropper, brush.
Preparation of seeds for testing
The seeds are conditioned prior to staining.
Premoistening the seeds:- Premoistening is preliminary to staining for most of the species, as imbibed seeds are generally less fragile than dry seeds. In addition staining is more even in color this facilitates evaluation. Pre moistening is done by:Soaking in water:- seeds
should be fully immersed in water and left until completely imbibed. If the
soaking period is more than 24 hrs, the water should be changed.
Exposure of tissues:-
In many species it is necessary to expose the tissues prior to staining to
allow easy penetration of the Tz solution and to facilitate evaluation. The
seed coats can be opened and internal tissues can be exposed by the following
techniques:
a)
Piercing
the seed: Pre-moisten or hard seeds should be pierced using a
needle or sharp scalpel at a non-essential part of the seed.
b)
Cutting
the seed:- The embryo and other essential tissues
are exposed by bisecting the pre-moistened seeds longitudinally and
transversally. The longitudinal cut should be made through the middle of the
embryo axis.
c)
Excision
of the embryo: - The embryo is excised with a dissecting
lancet and transferred to the tetrazolium solution.
d) Removal of the seed coat:-
When cutting techniques are inappropriate, the whole seed coat must be removed.
Care being taken to avoid embryo damage.
Staining:-Adequately
prepared seed samples should be completely immersed in Tz solution of
prescribed concentration in beakers or petri dishes. Use 1% solution for seeds
that are bisected, whereas for those seeds whose embryo is bisected the
solution of 0.1% concentration is used. The seeds should be stained in dark
light. The temperature between 20-45o C has no effect on the
accuracy of the Tz test, but the rate of staining is higher at high
temperatures. At the end of staining period, the solution is siphoned off and
the seeds rinsed with water and examined for staining pattern.
General Procedure:-
I.
Required number of seeds should be
soaked in water for the prescribed period or overnight at room temperature.
II.
Water soaked seeds are to be prepared
for quick penetration of tetrazolium.
III.
For staining purpose, the prepared seeds
should be soaked in tetrazolium solution of prescribed concentration for the
species in beakers/petri dishes. For quick reaction, place the sample at 35-38oC.
IV.
When the coloration has developed after
prescribed time, the Tz solution should be drained out and the seeds should be
rinsed 2-3 times with water. Seeds are then evaluated into viable and
non-viable categories on the basis of staining pattern.
Evaluation:- The
main purpose of the Tz test is to
distinguish the viable and non-viable seeds. Viable seeds are those that stain
completely, or if partly then essential structures are stained fully.
Non-viable seeds remain completely unstained. At the examination, each seed is
evaluated as viable or non viable on the basis of staining patterns of
different tissues. Approximate light and magnification are indispensable for
proper examination. Also knowledge of seed and seedling structure is a
prerequisite for conducting Tz test properly.
Calculation and
Reporting of Results
In
testing a sample, the number of viable seeds are counted in each replicate and
averaged over replication. The seed viability is expressed in percentage by
number and calculated to the nearest whole number as follows:
Viability
(%) = Average number of viable seeds / Number of seeds per replicate X 100
The
result is reported as “Tetrazolium test-% of seeds were viable”
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