PRACTICAL-1
EXPERIMENT 1
DETERMINATION OF SEED VIABILITY BY TETRAZOLIUM TEST
Principle: Viability refers to ability of the seed for its germination, emergence and
development of seedling for successful establishment of a crop. Seed viability study by
tetrazolium (TZ) test is superior over simple
germination test, where dormant seed could
not be accounted as viable. In TZ test method,
tetrazolium salt solution is imbibed by seed
and interacts with the reduction process of
living tissue by accepting hydrogen from
dehydrogenases. All living cells of the seed
which respire can reduce a colourless solution
of 2, 3, 5triphenyltetrazolium chloride (TZ)
into a red colour compound, called triphenylformazan. The reaction is as [Link]-
phenyl formazon is non-diffusible. Therefore, living and non-living tissue could be easily
identified by TZ test from which viability per cent could be determined.
Requirements: Seeds – Maize, wheat, mungbean, cowpea,
2,3,5Triphenyltetraolium chloride (TZ) solution (1%),
Petridish, Forcep, Distilled water, pH meter
Procedure: Seeds of maize, mungbean and cowpea were soaked in distilled water
overnight. Dicot seeds (25 each type) were de-coated and embryoed cotyledons were
placed in petridishes separately having some amount of distilled water to prevent dryness.
Monocot seeds (25 each type) were cut longitudinally and one half of each seed were
kept in petridishes separately as before. Water from petridishes were decanted and
replaced with 1% TZ salt solution (pH 6-7). It is now kept under normal laboratory
condition for one hour.
(A)Overnight seed soak (B) cutting of seed (C) dissect the seed
(D)dipped into TZ solution (E) kept of one hour (F)counting
Results: Characteristic red colouration were observed in the viable embryos, whereas
non-viable embryos failed to take colour. Embryos whose 50% areas or more than 50%
areas were stained red, considered as viable and results are presented in the following
table. Viability of the seed is expressed as per cent.
Table. 1. Viability study of seeds
Sl. Seeds No. of No. of seeds whose 50 % or Viability
No. seeds more than 50% area of embryo were (%)
taken stained
1 MAIZE 10 4
2 WHEAT 12 8
3 PEA 15 10
Conclusion:
EXPERIMENT 2
DETERMINATION OF SEED VIABILITY BY USING HYDROGEN PEROXIDE
Introduction
The H2O2 (hydrogen peroxide) method is favored for its objectivity and simplicity. This
technique involves clipping the seed coat and incubating the seeds in a dilute H2O2
solution for one week. At the end of this period, seeds with radicles longer than 1 mm are
counted, marking the conclusion of the test. Unlike the tetrazolium, X-ray, and excised
embryo methods, the H2O2 test measures actual growth, making it a preferred choice.
Materials
Petri dishes, filter paper, magnifying glass, dropper and bottle, solution of tetrazolium,
dissecting needles, forceps
Procedure
1. Soaking
Randomly select 300 seeds and soak them in 100 ml of 1.0% H2O2 at room temperature
overnight. For the test, you will need four replications of 50 seeds each, with extra seeds
included to replace any that are damaged during cutting.
2. Cutting
Using a single-edge razor blade, cut the tip of the radicle end of each seed just enough to
expose the radicle tip without damaging it. The cut should be made at an angle rather
than straight across to minimize damage. Place the seeds in water after cutting to prevent
desiccation. Note that some radicles may curl during elongation; their total length should
be considered for classification. If the embryos separate from the seed coats during
soaking, try to match the free embryos with empty seeds to avoid errors in classification.
Very long embryos should be classified as "evident," but small embryos must be excluded
from the tally. If many embryos cannot be classified, repeat the test.
3. Incubation
Pour 150 mL of 1.0% H2O2 into each of four beakers (200 mL for larger seeds). Add 50
cut seeds per beaker and incubate in the dark at 20-25°C to allow for embryo elongation.
4. Assessment
After 3 days of incubation, remove and count any seeds with "evident" radicles. Return
the remaining seeds to a fresh H2O2 solution. On day 7, remove and assess all seeds, then
terminate the test. The data is usually expressed as the proportion of "evident" germinants
to the total number of seeds used in the test.
120 Control
A
% germination
100 5 mM H2O2
80
10 mM H2O2
20 mM H2O2
60
40
20
0
0 12 24 36 48
Time (h)
0,1
a
B a
b
FW (g/Plant)
b bc
c c c
0,05
0 0 5 10 20 0 5 10 20
Darkness Photoperiod
H2O2[mM]
Fig 1.- Effect of H2O2 pre-treatment on percentage of germination of pea seeds incubated
in darkness (A) and in the following growth of pea seedling after 48 h in darkness or with
a 12 h photoperiod (B). Pea seeds were previously imbibed in H2O (control) or with different
H2O2 concentrations for 24 h. After this period, seeds were incubated in the presence of
dH2O in darkness or with a 12 h-photoperiod up to 48 h. Different letters indicate statistical
significance according to Tukey’s test (P< 0.05).