Seed Quality Tests

1. Tests for Estimation of seed moisture content:

Moisture content of the seed is determined by low constant temperature oven method as per the ISTA (1996).  About five grams of seed sample was taken at random from each treatment in two replicates, ground and dried in an oven at a temperature of 103 ± 1⁰C for 17 hours.  The seed moisture content was determined on wet weight basis using the formula and expressed in percentage.

Moisture content (%) = (M₂ – M₃ / M₂ – M₁) x 100        

Where,             M₁        =          Weight of the container without seed

M_{2          =}              Weight of the container + seed before drying.

                        M₃        =          Weight of the container + seed after drying.

Specifications for Moisture Test of Different species using Air-Oven method

Scientific name	Grinding	Drying time(hour)	Drying temperature (0C)	Moisture content above which pre drying is done
Allium sp.	-	17	103	-
Arachis hypogaea	C	17	103	17.0
Avena sativa	F	2	130	17.0
Brassica sp.	-	17	103	-
Cicer arietinum	C	1	130	17.0
Daucus carota	-	1	130	-
Glycine max	C	17	103	10.0
Gossypium sp.	F	17	103	17.0
Hordeum vulgare	F	2	130	17.0
Lens culinaris	C	1	130	17.0
Lycopersicon sp.	-	1	130	-
Oryza sativa	F	2	130	13.0
Pennisetum glaucum	F	1	130	17.0
Phaseolus sp.	C	1	130	17.0
Pisum sativum	C	1	130	17.0
Secale cereale	F	2	130	17.0
Sorghum sp.	F	1	130	17.0
Trifolium sp.	-	1	130	-
Triticosecale sp.	F	2	130	17.0
Triticum sp.	F	2	130	17.0
Vicia sp.	C	1	130	17.0
Vigna sp.	C	1	130	17.0
Zea mays	F	4	130	17.0

2. Test weight (g): Test weight (g) is determined by counting manually 1000 seeds of eight replications from each genotype and weighed up to two decimals and the weight is expressed in grams.

3. Seed density (g/cc): The seed density is determined by water displacement method. Twenty ml of Toluene solution was taken in a measuring jar and five grams of seed sample was put into the jar. The increase in volume of the toluene solution is recorded and the density is calculated using the following formula and expressed in gram per cubic centimeter.

Seed density (g/cc)  =      Seed weight (g)

                                       Seed volume (cc)

4. Germination test (%): Germination test is conducted by using ‘between paper’ towel method as per ISTA (1996). Four replicates of 100 seeds from each genotype are placed equidistantly on moist kraft paper. The rolled towels should be incubated at 25±1⁰C for 14 days. The first and final counts were recorded on fifth and fourteenth day, respectively. The germination is recorded based on the normal seedling on the final count and expressed in percentage.

a. Top of paper method:  Two moist Whatmann filter papers (No 1) are placed in a glass petridish of 20 cm diameter. Fifty seeds of four replicates are sown and incubated at the required temperature. Cover the lid to minimize the evaporation loss. Incubate the petriplates in the seed germinator, maintained at the required temperature levels (25 ⁰C)as per the treatment.

b. Between paper method: One hundred seeds of four replicates should be placed in between two layers of moist Kraft paper and then rolled. Place the rolled towels in slant position in the germinator maintained at required temperature as per the treatment.

5. Seedling length (cm): Ten seedlings from each replication kept for germination were taken at random on final count. The seedling length is measured from the tip of the primary root to the tip of the primary leaf and mean of 10 seedlings is calculated and expressed in centimeters.

6. Mean seedling dry weight (mg): The seedlings used to measure seedling length are dried in a hot air oven maintained at 80±2⁰C for 24 hours. Then cooled over silica gel and weighed. The mean dry weight of seedlings and expressed in milligrams.

7. Electrical conductivity (dSm-¹): Twenty-five seeds of three replicates are soaked in 25 ml distilled water for 24 h at 25 ±1⁰C. At the end of soaking period, decant the steeped water (seed leachate)  and measure electrical conductivity of the seed leachate with the help of Conductivity Bridge (Model D1909/DS-7007) and expressed in dSm^{-   1}

8. Viability tests:

Tetrazolium test: Triphenyl tetrazolium chloride solution (0.5%) is prepared by dissolving 0.5g of tetrazolium salt in 100ml of phosphate buffer with a pH of 7.0. Seeds are soaked in water, after the removal of seed coat, the embryos are (100X3) immersed in TZ solution and incubated in dark for 8 hours. Thereafter the excess TZ solution is decanted and seeds are evaluated as viable and unstained as non-viable and expressed in percentages as per ISTA (1996).

9. Total dehydrogenase activity: The seeds are soaked in water and after removal of seed coat seeds (100X3) are immersed in 2 ml of 0.5 per cent Tetrazolium solution and incubated at 25 ±1⁰C in dark for 6h and then washed thoroughly with distilled water. The red color (Formazan) developed is eluted from the stained embryos by soaking in 5 ml of 2-Methoxy Ethanol (Methyl Cello Solve) in a screw caped vial until all the seeds discoloured. The extract is decanted and the colour intensity is measured in Spectrophotometer (Model SL171) at 480 nm with suitable blank. The dehydrogenase activity is expressed in terms of absorbance values.

10. Seed health test: Detection and identification of seed mycoflora is done by blotter paper method ISTA (1996). Seeds (100X3) are placed equidistantly in sterile glass petridishes of 9 cm diameter containing three moist blotter papers (Whatman No.1). Then the incubate petridishes at 20⁰C for eight days with 12 hours light and 12 hours dark cycles. After incubation, seeds are examined under stereo binocular microscope for the presence of seed infection on the seeds and recorded as number of seeds infected by pathogen fungi and expressed in per centage.

11. Field emergence (%): The seeds are (100X4) sown on a well-prepared, raised seedbed and optimum soil moisture is maintained by watering. Germination count is taken on final count from the day of sowing and the germination per cent is calculated taking into account the number of normal seedlings emerged out.

12. Vigour tests

According to ISTA “the sum total of those properties of the seed which determines the potential level of activity and performance of the seed or seed lot during germination and seedling emergence”.

Physical tests	Performances	Stress tests	Biochemical tests
seed size 1000 seed weight Physical soundness X-ray test	First count Speed of germination Seedling growth rate Seedling dry weight	Cold test Cool germination test Brick gravel test Paper piercing test Hot water/Compacted soil test Accelerated ageing test Controlled deterioration Low/high pH test	GADA test T Z test RQ test Mitocandrial activity ATP test Membrane integrity

Seedling vigour index: Seedling vigour index is calculated by using seedling growth parameters and expressed as a whole number as suggested by Abdul-Baki and Anderson (1973) and it is given below:

Vigour index - I = Germination (%) x Mean seedling length (cm) 

Vigour index – II = Germination (%) x Mean seedling dry weight (mg)

a. Hiltner Test (Brick gravel test): The test i developed by Hiltner in Germany in 1917. He observed that the seeds of cereal crops affected by Fusarium disease were able to germinate in regular test but were not able to emerge from brick gravels of 2-3 mm size. Compared to this, healthy seeds were able to emerge from the brick gravel (Robersts, 1972). The principle is that the weak seedlings are not able to generate enough force to overcome the pressure of brick gravels, so this method can be used to differentiate vigour levels in cereal seeds. Perry (1984b) found this method reproducible and associated with field emergence in case of wheat

b. Paper Piercing Test: The principle of paper piercing test is similar to that of brick gravel test. High vigour seed lots are expected to produce strong seedlings which can pierce a particular type of paper while seedlings of poor vigour lots may not be able to pierce the paper. Therefore, the seedlings which emerge by piercing the paper are more vigorous than those which are not able to emerge through the paper.

c. Cold Test: The cold test has been developed in USA to evaluate the seed vigour of maize (corn). In USA when the corn is planted in late spring, the soil is humid and cold. The weak seeds do not germinate and establish. Therefore to stimulate the actual field conditions witnessed at the time of corn planting, cold test has been developed. The test aims to differentiate between weak and vigorous seed lots by subjecting them to low temperature prior to germination at optimum temperature. The test has been criticized for using field soil which greatly varies from place to place.

d. Speed of germination:

Speed of Germination = no.of normal seedlings + - - - - + no.of normal seeds. +.........+ no.of normal seeds

                                                       First day                                   Second day                          Final day

e. Accelerated Ageing Test: Accelerated ageing response (%): Ten gram of seed is subjected for accelerated ageing in an ageing chamber maintained at 40±1⁰C at 90±2⁰ per cent RH (Delouche and Baskin, 1973) for a period of 7 days. The seeds are then removed and dried under shade for 48h. Then the accelerated aging (AA) response is determined by germination test as per ISTA (1996) outlined under section.

13. Tests for identification of mechanically damaged seeds

a. Fast green test

The fast green test reveals physical fractures in the seeds. Seeds are soaked in a 0.1% fast green solution for only 15-30 seconds. During this period, the fast green penetrates any area of the seed coat which has been fractured and stains the endosperm green. After the soaking period, the seeds are washed and the fractures then become apparent (visible) in the seed coat. The stained seeds are counted and expressed in percentage.

b. Ferric chloride test

Mechanically injured legume seeds turn black when placed in a solution of ferric chloride (Hardin 1980). The seeds (100X4) are immersed in 20% solution of ferric chloride (FeCl3) for 15 minutes, the seeds are washed and all black stained seeds are separated and expressed in percenatge.

c. Indoxyl acetate test

Any damage to seeds coats, paricularly in soybeans can be detected. Seeds are soaked for 10 seconds in a 0.1% solution of indoxyl acetate for 10 seconds and allowed to air dry. Lesions in the seed coats that are difficult to detect become visible because they turn purplish green against the yellow or white seed cotyledon background.

d. Vital coloring test:

The vital coloring method is the differential coloration of live versus dead tissues when exposed to certain dyes. An early method sulfuric acid is used. More recently, indigo carmine and other aniline dyes been used. This dye stains dead tissue blue, but it is incapable of penetrating live tissue, which remains unstained. Gadd suggested that it is particularly useful for determining viability of forest tree seeds.
By :
Mohammad Safar Alizada (Noori)
Sr. MSc.(Agri) in Seed Science and Technology, UAS, Bangalore,India