Flow Chart for Seed Germplasm Conservation
II. Testing for Seed Viability
Page | 174 Recording and Calculation of Seed Moisture Content
Accession no.
Replicate/
Moisture Bottle no.
Wt of empty Moisture
Bottle with lid (g)
Wt of Moisture
Bottle with lid + seed
before drying (g)
Wt of Moisture
Bottle with lid + seed after drying (g)
Moisture content % (wb)
W1 W2 W3 (W2-W3)/
(W2-W1) × 100
Average (R I + R II)/2
R I
R II
R I
R II
R I
R II
R I
R II
Example:
Calculation:
Rep 1: % Moisture content = (W2-W3)/(W2-W1) × 100 Rep 2: Moisture content =(W2-W3)/(W2-W1) × 100
Moisture content (fresh-weight basis) = (%MC Rep.1+ %MC Rep.2 )/2
Page | 175 Standard Germination Test
The germination test is conducted as per the rules and procedures prescribed by ISTA.
However, a compromise in seed quantity is permitted since availability of seed in submitted samples of germplasm is significantly lesser than commercial samples received in seed testing laboratories. Twenty five seeds in two replications are used in exceptional cases where seed quantity is extremely low. Two basic methods are used for testing the germination of germplasm seeds.
Top of Paper Method: This method is adopted for small seeded crops such as mustard, amaranth, etc. Two layers of filter papers, moistened with distilled water, are placed in petri plates that are labeled with accession number, replication number and date of plating. Seeds are plated on the paper and the petri plates are incubated in germinators maintained at high humidity and appropriate /optimum temperature as recommended by ISTA (Table 1).
Germination evaluation is done according to ISTA rules (ISTA, 2019).
Between Paper Method: This method is suited for larger seeded crops such as green gram, chickpea, etc. Seeds are germinated between two layers of moist crepe papers, by arranging them in rows at regular intervals and leaving sufficient gap on both sides. It is then over- layered with a second layer of moistened crepe paper, followed by a wax paper. The required labelling is done on the wax paper and they are rolled loosely, with the wax paper on the outer side. The paper rolls are placed in trays containing distilled water, to keep the papers moist during the test duration. The tray is placed in germinator maintained at recommended temperature.
Germination testing (Top of paper method)
Germination testing (Between paper method)
Page | 176 Sand is also used as germination medium in case of large sized seeds and also when fungal infection or phytotoxicity of paper medium is anticipated
Seedling Evaluation
Germination evaluation is done according to ISTA rules. Seedlings are assessed on the day designated for final count and are classified as normal seedlings, abnormal seedlings, fresh ungerminated seeds and dead seeds.
Abnormal seedlings
Seedlings with the following defects are classified as abnormal-
Damaged: seedlings with any of the essential structures missing or so badly and irreparably damaged that balanced development cannot be expected. For eg., missing, broken, or split primary root, Shoot that is split right through or missing, separated or missing cotyledons.
Absence of seminal roots in the case of monocots is also considered abnormal.
Deformed or unbalanced: seedlings with weak development or physiological disturbances or in which essential structures are deformed or out of proportion. For eg., stubby, stunted, split or glassy roots and shoots, necrotic cotyledons and deformed terminal bud/leaves.
Decayed: seedlings with any of their essential structures so diseased or decayed as a result of primary infection that normal development is prevented.
Seedlings which are decayed by fungi or bacteria are classified as normal, if it is evident that the parent seed is not the source of infection, and if it can be determined that all the essential structures were present.
A) Seedling evaluation B) Normal and abnormal seedlings Fresh Ungerminated seeds
When 5% or more of fresh ungerminated seeds are present, their viability should be determined through quick viability testing, as descibed in section III. A high percentage of
Page | 177 fresh ungerminated seed is an indication of dormancy being prevalent in the sample. ISTA has recommended specific dormancy breaking treatments for each species, based on the nature of dormancy. The most common type of dormancy in agri-horticultural crops is the physiological dormancy. Physical dormancy is also prevalent in certain families of cultivated crops like fabaceae, malvaceae and anacardiaceae. The dormancy breaking treatments recommended by ISTA for major crop species is mentioned in Table 4. After exposing the seeds to the relevant treatment, the germination test should be repeatedusing standard procedure, for noting the final viability percentage.
Reporting of results
The result of the germination test is expressed as percentages by number of normal and abnormal seedlings and hard, fresh and dead seeds. The percentages are rounded to the nearest whole number. The final viability percentage is the average percentage of normal seedlings in the sample.
Table 4: Germination methods for some agricultural and horticultural seeds
Species Substrata Temp oC Final
Count
Recommendation for breaking dormancy
Abelmoschus esculentus(Okra) TP;BP 20-30 21 --
Arachis hypogaea (Groundnut) BP;S 20-30;25 10 Remove shells,
Allium cepa (Onion) BP;TP 20;15 12 Prechill
Avena sativa (Oat) BP;S 20 10 Preheat (30-35°C;
prechill, GA3) Beta vulgaris (Sugar beet) TP;BP;S 20-30;
15-25
14 Prewash 2-4 hours in running water
Brassica juncea (Sarson) TP 20-30;20 7 Prechill; KNO3
Brassica juncea (Rape seed) TP 20-30;20 7 Prechill
Brassica oleracea (Cabbage, Cauliflower)
TP 20-30;20 10 Prechill, KNO3
Cajanus cajan (Red gram) BP;S 20-30;25 10 --
Capsicum sp. (Chilli) TP;BP 20-30 14 KNO3
Cicer arietinum (Bengal gram) BP;S 20-30;20 8 --
Corchorus sp .(Jute) TP;BP 30 5 --
Cucumismelo (Muskmelon) BP;S 20-30;25 8 Low moisture
Cucumissativus (Cucumber) TP;BP;S 20-30;25 8 -- Cucurbita moschata (Pumpkin) BP;S 20-30;25 8 -- Cucurbita pepo (Summer squash) TP;BP;S 20-30;20 14 --
Daucuscarota (Carrot) TP;BP;S 20-30;20 14 --
Glycine max (Soybean) BP;S 20-30;25 8 --
Page | 178
Gossypium Sp.(Cotton) BP;S 20-30;25 12 --
Helianthus annus (Sunflower) BP;S 20-30;25;
20
10 Preheat, prechill
Hordeum vulgare (Barley) BP;S 20 7 Preheat (30-35°C)
prechill, GA3,
KNO3
Lactuca sativa (Lettuce) TP;BP 20 7 Prechill
Lens culinaris (Lentil) BP;S 20 10 Prechill
Linum usitatissimum (Linseed) TP;BP 20-30;20 7 Prechill Lycopersicon lycopersicum
(Tomato)
TP;BP 20-30 14 KNO3
Nicotiana tabacum (Tobacco) TP 20-30 16 KNO3
Oryza sativa (Paddy) TP;BP;S 20-30;25 14 Preheat (50°C), soak in H2O or HNO3 24 hr Pennisetum typhoides
(Pearlmillet)
TP;BP; 20-30 7 --
Pisum sativum (Pea) BP;S 20 8 --
Ricinus communis (Castor) BP;S 20-30 14 --
Sesamum indicum (Sesame) TP 20-30 --
Solanum melongena (Brinjal) TP;BP 20-30 14 --
Sorghum vulgare (Jowar) TP;BP 20-30 14 --
Triticum aestivum (Wheat) TP;BP;S 20 8 Preheat (30-35°C)
prechill, GA3
Vicia faba (Broad bean) BP;S 20 14 Prechill
Vigna mung o(Black gram) BP;S 20-30;25;
20
7 --
Vigna radiata (Green gram) BP;S 20-30;25 7 --
Vigna unguiculata( Cowpea) BP;S 20-30;25 8 --
Zea mays (Maize) BP;S 20-30;25;
20
7 --
TP: Top of Paper; BP: Between paperm, S: Sand, TS: Top of Sand, GA3: Gibberellic acid (0.05 to 0.1%) KNO3: Potassium nitrate (0.2%), HNO3: Nitric acid (1 N)
Page | 179 DATA SHEET FOR RECORDING GERMINATION RESULTS
Crop/species: Temperature:
Accession number: Light:
Date of storage: Special treatments:
Date of testing: Incubation time:
Substrate:
Replication Days Normal seedlings Total Remarks
I II III IV
No. of seeds tested Date
1 2 3 4 5 6 7 Abnormal
Hard/Fresh Ungerminated Dead
Speed of Germination