Germination Testing: Principles and Procedure
The purpose of laboratory testing of seed germination is to assess seed quality or viability and to predict performance of the seed and seedling in the field. A NOTIFIED laboratory under SEEDS ACT or qualified laboratory of 1STA for testing seeds must test seed processed for sale. The ultimate aim of testing the germination in seed testing laboratory is to obtain information about the planting value of the seed sample and by inference the quality of the seed lot. In addition, the laboratory germination results are also required for comparing the performance potential or superiority of the different seed lots. In general, the farmers, seeds men and public agencies use the germination results for the following purposes:
1. Sowing purposes, with a view to decide the seed rate to achieve desired field establishment.
2. Labeling purposes.
3. Seed certification purposes.
4. Seed Act and Law Enforcement purposes.
In seed testing germination has been defined as "the emergence and development from the seed embryo of those essential structures which, for the kind of seed tested indicate its ability to develop into a normal plant under favorable, conditions in soil". The seedlings devoid of an essential structure; showing weak or unbalanced development; decay or damage affecting the normal development of seedling are not considered in calculating the germination percentage. Factors that can affect the performance of seed in germination tests include; diseased seed, old seed, mechanically damaged seed, seed stored under high moisture, and excessive heating of seed during storage or drying.
In most cases a seed treatment will improve germination of seed only if the poor quality is due to seed-borne disease. Several different kinds of testing are available depending on the type of seed to be tested, the conditions of the test, and the potential uses of the seed. The most common tests are the cold germination test, accelerated aging test, the tetrazolium test and warm germination test. Each test is designed to evaluate various qualities of the seed.
1. Germination Testing
The most common test is a warm germination test because it is required by seed laws to appear on the label. The percentage of germinating seed in a Warm germination test must be printed on the label of the seed if it is to be sold as seed. The Warm germination test reflects the field emergence potential of a seed lot under ideal planting conditions. Usually 400 seed from each seed lot are placed under moist conditions on blotters, rolled towels, or sand and maintained about 75to 85 degrees F for about seven days in most of the cases. At the end of this period the seedlings are categorized normal, abnormal, or diseased, and dead or hard seeds. The percentage germination is calculated from the number of normal seedlings from the total number of seeds evaluated. The method of testing germination is discussed below.
The first and foremost step is to draw a true representative sample from the seed lot. To obtain a random sample for testing it is always best to take samples from different parts of the bag or container. If the seed to be tested is from a seed lot that contains more than one bag, samples must be taken from several bags. A good rule of thumb for determining how many bags to sample is to take samples from a number of bags that represents the square root of the lot size. For example if the lot contains 100 bags, the sample at least three bags. If the lot contains 100 bags, the sample at least 10bags. The sample thus drawn is further divided and the required numbers of seeds are the taken to perform the actual test.
2. Essential equipments and supplies for germination test
The following pieces of equipments and supplies are essential to carry forward the
Germination tests in the seed testing laboratories.
a) Seed Germinator
The seed germinators are the essential requirement for germination testing for maintaining the specific conditions of temperature, relative humidity and light. The seed germinators are generally of two types namely: Cabinet germinator and walk in germinator.
The cabinet seed germinators are essential under the situations, where various kinds of seeds that require different sets of conditions, are being handled in the laboratory. The number of the pieces of the germinators required by the laboratory will depend on the number of seed samples and the species being analyzed by the laboratory.
The seed testing laboratories that handle large number of seed samples and require maintaining only fewer (2-3) sets of temperature conditions, the walk-in-germinators are preferred. Such germinators are more useful for conducting the germination tests in sand media, which require large germination space.
b) Counting devices
The counting devices include the counting boards, automatic seed counter and vacuum seed counter. These devices are required to aid germination testing by minimizing the time spent on planning the seeds as well as to provide proper spacing of the seed on germination substrata. Counting boards are suitable for medium and bold sized seeds, while vacuum counter can be, used for small sized seeds. In the absence of counting devices, the work may be accomplished manually.
c) Other equipments:
The other equipments required for germination testing include the refrigerators, scarifier, hot water bath, incubator, forceps, spatula, germination, boxes, plastic plates, roll- towel stands and plastic or surgical trays, etc. A large oven with temp. Range 100 -200 C is also required for sterilizing the sand.
d) Miscellaneous Supplies, Glassware and Chemicals
Germination paper (Creppe Kraft paper or towel paper, sunlit filter paper and blotters) and sand are the basic supplies required for germination tests. In addition, the laboratory may also require some glassware, such as Petridishes, beakers, funnel, measuring cylinders, muslin cloth, rubber bands and tubes etc. and certain chemicals like Potassium nitrate, Thiourea, Gibrellic acid, and Tetrazolium chloride for specific purposes. Voltage stabilizers are required for the supply of the constant electric current. The voltage stabilizers are essential for costly germinators air-conditioners and refrigerators. Under the situations of erratic power supplies and breakdowns, electricity generators are also required.
3. Care of equipments:
The seed analyst must ensure that:
1. All the equipments are in proper working condition
2. The germinators are maintaining correct temperature
3. The relative humidity inside the germinator is maintained 90--98%
4. The phytosanitary conditions of the germinators and germination trolleys are adequate
5. The germinators are disinfected periodically by flushing with hot water; solution of
Potassium permanganate or chlorine water
6. The temperature and the R.H. of the walk-in-germinators are recorded daily and
displayed on a chart and .
7. The floor, ceiling and walls of the walk-in-germinator are devoid of cracks, crevices;
Evenly plastered and duly painted to avoid contamination by fungus, bacteria or
4. Handling of substrata .
The accuracy and reproducibility of the germinator result are very much dependent on the quality of the substrata (paper and sand) used for germination testing. The germination substrata must meet the following basic requirements:
1. It should be non-toxic to the germinating seedlings. .
2. It should be free from mould sand other microorganisms. .
3. It should provide adequate, aeration and, moisture to the germinating seeds.
4. It should be easy to handle and use. .
5. It should make good contrast for judging the seedlings .
6. It should be less expensive..
a) Paper substrata
The paper substrata are used in the form of top of paper (TP) or between paper (BP)
tests. In most of the laboratories, paper-toweling method (Roll towel test) is most commonly used for medium sized and bold seeds. The paper substrata are not reusable.
b) Sand substrata
The sand substrata have advantage of being relatively less expensive and reusable. The results in sand media are more accurate and reproducible in comparison with 'roll towel' tests especially in case of seed lots that are aged or heavily treated with chemicals. The sand should be reasonably uniform and free from very small and large particles. It should not contain toxic substances and its pH should be within the range of 6.0- 7.5. The sand should be washed, sterilized and graded with a sieve set having holes of 0.8 mm diameter (upper sieve) and 0.05mm diameter (bottom sieve). The sand retained on the bottom sieve should only be used.
5. Testing of Substrata
Phytotoxicity: The substrata should be tested for its phytotoxicity, capillary rise, moisture holding capacity and bursting strength, etc., before accepting the supplies in the laboratory. Periodic checks of the quality of the substrata should also be made in the laboratory.
By germinating the seeds of Brassica, Onion, Chillies or Berseem and studying the phytotoxic symptoms on the germinating seedlings can check the phytotoxicity of the paper or sand substrata.
I.. The paper should be cut into circles and rectangles or squares of the
desired size according to the size and shape of the containers.
2. Place 2-4 circles/rectangles of the paper to be tested in the Petri dishes or
3. Moisten the paper with tap water using only enough water to saturate the paper.
Excess water should not be used.
4. Arrange 25 seeds of Brassica, Onion, Chillies or Berseem properly spread over
the moist paper.
5. Cover the dishes with lids.
6. Conduct a control test as outlined above (step 1-4) using paper of accepted
quality such as 'Waterman' or 'Sunlit' brand filter paper.
7. Transfer the test to the prescribed temperature conditions of the species used at
8. Evaluate the test 1-2 days before the date of the first count of the crop specified
9. Check the phytotoxic symptoms on the seedlings.
10. Compare the seedlings with those grown on the non-toxic paper (control test).
The phytotoxic symptoms include shortened roots; discolored root tips; root raised from the paper; inhibition of root hairs development and root hairs bunched. The symptoms are more pronounced at an early stage of root growth. The phytotoxic symptoms are also evident in the plumular areas in the form of thickened or flattened plumules or, oleoptiles.
The phytotoxicity of the sand substrata can also be measured by the procedure outlined
as above. However, care need to be exercised that the sand substrata should be moistened with the measured quantity of the water and the seeds are planted on the ton of sand (TS)
The pH of the substrata can be measured with the help of pH paper or pH meter as follow:
1. Soak the paper or sand in water for 16-18 hrs.
2. Decant the water.
3. Measure the pH with litmus paper or pH meter.
If the sand substrata are found to be acidic or alkaline, wash it thoroughly with the
water and sterilize before use.
1. Cut four strips of germination paper10 mm wide; two in machine direction and
the other two in cross machine direction.
2. Take distilled water in small glass beakers.
3. Immerse one end of each strip in the water to depth of 20 mm.
4. Wait for 2 minutes and then measure the height to which water has risen in the
strip to the nearest mm.
5. Commute the average for the two strips cut in machine direction or cross machine direction separately.
6. The lower value of the two averages should be considered as capillary rise.
Bursting strength: The bursting strength of the paper is measured with equipment however, it can be checked as follows:
1. Hold the two ends of the germination paper and exert the pressure by
stretching the paper with mid force.
2. Soak the paper in water for 1-2 hours. .
3. The paper of desired bursting strength would not tear off easily. .
6. Test conditions
a) Moisture and aeration:
The moisture requirements of the seed will vary according to its kind. Large seeded species require more water than the small seeded species. It is essential that the substratum must be kept moist through out the germination period. Care need to be taken that the sub- stratum should not be, too moist. The excessive moisture will restrict the aeration and may cause the rotting of the seedlings or development of watery seedlings. Except the situations where a high moisture level is recommended (e.g. Paddy and jute), the sub stratum should not be so wet that a film of water forms around the seeds. In situations where low level of moisture is recommended (e.g. Cucurbitaceous seeds) , the moist substratum should be pressed against the
Dry blotters or towel paper, to remove excess moisture. The water used for moistening
the substratum must be free from organic and inorganic impurities. Normally the tap water is used. However, it is essential to measure the pH of water before its use. The pH of the water should be in the range of 6.5- 7.5. Under the situations where pH of the water is not satisfactory, distilled water or deionized water may be used. Under such situation care need to be exercised to aerate the tests frequently to provide oxygen supply to the germinating seedlings because oxygen level in distilled water is very low.
The initial quantity of water to be added to the substratum will also depend on its nature and dimensions. Subsequent watering, if, any may be left to the discretion of the analyst but it should be avoided as far as possible because it may cause the variation in germination results. In order to reduce the need for additional watering during the germination period, the relative humidity of the air surrounding the seeds should be kept at 90-95 % to prevent loss of water by evaporation.
Special measures for aeration are not usually necessary in case of top of paper (TP) tests. However, in case of 'Roll towel' tests (BP) care should be taken that the rolls should be loose enough to allow the presence of sufficient air around the seeds. In case of sand media, the sand should not be compressed while covering the seeds.
The temperature is one of the most important and critical factors for the laboratory
germination tests. The temperature requirement for germination is specific according to the kind of crop or species. This can vary within the species and with the age of seeds. At very low or high temperatures, the germination is prevented to a larger extent. The temperature should be uniform through out the germinator and the germination period. The variation in temperature inside the germinator should not be more than 1DC. The prescribed temperature for germination of agricultural, vegetable or horticultural seeds, provided in the Rules for Seed Testing can be broadly is classified into two groups, viz. constant temperatures and alternate temperatures.
Constant temperature: Wherever, the constant temperatures are prescribed or recommended for the germination tests, the tests must be held at the specific temperature during the entire germination period.
Alternate temperature: Wherever, the alternating temperatures prescribed, the lower
Temperature should be maintained for 16hours and the higher for 8 hours; a gradual changeover lasting3 hours is usually satisfactory for non-dormant seeds. However, a sharp changeover lasting1 hour or less, or transfer of test to another germinator at lower temperature may be necessary for seeds, which are likely to be dormant.
Seeds of most of the species can germinate. In light or darkness it is always better to illuminate the tests for the proper growth of the seedlings. Under the situations where light is essential for germinations, tests should be exposed to the natural or "artificial source of light. However "are must be made to ensure that an even intensity is obtained over the entire substrate and that any heating from the source does not affect the prescribed temperature. .
Seeds that require light for germination must be illuminated with cool fluorescent light
For at least 8hours in every 24 hours cycle. Under the situation where testing of the seed is required to be undertaken alternating "temperatures together with light, the tests should be illuminated during high temperature period. .
7. Laboratory procedures
The working sample or germination test consists of 400 pure seeds and randomly drawn either manually or with the help of counting devices. The seed for germination test must be drawn as follows in accordance with the following two situations:
a) When both purity and germination tests are required. .
1. Seeds for germination tests must be taken form the pure seed fraction after Conducting the physical purity analysis.
2. The counting of the seed must be made without discrimination as to the size and appearance.
b) Only germination test is required.
1. If, the percentage of pure seed is estimated or determined to be above 98 percent, the pure seed for germination test shall be taken indiscriminately from a representative portion of the submitted sample;
2. If, the pure seed is found to be less than 98 percent, the seeds for germination test must be obtained by separating the sample into two components namely
(a) The pure seed and
(b) Seeds of other species and inert matter.
For this purpose, at least one-fourth of the quantity required for regular purity analysis must be used after proper mixing and dividing the submitted sample.
Number of Replications:
Four replication of 100 seeds, A minimum of 3 replication of 100 seeds may be used under unavoidable situations or Eight or six replications of 50 seeds or Sixteen/twelve replication of 25 seeds according to the kind of and size of containers.
· Check the quality of germination paper before accepting the supplies.
· Measure the pH, capillary rise, bursting strength and phytotoxicity of the germination paper.
· Store the germination paper under hygienic conditions and protect it from dust and Micro flora.
· Very old stock should not be used as they often get contaminated. Such paper usually shows phytotoxic symptoms.
Between Paper (BP) Media (Roll Towel Test):
1. Soak the towel paper in water.
2. Remove the water.
3. Wash the paper with running water.
4. Remove extra moisture by pressing the soaked paper by hand and holding it in plastic/ Surgical trays placed on the table top in slanting position.
5. Place two layers of wet paper toweling as substratum.
6. Check Test number provided on the Analysis Card sample and label tally each other.
7. Record the test number, crop and date of putting on the wax paper or tag.
8. Arrange seeds spaced properly.
9. Place one layer of wet towel paper over the seed.
10. Turn up two inches of the bottom edge.
11. Roll firmly from left to right and secure with rubber band in the center.
12. Place the prepared roll towel in roll towel stand or baskets.
13. Transfer the basket or roll towel stand in the
germinator maintained at the desired Temperature.
Top of Paper (TP) Media:
1. Paper of known quality such as 'Sunlit' or 'Whatman' filter paper should be used.
2. Crepe Kraft (towel) paper or blotter paper of unknown quality should not be used for Top of paper tests.
3. The paper should be cut in the form of circles/squares or rectangles according to the Size and shape of petridish/container.
4. Put 2-3 layer of filter paper in the petridish/germination box having airtight lids.
5. Put enough water to moisten the filter paper.
6. Hold the petridish /germination box in slanting position in order to drain out the Extra moisture.
7. Record the test number and date of putting on the lid of the container on die paper Slip.
8. Space the counted seeds on the moist blotter/filter paper.
9. Cover the lid
10. Transfer the test in the germinator maintained at the desired temperature.
1. Properly graded and sterilized sand free from impurities and toxic chemicals should be Used.
2. Sand should not be stored in the stores where fertilizers and chemicals are stored.
3. Grade,the sand with a sieve set of 0.8mm x 0,05 mm (mesh).
4. Sand retained over Q05 mm sieve should only be used.
5. After each test,the sand should be dried and sterilized.
6. If required,the sand may be washed before sterilization.
7. If the sand found to be heavily contaminated or changed in colour after repeated use it Should be replaced with fresh stocks.
8. The pH of the sand should be within the range of 6.0 - 7.5.
9. The sand should also be checked if its phytotoxicity.
10. Determine the, moisture holding capacity of the sand
11. Put required quantity of water to moisten the sand.
12. The moisture level of the sand will vary according to the kind of seed.
13. Place moist sand in plastic germination boxes.The depth of sand bed should be Approximately 2".
14. Space the counted seed on the sand bed contained in the germination boxes. .
15. Cover the seed with moist sand layer, approximately 1/4" in thickness.
16.Put the cover on the germination boxes and place them under prescribed controlled Temparature conditions.
After placing the seeds on the prescribed substrata, the test should be transferred to the controlled temperature condition maintained in the cabinet or walk-in-germinator for prescribed period, which varies according to the species(1STA Seed Testing Rules). In the Rules for Seed Testing, two kinds of temperature conditions are provided. A single numerical indicate the constant temperature and numerical separated by a dash(-) indicate an alternating temperature. If temperatures can not be conveniently altered over weekends or holidays, the tests must be kept at the lower temperature. The daily alternation of temperature either brought out manually by transferring the test from one germinator to another or by changing the temperature of the chamber (Automatic Seed Germinator).
9. Methods to improve germination
Hard seeds: For many species where hard seeds occur, some special treatment is essential. This treatment may be applied prior to the commencement of the germination test or, if it is, suspected that the treatment may adversely affect non-hard seeds, it should be carried out on the hard seeds remaining after the prescribed test period. The treatments are as below:
Soaking: Seeds with hard seed coats may germinate more readily after soaking for up to 24-48 hours in water or for Acacia spp. after plunging seeds in about three times their volume of near boiling water until it cools. The germination test is commenced immediately after soaking.
Mechanical scarification: Careful-piercing, chipping, filing or sand papering of the seed coat may be sufficient to break the dormancy condition. Care must be taken to scarify the seed coat at a suitable part in order to avoid damaging the embryo. The best site for mechanical scarification is that part of the seed coat immediately above the tips of the cotyledons.
Acid scarification:Treating with in concentrated Sulphuric acid (H2SO4)is effective with some species (e.g. Macroptilium sp., Brachiaria sp., Sesbania sp.). The seeds are moistened with in the acid until the seed coat becomes pitted. Digestion may be rapid or take more than one hour, but the seeds should be examined every few minutes. After digestion, seeds must be thoroughly washed in running water before the germination test is commenced. In the case of Oryza sativa scarification may be performed by soaking the seed in one normal nitric acid (HNO3) for 24 hours (after preheating at 50 °C).
Naturally occurring substances in the pericarp or seed coat, which act as inhibitors of germination may be removed by washing the seeds in running water at a temperature of 25°C before the germination test is made. After washing, the seeds should be dried back at a maximum temperature of 25°C (e.g. Beta vulgaris). Germination of certain species is promoted by removing outer structures such as involucre of bristles or lemma and palea of certain Poaceae(Gramineae).
Disinfection of the seed: For samples of Arachis hypoagea and Beta vulgaris only, a fungicide treatment may be applied before planting the seed for germination then the seed lot is known not to have received such a treatment.When a fungicide pretreatment is used the name of the chemical the percentage of active ingredients and the method of treatment shall be reported on the certificate.
Prechilling : In some seeds having physiological dormancy pre chilling is required for inducing germination. Replicates for germination are placed in contact with the moist .substratum and kept at a low temperature for an initial period before they are removed to the temperature as shown in (ISTA Seed Testing Rules -Table 2). Agricultural and vegetable seeds are kept at a temperature between5°C and 10°C for an initial period up to,7 days.Tree seeds are kept it a temperaturebetween3°C and,5°C, for a period, varying with the species,from 7 days to 12 months.In some,cases it may be necessary to extend the prechilling period or to rechill. The prechilling period is not included in the germination test period but both the duration and the temperature should be reported on the analysis card.
Pre-drying: The replicates for germination should be heated at a temperature not exceeding 40°C with free air circulation for a period of up to 7 days before they are placed under the prescribed germination conditions. In somecasesit maybe necessary to extend the pre-drying period. Both the duration and the temperature should be reported on the Analysis Certificate.
Potassium nitrate (KNO3):The germination substratum may be moistened with a 0.2% solution of KNO3, as indicated in (1STA Seed Testing Rules -Table 2). The substratum is saturated at the beginning of the test but water is used for moistening it there after .The use of this treatment should be noted on the analysis certificate.
The procedure for preparing solutions and soaking blotters is as follows:
i). Preparation of stock KNO3, solution(2%): Place20 GmsKNO3 crystals in 1000ml water shake until dissolved.This must be diluted before being used to soak blotters.
ii). Preparation of 0.2% KNO3 solution for soaking blotters: Add 90 ml water to 10 ml of stock solution.
iii). Procedure for soaking blotters: a. Take the blotters representing the sample and place into, the prepared solution,(0.2%)-one at a time. b. Turn blotters over in one movement, but ensuring that they are still free moving in the solution. c. Remove one at a time, in order of placing in solution and place on tray.
Gibberellic acid (GA3): Moisten the germination substratum with 50 ppm solution of GA,which can be prepared by dissolving 500 mg of GA3 in 1000 ml of water. Place the seed for germination under prescribed temperature conditions.
10. Duration of testing
The duration of the test is determined by the time prescribed for the, final count (1STA Seed Testing Rules, Table 2) but the chilling, periods before or during the test, which is required to break dormancy, is not included in the test period. If at the end of the prescribed test period some seeds have just started to germinate, the test may be extended for an additional period up to 7 days. A test may be terminated prior to the prescribed time when the analyst is satisfied that the maximum germination of the sample has been obtained. The time for the, first count is approximate and a deviation of 1-3 days is permitted. The First count may be delayed to permit the development of root hairs in order to be certain that root development is normal, or may be omitted. Intermediate counts may be at the discretion of the analyst to remove seedlings, which have reached a sufficient state of development for evaluation, to prevent them becoming entangled. But the number of intermediate counts should be kept to a mini- mum to reduce the risk of damaging any seedlings that are not sufficiently developed.
Seedlings may have to be removed and counted at more frequent intervals during the prescribed period of the test when a sample contains is infected with 'fungi or bacteria. Seeds that are obviously dead and decayed, and may, therefore, be a source of contamination for healthy seedlings, should be removed at each count and the number recorded.
12. Evaluation of germination test
The germination tests need to be evaluated on the expiry of the germination period, which varies according to the kind of seed. However, the seed analyst may terminate the germination test on or before the final count day or extend the test beyond the period de-pending on the situation
First and second counts are usually taken in case of Top of Paper(TP)and Between Paper(BP)media; however a single final count is made in case of and tests. At the first and subsequent counts only normal and dead seeds (which are source of infection) removed and recorded.
In evaluating the germination test the seedling and seeds are categorized into
Normal seedlings,abnormal seedlings,dead seeds,fresh ungerminated and hard seeds.It may also be necessary to remove the seed coat and separate the cotyledons In order to examine the plumule in species where essential structures are still enclosed at the end of the test.
a)NormalSeedlings: It is necessity to separate normal seedling, which are counted in the percentage germination, from any abnormal seedlings .To achieve uniformity in evaluating normal seedlings, they must conform to one of the following definitions:
a. Seedlings which show the capacity for continued development into normal, plant. when grown in good quality soil and under favourable conditions of water supply, temperature and light.
b. Seedlings that possess all the following essential structures when tested on artificial substrata:
i) A well-developed root system including a primary root except for those plants (e.g. ceftt 1 in species of Gramineae) normally producing seminal roots of which there still are at least two.
ii) A well-developed and intact hypocotyl without damage to the conducting tissues.
iii) An intact plumule with a well developed green leaf, within or emerging through the coleoptile, or an intact epicotyl with a normal plumular bud.
iv) One cotyledon for seedlings of monocotyledons and two cotyledons and seedlings of dicotyledons.
c. Seedling with the following slight defects provided they show vigorous and
balanced development of the other essential structures:
i. Seedlings of Pisum, Vicia, Phaseolus,Lupinus, Vigna, Glycine, rachis,Gossypium, Zea and all species of Cucurbitaceae, with a damaged primaryroot but with several secondary roots of sufficient length and vigour to support the seedlings in soil.
ii. Seedlings with superficial damage or decay to the hypocotyl, epicotyl or cotyledons, which is limited in area and does not affect the conducting tissues.
iii. Seedlings of dicotyledons with only one cotyledon.
d. Seedlings of tree species having epigeal germination when the radicle is four times the length of the seed provided all structures that have developed appear normal.
e. Seedlings which are seriously decayed by fungi or bacteria, but only when it is clearly evident that the parent seed is not source of infection and it can be determined that all the essential structures were present.
b) Abnormal Seedlings: Abnormal seedlings are those, which do not show the capacity for continued development into normal plants when grown in good quality soil and under favorable conditions of water supply, temperature and light.
Seedlings with the following defects shall be classed as abnormal :
i. Damaged seedlings; seedlings with no cotyledons; seedlings with constrictions, splits, cracks or lesions which affect the conducting tissues of the
epicotyl, hypocotyl or root; seedlings without a primary root of those species where a primary root is an essential structure, except for Pisum, Vicia, Lupinus,Vigna, Glycine, Arachis, Gossypium, Zea and all species of Cucurbitaceae, when several vigorous secondary roots have developed to, support the seedlings, in soil.
ii. Deformed seedlings: Seedlings with weak or unbalanced development of the essential structures such as spirally twisted or stunted plumules, hypocotyls or epicotyls; swollen shoots and stunted roots; split plumules or coleoptiles without a green leaf; watery and glassy seedlings, or without further development after emergence of the cotyledons.
iii)Decayed seedlings: seedlings with any of the essential structures so diseased or decayed that normal development is prevented, except when there is clear evidence to show that the cause of injection is not the seed itself.
iv.Seedlings showing cotyledon development from the micropyle, or radicle development from a part of the seed other than the micropyle.
Special categories of abnormal seedlings The 3 main categories of abnormality, damage, deformity and decay, outlined in the previous section, can be further classified into categories as follows:
a. No roots, in Avena, Hordeum, secale and Triticum or one seminal root only.
b.Primary root (or seminal roots in Gramineae ) short and stunted.
c. Primary root thin and weak, too short or too long.
d. Primary root short and stunted, or short and weak, or spindly, secondary roots weak.
e. No primary root or no well developed secondary roots.
f. Seminal roots short and weak,or spindly, or watery.
g. primary root split longitudinally , or damaged with secondary roots weak,
h. Radicle with no hair roots.
i. Radical or primary root brown in colour.
a. Hypocotyl short and thick, or twisted, or curled over. Or watery.
b. Epicotyl or stem with constriction,grainy lesion, or open split likely to interfere with the conducting , tissues.
c. Hypocotyl with construction ,grainy lesions or open split likely to lesions or interface with the conducting tissues .
d. Epicotyl or stem short and thick, or twisted around the main axis,
e.No terminal bud.
f. Two shoots which are short and weak,or spindly.
g. No primary leaves, with or without terminal or auxiliary buds, or with more than half The total area of the primary leaves missing or not capable of functioning normally, or With one primary leaf and evidence of damage to the shoot apex.
a. No green leaves
b. Short leaves extending less than half the length of coleoptile.
c. Leaves shattered or split longitudinallyand/or coleoptile with a split easily visible to The naked eyeor abnormal coleoptiledevelopment due to damage
d. Plumule spindly,or pale, or watery
e. Plumule short and thick, usually with short or stunted seminal roots.
b. One,with evidence of damage to the shoot apex.
c. Poorly developed leaf-like cotyledon in Allium, without a definite bend, or "knee".
d. Enlarged, with short hypocotyl.
e. Physiological necrosi
f .Grey in colour
g.Swallow and blackned.
h. More that half the total area broken off, or covered with spots or darkened areas, or with open splits if development as a whole is out of proportion compared with that of a normal seedlings germinated at the same time.
a. Decayed cotyledons.
b. Decayed hypocotyl.
c. Decayed epicotyl or stem.
d. Decayed plumule, or decay at point of attachment between seedlings and endosperm or Discoloration of the coleoptile which has penetrated to the leaves.
e. Decayed primary root (except secondary infection by Phoma betae) or seminal roots in the gramineae.
f.Decay or discoloration at point of attachment between cotyledons and seed lings axis,or adjacent to the shoot apex.
g.Completely delayed seedling.
f) Other abnormalities:
a. Seedlings short and weak,or spindly, or watery.
b. Frost damaged seedlings with grainy coleoptile or a plumule, which is weak and Spirally twisted.
c. Entirely white seedling in the Gramineac and Liliaceae.
d. completely ahattered seedling.
12. Caluculation and expression of result
results are expressed as percentage by number.Germination rate is the average number of seeds that germinate over the five-day and 10-day time period.
Germination (%) = Number seeds germinated
----------------------------------------- x 100
Number seeds on tray
When four 100-seed replicates of a test are within the maximum tolerated range the average represents the percentage germination to be reported on the Analysis Certificate.The average percentage is calculated to the nearest whole number.The total % of all the category of seeds (normal,abnormal,dead hard,fresh un germinated) should be 100.
The result of a test shall be considered unsatisfactory and shall not be reported and a second test shall be made by the same or an alternative method, under the following circumstances:
a) When dormancy is suspected (fresh ungerminated seeds).
b) When the result may not be reliable because of phytotoxicity or spread of fungi or Bacteria.
c) When there is difficulty in deciding the correct evaluation of a number of seedlings.
d) When there is evidence of errors in test conditions, seedling evaluation or counting.
e) When the range for the 100-seed replicates exceeds the maximum tolerated range
14. Reporting of result
The following items shall be entered in the appropriate space of the analysis certificate