Interactive data analysis platform for germination analysis
International Rule for Seed Testing (ISTA) 2016
Dr. Mohammadreza Labbafi
Table of Contents
Four hundred seeds are taken at random from the well mixed pure seed and spaced uniformly and adequately apart on the moist substrate. Care must be taken to ensure that there is no selection of seeds thus causing biased results. Replicates of 100 seeds are normally used, spaced sufficiently far apart on the seed bed to minimize the effect of adjacent seeds on seedling development. To ensure adequate spacing, split replicates of 50 or even 25 seeds may be necessary, particulary where there are seed-borne pathogens or saprophytes present. When seeds grown on paper substrates are heavily infected, it may be necessary at an intermediate count to transfer remaining seeds and seedlings to fresh media.
Multigerm seed units, except for Arachis, are not broken up for the germination test but are tested as though they were single seeds.
For Arachis, although a pod is a pure seed unit, seed must be removed from the pod before use in a germination test.
The ISTA germination test is based on 400 seeds. In certain circumstances it may be necessary to test fewer than 400 seeds. In such cases, at least 100 seeds must be tested in replicates of 25 or 50.
At the request of the applicant, a germination test can be carried out on 200 seeds, for issuance on a Blue International Seed Sample Certificate only. In this case, the number of tested is less than 400 and must be reported under �Other Determinations�.
When due to counting errors more than 5 seeds are lost or found during a germination test (i.e.� 1.25 % for a total of 400 seeds), then the must be repeated.
If there are up to 5 seeds lost or found as extra in the test, then each replicate must be adjusted to 100 by calculation. For example, if one replicate had 80 normal seedlings, 10 abnormal seedlings and 9 dead seeds, with one seed missing, then the result must be adjusted to 100 with the following proportional calculation: 80*100:99 normal seedlings, 10*100:99 abnormal seedlings and 9*100:99 dead seeds.
Note: if the submitted sample is smaller than prescribed, the sampler must be notified accordingly and analysis withheld until sufficient seed is received in a single submitted sample, except that in the case of very expensive seed, the analysis may be completed to the extent possible, and the following statement inserted on the certificate:
�The sample submitted weighed only�.g and is not in accordance with the International Rules for Seed Testing�.
Or, in the case of pelleted seeds:
�The sample submitted contained only � pellets (seeds) and is not in accordance with the International Rules for Seed Testing�.
Permitted substrates, temperatures, duration of tests and additional directions, including recommended procedures for breaking dormancy, are indicated. Substrates, temperatures and duration of test indicated are prescriptive and no others may be used.
The seeds are germinated on top of one or more layers of paper which are placed:
- On the Jacobsen apparatus;
- Into transparent boxes or Petri dishes. The appropriate quantity of water is added at the beginning of the test and evaporation may be minimized by a tightly fitting lid or by enclosing the dishes in plastic bags;
- Directly on trays in germination incubators. The relative humidity in the incubators must then be maintained at a level that prevents tests drying out. Moistened porous paper or absorbent cotton can be used as base for the substrates.
The seeds are germinated between two layers of paper. This may be achieved:
- By loosely covering the seeds with an additional layer of filter paper;
- By placing the seeds into folded envelopes which may be placed in a flat or upright position;
- By placing the seeds in rolled paper towels (the rolls must be placed in an upright position).
The substrates are kept in closed boxes, wrapped in plastic bags or placed directly on trays in a cabinet germinator, provided the relative humidity in the germinator can be maintained very near saturation.
The seeds are placed in a pleated, accordion-like paper strip with 50 pleats, usually two to a pleat. The pleated strips are kept in boxes or directly in a wet cabinet, with a flat strip often wrapped around the pleated paper to ensure uniform moisture conditions. This method may be used an alternative where TP or BP are prescribed.
Sand and organic growing media are used as follows:
The seeds are pressed into the surface of the sand or the organic growing medium.
The seeds are planted on a level layer of moist sand or the organic growing medium and covered with 10-20 mm of uncompressed substrate, depending on the size of the seed. To ensure good aeration it is recommended that the bottom layer be loosened by raking before sowing.
Sand or organic growing media may be used instead of paper, even if not prescribed:
- When the evaluation of a diseased sample proves impracticable because of the spread of infection between seeds and seedlings on paper substrate;
- For investigative purposes and to confirm evaluation of seedlings in cases of doubt;
- When seedlings show phytotoxic symptoms.
The seeds are germinated on top of a moistened sheet of crepe cellulose paper which is covered with a 2 cm layer of dry sand. Crepe cellulose paper is a multi-layered paper pad, e.g. Versa-Pak.
Soil is generally not recommended as a primary growing medium. However, it may be used as an alternative to organic growing media when seedlings show phytotoxic symptoms or if evaluation of seedlings is in doubt on paper or sand.
Precautions must be taken to ensure that the medium cannot dry out and contains sufficient water for the whole test period. Subsequent watering should be avoided wherever possible, as it is likely to increase variability between replicates and between tests. However, it may be necessary to add water at intermediate counts.
Special measures for aeration are not necessary for TP and PP tests enclosed in boxes or Petri dishes. For BP, however, care must be taken that envelopes and rolled paper towels are loose enough to allow for sufficient air around the seeds. For the same reason, sand and organic growing media must not be compressed.
The temperature prescribed in Table for the germination of a species are those to which the seed is exposed on or inside the substrate. They should be as uniform as possible throughout the germination apparatus, incubator or room germinator. For any test, whether in darkness or under artificial light or in indirect daylight, variation from the prescribed temperature must not be more than � 2 C.
Where alternating temperatures are indicated, the lower temperature should be maintained for 16 h and the higher for 8 h. A gradual changeover lasting no more than 3 h may be satisfactory, but a sharp changeover lasting one hour or less may be necessary for breaking dormancy.
Where a temperature range is given, no tolerances may be applied to the upper or lower temperatures. For example, when a prechilling temperature of 5 to 10 C is prescribed, this means that the allowed temperature range is 5 to 10 C, and not 5 � 2 C to 10 � 2 C.
Seeds of most of the species in Table will germinate either in light or in darkness. However illumination of the substrate from an artificial source or by indirect daylight is generally recommended, as better developed seedlings, which are more easily evaluated are produced. Seedlings grown in complete darkness are etiolated and white and therefore more sensitive to attack by micro-organisms. Beside, certain defects, such as chlorophyll deficiency, cannot be detected.
In certain cases (e.g. some tropical and subtropical grasses), light may promote germination of dormant samples. In such cases, the light should be between 750 and 1250 lux from cool white lamps. There are also a few species (e.g. Phacelia tanacetifolia) which must be germinated in darkness, as light may be inhibitory. Specific recommendations for light or darkness are given in the last column of Table.
When alternative methods are indicated in Table, one of them (any combination of substrate and temperature) must be used. The choice of method will depend largely on the facilities and experience of the testing laboratory and to some extent on the provenance and condition of the sample.
For various reasons (e.g. physiological dormancy, hardseededness, inhibitory substances) a considerable number of hard or fresh seeds may remain at the end of the germination test. More complete germination may be obtained by retesting after one or a combination of the procedures listed. These procedures may be applied to the original test, if dormancy is suspected. The period of treatment is not included in the germination test period. Precise details and duration of the dormancy-breaking procedure must be reported on the ISTA Certificate.
For some tree and shrub seeds, where it is known from experience that a proportion of the seeds will not germinate because of dormancy, a second test incorporating a special dormancy-breaking procedure is prescribed which preferably should run concurrently with the normal test (double test).
Prechilling: The replicates for germination are placed in contact with the moist substrate and kept at a low temperature for an initial period before they are moved to the temperature. Agricultural, vegetable, flower, spice, herb and medicinal seeds are usually kept at a temperature of 5 to 10 C for an initial period of up to 7 days. In some cases it may be necessary to extend the prechilling period or to rechill.
Tree and shrub seeds are usually prechilled at a temperature of 1 to 5 C for a period, ranging with the species, from 2 weeks to 12 months prior to the germination test, but care must be taken to avoid freezing. For seeds where a long period of prechilling is required and a germination test cannot be completed within two months, quick visibility tests are recommended. For some tree and shrub species with a varying degree of dormancy, duplicate tests with and indicated in Table 5A, which should if possible be set to germinate at the same time.
The non-imbibed seeds of the replicates for germination are heated at a temperature of 30 to 35 C with free air circulation for a period of up to 7 days before they are placed under the prescribed germination conditions. In some cases it may be necessary to extend the preheating period.
For certain tropical and subtropical species, preheating temperatures of to 50 C may be used (e,g. Arachis hypogaea: 40 C; Oryza sativa: 50 C).
For some temperate herbage grass species, the seed submitted for testing is stored at a temperature of 15 to 25 C with free air circulation before they are tested. A prestorage period of up to one year can be used.
The tests should be illuminated during at least 8 h in every 24 h cycle and during the high temperature period when the seeds are germinated at alternating temperatures. The quality and intensity of light may be important. The light intensity should be between 750 and 1250 lux from cool white lamps. Illumination is recommended especially for certain tropical and subtropical grasses (e.g. Chloris gayana, Cynodon dactylon).
Where a high proportion of fresh ungerminated seeds is found at the end of the standard test (e.g. in Trifolium spp.), retesting in a sealed polyethylene envelope, of just sufficient size to hold the test satisfactorily, will usually induce these seeds to germinate.
The GA3 treatment is recooended mainly for Avena sativa, Hordeum vulgare, Secale cereal, Triticosecale, �Triticum aestivum and Valerianella locusta. The germination substrate is moistened with 0.05 % mg GA3, prepared by dissolving 500 mg GA3 in 1 litre of water. When dormancy is weaker, 0.02 % may be enough; when it is stronger, up to 0.1 % may be used routinely. If it is necessary to use concentrations higher than 0.1 %, care must be taken to ensure that the development of seedlings is not adversely affected. When a concentration higher than 0.08 % is required, dissolving the GA3 in a phosphate buffer solution is recommended. The buffer solution is prepared by dissolving 1.7799 g of Na2HPO4. 2H2O and 1.3799 g of NaH2PO4. H2O in 1 L of distilled water.
Instead of water, 0.2 % KNO3 solution, prepared by dissolving 2 g KNO3 in 1 L of water, is used to saturate the germination substrate at the beginning of the test. Water is used for moistening thereafter.
The seeds are soaked in concentrated sulphuric acid (H2SO4) 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 (e.g. Brachiaria spp.). In the case of Oryza sativa, scarification may be performed by soaking the seed in 1 M nitric acid (HNO3) for 24 h (after preheating at 50 � 2 C).
The seed is pierced, filed or sandpapered to improve permeability to moisture and gasses. Care must be taken to scarify the seed coat at a suitable place in order to avoid damaging the embryo and the resulting seedling. The best places are either immediately above the tips of the cotyledons or to the sides of the cotyledons.
For many species where hard seeds occur, no attempt is made to germinate them and percentage found is reported. Where a fuller assessment is required on the request of the customer, some special procedure for removing hardseededness is essential. This procedure may be applied prior to the commencement of the germination test, or, if it is suspected that the procedure may adversely affect non-hard seeds, it should be carried out on the hard seeds remaining after the prescribed test period.
Seeds with hard seed coats may germinate more readily after soaking for up to 24-48 h 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.
Careful piercing, chipping, filing or sandpapering of seed coat may be sufficient.
This procedure is effective with some species (e,g Desmodium spp., Macroptilium spp., Stylosanthes guianensis).
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 � 2 C before the germination test is made. After washing, the seeds must be dried at a temperature of 20 to 25 C (e.g. Beta vulgaris). Pelleted seed must not be prewashed.
Germination of certain species is promoted by removing outer structures such as involucre of bristles or lemma and palea of certain Poaceae.
For samples of Arachis hypogaea and Beta vulgaris only, a fungicide treatment may be applied by the laboratory before planting the seed for germination, when the seed lot is known not to have received such a treatment. Results are reported under �Germination� in the spaces provided.
For samples of other species, laboratory-applied fungicide treatments are not covered by the ISTA Rules. Germination test results for other species treated with laboratory-applied fungicide must be reported under �Other determinations� and followed by: �This method is not covered by the International Rules for Seed Testing�. In addition, a test without applying a laboratory fungicide treatment must also be conducted and the results reported under �Germination� in the spaces provided.
When a fungicide pretreatment is used, the name of method of treatment must be reported on the ISTA Certificate under �Other determinations�.
The duration of the test for individual species is indicated in Table. The duration of the treatment required to break dormancy (3) before or during the test is not taken as part of the germination test period.
If it seems advisable, when for example some seeds have just started to germinate, the prescribed test period may be extended by:
a) 7 days;
b) up to half the prescribed period;
c) up to 21 days for Lolium spp.;
d) up to 32 days for Festuca spp. (except F. arundinacea and F. pratensis);
e) up to 42 days for Poa spp. (except P. bulbosa);
f) up to 54 days for Poa bulbosa.
If, on the other hand, the maximum germination of the sample has been obtained before the end of the prescribed test period, a test may be terminated. At the request of the applicant the germination test may be terminated when the sample reaches predetermined germination percentage.
The time of the first count is approximate but must be sufficient to permit the seedlings to reach a stage of development which allows for accurate evaluation. The times indicated in Table refer to the highest temperatures. If a lower temperature is chosen, the first count may have to be postponed. For tests in sand organic growing media or soil lasting not more than 7-10 days, the first count may be omitted. Intermediate counts to remove seedlings which are sufficiently well developed are recommended in order to make counting easier and to prevent them from affecting the development of other seedlings. Number and date of intermediate counts may be left to the discretion of the analyst, but should be kept at a minimum to reduce the risk of damaging any seedlings which are not sufficiently developed. When samples are tested on paper, ungerminated seed and seedlings requiring additional time to reach the stage of development that allows for accurate evaluation, can be transferred to fresh substrate at intermediate counts. In doing so, care must be taken to ensure the integrity of replicates and to avoid any damage to the transferred seeds and seedlings.
Every seedling must be evaluated in accordance with the general principles laid down. For evaluation, the essential structures must be sufficiently developed to permit detection of any abnormality.
At the end of the germination test, the classification of ungerminated seeds must be determined as prescribed.
Seedlings which have reached a stage when all essential structures can be accurately assessed must be removed from the test at the first and any other intermediate counts. Badly decayed seedlings should be removed in order to reduce the risk of secondary infection, but doubtful seedlings with other defects must be left on the substrate until the final count, unless it is obvious that they will never develop into normal seedlings, e.g. broken seedlings and white seedlings.
When a unit produces more than one normal seedling, only one is counted for determining the germination percentage. On request the number of normal seedlings produced by 100 units, or the number of units which have produced one, two or more than normal seedlings may also be determined.
At the end of a germination test, hard seeds are counted and reported as such on the ISTA Certificate.
When 5 % or more of fresh seeds are believed to be present, their potential to germinate must be determined by dissection, tetrazolium or excised embryo. Those determined to have the potential to germinate are reported as fresh. Those determined not to have the potential to germinate are reported as dead.
After this determination, if there is any doubt as to whether the seed is fresh or dead, it must be classified as dead. If not already applied, measures described in 3 must be taken to break dormancy if 5 % or more of fresh ungerminated seeds are found.
Obviously dead (soft, mouldy) seeds are counted and reported as such on the ISTA Certificate. If it can be seen that a seed has produced any part of a seedling (e.g. the tip of the primary root) even though decayed at the time of assessment, it is counted as an abnormal seedling and not as a dead seed.
Upon request of the customer, the number of empty, embryoless or insect-damaged seeds may be determined and reported under �Other Determinations� on the ISTA Certificate.
To detect these other categories of seeds, the following methods may be used:
a) Before the germination test:
- X-ray test, which is conducted in the replicates used for the germination test;
- cutting test, which is performed on four separate replicates of 100 seeds, soaked for up to 24 h at room temperature. Each seed is cut along its longitudinal axis and the content examined and classified as full, empty, embryoless or insect-damaged;
b) After the germination test:
- cutting test or X-ray test of apparently fresh ungerminated seeds.
When a tetrazolium test is performed, the percentage of empty and insect-damaged seeds can also be determined during preparation and evaluation.