II. GERMINATION TEST:

Germination testing is considered as the most important quality test in evaluating the planting value of a seed lot. The ability of seeds to produce normal seedlings and plants later on is measured in terms of germination test. Testing of seeds under field conditions is normally unsatisfactory as the results cannot be reproduced with reliability. Laboratory methods then have been conceived wherein the external factors are controlled to give the most uniform, rapid and complete germination. Testing conditions in the laboratory have been standardized to enable the test results to be reproduced within limits as nearly as possible as those determined by random sample variation.

Objective

The ultimate objective of seed germination testing is to obtain information with respect to the planting value of the seed and to provide results which could be used to compare the value of different seed lots.

Definition

Germination of a seed lot in a laboratory is the emergence and development of the seedling to a stage where the aspect of its essential structures indicates whether or not it is able to develop further into a satisfactory plant under favourable conditions in soil (ISTA, 1985). These essential structures are a well-developed and intact root system, hypocotyl, plumule and one or two cotyledons according to the species Seedlings cannot be evaluated in a germination test until these essential structures are clearly identifiable and the reported percentage germination expresses the proportion of seeds which have produced normal seedlings within the period specified for each species.

General Principles

Germination tests shall be made with seeds from the pure seed fraction of a purity test. A minimum of four hundred seeds are required in four replicates of 100 seeds each or eight replicates of 50 seeds each or 16 replications of 25 seeds each depending on the size of seeds and size of containers of substrate.

The seeds shall receive no pretreatments excepting those recommended in the following Table.

TABLE-1

Crop	Media	Temperature	1^st count	Final count	Addl.directions
Paddy	BP, TP,S	20-30,25	5	14	Preheat(50° C) Soak in water or HNO3 24 hours
Ragi	TP, BP	20-30	4	8	0.2% KNO3(2 to 3 hours)
Maize	BP, S	20-30, 25	4	7	-
Cumbu	TP, BP	20-30	3	7	0.2% KNO3(2 to 3 hours)
Sorghum	TP, BP	20-30, 25	4	10	Prechill
Bengal gram	BP, S	20-30, 20	5	8	-
Blackgram	BP, S	20-30, 25	4	7	-
Cowpea	BP, S	20-30, 20	5	8	-
French bean	BP, S	20-30, 25,20	5	9	-
Greengram	BP, S	20-30, 25	5	8	-
Horsegram	BP	30	3	5	-
Peas	BP,s	20	5	8	-
Redgram	BP, S	30	4	6	-
Castor	BP, S	20-30	7	14	-
Groundnut	BP, S	20-30, 25	5	10	Remove shells, Pre heat 40° C
Gingelly	TP	20-30	3	6	-
Soya bean	BP, S	20-30,25	5	8	-
Sunflower	BP, S	20-30, 25	4	10	Ethrel 25ppm 48 hrs
Cotton	BP, S	20-30, 25	4	12	Hotwater(85°C-1 minute)
Sun hemp	BP, S	20-30	4	10	-
Cluster bean	BP	20-30	5	14	-
Oat	BP, S	20	5	10	Preheat30-35°c prechill
Dhaincha	TP,BP	20-30	5	7	Rub seed coat on paper
Sugar beet	TP,BP,S	20-30;15-25	4	14	Prewash multigerm 2hrs,monogerm 4hrs
Ashgourd	S	30-35	5	14	Light
Bittergourd	BP,S	20-30,30	4	14	-
Bottlegourd	BP,S	20-30	4	14	-
Cucumber	TP,BP,S	20-30,25	4	8	-
Pumpkin	BP,S	20-30,25	4	8	-
Ridgegourd	BP,S	30	4	14	-
Snakegourd	S	30-35	-	14	Dark,GA3 500 ppm 24hrs Remove seed coat
Watermelon	BP,S	20-30:25	5	14	-
Brinjal	TP,BP	20-30	7	14	-
Chilli	TP,BP	20-30	7	14	Kno3
Bhendi	BP,S	20-30	4	21	-
Tomato	TP,BP	20-30	5	14	Kno3
Onion	TP,BP	20-15	6	21	Prechill
Amaranthus	TP	20-30	-	8	Light
Coriander	TP,BP	20-30,20	7	21	-
Spinach	TP,BP	15-10	7	21	Prechill
Carrot	TP,BP	20-30,20	7	14	-
Radish	TP,BP	20-30,20	4	10	Prechill
Turnip	TP	22-30,20	5	7	Prechill,Kno3
Field bean	BP,S	20-30,25	4	10	-
Cabbage	TP	20-30,20	5	10	Prechill,Kno3
Knol-Khol	TP	20-30,20	5	10	Prechill,Kno3
Cauliflower	TP	20-30,20	5	10	Prechill,Kno3

NOTE: (1) Prechilling: The replicates for germination are placed in contact with the moist substratum and kept at low temperature(between 5° c and 10° c)for upto seven days for all agricultural and vegetable seeds.

(2) Potassium nitrate(Kno3): Instead of water 0.2%Kno3 Solution (prepared by dissolving 2g Kno3 in one litre of water) is used to saturate the germination substratum at the beginning of the test. Water is used for moistening thereafter.

(3) Gibberellic acid(GA3): Required concentration should be prepared. For preparing 1000ppm solution dissolve 1gmGA3 in 1000ml of water, for 500ppm dissolve 500mg in 1000ml of water and for 100ppm,100mg should be dissolved in1000ml of water. When concentration of GA3 is not mentioned ,any concentration ranging from 100 to 500ppm should be used. Seeds should be soaked in required concentration of GA3 for 17hrs at room temperature, dried on the laboratory table and put for germination.

The seeds arranged in replicates are tested under favorable moisture conditions and in accordance with the methods prescribed in the above Table. After the period indicated in the table the replicates are examined and counts are made.

General Requirements for Germination

Seeds require certain conditions for normal germination. The most important requirements are substrata, moisture, temperature and light.

Suitable substratum

The substrata serve as a moisture reservoir and provide a surface or medium for which the seeds can germinate and the seedlings grow. The commonly used substrates are paper, sand and soil.

Paper substrate

Most widely used paper substrates are filter paper, blotter or towel (kraft paper), these are easy to handle versatile and comparatively cheap.

Sand

It may be necessary to wash and sterilise the sand before use. For reuse of sand it must be washed, dried and resterilized. Sand which has been used for testing chemically treated samples, should preferably be discarded without being reused, if however, it is reused it should be ascertained that chemicals which may have accumulated in the sand do not cause phytoxic symptoms.

Soil

Soil should be of good quality, non-caking and free from any large particles. It must be reasonably free from weed seeds, bacteria, fungi, nematodes or toxic substances, which might interfere with the germination of seeds, the growth of seedlings or their evaluation. Soil should allow adequate aeration for germination when water is added with a pH of 6.0-7.5. Before use soil may require sterilisation and soil is not recommended for reuse.

Specification of Germination paper:

Germination paper should preferably possess a creaped surface. The paper should have an open, porous formation and he free from impurities or toxic substances that may affect seed germination. It should be free of fungi or bacteria which might interfere with the growth or evaluation of seedlings. It should hold sufficient moisture during the period of test and should possess sufficient strength to resist wear and tear during handling. The texture should be such that the roots of germinating seedlings will grow on and not into the paper.

The paper shall meet the following requirements.

Type of Paper	Basis Mass (g/m2)	Bursting Strength (kg/cm2)	Capillary rise (in mm). Min	PH	Ash, % by mass (Max.)
Filter paper	130-135	1.0	30	6.0 to 7.5	1.20
Towel paper	95-100	2.0	30	6.0 to 7.5	1.50

In this, test, comparison shall be made between germination papers of unknown quality and known acceptable quality. Pieces of paper should be cut to size and placed in petridishes or plastic boxes. Petridishes or plastic boxes should be lined with two thickness of such paper. The papers should be saturated with tap water and seeds of Brassica species or onion should be germinated. Evaluation may be done by comparing the development of the seedlings grown on unknown quality of paper and those grown on the known quality of paper. The evaluation of seedlings shall be made after 3 days in case of Brassica and after 6 days incase of onion. If paper of unknown quality contains toxic substances, the root tips will be shortened and sometimes discoloured, root hairs ‘bunched’ and sometimes plumules shortened.

Determination of Capillary Rise

Cut ten strips of paper each 10 mm wide, five in the machine direction of the paper and five in the cross-machine direction. Immense each strip in distilled water at 27 ± 2 °C to a depth of 20 mm at the end of the strip. After 2min measure the height to which the water has risen in the strip, to the nearest 1 mm. Calculate the average of the 5 strips in the cross machine direction. The lower of these two averages shall be taken as the result for the test.

SPECIFICATION FOR SAND

Sand shall be clean and free from clay like material dirt, crushed stones or pebbles. The particle size of the sand shall be such that whole of the material shall pass through 850 micron IS sieve (aperture 0.85 mm) but shall be retained on a 45 micron IS sieve (aperture 0.045 mm). It should not contain toxic materials to cause injury to seedlings. The pH should be within the range from 6.0 to 7.5. The specific conductance should be within the range from 0.01 x 0.02 ms/cm2.

Biological Test for Toxic Materials:

In this test, comparison shall be made between sand of unknown quality and sand of known pre tested quality. Sand should be placed in petri dishes or boxes to form a uniform layer 2 cm deep. It should be moistured to its 50% water holding capacity, Seed of onion or Brassica species should be placed and cover to a depth of 1 cm of moist sand. Evaluation may be done by comparing the development of the seedlings grown on the unknown quality and those grown on known quality as described for testing the germination paper.

Storing of Samples

The official samples after testing should be stored in controlled storage (3° C and 50% RH) for a minimum period of two years from the date of grant/extension of the certificate, unless required for longer period. Every care should be taken to protect the samples from insects and rodents.

Adequate Moisture of Water

High concentration of water at cellular level is necessary for the seed to start germination. Moisture is supplied to the seeds through the substratum. Generally, the moistened substrata is sufficient to rehydrate to 30-80 per cent. However, the moment the radicle emerges, additional moisture contributes better seedling growth. In the case of vegetable seeds, care is necessary in moistening the substrata. Too much water would allow fungal growth and decay of seeds.

The general specifications for water are: It should be free from organic or inorganic impurities. The pH value should be within the range of 6.0 to 7.5. If the usual water supply in the laboratory is not satisfactory, distilled, de-ionised water may be used. To ensure the quality of water being used, an analysis should be obtained from time to time.

Favorable temperature

Seeds of most of agricultural and horticultural crops germinate in the temperature range of 10° C –35° C Some seeds germinate better at constant temperature. Others require an alternating temperature.

Temperature control is also necessary to overcome dormancy wherever it occurs. Exposure of seeds to the temperature at 40°C or higher, alternation of temperature, low temperature applications are the easiest and safest method to overcome seed dormancy although methods to overcome dormancy by chemical treatments do exist.

Therefore, the temperatures prescribed in the above Table should be determined at the level of the seeds on the substrate.

Temperatures should be as uniform as possible throughout the germination apparatus and care should be taken that the temperature of tests does not exceed the level prescribed in the Table and should not be more than ± 1° C.

Where alternating temperatures are indicated, the lower temperature should usually be maintained for 16 hours and the higher for 8 hours. If alternation of temperatures cannot be controlled over week-ends or public holidays, the test should be kept at lower temperature

Light:

There are crops for which light is not required during germination test. However, presence of light is desirable to enable the evaluation of seedlings easier and with greater certainty. Other crops like lettuce and tobacco require light during germination on the test.

Seeds of most of the species in the Table will germinate either in light or in darkness. However, illumination of the substrate from artificial source or by day light is generally recommended for better seedling development to avoid etiolation and also to detect seedlings having chlorophyll deficiency.

Specific recommendations for light or darkness, respectively, are given in the additional directions column of Table.

Procedures

Working sample

Four hundred seeds are counted at random from the well-mixed pure seed. Replicates of 100 seeds are normally used, spaced sufficiently far apart on the seed bed to minimise the effect of adjacent seeds on seedling development. To ensure adequate spacing, split replicates of 50 or even 25 seeds may be necessary, particularly where there is seed-borne disease.

Testing four hundred seeds is recommended on seed law enforcement, seed certification and service samples.

Methods using paper:

Paper substrates are used for the following methods:

a) TP (Top of paper): As the name indicates, the seeds are placed directly on one or more layers of moist filter or blotter papers in petridishes. These petridishes are tightly covered with lid and placed inside the germination cabinet. The relative humidity in the cabinet must then be maintained to 95-99% to prevent drying out during test period.

b) BP(Between paper):The seeds are germinated between two layers of paper. This may be achieved by loosely covering the seeds with an additional layer of paper or by placing the seeds in rolled towels. The rolled towels are to be placed inside the germinator in an upright position.

Methods using sand

The seeds are planted on a leveled layer of moist sand and covered with 10-20 mm of uncompressed sand depending on the size of the seed. To ensure good aeration it is recommended that the bottom layer of sand be loosened by raking before sowing.

Sand may be used instead of paper, even if not prescribed in Table when the evaluation of a diseased sample proved impracticable because of the contamination of the paper substrate.

Moisture and aeration

The substrate must all times contain sufficient moisture to meet the requirements for germination. However, moisture content must not be excessive, or aeration maybe limited. The initial quantity of water to be added will depend on the nature and dimensions of the substrate and also on the size and species of the seed to be tested.

The amount of water to be added to the sand can be calculated as follows

Ml of water to be 118.3 ml of sand

Added to each 100 = ------------------------------------- x (20.2 – 8.0)

gram of sand Wt. Of 118.3 ml of sand in gm

The amount of water provided by this formula is satisfactory for seeds of the size of mustard, for larger seeds slightly more and for smaller seeds slightly less water should be added.

Subsequent watering should be avoided wherever possible as it is likely to increase the variability between replicates and between tests. Therefore, precautions should be taken to ensure that the substrate may not dry out and that sufficient water is supplied continuously during the test period.

Pretreatments for Germination

For various reasons (e.g physiological dormancy, hard seededness, inhibitory substances) a considerable number of hard or fresh seeds may remain at the end of the germination test. In order to prevent these non-germination and to have complete germination various kinds of pretreatments are recommended and given in Table. These pretreatments include dry storage, prechilling (treat the moist seeds at a temperature of 5° – 10 C for about seven days) preheating (at 30-35 C) light (750-1250 lux from cool white lamps for 8 hrs per day)potassium nitrate (0.2% KNO3) gibberellic acid (GA3 0.05-1%) application etc.

For hard seeds, puncturing the seed with a needle away from embryo, mechanical scarification and acid scarification are recommended. Similarly for removing inhibitory substances, pre wash the seeds by running water at a temperature of 25 C and bring back these pre washed seeds to its original moisture content.

Duration of the Test

Duration of the test for individual species is indicated in table. The duration of the treatment required to break dormancy before or during the test is not taken as part the germination test period.

The time of first count is approximate, but must be sufficient to permit the seedlings to reach a stage of development which allows for accurate evaluation. The time indicated in table refer to the highest temperatures. If lower temperature is choosen, the first count may have to be postponed. The tests lasting 7-10 days, intermediate counts to remove seedlings which are sufficiently well developed are recommended in order to make counting easier to prevent them from affecting the development of other seedlings. The first count may be omitted, if the first test is conducted in sand. If the maximum germination of the sample has been obtained before the end of the prescribed test period, a test may be terminated.

The seed testing laboratory on request of producer may release the result of seed germination on the basis of first count if the sample in question meets the minimum limits of germination for certification / labeling.

Seedling Evaluation

Seedlings which have reached a stage when all essential structures can be accurately assessed, shall be removed from the test at the first or any other intermediate counts. Badly decayed seedlings should be removed in order to reduce the risk of secondary infection, but abnormal seedlings with other defects should be left on the substrate until the final count.

Categories of seedlings

Normal Seedlings

Normal seedling is one which shows the capacity for continued development into mature plant when grown in good quality soil and under favourable conditions of water supply, temperature and light.

According to the International Seed Testing Association (1985) seedlings to be classified as normal seedling, must conform with one of the following categories:

a)Intact seedlings: Seedlings with all their essential structures, well developed complete in all proportion and healthy.

b) Seedlings with slight defects: Seedlings showing certain slight defects of their essential structures provided they show an otherwise satisfactory and balanced development comparable to that of intact seedlings of the same test.

c) Seedlings with secondary infections: Seedlings which are seriously infected 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.

Abnormal seedlings:

An abnormal seedling is one which does not have the capacity to develop into a normal plant when grown in the soil under favourable conditions because one of more of the essential structures is irreparably defective.

Three major classes of abnormal seedlings are:

a) Damaged Seedlings: Seedlings with any of the essential structures missing or so badly damaged that balanced development does not occur. The damage to the embryo in the seed usually results from external cause i.e. mechanical handing.

b) Deformed or unbalanced seedlings: Seedlings with weak and unbalanced development which may be caused by internal disturbances of physiological biochemical character. Such internal disturbances, however, are often due to the earlier external disturbances such as unfavourable growing conditions of the parent plants, poor ripening conditions for the seed, premature harvesting, effect of herbicides or pesticides and inappropriate storage conditions or ageing of the seed.

c) Decayed seedlings: Seedlings with any of their essential structures so diseased or decayed as a result of primary infection that normal development is prevented. These may result from the external or internal seed borne diseases.

Multigerm seed units

Seeds which are capable of producing more than one seedling. Several types of seed units can produce more than one seedling e.g. unseparated schizorcarps of umbeliferae, clusters of Beta vulgaris, fruits of tectona grandis, polyembryonic seeds. In such cases only one normal seedling is counted for determining the germination percentage.

Ungerminated seed

Seeds which have not germinated by the end of test period when tested under the conditions prescribed in Table are classified as follows.

a) Hard seeds: Seeds which do not absorb moisture till the end of the test period and remain hard.

b) Fresh seeds: Seeds which are neither hard nor have germinated but remain clean and firm and apparently viable at the end of the test period. The viability of the fresh seeds may be determined by tetrazolium test.

c) Dead seeds: Seeds at the end of the test period are neither hard nor fresh nor have produced any part of a seedling. Often dead seed collapses and a milky paste comes out when pressed at the end of the test.

Retesting

If the results of a test are considered unsatisfactory it shall not be reported and a second test shall be made by the same method or by alternative method under the Replicates performance is out of tolerance. Results being inaccurate due to wrong evaluating of seedlings or counting or errors in test conditions. Dormancy persistance or phyto toxicity or spread of fungi or bacteria.

Reporting Results

The result of the germination test is calculated as the averages of 4 x 100 seed replicates. It is expressed as percentage by number of normal seedlings. The percentage is calculated to the nearest whole number.

The percentage of abnormal seedlings, hard, fresh and dead seeds is calculated in the same way. These should be entered on the Analysis Certificate under appropriate space. If the result is nil for any of these categories it shall be reported as ‘o’ instead of leaving the appropriate column blank.

Use of Tolerances:

For the use of tolerances, appropriate table given in hand book of seed testing should be used.