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  

           insects.

 

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  

                              plastic containers.

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

                               test crop.

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.

 

Capillary Rise:

 

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.

 

 

b) Temperature

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.

 

 

c) Light          

 

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.

 

Paper Substrata:

          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.

 

Sand Substrata(s):

 

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.

8.Germination Environment

 

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).

 

Inhibitory        Substances:

                           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 25C before the germination test is made. After washing, the seeds should be dried back at a maximum temperature of  25C  (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 between5C and 10C for an initial period up to,7 days.Tree seeds are kept it a temperaturebetween3C and,5C, 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 40C 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.

 

Chemical         Treatments:

  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) Roots:!

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.

 

 

b)Hypocotylsand epicotyl:

 

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.

 

c) Coleoptile(Gramineae):

 

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.

 

d)Cotyledons(Dicotyledonous species):

a.None

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.

 

e)Decay:

 

 

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.

 

13.             Retesting

 

      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