Germination Testing: Principles and Procedure
Thepurpose of laboratory testing of seed germination is to assess seed quality orviability and to predict performance of the seed and seedling in the field. ANOTIFIED laboratory under SEEDS ACT or qualified laboratory of 1STA for testingseeds must test seed processed for sale. The ultimate aim of testing thegermination in seed testing laboratory is to obtain information about theplanting value of the seed sample and by inference the quality of the seed lot.In addition, the laboratory germination results are also required for comparingthe performance potential or superiority of the different seed lots. In general, the farmers, seeds men and publicagencies use the germination results for the following purposes:
1. Sowingpurposes, with a view to decide the seed rate to achieve desired field establishment.
2.Labeling purposes.
3. Seedcertification purposes.
4. SeedAct and Law Enforcement purposes.
In seedtesting germination has been defined as "the emergence and developmentfrom the seed embryo of those essential structures which, for the kind of seed tested indicate its abilityto develop into a normal plant under favorable, conditions in soil". Theseedlings devoid of an essential structure; showing weak or unbalanceddevelopment; decay or damage affecting the normal development of seedling arenot considered in calculating the germination percentage. Factors that canaffect the performance of seed in germination tests include; diseased seed, oldseed, mechanically damaged seed, seed stored under high moisture, and excessiveheating of seed during storage or drying.
In mostcases a seed treatment will improve germination of seed only if the poorquality is due to seed-borne disease. Several different kinds of testing areavailable depending on the type of seed to be tested, the conditions of thetest, and the potential uses of the seed. The most common tests are the coldgermination test, accelerated aging test, the tetrazoliumtest and warm germination test. Each test is designed to evaluate variousqualities of the seed.
<![if !supportLists]>1.<![endif]>Germination Testing
Themost common test is a warm germination test because it is required by seed lawsto appear on the label. The percentage of germinating seed in a Warmgermination test must be printed on the label of the seed if it is to be soldas seed. The Warm germination test reflects the field emergence potential of aseed lot under ideal planting conditions. Usually 400 seed from each seed lotare placed under moist conditions on blotters, rolled towels, or sand andmaintained 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, ordiseased, and dead or hard seeds. The percentage germination is calculated fromthe number of normal seedlings from the total number of seeds evaluated. Themethod of testing germination is discussed below.
The firstand foremost step is to draw a true representative sample from the seed lot. Toobtain a random sample for testing it is always best to take samples fromdifferent parts of the bag or container. If the seed to be tested is from aseed lot that contains more than one bag, samples must be taken from severalbags. A good rule of thumb for determining how many bags to sample is to takesamples 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 arethe taken to perform the actual test.
2. Essential equipments andsupplies for germination test
The following pieces of equipments and supplies areessential to carry forward the
Germinationtests in the seed testing laboratories.
a) Seed Germinator
The seed germinators are the essential requirement forgermination 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 ingerminator.
The cabinet seed germinators are essential under thesituations, where various kinds of seeds that require different sets ofconditions, are being handled in the laboratory. The number of the pieces ofthe germinators required by the laboratory will depend on the number of seedsamples and the species being analyzed by the laboratory.
The seed testing laboratories that handle large number ofseed samples and require maintaining only fewer (2-3) sets of temperatureconditions, the walk-in-germinators are preferred. Such germinators are more useful for conductingthe 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 toaid germination testing by minimizing the time spent on planning the seeds aswell as to provide proper spacing of the seed on germination substrata.Counting boards are suitable for medium and bold sized seeds, while vacuumcounter 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 waterbath, incubator, forceps, spatula, germination, boxes, plastic plates, roll-towel stands and plastic or surgical trays, etc. A large ovenwith temp. Range 100 -200 C is also required for sterilizing the sand.
d) Miscellaneous Supplies,Glassware and Chemicals
Germination paper (Creppe Kraftpaper or towel paper, sunlit filter paper and blotters) and sand are the basicsupplies required for germination tests. In addition, the laboratory may alsorequire some glassware, such as Petridishes, beakers,funnel, measuring cylinders, muslin cloth,rubberbands and tubes etc. andcertain chemicals like Potassium nitrate, Thiourea, Gibrellic acid, and Tetrazoliumchloride for specific purposes. Voltage stabilizers are required for the supplyof the constant electric current. The voltage stabilizers are essential forcostly germinators air-conditioners and refrigerators.Under the situations of erratic power supplies and breakdowns, electricitygenerators are also required.
3. Care ofequipments:
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 ismaintained 90--98%
4. The phytosanitary conditionsof the germinators and germination trolleys are adequate
5. Thegerminators are disinfected periodically by flushing with hot water; solutionof
Potassium permanganate or chlorine water
6. Thetemperature and the R.H. of the walk-in-germinators are recorded daily and
displayed on achart and .
7. Thefloor, ceiling and walls of the walk-in-germinator are devoid of cracks,crevices;
Evenly plastered and duly painted to avoidcontamination by fungus, bacteria or
insects.
4. Handling ofsubstrata .
Theaccuracy and reproducibility of the germinator result are very much dependenton the quality of the substrata (paper and sand) used for germination testing.The germination substrata must meet the following basic requirements:
1. Itshould be non-toxic to the germinating seedlings. .
2. Itshould be free from mould sand other microorganisms. .
3. Itshould provide adequate, aeration and, moisture to the germinating seeds.
4. Itshould be easy to handle and use. .
5. Itshould make good contrast for judging the seedlings .
6. Itshould be less expensive..
a) Paper substrata
The papersubstrata 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 mostcommonly used for medium sized and bold seeds. The paper substrata are notreusable.
b) Sand substrata
The sandsubstrata have advantage of being relatively less expensive and reusable. Theresults in sand media are more accurate and reproducible in comparison with'roll towel' tests especially in case of seed lots that are aged or heavilytreated with chemicals. The sand should be reasonably uniform and free fromvery small and large particles. It should not contain toxic substances and itspH 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 (uppersieve) and 0.05mm diameter (bottom sieve). The sand retained on the bottomsieve should only be used.
5. Testing of Substrata
Phytotoxicity: Thesubstrata should be tested for its phytotoxicity,capillary rise, moisture holding capacity and bursting strength, etc., beforeaccepting the supplies in the laboratory. Periodic checks of the quality of thesubstrata should also be made in the laboratory.
By germinating the seeds of Brassica,Onion, Chillies or Berseemand studying the phytotoxic symptoms on thegerminating seedlings can check the phytotoxicity ofthe paper or sand substrata.
I.. Thepaper should becutinto circlesand rectangles or squares ofthe
desiredsize according to the size and shape ofthe containers.
2. Place2-4 circles/rectangles of thepaperto betestedin thePetri dishesor
plasticcontainers.
3. Moisten the paper with tap water usingonly 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 moistpaper.
5. Cover the dishes with lids.
6. Conduct a control test as outlinedabove (step 1-4) using paper of accepted
qualitysuch as 'Waterman' or 'Sunlit' brand filter paper.
7. Transfer the test to the prescribedtemperature conditions of the species used at
testcrop.
8. Evaluate the test 1-2 days before thedate of the first count of the crop specified
9. Check the phytotoxicsymptoms on the seedlings.
10. Compare the seedlings with those grown onthe non-toxic paper (controltest).
The phytotoxic symptoms include shortened roots; discoloredroot tips; root raised from the paper; inhibition of root hairs development and root hairsbunched. The symptoms are more pronounced at an early stage of rootgrowth. The phytotoxicsymptoms are also evident in the plumular areas inthe form of thickened or flattened plumules or, oleoptiles.
The phytotoxicity of the sand substrata can also be measured bythe procedure outlined
asabove. However, care need to be exercised that the sand substrata should bemoistened with the measured quantity of the water and the seeds are planted onthe ton of sand (TS)
The pH ofthe substrata can be measured with the help of pH paper or pH meter as follow:
<![if !supportLists]>1.<![endif]>Soakthe paper or sand in water for 16-18 hrs.
<![if !supportLists]>2.<![endif]>Decantthe water.
<![if !supportLists]>3.<![endif]>Measurethe pH with litmus paper or pH meter.
If the sand substrata are found to beacidic or alkaline, wash it thoroughly with the
water andsterilize before use.
Capillary Rise:
1. Cut four strips of germination paper10mm wide; two in machine direction and
the othertwo in cross machine direction.
2. Take distilled water in small glassbeakers.
3. Immerse one end of each strip in thewater to depth of 20 mm.
4. Wait for 2 minutes and then measure theheight to which water has risen in the
strip tothe nearest mm.
5. Commute theaverage forthe twostripscut in machine directionor cross machine directionseparately.
6. The lower value of the two averagesshould be considered as capillary rise.
Bursting strength: The bursting strength of the paperis measured with equipment however, it can be checkedas follows:
1. Hold the two ends ofthe germination paperand exert thepressureby
stretchingthe paper with midforce.
2. Soak the paper in water for 1-2 hours. .
3. The paper of desired bursting strengthwould not tear off easily. .
6. Test conditions
a) Moisture and aeration:
The moisture requirements of the seed will vary accordingto its kind. Large seeded species require more water than the small seededspecies. It is essential that the substratum must be kept moist through out thegermination 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 therotting of the seedlings or development of watery seedlings. Exceptthe situations where a high moisture level is recommended (e.g. Paddy andjute), the sub stratum should not be so wet that a film of water forms aroundthe seeds. In situations where low level of moisture is recommended(e.g. Cucurbitaceous seeds) , the moist substratum shouldbe pressed against the
Dryblotters or towelpaper, to remove excess moisture. The water used for moistening
thesubstratum must be free from organic and inorganic impurities.Normally thetap water is used. However, it is essential to measure the pH of water before its use. The pH of the water should beinthe rangeof 6.5- 7.5.Underthe situationswhere pH ofthewater is notsatisfactory, distilled water or deionized water may be used. Under such situation care needto be exercised to aerate thetests frequently toprovide oxygen supplytothe germinating seedlings because oxygenlevel in distilled water is very low.
Theinitial quantity ofwater to be added to thesubstratum will also depend on itsnature and dimensions. Subsequent watering, if, any may beleft tothediscretion of theanalyst but it shouldbeavoided as faras possible because itmay causethe variation in germination results. In order to reducethe need for additional watering during the germination period, the relative humidity oftheair surrounding the seedsshould be kept at 90-95 % to prevent loss of water by evaporation.
Specialmeasures for aerationare not usually necessary in case of topof paper (TP) tests. However, in case of 'Rolltowel' tests (BP) care should be taken that the rolls should be loose enough to allowthe presence ofsufficient air around the seeds. In case of sand media, thesand should not be compressed while covering the seeds.
b) Temperature
Thetemperature is oneof themost important and critical factorsfor the laboratory
germination tests. The temperature requirement for germination isspecificaccording to the kindof crop or species. This can varywithin the species and with the age ofseeds. At verylow orhigh temperatures, thegermination is prevented toa larger extent. The temperatureshould be uniformthroughout the germinator and the germinationperiod. The variation in temperature inside the germinator should not be more than 1DC. The prescribed temperature for germinationof agricultural, vegetable orhorticultural seeds, provided intheRules for Seed Testing can bebroadlyis classifiedinto two groups, viz.constant temperatures and alternatetemperatures.
Constant temperature: Wherever, the constant temperatures are prescribed or recommended forthe germination tests, the tests must be held at the specific temperature during the entire germinationperiod.
Alternate temperature: Wherever, the alternatingtemperatures prescribed, the lower
Temperatureshould be maintained for 16hours and the higher for 8 hours; a gradual changeover lasting3hours is usually satisfactory for non-dormant seeds. However, a sharpchangeover lasting1 hour or less, or transfer of test to another germinator atlower temperature may be necessary for seeds, which are likely to be dormant.
c) Light
Seeds ofmost of the species can germinate. In light or darkness it is always better to illuminate the tests for the propergrowth of the seedlings. Under the situations where light is essential forgerminations, tests should be exposed to the natural or "artificial sourceof light. However "are must be made to ensure that an even intensity isobtained over the entire substrate and that any heating from the source doesnot affect the prescribed temperature. .
Seeds thatrequire light for germination must be illuminated with cool fluorescent light
For at least 8hours in every 24 hours cycle. Under the situation where testingof the seed is required to be undertaken alternating "temperaturestogether with light, the tests should be illuminated during high temperatureperiod. .
7. Laboratory procedures
The working sample or germination test consists of 400pure seeds and randomly drawn eithermanually or with the help of counting devices. The seed for germination testmust be drawn as follows in accordance with the following two situations:
a) Whenboth purity and germination tests are required. .
1. Seeds for germination tests must be takenform the pure seed fraction after Conducting thephysical purity analysis.
2. Thecounting of the seed must be made without discrimination as to the size andappearance.
b) Onlygermination test is required.
1. If, the percentage of pure seed is estimated ordetermined to be above 98 percent, the pure seed for germination test shall be taken indiscriminately from arepresentative 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 intotwo components namely
(a) Thepure seed and
(b) Seedsof other species and inert matter.
For this purpose, at least one-fourth of the quantityrequired for regular purity analysis must be used after proper mixing anddividing the submitted sample.
Number of Replications:
Four replication of 100 seeds, A minimum of 3 replicationof 100 seeds may be used under unavoidable situations or Eight or sixreplications of 50 seeds or Sixteen/twelve replication of 25 seeds according tothe kind of and size of containers.
Paper Substrata:
<![if !supportLists]>·<![endif]>Check the quality of germination paper beforeaccepting the supplies.
<![if !supportLists]>·<![endif]>Measurethe pH, capillary rise, bursting strength and phytotoxicity of the germination paper.
<![if !supportLists]>·<![endif]>Storethe germination paper under hygienic conditions and protect it from dust andMicro flora.
<![if !supportLists]>·<![endif]>Veryold stock should not be used as they often get contaminated. Such paper usuallyshows phytotoxic symptoms.
Between Paper (BP) Media (Roll TowelTest):
1. Soakthe towel paper in water.
2. Removethe water.
3. Washthe paper with running water.
4. Removeextra moisture by pressing the soaked paper by hand and holding it in plastic/ Surgical trays placed on the table top in slanting position.
5. Placetwo layers of wet paper toweling as substratum.
6. CheckTest number provided on the Analysis Card sample and label tally each other.
7. Recordthe test number, crop and date of putting on the wax paper or tag.
8. Arrangeseeds spaced properly.
9. Placeone layer of wet towel paper over the seed.
10. Turnup two inches of the bottom edge.
11. Rollfirmly from left to right and secure with rubber band in the center.
12. Placethe prepared roll towel in roll towel stand or baskets.
13. Transfer the basket or roll towel stand in thegerminator 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 unknownquality should not be used for Top of paper tests.
3. The paper should be cut in the form of circles/squaresor 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 /germinationbox in slanting position in order to drain out the Extra moisture.
7. Record the test number and date of putting on the lidof the container on die paper Slip.
8. Space the counted seeds on the moist blotter/filterpaper.
9. Cover the lid
10. Transfer the test in the germinator maintained at thedesired temperature.
Sand Substrata(s):
1. Properly graded and sterilized sand free fromimpurities and toxic chemicals should be Used.
2. Sand should not be stored in the stores where fertilizersand chemicals are stored.
3. Grade,thesand 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,thesand should be dried and sterilized.
6. If required,thesand may be washed before sterilization.
7. If the sand found to be heavily contaminated or changedin colour after repeated use it Shouldbe 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 quantityof water to moistenthesand.
12. Themoisture level of thesandwill vary according to the kind ofseed.
13. Place moist sand in plastic germination boxes.The depth of sandbed 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 underprescribed controlled Temparature conditions.
8.Germination Environment
After placing the seeds on the prescribed substrata, thetest should be transferred to the controlled temperature condition maintainedin the cabinetor walk-in-germinator for prescribed period,which varies according to the species(1STA Seed Testing Rules). In the Rulesfor Seed Testing, two kinds of temperature conditions are provided. A singlenumerical indicate the constant temperature and numerical separated by a dash(-) indicate an alternating temperature. If temperaturescan not be conveniently altered over weekends or holidays, the tests mustbe kept at the lower temperature. The dailyalternation oftemperatureeither brought out manuallyby transferring the test from onegerminator to another or by changing the temperature of the chamber(Automatic Seed Germinator).
9. Methods to improve germination
Hard seeds: For manyspecies where hard seeds occur, some special treatment is essential. This treatmentmay be applied prior to the commencement of the germination test or, if it is, suspected that the treatment mayadversely affect non-hard seeds, it should be carried out on the hard seeds remaining after the prescribed test period. The treatmentsare as below:
Soaking: Seeds with hard seed coats may germinate more readily after soaking forup to 24-48 hours in water or for Acacia spp. after plunging seeds in about three times their volume of near boilingwater until it cools. The germination test is commencedimmediately after soaking.
Mechanical scarification: Careful-piercing,chipping, filing or sand papering oftheseed coat may besufficient to break the dormancycondition. Care must be taken to scarifythe seed coat at a suitable part in order to avoid damaging the embryo. The best site for mechanical scarificationis that part of the seed coat immediately above the tips of the cotyledons.
Acid scarification:Treatingwithin concentrated Sulphuricacid (H2SO4)is effective with some species (e.g. Macroptilium sp., Brachiaria sp., Sesbania sp.). The seeds are moistened with in the acid until theseed coat becomes pitted. Digestion maybe rapid or take more thanone hour, but the seeds should be examined every few minutes. After digestion,seeds must be thoroughly washed in running water before the germination test iscommenced. In the case of Oryza sativa scarification may be performed bysoaking the seed inonenormal nitric acid (HNO3) for 24 hours (after preheating at50 °C).
Inhibitory Substances:
Naturally occurringsubstances in the pericarp or seed coat, which act as inhibitors of germination may be removed by washing theseeds in running water at a temperatureof 25°C before the germination test is made. After washing, the seeds should bedried back at a maximum temperature of 25°C(e.g. Beta vulgaris).Germination of certainspecies is promoted by removing outer structures such as involucreof 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 thenthe seed lot is known not to have received such a treatment.Whena fungicide pretreatment is used the name of the chemical the percentage of active ingredients and the method oftreatment shall be reported on the certificate.
Prechilling : In some seeds having physiological dormancy pre chilling isrequired for inducinggermination. Replicates for germination are placed in contact with themoist .substratum and kept at alow temperature for an initial period before they are removed to the temperatureas shown in (ISTA Seed Testing Rules-Table 2). Agricultural and vegetable seedsare kept at a temperature between5°C and 10°C for an initial period up to,7 days.Tree seeds are kept it a temperaturebetween3°Cand,5°C, for a period, varying with the species,from7 days to 12 months.In some,casesit may be necessary to extend the prechilling period or to rechill.The prechilling period is not included in thegermination test period but both the duration and the temperature should bereported on the analysis card.
Pre-drying: The replicates for germination shouldbe heated at a temperature not exceeding 40°C with free air circulation for aperiod of up to 7 days before they are placed under the prescribed germinationconditions. In somecasesit maybenecessary 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 (1STASeed Testing Rules -Table 2). Thesubstratum is saturated at the beginning of the test but water is used formoistening it there after .The use of this treatment should be noted on theanalysis certificate.
The procedure for preparing solutions and soaking blottersis 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 solutionfor soaking blotters: Add 90ml water to 10 mlof stock solution.
iii). Procedure for soaking blotters: a. Take the blotters representing the sampleand place into, the prepared solution,(0.2%)-one atatime. b. Turn blotters overinone movement,but ensuring that they are stillfree movingin the solution. c. Remove one at a time, in order of placing in solutionand place on tray.
Gibberellic acid (GA3): Moisten thegerminationsubstratum with 50 ppm solution ofGA,which can be prepared by dissolving 500 mgof GA3 in 1000 ml of water. Place the seed for germinationunder prescribed temperature conditions.
10. Duration of testing
Theduration of the test is determined by the time prescribed for the, final count (1STA Seed Testing Rules,Table2) but the chilling, periods before or duringthetest, whichis required to break dormancy, is not included in the test period. If at the end of the prescribed test period someseeds havejust started togerminate, the testmaybe extendedfor an additional period upto 7 days. A test may be terminatedprior to the prescribed timewhen the analyst is satisfied that the maximum germination of the sample has been obtained.The time for the, firstcount is approximate and a deviation of 1-3 days is permitted. The First count may bedelayed topermit thedevelopment ofroot hairsinorder to becertainthat root development is normal, or may be omitted. Intermediate counts may beat the discretion of the analyst to removeseedlings,which havereached asufficient stateof development for evaluation, to preventthem becoming entangled. But thenumberof intermediatecounts should be kepttoa mini- mum toreduce therisk ofdamagingany seedlings that arenot sufficiently developed.
Seedlingsmay have to be removed and counted at more frequent intervals during the prescribed period of the test whena sample contains is infected with'fungior bacteria. Seeds thatare obviously dead and decayed, and may, therefore, be a source of contamination for healthy seedlings, should be removed ateach count and the number recorded.
12. Evaluation of germination test
Thegermination tests need to be evaluated on the expiryofthe germination period, which varies according tothe kind of seed. However, the seed analyst may terminate the germination test onor before the final count day or extend the test beyond the period de-pending onthe situation
First and second counts are usually takenin 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 subsequentcounts only normal and dead seeds (which are source of infection) removed and recorded.
In evaluating thegermination test the seedling and seeds are categorized into
Normal seedlings,abnormalseedlings,dead seeds,fresh ungerminated and hard seeds.Itmay also be necessary to remove the seed coat and separate the cotyledons Inorder to examine the plumule in species whereessential structures are still enclosed at the end of the test.
a)NormalSeedlings: It is necessity to separatenormal seedling, which are counted in the percentage germination, fromany abnormal seedlings .To achieve uniformityin evaluating normal seedlings, they must conform to one of the following definitions:
a. Seedlings which show thecapacity for continued development into normal, plant. whengrown in good quality soil and under favourable conditionsof water supply, temperature and light.
b. Seedlings thatpossess all the following essential structures when tested on artificialsubstrata:
<![if !supportLists]>i)<![endif]>Awell-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.
<![if !supportLists]>ii)<![endif]>A well-developed and intact hypocotyl without damage to the conducting tissues.
<![if !supportLists]>iii)<![endif]>An intact plumulewith a well developed green leaf, within or emerging through the coleoptile, or an intact epicotylwith a normal plumular bud.
<![if !supportLists]>iv)<![endif]>One cotyledon for seedlings of monocotyledons andtwo cotyledons and seedlings of dicotyledons.
c. Seedling withthe following slight defects provided they show vigorous and
balanced developmentof the other essential structures:
i. Seedlingsof Pisum, Vicia, Phaseolus,Lupinus, Vigna, Glycine, rachis,Gossypium, Zea andall species of Cucurbitaceae, with a damaged primaryrootbut with several secondary roots of sufficientlength and vigour to support the seedlings in soil.
ii. Seedlings with superficialdamage or decay to the hypocotyl, epicotyl or cotyledons, which is limited in area and does not affect the conductingtissues.
iii. Seedlings of dicotyledonswith only one cotyledon.
d. Seedlings of tree specieshaving epigeal germination when the radicle is four times the length of the seed providedall structures that have developed appear normal.
e. Seedlings which areseriously decayed byfungi or bacteria,but only whenitis clearly evident that theparent seed is not source ofinfectionand it can bedetermined that all the essential structures were present.
b) Abnormal Seedlings: Abnormalseedlings are those, which donot show the capacity for continued developmentinto normal plantswhengrown ingoodquality soilandunder favorable conditions ofwater supply, temperature and light.
Seedlings with the following defectsshall be classed as abnormal :
i. Damaged seedlings; seedlings with no cotyledons; seedlings withconstrictions, splits, cracks or lesions which affect the conducting tissues ofthe
epicotyl, hypocotyl or root;seedlings without a primary root ofthose species where a primary root is anessentialstructure, exceptfor Pisum, Vicia, Lupinus,Vigna, Glycine, Arachis, Gossypium, Zea and all speciesof Cucurbitaceae, whenseveralvigorous secondary roots havedeveloped to, support the seedlings, in soil.
ii. Deformedseedlings:Seedlings with weak or unbalanced development of the essential structures such as spirallytwisted or stunted plumules,hypocotyls or epicotyls; swollenshoots and stuntedroots; split plumules orcoleoptiles without agreen leaf; watery and glassyseedlings, or without further development after emergence ofthe cotyledons.
iii)Decayedseedlings: seedlings with any of the essential structures so diseased or decayedthat normal development is prevented,except when thereisclear evidence to show thatthe cause of injection is not the seed itself.
iv.Seedlings showing cotyledondevelopment from the micropyle, or radicle development from a part of the seed otherthan the micropyle.
Special categories ofabnormal seedlings The3main categories of abnormality,damage, deformity and decay,outlined in the previous section, can befurther 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 andstunted, 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 brownin colour.
b)Hypocotylsand epicotyl:
a. Hypocotylshort and thick, or twisted, or curled over. Or watery.
b. Epicotyl or stem withconstriction,grainy lesion, or open splitlikelyto interfere with the conducting , tissues.
c. Hypocotyl with construction ,grainy lesions or open split likely to lesions or interface with theconducting tissues .
d. Epicotylor stem short and thick, or twisted around the main axis,
e.No terminal bud.
f. Two shoots which are short andweak,or spindly.
g. No primary leaves, with or without terminal or auxiliary buds,or withmore thanhalf Thetotal area ofthe primary leaves missing or notcapable of functioning normally, or With one primary leaf and evidence ofdamage tothe shoot apex.
c) Coleoptile(Gramineae):
a. No greenleaves
b. Short leavesextendingless than halfthelength of coleoptile.
c. Leaves shattered orsplitlongitudinallyand/orcoleoptile witha split easily visible to The naked eyeorabnormal coleoptiledevelopment due to damage
d. Plumulespindly,or pale, or watery
e. Plumule short and thick, usuallywith short orstunted seminalroots.
d)Cotyledons(Dicotyledonousspecies):
a.None
b. One,with evidence of damage to the shoot apex.
c. Poorly developed leaf-likecotyledon in Allium, without a definite bend, or "knee".
d. Enlarged, with short hypocotyl.
e. Physiological necrosi
f .Grey in colour
g.Swallowand blackned.
h. More that halfthe total area broken off, or covered with spots or darkened areas, or with opensplits if development as a whole is outof proportion compared withthat of a normal seedlingsgerminated at the same time.
e)Decay:
a. Decayed cotyledons.
b. Decayed hypocotyl.
c. Decayed epicotylor stem.
d. Decayed plumule,or decay at pointof attachment between seedlings and endosperm or Discoloration of the coleoptile which haspenetrated to theleaves.
e.Decayed primary root(exceptsecondary infection by Phoma betae) orseminal roots in the gramineae.
f.Decay or discoloration at point of attachment between cotyledons and seed lings axis,oradjacent 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 coleoptileor a plumule, which is weak and Spirally twisted.
c. Entirely white seedling in the Gramineacand Liliaceae.
d. completely ahatteredseedling.
12. Caluculation and expression of result
results are expressed as percentage by number.Germination rateis the average number of seeds that germinate over the five-day and 10-day time period.
Germination (%) = Number seedsgerminated
----------------------------------------- x100
Number seedson tray
When four 100-seed replicatesof a test are within the maximum tolerated range the average represents the percentage germination to be reportedon the Analysis Certificate.Theaverage percentage is calculated tothe nearest whole number.The total % of all the category of seeds (normal,abnormal,deadhard,fresh un germinated) should be 100.
<![if !supportLists]>13.<![endif]>Retesting
The result of a test shall be considered unsatisfactoryand shall not be reported and a secondtest shall be made by the same oran alternative method, under the following circ*mstances:
a) When dormancy is suspected (fresh ungerminated seeds).
b) When the result may notbe reliable because of phytotoxicity or spreadof fungi or Bacteria.
c) When there isdifficulty 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
