HortTechnology - Oct./Dec. 1991

Seed Testingand Quality-Assurance

Eric E. Roos andLoren E. Wianer

Additional index words. seed purity,germination, seed vigor

H igh-quality seed is the basisfor profitable and productiveagriculture, be it a large com-

mercial field operation, a small family-owned farm, or a greenhouse-basedoperation producing bedding plantsor specialty horticultural crops. As-sessing seed quality and assuring thebuyer of the validity of the seed label(Fig. 1) has been an important missionof the various seed regulatory agenciesof state and federal governments andservice-testing provided by commercialseed-testing laboratories around theworld. Based on validity of labeling,commercial sales of seed have been aprofitable business to both the seedproducer and the consumer.

In the United States and Canada,seed-testing rules have been developedand standardized by the Assn. of OfficialSeed Analysts (AOSA) and in othercountries around the world by the Inter-

USDA/ARS National Seed Storage Laboratory FortCollins, CO 80523.

national Seed Testing Assn. (ISTA).The first state seed-testing laboratory inthe United States was established in1876 at the Connecticut AgriculturalExperiment Station, and by 1930, 44states had established seed-testinglaboratories (Justice, 1961). In 1897,the first seed-testing rules in NorthAmerica were prepared and publishedby the USDA in a circular entitled “Rulesand Apparatus for Seed Testing” as un-official guidance for seed analysts (Jus-tice, 1961). The formation of the AOSAin 1908 and the passage of the U.S.Federal Seed Act in 1939 resulted in theformalized procedures for testing seedsthat are acknowledged and used in theUnited States.

To meet the demands of newtechnology and new crops, the rules andprocedures are reexamined and revisedannually. For example, the developmentof seed-coating technology has led tochanges and additions to the “Rules forTesting Seeds” as published by the AOSA(1988) and the “International Rules forSeed Testing” published by the ISTA(1985).

Assessment of seed quality includestests for purity (physical and genetic),viability (germinable plus dormant), andvigor. purity and germination normallyare included as information on the seedlabel, along with a statement of thevariety or cultivar. Vigor test results arenot included as part of the regulatorylabeling requirement. They are usedextensively for in-house quality controlby seed producers. A purchaser of seedmay ask for vigor test results, or canobtain them from a commercial labo-ratory. Assurance of seed quality by acommercial producer involves continualmonitoring of viability and/or vigorduring all phases of harvesting, condi-tioning, and storage before the sale andshipping of the seed. Mechanical dam-age, seed contamination, and deteriora-tion all must be avoided if high quality isto be retained.

Purity testsThe concept of seed purity in-

cludes both physical purity and geneticpurity. Physical purity includes thepresence or absence of soil, plant de-bris, weed seed, and other contami-nants. Genetic purity relates to thepresence of the correct cultivar anddegree of contamination with othercultivars, or, in the case of hybrid seed,the presence or absence of self-polli-nated inbreds or incorrect crosses.

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To determine physical purity, arandom sample of the seed lot must beexamined carefully by hand, often us-ing a microscope. Each seed is ob-served by a trained analyst. This test isvery tedious and time-consuming, butis necessary to evaluate the degree ofcontamination by noxious weeds,common weeds, and other crop seeds.

Genetic purity is determined ei-ther by morphological characteristicsof the seed or seedling, or by usingvarious chemical tests (Table 1) suchas the fluorescence test and the phenoltest (AOSA, 1988), both of which canbe used to identify certain closely re-lated cultivars or species. On a moresophisticated level, electrophoresis ofseed proteins and enzymes can be usedto separate cultivars that may differ byonly one or a few genes. It should bestressed that in many instances achemical test cannot identify an un-known cultivar; rather, the test can saywhat it is not. If it is known that thesample is either one cultivar or an-other, the test may make the distinc-tion. More than one test often is neededto identify cultivars.

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Seed viability normally is evaluatedby means of the standardgerminationtest. Although viability refers to seedsthat are alive, in this test the term germi-nation is used to count specifically thoseseeds that are capable of producing“normal” seedlings. A representativesample of at least 400 seeds (four repli-cations of 100 seeds) is planted on specialblotter paper, germination toweling, insand, or in some other suitable substrate.The sample is placed in a germinatorunder controlled conditions of tem-perature, light, and water, and isevaluated after 7 to 21 days (or longer,depending on the species being tested)for germinability. Seedlings are classi-fied as being normal or abnormal (Fig.2), depending on the presence or ab-sence of all essential structures (roots,shoots, cotyledons, etc.). Descriptionsof what constitutes a normal or ab-normal seedling is an integral part ofthe seed testing rules. Seed analysts aretrained in interpretation of these de-scriptions and often participate in seedschools or in “referee tests,” whichcontribute to standardization amongvarious laboratories of these subjectiveanalyses. The Society of CommercialSeed Technologists requires newcommercial analysts to complete a 2-year training program and pass a rigor-ous written and practical test beforeconferring the title of Registered SeedTechnologist. The AOSA has a similarprogram of training and testing thatleads to certification of seed analysts.

When dormancy is encounteredor when a quick test is desired, thetopographical tetrazolium (TZ) testcan be employed. Depending on thespecies, seeds are placed into TZ so-lution either as whole seeds or afterbeing bisected with a scalpel. Wholeseeds are placed in 1.0% TZ solutionand bisected seeds are placed into 0.1%

Viability tests

TZ solution. Staining time varies from0.5 to 8.0 h, depending on the seedpreparation. The colorless TZ solutionis taken up by the seed tissue and, ifdehydrogenase enzyme activity is present(indicative of cell viability), the TZ solu-tion is reduced to form an insoluble redstain in the respiring tissue. This redcolor then is quantified subjectively andrelated to seed viability. This test also isused to detect seed vigor (see below).

Another test that can detect emptyseeds, shriveled embryos, and/or insectdamage is the X-ray test. Initially usedfor tree seeds (which have a long ger-mination period), the X-ray test canexamine a seed lot within a few secondsor minutes and reveal informationabout potentially dead or empty seeds.The seeds are placed directly over theX-ray film (or Polaroid film) and ex-posed to a brief dose of soft X rays.After developing, the film is read di-rectly to interpret damaged or abnor-mal seeds.

Vigor testsSeveral vigor tests have been de-

veloped over the years to measure seedperformance under a wide range offield conditions. These tests also maybe applicable to predict seed storage(Roos, 1989). The basis for vigor test-ing is the assumption that seeds un-dergo a sequential loss of cell function,which culminates in the loss of ger-minability (Fig. 3). While this schemeprovides a simplistic illustration of whatis thought to occur, the exact sequenceof events is not known. Exact proce-dures for conducting most of thesetests have been summarized in the SeedVigor Testing Handbook, published byAOSA (1983).

Accelerated aging is one of themost widely used tests for predictingstorability and field performance, as itcan be applied to a wide range ofspecies. Seeds are exposed to very high

American Society for Horticultural Science

humidity (95% to 100%), at a tempera-ture of 40 to 45C (specific tempera-tures vary for different species), for aperiod of 2 to 4 days (again, specifictime periods vary for different species).At the end of the aging period, seedsare subjected to the standard germina-tion test and the results are comparedwith control (unaged) samples fromthe same seed lot. The chief difficultyin getting repeatable results is due tononuniform uptake of water duringthe exposure to high humidity.

Controlled deterioration (Mat-thews, 1980) is similar to acceleratedaging, except that seeds are first partiallyimbibed on moistened filter paper topredetermined moisture levels (thusavoiding the problem of uniform wateruptake). Moisture content is checked byfrequent weighing during this imbibi-tion. The seeds then are sealed in con-tainers and held for 24 h at 10C to

HortTechnology - Oct./Dec. 1991

equilibrate before being incubated in a45C water bath for 1 day. Seeds then aregerminated (until radicle emergence) at20C and compared with unaged con-trols.

The cold test, which is used pri-marily for predicting field emergenceof maize seed, is used routinely bymajor seed companies for their evalu-ation of seed lots. This test was de-veloped in the 1940s and involves theuse of soil, taken from the field, thatcontains various microorganisms. Seedsare planted in plastic boxes containingthe appropriate substrate and mois-ture and held at 10C for 7 days andthen transferred to another chamber at25C. Seedling counts are made 4 dayslater.

The cool germination test was de-veloped for sensitive species that couldnot tolerate the low temperature of thecold test. Soil is not used, and the

temperature is a constant 18C: other-wise, the test is identical to the stan-dard germination test. This test wasdeveloped specifically for testing cot-ton seed lots.

The conductivity test measureselectrolytes leached from the seed dur-ing water uptake. It was proposedinitially as a seed vigor test for predict-ing field emergence of peas. Theprinciple of the test assumes that seeddeterioration is manifested by loss ofcell membrane integrity. If this as-sumption is correct, this test shouldhave wide application to differentspecies.

Seedling growth rate is simply acomparison of root and/or shootlength or seedling weight after ger-mination (under controlled conditionsand time period). Thus, it measuresthe metabolic efficiency of mobiliza-tion of storage reserves by the seed. It

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is applicable to essentially all seed spe-cies; however, precise control of envi-ronmental conditions is needed toachieve reliable results.

The seedling vigor classificationtestis conducted in the same manner asthe standard germination test. A fur-ther separation of “normal” seedlingsis performed to identify “strong” and“weak” seedlings. Typically, weak seed-lings may have no primary root (al-though secondary roots have devel-oped), breaks or lesions in the cotyle-dons and/or hypcotyl, partial decay ofthe primary leaf, or are generally smalland poorly developed.

is “FIRST THE SEED,” and to thehorticultural crop producer that meansthe difference between harvesting aprofitable crop or not. Quality assur-ance means different things to differ-ent people. For people working with

Tetrazolium testing was devel-oped originally as a quick estimate ofseed viability, but can be used as avigor test. The principle of the test isthe measurement ofnonspecific dehy-drogenase enzyme activity, which re-sults in a reduction of the soluble,colorless, tetrazolium salt to a red,insoluble, formazan that stains the re-spiring seed tissues. The TZ test can beused to assess vigor by critically evalu-ating the staining patterns of tissues.Although the TZ test has wide applica-tion, its subjectiveness makes it ofquestionable value for detecting smalldifferences in vigor.

Quality assuranceThe American Seed Trade theme

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seed certification programs, it meansbeing sure that the seed is inspected forgenetic variability in the field, in thelaboratory, and, after certification iscomplete, through grow-out tests. Forseed analysts it means conducting teststo assure trueness-to-type, freedomfrom contamination, and the ability toproduce normal, healthy plants in thefield. Several state seed certificationagencies have set up specialized pro-grams for quality assurance and areproviding this service to the seed pro-ducers.

Most quality assurance programsbegin during seed production in thefield. The use of high-quality, geneti-cally pure planting seed, proper isola-tion distances, and weed- and crop-free soil are all beginning steps in aquality-assurance program. Using theproper seeding rates, row spacing,fertility levels, and irrigation schedul-ing is essential in producing high-quality seed.

A seed has its highest quality whenit is physiologically mature. This stage ofdevelopment usually is defined as thatpoint during development when theseed has attained maximum dry weight.The quality of the seed at physiologicalmaturity depends on the environmentprevailing during seed development. Theseed producer can control some of theenvironmental factors that affect qual-ity, such as soil moisture, fertility, diseaseand insect stresses, and uniformity ofstand.

quire systematic sampling and testing ofthe seed during production, condition-ing, and storage. During seed produc-tion, a seed sample should be takenwhen the crop has reached physiologicalmaturity. This sample is used to estab-

Once the seed has been separatedfrom the mother plant by the forma-tion of an abscission layer, its quality isinfluenced by the environment, anddeterioration begins. Crops such asbeans (Phaseolus vulgaris L.) can bedamaged easily during harvesting andhandling. Factors such as moisturecontent during harvesting, mechani-cal damage during combining, andimproper drying techniques all canlead to poor-quality seed. This damagecan cause loss of vigor and/or viabil-ity, making the seed unusable forplanting purposes. To prevent qualitylosses, seed companies have developedquality-assurance programs to evaluate,monitor, and minimize the loss of quality.

Quality assurance programs re-

lish the level of quality and the seedmoisture content before harvesting andconditioning. Once the proper mois-ture content has been reached for har-vesting, another seed sample should betaken. This sample will indicate if seedquality has been lost during ripening.During harvesting, the seed should bechecked for cracked or broken seeds todetermine that the harvest equipment isproperly adjusted. There are several quicktests that can be used in the field to checkseed damage. Once the harvestingequipment has been adjusted properly,a sample should be taken to assess thequality after harvesting and to checkseed moisture again. If the seed mois-ture content is not acceptable for tem-porary storage the seed must be driedbefore seed conditioning, preferably atlow temperature to avoid loss of viabilityand vigor. After drying, a seed sampleshould be taken to determine if seed hasbeen damaged during drying. Oftenseed is not conditioned immediatelyand is put into temporary bulk storage.Movement into bulk storage usuallyrequires the use of an elevator, whichcan damage the seed, therefore, a seedsample should be taken to determine ifquality is lowered.

Before conditioning, genetic pu-rity tests should be conducted to deter-mine if the seed meets the cultivar de-scription. In most quality assuranceprograms the plants in the field wereinspected to determine trueness-to-kind.However, cultivar purity tests of theseed will point out discrepancies duringproduction that cannot be observed inthe field. One of the common geneticpurity tests that is conducted is a seed-ling grow-out. This evaluation is usedroutinely for vegetable crops. In addi-tion to grow-out tests, many seed com-panies are now using electrophoreticfingerprinting. As cultivars become morespecific in their environmental conditionsfor growth, these evaluationswill becomemore necessary for a quality-assuranceprogram.

Seed conditioning is an essentialstep in making seed of genetically supe-rior cultivars available to crop produc-ers. Some objectives of seed-condi-tioning are to remove contaminants,upgrade quality, improve plantability,apply seed treatment, and package theseed (Fig. 4). Each of the steps in thisflow diagram requires specializedequipment that performs specific condi-tioning functions. However, as the seedpasses through each piece ofequipment,

American Society for Horticultural Science

it can be damaged. Consequently, aseed sample should be taken at eachpoint along the way to assure that a pieceof equipment is not reducing quality.

The work of a quality-assuranceprogram is not completed when theseed is put into storage. If seed remainsin storage more than a few months, itshould be sampled and checked todetermine if its vigor or viability haschanged. It is also important that allstorage areas be kept free ofinsects androdents.

The quality-assurance program,as outlined, creates a large amount ofdata that can be helpful in determiningwhere and why seed quality has beenlost. When samples are evaluated usingthe various tests outlined above, a seedcompany can make the necessarymodifications in their productionpractices, conditioning procedures, andstorage facilities to produce and main-tain a high-quality product, THESEED.

HortTechnology - Oct./Dec. 1991

ConclusionsThe availabil i ty of high-

quality seed does not just happen, butresults from the ability of the seedanalyst to determine the purity, vi-ability, and vigor of each seed lot andthe meticulous attention by the seedproducer to overcome problems dur-ing seed development, harvesting,conditioning, and storage. Seed test-ing has become a highly technical fieldthat requires specialized knowledgeand training. New tests for geneticpurity (cultivar identification) and vigorare under development and will requiresophisticated instruments and highlytrained analysts.

Literature CitedAssociation of Official Seed Analysts. 1983.Seed vigor testing handbook. AOSA,Springfield, Ill. AOSA Contrib. 32.

Association of Official Seed Analysis. 1988.Progress report on the AOSA cultivar pu-rity testing handbook. Nwsl. Assn. OfficialSeed Analysts 62(3):1-90.

Association of Official Seed Analysts. 1988.Rules for testing seeds. J. Seed Technol.12(3):1-109.

Copeland, L.O. and M.B. McDonald.1985. Principles of seed science andtechnology. 2nd ed. Burgess, Minneapo-lis.

Delouche, J.C. and C.C. Baskin. 1973. Ac-celerated aging techniques for predictingthe relative storability of seed lots. SeedSci. Technol. 1:427-452.

International Seed Testing Association.1985. International rules for seed testing.Seed Sci. Technol. 13:299-355.

Justice, O.L. 1961. The science of seedtesting, p. 407413 In: A. Stefferud (ed.).Seeds, the yearbook of agriculture. U.S.Govt. Printing Office, Washington, D.C.

Matthews, S. 1980. Controlled deteriora-tion: A new vigor test for seeds, p. 647-660. In: P.D. Hebblethwaite (ed.). Seedproduction. Butterworths, Boston.

Roos, E.E. 1989. Long-term seed storage,p. 129-158. In: J. Janick (ed.). Plantbreeding reviews. Timber Press. Portland,Ore.

Vaughan, C.E, B.R. Gregg, and J.C.Delouche (eds.). 1968. Seed processing andhandling. Seed Technology Laboratory,Mississippi State Univ., State College.Hdbk. 1.

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