This article is from ECHO Asia Note #11
Introduction and Background
Saving your own seeds is a cost-effective way to access crop seed forfuture planting and to help maintain the planet's plant biodiversity.Whether you plant your own saved seeds, give them away to friendsand neighbors, or distribute them through your organization, knowingthe viability of your seeds is important. As a follow-up to the article inthe January 2011 EC HO Asia Notes, Issue 8, titled "Building your ownseed germination chamber for testing seed viability"( www.echonet.org/repository#1003:d:Build Your Own SeedGermination Cabinet), this article will explore the details of severallow-cost methods for testing seed viability.
Seed viability is a measure of the percentage of seeds that are aliveafter storage. The greater the viability of your seeds, the fewer seedswill be needed to establish a desired number of plants in the field ornursery.
Seed viability can be tested in many easy ways. A seed germinationtest is probably the most simple: seeds are given the neededresources (air, water, warmth, and light) to germinate and grow into aseedling. Simply place seeds in the soil or in a pot of soil and see howmany grow. However, one disadvantage of using soil, pots, andoutdoor resources is environmental fluctuation that can cast doubt onthe true viability of the seeds (did the seeds fail to germinate becausethey were dead, or because they were watered erratically, fell victimto fungal attack, got too hot, etc.?).
A dedicated seed germination cabinet like the one described in theaforementioned article (Figure 1) is a great way to provide constantlight and temperatures to germinating seeds, and is another goodoption. However, even without a cabinet and with very few resources,you can still conduct a reliable seed viability test.
Procedures
The various methods for determining seed viability all serve to provideseeds with a substrate that makes water available at the properamount for the seeds to imbibe (take up into themselves) and togerminate. Choose a method based upon your available resources,and remember that you can always use several different methods tocompare results.
All seeds have specific light and temperature requirements, but ageneral rule of thumb is that most seeds will germinate when thetemperature is between 20 and 30ºC , sufficient water is present in thesubstrate, and some amount of light is given to the seeds (See Table5.1 in "Manual of Seed Handling in Genebanks" for specific seedrequirements and more in-depth coverage of seed viability testing).
Seeds for the test should be randomly selected from your entire lot ofseeds. International standards for seed testing suggest that 200 seedsbe used ingermination test. If this quantity of seeds is difficult toattain, 100 or even 50 seeds may be used. Divide your total numberof available seeds by two, so that you will have two replications foryour germination test. If you have an abundance of seeds (lettuce,cabbages, tomatoes, etc.), then four replications of 100 seeds willprovide very robust results.
Control of Pathogens
Pathogenic contamination by fungi, molds, and bacteria is common inseed germination tests and will often result in poor seed germinationand false conclusions. Aseptic technique is an important way to reducecontamination by killing and minimizing the presence of pathogens.Clean and disinfect all work surfaces with a 70 to 95% alcohol solutionor a 20% bleach solution (don't forget to wash your hands!).Germination containers and forceps should be soaked in a 20% bleachsolution for 10 to 15 minutes, or surface sterilized with alcohol.Prevent seeds from touching each other, regardless of what substrateis used, and promptly remove damaged or decaying seeds, recordingthe number of contaminated seeds.
The easiest way to prevent contamination is by surface sterilization ofseeds to be used in the experiment. The concentration of householdbleach is usually between 5 and 6% sodium hypochlorite by volume.Prepare a 1% sodium hypochlorite solution by adding 80mL of distilledwater to 20mL of household bleach. Soak seeds for 3 to 5 minutes (3minutes for small seeds and up to 5 minutes for larger, tough seeds,like beans) in enough bleach solution for adequate coverage (Figure2). Thoroughly rinse seeds 3 to 5 times in distilled water beforeintroducing them to the substrate.
Top of Paper Method
In the top of paper method, seeds are placed on top of substratepaper in containers with snug-fitting lids (to prevent moisture loss).Glass or petri dishes work well for this method. The EC HO Asia SeedBank uses rectangular plastic boxes with snug-fitting lids. Use apermanent marker to label the containers with the type of seed beingtested and the replication number. Sterilize containers as outlinedabove, and cut substrate paper (we use a double-folded paper towel)to fit in the container (Figure 3).
With a sterilized hand or an inverted funnel, snug substrate paper intothe container (Figure 4). Add an appropriate amount of distilled water(if distilled water is not available, use boiled and cooled water) tocompletely moisten the paper without soaking it (Figure 5). We havefound that typical containers with a double-folded paper towel requirefrom 2 to 6 ml of water, depending upon the size.
Spread the seeds uniformly on the moistened substrate, ensuring thatnone of the seeds touch each other (Figure 6). Close the lid, and placethe container inside a loosely fitting sandwich bag to help ensureadditional moisture retention (Figure 7). You want to prevent loss ofmoisture, but still allow diffusion of oxygen, which the seeds will needwhen germinating and respiring.
Place the containers in your seed germination cabinet, or in a warmarea where some light is present. Every day, or every other day,count, record, and remove germinated seeds. Germination is definedas the clear and unobstructed emergence of the radicle, or seedlingroot, from the seed coat (Figures 8, 9).
Substrates will most likely need to be re-moistened during the courseof the test; be sure to give containers with the same seed types anequal amount of water, making note of the amounts. Most seedgermination experiments should be run for up to 14 days but somegrass species may require up to 28 days to germinate. You maydecide to run your experiment shorter or longer depending upon thetype of seed, and if all seeds germinate. During the test, carefullyremove and discard any seeds that show signs of contamination ordecay.
Between Paper Method
In the between paper method, ordinary paper towels are used as thesubstrate and also act as the container to hold the seeds. Cut a papertowel to size to adequately hold all the seeds of the replication in neatrows on the towel. Using a pencil or permanent marker, label oneedge of the paper towel with the type of seed being tested and thenumber of the replication. Moisten (but do not soak) the paper towelwith distilled water.
Next, place the seeds in rows on the moist paper towel, leaving a 3 cmgap from the top and side edges, and ensuring that the seeds do nottouch each other. Any extra space should be at the bottom of thepaper towel, which will be immersed in water. Cover the seeds withanother moist paper towel and roll the paper towels from the nonlabelededge (Figure 10). Use a paper clip or rubber band to hold thenewly formed tube together. The seeds will be inside the tube, held byfriction between the two layers of moist paper towel.
Place the bottom of the paper towel tube in a deep-bottomed plastictray with enough water to ensure that all paper towel tubes are incontact with the water (Figure 11). Place the tray of paper towel tubesin your seed germination cabinet or in a warm area where some lightis present. Count, record, and remove germinated seeds by gentlyunrolling the paper towel tubes and re-rolling after recording.
Be sure to maintain sufficient water level in the tray; you may alsohave to spray the tops of the paper towel tubes to keep them moist.Similar to the top of paper method, maintain and continue theexperiment for up to 14 days (you may decide to run your experimentshorter or longer depending upon the type of seed, and if all seedsgerminate). Take particular care to remove and discard any seeds thatshow signs of contamination or decay.
Sand Germination Method
In the sand germination method, moist sand or other porous media isused as the germinating substrate for the seeds. Start with clean, finesand that you purchase or steam pasteurize (if possible), and packinto deep-bottomed plastic trays with adequate drainage. Water thesand with distilled water until moist but not soaked (you do not wantthe sand to evacuate the tray through the drainage holes).
Create equidistant holes in the sand (a pencil or permanent markerworks great for this), as deep as the length of the seeds you aretesting and spaced three times the length of the seeds. If you aretesting more than one type of seed, use a small wooden stake orplastic spoon to mark each row, with the name of the seed beingtested and the replication number. Place one seed in each hole, coverthem with sand, and water the entire tray. Be sure to water the traygently so as not to dislocate the seeds (Figure 12). Place the tray inyour seed germination cabinet or in a warm area where some light is
present.
Count and record germinated seeds daily or every other day.However, it is not necessary to remove germinated seeds unless theyare crowding each other out. If you choose to remove seedlings,snipping the seedlings at the base will kill the seedling and preventdislodging non-germinated seeds from the sand.
Keep the sand substrate moist during the course of the experiment,but do not over-water (Figure 13). As per the other two methods,maintain and continue the experiment for up to 14 days, dependingupon the type of seed and if all of your seeds germinate.
Analyzing the Results
Use the sample data collection sheet (Figure 14) to count and recordgerminated seeds over the course of your test. Record the totalnumber of seeds you started with for each replicate. Also record thenumber of germinated seeds and the number of seeds removed fromthe test due to contamination or decay (these latter we will not countas viable).
Percent total germination is a measure of the overall viability of anaccession of seeds, and can approximate the number of seeds that willgrow into plants when you plant them. At the end of the experiment,add the number of seeds that germinated each day of the trial, dividethat sum by the total number of seeds that began the test, andmultiply by 100 to calculate the parameter of percentage totalgermination.
Find the percentage total germination for each replication, so that youcan see whether or not the replications differ. If they differ a lot, itwould be a good idea to conduct the test again with a greater numberof seeds and more replications.
Data that you collect can be used to estimate the length of time aparticular seed accession takes to have 50% of its seeds germinate.You can calculate the "mean number of days to 50% germination" asfollows: for each day, multiply the number of seeds that germinatedby the day you counted them, then add all of those values and dividethat number by the total number of germinated seeds. For example, if12 seeds germinated on day 1, 8 seeds germinated on day 3, and 4seeds germinated on day 5, for a total germination of 24 seeds, meannumber of days to 50% germination would be calculated as: (1x12 +3x8 + 5x4)/24 = 2.3 days.
Conclusion
Testing the viability of your seeds by conducting a seed germinationtest is an important way to deduce the quality of your seeds, todetermine the efficacy of your seed storage methods, and to help youplant the proper amount of seeds. By conducting these simple seedviability tests, you can increase your seed saving efficacy and help toempower farmers in the saving and planting of important geneticdiversity.
Additional Resources
Gosling, P.G. "Viability testing." Chapter 24: Seed Conservation.London: Kew Royal Botanical Gardens. Available:www.kew.org/science-research-data/kew-in-depth/msbp/publicationsdata-resources/technical-resources/seed-conservation-sciencepractice/SC TSIP_chapter24.htm.
Rao, N. K., J. Hanson, M. E. Dulloo, K. Ghosh, D. Nowell, and M.Larinde. 2006. Handooks for Genebanks No. 8: Manual of SeedHandling in Genebanks. Rome: Biodiversity International. Available:http://www.bioversityinternational.org/index.php?id=19&user_bioversitypublications_pi1[showUid]=3020
Douglas, J. L., J. M. Grabowski, and L. E. Daughtry. 2003. "How to usea ragdoll test to estimate field germination." Plant Solutions forConservation Needs. Plant Note #5. Available:ftp://ftp-fc.sc.egov.usda.gov/GA/PMC /JLW/ragdoll.pdf..
