CPC Best Plant Conservation Practices

to Support Species Survival in the Wild

Seed Banking

Cleaning, Processing, Drying, and Storing Orthodox Seeds

Key Messages about Cleaning, Processing, Drying, and Storing Orthodox Seeds
  • Careful documentation and accession information will ensure the value of the conservation collection.
  • Evaluate initial quality of seeds and keep record of the number of apparently good seeds in the accession. These data can be used to evaluate if the seed health changes after storage.
  • For seeds that are tolerant of drying and freezing, dry to appropriate moisture targets to maintain appropriate relative humidity during storage. Store seeds at subfreezing temperatures.
  • Monitor storage conditions and seed viability periodically.

Figure 1.5 – Stages in seed cleaning process. (a) inflorescence of San Diego thornmint; (b) large chaff separated from seeds; (c) moderately cleaned seed with large chaff removed. This stage is appropriate for long-term storage. Moderate cleaning can save time so that seeds can be processed quickly, dried, and placed into cold storage; (d) meticulously cleaned seed. This level is appropriate for curation packages that require seed testing.

Evaluate the quality of seeds.

  • Determining viability of the collection can begin with visual assessment. If you have fewer than 100 seeds, examine seeds under a microscope to estimate whether they are empty or filled. With such a small sample, it is best not to destructively sample any seeds.
  • If you have an adequate seed accession (500 seeds or more), dissect 5–10 seeds under a dissecting microscope and assess percentage of the sample with filled, intact embryos. Extrapolate the percentage of the accession that is likely to be filled.
  • If more than 50% of your seeds are hollow, this may indicate herbivory or low reproductive viability. Consider making another collection of this population.
  • Report percent viability and how it was determined with accession records sent to the seed bank.

Divide seeds for storage.

  • Divide seeds according to CPC Best Practice Section, “Splitting Samples for Safety Duplication Storage and Testing.” Recall that your accession is subdivided into two long-term storage packages with maternal lines separated (~40% of accession to each primary and backup facility) and two curation packages (~10% of accession to each primary and backup facility).
  • Recall that the precision of cleaning differs between the long-term storage pack-ages and the curation packages. Recall that the precision of cleaning differs between the long-term storage packages and the curation packages (see Figure 1.5 above). Stored seed may be moderately cleaned, while seeds in the curation packages require thorough cleaning and each should contain a total of at least 50 seeds from all maternal lines or approximately 10% of the accession. These are needed for testing seed viability, germination, and/or longevity in storage. Place the curation package labeled “Curation” into the large envelope used for long-term storage sample (see step 4 in Figure 1.4).

Assemble the storage package.

  • To estimate the total number of seeds in the storage packages, clean, count and weigh up to 25 maternal lines (a subset of the total in your accession).
    • Provide a seed count on maternal line packages and record seed counts and weights on the Maternal Line Count Example Form.
    • Measure the mass of the cleaned maternal line with the discarded chaff.
  • Extrapolate to other maternal lines and to total accession seed count using the Maternal Line Count Example Form.
  • Indicate on seed packages actual counts like this: “100 seeds.” Indicate estimated counts on seed packages like this: “~ 1000 seeds.”

Assemble the curation package.

  • The curation package allows for easy, quick removal from a large storage package that remains in a freezer. Seeds in the curation package can be easily used to test seed viability after storage – thus validating the health of the whole accession. Without a curation package, the entire storage package and each maternal line envelope would need to be retrieved from the freezer for seed testing. In the time required to remove seeds from each maternal line envelope, the whole accession is subject to warming, which may reduce its longevity.
  • Count and report seeds in the curation packages.
  • Place counted, clean seeds into Uline S-11591 2 x 3.5” flat, glassine bags.
  • NLGRP will measure mass of 10 individual seeds. Estimate mass and seed numbers of the storage samples.
    • Knowing seed mass can provide good diagnostic information that can allow estimates of the number of seeds in a whole sample, establish the appropriate medium for germination testing, indicate differences among populations, or indicate maturity or seed fill (that is, if the seed does have an embryo).
    • Measurements of individual seeds are much more useful than a single measurement of a bulked sample because it provides a good characterization of the variation within the sample. Individual mass measurements require a precision balance. An electronic balance has a 0.1 µg resolution, which is almost sufficient for dust-sized seeds. NLGRP will provide seed mass in milligram units (grams multiplied by 1000).
  • The Primary Institution may elect to keep curation packages separated by maternal lines. This can increase opportunities for equalizing family lines for restoration.

Process seed as quickly as possible.

  • Maintain seeds while processing in a cool, dry location. Avoid exposure to high humidity, heat, or direct light. Periodically check the seeds for insect damage.

Dry to appropriate moisture targets to maintain appropriate relative humidity during storage.

  • Moisture target is set by storage temperature and risk of failure of storage temperature. Target should be no more than 25% RH (lower risk of failure) and no less than 10% RH (higher risk of failure) at the intended storage temperature. See the box “Recommended RH and Temperatures for Drying Seeds in Seed Lab” (Walters 2004).
  • Keep all maternal lines of an accession together for drying. Spread them out so that drying is effective.
  • Conditions needed to dry to the moisture target are constrained by drying time and drying temperature.
  • Drying time should be commensurate with drying temperature, ranging from less than 1 week for drying at 25°C and less than 1 month for drying at 5°C.

Testing the Relative Humidity of Seed Accessions Prior to Freezing

Authors: Stacy Anderson, Tobin Weatherson, Joe Davitt - San Diego Zoo Native Plant Seed Bank We are all taught in school that water expands when... Read More

Maintain seeds at temperatures below 25°C.

  • Drying temperatures that exceed 25°C can damage seeds.

Continuously monitor relative humidity and temperature.

  • Continuously monitor RH and temperature of drying conditions. (See recommended equipment list and sources below.)

Figure 1.6 – Silica gel is orange when activated and turns green when deactivated.

Dry seeds using desiccators and drying agent.

  • The simplest way to dry seeds is to place seeds in a desiccator with silica gel. Monitor the RH regularly. Replace silica gel as needed.
  • A more precise method for drying seeds is to use salts in a desiccator. This will allow drying to a particular RH percentage. Lisa Hill from the USDA-ARS National Laboratory for Genetic Resources Preservation demonstrates this method below.

How to Prepare a Saturated Salt Solution to Dry Seeds

Lisa Hill - National Laboratory for Genetic Resources Preservation (USDA-ARS) Drying seeds to appropriate relative humidity is the first step to long term seed storage.... Read More

Relative humidity will drop as temperatures chill.

  • Relative humidity influences internal seed moisture.
  • Recall that target storage RH should be no more than 25% RH (lower risk of failure) and no less than 10% RH (higher risk of failure) at the intended storage temperature. Drying RH to achieve target storage RH is approximately 25%-35% RH if drying at 25°C and 15%-25% RH if drying at 5°C (Walters 2004, unpublished 2019). See Table 1.1.
  • It is possible to use multiple steps to ensure rapid drying and desired accuracy of target RH. For example, drying over silica gel to achieve very low RH with a final overnight adjustment at room temperature over a saturated potassium acetate or calcium chloride solution would be fine.
  • It is more important to get seeds dried and placed at storage temperature than it is to achieve precise drying RH.

 

Why does CPC recommend drying seeds at 25-35% RH and 25C for long term storage in the freezer?

Dr. Christina Walters and Lisa Hill, National Laboratory for Genetic Resources Preservation, Fort Collins Because research has shown that seeds will last best when stored at a... Read More

Quickly transfer seeds to storage envelopes.

  • Quickly transfer seeds from desiccators to foil envelopes, and seal and move them to subfreezing temperatures.
  • Maintain target moisture using suitable, moisture-proof containers. CPC recommends using sealable foil envelopes (see Equipment List and Sources).
  • Seed storage containers must be resistant to breaks, tears, or punctures from sharp seed parts.
  • Storage containers must have a maximum water vapor transmission rate (WVTR) of 0.005 g H2O/m2/day, measured directly or reported by manufacturer using standard conditions. Lower WVTR is recommended for locations at sea level with high ambient RH.
  • Clearly label storage envelopes with the name of the plant, the accession number, collection date, the date placed into storage, the number of maternal lines, and the estimated number of seeds.

Recommended Relative Humidity and Temperatures for Drying Seeds in the Seed Lab

Orthodox seeds should be dried quickly so that they will have approximately 12% water content at the intended storage temperature.

The RH % target should be no more than 25% RH (lower risk of failure) and no less than 10% RH (higher risk of failure) at the intended storage temperature.

TABLE 1.1 Recommended drying conditions for seeds stored in moisture-proof containers at various temperatures. The given drying temperature and RH combinations give a storage RH of 20% at the indicated storage temperature.

Drying Temperature (°C) Drying Relative Humidity for Storage at 15°C Drying Relative Humidity for Storage at 5°C Drying Relative Humidity for Storage at –20°C
25 28% 33% 35%
15 20% 26% 35%
5 14% 20% 32%

Drying seeds at temperatures less than the storage temperature is not cost-effective and therefore is strongly discouraged.

Note that practitioners should feel comfortable achieving RH targets ± 3% while drying seeds.

Walters (2004, unpublished data 2019)

Mail duplicate seed accession to backup facility.

  • Contact NLGRP prior to mailing the duplicate processed, dried, and packaged seed accession.
  • Mail seeds in container that can protect them from compression and temperature fluctuations. DO NOT FREEZE or place cold packs into seed container prior to shipping unless NLGRP recommends this for your specific accession.

Store orthodox seeds at subfreezing temperatures.

  • Hold samples at < –20°C ± 2 °C.
  • Samples may be warmed periodically for viability monitoring or distribution. Sample exposure to room temperature should be kept to a minimum, be recorded, and not exceed 5 days in any year.
  • There should be concerted attempts to avoid warming the entire sample when only a small subsample is needed. Warming may decrease the lifespan of the seeds.
  • For walk-in freezers, observe human safety requirements by OSHA. Don’t stay in the freezer longer than 5 minutes.

Monitor storage temperatures continuously.

  • Monitor storage temperature continuously with equipment that will send automatic alarms when temperatures are out of range.
  • Maintain incident records when storage temperature warms above –15°C (for freezer storage), –60°C (for –80°C storage) and –150°C (for cryostorage).
  • Determine whether or not a management action is required (for example, replacing equipment, checking power sources, etc.).
  • Place contact numbers for service onto front of freezer.

Test seed viability after several years in storage.

  • When you test seed viability after several years in storage, check the RH of bags under storage conditions. (See equipment suggestions below.)
  • To confirm RH is holding steady, spot-check the RH in a subset of newly sealed bags that contain seeds held at storage temperature (–20°C). Spot-check the RH of some bags after they are stored for 5, 10, 15, and 20 years.
  • Replace storage containers within 30 years of banking.
  • See CPC Best Practice Section “Collecting and Maintaining Exceptional  Species in Tissue Culture and Cryopreservation” for recommendations for storing seeds that are intolerant of desiccation and/or freezing.
Blotter Paper and Germination Paper
Seed Separation Devices

Agriculex Column blower (to separate seeds from chaff quite extensively Agriculex), (519) 837-0871

Desiccators

SP Scienceware stackable desiccator chambers by Bel-Art are available from Cole-Parmer, Zoro, and others.

Foil Laminate Bags

Protective Packaging Ltd. has foil laminate bags of different sizes. One popular size is PP01625. Protective Packaging Ltd., +44 (0) 161 976 2006

Glenroy(262) 255-4422. Must be a USA company for orders over 30,000.

Flex-Pak Packaging Products, Inc., (630) 761-3335

Sealer for Bags

Rennco heat sealer (recommended model is LS-18-120) to seal barrier foil envelopes, rather than using an iron, to ensure a fully sealed package. The optimal temperature is 177o C, according to the engineer who tested the specifications on CPC packaging material. Relatively inexpensive, used heat sealers can be found online.

Temperature and Relative Humidity Sensors

Wireless (real-time, live)
OmniSense, (843) 522-0350

You will need to purchase a gateway and sensors individually or you may purchase together in the DriFi Restoration Moditoring Kit. There is a US $20 monthly data access fee.

Data Loggers
HOBO Temp/RH 2.5% Data Logger (most accurate and longest lasting) Onset, (800) LOGGERS.

iButtons (small, about the size of a quarter)
Maxim Integrated
iButtonLink Technology, (262) 662-4029

Humidity/temperature pen (inexpensive, with a digital display)
Fisher Scientific, (800) 766-7000

International Standards

Reference for CPC Guidelines
FAO Genebank Standards for Plant Genetic Diversity (FAO 2014)
Standards for Safety Duplication
4.2.1 All seed samples should be dried to equilibrium in a controlled environment of 5–20°C and 10–25 percent of relative humidity, depending upon species.
4.2.2 After drying, all seed samples need to be sealed in a suitable airtight container for long-term storage; in some instances where collections that need frequent access to seeds or likely to be depleted well before the predicted time for loss in viability, it is then possible to store seeds in non–airtight containers.
4.2.3 Most original samples and safety duplicate samples should be stored under long-term conditions (base collections) at a temperature of –18 ± 3°C and relative humidity of 15 ± 3 percent.
4.2.4 For medium-term conditions (active collection), samples should be stored under refrigeration at 5–10°C and relative humidity of 15 ± 3 percent.
MSB Partnership Collections (Millennium Seed Bank Partnership 2015)
Processing
Seed collections are accessioned, dried and processed according to recognized protocols or guidelines:
2.1 Unique accession reference number is assigned to all incoming material.
2.2 Collections are placed in cool/ambient drying conditions of 15% RH ± 3% within 4 weeks of collection (Immature seeds are ripened before drying; microscopic seeds (e.g. orchids) are dried for a maximum of 1 week).
2.3 Collections are cleaned to remove empty, poorly developed and insect-infested seeds and debris.
2.4 Purity is assessed by X-ray and/or cut test.
Storage and Duplication
3.1 Seed collections are banked as soon as possible after drying to equilibrium with 15% RH ± 3% (cool/ambient temperature), and within 6 months of collection (microscopic seeds are banked within 1 week of drying).
3.2 Collections are held in air-tight (hermetic) containers.
3.3 Collections are stored at –20°C ± 3°C.
3.4 Collection size is monitored to ensure that sufficient potentially viable seeds are available for effective management and distribution to users.

Basey, A. C., J. B. Fant, and A. T. Kramer. 2015. Producing native plant materials for restoration: 10 rules to collect and maintain genetic diversity. Native Plants Journal 16: 37–52.

Dickie, J. B., and H. W. Pritchard. 2002. Systematic and evolutionary aspects of desiccation tolerance in seeds. Pages 239–259 in M. Black and W. Pritchard, editors. Desiccation and plant survival. CABI, Wallingford, UK.

Food and Agriculture Organization of the United Nations (FAO) 2014. Genebank standards for plant genetic resources for food and agriculture. Rome, Italy. http://www.fao.org/3/a-i3704e.pdf.

Guerrant, E. O., Jr., K. Havens, and M. Maunder, editors. 2004. Ex situ plant conservation: supporting species survival in the wild. Island Press, Washington, DC.

Hong, T. D., S. H. Linington, and R. H. Ellis. 1998. Compendium of information on seed storage behaviour. Royal Botanic Gardens, Kew, UK.

Millennium Seed Bank Partnership (MSB). 2015. Seed conservation standards for “MSB Partnership Collections.” Royal Botanic Gardens, Kew, UK.

Pérez, H., E., and J. G. Norcini. 2010. A new method of wiregrass (Aristida stricta Michaux) viability testing using an enhanced forceps press test. Natural Areas Journal 30:387–391.

Seed Conservation Hub. Accessed August 3, 2017.

Walters, C. 2004. Guidelines for seed storage. Pages 442–453 in E. O. Guerrant, Jr., K. Havens, and M. Maunder, editors. Ex situ plant conservation: supporting species survival in the wild. Island Press, Washington, DC.

Walters, C., P. Berjak, N. Pammenter, K. Kennedy, and P. Raven. 2013. Preservation of recalcitrant seeds. Science 22: 915–916.

Wieland. G. D. 1995. Guidelines for the management of orthodox seeds. Center for Plant Conservation, St. Louis.

Wyse, S. V., and J. B. Dickie. 2017. Predicting the global incidence of seed desiccation sensitivity. Journal of Ecology. doi: 10.1111/1365-2745.12725.

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