Seed Cleaning

IPGRI Handbook for Genebanks No. 6. IPGRI, Rome. FAO/IPGRI, 1994. Genebank standards. FAO and IPGRI, Rome. Linington, S. H. 2003. The design of seed banks. Pp. 591-636 in Seed conservation: Turning science into practice. (R.D. Smith, J.B. Dickie, S.H. Linington, H.W. Pritchard and R.J. Probert, eds.). Royal Botanic Gardens, Kew, UK. 96 7. GERMPLASM DISTRIBUTION What is germplasm distribution? Germplasm distribution is the supply of representative samples of seed accessions from a genebank in response to requests from germplasm users. In general, seeds are ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

distributed only from active collections (see Flowchart 7.1). Why is germplasm distributed? The purpose of conserving germplasm in a genebank is either to improve crop varieties through plant breeding and related research activities or to restore diversity lost on farms and in natural habitats in order to meet the needs of farmers and communities. This contributes directly to improving the livelihoods of poor people and to protecting the environment. In the past, not enough emphasis was given to germplasm distribution. It is now widely recognized that the utilization of germplasm should ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

drive its conservation. Genebanks must be more proactive in establishing links with germplasm users, breeders, researchers, farmers and other groups. How should germplasm be distributed? Germplasm should be distributed in a way that ensures it reaches its destination in good condition. Environmental conditions during transport can be detrimental to seed quality, so seeds should be carefully packed and distributed in sealed moisture-resistant envelopes for protection during transit (see below). The scope and extent of distribution varies with each genebank. Germplasm may be distributed within or outside the country, depending on the genebank’s mandate and whether its ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

collection is national, regional or global. 1. Introduction 2. Germplasm acquisition and registration 2.1 Germplasm acquisition 2.2 Germplasm registration 3. Seed cleaning 4. Seed moisture content determination and drying 4.1 Seed moisture content determination 4.2 Seed drying 5. Seed quality testing 5.1 Seed viability testing 5.2 Seed health testing 5.3 Seed testing for inadvertent introduction of transgenes 6. Seed packaging and storage 6.1 Seed packaging 6.2 Seed storage 7. Germplasm distribution 8. Germplasm monitoring and regeneration 8.1 Germplasm monitoring 8.2 Germplasm regeneration 7. Distribution 97 Manual of Seed Handling CHECK IF IMPORT PERMIT IS NEEDED AND WHETHER THE REQUESTOR HAS SENT IT CHECK IF REQUESTOR PROVIDED THE LIST OF ACCESSION NUMBERS CHECK AVAILABILITY OF REQUESTED ACCESSIONS FOR DISTRIBUTION ON STANDARD MTA Are accession numbers specified? SELECT ACCESSIONS BASED NO YES CHECK IF THE MATERIAL CAN ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

BE DISTRIBUTED USING A TAILORED MTA INFORM REQUESTOR THAT MATERIAL CANNOT BE DISTRIBUTED CHECK IN THE DATA FILE WHETHER ENOUGH SEEDS ARE AVAILABLE FOR DISTRIBUTION Is enough seed available? INFORM REQUESTOR THAT REQUESTED ACCESSION NEEDS TO BE REGENERATED NO YES REGISTER THE REQUEST PREPARE THE RELEVANT MTA TO BE USED PREPARE THE SAMPLES FOR DISTRIBUTION PREPARE THE LIST OF ACCESSIONS TO BE DISPATCHED OBTAIN FROM DATA FILES THE MINIMUM PASSPORT DATA FOR EACH ACCESSION CHECK WHETHER ANY ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

SPECIAL TREATMENTS AND/OR PHYTOSANITARY CERTIFICATE IS NEEDED CHECK WHETHER IT IS IN-COUNTRY OR EXPORT DISTRIBUTION Can be distributed on Standard MTA? Can be distributed using a tailored MTA? Does it involve seed export? OBTAIN IMPORT PERMIT Are special treatments required? Has Import Permit been received? Is Import Permit necessary? DISPATCH THE SAMPLES FILE THE INFORMATION YES NO NO YES NO YES YES NO YES YES YES NO NO Is a phytosanitary certificate needed? SEND SAMPLES TO PLANT QUARANTINE FOR SPECIAL TREATMENTS AND PHYTOSANITARY CERTIFICATE Flowchart 7.1. Germplasm distribution. 98 Handbooks for Genebanks No. 8 Procedures for in-country seed distribution Step 1: Decide whether the accession can be distributed • Check the inventory database to see if the seed quantity in the genebank is sufficient ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

for distribution

Seed Cleaning

Samples are prepared for safety duplication in the same way as the base collection: • Seeds should be dried to a moisture content of 5±2% depending on species. • Seeds should be clean and healthy. • Percent germination should be greater than 85%. • Seeds should be hermetically sealed in appropriate containers. Sample size can be smaller, but it should be sufficient to conduct at least three regenerations (with the safety factor built in). To save time, samples for safety duplication ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

may be prepared simultaneously when processing seeds for base collection. Specific agreements should be made with the recipient institute for holding the duplicate of a collection. Ideally, duplicate collections should be held in the same conditions as base collections to ensure long-term survival, although several types of duplication are recognized:

 • Black box: when the sole responsibility of the recipient genebank is to maintain the duplicates without handling them. Beyond providing the best possible storage conditions, the recipient institute has no further responsibility for the samples. It is the originator‘s responsibility to establish a viability monitoring scheme and regenerate the collection when necessary. If the storage conditions for the backup collection are the same as for base collection, loss of viability can be predicted from the results of base collection-monitoring. Following regeneration of the sample in the base collection, the originator also replaces the safety duplicate. For black-box duplication outside the country, special permission is required to export ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

seeds without phytosanitary certificates from the originating country. Similarly, the phytosanitary authority in the destination country must permit the recipient to import seeds without the routine quarantine examination. • Base: maintained under suitable condition for long-term storage and incorporated into the recipient collection. • Active: when the duplicate collection is incorporated into the recipient’s collection, and is therefore subject to regeneration, multiplication and distribution by the recipient. 95 Manual of Seed Handling Archive collection Genebanks may choose to store germplasm samples that do not need to be represented in a base collection or distributed as an ‘archive collection’. These samples are maintained under optimal conditions for long-term survival but with no further investment in monitoring and regeneration. Germplasm included to archive ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

collection can be: • experimental lines bound by intellectual property (IP) rights— samples can be held as black-box collections and returned upon request to the IP holder; • germplasm that lies outside the genebank mandate—samples can be stored temporarily until another genebank with a relevant mandate is identified; • accessions identified as duplicates following rationalization of an existing base collection; and • accessions no longer needed in the collection following a reassessment of the genebank’s mandate or material disbanded due to lack of funding. Documentation Proper documentation of seed packaging and storage procedures allows rapid accession of new samples; response to queries regarding conserved germplasm; and monitoring of ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

the quality and quantity of stored material to carry out regeneration and distribution. Suggested descriptors include the following: • Storage conditions/collection type • Type of container, if this varies in the genebank • Number of containers • Total amount of seeds stored (by weight or number) • Date of storage • Location in genebank • Minimum amount of seed allowed (base unit) for dissemination/ regeneration • Location of safety duplicate, if available Further reading Cromarty A.S, Ellis, R.H. and Roberts, E.H. 1982. The design of seed storage facilities for genetic conservation. ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

IBPGR, Rome. Engels, J.M. and Visser, L. (eds.). 2003. A guide to effective management of germplasm collections.

Seed Processing

A genebank may opt to maintain one sample of each accession for both conservation (in a base ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

collection) and utilization (in an active collection) as long as the maintenance cost is not too high. If the genebank maintains both base and active collections, it is more cost effective to store only those accessions in the active collection that are being used by breeders and other users. (For further information, see Engels and Visser, 2003.) Type of storage facility The two commonly available ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

options for seed storage are walk-in cold stores and freezers. The choice depends on the number of accessions to be stored, seed size and storage temperatures selected. When collections are small and sub-zero temperatures are required, chest or upright freezers are a cheaper option for seed storage. How is storage space organized? The organization of storage space depends on the type of storage facility and the type of containers used in the genebank. In view of the cost of maintaining cold storage, the space should be optimized so that a maximum number of seed accessions can be stored. Walk-in cold storage If the genebank has a walk-in cold store, the best option is to use 93 Manual of ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

Seed Handling moveable racks that maximize storage space. Each rack is divided into a number of shelves. The distance between each shelf will depend of the size of containers. Small containers or aluminium foil bags can be held in boxes or trays and placed on the shelves. A coding system can help genebank staff to locate accessions readily for sample retrieval; coding can be computerized in a database or stock inventory system. For example, ‘A010201’ could be used to indicate the following location: • Room number (if more than one storage room is being used): A • Rack number: 01 • Shelf number: 02 • Tray/box number: 01 Chest or upright freezers For genebanks using chest or upright ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

freezers, containers that fit onto shelves or boxes holding individual small containers can be used to store accessions. As for cold storage, a coding system to help locating accession can be established, including freezer number, row number and box number. Storing seed samples Step 1: Check the number of seeds in the accession 1. Weigh the seeds of each accession. Convert the seed weights to numbers by using the 100-seed or 1000-seed weight as described in the previous section. 2. Verify if the sample contains more than the required number of seeds for a genetically homogenous sample (3000–4000 seeds) or a genetically heterogenous sample (4000–12 000 seeds). 3. If the sample contains less than the required amount, either proceed directly to regeneration or store temporarily in the genebank and regenerate at the earliest opportunity (see Chapter 8). Step 2: Identify a location for storage The next step is to determine the location inside the storage room or freezer in which the accession will be stored. 1. ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

2. Assign the space where the accession is to be placed. If the accession is stored in more than one container, keep them all together. Step 3: Place seeds in store 1. Make a list of assigned spaces where each accession will be placed. 2. Place the containers in the storage room or freezer in their assigned locations. 94 Handbooks for Genebanks No. 8 Step 4: Enter data into the database 1. Enter the data regarding storage location, date and number of containers in the inventory file. Safety duplication (security backup collection) Safety duplication means a genetically identical sub-sample of the accession is stored at another location (preferably outside the country) to provide insurance ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

against loss of material. Safety duplication includes both the duplication of material and its related information.

Seed Processing

Some precautions • Do not mix seeds harvested in different seasons as the quality and longevity of the samples can be different. Assign batch numbers (indicating season of harvest, site or field number and generation number) to differentiate the seed lots. • Keep seeds from different seasons in separate containers or within the same container using separate cloth or re-sealable plastic bags if the container can accommodate them. • Remember that containers removed from cold storage or freezers should be allowed to warm to room temperature before opening to avoid condensation of water on the seed surface. This may take several hours, especially for large seeds and those removed from sub-zero temperatures. • The self-adhesive labels and ink used for labelling must be water resistant and very durable. To ensure long-term conservation and continued availability of high-quality seeds for utilization, seeds packed in moisture-proof containers should be stored under controlled environmental conditions as described in the following section. Table 6.1. Model table for recording information on seed packaging. Date of packaging: Name of staff: Accession number Type of container Container number ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

Weight of container empty Weight of container and seeds Weight of seeds 1 2 3 91 Manual of Seed Handling Further reading FAO/IPGRI, 1994. Genebank standards. FAO and IPGRI, Rome. 6.2 Seed storage What is seed storage? Seed storage is the preservation of seeds under controlled environmental conditions that maintain seed viability for long periods. The longevity of seeds ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

depends on initial seed quality, moisture content and temperature during storage. In general, low moisture content and low temperature reduce the loss of seed viability. Different combinations of moisture content and temperature can be used to prolong seed viability during storage. Types of storage Two types of seed stores are used for conservation of genetic resources: those holding seed samples for long-term security—referred to as base collections—and those holding seed samples for vimmediate use— referred to as active collections. The temperature, RH, seed moisture content, containers and distribution arrangements of these stores vary. Base collections A base collection is a set of accessions in which each is distinct and as close as possible to the original sample in terms of genetic integrity. Normally, seeds are not distributed from base collections directly to users but are only used to regenerate active collections (FAO/IPGRI, 1994). Base collections are stored for long periods at below 0°C—usually at -18° to -20°C—to maintain seed viability. Engels and Visser (2002) introduced the term ‘most-original sample’ (MOS) to qualify the samples in base collections. A MOS consists of seeds that have undergone the lowest number of regenerations since the material was acquired by the genebank; it may be a sub-sample of the original seed lot or a seed sample from the first regeneration cycle if the original seed lot required regeneration before storage. Active collections Active collections consist of accessions that are immediately available for distribution. ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

These accessions are accessed frequently and maintained in conditions that ensure at least 65% viability for 10–20 years (FAO/IPGRI, 1994). Combinations of temperatures and moisture contents for storage of active collections that can assure viability above 65% for 10–20 years are given in Table 6.2. It is more practical to use a lower moisture content and store at a higher temperature to save on refrigeration costs. However, when drying 92 Handbooks for Genebanks No. 8 to the low moisture contents is not possible, storage with higher moisture but at a lower temperature can be considered. Table 6.2. Suggested storage temperature and moisture content for active collections (source: ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

Bioversity, unpublished). Temperature (°C) Storage characteristics Poor (e.g. onion) Good (e.g. barley) Moisture content (% wet basis) 25 3 7 20 3.5 7.5 15 5.0 8.0 10 6.0 9.0 5 7.0 10.0 0 8.0 11.0 Organization of collections The underlying principle of maintaining a base collection or MOS is that at least some seed of the original sample should be kept aside under the best possible conditions to ensure safe long-term survival. This may be achieved by keeping seeds for distribution physically separate (as active collections) from the original sample but there is no absolute requirement to do so.

Seed Processing

For materials showing large morphological variation (genetically heterogeneous accessions), an accession should consist of at least 4000 seeds, but 12 000 seeds are ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

preferred. In genebanks, it is easier to work in weights, but seed number can easily be converted from weights if the 100-seed or 1000-seed weight is known. For example, to determine the number of seeds in a sample for which the 100-seed weight is known: Number of seeds in the sample = Sample weight (g) x 100 Weight of 100 seeds (g) Laminated aluminium foil packets are the most commonly used containers in genebanks as they occupy little space and it is easy to reseal them. Aluminium foil packets to be used in genebanks should have the following specifications: • an outer layer of 17 g m-2 Melinex, 4 g m-2 lacquer; • a middle layer 33 g m-2 (12 µm) aluminium foil, 4 g m-2 lacquer; and • an inner layer of 63 g m-2 polyethylene. 89 Manual of Seed Handling How should seeds be packaged? Packaging is best carried out in an air-conditioned room where the RH is ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

controlled. It is important to ensure that seeds taken from the drying room are exposed to ambient air for the shortest time possible so that they do not re-absorb water. 1. Decide on the most suitable container for storing the seeds. Different types of containers can be used depending on the size and shape of the seeds, and the purpose of conservation (whether for base or active collections—see section 6.2). 2. Prepare and label the containers for each accession; computergenerated self-adhesive labels and barcodes11 are now being used in many genebanks. Bar-coding ensures that information is accurate and that no mistakes occur during transcription. Prepare a label to include with the seeds inside the container. Labels should contain the following information: • Accession number • Genus and species • Container number • Weight of seeds • Date of storage 3. Weigh out ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

each empty labelled container. 4. Fill the containers with the seeds and weigh again. Calculate the actual weight of the seeds. 5. Add the label and seal the container immediately to protect the seeds from high ambient RH. 6. Check the quality of each container after sealing it by making a visual examination to ensure that there is no leakage. 7. Any container found to be below standard should be replaced immediately. 8. Move the containers into the storage room. 11 Bar-coding is a computerized coding system that uses a printed pattern of bars of varying widths to uniquely identify accessions. Bar-codes are read by optically scanning the printed pattern and using a computer program to decode the pattern. The data contained in a bar-code can vary: in its simplest form, it can ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

be just an accession number, while in other cases, the bar-code can hold more elaborate passport and inventory details. Bar-coding provides enormous benefits to genebanks by allowing data capture that is faster and more accurate to minimize errors and facilitate inventory management. Example: Sample weight = 275 g Weight of 100 seeds = 12.5 g Total number of seeds in the sample = 275 x 100 = 2200 12.5 90 Handbooks for Genebanks No. 8 9. Enter relevant data about each accession into the data file. A model for recording container and seed weight is shown in Table 6.1. Reference samples (seed herbaria) Seed herbaria are useful to check physical attributes of seeds without having to open sealed containers. Pack a small sample (five to ten seeds or pods of legumes, or 5 g of cereals) of the original seeds separately in a transparent re-sealable plastic envelope or glass vial to verify genetic ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

integrity after regeneration and during seed transfer. The samples can be stored in a cabinet with closely spaced shelves. Ensure that seeds of the original sample are never completely exhausted so that they may serve as a reference for identification. 

Seed Processing

Dry seeds will reabsorb moisture from more humid ambient air. Therefore, seeds should be packaged in waterproof containers and hermetically sealed without delay following removal from the drying room or cabinet. Types of container Different types of container are available for packaging; the choice depends on storage conditions and species. It is important that the packing material be completely impermeable to water and suitable for long-term use. Frequently used containers include glass bottles, aluminium cans, laminated aluminium foil packets and plastic bottles. Different types of container each have advantages and disadvantages. Glass bottles are good ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

but can easily break. Aluminium cans are difficult to reseal once they have been opened. Aluminium foil packets can be resealed and occupy less space than other containers, but seeds with 1. Introduction 2. Germplasm acquisition and registration 2.1 Germplasm acquisition 2.2 Germplasm registration 3. Seed cleaning 4. Seed moisture content determination and drying 4.1 Seed moisture content determination 4.2 Seed drying 5. Seed quality testing 5.1 Seed viability testing 5.2 Seed health testing 5.3 Seed testing for inadvertent introduction of transgenes 6. Seed packaging and storage 6.1 Seed packaging 6.2 Seed storage 7. Germplasm distribution 8. Germplasm monitoring and regeneration 8.1 Germplasm monitoring 8.2 Germplasm regeneration 6. Seed storage 87 Manual of Seed Handling VISUALLY CHECK THE CONTAINERS PREPARE INSIDE AND OUTSIDE LABELS WEIGH ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

OUT SAMPLE OF SEEDS TO FIT THE CONTAINER Is the container below standard? DISCARD IT YES CHECK WHETHER THE ACCESSION SHOULD BE PLACED IN MORE THAN ONE CONTAINER LABEL THE CONTAINER(S) SEAL THE CONTAINERS Will the accession be placed in one container? PRINT OUT THE ADDITIONAL LABELS NEEDED NO VISUALLY CHECK IF EACH CONTAINER IS CORRECTLY SEALED Does the container have a lEAk PLACE THE CONTAINERS IN THE STORAGE ROOM FILE DATA OF EACH ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

ACCESSION NO YES NO YES Flowchart 6.1. Seed packaging. PROCEED TO FILL THE CONTAINERS AND ADD THE INSIDE LABEL sharp projections can pierce them and moisture can leak inside. Plastic bottles and aluminium cans with lids are moisture resistant but not moisture proof unless they have a tight rubber seal. They should be used with caution if the RH of the storage room is not controlled. 88 Handbooks for Genebanks No. 8 Testing quality of containers The quality and sealing capacity of containers can be tested as follows: 1. Fill the containers with regenerated self-indicating silica gel and seal it in the same way in which seeds are stored. 2. Accurately determine the weight of the containers with an analytical balance. 3. Hold the containers over water (but not touching it) in a desiccator for about a week. 4. Remove the containers from the desiccator and allow the surface to dry. 5. Weigh the containers, record the change in weight and examine the colour of the silica gel. • If the weight of the containers remains constant, then they are moisture proof and the seal is good. • If ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

the weight of the containers increases and the silica gel has turned pale blue or pink, then they are of poor quality or the seal is leaking moisture. 6. Adjust the seal and repeat the test to confirm the quality of the containers. A container’s quality can also be tested by filling it with water and holding it over silica gel in a desiccator or in a ventilated oven at 40°C for one to two weeks. A change in the container’s weight indicates poor quality or leakage in sealing. How many seeds should be packed? The number of seeds to be packed for storage will depend on the species being conserved and how often seeds will be removed for monitoring, distribution or regeneration. The FAO/IPGRI Genebank Standards (1994) recommend that for material which shows little morphological variation (genetically homogenous accessions), 3000 seeds are acceptable, but 4000 seeds are preferable to represent each accession. 

Seed Cleaning

84 Handbooks for Genebanks No. 8 Germplasm is most at risk from gene flow during regeneration (see Chapter 8) and controlling gene flow is essential to ensure genetic integrity. To reduce the risk in crops where transgenes are commonly part of new cultivars, it is recommended that regeneration be carried out in isolation from any areas where transgenic crops are likely to be grown. Information on crops’ transgenic status is essential to determine what measures, if any, are needed to confirm that germplasm is free of transgenes. It is recommended ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

that: • all results be made publicly available as soon as they have been confirmed; • all procedures and supporting information be presented; • the appropriate authority in the country of origin be informed in cases where transgenes are detected; and • for commercially released genetically modified crops and crops in experimental development, genebanks maintain a database of crops and their status in transgenic research. Once an accession has either been determined to not require testing or has tested negative, follow appropriate regeneration and maintenance procedures to maintain genetic integrity, as for all accessions. Procedures for testing for presence of GMOs The two basic methods to detect the presence of a gene/transgene are ELISA and PCR amplification. Both methods have already been described and are robust, although each has advantages and disadvantages. For example, ELISA detects the presence of a gene product (protein) and thus requires an expressing gene. Test kits are commercially available for most commercial events, which ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

can be used in the field. On the other hand, PCR can detect non-expressing gene sequences, in almost all tissues, but it is more difficult to perform and therefore not practical in the field. In most cases, the detection of a positive result using one method should be confirmed with a second method. If the materials are being analyzed at the molecular level for fingerprinting or diversity studies, an additional test for the presence of a transgene can be performed at minimal cost. The genes/transgenes that should be used in such tests include the current commercialized major events for the species. These can normally be found on the Internet and are indicated in the tests provided by commercial testing services (either as ELISA kits or PCR services). These will change as new transgenic events are introduced into the market or events become obsolete and are 85 Manual of ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

Seed Handling removed, although the need to test may continue for some time. The number of seeds in any accession may limit the level of detection. More information and technical guidance on sampling and detection of GMOs can be found at www.europa.eu.int/comm/environment/ biotechnology/pdf/recom2004_787.pdf. An updated list of validated methods is also available at http://biotech.jrc.it. Documentation Suggested descriptors to document accession-level information on the presence of transgenes include the following: • Source of the material for testing • Type of material (leaf, seedling, seed) • Number of plants sampled and tested per replicate • Number of ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

replicates • Transgenes tested • Method of testing • Date of test • Duration of test, if appropriate • Transgenes identified • Incidence of each transgene (%) 86 6. SEED PACKAGING AND STORAGE 6.1 Seed packaging What is meant by seed packaging? Seed packaging involves placing a counted or weighed sample of seeds into a container, which is then hermetically sealed for subsequent storage (see Flowchart 6.1). Why are seeds packaged? Seeds are packaged to: • prevent absorption of water from the atmosphere after drying; • keep accessions separate and avoid mixing them; and • prevent ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

contamination from insects and diseases. When should seeds be packaged? The best time to package seeds is immediately after moisture content has been determined to be within the required limits for safe storage. 

Seed Cleaning

There are a number of reasons why it is important to detect the presence of a gene/transgene in a genebank accession. While not an exhaustive list, these include: • regulatory issues, especially related to phytosanitary or biosafety, where the country of import, and potentially the country of ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

export, requires reporting on the presence of such genes; • situations in which the presence of such a gene/transgene could affect intellectual property rights either in the country where the genebank is located or in a country where the accession is to be sent; and • social issues necessitating that genetic identity be stated or that certain genes/transgenes should be limited. 

When should one detect the presence of a gene/transgene? It is generally accepted as unwise for crops containing transgenes to be incorporated into germplasm collections. The risk of inadvertent inclusion of transgenes can be classified as follows: • High probability: typically out-crossing crops with sexually compatible relatives on which extensive research in the field or commercial release is ongoing. • Low ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

probability: typically crops which are highly self-pollinating, multiplied vegetatively or crops for which genetic engineering has either not been done or is in its very early stages. 83 Manual of Seed Handling • Medium probability: the remainder of crops. • Immediate attention: crops with transgenes that are already commercially distributed. • Near-future attention: experimental field work is ongoing or expected within one to three years. • Long-term attention: crops for which no significant work has been done in the field. Genebanks should take proactive steps to limit the risk of exotic genes, including transgenes, in their ex situ collections. Accessions that do not require testing include: • species where no transgenic events (commercial or research) have occurred; • accessions for which there were no commercial transgenes present at the time of acquisition (such as maize ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

prior to 1996) or no transgenics near the collecting site; and • accessions for which there have been transgenic events, but good management practices have been followed in the accession process. In 2004, the Genetic Resources Policy Committee (GRPC) and the Science Council of the CGIAR organized a technical workshop to explore the ways and means to handle unintentional presence of transgenes in germplasm collections, with the goal of providing technical inputs into a process that would enable CGIAR Centre genebanks to draw up procedures aimed at preventing the unintentional introgression of transgenes into the collection. Further to this workshop, a guiding principle was prepared and adopted by the GRPC. For more information on this subject consult the Policies and Ethics section of Bioversity's website (www. ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

bioversityinternational.org/About_us/Policies_and_Ethics/index. asp; last visited 20 December 2006). These guiding principles were also considered at the Third Session of the Intergovernmental Technical Working group on Plant Genetic resources for Food and Agriculture held at FAO Rome October 26-28 October 2005. Further information about that meeting is available from the Commission web site http://www.fao.org/waicent/FaoInfo/Agricult/AGP/AGPS/ pgr/ITWG3rd/docsp1.htm Procedures to prevent unintentional gene flow from genetically modified organisms (GMOs) Transgenes and conventional genes are subject to the biological processes of mutation, gene flow, introgression, recombination and natural selection. Therefore, best practices for preventing introgression of conventional genes also provide an appropriate basis for preventing ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

introgression of transgenes. 

Seed Cleaning

PCR is especially useful to detect diseases because of its speed and accuracy, but it is an expensive technique—it can be used to detect any organism that has DNA by using positive and negative controls for comparison. Once the sequence of the organism is known, specific probes can be made to detect strains of pathogens. Nucleic acid hybridization assays (called southern and northern blotting), in which DNA or RNA is transferred from an electrophoresis gel onto a membrane and then the nucleic acids are detected with a labelled probe, can also be used. 

The nucleic acid spot hybridization (NASH) technique, in which a labelled DNA pathogen hybridizes directly to the pathogen DNA immobilized on a nylon membrane, can also be used 81 Manual of Seed Handling without going through the PCR stage. These techniques are constantly being refined and new procedures are becoming available for specific pathogen detection. For more information, ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

refer to Albrechtsen (2005). Serological and other methods Enzyme-linked immunosorbent assay (ELISA) ELISA is a diagnostic method that uses proteins called antibodies to detect plant pathogens. This assay is based on the ability of an antibody to recognize and bind to a specific antigen—a substance associated with a plant pathogen. The antibodies used in diagnostics are highly purified proteins produced by injecting a warm-blooded animal (like a rabbit) with an antigen associated with a particular plant disease. The animal reacts to the antigen and produces antibodies, which recognize and react only with the proteins associated with the causal agent of that plant disease. Colour changes on the unit’s surface indicate a positive reaction (disease present). There are many different types of ELISAs that can detect the presence of protein. A detailed description of these is beyond the scope of this publication and genebank staff members are advised to refer to Albrechtsen (2005). However, the general procedures for two most common methods—antigen-coated plate (ACP-ELISA) ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

and tissue-blot immunoassay (TBIA)—are given in Annex II. For more details, refer to Lin et al. (1990). Indicator plant method This is especially useful for detecting bacteria and viruses. Seed extracts are prepared and inoculated on indicator plants like tobacco. The pathogens are identified based on the symptoms that develop. Indicator plants can also be used to separate different viruses by virus-host specificity. Documentation Suggested descriptors to document accession-level information on seed health-testing include the following: • Source of the material for testing • Type of material (leaf, stem, root, seeds) • Number of plants sampled and tested per replicate • Number of replicates • Organisms tested for • Method of testing • Date of test • Duration of test, if appropriate • Diseases identified • Incidence of each disease (%) 82 Handbooks for Genebanks No. 8 Further reading Albrechtsen, S.E. 2005. Testing methods for seed-transmitted viruses: Principles and protocols. Oxford University Press, Oxford, UK. ISTA. 2005a. International Rules for Seed Testing. Edition 2005. ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

International Seed Testing Association, Bassersdorf, Switzerland. Lin, N.S., Hsu, Y.H. and Hsu, H.T. 1990. Immunological detection of plant viruses and mycoplasm-like organisms by direct-tissue blotting in nitrocellulose membranes. Phytopathology, 80: 824–828. 5.3 Seed testing for inadvertent introduction of transgenes What are transgenes? Transgenes are genes that are introduced into another organism or species through recombinant DNA techniques. Transgenic plants carry transgenes in their genomes and transmit them to their progeny through normal reproduction. Why determine the presence of a gene/transgene? One of the most important components of proper genebank management is testing for the presence of a gene or phenotype. This is critical for various phytosanitary requirements, but is also becoming important for the detection of transgenes. 

Seed Cleaning

Staining pattern after tetrazolium test in monocot seeds. Numbers 1–4 are germinable seeds and numbers 5–9 are nongerminable seeds (adapted from AOSA, 2005). 1 2 3 4 5 6 7 8 9 78 Handbooks for Genebanks No. 8 Why is seed health testing important? Crops are frequently infected with a range of common seed-borne pathogens that may not be visible or easily recognized during seed collection. Seed-borne inoculums reduce storage longevity and cause poor germination or field establishment. Seed-borne inoculums also promote disease in the field, reducing the value of crops. ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

Exchange of infected seeds may allow spread of diseases and pests into new regions. Genebanks should ensure that seeds prepared for conservation are free from seed-borne diseases and pests. Common seed-borne pests and pathogens There are four main types of common organisms that are carried in seeds and affect a wide range of crops: • Fungi • Bacteria • Viruses • Insects Specific methods for detecting pathogens vary by organism and host, and specific methods are required for accurate identification of most pathogens. Methods of detecting pests and pathogens Seed-health standard Examine a representative sample of seeds for the presence of pathogens using one or more of the following methods. Usually, a sample of 400 seeds in replication of 100 seeds each is drawn for examination. Sample size can be decreased for small seed lots. If the percentage of seeds infected is greater than 5%, the seed lot can be considered unsuitable for conservation. Visual examination The simplest method to detect diseases and pests is to examine dry seeds with the naked eye or under a low-powered microscope. This method reveals freely moving insects, eggs, mites, fungal ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

fructifications like sclerotia, galls, smut balls, bacterial masses and infected plant debris. Examination of dry seeds under ultraviolet or near-ultraviolet light reveals infections of certain fungi and bacteria through emission of fluorescence. Seedling evaluation Seeds should be planted in sterilized soil in a screenhouse. Seedlings should be observed immediately after germination and any plants exhibiting virus-like symptoms such as leaf mottling, curling or yellowing should be sampled and tested for viruses (see 79 Manual of Seed Handling below). Seedlings infected with bacteria or fungi may die and should be examined further in a laboratory, and the samples should be plated for identification of the pathogen (see below). If infection is suspected but no symptoms have been observed after the ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

second true leaf has emerged, it may be necessary to carry out serological tests for latent or symptom-less infection by viruses. 

Seed Processing

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Documentation Documenting viability data is crucial for the efficient management of germplasm collections as it enables genebank staff to make informed decisions regarding the timely regeneration of material (see Chapter 8). Suggested descriptors to document accessionlevel information on viability (germination) testing include the following: • Number of seeds tested per replicate • Number of replicates • Method of germination testing • Date of germination testing • Duration of testing (or days of first and final counts) • Number of germinated seeds at first count Table 5.3. Concentration, temperatures and period of staining with tetrazolium solution (for Annex I crops of the International Treaty on PGRFA). Crop Species Preconditioning Staining Barley Hordeum vulgare Imbibe or soak, 6–18h 0.5%, 3h, 30°C Beans Phaseolus spp. Imbibe 18–24h, then soak, 2–3h 0.5–1%, 6–24h, 30°C Brassica Brassica spp. Imbibe or soak, 16–18h 0.5–1%, 3–6h, 30°C Chickpea Cicer arietinum Imbibe or soak, 18h 1%, 6–24h, 30°C Cowpea Vigna unguiculata Soak, 22h 0.5–1%, ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

16–24h, 30°C Eggplant Solanum melongena Imbibe or soak, 18h 0.5–1%, 6–24h, 30°C Faba bean Vicia faba Soak, 22h 0.5–1%, 16–24h, 30°C Finger millet Eleusine corocana Soak, 18h, 5°C 0.5%, 3h, 30°C Lentil Lens culinaris Imbibe, 18h, then soak, 2–3h 1%, 6–24h, 30°C Maize Zea mays Imbibe or soak, 18h 0.5–1%, 2–6h, 30°C Pea Pisum sativum Imbibe 18–24h, then soak, 2–3h 0.5–1%, 6–24h, 30°C Pearl millet Pennisetum glaucum Imbibe or soak, 6–18h 0.5–1%, 6–24h, 30°C Rice Oryza sativa ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

Imbibe or soak, 18h 0.5%, 3h, 30°C Rye Secale cereale Imbibe or soak, 6–18h 0.5%, 2–3h, 30°C Sorghum Sorghum bicolor Imbibe, 16h, 30°C 0.5–1%, 0.5–1h, 40°C Sugar beet Beta vulgaris Imbibe or soak, 16–18h 1%, 24–48h, 30°C Sunflower Helianthus annuus Imbibe or soak, 18h 0.5–1%, 3–6, 30°C Triticale Triticosecale Imbibe or soak, 6–18h 0.5%, 2–4h, 30°C Wheat Triticum aestivum Imbibe or soak, 6–18h 0.5%, 2–4h, 30°C 76 Handbooks for Genebanks No. 8 • Dormant/hard seeds at first count (%) • Special treatments for dormancy breaking (if any) • Final germination (% normal seedlings) • Abnormal germination (%) • Dead seeds (%) • Tolerance levels for statistical accuracy Further reading Association of Official Seed Analysts. 2005. Rules for testing seeds. Association of ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

Official Seed Analysts, USA. Baskin, C. C. and Baskin, J. M. (1998) Seeds: Ecology, Biogeography and Evolution of Dormancy and Germination. Academic Press, San Diego, USA. Ellis, R.H., Hong, T.D and Roberts, E.H. 1985. Handbook of seed technology for genebanks. Volume 1: Principles and methodology. Handbooks for Genebanks. No. 2. IBPGR, Rome, Italy. FAO/IPGRI, 1994. Genebank standards. FAO and IPGRI, Rome, Italy. ISTA. 2003. ISTA Handbook for Seedling Evaluation. International Seed Testing Association. Bassersdorf, Switzerland. Figure 5.9. Staining pattern after tetrazolium test in dicot seeds. Illustrations depict both sides of seeds. Numbers 1–6 are germinable seeds and numbers 7–15 are nongerminable seeds (adapted from AOSA, 2005). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 77 Manual of Seed Handling ISTA. 2005. International Rules for Seed Testing. Edition 2005. International Seed Testing Association, Bassersdorf, Switzerland. Smith, R.D., Dickie, J.B., Linington, S.H., Pritchard, H.W and Probert, J.R. (eds.). 2003. Seed conservation: Turning science into practice. Royal Botanic Gardens, Kew, UK. 5.2 Seed health testing What is seed health? Seed health refers to the disease status of a seed sample and the presence or absence of disease-causing organisms and pests. What is seed health testing? Seed health tests determine the status of a seed sample, seed lot or accession with regard to diseases affecting that crop or wild species. Figure 5.10. ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )