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.