Crop Genebank Knowledge Base

Transgene detection

View section on seed testing for inadvertent introduction of transgenes by clicking on the icon above. (0.7MB)

Page compiled by: Bioversity International/ILRI, Addis Ababa, Ethiopia (Alexandra Jorge); ILRI, Addis Ababa, Ethiopia (Jean Hanson) including information extracted from: Rao NK, Hanson J, Dulloo ME, Ghosh K, Nowel D and Larinde M. 2006. Manual of seed handling in genebanks. Handbooks for Genebanks No. 8. Bioversity International, Rome, Italy. 147pp.

(More information on Transgenes can also be found at the genetic identity page under Management strategies).

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. 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 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.
Social issues requiring genetic identity to be stated or that certain genes/transgenes be limited.

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 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.
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 prior to 1996) or no transgenics near the collecting site.
Accessions for which there have been transgenic events, but good management practices have been followed in the accession process.

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 introgression of transgenes.

Germplasm is most at risk from gene flow during regeneration (see also information on Regeneration) 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 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.
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 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 material is 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 removed, although the need to test may continue for some time. The number of seeds in any accession may limit the level of detection.

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 replicates.
Transgenes tested.
Method of testing.
Date of test.
Duration of test, if appropriate.
Transgenes identified.
Incidence of each transgene (%).

References and further reading

Guiding principle of procedures aimed at preventing the unintentional introgression of transgenes into the collection was prepared and adopted by the Genetic Resources Policy Committee (GRPC) and the Science Council of the CGIAR in 2004 (and revised by FAO in 2005). For more information on this subject consult the document available from: http://www.cgiar.org/corecollection/docs/Guiding_Principles_Ex_Situ_Collections_Apr_2007.pdf or the FAO website at: www.fao.org/biotech/news_list.asp?thexpand=1&cat=131. Date accessed: 26 March 2010.

More information and technical guidance on sampling and detection of GMOs is available from: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32004H0787:EN:HTML. Date accessed: 26 March 2010.

An updated list of validated methods is also available from: http://mbg.jrc.ec.europa.eu/. Accessed 24 March 2010.

CIMMYT. 2005. Laboratory Protocols: CIMMYT Applied Molecular Genetics Laboratory. Third Edition. Mexico, D.F.: CIMMYT.
ISBN: 9686923306. Available from: www.cimmyt.org/english/docs/manual/protocols/abc_amgl.pdf. (1.9MB).

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