Genetic modification refers to techniques used to manipulate the genetic composition of an organism by adding specific useful genes. A gene is a sequence of DNA that contains information that determines a particular characteristic/trait. All organisms have DNA (genes). Genes are located in chromosomes. Genes are units of inheritance that are passed from one generation to the next and provide instructions for development and function of the organism. Crops that are developed through genetic modification are referred to as genetically modified (GM) crops, transgenic crops or genetically engineered (GE) crops.
The main steps involved in the development of GM crops are:
- Isolation of the gene(s) of interest: Existing knowledge about the structure, function or location on chromosomes is used to identify the gene(s) that is responsible for the desired trait in an organism, for example, drought tolerance or insect resistance.
- Insertion of the gene(s) into a transfer vector: The most commonly used gene transfer tool for plants is a circular molecule of DNA (plasmid) from the naturally occurring soil bacterium, Agrobacterium tumefaciens. The gene(s) of interest is inserted into the plasmid using recombinant DNA (rDNA) techniques. For additional information see Plasmids link
- Plant transformation: The modified A. tumefaciens cells containing the plasmid with the new gene are mixed with plant cells or cut pieces of plants such as leaves or stems (explants). Some of the cells take up a piece of the plasmid known as the T-DNA (transferred-DNA). The A. tumefaciens inserts the desired genes into one of the plant’s chromosomes to form GM (or transgenic) cells. The other most commonly used method to transfer DNA is particle bombardment (gene gun) where small particles coated with DNA molecules are bombarded into the cell. For additional information see Plant Transformation using Agrobacterium tumefaciens and Plant Transformation using Particle Bombardment links.
- Selection of the modified plant cells: After transformation, various methods are used to differentiate between the modified plant cells and the great majority of cells that have not incorporated the desired genes. Most often, selectable marker genes that confer antibiotic or herbicide resistance are used to favor growth of the transformed cells relative to the non-transformed cells. For this method, genes responsible for resistance are inserted into the vector and transferred along with the gene(s) conferring desired traits to the plant cells. When the cells are exposed to the antibiotic or herbicide, only the transformed cells (containing and expressing the selectable marker gene) will survive. The transformed cells are then regenerated to form whole plants using tissue culture methods.
- Regeneration into whole plants via tissue culture involves placing the explants (plant parts/cells) onto media containing nutrients that induce development of the cells into various plant parts to form whole plantlets (Figure 1). Once the plantlets are rooted they are transferred to pots and kept under controlled environmental conditions.
- Verification of transformation and characterization of the inserted DNA fragment. Verification of plant transformation involves demonstrating that the gene has been inserted and is inherited normally. Tests are done to determine the number of copies inserted, whether the copies are intact, and whether the insertion does not interfere with other genes to cause unintended effects. Testing of gene expression (i.e., production of messenger RNA and/or protein, evaluation of the trait of interest) is done to make sure that the gene is functional.
- Testing of plant performance is generally carried out first in the greenhouse or screenhouse to determine whether the modified plant has the desired new trait and does not have any new unwanted characteristics. Those that perform well are planted into the field for further testing. In the field, the plants are first grown in confined field trials to test whether the technology works (if the plants express the desired traits) in the open environment. If the technology works then the plants are tested in multi-location field trials to establish whether the crop performs well in different environmental conditions. If the GM crop passes all the tests, it may then be considered for commercial production.
- Safety assessment. Food and environmental safety assessment are carried out in conjunction with testing of plant performance. Descriptions of safety testing are described in the Food Safety Assessment and Environmental Safety Assessment links.
Figure 1: Regeneration of transgenic banana using tissue culture method