Creation of GMOs

Genetically Modified (GM) crops are typically created by integrating foreign DNA into a plant’s genome. The process of inserting DNA into an organism is called transformation. Several methods can be used to transform plants, as described below. Even under ideal conditions a transformation event is relatively rare. To assist with the identification of successfully transformed plants, the gene of interest is often transformed in along with a marker gene. Antibiotic resistance genes are often used as marker genes.

Transformation using Agrobacterium tumefasciens

Arguably the simplest and most preferable transformation method is using a species of bacteria, Agrobacterium tumefasciens. Agrobacterium naturally transforms its host plants with DNA that causes tumors or galls to grow on the host. It accomplishes this by altering hormone levels in the host plant. The tumorous growth produces ideal tissue for the bacteria to infect. In order to use Agrobacterium for plant biotechnology, researchers replace the tumor-inducing piece of DNA, or plasmid, with DNA that encodes the genes they want to engineer into the host plant. Depending on the host species, this type of transformation can be very simple or can be quite challenging. A general advantage of this transformation strategy is that it typically leads to only one or a few copies of the engineered DNA being introduced to the plant genome, which helps to ensure stable gene expression (i.e. the engineered genes will usually not be switched off by plant defense mechanisms). This transformation strategy also keeps DNA constructs intact, and both of these things make it easier to characterize engineered changes and obtain regulatory approval more quickly. A disadvantage of this approach is that the actual protocol often needs to be tailored to a particular plant species, and sometimes a new strain of Agrobacterium needs to be developed in order to infect particular plant species. GM plants produced by this method do not still have agrobacterium in them.

Particle bombardment

Particle bombardment, also known as the "gene gun”, is another common method for plant transformation. This strategy entails coating tiny gold or tungsten beads with the desired DNA construct. The particles are then shot at small pieces of plant tissue. This "bombardment" will lead to many copies of the DNA construct inside the plant cells, some of which will be integrated in the plant genome. An advantage of this method is that it can be used on many different plant species, including species that have not been previously transformed. Nonetheless, this method has fallen out of favor because it often leads to numerous copies of the engineered DNA construct being present in the genome, which in turn leads to unstable and unpredictable expression of the gene of interest. Most transgenic lines that suffer from these ill effects would never make it to production because all plants transformed using any method go through several rounds of traditional breeding after transformation to achieve a desirable field (as opposed to laboratory) variety.

Protoplast transformation

Plants can also be transformed by turning their cells into protoplasts, transforming the protoplasts, then regenerating these protoplasts into individual plants. Plant cells are encased by cell walls made of sugar polymers. Protoplasts are cells that have been stripped of their cell walls by enzyme treatments. Protoplasts can be made to uptake DNA in their surrounding solution by shocking them with electricity or treating them with various chemicals such as polyethylene glycol that induce osmotic stress. These protoplasts can then be regenerated into plants, often times with assistance from appropriate hormone treatments. Protoplast regeneration is a challenging and tedious process, so this method is typically only used for certain applications. When attempting to perform very precise genetic manipulations, this method is often preferred. For example, researchers have explored using site-specific recombination to pick a spot in a particular plant chromosome to integrate their DNA construct. This method is rather delicate and works most efficiently when used with protoplast transformation. A very limited number of plants, as far as we know only some species of moss, can undergo homologous recombination, a process that also enables "gene targeting" or integration at a previously designated location. Protoplast transformation is also the method of choice for this type of transformation.

Other transformation methods

The three methods mentioned above are the predominant methods used to transform plants. However, other methods can be used. Plant viruses are another method that can be used, however this is typically more difficult a species-specific.

Markerless transgenics

Typically, the use of marker genes is necessary to select for plants that have been successfully transformed. These marker genes typically take the form of an antibiotic or herbicide resistance gene (i.e. kanamycin, hygromycin or others). An alternative is to use a markerless insertion system. In some organisms homologous recombination can be used to first introduce and then excise a selectable marker with the use of a counter-selectable marker, however this approach has been unsuccessfully in many plant species. Markerless transgenics can be made in several ways. One way is to simply screen many progeny plants with PCR to look for plants that have the desired transgene. A second way is to use a selectable marker to select for the first transformation event, then use an introduced DNA recombination systems (such as Cre-Lox) to excise the selectable marker. The recombination system (from an alternate transformation event) can be segregated away from the desired transgene, resulting in a markerless transformant. The creation of markerless transgenics prevents the need to have a commercial plant containing and/or expressing an slectable marker, eliminating any risk associated with a marker and allowing for sequential transgenetics. Even is a marker gene is present in the genome, it may not be significantly expressed in a plant if that plant has not been under selection for one or more generations due to gene silencing.

Gene expression

A critical part of creating a plant with a desired trait from a transgene is to not only introduce that gene into the plant but also to ensure that the gene is expressed. Typically transgenes are integrated somewhat randomly in the genome and the location of the insertion can affect the transcription level. Alternatively, genes may be subject to gene silencing after one or more generations. Histrocially, strong promoters (such as 35S) have been used to drive transgene expression. An alternative to this is use a promoter of a strength suited to the desired expression level of a particular transgene. In addition, one can use tissue-specific promoters that express the gene only/predominately in a target tissue. For example, if a transgene prevents herbivory, it may be only necessary to express it in leaves rather than in the entire plant (as is typically the case with a strong, constutive promoter such as 35S). To accomplish this, a tissue-specific promoter can be used. This can greatly reduce / eliminate expression of transgenes in particular tissues. This technology can be use to reduce risks associated with transgenes expression in tissues used for human consumption when the transgene does not need to be expressed there - for example, if only the corn leaf is a target for pests, an anti-herbivory gene (such as Bt) need only be expressed in the leaves and not the corn ear.

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