How genetically modified crops are produced

The genetic information of an organism, encoded by the sequence of nucleotide bases within its DNA, determines the proteins it can manufacture and, through these, the physical and biological nature of the organism. Protein synthesis can be subdivided into two distinct processes: transcription and translation (Figure 13.1). Transcription is the copying of the information from a segment of DNA, often a gene encoding for a single protein, to a strand of mRNA. Translation, which takes place in association with ribosomes, involves the 'reading' of the nucleotide sequence in the mRNA molecule and the construction of the amino acid chain which forms the primary structure of the protein. Subsequent to this, the amino acid chain will fold, cross-)ink and may undergo post-)ranslational processing to form the functional protein.

GM crops usually have one or more genes added to their genetic make- up and so produce one or more extra proteins. It is these proteins that cause the crop to have different properties to those of the unmodified crop. In some cases the modification may involve

Table 13.1 Major herbicide-tolerant genetically modified crops worldwide, as of 2004 (adapted from Christou and Klee, 2004).

Crop

Trait (gene)

Country

Soybean

Glyphosate tolerance (CP4 EPSPS)

Argentina, Australia, Brazil, Canada, China, Czech Republic, EU, Japan, Korea, Mexico, Russia, Switzerland, South Africa, Taiwan, UK, Uruguay, USA

Soybean

Glufosinate tolerance (pat)

Canada, Japan, USA

Soybean

Glufosinate tolerance (bar)

USA

Maize

Glyphosate tolerance (Maize EPSPS)

Argentina, Australia, Canada, China, EU, Japan, Korea, Philippines, Taiwan, USA

Maize

Glyphosate tolerance (CP4 EPSPS)

Argentina, Australia, Canada, EU, Japan, Philippines, Taiwan, South Africa, USA

Maize

Glyphosate tolerance (CP4 EPSPS, gox)

Canada

Maize

Glufosinate tolerance (bar)

Canada, Japan, Philippines, Taiwan, USA

Maize

Glufosinate tolerance (pat)

Argentina, Australia, Canada, European Union, Japan, Philippines, Taiwan, USA

Oilseed rape

Glyphosate tolerance (CP4 EPSPS, gox)

Australia, Canada, China, EU, Japan, Philippenes, USA

Oilseed rape

Glufosinate tolerance (pat)

Australia, Canada, European Union, Japan, USA

Oilseed rape

Bromoxynil tolerance (bxn)

Australia, Canada, Japan, USA

Cotton

Glyphosate tolerance (CP4 EPSPS)

Argentina, Australia, Canada, EU, Japan, Philippines, South Africa, USA

Cotton

Bromoxynil tolerance (bxn)

Australia, Canada, Japan, USA

Cotton

Sulfonylurea tolerance

(als)

USA

Sugar beet

Glufosinate tolerance (pat)

Canada, Japan, USA

Sugar beet

Glyphosate tolerance (CP4 EPSPS, gox)

Australia, Philippines, USA

Sugar beet

Glyphosate tolerance (CP4 EPSPS)

Philippines, USA

Wheat

Glyphosate tolerance (CP4 EPSPS)

USA

Rice

Glufosinate tolerance (bar)

USA

Flax

Sulfonylurea tolerance

(als)

Canada, USA

Table 13.2 Adoption of GM crops in the European Union (EU) versus worldwide herbicide-tolerant (HR) crops; italics indicate approval for commercial release of new GM crops (from Madsen and Sand0e, 2005).

Year

European Union

H R crops world area

1986

HR marker gene in tobacco field tested in France and USA

1994

HR tobacco approved to be used as conventional tobacco

1996

GM soybean imported into the EU

HR tobacco, oilseed rape, soybean and chicory approved for breeding/import

0.6 M ha

1997

HR maize, oilseed rape and carnations approved to be used as conventional crops

6.9 M ha

1998

Stop for GM crops under debate in UK

Stakeholders in Denmark agree on a voluntary pause for commercial use of GM crops in 1999 HR oilseed rape, maize and carnation approved as conventional crops

20.1 M ha

1999

25 June: moratorium (suspension of new approvals for marketing of GMOs)

31.0 M ha

2000

H 5.9 M ha

2001

H 0.6 M ha

2002

New directive on the deliberate release of GM crops into the environment in force (EU Directive 2001/18/EC)

44.2 M ha

2003

October, Regulation on the traceability and labelling of GMOs and the traceability of food and feed products produced from GMOs (Regulation [EC] No. 1829/2003 and No. 1830/2003) published

49.7 M ha

2004

April, Regulation on the traceability and labelling of GMOs and the traceability of food and feed products produced from GMOs fully into force

GMO, genetically modified organism.

GMO, genetically modified organism.

over-expressing one of the genes, in which case it will produce more of one of its proteins, or under-expressing one of its genes, in which case the plant will produce less or none of one of its proteins.

Since the early 1980s scientists have developed a number of ways by which genes from other organisms may be added to the genetic material of the plant. The use of the microorganism Agrobacterium tumefaciens has proved useful in modifying a number of commercially important crops (Komari et al., 2004). A. tumefaciens is a common soil bacterium that is a plant pathogen, causing galls to be produced on the infected plants. It accomplishes this in nature by transferring some of its genes to plant cells. These genes are transferred in a plasmid, a circular DNA molecule, and in the case of A. tumefaciens the plasmid causing gall formation is termed the Ti (tumour inducing) plasmid. Using this naturally occurring system of genetic modification, scientists have successfully transformed a number of crop species (Komari et al., 2004). The gene of interest is first inserted

NUCLEUS

Chromosomal DNA

transcription of small subunit gene mRNA

HIGHER PLANT CELL

CYTOPLASM

nuclear pore mRNA

mRNA

ribosome

ribosome

Signal segment

H2 N-Met-Cys-Met aminoacyl-fRNAs fRNAs small subunit

-Tyr-COOH

Precursor of small subunit of RuBP carb.

CHLOROPLAST

CHLOROPLAST

transcription of large subunit gene

H2 N-Met H2 N-Met Cys-Met q)*' membrane pore specific for signal segment transcription of large subunit gene

Tyr-COOH

mRNAs

specific protease

-Tyr-COOH

Ribosome aminoacyl-iRNAs iRNAs

Small subunit

Large subunit

RuBP carboxylase (8 x small subunit + 8 x large subunit)

Figure 13.1 Diagrammatic representation of protein synthesis and its cellular location. The example given is for RuBisCo (reproduced from Goodwin and Mercer, 1983).

into the Ti plasmid of A. tumefaciens and the transformed bacteria are used to infect pieces of plant tissue. Successful infection will result in the Ti plasmid (and the added gene) being transferred to the plant genome. Due to totipotency, the plant tissue can be regenerated to a whole plant, in which each cell will contain the added gene. In order for this to be successful, the genes in the Ti plasmid that are responsible for tumor initiation are first removed. The introduced genes are integrated into the nuclear DNA of the plant in the same way as takes place when A. tumefaciens infects plants in the field.

Although this method has proved very successful for a number of dicotyledonous plant species, until recently it had not been so for monocotyledons, as A. tumefaciens would not infect them. However, techniques have now been developed that enable agrobacte-rium-mediated transformation of rice (Tyagi and Mohanty, 2000), maize (Ishida et al., 1996), barley and wheat (Cheng et al., 1997). It has also proved problematic with soybean, as regeneration from cell cultures is often not possible (McCabe et al., 1988). An alternative method of introducing genes has been used for these species. This involves coating tungsten particles with DNA containing the gene of interest and using a microprojectile gun to shoot the particles and DNA into plant cells. This method, often referred to as the 'Shotgun' method, has allowed transformation of plant species where A. tumefaciens is ineffective as a vector. DNA that has entered the cell incorporates into the nuclear DNA and is then passed on as the cell divides.

In addition to these methods, DNA can also be introduced to protoplasts (plant cells that have had their cell wall removed) using electroporation or chemical methods to open up the plasma membrane and allow DNA to enter the cell. Alternatively, microinjection can be used. The protoplast will grow back its cell wall and can be regenerated to a plant whose cells all contain the added gene.

In addition to the gene conferring the required trait a number of other genes and pieces of genetic information are added. These include a promoter (a molecular switch to ensure that the required gene will be transcribed in the transformed plant) and a stop sequence that ensures transcription stops at the end of the required gene. In addition, a marker gene is also added as a way of screening cell cultures to determine if transformation has been successful. For many GM crops the marker gene has been one which confers antibiotic resistance to the successfully transformed plant tissue, allowing easy selection for transformed tissue by culturing it in vitro in the presence of antibiotics. A commonly used gene is NPT II, which produces the enzyme neomycin phosphotransferase giving resistance to the aminoglycoside antibiotics (Komari et al., 2004). The gene encoding for the enzyme hygromycin phosphotransferase has also been used as a marker (Bilang et al., 1991).

There has been some concern regarding the use of antibiotic resistance genes, especially where they remain in the GM plant when it is at the stage of farm-scale evaluation and commercial production. Alternative methods (for instance, incorporation of the pmi gene encoding phosphomannose isomerise, ensuring that on a mannose-only culture medium only transformed tissue survives) have received limited use and may prove more publicly and environmentally acceptable (Joersbo, 2001) . The herbicide tolerance gene bar has also been used as a marker gene (Vasil, 1996) and, where herbicide tolerance is the trait being introduced, the presence of a marker gene would appear unnecessary, as a simple screen in the presence of the herbicide to which tolerance is introduced should successfully select for transformed tissue by killing untransformed cells. The process of producing a GM-HT crop is summarised in Figure 13.2.

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