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Methods for generating genetically modified (GM) plants1 are generally inefficient, and only a very small percentage of cells are successfully genetically modified (or ‘transformed’) with the gene(s) of interest. Marker genes are used to help find the transformed cells, and ultimately produce GM plants, that contain the gene(s) of interest, conferring a desired trait or traits in the plant (Miki & McHugh 2004).
For plant transformation, marker genes are often combined in the same piece of DNA as the gene(s) of interest, such that they are transferred together (Lee & Gelvin 2008). Marker genes may also be used on separate pieces of DNA, as often both types of introduced DNA are taken up and integrated into the genome in the same cells during the transformation process. Thus, the presence of the marker gene is an indirect indicator for the presence of the gene of interest.
There are two types of marker genes: selectable marker genes which confer resistance to a selective agent (such as an antibiotic or herbicide); and reporter genes which produce products that can be detected visually or by biochemical assays.
When assessing potential risks to the health and safety of people and the environment that may be posed when dealing with GM plants, the Gene Technology Regulator considers any introduced genetic material including any marker genes.
Information is provided here on the most commonly used antibiotic resistance selectable marker genes and reporter genes used in GM plants. The potential for these genes to pose risks to the health and safety of people and the environment is also addressed.
Herbicide tolerance genes are not covered here – these genes are assessed in detail in the risk assessment and risk management plans prepared for relevant licence applications.
Antibiotic resistance marker genes expressed in plants
Genes encoding resistance to antibiotics are a common type of selectable marker in GM plants. Antibiotics are usually lethal to plant cells as they block specific metabolic processes. The presence of an introduced antibiotic resistance gene allows a transformed (ie GM) cell or plant to survive in the presence of the corresponding antibiotic.
Generally, following the process for introduction of the new genes to the plant cells or tissues, the plant cells or tissues are placed on a synthetic medium containing the antibiotic as well as nutrients and hormones that promotes the growth of plants from single transformed cells.
The most common antibiotic resistance genes used for the selection of transformed plant cells are the nptII and hph genes – these genes are considered in detail below.
In addition to antibiotic resistance genes used for selection in plants, other antibiotic resistance genes may also be present in some GM plants. These are antibiotic resistance genes that were used for selection of bacteria carrying the genes of interest prior to their introduction into plant cells. These genes for selection of bacteria are not expressed in the GM plants, as the genetic elements that control expression in bacteria do not work in plants. The lack of expression of the bacterial antibiotic resistance genes in the GM plant means that no toxicity/allergenicity consideration of these bacterial antibiotic resistance proteins are required in a risk assessment for the GM plant.
Neomycin phosphotransferase II gene (nptII)
The nptII gene, derived from E.coli strain K12 (Beck et al. 1982), codes for an aminoglycoside 3’-phosphotransferase enzyme (APH(3')II or NPTII) that inactivates kanamycin and structurally related antibiotics such as neomycin, paromycin, ribostamycin, butirosin, gentamicin B, and geneticin G418, which would normally inhibit protein synthesis.
Hygromycin phosphotransferase gene (hph, hpt)
The hph (also abbreviated as hpt) gene confers resistance to the antibiotic hygromycin B. Hph genes have been isolated from E.coli (also referred to as the aph(4) gene) and Streptomyces hygroscopicus (aph(7)) (Kuhstoss & Rao 1983; Leboul & Davies 1982; Rao et al. 1983). The encoded hygromycin phosphotransferase (HPH) enzymes inactivate hygromycin B (Pardo et al. 1985; Rao et al. 1983). The gene from E.coli is used most often in GM plants.
Consideration of potential risks from nptII and hphTop of page
Although antibiotic resistance genes play no role in the desired phenotypes of the GM plants in the field, they usually remain in the plant genomes. In this context, they potentially pose two risks: (i) their protein products may directly or indirectly have a negative effect on people and/or animals that consume the plant material, and (ii) plants possessing these genes may cause environmental harm (eg have increased weediness leading to harm to the environment).
There is no evidence that the NPTII and HPH proteins are toxic or allergenic. Bioinformatic analyses have failed to find homology to any known toxins or allergens (EFSA 2009; Lu et al. 2007). Further, neither protein is known to be involved in the production of a toxic or allergenic compound. Toxicity experiments with animals (mainly mice and rats), often involving the feeding of exaggerated doses of these proteins by gavage (use of a small tubing to insert food), have failed to establish any deleterious effects of either NPTII (Flavell et al. 1992; Fuchs et al. 1993) or HPH (Lu et al. 2007; Zhuo et al. 2009). GM foods containing the NPTII or HPH proteins have been approved for sale in Australia (FSANZ 2004; FSANZ 2010).
Dietary intake of the protein products of antibiotic selection genes could conceivably reduce the therapeutic efficacy of antibiotics taken orally. This is especially important in regards to the nptII gene, as kanamycin/neomycin has human and animal therapeutic importance (EFSA 2009). Hygromycin is not used with humans, but may be used in animals such as pigs and poultry. However, like most proteins, NPTII and HPH are rapidly inactivated in simulated mammalian gastric juice (FSANZ 2004 Ref 6487, Fuchs 1993). Therefore, under normal digestion, it would be expected that any antibiotic resistance protein would be degraded before it could inactivate the corresponding antibiotic, negating any possible interference with oral administration of the antibiotic (EFSA 2009).
It has been suggested that the transfer of antibiotic resistance genes from GM plant material to intestinal bacteria or other microorganisms could lead to antibiotic resistant populations of these organisms (Flavell et al. 1992). However, evidence strongly suggests that such ‘horizontal gene transfer’ from plants to bacteria is extremely rare (Keese 2008). Most genetic material (DNA) is likely degraded in the stomach and intestines. The feeding of GM soybeans to humans has shown that although a small proportion of DNA may survive the stomach and small intestine, what remains is degraded in the large intestine (Netherwood et al. 2004). Similarly, experiments involving the feeding of GM plant material to animals such as chickens has suggested that most DNA fails to survive the digestion in the stomach (Chambers et al. 2002). In addition, these genes were originally isolated from bacteria which are widespread in the environment, including in the gastro-intestinal tract of people and animals. Transfer of these genes between bacteria is far more likely than transfer from GM plants to bacteria.
No feasible pathway links a plant possessing either the nptII or hph gene and environmental damage. Given that NPTII and HPH are naturally widespread in the environment, organisms in the environment are already exposed to these proteins. A GM plant with such a gene would only have a selective advantage and potentially become a weed in the presence of the antibiotic, and this is unlikely to occur in a natural situation (Nap et al. 1992). The European Food Safety Authority concluded that the use of the nptII and hph genes as selectable markers in GM plants (and derived food or feed) does not pose a risk to human or animal health or to the environment (EFSA 2009).
Reporter genes expressed in plants
Reporter genes code for molecules that can be readily identified visually or by biochemical assays, allowing the identification of cells/tissue expressing the introduced protein. These genes are commonly used as “reporters” of gene expression, by linking them to other genes or promoters in GM plants so that they are expressed in the same pattern as the linked gene or promoter. They can also be used as selection markers in plant transformation. The proteins of reporter genes are non-toxic to plant tissues, enabling their constitutive or regulated expression (temporal and/or spatial) in plants. The uidA (or gus) and gfp genes are commonly used reporter genes.
β-glucuronidase (uidA, gusA, gus) gene
The uidA (or gus) gene from E.coli encodes the enzyme β-glucuronidase (GUS), which enables E.coli to metabolise β-glucuronides as a source of carbon and energy. GUS expression from an introduced uidA gene can then be detected in GM plant tissue, in a process that kills the plant cells, using a substrate of the GUS enzyme which produces a coloured product when cleaved by GUS. Using differing substrates enables either the measurement of the amount of protein present or visualisation of the expression pattern (ie distribution) in intact plant tissue (Jefferson & Wilson 1991).
The gus gene and its associated protein is found in a wide range of organisms. In addition to E. coli, the gus gene is found in many other bacteria, including other microorganisms of the digestive tract and many soil bacteria. GUS activity is very common in almost all tissues of vertebrates, with high activity in the kidney, liver and spleen. GUS activity is also present in invertebrates such as molluscs, nematodes and insects (Gilissen et al. 1998). Low GUS activity has been detected in over 50 different plant species including a number of human food sources such as carrot, parsley and celery (Hu et al. 1990; Gilissen et al. 1998).
Green fluorescence protein (gfp) gene
The gfp gene, derived from the jellyfish Aequorea victoria, encodes the green fluorescent protein (GFP). GFP emits a green light when exposed to blue or ultraviolet light. Although its physiological role is unclear, GFP contributes to the bioluminescence of these jellyfish.
GFP is valuable as a marker of gene expression in both GM plant cells and GM animal cells (Elliott et al. 1999). Expression of GFP can be seen in living tissue through exposure to ultraviolet or blue light, avoiding the need to destroy the tissue. This makes it useful for observing the intracellular location and movement of linked proteins within living cells (Kallal & Benovic 2000; Leffel et al. 1997). Mutation of the gfp gene sequence has resulted in the development of a number of variants with useful properties.
Consideration of potential risks from uidA and gfp
The GUS protein is not considered to be toxic by the United States Environmental Protection Agency, which has exempted it from a requirement to establish a tolerance level (EPA 2001). It does not demonstrate any oral toxicity when administered at high doses to rodents and is rapidly degraded in gastric fluids (EPA 2001). In this respect, there have been no reports of toxicity or allergenicity in humans related to its digestion. The uidA gene was isolated from E.coli, which is found in the human digestive tract, as well as in soil and water ecosystems. Further, genes coding for GUS proteins are found in a range of vertebrate and invertebrates, including humans, and microorganisms other than E.coli (Gilissen et al. 1998).
Likewise, the GFP protein is not regarded as toxic to humans or other organisms. Humans are not known to consume the jellyfish A. victoria, and as such people have not been exposed to the GFP protein through food. Feeding of the protein to rats failed to show any toxic or allergenic reaction, and the protein is rapidly degraded in gastric digestion experiments (Richards et al. 2003). GFP is unlikely to be a food allergen as it does not display a number of characteristics common to known food allergen proteins, such as glycosylation and stability in the digestive tract.
The amino acid sequences of both the GUS and GFP proteins are not related to those of any known toxins or allergens, and the enzymatic activities of both proteins are not known to produce any toxic or allergenic compounds.
There are no reports of a GM plant expressing the GUS protein or GFP causing environmental harms associated with increased weediness. The reactions catalysed by the GUS and GFP proteins are not known to be associated with any biochemical process related to plant weediness and therefore expression of these proteins in GM plants is not expected to increase the weediness of these plants.
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1Information on plant transformation methods can be found in the risk assessment reference document Methods of Plant Genetic Modification available on the OGTR website at the "Documents relating to the Risk Assessment process" page.