Positive Selection Marker

There are sometimes different and confusing definitions in using the terms "positive selection marker" and "negative selection marker". At present, positive selection systems are those that enable the growth of transformed cells, whereas negative selection systems kill the transformed cells (see Sect. 3.3.4).

3.3.1.1 Antibiotics

The most widely used selection marker systems are based on aminoglycoside-modifying enzymes. These amino glycoside-modifying enzymes confer resistances against antibiotics as kanamycin, neomycin, gentamycin, paramomycin, streptomycin and spectinomycin.

Fig. 3.2 Overview of selection systems

- Positive selection marker systems by using:

o Toxic antibiotics o Toxic herbicides o Metabolic analogues o Non-toxic agents

  • Negative selection marker
  • Alternatives o Selectable marker gene elimination by:
  • Co-transformation
  • Recombinase induced elimination
  • Homologue recombination o Screenable marker genes o Marker-free transformation

Neomycin Phosphotransferase II

Within the aminoglycoside-modifying enzymes the neomycin phosphotransferase II (nptII), originated from transposon Tn5 of Escherichia coli K12 (Garfinkel et al. 1981), is the most used selectable marker gene. There are many advantages in using nptII in comparison with other selectable marker genes:

  • The gene confers resistance against different antibiotics: kanamycin, neomycin, paramomycin, geneticin.
  • The gene is efficient in model plants such as Arabidopsis, Petunia or Nicotiana tabaccum but also in most of the cultivated plants, both in monocots and dicots, in legumes and Gramineae.
  • Reproducible protocols are available for the transformation of most of these crops.
  • NPTII is available in combination with various regulation sequences, e.g. promoters.
  • There are mutated forms of the nptII gene that encode enzymes with reduced activity.
  • By using an intron-containing nptII gene only eukaryotic organisms will be able to process the gene (Paszkowsky et al. 1992; Maas 1997; Libiakova 2001). Accordingly, the potential risk of horizontal gene flow of antibiotic resistance genes from transgenic plants to bacteria is eliminated.
  • NPTII can be used not only as a selectable marker but also as a scorable marker, as a reporter gene for studying gene expression and regulation. In vitro assays (ELISA or use of radioisotopes) for quantitative or semi-quantitative analysis of the NPTII activity are available (McKenzie 2000; Ziemienowicz 2001).
  • The patent on the nptII coding sequence combined with regulatory sequences will expire soon (Konig et al. 2003).

Most of the first-generation transgenic crops contain nptII, and to this date nptII is the best studied selectable marker with regard to safety. Already in 1994, the use of nptII as a marker and as a food additive for transgenic tomatoes, oilseed rape, and cotton was evaluated by the United States Food and Drug Administration (FDA). The FDA found the use of nptII as a selection marker safe (FDA 1994). The conclusion was based on data from Calgene (1993), Redenbaugh et al. (1994), Fuchs et al. (1993), Nap et al. (1992), Flavell et al. (1992), Kasid et al. (1990) and Blease et al. (1990), among others. But unattached, without reference of scientific evaluation, the presence of antibiotic resistance genes, mainly NPTII, increases public and consumer criticism and still is dogged by controversy.

Mainly the concerns about the potential spread of antibiotic resistance genes through horizontal gene transfer led to the final recommendation that antibiotics widely used for clinical or veterinary use may not be used as selectable markers in plants (Miki 2004; FDA 1998). Also in Europe the use of antibiotics as selection marker was acknowledged as a problem and resulted in Directive 2001/18/EC, which requires the step by step phasing out of antibiotic resistance genes which may have adverse affects on human health and environment by the end of 2004 (EFSA 2004). However the European GMO Panel came to the conclusion in 2004 that the use of the nptII gene as selectable marker in GM plants (and derived food or feed) does not pose a risk to human or animal health or to the environment. These safety assessments were confirmed by the EFSA in 2007 again in the light of all relevant reviews and expert consultations: Ramessar et al. (2007), Goldstein et al. (2005), Miki and McHugh (2004), Working Party of the British Society for Antimicrobial Chemotherapy (Bennett et al. 2004), FAO/WHO Consultation on Foods Derived from Biotechnology (FAO/WHO 2000), Scientific Steering Committee of the European Commission (SSC 1999), Zentrale Kommission fur die Biologische Sicherheit, DE (ZKBS 1999), The Advisory Committee on Novel Foods and Processes, UK (ACNFP 1996), Nap et al. (1992).

But again, in contrast, in 2005 the WHO classified kanamycin and neomycin as critically important antibiotics (WHO 2005). To sum up there is no recommendation of a general ban of antibiotic markers, only a restricted use, but there are disagreements concerning the classification of antibiotics (mainly for kanamycin) whether they are of high, minor or no therapeutic relevance in human medicine.

Hygromycin Phosphotransferase

Cloning of the hygromycin phosphotransferase (hph) gene and fusion with eukary-otic promoters resulted in the development of vectors that permit selection for resistance to hygromycin B in both prokaryotic and eukaryotic cells (Elzen et al.

1985). Besides kanamycin, hygromycin B is the most frequently used antibiotic for selection. In comparison with kanamycin, hygromycin is more toxic and therefore kills sensitive cells faster. However, hygromycin is the preferred antibiotic resistance marker for the selection of monocotyledonous plants, although it is not user-friendly. Extreme care has to be taken when handling hygromycin B as it is very toxic by inhalation, in contact with skin and if swallowed.

Antibiotic Resistance Genes Beside nptII and hph

There are a lot of other marker genes, for example antibiotics like streptomycin (Maliga et al. 1988), spectinomycin (Svab and Maliga 1993), bleomycin (Hille et al.

  1. and chloramphenicol (de Block et al. 1984), which have been used in plant transformation experiences or are at least part of used transformation vectors. But most of them are under the control of a bacterial promoter and have been used for selection in bacteria not specified for selection in plants. In the end the genes are mostly integrated outside the left and right border regions of the used transformation vectors and therefore not part of the transgenic plants.
  2. 3.1.2 Herbicides

Millions of hectares are being planted with transgenic herbicide resistant plants (see also Chap. 9), meanwhile often "stacked" with insect resistance in the same seeds to enhance their value. The database summary Global status of approved genetically modified plants of AGBIOS (2009) shows 80 records for the trait herbicide tolerance. So, by far, herbicide tolerance is still the most used selection criteria. The advantages of the systems are the usage of the herbicide tolerance both as a desired trait in the field and as a selection marker (Goldstein et al. 2005) during developmental period. The most used systems comprise 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, resistance to glyphosate), phosphinothricin acetyl transferase (bar/pat, resistance to glufosinate) acetolactate synthase (ALS, resistance to chlorosulfuron) and bromoxinil nitrilase (Bxn, resistance to bromoxinil) in descending order of AGBIOS records. In 2006 glyphosate-resistant crops have grown to over 74 million hectares in five crop species in 13 countries (Dill et al. 2008).

Meanwhile new and improved glyphosate-resistant crops are being developed. These crops will confer greater crop safety to multiple glyphosate applications and these glyphosate-resistant plants are expected to continue to grow in number and hectares planted. But there is no guarantee that new molecular stacks conferring resistance to glyphosate and ALS-inhibiting herbicides or glyphosate with glufosi-nate will prevent the development of resistant weeds in the future. There are already several weed biotypes with confirmed resistance to glyphosate. So the question arises whether the presence of herbicide selection markers like glyphosate resistance in some years may be undesirable when the trait is no longer necessary or inapplicable for product function. Apparently, the same conclusion is valid for herbicide resistance marker genes as for other marker genes, needed in the first place but undesirable shortly afterwards.

There are alternatives to the most used herbicide resistance genes, for example selectable marker genes which mediate resistance against the herbicides cyanamide (Weeks et al. 2000), Butafenacil (Li et al. 2003; Lee et al. 2007), Norflurazon (Inui et al. 2005; Arias et al. 2006; Kawahigashi et al. 2007) or Gabaculine (Gough et al. 2001). However, by today, most of these alternatives have not been subjected to regulatory consideration for international approvals.

3.3.1.3 Metabolic Analogous, Toxic, Non-Toxic Agents

Many other new approaches comprise manipulating the plant's metabolic or bio-synthetic pathways. This is done by using metabolic analogous, toxic agents, non-toxic elements such as phytohormones, or carbon supplies which are natural to the plant. There is a wide range of used genes. XylA, dog, ipt, tps and manA are only a choice of new genes which were used to develop additional selection systems.

2-Deoxyglucose-6-Phosphate Phosphatase

The deoxyglucose (DOG) system is based on the sugar 2-deoxyglucose (2-DOG) which is phosphorylated by hexokinase yielding 2-DOG-6-phosphate (2-DOG-6-P)

in plant cells. 2-DOG-6-P is toxic to plants, since it inhibits respiration and cell growth. Over-expression of the gene enzyme 2-deoxyglucose-6-phosphate phosphatase (dogR1) in plant cells results in resistant plants (Kunze et al. 2001). Transgenic potato plants have been tested under field conditions with the result that no differences were found between the transgenic plants and the control plants. Whether the system can be applied without safety concern in the future has to be investigated further (GMO-Safety 2005).

Xylose Isomerase

The xylose isomerase (xylA) system is based upon selection of transgenic plant cells expressing the xylA gene from Streptomyces rubiginosus, which encodes xylose isomerase, on medium containing xylose (Haldrup et al. 1998). In contrast to antibiotic or herbicide selection, the system is generally recognized as safe because it depends on an enzyme which is already being widely utilized in specific food processes, especially in the starch industry. But to this day selectable markers like xylA have not yet appeared in approved food plants.

Isopentenyl Transferase

The enzyme isopentenyl transferase (ipt) is a more often used selection marker. The gene, encoded by the T-DNA of Agrobacterium tumefaciens, catalyzes the synthesis of isopentyl-adenosine-5'monophosphate, which is a precursor of the phytohor-mone cytokinin. Over-expression of ipt by using the gene under the control of a constitutive promoter yields enhanced cytokinin levels in transgenic plants. Cyto-kinins stimulate organogenesis; therefore due to the enhanced cytokinin concentrations the regeneration of transformed shoots is promoted. The combination of the ipt gene together with the kanamycin selection system enhances the transformation efficiency(Ebinuma et al. 1997; Endo et al. 2001).The system, also called the MAT system, is usable as a visible selection system since the transformed shoots lose their apical dominance and the ability to root. These abnormal morphologies of the shoots, so-called "extreme shooty phenotype" (ESP) prevented the development of ipt as a selectable transformation marker in practice, because it is only usable in combination with inducible artificial promoter systems (Kunkel et al. 1999; Zuo et al. 2002) or with marker elimination systems (Ebinuma et al. 2000, 2001). The use and removal of ipt were demonstrated in different plant species but the efficiency of the system was low, therefore further optimization of the selection system is required. Recently new publications (Rommens et al. 2004, 2006; Bukovinszki et al. 2006; Richael et al. 2008) give hope for an improved system. New methods (e.g. "All-native DNA transformation") for the production of trans-genic plants utilize isopentenyl transferase cytokinin genes in negative selection against backbone integration (e.g. see Sect. 3.4.3 and Chap. 4).

Phosphomannose Isomerase

The manA gene codes for the enzyme pmi (phosphomannose isomerase). Many plants are normally not able to use the sugar mannose as a source of carbohydrate. When plants are forced to grow on mannose as the only carbon source they first convert mannose to mannose-6-phosphate, which is no longer utilizable for the plants. Transformed with the pmi gene, the plant converts mannose-6-phosphate to fructose-6-phospate, which can be used in the plant metabolic pathway from there. Thus mannose can function as the only carbon source (Joersbo et al. 1998; Privalle et al. 1999). Species which have been successfully transformed using mannose as selective agent, among others, are sugar beet (Joersbo et al. 1998; Lennefors et al. 2006), sunflower, oilseed rape, pea, barley (Joersbo et al. 1999, 2000), sorghum (O'Kennedy et al. 2006), sugarcane (Jain et al. 2007), rice (Lucca et al. 2001; Ding 2006), tomato and potato (Bnza et al. 2008), apple (Degenhardt 2006), papaya (Zhu 2005), torenia (Li et al. 2007) and citrus (Ballester et al. 2008). Ballester and co-workers compared various selection systems with the same Citrus genotypes: nptII, ipt and pmi systems. The highest transformation rates were obtained with the pmi/mannose system, which indicates that this marker is also an excellent candidate for citrus transformation. So at the moment, beside the kanamycin and the glyphosate selection system, the pmi system is the most successful one. Regulatory approvals have been received for environment, food and feed with transgenic maize varieties in Mexico, Australia, Japan, Canada and the United States.

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