All GM wheat and barley lines in Group 2 contain a barley gene AlaAT (Muench & Good 1994). The AlaAT gene encodes a hypoxia-induced cytoplasmic alanine aminotransferase (AlaAT), with a deduced amino acid sequence of 482 residues. AlaAT catalyses the reversible reaction of pyruvate and glutamate to alanine and 2-oxoglutarate. AlaAT has been well studied in animals, where it is most commonly associated with the liver. In plants, two to six AlaAT isozymes have been identified and localised to various subcellular locations. AlaAT enzymes play key roles in both carbon and nitrogen metabolism, including general amino acid metabolism (Liepman & Olsen 2003).
AlaAT also plays more specialised roles in the C4 pathway of photosynthesis (Son & Sugiyama 1992) and in plant responses to hypoxia (Good & Crosby 1989; Ricoult et al. 2006; Miyashita et al. 2007) and nitrogen stress (Muench et al. 1998). AlaAT has also been implicated in seed storage protein production in rice (Kikuchi et al. 1999). Although alanine is accumulated in response to drought in some plants, this accumulation does not coincide with an induction of AlaAT (Good & Zaplachinski 1994). Similarly AlaAT does not respond to salt, cold or heat stress in maize (Muench et al. 1998).
In canola, over-expression of the barley AlaAT gene under the control of a canola root specific promoter resulted in an increased biomass and seed yield under low nitrogen conditions in both the field and laboratory (Good et al. 2007). In the field trials the increase in seed yield was 33 42%. When grown hydroponically, the GM canola plants over-expressing AlaAT had higher levels of alanine in the roots, and less glutamine and glutamate in the shoots, than control plants. In response to these altered amino acid levels, the GM canola plants increased the rate of nitrate influx (Good et al. 2007).
GM rice plants over-expressing the same barley AlaAT gene also showed an increase in biomass (30-34%) and grain yield (31-54%) in the laboratory (Shrawat et al. 2008). In this study, expression was driven by the OsAnt1 promoter, which shows strong expression in roots, and plants were well supplied with nitrogen. The increase in biomass primarily depended on the accelerated formation of tillers. Hydroponically grown GM rice plants also showed more vigorous growth, produced bushier, finer and more branched root systems, showed changes in the amount of several amino acids, and had higher total nitrogen content than control plants. For example, glutamine, glutamate and asparagine levels were increased in both roots and shoots of the GM plants, whereas arginine levels were only increased in shoots. The increase in total nitrogen content was attributed to an increase in nitrogen uptake efficiency (Shrawat et al. 2008).
The introduced genes for improved water use efficiency, carbon assimilation and photosynthesis, and the encoded proteins
The genes in Group 4 (genes 6-31, Table 1) include 19 genes encoding transcription factors, two genes encoding metabolic enzymes, four genes encoding proteins involved in calcium-binding or peptide growth and one with unclear functions.
A detailed description for each of these 26 genes can be found in Section 5.2 of the RARMP for DIR 100 (http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/dir100-3/$FILE/dir100rarmp.pdf). A general discussion of stress responses and the role of transcription factors can also be found in Section 5.2 of that RARMP. The purpose of the genetic modifications in wheat using these genes is to enhance the agronomic performance of GM wheat in heat and drought prone environments through enhancing water use efficiency, carbon assimilation and photosynthesis. The applicant states that some GM plants carrying multiple genes (2-5 genes) from this group may also be tested in field trial. These will be generated either by co-bombardment of a mixture of candidate genes or by conventional crossing of GM lines containing Group 4 genes. These GMOs would only be included in the field trial if they pass phenotypic screening at the T1 stage in controlled environment facilities.
The introduced genes for enhanced fibre content and the encoded proteins
The Group 5 GM wheat lines each contain one of the two genes (designated CME A and CME B) from barley that encode carbohydrate metabolism enzymes.
Both CME A and CME B proteins are large integral membrane proteins thought to be located in the Golgi apparatus. Wheat has homologues of these proteins, which are normally expressed in the wheat grain and have 93% and 98% identity with CME A and CME B from barley, respectively (information provided by the applicant). Over-expression of either CME A or CME B under the control of the endosperm specific Bx17 promoter in the GM wheat lines is expected to alter the carbohydrate composition in the GM wheat seeds.
The introduced Lr34 gene and the encoded proteins
The Group 6 GM wheat lines contain a wheat gene Lr34 for enhanced resistance to certain fungal diseases. The Lr34 gene encodes an adenosine triphosphate (ATP)-binding cassette (ABC) transporter, which belongs to the subfamily G of the ABC transporters (ABCG) (Krattinger et al. 2011), formerly known as pleiotropic drug resistance (PDR) subfamily (Crouzet et al. 2006; Krattinger et al. 2009).
ABC transporter proteins form one of the largest protein families and are found in all living organisms (Lorkowski & Cullen 2002). They play many roles in metabolism and have been shown to function as PDR proteins and as transporters of metal ions, lipids, peptides, and several other molecules (Gottesman & Ambudkar 2001). Full-size ABCG transporters share a conserved structure consisting of two cytosolic nucleotide binding domains (NBDs) and two transmembrane domains (TMDs) (Jasinski et al. 2009).
Full-size ABCG transporters are unique for plants and fungi (Jasinski et al. 2009) and possibly transport a wide set of structurally and functionally diverse molecules (Rea 2007). Other genomic studies demonstrate that full-size ABCG proteins are widespread in plants; 15, 23 and 19 members have been identified in thale cress (Arabidopsis thaliana), rice (Oryza sativa) and Barrel Medic (Medicago truncatula), respectively (Crouzet et al. 2006; Jasinski et al. 2009). Therefore, Krattinger et al. (2011) predicted that up to 60 full-size ABCG transporter genes may exist in hexaploid wheat with its three homeoeologous genomes.
Members of the ABCG family are involved in plant response to biotic stress such as fungal and bacterial pathogens (Stukkens et al. 2005; Kobae et al. 2006; Stein et al. 2006), as well as abiotic stresses such as salinity, cold and iron deficiency (Crouzet et al. 2006). The Lr34 gene is located on wheat chromosome 7D and has been shown to confer durable, race non-specific resistance in adult wheat plants against leaf rust (Puccinia triticina), stripe rust (Puccinia striiformis) and powdery mildew (Blumeria graminis) (Lagudah et al. 2006; Krattinger et al. 2009). The exact defence mechanism of Lr34 and other similar plant ABCG transporters is still unclear. However, it is possible that the Lr34 resistance is either the result of senescence-like process, or exporting metabolites that affect fungal growth (Krattinger et al. 2009).
The antibiotic resistance marker genes (nptII and hpt) and the encoded proteins
All GM wheat lines from Groups 1, 3, 5 and 6, and some GM wheat lines from groups 2 and 4 contain the antibiotic resistance gene nptII. This gene, encoding the enzyme neomycin phosphotransferase type II, was derived from E. coli and confers resistance on the GM plant to antibiotics such as kanamycin or neomycin.
The GM barley lines from Groups 2 and 3 contain the hpt gene from E. coli, which confers resistance to the antibiotic hygromycin B. The hpt gene, also called hph gene in some literature, encodes the hygromycin phosphotransferase (HPT or HPH) enzyme which catalyses the phosphorylation of the 4 hydroxy group on the hyosamine moiety, thereby inactivating hygromycin (Rao et al. 1983).
Both the nptII and hpt genes were used as selectable markers in the early laboratory stages of development of the plants to enable selection of plant cells containing the desired genetic modification.
The herbicide resistance marker gene (bar) and the encoded protein
Most GM wheat lines in Group 4 contain the herbicide tolerance marker gene bar. The bar gene, also called pat gene in some literature, was isolated from Streptomyces hygroscopicus, a common saprophytic, soil-borne bacterium (Thompson et al. 1987). The bar gene encodes the phosphinothricin acetyl transferase (PAT) protein, which confers tolerance to glufosinate ammonium, the active component in a number of herbicides. The bar gene was used as a selectable marker in the early laboratory stages of development of the plants to enable selection of plant cells containing the desired genetic modification.
Toxicity/allergenicity of the proteins encoded by the introduced genes
The introduced genes of interest (Groups 2, 4, 5 and 6)
All of the genes of interest introduced into the GM plants were isolated from wheat or barley. Although wheat and barley contain a number of anti-nutritional factors and allergens that, in extreme cases, may have a toxic effect (OGTR 2008a; OGTR 2008b), the proteins encoded by the introduced genes are not expected to have any toxic or allergenic effects as they are widely consumed as both human food and animal feed without any adverse effects. As all introduced genes of interest are derived from wheat or barley, the encoded proteins are already present in non-GM plants, albeit possibly at lower or higher levels and with varied expression patterns. On this basis, people and other organisms have a long history of exposure to the introduced genes and their products.
A comprehensive search of the scientific literature yielded no further information to suggest that the encoded proteins are toxic or allergenic to people, or toxic to other organisms. Bioinformatics analysis may assist in the assessment process by predicting, on a purely theoretical basis, the toxic or allergenic potential of a protein based on similarity to known toxins and allergens. The results of such analyses are not definitive and are used to identify those proteins requiring more rigorous testing (Goodman et al. 2008).
For AlaAT, it is possible that the GM wheat and barley plants will produce altered levels of some metabolites in both below and above ground tissues. For example, levels of the metabolites involved in the reaction catalysed by alanine aminotransferase (pyruvate, glutamate, alanine and 2-oxoglutarate) could be altered, as well as amino acids such as glutamine and asparagine. These metabolites are ubiquitous in nature and consumed widely by humans in both natural products and dietary supplements, although as with most substances, very high levels of intake are not recommended. In particular, high levels of glutamate (in the order of 1000 mg/kg body weight) have been associated with neurotoxicity in animals (FSANZ 2003a; Olney & Ho 1970; Barinaga 1990). Glutamate is commonly added to processed foods in the form of mono-sodium glutamate (MSG), the safety of which has been debated for decades (Barinaga 1990). However, MSG still remains on the United States Food and Drug Administration list of additives generally recognised as safe10.
No studies on the toxicity or allergenicity of the GM wheat and barley lines have been undertaken to date as the proposed trial is still at an early stage.
The selectable marker genes
The antibiotic selectable marker genes nptII and hpt were isolated from the common gut bacterium E. coli, while the herbicide selectable marker gene bar was isolated from the soil-borne bacterium S. hygroscopicus. The nptII, hpt and bar genes have been used extensively as selectable markers in the production of GM plants (Miki & McHugh 2004). As discussed in previous DIR RARMPs, regulatory agencies in Australia and in other countries have assessed the use of these genes in GM plants as not posing a risk to human or animal health or to the environment.
For the nptII gene, more detail can be found in the RARMPs for DIR 070/2006 and DIR 074/2007 (available at <http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/dir070-2006> and <http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/dir074-2007> or by contacting the OGTR). The most recent detailed international evaluation of nptII in terms of human safety was by the European Food Safety Authority, which concluded that the use of the nptII gene as a selectable marker in GM plants (and derived food or feed) does not pose a risk to human or animal health or to the environment (EFSA 2009).
For the hpt gene, more detail can be found in the RARMPs for DIR 073/2007 and DIR 077/2007 (available at <http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/dir073-2007> and <http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/dir077-2007>). The HPT protein is easily digested by simulated gastric juices and the amino acid sequence contains no similarities to known allergens (Lu et al. 2007). The European Food Safety Authority concluded that inclusion of the hpt gene in GM plants would not significantly affect the health of humans or animals (EFSA 2004).
The bar gene has been discussed most recently in the RARMP for DIR 108. Food Standards Australia New Zealand (FSANZ) has approved the use of food derived from a range of GM plants containing either the bar or pat gene, including GM cotton, corn, canola, rice and soybean, concluding that the PAT protein is not toxic (for example ANZFA 2001a; ANZFA 2001b; ANZFA 2001c; FSANZ 2003b; FSANZ 2005; FSANZ 2008). The studies submitted in support of the food uses for this protein indicate that it has none of the properties associated with protein toxins or allergens. A number of GM crops, including food crops, containing the bar or pat gene encoding the PAT protein, have been approved for commercial release both in Australia (DIR 021/2003, DIR 062/2005, DIR 091 and DIR 108) and overseas. No adverse effects on humans, animals or the environment have been reported from any releases (CERA 2011).
The regulatory sequences
Promoters are DNA sequences that are required in order to allow RNA polymerase to bind and initiate correct gene transcription. Also required for gene expression in plants is a transcription termination region, including a polyadenylation signal. Other sequences, such as introns, may contribute to the expression pattern of a given gene. Promoters and terminators used in the GM wheat and barley lines for controlling the expression of genes of interest are listed in Table 2 and are detailed below.
Regulatory sequences for expression of the RNAi constructs (Groups 1 and 3)
Two promoters from the wheat high molecular weight glutenin subunit genes Bx17 and Dx5 were used to control the expression of the GWD RNAi (Group 1) and SBE RNAi (Group 3) constructs respectively in the GM wheat and barley lines. Both the Bx17 and Dx5 promoters are shown to be endosperm-specific (information provided by applicant).
Separation of the sense and anti-sense arms of RNAi constructs with a spliceable intron has been shown to increase the effectiveness of silencing (Smith et al. 2002). The intron used in the GWD RNAi constructs is from the rice starch branch enzyme I gene (GeneBank accession number D10838), while the introns for the SBE RNAi constructs are from either the rice tubulin gene (GeneBank accession number AJ488063.1) or the TaSBE IIa gene.
The mRNA termination region for all RNAi constructs in the GM wheat and barley is derived from the nos gene from A. tumefaciens (Depicker et al. 1982; Bevan 1984). The nos terminator has been used in a wide variety of constructs used for plant genetic modifications (Reiting et al. 2007).
Regulatory sequences for expression of other genes of interest (Groups 2, 4, 5 and 6)
In Group 2 GM wheat and barley lines, the AlaAT gene is under the control of a tissue specific promoter OsAnt1 derived from rice. OsAnt1is active in roots and, at a lower level, vascular tissue of the stems and leaves (Shrawat et al. 2008). The mRNA termination region for the AlaAT gene is the nos terminator.
The introduced genes in Group 4 GM wheat lines are driven by different promoters with different tissue specificity (listed in Table 2). The terminator from the rice RuBisCo small subunit gene is used as the mRNA termination region for all these genes.
The Bx17 promoter and nos terminator are also used to control the expression of the CMEA and CME B genes in GM wheat lines in Group 5. The Group 6 GM wheat lines contain the wheat Lr34 gene under the control of its native promoter and terminator.
Regulatory sequences for expression of the selectable marker genes
Expression of the nptII gene in GM wheat plants is controlled by either the rice Actin 1 (Act1) gene promoter (McElroy et al. 1990) in combination with the nos terminator, or the cauliflower mosaic virus (CaMV) 35S gene promoter (Odell et al. 1985) with either the nos terminator or the CaMV 35S terminator.
Expression of the hpt gene in GM wheat and barley plants is controlled by either the CaMV 35S promoter in combination with the nos terminator or the CaMV 35S terminator, or the maize Ubiquitin-1 (Ubi-1) gene promoter (Christensen et al. 1992) with the nos terminator.
Most of the GM wheat lines in Group 4 contain the bar gene, which is controlled by either the maize Ubi1 promoter or the rice Act1 promoter with the nos terminator in either case.
Both the Act1 and Ubi-1 are constitutive promoters and direct the marker genesto be expressed in most plant tissues and throughout the plant lifecycle. Humans, animals and other organisms are commonly exposed to maize and rice as they have been consumed safely by humans and animals for centuries. Although CaMV and A. tumefaciens are plant pathogens, the regulatory sequences comprise only a small part of its total genome, and are not in themselves capable of causing disease.
Method of genetic modification
Two different methods were used to generate the GM wheat and barley lines for the proposed release – biolistic transformation (some wheat lines in Groups 1 and 2, and all wheat lines in Groups 4, 5 and 6) or A. tumefaciens-mediated transformation (some wheat lines in Groups 1 and 2, all wheat lines in Group 3 and all barley lines in Groups 2 and 3).
Biolistic transformation (Pellegrineschi et al. 2002) involved coating very small gold particles with two transformation constructs (in the form of plasmid or DNA fragment), one containing a plant selectable marker and a second containing the gene of interest. The particles were then ‘shot’ into intact immature embryos from T. aestivum cultivars Bobwhite, Frame or Gladius. Genetically modified plant tissues were recovered by survival on tissue culture media containing one of the selective agents geneticin (G418; for nptII selectable marker), hygromycin (for hpt selectable marker) and Bialaphos (for bar selectable marker).
A. tumefaciens-mediated transformation was used to generate the GM barley lines, as well as some GM wheat lines. A. tumefaciens is a common gram-negative soil bacterium that causes crown gall disease in a wide variety of plants (Van Larebeke et al. 1974), through transfer of DNA (transfer-DNA or T-DNA, located between specific border sequences on a resident plasmid) from A. tumefaciens to the plant genome. Disarmed Agrobacterium strains have been constructed specifically to facilitate genetic modification of plants with desired genes without causing disease. The disarmed strains used for genetic modification do not contain the genes responsible for the overproduction of auxin and cytokinin (iaaM, iaaH and ipt), which are required for tumour induction and rapid callus growth (Klee & Rogers 1989). Agrobacterium plasmid vectors used to transfer T-DNAs contain well characterised DNA segments required for their replication and selection in bacteria, and for transfer of T-DNA from Agrobacterium and its integration into the plant cell genome (Bevan 1984; Wang et al. 1984).
To generate the GM wheat and barley lines in the current application, immature embryos from wheat cultivars Bobwhite or NB1, or barley cultivar Golden Promise were infected with A.tumefaciens carrying the gene of interest (Tingay et al. 1997; Matthews et al. 2001). Following co-cultivation and callus induction steps, the wheat and barley calli were induced to form plantlets on media containing an antibiotic (such as Timentin) to eliminate A. tumefaciens and one of the selective agent G418 (for nptII selectable marker) or hygromycin (for hpt selectable marker).
Both biolistic and Agrobacterium-mediated transformation have been widely used in Australia and overseas for introducing new genes into plants and are not known to cause any adverse effects on human health and safety or the environment.
Characterisation of the GMOs with RNAi constructs (Groups 1 and 3)
Stability and molecular characterisation the GMOs
For the Group 1 GM wheat lines (GWD RNAi), the copy numbers of the introduced RNAi construct and the exact locations within the genome are not known. The copy numbers of some of the Group 3 GM wheat and barley lines (SBE RNAi) have been determined by Southern blotting. The wheat lines 85.2c, 212 and YDH7 carry two inserts of the SBE IIa RNAi construct. The wheat line X5.3-1 has two copies of the SBE IIa RNAi construct inserted at different loci. The wheat line UW89o.2 has a single copy insertion of theSBE IIa RNAi construct. The barley line BC10.5 has one insert of SBE IIa construct and two inserts of the SBE IIb constructs. However, the applicant states that the exact locations of the inserted constructs in the genome are not known.
For all GM lines in this application, the stability of the genotype has been monitored by following the presence of the introduced construct(s) in each generation grown. For GWD RNAi lines, the introduced construct has been followed for five generations by PCR analysis, demonstrating that the introduced construct is stably inherited. The SBE RNAi lines have been selected by single seed descent (SSD) method and observed for at least four generations. Integration of the SBE RNAi construct was found to be stably inherited in all the GM lines as shown by PCR analysis.
Characterisation of the phenotype of the GM wheat and barley
As the GM wheat and barley lines are based on RNAi technology, no new proteins are produced. The RNAi constructs are under the control of the endosperm-specific Bx17 or Dx5 promoters, thus expression is only expected to occur in the seed as the promoter is not active in other tissues (see Section 98). The applicant stated that expression of the introduced RNAi constructs in the seed of some GM lines has been assessed from glasshouse grown plants.
The purpose of the proposed trial for GM wheat and barley lines carrying an RNAi construct is to assess whether the expression of the introduced RNAi constructs can silence or reduce the expression of the target genes. Phenotypes associated with the genetic modification in these lines will be examined under field conditions.
