5.1 Introduction to the GMOs
19. The GM wheat lines contain gene constructs designed to decrease expression of five different families of wheat genes which contribute to grain composition (Table 1). The decrease in endogenous gene expression is brought about by a mechanism known as gene silencing or RNA interference (RNAi). RNAi is a plant defence against infecting RNA viruses, and works by using RNA sequences identified by a plant as foreign to recognise matching sequences, which are then destroyed by enzymes (reviewed by Baulcombe 2004). In viral infections, this mechanism means that once a sequence has been recognised as belonging to a virus, any matching sequences belonging to replicating viruses are quickly destroyed.20. For a transgene to induce RNAi against an endogenous gene, the transcript from the transgene must mimic the structure of the double-stranded RNA viruses which naturally induce RNAi, using sequences from the gene to be silenced (the target gene) (reviewed by Waterhouse & Helliwell 2003). RNAi transgene constructs typically consist of two copies of a fragment of the target gene, arranged to give rise to a single transcript with one forward orientation copy of the target gene sequence followed by one reverse orientation copy. Because the transcript contains identical gene fragments in opposite orientations, they are complementary and naturally base-pair into a double-stranded RNA structure. The double-stranded RNA structure is recognised as being virus-like by the cellular RNAi machinery, which then cuts the transcript into fragments of 21-24 nucleotides (nt). The RNA fragments become sequence guides for enzymes which destroy complementary RNA sequences, including any endogenous transcript with sequence closely matching the transgene. Through this pathway, there is a strong decrease in levels of endogenous transcripts highly similar to the RNAi construct. Because the transcript from the construct is destroyed in this process, no proteins are produced.
21. In addition, each GM wheat line contains the antibiotic resistance selectable marker gene, neomycin phosphotransferase II (nptII). This gene, encoding the enzyme neomycin phosphotransferase, was derived from Escherichia coli and confers resistance to antibiotics such as kanamycin and G-418 on the GM plants. The wheat lines also contain a bacterial selectable marker, β-lactamase (bla), a gene derived from E. coli which confers resistance to the antibiotic ampicillin. Expression of the bla marker is controlled by sequences which only direct transcription of the gene in bacterial cells, and therefore is not expressed in the GM wheat lines.
Table 1. The genes used to genetically modify wheat
| Gene | Database identification number (database name) | Function of protein | Source | Intended purpose |
|---|---|---|---|---|
| three α-gliadin family members | TC219947 TC89936 TC220528 (DFCI Wheat gene index) | Family of grain storage proteins known to influence grain quality parameters | Wheat | Altered grain protein composition |
| four γ-gliadin family members | TC57438 TC57118 TC51146 TC57070 (DFCI Wheat gene index) | Family of grain storage proteins known to influence grain quality parameters | Wheat | Altered grain protein composition |
| Starch Metabolic Enzyme A (SME A) | CCI | CCI | Wheat | Altered grain starch composition |
| Starch Metabolic Enzyme B (SME B) | CCI | CCI | Wheat | Altered grain starch composition |
| Starch Enzyme I (SEI) | CCI | CCI | Wheat | Altered grain starch composition |
| Neomycin phospho-transferase II | AAF65403 (Genbank) | Kanamycin resistance | E. coli | Selectable marker |
22. Short regulatory sequences (promoters and transcription termination sequences) that control expression of the introduced RNAi constructs are also present in the GM wheat lines. These are derived from wheat and Oryza sativa (rice), the plant virus Cauliflower mosaic virus (CaMV), and the bacterium Agrobacterium tumefaciens.
5.2 The introduced RNAi constructs and their associated effects
23. The RNAi constructs in the current application contain fragments, rather than entire coding sequences, of the different target genes. However, the complete coding sequences of the nptII and bla marker genes are present in various constructs.5.2.1 The α-gliadin and γ-gliadin gene families
24. The wheat grain consists of a seed coat enclosing the wheat embryo and the endosperm, a large storage organ which provides energy to the embryo following germination. Starch is the major component of wheat grains, and proteins comprise approximately 11-19% of the grain mass (Bordes et al. 2008).25. As with all cereal-seed proteins, wheat grain proteins have historically been grouped chemically on the basis of solubility: the water-soluble albumins, dilute saline-soluble globulins, dilute acid or alkali soluble glutelins (known in wheat as glutenins), and alcohol-soluble prolamins (so named because they are rich in the amino acids proline and glutamine) (reviewed by Gianibelli et al. 2001). The prolamins of wheat are also known as gliadins, a large class of proteins which are divided into groups according to mobility in acid polyacrylamide gel electrophoresis into α,β, γ and ω groups (from lowest to highest molecular weight) (reviewed by Gianibelli et al. 2001). Protein sequence comparisons have revealed that there is a common evolutionary origin of wheat gliadins and glutenins, which have more recently been described as a combined group, the prolamin superfamily (Shewry & Halford 2002).
26. Proteins of the prolamin superfamily account for approximately 80% of protein present in wheat flour, with approximately equal amounts of glutenins and gliadins (Gras et al. 2001). α-gliadins are the most abundant group of seed storage proteins in wheat, making up 15-30% of wheat seed protein (Gu et al. 2004). These proteins give rise to the unique dough-forming properties of wheat flour. Upon adding water to wheat flour to make dough, the insoluble gliadins and glutenins form a viscoelastic protein mass known as gluten. Gliadins contribute to the extensibility of dough, glutenins the elasticity, and the ratio of gliadins to glutenins is an important determinant of dough properties.
27. Gliadin genes are clustered at complex loci present in each wheat genome (A, B and D). The homologous group 1 chromosome loci Gli-A1, Gli-B1 and Gli-D1, contain all ω- and most γ-gliadins, and the homologous group 6 chromosome loci Gli-A2, Gli-B2 and Gli-D2 contain all α-, some β- and some γ-gliadins (reviewed by Gianibelli et al. 2001). The number of wheat γ-gliadin genes has been estimated at 15-40 (Sabelli and Shewry 1991, cited by Zhang et al. 2003). There are estimated to be up to 150 α-gliadin genes in wheat (Anderson et al. 1997), approximately 50% of which are thought to be psuedogenes (Anderson & Greene 1997), however only a small number can be resolved by electrophoresis methods. The size of the γ-gliadin and α-gliadin families varies between cultivars and each group includes some highly similar, recently duplicated sequences which are expected to be impossible to separate by physical methods such as electrophoresis (Anderson et al. 1997; Anderson et al. 2001).
5.2.2 Effects of α- and γ-gliadin silencing
28. The DIR 092 application includes four GM wheat lines containing RNAi constructs designed to reduce expression of γ-gliadin genes, and two GM wheat lines containing RNAi constructs designed to reduce expression of α-gliadin genes. There are two major aims driving global research efforts related to gliadins. Firstly, understanding the contribution of specific groups of gliadins to grain product quality may enable manipulation of dough characteristics to suit specific end uses. Secondly, gliadins are known to be major inducers of celiac disease, an inflammatory disorder of the small intestine triggered by gluten, affecting approximately one in 200 people (reviewed by Sollid 2002). Better understanding of the role of specific gliadins in celiac disease may lead to development of wheat products suitable for celiac sufferers. However, due to the complex, closely linked nature of the loci encoding the large gliadin sub-families and the hexaploid nature of the wheat genome, dissection of the roles of gliadin sub-families has been difficult. A simple alternative to breeding approaches is the use of RNAi to silence these groups of genes.29. Sequences are not known for all α- and γ-gliadins, but sequence comparisons have established that there is high similarity within each sub-family (Anderson & Greene 1997; Anderson et al. 2001). As the RNAi mechanism depends upon sequence homology, it is likely that cross-silencing of many family members would result from an RNAi construct designed against conserved domains of well chosen family members. The γ-gliadin RNAi construct in the proposed dealing uses a chimeric hairpin containing sequences of four phylogenetically diverse γ-gliadin cDNA clones (by comparison to Anderson et al. 2001). Similarly, the α-gliadin construct contains two stretches of sequence from each of three phylogenetically diverse α-gliadin cDNA clones (by comparison to Anderson & Greene 1997).
30. Previous studies which used RNAi to alter α- and γ-gliadin gene expression have been successful in broadly reducing levels of the targeted gliadin sub-families (Folck et al. 2003; Wieser et al. 2006; Gil-Humanes et al. 2008). In these α-gliadin RNAi lines, compensatory changes in levels of other grain proteins were observed, such that total grain protein levels are constant. Similar changes have been observed in α- and γ-gliadin RNAi lines in the proposed release (information supplied by the applicant).
5.2.3 The Starch Metabolic Enzyme A, Starch Metabolic Enzyme B and Starch Enzyme I genes and effects of their silencing
31. The DIR 092 application includes GM wheat lines carrying RNAi constructs designed to reduce expression of Starch Metabolic Enzyme A (SME A, four lines) and Starch Metabolic Enzyme B (SME B, four lines). Also included are two cross-derived lines of wheat, the product of crosses between a single Starch Enzyme I (SE I) RNAi line and two different SME B RNAi lines. RNAi constructs targeted against these three genes reduces their expression, resulting in changes to grain starch composition. The specific identities of these genes and the phenotypes resulting from their silencing have been declared CCI and are not discussed further in this Section.5.2.4 Toxicity/allergenicity of the effects associated with the introduced RNAi constructs
32. Several types of allergic and immune reactions to wheat products have been recorded, with bakers asthma and celiac disease being the best characterised. Bakers asthma is a respiratory allergy to inhaled flour and dust from grain processing, which is one of the most important occupational allergies in many countries (reviewed by Tatham & Shewry 2008). Celiac disease is an inflammatory disorder of the small intestine triggered by gluten consumption resulting in poor nutrient absorption, which affects approximately one in 200 people (reviewed by Sollid 2002). Other less well studied reactions to wheat include dietary allergy and pollen allergy. A variety of wheat proteins contribute to these adverse reactions, and identification of specific problem proteins is complicated by similar allergy/intolerance responses being induced by different proteins in different individuals.33. The most important contributor to bakers asthma is a group of α-amylase inhibitors found in wheat grains which inhibit mammalian and insect α-amylase enzymes (reviewed by Tatham & Shewry 2008). A wide variety of other proteins have been shown to bind to immunoglobulin E (IgE) from bakers asthma patients, indicating an involvement in the allergy, including wheat germ agglutinin, peroxidase, thioredoxin, α-, β-, γ-, and ω-gliadins, α- and β-amylase, acyl CoA oxidase, glycerinaldehyde-3-phosphate dehydrogenase, triosephosphate isomerase and serpin (reviewed by Tatham & Shewry 2008). Experiments involving testing patient serum IgE for immunoreactivity to blotted wheat proteins indicated individuals react to unique sets of 10 to 50 proteins, and only the more commonly occurring of these have been identified (reviewed by Tatham & Shewry 2008). Some of these proteins are also known to be involved in dietary allergy to wheat, with gluten proteins recognised as the most important contributors.
34. Celiac disease has been characterised as an intolerance to various epitopes (portions of proteins to which antibodies react) derived from gluten proteins including α- and γ-gliadins, and low molecular weight glutenin subunits (reviewed by Sollid 2002). It is thought to be initiated through a T-cell response to specific gluten peptides, often α-gliadins, in the small intestine. This leads to expression of the enzyme tissue transglutaminase which damages the intestinal mucosa and also deamidates further gluten proteins, leading to an enhanced T-cell response (reviewed by van Herpen et al. 2006). The specific range of epitopes against which a celiac patient reacts varies between individuals, however α-gliadins are particularly common.
35. In the GM wheat lines modified for grain composition, the use of RNAi has the direct effect of reducing the expression of endogenous transcripts, without the expression of novel proteins. However, secondary effects of silencing the target genes can alter expression of untargeted proteins. Data provided by the applicant shows that secondary effects occur in the gliadin silencing lines: in the γ-gliadin RNAi lines, γ-gliadin expression is strongly reduced, and total grain protein concentration is maintained at normal levels through compensatory increases in levels of α-gliadins and high and low molecular weight glutenins. The applicant reports similar compensatory mechanisms in α-gliadin silencing lines. The groups of seed storage proteins among which these changes are seen include known epitopes to which celiac and wheat allergy sufferers react, raising the possibility that these GM wheat lines may have increased allergenicity/immunogenicity for some people upon consumption or inhalation of flour.
36. The silencing of SME A, SME B and both SME B and SE I results in changes to the starch composition of the GM grains. Humans are exposed to vast amounts of dietary starch, from a range of sources varying in the parameters changed in the GM lines. Because current starch intakes result in no allergenic or toxic effects, it is highly unlikely that the changes occurring in the silencing lines would result in altered toxicity or allergenicity of the GM wheat lines compared to parental cultivars.
37. No studies on the toxicity or allergenicity of the GM wheat lines and their products have been undertaken to date as the proposed trial is at an early stage, and the proposed rat and pig nutritional studies will not address this issue. Such studies would have to be conducted if approval was sought for the GMOs or their products were to be considered for human consumption in Australia.
5.2.5 The plant antibiotic resistance marker gene nptII and the encoded protein
38. The GM wheat lines contain the antibiotic resistance selectable marker gene, neomycin phosphotransferase II (nptII). This gene, encoding the enzyme neomycin phosphotransferase, was derived from E. coli and confers resistance to antibiotics such as kanamycin or G-418 on the GM plant. The nptII gene was used as a selective marker to identify transformed plant tissue during initial development of the GM plant lines in the laboratory.39. The nptII gene is used extensively as a selectable marker in the production of GM plants (Miki & McHugh 2004). As discussed in previous DIR RARMPs, and in more detail in the RARMPs for DIR 070/2006 and DIR 74/2007 ( available by contacting the OGTR), regulatory agencies in Australia and in other countries have assessed the use of the nptII gene in GMOs as not posing a risk to human or animal health or to the environment. The most recent 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 2007).
5.2.6 The bacterial antibiotic resistance marker gene bla and the encoded protein
40. The GM wheat lines contain the β-lactamase (bla, also known as amp) antibiotic resistance marker gene. The bla gene is derived from E. coli (Spanu et al. 2002) and encodes the β-lactamase enzyme, which confers ampicillin resistance.41. The β-lactamase enzyme is widespread in the environment and in food. Naturally occurring ampicillin-resistant microorganisms have been found in mammalian digestive systems (Spanu et al. 2002). The bla gene was originally isolated from antibiotic resistant strains of E. coli found in hospital patients.
42. The bla gene in the GM wheat lines is under the control of its own bacterial promoter and terminator from E. coli and therefore is not expressed in the GM wheat plants. The gene was used in the laboratory prior to the production of the GM wheat lines.
43. A number of GM food crops containing the bla gene have been approved for limited and controlled release both in Australia (DIRs 019/2002, 026/2002, 028/2002, 051/2004, 052/2004, 070/2006 and 071/2006) and overseas. No adverse effects on humans, animals or the environment have been reported from these releases.
5.3 The regulatory sequences
5.3.1 Regulatory sequences for expression of the wheat RNAi constructs
44. Promoters are DNA sequences that are required in order to allow RNA polymerase to bind and initiate correct transcription. The regulatory sequences included in each RNAi construct are described in Table 2. Two different promoters control the expression of RNAi constructs in the GM wheat lines: the wheat Bx17 high molecular weight glutenin subunit promoter and the wheat Dx5 high molecular weight glutenin promoter. Both the Bx17 and Dx5 promoters are thought to be endosperm specific (information provided by applicant). Published characterisation of marker gene expression from approximately 1200 nt of the Dx5 promoter confirmed that it directs endosperm-specific expression (Lamacchia et al. 2001). However, a much shorter sequence from the Dx5 promoter is used in the GM wheat lines, which may result in a different expression pattern.45. Separation of the inverted-repeat arms of RNAi constructs with a spliceable intron has been shown to increase the effectiveness of silencing (Smith et al. 2002). Various introns are used to separate the arms of the RNAi constructs in the GM wheat lines, and are described in Table 2. These sequences are derived from rice and wheat intron sequences.
46. Also required for gene expression in plants is an mRNA termination region, including a polyadenylation signal. The mRNA termination region for all RNAi constructs in the GM wheat lines is derived from the Agrobacterium tumefaciens nos terminator (Bevan 1984).
Table 2. Gene constructs used to generate the GM wheat lines proposed for release
| Construct | RNAi construct | Plant selection marker | Bacterial selection marker | Transformation method | |||
|---|---|---|---|---|---|---|---|
| Promoter | Inverted repeat target sequence | Intron | Terminator | ||||
| pBx17GLdup | pBx17 | 272-288 nt of each of four γ-gliadins | Rice SBEI intron 9 | nos | - | bla | Biolistic |
| pBx17Adup | pBx17 | 117-228 nt of two parts of each of three α-gliadins | Rice SBEI intron 9 | nos | - | bla | Biolistic |
| SME A-IRBx17casNOT | pBx17 | 572 nt of SME A | Rice SBEI introns 4 and 9 | nos | - | bla | Biolistic |
| pBx17SME B casNOT | pBx17 | 581 nt SME B | Rice SBEI introns 4 and 9 | nos | - | bla | Biolistic |
| pCMneoSTLS2 | - | - | - | - | p35S:STLS2 intron 9:NPTII:35S | bla | Biolistic |
| CCI | pDx5 | exons 1-3 of SE I | SE I intron 3 | nos | pActin:actin intron:NPTII:nos | bla | Agrobacterium-mediated |
Although A. tumefaciens is a plant pathogen, the regulatory sequence comprises only a small part of its total genome, and is not capable of causing disease.
5.3.2 Regulatory sequences for the expression of the nptII gene
47. A nptII transformation marker plasmid pCMneoSTLS2 (Maas et al. 1997) was used, in addition to the plasmids encoding RNAi constructs, for those GM wheat lines generated by biolistic transformation (see Table 2). The nptII gene is under the control of the CaMV 35S promoter and 35SpA terminator sequence. CaMV 35S is a constitutive promoter and directs the nptII gene to be expressed in most plant tissues and throughout the plant lifecycle. Although CaMV is a plant pathogen, the regulatory sequence comprises only a small part of its total genome and is not capable of causing disease. The 35S promoter and nptII coding sequence are separated by intron 9 of the potato stls2 gene, so as to ensure expression only in eukaryotic cells.48. The line generated by Agrobacterium-mediated transformation contains a nptII transformation marker as part of the T-DNA transformed into the plant (see below). The expression of the nptII gene is controlled by the rice Actin promoter, which is separated from the nptII coding sequence by a rice Actin intron. Expression is terminated by the A. tumefaciens nos terminator. Although A. tumefaciens is a plant pathogen, the regulatory sequence comprises only a small part of its total genome, and is not capable of causing disease.
5.4 Method of genetic modification
49. Two different methods were used to generate the GM wheat lines in the proposed release (see Table 2). Biolistic transformation (Pellegrineschi et al. 2002) involved coating very small gold particles with two transformation constructs, one containing a plant selectable marker (pCMneoSTLS2, common to all lines generated using this method) and a second containing the RNAi construct. The particles were then ‘shot’ into embryos from T. aestivum cultivar Bobwhite 26. Genetically modified plant tissues were recovered by survival on tissue culture media containing the selective agent G-418.50. A. tumefaciens-mediated transformation was used to generate the one parent of the cross-derived double RNAi 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). Plants can be genetically modified by the transfer of DNA (transfer-DNA or T-DNA, located between specific border sequences on a resident plasmid) from A. tumefaciens through the mediation of genes from the virulence region of Ti plasmids.
51. Disarmed Agrobacterium strains have been constructed specifically for plant transformation. The disarmed strains 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).
52. To generate the line in the current application, the Agrobacterium strain LBA4404 was transformed with a disarmed T-DNA-containing plasmid encoding a plant selectable marker and an RNAi cassette (see Table 2). The Agrobacterium was injected under the scutellum of developing embryos of the wheat cultivar NB1 and, after incubation, callus tissue was cultured from transformed embryo cells, which was regenerated into plantlets in tissue culture in the presence of the selective agent. The initial transformation events were performed by CSIRO’s collaborators at Biogemma PLC in the United Kingdom and transformed wheat seeds were imported into Australia under an Australian Quarantine Inspection Service (AQIS) permit.
53. The current application involves 16 GM wheat lines, which were generated from 15 independent transformation events. For each of these independent events the introduced RNAi constructs are expected to be located at different sites in the wheat genome. Fourteen of the GM wheat lines are the product of single transformation events. The other two lines were generated by crossing a line with an additional transformation event to each of two transformation events already included in the proposed trial as single transformation events. Thus the 15th transformation event is part of the proposed trial only in combination with two other transformation events.
54. 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.
5.5 Characterisation of the GMOs
5.5.1 Stability and molecular characterisation
55. Constructs used to generate the GM wheat lines were sequenced prior to transformation.56. Molecular characterisation of the GM wheat lines has not been carried out, as the project is in early stages. The genomic locations of the introduced DNA has not been characterised, and the number of copies of the transgenes present in each line is unknown.
57. The applicant states that the transgenes are stably inherited (as monitored by PCR assays) over four generations of single-seed descent for lines generated by biolistic transformation, and for three generations of single-seed descent for cross-derived lines.
5.5.2 Expression of the introduced RNAi constructs in the GM wheat lines
58. Expression of the introduced RNAi constructs has not been investigated, except through assessment of resultant phenotypes (see below). Transcripts generated by RNAi constructs are cleaved by RNase enzymes as part of the process of RNAi, and so measurement of whole transcripts is typically not used as a measure of their expression levels. However, quantification of the 21-24 nt RNA molecules produced during transcript cleavage is often used to gauge the effectiveness of silencing, as is quantification of target gene transcript levels. Neither of these alternative measures have been employed by the applicant.5.5.3 Characterisation of the phenotypes of the GM wheat lines
Gliadin RNAi lines
59. Seed proteins from T21 and T3 γ-gliadin and α-gliadin RNAi lines have been quantified using high performance liquid chromatography. In the γ-gliadin RNAi lines, total grain protein levels are unchanged, γ-gliadins are reduced by approximately 64-87%, and compensatory increases in other classes of seed storage proteins (α-gliadins and high and low molecular weight glutenins) were observed. In the α-gliadin RNAi lines, α-gliadin content is reduced by 3-41%. In one of the lines compensatory increases in high and low molecular weight glutenins are seen, while no changes in other classes were observed in the second line. Plant height, tiller number and 100 seed weight do not vary between these lines and non-GM lines.
SME A RNAi lines
60. SDS-PAGE analysis of proteins isolated from starch granules of T3 SME A RNAi lines showed a strong reduction in the amount of SME A protein present. This result is supported by activity assays which showed that the endosperm proteins of RNAi lines have approximately 20% of the SME A enzyme activity non-GM lines. Analysis of grain starch showed that total starch content is unchanged, and a small increase in amylose content is observed. Further details of analysis of grain composition in the SME A RNAi lines have been declared CCI and are not discussed further in this section.
SME B RNAi lines
61. Analysis of endosperm proteins in T3 SME B RNAi lines by western blotting showed that SME B protein was reduced to undetectable levels. Amylose content and chain length distribution of debranched amylopectin are unchanged. Further details of analysis of grain composition in the SME B RNAi lines have been declared CCI and are not discussed further in this section.
62. Observation of some secondary vegetative phenotypes has been carried out for the SME B RNAi lines, however this information has been declared CCI and is not discussed further in this section.
SME B and SE I RNAi lines
63. Some characterisation of lines carrying RNAi constructs against both SME B and SE I has been carried out. The details of this characterisation have been declared CCI and are not discussed further in this section.
1The generation of a GM plant is identified by the letter T (transgenic) followed by a numeral indicating the number of generations for which it has been maintained, beginning at T0 for the original transformed plant.