5.1 Introduction to the GMOs
31. The GM wheat lines contain partial sequences of the SEI gene (details of which are CCI) and the GM barley line contains partial sequences of the SEI and SEII genes (details of which are CCI), which are both involved in starch biosynthesis. The partial gene sequences are part of RNA interference (RNAi) constructs used to genetically modify wheat and barley (see Section 5.3, this Chapter for more detailed information). The RNAi constructs were designed to down-regulate or silence endogenous wheat or barley genes involved in starch biosynthesis. Evaluation of the GM wheat and barley lines in contained facilities has shown altered grain starch composition as a result of the genetic modification. Background information on grain starch and its synthesis is found in Section 5.2 of this Chapter.
The GM wheat and barley lines
32. All the wheat and barley lines proposed for release have been genetically modified to produce grains with altered grain starch composition compared to their non-GM parent lines. A summary of the genes or partial genes contained within the introduced construct is in Table 2.
Figure 2. Diagram of some of the proposed containment measures (not drawn to scale). The diagram is based on the first year planting regime.
33. The GM wheat line 85.2c contains partial sequences of the SEI gene in a genetic background of NB1.
34. The wheat line 50.1b which itself is not proposed for release in this trial, was generated as a parental line for the two other GM wheat lines proposed for release. The 50.1b line contains partial sequences of the SEI gene in a genetic background of NB1. The applicant has indicated that the 85.2c and 50.1b lines exhibit the same phenotype.
35. The GM wheat line 212 was generated by crossing line 50.1b with a SEIII (details of which are CCI) triple null.
36. Similarly, the GM wheat line YDH7 was generated by crossing line 50.1b with a SEIV (details of which are CCI) triple null.
37. The GM barley line BC10.5 contains partial sequences of both the SEI and SEII genes. This line was generated through the hybridisation of parental lines containing each of the SEI or SEII constructs. Both parental lines containing the individual constructs are in a genetic background of Golden Promise.
Table 2. The genes or partial genes used to modify wheat and barley.
|Gene sequences||Accession No (Genbank)||Protein encoded||Protein involved in||Source||Intended purpose|
|SEI (partial sequences)||-||Starch Enzyme I||Starch biosynthesis||Wheat||Altered starch composition|
|SEII (partial sequences)||-||Starch Enzyme II||Starch biosynthesis||Wheat||Altered starch composition|
|nptII||AAF65403||neomycin phosphotransferase type II||kanamycin and neomycin resistance||Escherichia coli||Selection of transformants|
|Escherichia coli||Selection of transformants|
38. The GM wheat lines also contains the antibiotic resistance selectable marker gene, neomycin phosphotransferase type II (nptII). The GM barley line contains the antibiotic resistance gene hygromycin phosphotransferase (hpt), details of these genes are in Sections 5.3.4 and 5.3.5.
5.2 Background on grain starch and its synthesis
Starch and its function
39. Starch synthesis is highly conserved in plants and has a crucial role in plant physiology and reproduction (Dinges et al. 2001). Starch serves as the main storage carbohydrate in plants and is synthesised in different tissues within the plant such as in the chloroplast of leaves and in the amyloplast of storage tissues such as cereal seeds and tuberous tissue (Boyer 1996; Smith 2007).
40. Starch has been an important food component for humans and as a feed source for animals for centuries. The composition of starch plays an important role in determining the nutritional quality and physical properties of food products derived from cereal grains. The synthesis of starch and products derived from it, have been described extensively in the scientific literature and has been covered in various reviews [see for example James et al (2003) Ball and Morell (2004), Jobling (2004), Tetlow et al (2004a) and Van Hung et al (2006)].
41. In humans, starch is digested extensively in the small intestine and the resulting glucose is released into the blood stream and thus available as an energy source. Resistant starch (RS) is the component of starch that escapes digestion in the small intestine and passes into the large bowel. Resistant starch also contributes to total dietary fibre intake. However, the level of resistant starch in the diet of the general public is low, people in Australia only consume 10-20 % of the recommended daily intake. The lack of RS in the human diet is thought to contribute to the high rates of diet related diseases such as diabetes and colo-rectal cancer (Bird et al. 2000; CSIRO 2008). There are different types of resistant starch and they are present naturally at varying levels in a number of foods. The physical properties of RS have been made use of in the food industry, reviewed by Sijita et al (2006).
The starch biosynthetic pathway
42. Starch biosynthesis occurs in all higher plants; in cereals it occurs mainly in storage tissues such as the grain but also in the leaves (Boyer 1996). Transient starch produced during the day is used as a subsequent carbon source during non-light periods while starch synthesised in the storage tissues such as the cereal grains are produced for longer term carbohydrate storage (Tetlow et al. 2004a). Starch generally makes up approximately 65% of the wheat kernel (Hannah 2007).
43. Starch is an insoluble polymer made up of two D-glucose homopolymers; amylopectin and amylose. The amylopectin component of starch is approximately 70-80% with amylose making up the remaining 20-30%. Mutations in the biosynthesis pathway of either amylose or amylopectin can lead to the production of different ratios of amylose to amylopectin (Ball et al. 1996; Myers et al. 2000; Jobling 2004; Van Hung et al. 2006).
44. Amylopectin is a molecule with a high degree of structural organisation, it is made up of glucose molecules, ranging between 3x105 and 3x106 glucose units, joined via α-1-4 linkages generating linear chains of various lengths. In addition it has random α-1-6 branch points catalysed by starch branching enzymes (Myers et al. 2000; James et al. 2003; Van Hung et al. 2006).
45. Amylose is essentially a linear molecule made up of glucose molecules, ranging between 500-6000 glucose units, joined through α-1-4 linkages, it can also contain a small number of α-1-6 branches (Myers et al. 2000; James et al. 2003; Van Hung et al. 2006).
46. The synthesis of starch in the cereal endosperm involves three main classes of enzymes; the starch branching enzymes (SBE), starch synthases (SS) and starch debranching enzymes. In cereals some isoforms11 of these enzymes are unique to the endosperm (Ball et al. 1996; Myers et al. 2000). Figure 3 provides a diagram of the starch biosynthetic pathway in the cereal endosperm.
Starch branching enzymes
47. Starch branching enzymes (SBE) form the branches on the linear chain of glucose molecules by generating α-1-6 linkages through cleavage of α-1-4 linkage
Figure 3. A simplified diagram of starch synthesis in the amyloplast. Adapted from (Dupont 2008).
48. The co-ordination of branching, de-branching and starch synthases activity required for starch synthesis appears to be modulated through the physical association of these enzymes within the amyloplast. Such physical association of enzymes involved in the starch biosynthetic pathway have recently been established and could explain some of the pleiotropic effects observed as a result of the analysis of mutants in this pathway (Tetlow et al. 2004a; Hennen-Bierwagen et al. 2008).
49. The cereal endosperm has at least five isoforms of starch synthases, grouped according to their sequence conservation. These isoforms have a distinctive function in the synthesis of amylopectin (Ball & Morell 2004). A granular bound form, GBSSI has a role in amylose elongation (Nakamura et al. 1995).
50. Starch synthases use ADP-glucose (ADPG) to elongate linear chains by transferring the glucose moiety from ADGP to form α-1-4 linkages. ADGP is synthesised from glucose-1-phosphate and ATP by another important enzyme of the starch synthesis pathway adenosine 5'-diphosphate pyrophosphorylase (AGPase). ADPase is thought to be a rate limiting enzyme in starch biosynthesis (Stark et al. 1992; Hannah 2007).
Starch debranching enzymes
51. In addition to starch synthases and branching enzymes, debranching enzymes have also been indicated to play a role in starch synthesis. In plants two debranching types have been identified, the pullunases and isoamylases. Both enzyme types hydrolyse α-1-6 linkages but have different substrate specificities (Tetlow et al. 2004a).
5.3 The introduced constructs and associated end products
52. Wheat and barley were genetically modified using the SEI RNAi construct. This construct contains a tandem repeat of a partial sequence of the SEI gene. The partial sequences are arranged in opposite orientation (sense and anti-sense) and are separated by intron 3, also derived from the SEI gene. Expression is driven by the wheat derived Dx5 high molecular weight glutenin promoter (pHMWG) which is endosperm specific (Blechl & Anderson 1996). The termination sequence for the construct was derived from the nos gene from A. tumefaciens. The nptII gene is also present in this construct. The nptII gene expression is driven by the actin promoter from rice and has the nos termination sequence. The RNAi construct is flanked on either side by the left and right border sequences from A. tumefaciens. A small sequence from exon 4 from SEI is also present as a result of the cloning procedure.
53. Barley was also genetically modified using the SEII RNAi construct in addition to the SEI construct. The basic design of the SEII construct is similar in to the SEI RNAi construct except that the partial gene sequences and the intron 3 separating them were derived from the SEII gene. Expression of the construct is again driven by the Dx5 promoter and the nos termination sequence from A. tumefaciens. The SEII construct also contains the hpt gene which has the Cauliflower mosaic virus (35S) promoter and nos termination sequence. As above, the RNAi construct is flanked by the left and right borders from A. tumefaciens. A small sequence from exon 4 from SEII is also present as a result of the cloning procedure.
54. Transcription of the introduced RNAi construct will generate messenger RNA (mRNA) from the tandem unit (sense) and its complimentary inverted repeat (antisense). The annealing of the sense and anti-sense mRNAs produces a double stranded RNA (dsRNA) of which the intron is spliced out. The presence of a double stranded RNA triggers a conserved biological response to dsRNA; it is cut into small fragments of 21-25 base pairs. These small fragments guide specific enzymes to any RNA that has the same or closely similar sequence which they cleave and subsequently degrade (Ahlquist 2002). This is known as RNA interference (RNAi) and leads to the down-regulation of the targeted gene(s) (Hannon 2002). Degradation of the mRNA therefore prevents production of the targeted protein. The use of an intron between the inverted repeats has been shown to enhance RNAi silencing (Smith et al. 2002).
5.3.1 The genes encoding the starch branching enzymes SEI and SEII
55. The SEI and SEII genes were isolated from the wheat Aegilops tauschii (donor of the D genome in wheat) and Triticum aestivum, respectively.
56. The naturally occurring SEI and the SEII genes share 86% homology at the protein level and differ mainly at the amino terminus of the protein. This difference has been utilised to generate isoform specific anti-sera to these proteins.
57. The SEI sequences from wheat and barley share 98 % sequence homology at the protein level. The SEII sequences from wheat and barley share 95% homology at the protein level (National Center for Biotechnology Information 2001).
5.3.2 The effects associated with the introduced RNAi constructs for altered starch composition
58. The aim of the genetic modification is to suppress the expression of the corresponding endogenous genes. This is achieved through the incorporation of partial sequences of the target genes in the genetic construct and as such no new proteins are produced by the GM wheat and GM barley lines. Suppression of endogenous genes in the GM wheat and GM barley does not change the types of starches found in grains but alters starch composition of the grain. There is evidence in the literature that changes to the expression of the SEI and/or SEII genes can alter starch composition which in turn may enhance the nutritional properties of starch such as a higher resistant starch content. Resistant starch is the fraction of starch that remains undigested in the small intestine and, as a result the rate at which glucose enters the blood stream is reduced.
59. A change in the composition of starch can also have profound effects on starch physical properties and thus alter flour and dough properties. This can result in flour products with unique qualities that allow for new uses in food and also in other non-food related applications (Van Hung et al. 2006). For example, low amylose flours are seen as beneficial in the pasta and noodle industry as they result in the production of a higher quality product with better texture. In frozen foods its use is seen as beneficial in improving freeze-thaw stability and preserving flavour (Jobling 2004). More detail on the effects of altered starch composition on flour properties can be found in reviews by Jobling (2004) and Van Hung (2006).
5.3.3 Toxicity/allergenicity of the end products associated with the introduced RNAi construct for altered starch composition
60. Bioinformatic analysis may assist in the assessment process by predicting, on a purely theoretical basis, the toxic or allergenic potential of a protein. The results of such analyses are not definitive and should be used only to identify those proteins requiring more rigorous testing (Goodman et al. 2008).
61. No toxicity/allergenicity tests have been performed on the GM wheat or barley as the proposed trial is still at an early stage. Such tests would have to be conducted if approval was sought for the GMOs, or products derived from the GMOs, to be considered for general human consumption in Australia.
62. Based on consumption of cereals and other foods with altered starch composition, the changes in starch composition observed in the GM lines proposed for release are unlikely to be detrimental to human health. For example, rice has a number of naturally occurring varieties which have been consumed by humans for centuries. These different varieties have different starch composition which also results in different physical characteristics of the rice (Brand Miller et al. 1992).
5.3.4 The antibiotic resistance gene nptII and the encoded protein
63. The GM wheat lines contain the antibiotic resistance gene, neomycin phosphotransferase type II (nptII). This gene, encoding the enzyme neomycin phosphotransferase, was derived from E. coli and confers resistance on the GM plant to antibiotics such as kanamycin or neomycin. The nptII gene was used as a selective marker to identify transformed plant tissue during initial development in the laboratory of the GM wheat lines.
64. The nptII gene is used extensively as a selectable marker in the production of GM plants (Miki & McHugh 2004). As discussed in more detail in the RARMPs for DIR 070/2006 and DIR 074/2007 (or 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).
5.3.5 The antibiotic resistance gene hpt and the encoded protein
65. The GM barley line contains the hpt gene from E. coli which encodes an aminocyclitol phosphotransferase and confers resistance to the antibiotic hygromycin B.
66. The HPT enzyme catalyses the phosphorylation of the 4-hydroxy group on the hyosamine moiety, thereby inactivating hygromycin (Rao et al. 1983) and preventing it from killing cells producing HPT. The hpt gene was used as a selectable marker gene in the early laboratory stages of development of the plants to enable selection of plant cells containing the desired genetic modification.
67. The hpt gene is used extensively as a selectable marker in the production of GM plants (Miki & McHugh 2004). As discussed in the RARMP for DIR 073/2007 and more recently DIR 077/2007, the use of hpt, or other hygromyin B phosphotransferase encoding genes, as marker genes in GM plants has been assessed as not posing a risk to human health and safety or the environment. 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).
5.4 The regulatory sequences
5.4.1 Regulatory sequences for expression of the partial gene sequences for SEI and SEII
68. Promoters are DNA sequences that are required to allow RNA polymerase to bind and initiate correct transcription. The SEI and SEII partial gene sequences present in the GM wheat and barley are under the control of the endosperm specific high molecular weight glutenin promoter (HMWG-Dx5) obtained from wheat (Blechl & Anderson 1996).
69. The applicant has not tested plant tissues other than the endosperm to confirm that expression of the partial genes in the RNAi construct is indeed confined to the endosperm. However, the applicant has performed in silico analysis of expression patterns of the Dx5 promoter using information deposited in the wheat EST (expressed sequence tag) database of Gene Index Project (Computational Biology and Functional genomics Laboratory 2008) and the Dx5 unigene set at NCBI (NCBI 2009). These databases contain cDNA libraries which have been derived from all tissues of wheat. Out of a total of 1,034,368 ESTs, the ESTs derived from the Dx5 gene were only identified in developing heads or endosperm derived cDNA libraries. In the NCBI Unigene set for Dx5, transcripts were identified in developing heads or endosperm derived libraries. One transcript was identified in a library from an unspecified origin. Overall these analyses indicate that expression driven from the Dx5 promoter is confined to the endosperm.
70. Also required for gene expression in plants is an mRNA termination region, including a polyadenylation signal. The mRNA termination region for the SEI and SEII genes in the GM wheat and barley is derived from the nos gene from A. tumefaciens.
5.4.2 Regulatory sequences for the expression of the nptII and hpt gene
71. Expression of the nptII gene in GM wheat plants is controlled by the actin gene promoter from rice and the nos gene mRNA termination region from A. tumefaciens (Bevan 1984). Expression of the hpt gene in GM barley plants is controlled by the Cauliflower mosaic virus (CaMV) 35S promoter and the nos gene from A. tumefaciens (Bevan 1984).
5.5 Method of genetic modification
72. The GM wheat lines 85.2c and 50.1b (the parent line for the proposed lines 212 and YDH7, see below) were generated by A. tumefaciens-mediated transformation. The pSB11 vector which contained the SEI RNAi construct was co-electroporated with the vir plasmid pAL4404 into the disarmed A. tumefaciens strain LBA4404. The Agrobacterium was then injected under the scutellum of developing wheat embryos of the NB1 cultivar. The developing embryos were excised after a few days and transferred to a medium which induced the formation of the callus. The callus was then transferred to a medium containing the selective antibiotic to induce the formation of plantlets. The wheat cultivar NB1 which was selected because it is amenable to Agrobacterium-mediated transformation.
73. The GM wheat line 212 was generated through transformation of the NB1 line (giving GM line 50.1b) which was then conventionally bred with a SEIII triple null mutant line. The triple null SEIII was derived through hybridisation of the three wheat cultivars; Cadoux (noodle wheat), Vectis (soft biscuit wheat) and a chromosome engineered line of Chinese Spring, CS7AL-15.
74. The GM wheat line YDH7, was generated through conventional breeding of GM line 50.1b with a SEIV triple null mutant wheat which was derived through hybridisation between three exotic lines; Kanto 79, Chousen 30 and Turkey 116.
75. The GM barley line BC10.5 was generated by hybridisation of GM barley lines containing either one of the SEI or SEII RNAi constructs. The pSB11 vector containing either the SEI or SEII RNAi construct was electroporated into the disarmed A. tumefaciens strain Agl1 carrying the plasmid pWBVec8. Following the transformation process and plant regeneration, screening was performed in the presence of hygromycin to allow the identification of the GM plants containing the introduced RNAi construct(s). This method of transformation is used extensively to genetically modify plants (Valentine 2003) and has been discussed in previous RARMPs [most comprehensively for DIR 060/2005 (available at OGTR website or by contacting the OGTR)].
5.6 Characterisation of the GMOs
5.6.1 Stability and molecular characterisation
76. All constructs used for the generation of the GM lines were sequenced prior to transformation. The GM wheat lines contain one RNAi construct (SEI); the GM barley line contains two RNAi constructs (SEI and SEII). The copy number of the introduced RNAi construct was confirmed by Southern blot analysis. Lines 85.2c and 50.1b (the parental line for genetically modified lines 212 and YDH7) contained two copies of the SEI construct. The GM barley line was confirmed to contain one copy of the SEI construct and two copies of the SEII construct. The applicant states that the GM lines have not been analysed for the presence or absence of any vector sequences that may have been incorporated during the transformation process. In some instances parts of the vector beyond the left border can be transferred during the Agrobacterium transformation process (Zambryski 1988).
77. The GM lines have been observed for at least four generations and integration of the construct was found to be stably inherited in all the GM lines as shown by PCR analysis. All GM wheat and barley lines showed altered grain starch composition in the various generations as compared with non-GM wheat and barley.
78. The stably integrated lines were achieved through single seed descent selection (SSD). This is an established method for selecting for stable integration of the introduced genes in cereals as they are predominantly self-pollinating (Tigchelaar & Casali 1976). SSD4 (4th generation single seed descent) lines were grown to produce seed for the proposed release.
5.6.2 Expression of the introduced RNAi constructs in the GM wheat and barley
79. As the GM lines are based on RNAi technology, no new proteins are produced. The RNAi constructs are under the control of the endosperm specific promoter HMWG-Dx5, thus expression is only expected to occur in the seed as the promoter is not active in other tissues. The high molecular weight glutenin promoter is accepted as being very specific for endosperm expression (Blechl & Anderson 1996).
80. Expression of the introduced constructs in the genetically modified lines has been assessed from glasshouse grown plants. Protein expression analyses of the GM lines using specific antibodies to the SEI and SEII proteins indicate that both of the introduced RNAi constructs are expressed as indicated by reduced production of the SEI and/or SEII proteins.
81. Northern analysis on endosperm isolated from the GM wheat line 85.2c, 15 days after anthesis, indicates an absence of the SEI transcript; the SEII transcript was still detectable. No analysis has been performed to date on the GM lines 212 and YDH7. However, it should be noted that the GM line 50.1b is the parental line for both of these GM lines and GM lines 50.1b and 85.2c were derived from separate transformation events.
82. In the barley line BC10.5 real time PCR analysis shows a reduction in the transcript levels for SEI to approximately 17% compared to the parental line. The SEII transcript level was reduced by approximately 29% compared to the parental line. Thus the level of both transcripts was reduced but silencing was not complete.
5.6.3 Characterisation of the phenotype of the GM wheat and barley
83. One of the aims of the proposed trial is to assess the agronomic performance of the GM wheat and barley lines under field conditions to ascertain that the intended effect of altered grain starch composition does not result in unacceptable impacts on agronomic characteristics. Comparison between GM and non-GM plants under glasshouse conditions have shown no evidence that the introduced genes have affected their growth habit. Visual observation of the GM plants did not show any major variation in plant height. A sub sample of the seeds to be used in the field has been grown and the plant height and tiller number were measured. These measurements show that the GM wheat and barley lines are similar to the control lines for mean plant height at booting stage and number of tillers per plant. Two GM wheat lines showed a slight increase in mean grain number per spike (Table 3).
Table 3. Plant height (cm) and tiller number and mean number of grains/spike for the GM and non-GM lines
|Line||Mean plant height at booting stage* (±SD)||Mean number of tillers per plant (±SD)||Mean number of grains/spike (±SD)|
|NB1 (non-GM)||66.7± 5.8||7.3± 2.4||21.7± 1.5|
|85.2c||68.2± 3.3||6.7± 0.9||23.3± 2.1|
|212||69± 1.7||7.0± 0.8||20.0± 2.0|
|YDH7||66.5± 3.6||7.3± 1.9||27.3± 1.2|
|Golden Promise (non-GM)||63.0± 5.2||10.0± 0.8||30.7± 3.5|
|BC10.5||64.3± 2.3||10.7± 0.5||30.0± 6.6|
5.6.4 Biochemical characterisation of GM wheat and barley
84. The applicant has provided preliminary grain composition analyses of the GM lines which include data on total protein, ash, fat, glucose, sucrose, fructose and moisture content (see Table 4). These data showed some differences between the GM lines and the non-GM controls. However, the data is based on single samples and the significance of the differences has not been determined.
85. In the wheat lines containing the SEI RNAi construct a reduction in SEII protein was also observed. The applicant states there was no evidence of cross-silencing and the reduction of the SEII protein may have been the result of protein- protein interactions. Similar interactions in the starch synthesis pathway have been observed by others and as such these effects could be anticipated (Tetlow et al. 2008), see also Section 5.2.
Table 4. Grain composition of the GM and non-GM wheat and barley lines. No data provided due to very limited sampling, data are an indication only.
|Line||Moisture (% of flour)||Sucrose (% of flour)||Fructose (% of flour)||Glucose (% of flour)||Ash (% of flour)||Protein (% of flour)||Fat (% of flour)|
|Wheat 85.2c (SEI construct)||7.4||1.2||0.2||0||1.9||11||4.5|
|Wheat 212 (SEI in triple null SEIII mutant background)||8.2||0.8||0.1||0||1.8||12||3.4|
|Wheat YDH7 (SEI in triple null SEIV mutant background)||8.5||0.6||0||0||2.1||13||2.8|
|Golden Promise- barley control||5.5||1.6||0.1||0||2.5||12||5|
|Barley BC10.5 (SEI and SEII)||6.3||1||0.1||0.1||2.3||9.1||3.1|
11Isofrom: any of several different forms of the same protein as a result of changes in the DNA sequence, or different forms of a protein may also be produced from related genes, or may arise from the same gene by alternative splicing.