98. Each event compiled during hazard identification is characterised to determine which events represent a risk to the health and safety of people or the environment posed by, or as a result of, gene technology.
99. The criteria used by the Regulator to determine harm are described in Chapter 3 of the Risk Analysis Framework (OGTR 2007). Harm is assessed in comparison to the parent organism and in the context of the proposed dealings and the receiving environment. Wherever possible, the risk assessment focuses on measurable criteria for determining harm.
100. The following factors are taken into account during the analysis of events that may give rise to harm:
- the proposed dealings, which may be for the purpose of experimentation, development, production, breeding, propagation, use, growth, importation, possession, supply, transport or disposal of the GMOs
- the proposed limits
- the proposed controls
- characteristics of the non-GM parent
- routes of exposure to the GMOs, the introduced gene(s) and gene product(s)
- potential effects of the introduced gene(s) and gene product(s) expressed in the GMOs
- potential exposure to the introduced gene(s) and gene product(s) from other sources in the environment
- the biotic and abiotic environment at the site(s) of release
- agronomic management practices for the GMOs.
102. As discussed in Chapter 1, Section 5.2.5, the GM wheat lines contain the antibiotic resistance selectable marker gene, nptII. The nptII gene, encoding neomycin phosphotransferase type II, has already been considered in detail in the RARMP prepared for DIR 070/2006 and by other regulators and was found to pose no risks to either people or the environment. Therefore the potential effects of the nptII gene will not be further assessed for this application.
103. As discussed in Chapter 1, Section 5.2.6, the GM wheat lines also contain the antibiotic resistance selectable marker gene, bla. The bla gene, encoding β-lactamase, is not expressed in the GM wheat lines as it is linked to a bacterial promoter that does not function in plants, and therefore it will not be assessed further.
Table 4. Summary of events that may give rise to an adverse outcome through the downregulation or silencing of endogenous genes controlling grain composition.
| Hazard category | Event that may give rise to an adverse outcome | Potential adverse outcome | Identified risk? | Reason |
|---|---|---|---|---|
| Section 2.1 Production of a substance toxic/allergenic to people or toxic to other organisms | 1. Exposure to GM plant material containing the introduced RNAi constructs or their associated effects | Allergic reactions in people or toxicity in people and other organisms | No |
|
| Section 2.2 Spread and persistence of the GM wheat lines in the environment | 2. Expression of the introduced RNAi constructs improving the survival of the GM wheat plants | Weediness; allergic reactions in people or toxicity in people and other organisms | No |
|
| 3. Dispersal of reproductive (sexual or asexual) GM plant materials through various means, including animals and extreme weather conditions | Weediness; allergic reactions in people or toxicity in people and other organisms | No |
| |
| Section 2.3 Vertical transfer of genes or genetic elements to sexually compatible plants | 4. Expression of the introduced RNAi constructs and regulatory sequences in other wheat plants | Weediness; allergic reactions in people or toxicity in people and other organisms | No |
|
| 5. Expression of the introduced RNAi constructs or regulatory sequences in other sexually compatible plants | Weediness; allergic reactions in people or toxicity in people and other organisms | No |
| |
| Section 2.4 Horizontal transfer of genes or genetic elements to sexually incompatible organisms | 6. Presence of the introduced RNAi constructs, in other organisms as a result of gene transfer | Weediness; allergic reactions in people or toxicity in people and other organisms | No |
|
| Section 2.5 Unintended changes in biochemistry, physiology or ecology | 7. Changes to biochemistry, physiology or ecology of the GM wheat lines resulting from expression, or random insertion, of the introduced RNAi constructs | Weediness; allergic reactions in people or toxicity in people and other organisms | No |
|
| Section 2.6 Unauthorised activities | 8. Use of the GMOs outside the proposed licence conditions | Potential adverse outcomes mentioned in Sections 2.1 to 2.5 | No |
|
2.1 Production of a substance toxic/allergenic to people or toxic to other organisms
104. Toxicity is the adverse effect(s) of exposure to a dose of a substance as a result of direct cellular or tissue injury, or through the inhibition of normal physiological processes (Felsot 2000).105. Allergenicity is the potential of a protein to elicit an immunological reaction following its ingestion, dermal contact or inhalation, which may lead to tissue inflammation and organ dysfunction (Arts et al. 2006).
106. A range of organisms may be exposed directly or indirectly to the introduced RNAi constructs for modified grain composition and their associated effects. Workers cultivating the wheat would be exposed to all plant parts. Organisms may be exposed directly to the end effects of the introduced sequences through biotic interactions with GM wheat plants (vertebrates, insects, symbiotic microorganisms and/or pathogenic fungi) or through contact with root exudates or dead plant material (soil biota). Indirect exposure would include organisms that feed on organisms that feed on GM wheat plant parts or degrade them (vertebrates, insects, fungi and/or bacteria).
Event 1. Exposure to GM plant material containing the introduced RNAi constructs or their associated effects
107. Expression of the introduced RNAi constructs for altered grain composition could potentially result in the production of novel toxic or allergenic compounds in the GM wheat lines, or alter the expression of endogenous wheat proteins. If humans or other organisms were exposed to the resulting compounds through ingestion, contact or inhalation of the GM plant materials, this may give rise to detrimental biochemical or physiological effects on the health of these humans or other organisms.
108. Non-GM wheat is not known to be toxic to humans or other organisms. However, non-GM wheat flour can produce allergic and autoimmune responses in susceptible individuals on inhalation or ingestion. As discussed in Chapter 1, Section 5.2.4, celiac disease is an inflammatory response of the small intestine initiated by gliadins and low molecular weight glutenins. Allergies to gliadins are also well documented.
109. Although no toxicity studies have been performed on the GM wheat plant material, the introduced RNAi constructs are composed of partial gene sequences isolated from naturally occurring organisms that are already widespread and prevalent in the environment. It is not expected that any novel products would be produced as a result of the expression of the introduced RNAi constructs as they are likely to be degraded upon initiating RNAi, before transcription can occur. The components of the introduced RNAi constructs were isolated from non-GM wheat and rice (see Chapter 1, Section 6.5).
110. With the exception of the α- and γ-gliadin RNAi lines, no information was found to suggest that the changes brought about by the introduced RNAi constructs affect the production of endogenous wheat toxins and allergens (Chapter 1, Sections 5.1 and 5.2.4) and therefore exposure to the GM plant materials of these lines is not expected to adversely affect the health of humans or other organisms.
111. Data provided by the applicant on the characterisation of the α- and γ-gliadin RNAi lines has revealed decreased expression of the targeted gliadin proteins, and compensatory increases in expression of other gliadin and glutenin proteins compared to non-GM wheat. Allergens and celiac epitopes are known to occur in several grain protein fractions, notably in the low molecular weight glutenins, and the α-, γ- and ω-gliadins. In the γ-gliadin silencing lines the maximum observed percentage increase in each of these protein classes was 55% for low molecular weight glutenin subunits, 41% for α-gliadins, and 19% for ω-gliadins. In the α-gliadin silencing lines the maximum observed percentage increase in each class of protein was 27% for low molecular weight glutenin subunits, 9% for γ-gliadins, and 32% for ω-gliadins. It is important to note that this data does not reveal the extent to which individual proteins within these groups change. However, given the diversity of allergens present in wheat grains, these changes are highly likely to increase the content of allergenic proteins or celiac-triggering epitopes for some individuals, and decrease them for others. On this basis, the gliadin RNAi lines could potentially trigger adverse effects in celiac disease and wheat allergy sufferers at lower exposure levels than the non-GM wheat parental cultivar. It is not anticipated that the gliadin RNAi lines would initiate celiac disease or allergy symptoms in people who do not normally show these reactions to the non-GM parental wheat cultivar, as no novel proteins have been introduced in the GM wheat lines.
112. Wheat variety and growing conditions influence the specific gliadin and glutenin proteins expressed in grains (reviewed by Gras et al. 2001). Gliadin profiling is widely used to identify wheat cultivars, highlighting the high genetic variability between cultivars for the specific gliadin proteins expressed (reviewed by Gianibelli et al. 2001). High variability in overall gliadin and glutenin content was revealed by a survey of Spanish wheat varieties by Pena et al. (2005) which found four-fold variability in the gluten content of flour, more than five-fold variability in α-gliadin content, more than eight-fold variability in γ-gliadin content, and more than three-fold variability in low molecular weight glutenin content. Comparison of the levels of gliadins in the GM wheat lines and other wheat varieties is difficult because differences in the environmental conditions in which the lines were cultivated influence gliadin content, and also because reported measurement units differ. However, it appears likely that the protein levels observed in the GM wheat lines are within the range of natural variation observed between different non-GM wheat genotypes.
113. The high level of variability in the protein composition of different wheat varieties suggests that some wheats are low in allergens and irritants. However, individuals diagnosed with celiac disease or wheat allergy usually attempt to exclude wheat (specifically, gluten) from their diet. Due to their sensitivity, it is usually not considered safe to consume any wheat products, and for particularly sensitive celiacs, even the low level of gluten proteins in products labelled as “gluten free” can be of concern (reviewed by Hischenhuber et al. 2006). Thus, there is a very low probability of accidental consumption of flour from the GM wheat lines by sensitive celiac and allergy sufferers because they are already avoiding wheat products.
114. Celiac disease and wheat allergy are thought to mildly affect many more people than have been diagnosed, and who can show very low levels of symptoms (Hischenhuber et al. 2006). It is unclear how the changes in protein composition observed in the gliadin RNAi lines may affect such people, should they accidentally consume or inhale flour from the GM wheat lines. Although they are less likely to show adverse effects than sensitive celiac and allergy sufferers, prediction of whether or not allergy or celiac symptoms may be induced would depend on individual sensitivity and the amounts consumed. However, in light of the high variability in gliadin and glutenin content in different wheat varieties, it is likely that any accidental exposure to the altered gliadin and glutenin levels in the GM wheat lines would fall within the range of normal exposure from non-GM wheat products.
115. The proposed limits and controls of the trial (Chapter 1, Sections 3.2 and 3.3) would minimise the likelihood of exposure of people and other organisms to GM plant materials. The proposed trial site will be surrounded by a stock-proof fence, around which rodent baiting and trapping will be carried out. The area of the trial will be locked, and only approved staff with appropriate training will have access to the site. These measures will reduce inadvertent access by humans and prevent grazing livestock from entering the site, which minimises exposure of the public and animals to the GM plant material. Livestock would not be intentionally exposed as the GM plant material will not be used as feed, with the exception of planned nutritional studies on rats and pigs, from which no material will enter the human food or animal feed supply.
116. Contact with, or inhalation of, GM plant materials would be limited to trained and authorised staff. There is little potential for exposure of the public to GM plant material via ingestion, skin contact or inhalation as no GM plant material will be used as human food. The short duration (2009-2012) and small size (1 ha) of the proposed trial would also limit the potential for exposure to the GM plant material.
117. The applicant proposes to conduct other limited and controlled releases of GM wheat and barley at the DIR 092 release site (see Chapter 1, Section 6.2). Proposed release DIR 093 involves feeding GM wheat products to human volunteers in controlled nutritional experiments. Mixture of GMOs from these trials is possible through mechanical mixture, through replanting of trial sites in successive years and through pollen-mediated gene flow among the GM wheat lines. Pollen-mediated gene flow is discussed in Event 4 below.
118. Mechanical mixing at harvest has been reported to lead to potentially high levels of unintended presence of one wheat line in another wheat line. A study of wheat varietal purity in Colorado detected levels of herbicide-tolerant GM wheat comprising as much as 11% of seed thought to be non-GM wheat (Gaines et al. 2007). This occurred in a field in which GM wheat had not previously been grown which was sown with farm-saved seed, and was attributed primarily to use of the same harvest equipment for the two varieties, with a possible contribution from mixed seed saved from previous years. The applicant proposes to clean all sowing, harvesting and threshing equipment between processing of different GM wheat lines. Removal of residual wheat seed from this equipment will largely prevent mechanical mixture of seed lots between the proposed trials.
119. Mixing of seed can also occur through the growth of volunteer wheat in the field following dropping of seed at harvest, and this could lead to mixtures of GMOs from different trials if sites are replanted in successive years. For example, the Colorado study discussed above detected herbicide-tolerant GM wheat at levels of 0.25% and 4.2% in non-GM wheat harvested from two fields planted two seasons previously with the GM variety (Gaines et al. 2007). The applicant states that they aim to plant the GM wheat lines on different areas of the site each year so as to minimise build-up of soil diseases. However, if replanting of an area is necessary, the applicant proposes to restrict this to using areas initially planted in season one for the third season sowing. The applicant does not propose to plant GM wheat lines from other proposed releases over areas previously utilised for DIR 092. This proposed planting arrangement will greatly reduce the likelihood of seed mixture between trials through volunteer persistence.
120. The controls proposed by the applicant are considered effective measures to prevent mechanical mixture of seed and growth of volunteer wheat originating from one trial in an area planted to another trial. For these reasons, it is considered unlikely that seed-mediated mixing of GM wheat lines from DIR 092 with GM wheat lines proposed to be fed to humans in the course of proposed DIR 093 will inadvertently lead to human consumption of material from DIR 092 lines.
121. Conclusion: The potential for allergic reactions in people, or toxicity in people and other organisms as a result of exposure to GM plant materials with altered grain composition as a result of the introduced RNAi constructs is not an identified risk and will not be assessed further.
2.2 Spread and persistence of the GM wheat lines in the environment
122. Baseline information on the characteristics of weeds in general, and the factors limiting the spread and persistence of non-GM wheat plants in particular, is given in The Biology of Triticum aestivum L. em Thell. (Bread Wheat) (OGTR 2008). In summary, wheat shares some characteristics with known weeds, such as wind-pollination (although it is predominantly self-pollinating) and the ability to germinate or to produce some seed in a range of environmental conditions. However, wheat lacks most characteristics that are common to many weeds, such as the ability to produce a persisting seed bank, rapid growth to flowering, continuous seed production as long as growing conditions permit, high seed output, high volume seed dispersal and long-distance seed dispersal (Keeler 1989). In addition, wheat has been bred to avoid seed shattering and white wheats have little seed dormancy (OGTR 2008).123. Scenarios that could lead to increased spread and persistence of the GM wheat lines include expression of the introduced RNAi constructs conferring tolerance to abiotic or biotic stresses, or increasing the dispersal potential of GM plant materials. These events could lead to increased exposure of vertebrates (including people), invertebrates and microorganisms to the encoded proteins.
Event 2. Expression of the introduced RNAi constructs improving the survival of the GM wheat plants
124. If the GM wheat lines were to establish or persist in the environment they could increase the exposure of humans and other organisms to the GM plant material. The potential for increased allergenicity in people or toxicity in people and other organisms as a result of contact with GM plant materials containing the introduced RNAi constructs has been considered in Event 1 and was not considered an identified risk in the context of the proposed limits and controls.
125. If the expression of the introduced RNAi constructs for altered grain composition were to provide the GM wheat plants with a significant selective advantage over non-GM wheat plants and they were able to establish and persist in favourable non-agricultural environments, this may give rise to lower abundance of desirable species, reduced species richness, or undesirable changes in species composition. Similarly, the GM wheat plants could adversely affect agricultural environments if they exhibited a greater ability to establish and persist than non-GM wheat.
126. The impact of the genetic modification on survival of the GM wheat lines is uncharacterised under field conditions. The applicant has provided data demonstrating that the introduced RNAi constructs result in modified grain composition phenotypes in the GM wheat lines grown in glasshouse conditions. The applicant states that the genetic modifications are expected to result only in endosperm phenotypes because of the use of endosperm-specific promoters. Thus, other than grain composition, the phenotypes of the GM wheat lines have not been characterised in great detail. The RNAi constructs are composed of wheat and rice partial gene sequences, and do not result in the expression of novel proteins in the GM wheat lines.
127. The alteration of grain composition in the GM wheat lines could possibly have secondary effects on seed germination or seedling vigour. Carbohydrates and proteins are the major storage compounds present in cereal grains, accumulating in the endosperm. These energy reserves support germinating plants until they become photosynthetically active. Mobilisation of storage reserves can begin before germination is completed, with major mobilisation occurring after the completion of germination (reviewed by Nonogaki 2008). The composition of storage compounds is changed in the GM wheat lines, leading to the possibility of changed availability of energy to germinating seeds. Processes controlled before the point of germination, such as seed dormancy, which do not rely on energy reserves from the endosperm, are unlikely to be altered by the changes in grain composition in the GM wheat lines.
128. Starch reserves are mobilised by enzymes catalysing hydrolysis of the linkages between glucosyl residues, with α-amylases breaking down α--(1-4) linkages, and debranching enzymes breaking down α--(1-6) linkages (reviewed by Nonogaki 2008). The rate at which starch is acted upon by hydrolytic enzymes, or the total amount of energy available, could be changed as a result of changes to starch composition in some of the GM wheat lines. Effects on processes utilising the energy from storage starch, including germination and early seedling growth, could follow from these changes.
129. Protein reserves are made available by the action of proteinase and peptidase enzymes, some of which are pre-formed in the endosperm of mature grains, but most of which are secreted from the aleurone following germination (reviewed by Nonogaki 2008). In the α- and γ-gliadin RNAi lines, changes to total protein did not occur, but changes in the types of storage protein accumulating occurred which may alter the supply of energy to the seedling, affecting seedling vigour.
130. Modern wheat cultivars, some of which are bred for high vigour, are not recognised as a significant weed risk in Australia, and there have been no reports of bread wheat becoming an invasive pest in Australia or overseas. Additionally, the spread and persistence of the GM wheat plants would still be limited by lack of seed shattering, temperature, low intrinsic competitive ability, nutrient availability, pests and diseases and other environmental factors that normally limit the spread and persistence of wheat plants in Australia (Slee 2003; Condon 2004). However, if there were any developmental advantages conferred to the GM wheat lines as a result of altered grain composition, their persistence at the release site would be limited by the controls proposed by the applicant.
131. The proposed limits and controls of the trial (Chapter 1, Sections 3.2 and 3.3) would minimise the likelihood of the spread and persistence of the GM wheat lines proposed for release. The release would be of limited size and short duration and the applicant proposes a number of control measures, including destruction of all plant materials not required for further analysis, repeated post harvest irrigation of the site to encourage germination of remaining seed followed by herbicide treatments to destroy volunteers and post harvest monitoring of the proposed site.
132. Conclusion: The potential for increased weediness, allergenicity or toxicity due to expression of the introduced RNAi constructs for altered grain composition improving the survival of the GM wheat lines is not an identified risk and will not be assessed further.
Event 3. Dispersal of reproductive (sexual or asexual) GM plant materials through various means, including animals and extreme weather conditions
133. If the GM wheat lines were to be dispersed from the release site they could increase the exposure of humans and other organisms to the GM plant material and/or establish and persist in the environment. The effects of contact, inhalation or ingestion of the GM wheat lines have been assessed in Event 1 and were not an identified risk in the context of the proposed limits and controls. The potential for the introduced gene sequences to result in improved survival of the GM wheat lines in the environment was assessed in Event 2 and was not an identified risk.
134. Wheat lacks seed dispersal characteristics such as stickiness, burrs, and hooks, which contribute to seed dispersal via animal fur (Howe & Smallwood 1982). Seeds which survive chewing and digestion by animals are typically small and dormant (Malo & Suárez 1995). The GM wheat lines proposed for release are in white wheat parental backgrounds, which have large seeds with low dormancy and thin seed coats (Hansen 1994), and are therefore likely to be easily broken down in the digestive system of mammals.
135. The proposed release site will be surrounded by a 1.8 m fence with a locked gate, limiting the possibility of seed dispersal by any large animals or by unauthorised people accessing the site. Dispersal by authorised people entering the proposed trial site would be minimised by a standard condition of DIR licences which requires the cleaning of all equipment used at the trial site, including clothing. All GM plant material will be transported in accordance with the Regulator’s transport guidelines which will minimise the opportunity to disperse the GM material.
136. Rabbits favour soft, green, lush grass (Myers & Poole 1963) and select the most succulent and nutritious plants first (Croft et al. 2002). Although viable seeds from a variety of plant species have been found in rabbit dung, viable wheat seeds were not among them (Malo & Suárez 1995). In a study that looked at the germination of seeds on dung from cattle, red deer, sheep, hare, rabbit and red grouse, the number of germinations was least on rabbit dung (Welch 1985). Similarly, a study that looked at viable grass seeds in dung from cattle, pronghorn and rabbit, found few seedling populations of any species emerged from rabbit dung (Wicklow & Zak 1983).
137. Habitat modifications such as reduced plant cover have been reported to be a deterrent to the movement of mice (White et al. 1998; Central Science Laboratory 2001; AGRI-FACTS 2002; Brown et al. 2004) and therefore the proposed 10 m wide herbicide-treated zone of reduced plant cover around the trial site is expected to discourage dispersal by mice. Rodent baits and traps will be placed around the fence surrounding the site, which will further limit seed dispersal by rodents.
138. Wheat seed could also be transferred from the GM trial site via water run-off. However, irrigation of the site or rainfall will produce minimal water run-off as the site is reasonably flat (information supplied by the applicant).
139. Extremes of weather may cause dispersal of plant parts. However, control measures have been proposed by the applicant to minimise dispersal. These include locating the proposed release away from natural waterways to prevent dispersal in the event of flooding, and having an isolation zone in which there are no other wheat or related plants in the event of strong winds dispersing pollen or seeds.
140. Conclusion: The potential for allergenicity, toxicity or increased weediness due to the dispersal of reproductive (sexual or asexual) GM plant materials through various means including animals and extreme weather conditions is not an identified risk and will not be assessed further.
2.3 Vertical transfer of genes or genetic elements to sexually compatible plants
141. Vertical gene flow is the transfer of genetic information from an individual organism to its progeny by conventional heredity mechanisms, both asexual and sexual. In flowering plants, pollen dispersal is the main mode of gene flow (Waines & Hedge 2003). For GM crops, vertical gene flow could therefore occur via successful cross-pollination between the crop and neighbouring crops, related weeds or native plants (Glover 2002).142. Baseline information on vertical gene transfer associated with non-GM wheat plants can be found in The Biology of Triticum aestivum L. em Thell. (Wheat) (OGTR 2008). In summary, wheat plants are primarily self-pollinating and while natural hybrids with other species can occur at low frequencies, they are usually sterile.
Event 4. Expression of the introduced RNAi constructs and regulatory sequences in other wheat plants
143. Transfer and expression of the introduced RNAi constructs for altered grain composition to other wheat plants could increase the weediness potential, or alter the potential allergenicity and/or toxicity of the resulting plants.
144. All of the introduced regulatory sequences are expected to operate in the same manner as regulatory elements endogenous to the wheat plants. While the transfer of either endogenous or introduced regulatory sequences could result in unpredictable effects, the impacts from the introduced regulatory elements are likely to be equivalent and no greater than the endogenous regulatory elements.
145. As discussed in Event 1, allergenicity to people and toxicity to people and other organisms are not expected to be changed in the GM wheat plants by the introduced RNAi constructs, with the exception of the α- and γ-gliadin RNAi lines, in which levels of some allergens are likely to be increased. This will be the same if the introduced RNAi constructs are expressed in other wheat plants.
146. Wheat varieties range from 94 to 99% self-pollinating (Hucl 1996), and wind pollination is the predominant mode of outcrossing. Characteristics contributing to pollen shed, such as the proportion of florets with protruding anthers and pollen production, vary between cultivars and are influenced by environmental conditions (Waines & Hedge 2003; reviewed by OGTR 2008). The proportion of pollen shed outside the floret is reported to vary from 3 to 80%, depending upon variety (Beri & Anand 1971). Studies of pollen movement indicate that 90% of pollen grains disperse within 6 m of the source, however viable pollen grains have been detected at 1000 m (reviewed by Waines & Hedge 2003). Under field conditions wheat pollen has a viable lifespan of less than 30 minutes (OECD 1999). Environmental conditions including temperature, relative humidity and wind intensity have a great influence on pollen viability and pollen movement.
147. Gene flow rates in wheat have been studied from both experimental- and commercial-scale fields, with gene flow over long distances only detectable from commercial-scale fields (reviewed in OGTR 2008). The majority of gene flow from experimental scale fields occurs up to ten metres from the pollen source, and only low levels of gene flow have been detected as far as 300 m away (Matus-Cadiz et al. 2004). In a small scale study of pollen-mediated gene flow at Ginninderra Experimental Station, 0.055% cross-pollination was detected between adjacent rows of wheat, and 0.012% cross-pollination was detected from a pair of 8 m2 plots in seed harvested from a 2 m wide planting surrounding the plots at a distance of 1 m (Gatford et al. 2006). The plants to which gene flow was measured were all within 4.2 m of the pollen source plot. Higher levels of gene flow, at longer distances, have been detected from larger scale and commercial wheat plantings, and results indicate that gene flow levels are highly variable and can depend greatly on prevailing winds. Matus-Cadiz et al. (2007) successfully detected trace levels of pollen flow from a commercial field at 500 m, 630 m and 2.75 km. The likelihood of gene transfer declines rapidly as the distance from the pollen source increases.
148. The applicant proposes to prevent cultivation of non-GM wheat breeding lines within 500 m of the trial site, and prevent cultivation of non-GM, non-breeding lines of wheat within 200 m of the site. These measures are further discussed in Chapter 3, Section 4.1.1. Isolation from other wheat cultivation will greatly restrict the potential for pollen flow and gene transfer.
149. The applicant has also proposed that two other limited and controlled releases of GM wheat and barley lines be grown adjacent to the GM wheat lines proposed for release in this application (Figure 4). Given the close proximity of these trials to each other, a limited amount of cross pollination is likely to occur between the GM wheat lines of the three proposed releases. Additionally, the applicant proposes to save seeds from each harvest for replanting in subsequent years in order to generate sufficient grain for further analysis including nutritional experiments. This gives rise to the possibility of low-level cross pollination between GM wheat lines during the first year of the trial, and subsequent amplification of a cross pollination event through propagation in later years. GM wheat lines of DIR 093 are proposed to be fed to humans in controlled nutritional studies, so the potential for cross pollination of DIR 093 plants by those from DIR 092 gives rise to the possible exposure of humans to the GM wheat lines of DIR 092.
Figure 4. Schematic representation of the arrangement of proposed trials at the release site in the first year using projected barley planting widths provided by the applicant.
150. The applicant proposes to surround each trial at the site with a 2 m wide planting of non-GM wheat, and for barley plantings of adjacent proposed DIRs to be used to separate the GM wheat plantings of different trials. Figure 4 shows a representation of the proposed planting scheme for one season of the trials, which reflects the relative positions and separation of the proposed trials, but not the specific dimensions or areas to be planted as this is yet to be determined. This scheme amounts to a minimum separation distance between the GM wheat lines of the different proposed DIRs of 4 m of non-GM wheat plus the width of the barley plantings. The level of gene flow which could potentially occur over this distance is difficult to predict, as measurements of gene flow depend greatly upon the size of pollen source plantings and environmental factors such as prevailing winds. However, data from the locality of this trial site (see above) indicates that cross pollination at distances of up to 4.2 m from a pair of 8 m2 plots of wheat occurs at a frequency of 0.012% and is expected to be even lower at greater distances. Thus, the measures proposed by the applicant would restrict gene flow between the proposed trials. Low levels of gene flow between the trials may lead to stacking of GM traits, which may give rise to GM wheat plants with altered weediness compared to any individual GM wheat line in the three proposed releases; or, may lead to material from DIR 093 proposed to be used in human nutritional experiments containing low amounts of material from the GM wheat lines from adjacent trials.
151. If cross pollination between the α- and γ-gliadin RNAi lines and the GM wheat lines in DIR 093 were to occur, then the humans in the nutritional study may be exposed to increased levels of endogenous wheat allergens. However, the amount of this increase is likely to be very small. As noted in Event 1, cultivated wheat varieties show substantial variability in the levels of allergenic protein classes, and the changes observed in the α- and γ-gliadin RNAi lines are likely to be within the natural range of variation for these protein classes. Any changes in levels of allergens or celiac epitopes will be greatly diluted in the GM wheat grain fed to human volunteers as a part of DIR 093 (if approved), because only low levels of cross pollination are likely over the distances involved. Additionally, it is highly unlikely that people with existing sensitivity to wheat products would volunteer for a nutritional study involving wheat products, and exposure to the GM wheat lines during the nutritional study would be limited to a small number of participants for relatively short periods of time (see the RARMP for DIR 093 for more detailed information).
152. The human nutritional experiments described in application DIR 093 are proposed to take place at CSIRO Human Nutrition in Adelaide, overseen by the CSIRO Human Nutrition Human Research Ethics Committee. This committee is registered with the NHMRC, a requirement of which is compliance with the National Statement on Ethical Conduct in Human Research (National Health and Medical Research Council et al. 2007), a set of guidelines published by the NHMRC. Important issues addressed in the guidelines are that ethics committees and researchers undertake full assessment of potential risks to human volunteers, and ensure that volunteers are properly informed about the trials they are consenting to participate in. The RARMP for DIR 093 will address the issue of potential impurity of material for human nutritional trials, including that which may occur as a result of cross-pollination from lines released under proposed DIR 092 which have undergone very limited characterisation.
153. Cross pollination between GM wheat lines of the different trials proposed to be released at the site must also be considered in relation to the stacking of GM traits possibly contributing to weediness of the resultant GM wheat lines. Among the traits in the other trials proposed for the release site is a trial of GM wheat lines modified for enhanced nutrient utilisation (DIR 094). If these lines were cross-pollinated by lines from proposed release DIR 092, the improved nutrient utilisation of the DIR 094 lines may contribute to the spread and persistence of the DIR 092 genetic modifications. Two lines proposed for release under DIR 093 have a slightly increased number of grains per spike. If these lines were cross-pollinated by lines from proposed DIR 092, seed production could be increased in the resultant lines, contributing to the survival and spread of the DIR 092 genetic modifications.
154. The combination of these traits with those in the GM wheat lines of DIR 092 is likely to contribute only incrementally to the potential weediness of the GM wheat plants, the spread and persistence of which would be limited by factors such as lack of seed shattering, temperature, low intrinsic competitive ability, pests and diseases and other environmental factors that normally limit the spread and persistence of wheat plants in Australia. The persistence of such lines would also be limited by measures proposed by the applicant to limit the persistence of the GM wheat lines at the release site.
155. The proposed limits and controls of the trial (Chapter 1, Sections 3.2 and 3.3) would restrict the potential for pollen flow and gene transfer to non-GM wheat plants. In particular, the applicant proposes to isolate the trial site from other plantings of wheat, and the majority of the pollen is expected to fall within the trial site or the 10 m herbicide-treated area directly surrounding the trial site. The applicant also proposes to perform post harvest monitoring of the site for twenty four months or until the site has been clear of volunteers for one growing season and to destroy any volunteer plants found at the site. These latter measures would ensure any remaining GM wheat seeds, or plants that were potentially the product of gene flow, in these areas would be destroyed.
156. Conclusion: The potential for allergenicity in people, or toxicity in people and other organisms or increased weediness due to the expression of the introduced RNAi constructs and regulatory sequences in other wheat plants as a result of gene transfer is not an identified risk and will not be assessed further.
Event 5. Expression of the introduced RNAi constructs and regulatory sequences in other sexually compatible plants
157. As identified in The Biology of Triticum aestivum L. em Thell. (Bread Wheat) (OGTR 2008), there are few species outside the Triticum genus that are sexually compatible with wheat and known to form hybrids under natural conditions, and these hybrids are usually sterile. Examples include Aegilops cylindrica, A. ovata, A. biuncialis and Secale cereale.
158. A botanical survey for species related to wheat was undertaken at Ginninderra Experimental Station on behalf of the applicant in August 2008, in which Elymus scaber (common wheatgrass), Hordeum marinum (sea barley) and Hordeum leporinum (barley grass) were identified at the site, and it was reported that other weedy Hordeum taxa could be expected to occur (information provided by the applicant).
159. As discussed in Chapter 1, Section 6.4, wheat is sexually compatible with many species within the genus Triticum, and in closely related genera such as Aegilops and Elytrigia. Although wheat can hybridise with Hordeum marinum (reviewed by Colmer et al. 2006), this requires substantial intervention (Pershina et al. 1998; Islam & Colmer 2008) and the resultant hybrids are usually infertile.
160. A search of the scientific literature did not detect specific reports of hybridisation between wheat and Elymus scaber or Hordeum leporinum. Hybridisation between wheat and other species in the Elymus and Hordeum genera have been recorded, and typically result in sterile hybrids (reviewed in OGTR 2008). A review of possible means of pollen-mediated gene flow from GM wheat to wild relatives in Europe concluded that there was a minimal possibility of gene flow from wheat to Horedum spp. or Elytrigia spp. (Eastham & Sweet 2002), Elytrigia being a genus very closely related to Elymus. Hybridisation would require synchronicity of flowering between the GM wheat lines and related species to enable cross-pollination and gene flow to occur.
161. The proposed limits and controls of the trial (Chapter 1, Sections 3.2 and 3.3) would restrict the potential for pollen flow and gene transfer to sexually compatible plants. In particular, the applicant proposes to isolate the trial site from other sexually compatible species, and the majority of the pollen is expected to fall within the trial site or the 10 m herbicide-treated area directly surrounding the trial site. The applicant proposes to heavily graze the area surrounding the release site, which the applicant states will prevent any relatives of wheat flowering at the same time as the GM wheat lines. The applicant proposes to inspect the area immediately around the 10 m herbicide-treated zone for Elymus scaber and Hordeum spp. at fortnightly intervals from September until the GM wheat lines have finished flowering each year, and any plants found will be destroyed.
162. Expression of the introduced RNAi constructs in other sexually compatible plants is also unlikely to give these plants a significant selective advantage.
163. Conclusion: The potential for allergenicity in people, or toxicity in people and other organisms or increased weediness due to the expression of the introduced RNAi constructs and regulatory sequences in other sexually compatible plant species as a result of gene transfer is not an identified risk and will not be assessed further.
2.4 Horizontal transfer of genes or genetic elements to sexually incompatible organisms
164. Horizontal gene transfer (HGT) is the stable transfer of genetic material from one organism to another without reproduction (Keese 2008). All genes within an organism, including those introduced by gene technology, are capable of being transferred to another organism by HGT. HGT itself is not considered an adverse effect, but an event that may or may not lead to harm. A gene transferred through HGT could confer a novel trait to the recipient organism, through expression of the gene itself or the expression or mis-expression of endogenous genes. The novel trait may result in negative, neutral or positive effects.165. Risks that might arise from HGT have been considered in previous RARMPs (eg DIR 057/2004 and DIR 085/2008), which are available from the OGTR website or by contacting the Office. From the current scientific evidence, HGT from GM plants to other organisms presents negligible risks to human health and safety or the environment due to the rarity of such events, relative to those HGT events that occur in nature, and the limited chance of providing a selective advantage to the recipient organism.
166. Baseline information on the presence of the introduced or similar genetic elements is provided in Chapter 1, Section 6.5. All of the introduced genetic elements are derived from naturally occurring organisms that are already present in the wider Australian environment.
Event 6. Presence of the introduced genetic material in other organisms as a result of horizontal gene transfer
167. Possible risks arising from HGT of the introduced genetic material to other organisms involves consideration of the potential recipient organism and the nature of the introduced genetic material. Risks that might arise from HGT from a GMO to another organism have been recently reviewed (Keese 2008).
168. HGT could result in the presence of sequences from the introduced RNAi constructs for modified grain composition in bacteria, plants, animals or other eukaryotes. The sequences comprising the RNAi constructs were isolated from wheat and other organisms already widespread in the environment (Chapter 1, Sections 5.1 and 6.5) and already available for transfer via demonstrated natural mechanisms. Importantly, the RNAi constructs include only parts of coding sequences rather the entire sequence of any protein. If HGT were to occur, it could only result in the expression of short protein fragments, which may or may not include functional domains of the proteins encoded.
169. The nature of the RNAi constructs is considered unlikely to change the potential frequency of HGT, which is known to occur at an extremely low frequency from plants to other eukaryotes or prokaryotes (Keese 2008). The nature of the RNAi constructs is also considered unlikely to result in harmful consequences, should HGT occur. The RNAi constructs differ from most other transgenes previously evaluated for intentional release in the arrangement of DNA sequences into an inverted repeat. Sequencing of the Arabidopsis genome has indicated that a significant proportion of plant genes are arranged in arrays of tandem repeats, and within this group there are many examples of local inversions (The Arabidopsis Genome Initiative 2000). During genome evolution, genome rearrangement, including the generation of inverted duplications, is a frequent occurrence (reviewed by Shapiro 2005). Transcribed inverted repeats of protein coding sequences appear to be the evolutionary origin of microRNAs, a conserved endogenous silencing mechanism which is involved in the regulation of hundreds, perhaps thousands, of plant genes (Axtell & Bowman 2008). This evidence shows that inverted repeat sequences, including those with functional promoters, are commonly available for HGT from plants.
170. A key consideration in the risk assessment process should be the safety of the protein product resulting from the expression of the introduced genes rather than HGT per se (Thomson 2000). If the introduced RNAi constructs or their end products are not associated with any risk then even in the unlikely event of HGT occurring, they should not pose any risk to humans, animals or the environment. Conclusions reached for Events 1 - 4 associated with the expression of the introduced RNAi constructs did not represent an identified risk. Therefore, any rare occurrence of HGT of introduced genetic material to other organisms is expected to be unlikely to persist and/or result in an adverse effect.
171. Conclusion: The potential for an adverse outcome as a result of HGT is not an identified risk and will not be assessed further.
2.5 Unintended changes in biochemistry, physiology or ecology
172. All methods of plant breeding can induce unanticipated changes in plants, including pleiotropy1 (Haslberger 2003). Gene technology has the potential to cause unintended effects due to the process used to insert new genetic material or by producing a gene product that affects multiple traits. Such pleiotropic effects may include:- altered expression of an unrelated gene at the site of insertion
- altered expression of an unrelated gene distant to the site of insertion, for example, due to the encoded protein of the introduced gene changing chromatin structure, affecting methylation patterns, or regulating signal transduction and transcription
- increased metabolic burden associated with high level expression of the introduced gene
- novel traits arising from interactions of the protein encoded by the introduced gene product with endogenous non-target molecules
- secondary effects arising from altered substrate or product levels in the biochemical pathway incorporating the protein encoded by the introduced gene.
Event 7. Changes to biochemistry, physiology or ecology of the GM wheat lines resulting from expression or random insertion of the introduced RNAi constructs
174. For the majority of the lines, no phenotypic differences between the GM wheat plants and the non-GM parental lines have been observed under glasshouse conditions, although the applicant indicates that lines have undergone very limited phenotypic characterisation beyond examination of grain properties. Considerations relevant to altered biochemistry, physiology and ecology, in relation to expression of the introduced gene, have already been discussed in Events 1 to 3, and were not considered identified risks.
175. Various biochemical pathways of the GM wheat plants could be changed by the expression of the RNAi constructs, resulting in the production of novel or higher levels of endogenous toxins, allergens or anti-nutritional compounds. Non-GM wheat can be toxic to animals if consumed in large quantities (due to nitrate poisoning), and wheat flour is allergenic to some people and may also give rise to celiac disease. For further discussion regarding the toxicity and allergenicity of non-GM wheat see The Biology of Triticum aestivum L. em Thell. (Bread Wheat) (OGTR 2008).
176. The outcome of random insertion of an introduced gene is impossible to predict. Such outcomes may include, for example, alteration to reproductive capacity, altered capacity to deal with environmental stress, production of novel substances, and changes to levels of endogenous substances. Additionally, unintended changes that occur as a result of gene insertions are rarely advantageous to the plant (Kurland et al. 2003).
177. Unintended secondary effects occurring as a result of altered grain composition could include changes in seed germination and seedling vigour (as discussed in Event 2), pest preference for grain, seed dormancy, timing of flowering and seed set, outcrossing tendency or disease susceptibility. While the GM wheat lines have not undergone thorough phenotypic analysis, it is expected that substantial changes in these parameters would have been detected in the time these lines have been under development.
178. In plants, RNAi constructs can give rise to off-target silencing effects, where small RNAs derived from the sequence directing RNAi closely match non-target sequences expressed in the same cells. Homology of as little as 20 nt can give rise to off-target silencing (reviewed by Small 2007). The strength of silencing of the non-target gene generally increases with greater lengths of homology and the strongest effects are expected to occur between highly homologous gene family members (Miki et al. 2005). This is expected to occur only in tissues in which the RNAi construct is expressed, which is thought to be restricted to the endosperm in the GM wheat lines (information supplied by applicant). Potential off-target silencing may be predicted if the sequence of the host genome is known, however this is not the case for wheat. Similarly to the effect of random insertions discussed below, any strong off-target silencing effect is likely to be detrimental to the plant and so likely to be detected during production of the GM wheat lines.
179. The likelihood of any pleiotropic effects causing adverse effects is minimised by the proposed limits and controls outlined in Chapter 1, Sections 3.2 and 3.3. In particular, the scale and duration of the trial would limit the potential for adverse effects. The proposed trial sites will be surrounded by a stock-proof fence, and access to the trial site will be via locked gates, which limits exposure of the public, and some animals to the GM plant material. Livestock would not be intentionally exposed as the GM plant material will not be used as feed. Rat and pig nutritional trials have been proposed by the applicant, and no material from these experiments will enter the human food or animal feed supply.
180. Conclusion: The potential for an adverse outcome as a result of altered biochemistry, physiology or ecology is not an identified risk and will not be assessed further.
2.6 Unauthorised activities
Event 8. Use of GMOs outside the proposed licence conditions (non-compliance)181. If a licence were to be issued, non-compliance with the proposed conditions of the licence could lead to spread and persistence of the GM wheat lines outside of the proposed release areas. The adverse outcomes that this event could cause are discussed in the sections above. The Act provides for substantial penalties for non-compliance and unauthorised dealings with GMOs. The Act also requires that the Regulator has regard for the suitability of the applicant to hold a licence prior to the issuing of a licence. These legislative provisions are considered sufficient to minimise risks from unauthorised activities.
182. Conclusion: The potential for an adverse outcome as a result of unauthorised activities is not an identified risk and will not be assessed further.
1Pleiotropy is the effect of one particular gene on other genes to produce apparently unrelated, multiple phenotypic traits (Kahl 2001).