116. 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.
117. The criteria used by the Regulator to determine harm are described in Chapter 3 of the Risk Analysis Framework (OGTR 2007a). 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.
118. 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.
The nine events that were characterised are discussed in detail later in this Section. They are summarised in Table 6 where events that share a number of common features are grouped together in broader hazard categories. None were considered to lead to an identified risk that required further assessment.
119. As discussed in Chapter 1 Section 5, the GM sugarcane plants would contain combinations of the reporter gene uidA, and the antibiotic resistance selectable marker genes bla and nptII. The bla gene, encoding β-lactamase, would not be expressed in the GM sugarcane plants as it is linked to a bacterial promoter that does not function in plants. It will therefore not be further assessed for this application. The uidA and nptII genes and their products have already been considered in detail in previous RARMPs and by other regulators. Since neither of these genes has been found to pose risks to either people or the environment, their potential effects will not be further assessed for this application.
Table 6. Summary of events that may give rise to an adverse outcome through the expression of the introduced genes and RNAi constructs for altered plant growth, enhanced drought tolerance, enhanced nitrogen use efficiency, altered sucrose accumulation or improved cellulosic ethanol production from sugarcane biomass.
|Hazard category||Event that may give rise to an adverse outcome||Potential adverse outcome||Identified risk?||Reason|
Production of a substance toxic/allergenic to people or toxic to other organisms
|Exposure to GM plant material containing the introduced genes or RNAi constructs, or their end products||Allergic reactions in people or toxicity in people and other organisms||No|
Spread and persistence of the GM sugarcane plants in the environment
|Expression of the introduced genes or RNAi constructs improving the survival of the GM sugarcane plants||Weediness; allergic reactions in people or toxicity in people and other organisms||No|
|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|
Vertical transfer of genes or genetic elements to sexually compatible plants
|Expression of the introduced genes, RNAi constructs and regulatory sequences in other sugarcane plants||Weediness; allergic reactions in people or toxicity in people and other organisms||No|
|Expression of the introduced genes, RNAi constructs or regulatory sequences in other sexually compatible plants||Weediness; allergic reactions in people or toxicity in people and other organisms||No|
Horizontal transfer of genes or genetic elements to sexually incompatible organisms
|Presence of the introduced genes or RNAi constructs in other organisms as a result of gene transfer||Weediness; allergic reactions in people or toxicity in people and other organisms||No|
Unintended changes in biochemistry, physiology or ecology
|Changes to biochemistry, physiology or ecology of the GM sugarcane plants resulting from expression, or random insertion, of the introduced genes or RNAi constructs||Weediness; allergic reactions in people or toxicity in people and other organisms||No|
Unintended presence in the environment of Agrobacterium tumefaciens containing the introduced genes or RNAi constructs
|Transfer of the introduced genes or RNAi constructs from Agrobacterium to other organisms||Weediness; allergic reactions in people or toxicity in people and other organisms||No|
|Use of the GMOs outside the proposed licence conditions||Potential adverse outcomes mentioned in Sections 2.1 to 2.6||No|
2.1 Production of a substance toxic/allergenic to people or toxic to other organisms
120. 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).
121. 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).
122. A range of organisms may be exposed directly or indirectly to the introduced genes or RNAi constructs for enhanced drought tolerance, enhanced nitrogen use efficiency, altered plant growth, altered sucrose accumulation and increased efficiency of post-harvest processing for cellulosic ethanol production and their end products. Workers cultivating the sugarcane would be exposed to all plant parts. Organisms may be exposed directly to the introduced genes or RNAi constructs and their end products through biotic interactions with GM sugarcane 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 sugarcane plant parts or degrade them (vertebrates, insects, fungi and/or bacteria).
Event 1. Exposure to GM plant material containing the introduced genes, RNAi constructs, or their end products
123. Expression of the introduced genes or RNAi constructs for enhanced drought tolerance, enhanced nitrogen use efficiency, altered plant growth, altered sucrose accumulation or increased efficiency of post-harvest processing for cellulosic ethanol production could potentially result in the production of novel toxic or allergenic compounds in the GM sugarcane plants, or alter the expression of endogenous sugarcane 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.
124. Non-GM sugarcane is not known to be toxic to humans or other organisms (OGTR 2008b). Although no toxicity studies have been performed on the GM sugarcane plant material, most of the introduced genes or RNAi constructs were isolated from naturally occurring organisms that are already widespread and prevalent in the environment, such as common food plants or naturally occurring bacteria (see Chapter 1, Section 6.5).
125. People and animals are exposed to most of the proteins produced by these genes through their diet and the environment. No information was found to suggest that any of the proteins encoded by the introduced genes or RNAi constructs are toxic or allergenic to people or other organisms (Chapter 1, Section 5.2.6).
126. It is not expected that any novel products would be produced as a result of the expression of the introduced gene fragments in the RNAi constructs as they are likely to be degraded upon initiating RNAi, before transcription can occur. These gene fragments are intended to silence their endogenous counterparts and therefore the level of these proteins in plant tissues would be lower than in non-GM sugarcane plants. Therefore, if these proteins had any toxicity potential then the respective sugarcane plants would be less toxic than the non-GM parent.
127. Sugarcane pollen may be an allergen (Chakraborty et al. 2001), although allergic responses to the commercial hybrid cultivars of sugarcane have not been reported in Australia. Due to the very limited quantities of pollen produced by sugarcane, it is expected that people would be exposed to very small quantities, if any, of pollen. As discussed above, the encoded proteins in the GM sugarcane are not considered to be toxic or allergenic.
128. 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. Human contact with, or inhalation of, GM plant materials would be limited to trained and authorised staff. The proposed trial sites are located on BSES research stations so access to the general public would be minimised. There is little potential for exposure of the public to GM plant material via ingestion, skin contact or inhalation as no GM plant material would be used as animal feed or human food. Livestock would not be intentionally exposed as the GM plant material would not be used as animal feed.
129. Researchers and technical staff conducting the trials would be exposed to the GM plant materials during all phases of the trial. Workers may come into contact with the proteins encoded by the introduced genes when the plant cells have been damaged, or via pollen. Sugarcane plants possess leaves with sharp edges and irritating hairs, and so workers typically wear appropriate clothing to reduce dermal contact. Exposure to the GM sugarcane is unlikely to lead to an adverse outcome as the GM sugarcane plants are unlikely to be any more toxic than non-GM sugarcane. No adverse effects have been reported from exposure of sugarcane workers to GM sugarcane plant material containing some of the same proteins released under licence DIR 070/2006.
130. After harvest the applicant proposes to destroy the GM sugarcane material, apart from retaining some plant material for research purposes and for new plantings within the trial. These measures would minimise exposure to the GM plant material.
131. Conclusion: The potential for allergic reactions in people, or toxicity in people and other organisms as a result of exposure to GM plant materials containing proteins encoded by the introduced genes or as a result of the RNAi constructs is not an identified risk and will not be assessed further.
132. As this is early stage research little is known about the GM sugarcane plants proposed to be released. Some of the genes and RNAi constructs have not previously been expressed in GM plants. If further information on the allergenicity or toxicity of the GM sugarcane was to become available during the proposed 15 year duration of the trial, the context of this assessment may change. Data on the toxicity or allergenicity of the GM sugarcane lines modified for altered plant growth, enhanced drought tolerance, enhanced nitrogen use efficiency, altered sucrose accumulation and increased efficiency of post-harvest processing for cellulosic ethanol production relating to toxicity and allergenicity would be able to inform future risk assessments and reduce the uncertainty associated with these genes and RNAi constructs in sugarcane.
2.2 Spread and persistence of the GM sugarcane plants in the environment
133. Baseline information on the characteristics of weeds in general, and the factors limiting the spread and persistence of non-GM sugarcane plants in particular, is given in The Biology of the Saccharum spp. (sugarcane) (OGTR 2008b). In summary, the document concludes that modern cultivars of non-GM sugarcane are not problematic weeds in Australia where sugarcane occurs almost exclusively as a managed agricultural crop.
134. Characteristics of sugarcane that may contribute to the likelihood of its persistence in the environment include its ability to regenerate by re-sprouting from underground buds or from vegetative cuttings containing viable buds. Sugarcane plants can also persist in the field for over 10 years (information supplied by the applicant).
135. Modern sugarcane cultivars are not invasive in natural undisturbed environments and are not recognised as weeds in Australia. The establishment, spread and persistence of sugarcane populations is likely to be limited by a complex interaction of factors including weed competition, pest infestation, disease infection, moisture stress and soil fertility (Bakker 1999; Hogarth & Allsopp 2000; OGTR 2008b).
136. Scenarios that could lead to increased spread and persistence of the GM sugarcane plants include expression of the introduced genes or 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 genes or RNAi constructs improving the survival of the GM sugarcane plants
137. If the GM sugarcane plants 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 has been considered in Event 1 and was not considered an identified risk.
138. If the expression of the introduced genes or RNAi constructs for enhanced drought tolerance, enhanced nitrogen use efficiency, altered plant growth, altered sucrose accumulation or increased efficiency of post-harvest processing for cellulosic ethanol production were to provide the GM sugarcane plants with a significant selective advantage over non-GM sugarcane 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 sugarcane plants could adversely affect agricultural environments if they exhibited a greater ability to establish and persist than non-GM sugarcane.
139. The impact of the genetic modifications on survival of the GM sugarcane plants is uncharacterised under field conditions. However, a number of predictions can be made based on knowledge of the gene functions and prediction of their effects when expressed in the GM plants. Predictions can also be made based on the observed phenotypes of some of the GM sugarcane lines released under licence DIR 070/2006, which contain similar constructs to some in the current application. The phenotypes of these lines are summarised in Chapter 1, Section 5.5.2.
140. Two of the genes in the current application are GA20-oxidases, which are enzymes catalysing a step in GA biosynthesis leading to production of biologically active GA. Increasing GA20-oxidase expression in potato, Arabidopsis, aspen and tobacco results in elongation of internodes, early flowering, taller plants and increased biomass (Coles et al. 1999; Carrera et al. 2000; Eriksson et al. 2000; Biemelt et al. 2004). Silencing of GA20-oxidases has generally opposite effects in Arabidopsis and potato, including decreased internode elongation, reduced stem elongation and late flowering (Coles et al. 1999; Carrera et al. 2000). Over-expression of a pumpkin GA20-oxidase in Arabidopsis, lettuce and Solanum dulcamara has been shown to have effects similar to those seen for GA20-oxidase silencing (Curtis et al. 2000; Niki et al. 2001; Radi et al. 2006). A third GA biosynthesis gene in the current application is a GA2-oxidase, which decreases levels of biologically active gibberellins. Over-expression of GA2-oxidase in Arabidopsis, tobacco, Nicotiana sylvestris and rice has been shown to result in dwarfism, reduced germination and delayed flowering (Sakamoto et al. 2003; Biemelt et al. 2004; Lee & Zeevaart 2005; Radi et al. 2006). These phenotypes are highly similar to the effects of reduced GA20-oxidase expression, highlighting the opposite biological functions of these two enzymes. Under DIR 070/2006, lines were released containing the same GA oxidases as are included in the current application.
141. The other genes to be used to alter plant growth in the GM sugarcane plants are homologs of Teosinte Branched1. Over-expression of the endogenous OsTB1 gene in rice led to a reduction in tillering while the converse phenotype resulted from silencing of the endogenous gene (Takeda et al. 2003). Similarly, over-expression of maize TB1 in GM wheat plants produced shorter plants with a reduced number of tillers and spikes but an increased number of leaves (Lewis et al. 2008). Sugarcane lines generated under DIR 070/2006 with increased OsTB1 or ShTB1 (named SoTB1 in that application) showed no alteration in phenotype, whereas lines containing RNAi constructs reducing ShTB1 expression were shorter with thinner stalks and increased tiller production (information provided by applicant).
142. Alteration of plant growth may result in improved competitiveness of the GM sugarcane compared to non-GM sugarcane. Potential changes could include: more vigorous growth improving the ability of sugarcane to establish in a competitive environment; increased plant height improving the ability of sugarcane to shade out other plants; shorter stature resulting in a decreased tendency to lodge; altered bud growth characteristics leading to stem pieces more readily shooting and establishing as new plants. Secondary effects of these changes, including potential effects on reproductive behaviour, are considered below. These changes could increase the potential weediness of the GM sugarcane plants.
143. One of the categories of GM sugarcane plants contains the ZmDof1 gene for enhanced nitrogen use efficiency, which if successful would confer improved growth in soil with low nitrogen levels. Expression of ZmDof1 in A. thaliana and potato has been shown to improve growth under low nitrogen conditions in the laboratory (Yanagisawa et al. 2004; Yanagisawa 2004). In an environment in which nitrogen availability was the main factor limiting the spread and persistence of sugarcane, expression of this gene for nitrogen use efficiency could increase weediness of the GM sugarcane plants. Dof proteins have also been shown be involved in numerous other processes including light responses, auxin responses, defence and seed germination (reviewed by Yanagisawa 2002). Indeed, an Arabidopsis mutant with a disrupted Dof gene (DAG1) produced seed with no dormancy and no requirement for light to induce germination (Papi et al. 2000). These mutants also had altered seed coat structure (Papi et al. 2002).
144. In environments where nitrogen availability is limiting, improved nitrogen use efficiency could increase the competitiveness and increase the potential weediness of the GM sugarcane plants compared to non-GM sugarcane.
145. Seven of the categories of GM sugarcane plants contain introduced gene constructs derived from five genes designed to alter sucrose accumulation. The identities of these genes has been declared CCI and they are not discussed further in this section.
146. It could be speculated that changes in sucrose accumulation could improve the competitiveness of the GM sugarcane compared to non-GM sugarcane, increasing its potential weediness. For example, plant growth requires sucrose as an energy source, and so altered sucrose accumulation could alter plant growth, potentially leading to some of the effects considered above for GM lines in which plant growth may be altered. However, the introduced gene fragments are currently poorly characterised, and it is highly speculative to consider their potential secondary effects.
147. Six of the categories of GM sugarcane plants contain introduced gene constructs designed to increase the efficiency of post-harvest processing of sugarcane biomass for cellulosic ethanol production. The identities of these genes has been declared CCI and they are not discussed further in this section.
148. Three of the categories of GM sugarcane plants contain introduced gene constructs for enhanced drought tolerance. In an environment in which water availability was the main factor limiting the spread and persistence of sugarcane, expression of the genes for enhanced drought tolerance could result in increased weediness of the GM sugarcane plants. The identities of two of these genes, WUE1 and WUE2 has been declared CCI and they are not discussed further in this section.
149. Expression of OsDREB1A and closely related genes from rice and Arabidopsis has been studied in a number of GM plants where improvements to drought tolerance have been shown. OsDREB1A and other rice DREB genes have been shown to be induced by dehydration, cold stress and high salt (Dubouzet et al. 2003), and are thought to play a role in initiating transcriptional responses to stress. Over-expression of OsDREB1A in Arabidopsis and rice plants leads to improved drought, salt and freezing tolerance, and growth retardation (Dubouzet et al. 2003; Ito et al. 2006). GM Arabidopsis plants expressing the Arabidopsis DREB1A gene from a constitutive promoter showed improved tolerance of drought, salinity and freezing stress, and severe growth retardation (Liu et al. 1998; Kasuga et al. 1999; Ito et al. 2006). However, when AtDREB1A was expressed from a stress-inducible promoter plant growth was normal and tolerance of stress was further improved (Kasuga et al. 1999).
150. Through unintended effects, the GM sugarcane plants may have increased weediness. For example, plant responses to a variety of stresses involve interconnected signalling and transcriptional controls. Homologues of the introduced genes for enhanced drought tolerance encode proteins which have been shown to enhance tolerance to other abiotic and biotic stresses such as cold, freezing, pathogens and salinity. Thus, it is possible that the introduced genes and gene fragments may give rise to GM sugarcane plants with enhanced tolerance of a range of stresses. The introduced genes or RNAi constructs, particularly those attempting to alter plant growth, could potentially affect fertility, flowering time and seed development (including germination) of the GM sugarcane lines as compared to commercially grown sugarcane, which could lead to increased spread and persistence of the GM sugarcane plants.
151. During the trial the applicant proposes to cross some of the GM plants to produce offspring containing more than one of the categories of genetic modification. This may create a plant with more than one gene for enhanced drought tolerance, or altered plant growth, or genes for both of these traits. This may result in a plant that has enhanced stress tolerance and earlier or increased flowering or seed production.
152. However, two issues should be considered. First it should be noted that the anticipated effects of the some of the introduced genes or RNAi constructs are derived from the available published literature on the genes and related members of their gene families. Thus, a much broader range of anticipated effects are considered than would likely result from any single introduced gene. A second consideration is that when a gene is expressed in different plant species the same effect on phenotype does not always eventuate. Therefore, the introduced genes or RNAi constructs may not confer any phenotypic changes or enhanced stress tolerance in the GM sugarcane plants.
153. It is possible that altered expression of a regulatory gene involved in plant responses to stress could enhance tolerance to several environmental stresses. However, it is unlikely that the introduced genes or RNAi constructs for enhanced drought tolerance, enhanced nitrogen use efficiency, altered plant growth, altered sucrose accumulation or increased efficiency of post-harvest processing for cellulosic ethanol production could alter all of the characteristics which limit the spread and persistence of sugarcane such as its low fertility and seed viability, poor ability of seedlings to establish and compete without human intervention, nutrient requirements, and susceptibility to pests and diseases and other factors that normally limit the spread and persistence of sugarcane plants in Australia (Bakker 1999; Hogarth & Allsopp 2000; OGTR 2008b).
154. Although alteration of plant growth by the introduced genes or RNAi constructs could potentially result in increased weediness of the GM sugarcane plants, it could also result in GM plants of lower fitness than other commercially available sugarcane varieties because of unintended effects of the introduced genes or RNAi constructs. For example, lines released under DIR 070/2006 expressing HvGA20ox-1 and-2 displayed increased plant height which in turn results in an increased susceptibility to lodging. In the same release, plants expressing PcGA2-ox1 had decreased plant height, and the applicant states that they may be less competitive as a result, due to being shaded more readily by competition. The trial would enable the applicant to assess the effect of the introduced genes or RNAi constructs on plant physiology and agronomic performance.
155. A further important consideration is the reduction in plant vigour routinely observed in sugarcane plants which have undergone tissue culture. The current application includes two categories of GM sugarcane expressing only marker genes, for the specific purpose of providing a baseline against which effects of the other genetic modifications may be measured, as comparison to non-tissue-cultured sugarcane is inappropriate. Data provided by the applicant from release DIR 070/2006 show that plant height, stalk number, stem diameter and cane yield are reduced in a population of nptII-expressing GM sugarcane compared to untransformed sugarcane. These effects are expected to generally decrease the competitiveness of GM sugarcane, in some cases by a substantial margin.
156. 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 sugarcane plants proposed for release. The release would be of limited size and at a limited number of locations. However, the proposed duration of the release gives rise to some uncertainty in the risk assessment (see below and Chapter 3, Section 4.1.1). The applicant proposes a number of control measures, including destruction of all plant materials not required for further analysis, and post harvest monitoring of the proposed sites for at least one year and until no sugarcane plants have been found on the sites for six months.
Conclusion: The potential for increased weediness, allergenicity or toxicity due to expression of the introduced genes or RNAi constructs for enhanced drought tolerance, enhanced nitrogen use efficiency, altered plant growth, altered sucrose accumulation or increased efficiency of post-harvest processing for cellulosic ethanol production improving the survival of the GM sugarcane plants is not an identified risk and will not be assessed further.
157. This field trial is part of early stage research, and therefore the effect of the introduced genes or RNAi constructs for altered plant growth, altered sucrose accumulation or increased efficiency of post-harvest processing for cellulosic ethanol production on the GM sugarcane lines is unknown in the field. The properties of progeny of crosses between the GM sugarcane and different categories of GM sugarcane or non-GM sugarcane different to the parental cultivar, are also unknown. Also, the ability of the GM sugarcane plants to withstand drought stress or have enhanced nitrogen use efficiency throughout different stages of their lifecycle, as compared to commercially available sugarcane cultivars, is unknown. Under current agricultural practices, climate and weather patterns the proposed controls are appropriate, however, over time if these were to change then the appropriateness of these controls is less certain. Data on the unintentional effects of the genes for altered plant growth, altered sucrose accumulation, increased efficiency of post-harvest processing for cellulosic ethanol production, enhanced drought tolerance and enhanced nitrogen use efficiency, particularly that relating to weediness, would inform future risk assessments and reduce the uncertainty of assessing risks associated with dealings with GM sugarcane containing these genes and RNAi constructs.
Event 3. Dispersal of reproductive (sexual or asexual) GM plant materials through various means, including animals and extreme weather conditions
158. If the GM sugarcane plants were to be dispersed from the release sites 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 sugarcane plants have been assessed in Event 1 and were not an identified risk. The potential for the introduced genes or RNAi constructs to result in improved survival of the GM sugarcane plants in the environment was assessed in Event 2 and was not an identified risk.
159. Although sugarcane can produce seeds, commercial sugarcane is propagated vegetatively, with stem pieces (setts) being planted in the field. Usually one bud per stem cutting is developed into a primary stalk which then gives rise to tillers (for more details see The Biology of the Saccharum spp. (sugarcane) (OGTR 2008b).
160. Viable sugarcane stems could be unintentionally dispersed during transportation. Sugarcane volunteers have been found growing along roadsides and railways in sugarcane cultivation areas. These volunteers are believed to have originated from stem cuttings displaced or fallen from vehicles, and generally consist of only a few groups of stems which do not become self perpetuating or result in further spread.
161. In the course of the dealings the applicant proposes to transport GM sugarcane setts between the release sites, cultivate GM sugarcane plants and collect GM plant materials for research purposes, laboratory research or new plantings within the trial. Accidental spillage or dispersal of GM plant materials, especially setts, in the course of these dealings could allow the GM sugarcane plants to spread and persist in the environment.
162. The applicant has proposed to transport the GM sugarcane plants and setts to and between BSES stations according to transport guidelines issued by the Regulator. The applicant has also proposed that transport of harvested material between field locations and crushing machinery within BSES stations would be in covered trailers, with sugarcane plant material being tied down. This constitutes a lower level of containment than is required by the Regulator’s transport guidelines, which require propagative material be contained within primary and secondary unbreakable containers. A standard condition of DIR licences requiring that all transport be according to the Regulator’s transport guidelines is included in the imposed licence conditions.
163. Spillage of setts during transport according to the Regulator’s transport guidelines would be rare, reducing the risk of dispersal of the GM sugarcane during transport. Further, the accounting procedures required by the Regulator’s guidelines would have the result that any spillage would be detected, initiating cleaning and monitoring of the site of the spill. Finally, appropriate environmental conditions are necessary for survival and persistence of any distributed setts. For example, soil-borne fungal infections are known to reduce sett germination, leading to the common practice of treating setts with fungicide prior to planting (FAO 2004).
164. During harvesting of GM sugarcane, accidental mixing of non-GM sugarcane with GM sugarcane may occur. At the field sites, the applicant proposes to separate GM sugarcane from any adjacent sugarcane by a guard row of non-GM cane and a 6 m isolation zone of bare ground or grass. This measure would clearly separate the GM sugarcane from neighbouring non-GM sugarcane, minimising the likelihood of accidental mixing at harvest. Additionally, the applicant has proposed to mark GM field corners with star pickets, sign GM plantings, and implement staff management procedures. In crossing facilities, the applicant proposes to label GM sugarcane with barcodes, and maintain GM sugarcane in clearly signed areas separate to areas used for non-GM sugarcane. These measures would minimise any potential mixing of non-GM sugarcane with GM sugarcane.
165. The applicant has proposed to cultivate GM sugarcane plants on seedling benches prior to planting in the field, using areas set aside at each BSES station at which field planting would occur. In these nursery facilities non-GM sugarcane plants would also be cultivated on separate benches to the GM sugarcane, and the GM sugarcane would be identified by barcode labels. Separation of GM and non-GM sugarcane within the nursery area and identification of GM plants would reduce the potential for human error leading to accidental dispersal of the GM sugarcane by workers mistaking them for non-GM plants.
166. The applicant proposes to thoroughly clean equipment used to plant and the harvest the GM sugarcane to prevent dispersal of GM plant materials to other locations and to meet domestic quarantine requirements. The applicant proposes to destroy all plant material other than that collected for future research or for new plantings within the proposed release. The sites would be monitored for volunteers after the final harvest and any volunteer sugarcane plants would be destroyed.
167. On completion of the trial the sugarcane plant material would be destroyed by a combination of harvesting, mulching, herbicide treatment and burning. The applicant has proposed that some material be left to rot in the field. This could lead to the possibility of dispersal of potentially viable stem pieces which may lead to the establishment of GM sugarcane.
168. Flooding may cause dispersal of plant parts. However, control measures have been proposed by the applicant to minimise dispersal by flooding. These include locating the proposed release sites at least 50 m away from natural waterways (with the exception of the BSES Meringa photoperiod facility) on land that is at minimal risk of flooding. The applicant states that BSES Meringa, Burdekin and Southern have no history of flooding, and BSES Woodford is in an elevated part of a flood-prone area and has experienced no significant flooding in the past 10 years. At BSES Woodford the applicant plans to use the highest part of the site for the trial, which has no history of flooding. The applicant states that flooding is rare at BSES Central, which is 5 km from the nearest major waterway and that the station was unaffected by significant floods in the area in the high rain experience in the most recent rainy season. The applicant also states that sugarcane plants are generally not uprooted by floodwaters, unless significant water flow occurs. Although the BSES Meringa photoperiod facility is within approximately 20 m of a small natural waterway, the risk of dispersal of sugarcane from this facility is limited because GM sugarcane at this location would be large plants in pots held within a large trolley (which would restrict movement of pots), and in addition BSES Meringa has no history of flooding. All other parts of the proposed crossing facility at BSES Meringa are more than 50 m from natural waterways.
169. As sugarcane can reproduce through vegetative cuttings, it is possible that pests such as feral pigs or other large animals could disperse viable materials. However, this is unlikely to occur because of the size and weight of the canes. Dispersal of cane material through these means has not been reported to date.
170. The sexual reproductive behaviour of sugarcane has been little studied in the field, as seed production is not important for sugarcane cropping. Conditions for seed production have been studied in breeding facilities, however, the relevance of these observations to field-grown sugarcane is limited by the optimisation of environmental conditions in breeding situations. Sugarcane plants can flower and produce seed in the field where appropriate conditions exist for floral induction and production of viable pollen. These factors are discussed further in Event 4. To summarise, the frequency of production of fertile seed decreases with increasing distance from the equator. BSES Meringa is the proposed location at which production of viable seed is most likely, and this station is not proposed to be used for field trials. The applicant proposes to monitor GM sugarcane at BSES Meringa for flowering, and contain inflorescences in pollen lanterns prior to spikelets opening to control distribution of pollen.
171. Sugarcane seed is adapted for wind dispersal by the retention of callus hairs (or fuzz) (Babu 1979). Sugarcane seed is short-lived, losing 90% of viability within 80 days at 28ºC unless it is desiccated (Rao 1980), with more recent data suggesting that seed remains viable for at least 2-3 months when stored at room temperature (Powell et al. 2008). Numerous animals, including mammals, birds and insects, are known to eat GM sugarcane plant materials, including flowers, pollen and seed. Of those, seed has the potential to produce a new GM sugarcane plant if it were still viable after excretion.
172. Generally, sugarcane seeds are hard to germinate and the seedlings require particular favourable environmental conditions to survive for the first three to four weeks after germination (Breaux & Miller 1987) and therefore require careful nurturing. Powell et al. (2008) studied the effect of temperature on sugarcane seed germination, finding that germination occurred at temperatures from 15 to 42oC, with optimum germination at 30 to 36oC. Combining this data with knowledge of seed longevity, the authors concluded that sugarcane seed, which is predominantly produced in winter, could survive until temperatures favoured germination only in north Queensland. Seedlings are occasionally observed in the field in the Herbert district and further north (Robert Birch, personal communication, 2008). A requirement for very specific environmental conditions for germination is suggested by the lack of reports of seed germination in more southern field sites, and by reported low numbers of seedlings observed in more northern regions. It is unknown whether field-germinated seedlings survive to maturity. Of the BSES stations proposed to be used for the trial, only BSES Meringa is located in an area where temperatures are thought to be conducive to sugarcane seed germination in the field. Within the proposed crossing facility at BSES Meringa the applicant proposes to contain open inflorescences in pollen lanterns and mature fuzz containing seeds in bags for drying seed, and so the dispersal of pollen or seed into the environment is considered very unlikely.
173. There is no data to suggest that seed viability or dispersal would be altered in the GM sugarcane plants compared to the non-GM parental sugarcane plants. However, seed production may be increased in some of the GM sugarcane plants compared to the non-GM parental sugarcane plants (see Event 2). Survival of any GM sugarcane seedlings would be limited by factors such as humidity, temperature, low intrinsic competitive ability, nutrient availability, pests and diseases and other environmental factors that normally limit the spread and persistence of sugarcane plants in Australia (see Event 2).
174. In the unlikely event that material is dispersed away from the proposed release sites, it is unlikely to be a source of potential harm because the GM plants are unlikely to establish and persist outside the release sites or to be toxic or allergenic (Events 1 and 2).
175. 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.
176. The assessment of dispersal of reproductive plant material has been prepared using the current context for the trial described in Chapter 1, including weather conditions and agricultural practices. This may alter over the proposed fifteen year period of the release. For example, cultivars used in the Queensland sugarcane industry have recently been changing in response to occurrence of smut. While there is published literature relating to older cultivars, there is less industry experience and published knowledge of flowering, seed production and seed viability of new cultivars. In addition, more cultivars are anticipated to be introduced in the near future. In this potentially changing context, it is uncertain whether parental non-GM sugarcane cultivars which may be used in the current application have an altered ability to disperse and whether the addition of any of the proposed genetic modifications will give an additional selective advantage.
2.3 Vertical transfer of genes or genetic elements to sexually compatible plants
177. 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).
178. Baseline information on vertical gene transfer associated with non-GM sugarcane plants can be found in The Biology of the Saccharum spp. (sugarcane) (OGTR 2008b). In summary, sugarcane pollen viability is extremely low under natural conditions, commercial sugarcane varieties show very low fertility and crossing to plants outside of the Saccharum genus has rarely been observed. Thus, it is highly unlikely that crossing with sexually compatible plants would occur.
Event 4. Expression of the introduced genes or RNAi constructs and regulatory sequences in other sugarcane plants
179. Transfer and expression of the introduced genes or RNAi constructs for enhanced drought tolerance, enhanced nitrogen use efficiency, altered plant growth, altered sucrose accumulation or increased efficiency of post-harvest processing for cellulosic ethanol production to other sugarcane plants could increase the weediness potential, or alter the potential allergenicity and/or toxicity of the resulting plants.
180. All of the introduced regulatory sequences are expected to operate in the same manner as regulatory elements endogenous to the sugarcane 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.
181. As discussed in Event 1, allergenicity to people and toxicity to people and other organisms are not expected to be changed in the GM sugarcane plants by the introduced genes or RNAi constructs. This will be the same if the introduced genes or RNAi constructs are expressed in other sugarcane plants.
182. Sugarcane is principally a wind pollinated out-crosser with a low frequency of self pollination. The initiation of flowering is strongly influenced by day length and other environmental conditions. Pollen viability and pollen movement are also strongly influenced by environmental conditions including temperature, relative humidity and wind intensity (discussed below). There is little information available on rates and distances of outcrossing for sugarcane.
183. Sugarcane is cultivated within close proximity to all of the proposed trial sites. At BSES Woodford, Southern, Central and Burdekin, non-GM sugarcane is cultivated within the BSES stations at a minimum distance of 10 m from the proposed trial sites. At BSES Southern, Central and Burdekin, commercial sugarcane is propagated in adjacent properties at a minimum distance of 20 m from the proposed trial sites. Within crossing facilities at BSES Southern and Meringa, the applicant proposes to enclose open GM sugarcane inflorescences in pollen-impermeable lanterns to separate them from non-GM sugarcane inflorescences. The genes for enhanced drought tolerance, enhanced nitrogen use efficiency, altered plant growth, altered sucrose accumulation or increased efficiency of post-harvest processing for cellulosic ethanol production could be transferred to these sexually compatible sugarcane plants.
184. Sugarcane does not flower with great consistency, and cultivars differ in their propensity to flower (Bonnett et al. 2007). The initiation of sugarcane flowering has largely been studied within the context of breeding programs, where unreliable flowering of many cultivars is a reported problem (Berding et al. 2004). Observations of the breeding collection at BSES Meringa indicated that an average of 38.2% of clones in the breeding collection flowered each year between 1978 to 1996 and the flowering was variable, ranging from 16% of clones in 1993 to 66% in 1984 (Cox et al. 2000). Shortening day length appears to control the initiation of flowering (Moore & Nuss 1987) and environmental factors appear to influence the extent of flowering. Under adequate irrigation, temperatures above 32oC impede flowering (Cox et al. 2000), and night-time temperatures above 18oC are required for floral development (Berding 1981). Sugarcane has been observed to flower throughout Queensland and as far south as the New South Wales border (Graham Bonnett and Juan Jose Olivares-Villegas, personal communication 2009), however the frequency of flowering in the Burdekin region and further south, which is generally thought to be low, has not been systematically recorded.
185. Sugarcane pollen viability is variable and is strongly influenced by temperature, being greatly reduced under night-time temperatures below 21oC (Berding 1981). In Queensland, this leads to a general reduction in levels of pollen viability the further south sugarcane is cultivated. The applicant has provided data showing that pollen viability on inflorescences sampled from commercial crops in the Mulgrave region was significantly higher than in samples from the Burdekin region, which showed very low pollen viability. Bonnett et al. (2007) also reported low levels of pollen viability in the Burdekin region. Viable pollen has been sampled from field-grown sugarcane from Bundaberg and at the New South Wales border (Graham Bonnett and Juan Jose Olivares-Villegas, personal communication 2009). Reports of seed germination from fuzz collected from sugarcane fields show that very low amounts of viable seed are produced in the Burdekin region (Bonnett et al. 2007), in Mackay (data provided by the applicant), and as far south as Bundaberg (Graham Bonnett and Juan Jose Olivares-Villegas, personal communication 2009). However, there are no reports of systematic field observations of the production of viable pollen or seed in the majority of sugarcane-growing regions, and how this is effected by seasonal variability. There is a particular lack of relevant data for areas south of the Burdekin region. Available reports reveal variability between sugarcane cultivars, and strongly indicate that the frequency of flowering and production of viable pollen is very low in most parts of Queensland, with sugarcane seed production most likely to occur north of the Burdekin region. Sugarcane pollen rapidly desiccates and is not viable beyond 20 minutes in open air (Venkatraman 1922) or 35 minutes at 26.5ºC and 65% relative humidity (Moore 1976). Standard cultivation practices for commercial sugarcane cultivation include harvesting prior to flowering to maximise sugar content. Although this practice is not strictly adhered to, it would limit the opportunity for gene flow to nearby commercial crops.
186. As discussed in Event 3, sugarcane seed remains viable for only several months (Rao 1980; Powell et al. 2008), and germination is unreliable (Breaux & Miller 1987). Survival of any GM sugarcane seedlings would be limited by factors that normally limit the spread and persistence of sugarcane plants in Australia.
187. North of the Burdekin region (ie at BSES Meringa) environmental conditions can be suitable for viable pollen production, seed germination and seedling survival. In these regions the GM sugarcane plants would be grown in pots and monitored for flowering. The applicant proposes that flowering plants would have their inflorescences removed shortly before spikelet opening, at which time the inflorescences would be transferred to a crossing shed, where they would be enclosed in pollen lanterns. The applicant has also proposed measures to control potential pollen transfer in the event that spikelets open prior to inflorescences being enclosed in pollen lanterns, including removal and destruction of any spikelets which open before pollen lanterns are applied to GM inflorescences, and removal and destruction of any open spikelets on nearby non-GM inflorescences which are not enclosed in pollen lanterns, preventing set of seed. These measures would minimise the dispersal of pollen and the potential for cross-pollination to lead to seed set, thus limiting potential gene flow to other sugarcane plants in crossing facilities.
188. During the trial the applicant proposes to cross some of the GM sugarcane plants to produce offspring containing more than one of the sugarcane genetic modifications, using plants from categories expected to have altered drought tolerance and altered plant growth. At BSES Woodford, Southern and Meringa, GM sugarcane may be grown under the limited and controlled releases DIR 070/2006 and DIR 078/2007, and at BSES Central under DIR 078/2007. These GM sugarcane plants contain introduced genes or RNAi constructs for altered plant growth, enhanced nitrogen use efficiency and altered sugar production. Nine of the eighteen genes in DIR 070/2006 are the same as used in the current application, in which their expression is controlled by a greater variety of promoters and terminators.
189. Low levels of gene flow both within and between the sugarcane trials, or deliberate experimental crossing within the proposed trial, may lead to stacking of GM traits, thus producing a GM sugarcane plant which possesses genes for enhanced drought tolerance, enhanced nitrogen use efficiency, altered plant growth, altered sucrose production, altered sucrose accumulation and increased efficiency of post-harvest processing for cellulosic ethanol production which may give rise to altered weediness compared to any individual GM sugarcane plant in the proposed release.
190. However, the combination of traits is likely to contribute only incrementally to the potential weediness of the GM sugarcane plants, the spread and persistence of which would be limited by factors such as low fertility and seed viability, poor ability to establish and thrive without human intervention, competition with other plants, soil type and fertility, and pests and diseases that normally limit the spread and persistence of sugarcane plants in Australia.
191. 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 sugarcane plants. The applicant also proposes to perform post harvest monitoring of the field planting sites for at least twelve months and until the sites have been clear of volunteers for six months and to destroy any volunteer plants found at the sites. This would ensure that any GM sugarcane seeds or plants that were potentially the product of gene flow remaining in these areas would be destroyed.
192. Conclusion: The potential for allergenicity in people, or toxicity in people and other organisms or increased weediness due to the expression of the introduced genes, RNAi constructs and regulatory sequences in other sugarcane plants as a result of gene transfer is not an identified risk and will not be assessed further.
193. As this is early stage research no characterisation of the GM sugarcane plants has been performed. The secondary effects of genes, and combinations of genes, is not known and it is possible that they may affect flowering, pollen production or seed viability. This could lead to a greater chance of gene flow in the future. New varieties of sugarcane may also be introduced over the long term of this application which may have altered flowering characteristics, especially in cooler climatic conditions. Predictions of how the climate may change over the proposed 15 year duration of this release suggest that changes in the reproductive capacity of sugarcane in the locations of the proposed release may occur over this period.
Event 5. Expression of the introduced genes, RNAi constructs and regulatory sequences in other sexually compatible plants
194. Sugarcane is a hybrid derived from S. spontaneum and S. officinarum, and these species as well as other members of the Saccharum genus are sexually compatible with commercial sugarcane hybrids. Sugarcane is also sexually compatible with other genera within the tribe Andropogoneae (for more detail see Chapter 1, Section 6.4) (OGTR 2008b). To summarise, genera for which hybridisation to sugarcane has been observed under experimental conditions are Erianthus, Miscanthus, Sorghum and Zea (maize). Possible artificial hybridisations to the genera Narenga, Imperata (blady grass), Sclerostachya and Miscanthidium have been reported, but not fully verified. Although hybridisation with Bambusa (bamboo) has been reported, this report is thought to be false.
195. Commercial sorghum and maize crops are not currently cultivated near the BSES stations at which trial sites are proposed (information supplied by applicant), however, for the planned station (for which the location is yet to be determined) proximity to sorghum and maize cultivation is unknown, as is the proximity of existing BSES stations to future maize and sorghum plantings. Wild sorghum species are weeds of Australian sugarcane crops (McMahon et al. 2000) and are widespread in Australia (Hnatiuk 1990).
196. Blady grass (Imperata cylindrica) is common throughout Queensland coastal areas, however the applicant has stated that no-one is aware of the presence of much (if any) blady grass in the vicinity of the BSES stations proposed to be used for the trial. The presence of blady grass at the planned BSES station proposed to be used in the release is unknown, as is the future distribution of this grass.
197. Although some other sexually compatible species are likely to occur in the areas of the proposed release, crosses of sugarcane with genera outside the Saccharum genus are extremely difficult even under experimental conditions. The extremely low numbers of known genuine progeny obtained in any crosses have been of low vigour and sterile. A hybrid between maize and sugarcane has been generated using maize pollen (Janakiammal 1938 as cited by Bonnett et al. 2008) and confirmed using molecular markers (Nair et al. 2006). However, this cross has proved difficult to replicate (Janaki-Ammal 1941). Hybrids between sorghum and sugarcane have been generated using sugarcane as both the female (Grassl 1980) and male parent (Nair 1999). However, these experiments used large numbers of florets and produced few hybrids which were male sterile (Grassl 1980) or lacked vigour and showed slow growth (Nair 1999). Hybrids with Imperata cylindrica have been reported from a controlled cross, but have not been confirmed by molecular analysis (Bonnett et al. 2008).
198. In one of the areas proposed for release (BSES Meringa), S. spontaneum, S. robustum and S. officinarum are grown as part of a germplasm collection (see Chapter 1, Section 6.4). These species are all sexually compatible with cultivated sugarcane. S. robustum is thought to be the ancestral species from which S officinarum is derived (D'Hont et al. 1998; Brown et al. 2007), and is so closely related that it has been proposed that it should be classified as S. officinarum (Irvine 1999). The potential for GM sugarcane in the proposed release to cross-pollinate these plants would be minimised by measures proposed by the applicant to control pollen dispersal, in particular, enclosure of inflorescences of GM sugarcane in pollen lanterns prior to pollen shed.
199. Naturalised populations of S. spontaneum have been recorded at several locations within sugarcane growing areas and along part of the Mulgrave river within the Cairns LGA (Bonnett et al. 2008). Naturalised S. spontaneum is considered the most likely species to naturally hybridise with cultivated sugarcane (Bonnett et al. 2008), and this appears to be the case for the GM sugarcane lines in the current application. However, recorded naturalised populations of S. spontaneum on the Mulgrave River are many kilometres from the nearest of the BSES stations proposed for the release, BSES Meringa (information supplied by applicant).
200. Hybridisation would require synchronicity of flowering between the GM sugarcane plants and related species to enable cross-pollination and gene flow to occur. S. spontaneum has been shown to flower synchronously with sugarcane in the Herbert River region (near Ingham), and predominantly asynchronously in the Mulgrave Region (near BSES Meringa, Olivares-Villegas et al. 2008). S. spontaneum is not known to occur in proximity to the more southern sites of the proposed release (at BSES Woodford, Southern, Central and Burdekin).
201. Expression of the introduced genes or RNAi constructs in other sexually compatible plants is also unlikely to give these plants a significant selective advantage. The proposed limits and controls of the trial (Chapter 1, Sections 0 and 3.3) would restrict the potential for pollen flow and gene transfer to sexually compatible plants. At the proposed release site in the Mulgrave region (BSES Meringa) the applicant proposes to move the inflorescence of flowering plants into a crossing shed prior to flowering and enclose the inflorescences with pollen-proof lanterns to reduce pollen dissemination.
202. Conclusion: The potential for allergenicity in people, or toxicity in people and other organisms or increased weediness due to the expression of the introduced genes, 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.
203. Currently there are limited incidences of sexually compatible species growing within the range of pollen flow from the proposed trial sites. There is also limited overlap of flowering times between current commercial varieties which may be used as the parent of the GM sugarcane plants and S. spontaneum. However, over the long term of this application the distribution of sexually compatible species may alter, or flowering times may change due to the introduction of new varieties of sugarcane, or changes in climatic conditions.
2.4 Horizontal transfer of genes or genetic elements to sexually incompatible organisms
204. 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.
205. Risks that might arise from horizontal gene transfer 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.
206. Baseline information on the presence of the introduced or similar genetic elements is provided in Chapter 1, Section 6.5. Most 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
207. 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) and considered in detail in a previous RARMP (DIR 085/2008) which is available from the OGTR website or by contacting the Office.
208. HGT could result in the presence of the introduced genes or RNAi constructs for enhanced drought tolerance, enhanced nitrogen use efficiency, altered plant growth, altered sucrose accumulation or increased efficiency of post-harvest processing for cellulosic ethanol production in bacteria, plants, animals or other eukaryotes. However, the introduced sequences were mostly isolated from organisms already widespread in the environment (See Chapter 1, Section 6.5) and already available for transfer via demonstrated natural mechanisms.
209. A key consideration in the risk assessment process should be the safety of the protein product resulting from the expression of the introduced genes or RNAi constructs rather than horizontal gene transfer per se (Thomson 2000). If the introduced genes and 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 genes or 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.
210. Conclusion: The potential for an adverse outcome as a result of horizontal gene transfer is not an identified risk and will not be assessed further.
2.5 Unintended changes in biochemistry, physiology or ecology
211. All methods of plant breeding can induce unanticipated changes in plants, including pleiotropy11 (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.
212. Such unintended pleiotropic effects might result in adverse outcomes such as toxicity or allergenicity, weediness, altered pest or disease burden, or reduced nutritional value as compared to the parent organism. However, accumulated experience with genetic modification of plants indicates that, as for conventional (non-GM) breeding programs, the process has little potential for unexpected outcomes that are not detected and eliminated during the early stage of selecting plants with new properties (Bradford et al. 2005).
Event 7. Changes to biochemistry, physiology or ecology of the GM sugarcane plants resulting from expression or random insertion of the introduced genes or RNAi constructs
213. No data is available on the phenotype of the GM sugarcane plants. Some phenotypic data is available for GM sugarcane containing some of the same genes or RNAi constructs released under DIR 070/2006 (refer to Chapter 1, Section 5.5.2 for details), however under the current release these genes and RNAi constructs would be combined with a greater variety of regulatory elements. 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.
214. Various biochemical pathways of the GM sugarcane plants could be changed by the expression of the introduced genes or RNAi constructs, resulting in the production of novel or higher levels of endogenous toxins, allergens or anti-nutritional compounds.
215. Sugarcane products (from either GM or non-GM plants) can be detrimental if fed to animals in large quantities due to the presence of anti-nutritional compounds including hydrocyanic acid and lignin (Leng 1991; OGTR 2008b). Sugarcane pollen may also be an allergen (Chakraborty et al. 2001), although allergic responses to the commercial hybrid cultivars of sugarcane have not been reported in Australia. Further discussion regarding the toxicity and allergenicity of sugarcane is provided in The Biology of the Saccharum spp. (sugarcane) (OGTR 2008b).
216. Unintended secondary effects occurring as a result of enhanced drought tolerance, enhanced nitrogen use efficiency, altered plant growth, altered sucrose accumulation or increased efficiency of post-harvest processing for cellulosic ethanol production could include changes in plant growth, seed germination, seed dormancy, timing of flowering and seed set, outcrossing tendency or disease susceptibility (as discussed in Event 2). For example, in describing phenotypes of GM sugarcane lines released under DIR 070/2006, the applicant has noted unexpected effects on tillering and bud maturation in lines expressing HvGA20ox-1 and HvGA20ox -2.
217. 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 nucleotides 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). Potential off-target silencing may be predicted if the sequence of the host genome is known, however this is not the case for sugarcane. Similarly to the effect of random insertions discussed below, any strong off-target silencing effect is likely to be detrimental to the plant.
218. 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).
219. 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 of the trial would limit the potential for adverse effects. Access to the proposed trial sites would be by private road, which limits exposure of the public to the GM plant material. Humans and livestock would not be intentionally exposed as the GM plant material will not be used as human food or animal feed.
220. 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.
221. As this is early stage research little is known about the GM sugarcane plants to be released. No primary screening would be carried out prior to planting at the trial sites. This assessment has been made based on the current context for the trial, so if this were to change over the fifteen year trial duration then there is uncertainty regarding the outcome of this assessment. Data regarding the phenotypes of the GM sugarcane lines would provide greater certainty to a future risk assessment.
2.6 Unintended presence of Agrobacterium tumefaciens containing the introduced genes or RNAi constructs, during release
222. A. tumefaciens is a soil-borne, Gram-negative bacterium that, in nature, causes crown gall in plants. The bacterium enters wounded cells of the host and causes surrounding host cells to proliferate irregularly and form a gall. The bacterium is confined to the cells of the gall. Eventually, degradation of the gall releases the A. tumefaciens back into the soil where it can live saprophytically for several years (Krimi et al. 2002; Escobar & Dandekar 2003).
223. For genetic modification, ‘disarmed’ strains of A. tumefaciens that cannot cause crown gall are used to transfer DNA to plant cells under controlled, optimized laboratory conditions. The strains used for genetic modification may also contain hypervirulent, attenuated tumour-inducing plasmids to increase cell transformation rates. A. tumefaciens has been shown to persist in in vitro plant tissues and shoots. Broad spectrum antibacterial compounds tend to have a bacteriostatic effect, suppressing, but not eliminating bacterial growth and when removed the bacteria may resume growth. In particular, Gram-negative bacteria (such as A. tumefaciens) are considered to be difficult to eradicate completely from in vitro cultures (Barrett et al. 1997; Leifert & Cassells 2001), although persistence of A. tumefaciens in some GM plants has not been detected (Charity & Klimaszewska 2005).
224. During Agrobacterium-mediated transformation of plant cells, the A. tumefaciens attaches to plant cell walls and a virulence system is activated in the bacterium, ultimately allowing the transfer and integration of bacterial DNA into the plant DNA (de la Riva et al. 1998). As with most bacterial endophytes, disarmed strains of A. tumefaciens would be expected to inhabit the intercellular spaces and xylem vessels of plant tissue (Rosenblueth & Martínez-Romero 2006) via the formation of surface-associated biofilms (Danhorn et al. 2008). This means it is highly unlikely that A. tumefaciens would be incorporated into plant reproductive cells. For this reason, A. tumefaciens may persist in vegetatively propagated GM plants (such as sugarcane) since there would be no opportunity for elimination of the A. tumefaciens in sexually produced generations.
225. The transfer of GM sugarcane plants, carrying A. tumefaciens, into the environment could result in the transfer of genes to non-target plants or other microorganisms (Leifert 2000). Possible risks associated with the use of A. tumefaciens for genetic modification of plants under laboratory conditions have also been considered in previous RARMPs concerning GM rose (DIR 060/2005), GM bananas (DIR 076/2007 and DIR 079/2007) and GM torenia (DIR 084/2008). The RARMPs for these assessments are available on the OGTR website or by contacting the OGTR.
Event 8. Transfer of the introduced genes or RNAi constructs from A. tumefaciens to other organisms
226. If A. tumefaciens containing an introduced gene construct was present in the GM sugarcane it could transfer the introduced genes or RNAi constructs via conjugation with a wild type strain or other bacteria and yeast naturally present in the soil at the site (Hammerschlag et al. 2000). This general possibility of horizontal gene transfer has already been discussed in Event 6 and was not considered to be an identified risk.
227. If the A. tumefaciens were present in GM sugarcane tissue it could also genetically modify cells of other plants. Although the conditions for A. tumefaciens infection and gene transfer to plants would exist in nature, the creation of a GM plant would be highly unlikely because it would be unlikely that the A. tumefaciens would genetically modify a cell or cells that would give rise to a new organism, and it is unlikely that conditions in nature would exist that would select for the survival of the infected GM plant cells.
228. The applicant proposes to generate the GM sugarcane plants using both biolistic and Agrobacterium-mediated genetic modification (Chapter 1, Section 5.4). The extent to which Agrobacterium persists in GM sugarcane is unclear, but is expected to be strongly reduced by inclusion of Timentin, and antibiotic acting against Agrobacterium, in tissue culture media. Arencibia et al. (1998) first reported Agrobacterium-mediated transformation of sugarcane, noting that Agrobacterium did not persist in lines characterised by Southern blotting. However, the transformation methods of Arencibia et al. differ from those described by the applicant. The applicant intends to PCR test each GM sugarcane plant for the presence of Agrobacterium, using primers specific to regions outside the T-DNA and would not release any plants positive for Agrobacterium. Given that the applicant proposes to transfer GM sugarcane plants directly from tissue culture to BSES stations for propagation on seedling benches, this measure is considered a necessary step to reduce the likelihood that plants transferred to the field would be carrying residual A. tumefaciens.
229. Conclusion: The potential for an adverse outcome resulting from the persistence in the environment of A. tumefaciens containing the introduced genes or RNAi constructs is not an identified risk and will not be assessed further.
2.7 Unauthorised activities
Event 9. Use of GMOs outside the proposed licence conditions (non-compliance)
230. 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 sugarcane plants 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.
231. Conclusion: The potential for an adverse outcome as a result of unauthorised activities is not an identified risk and will not be assessed further.
11Pleiotropy is the effect of one particular gene on other genes to produce apparently unrelated, multiple phenotypic traits (Kahl 2001).