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Risk Assessment and Risk Management Plan for DIR 070/2006 Limited and controlled release of GM sugarcane with altered plant architecture, enhanced water or improved nitrogen use efficiency Applicant: BSES Limited February 2007 PAGE INTENTIONALLY LEFT BLANK DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Executive Summary Introduction The Gene Technology Regulator (the Regulator) has made a decision to issue a licence for dealings involving the intentional release of genetically modified (GM) sugarcane lines which are modified for altered plant architecture, enhanced water use efficiency or improved nitrogen use efficiency into the environment, in respect of application (DIR 070/2006) from the BSES Limited. The DIR070/2006 licence permits the release of up to 2500 genetically modified (GM) sugarcane lines on a limited scale and under controlled conditions. The Gene Technology Act 2000 (the Act) and the Gene Technology Regulations 2001 (the Regulations) govern the process undertaken by the Regulator before a decision is made on whether or not to issue a licence. The decision is based upon a Risk Assessment and Risk Management Plan (RARMP) prepared by the Regulator in accordance with the Risk Analysis Framework and in consultation with a wide range of experts, agencies and authorities, and the public. More information on the comprehensive assessment required for licence applications to release a genetically modified organism (GMO) into the environment is available from the Office of the Gene Technology Regulator (OGTR) (Free call 1800 181 030) or at <http://www.ogtr.gov.au/>. The application BSES applied for a licence to release up to 2500 GM sugarcane lines into the environment under limited and controlled conditions. Up to 1900 of the GM sugarcane lines have been modified to alter plant size and shape, enhance water use efficiency (WUE)1, or improve nitrogen use efficiency (NUE)2. The remaining 600 GM sugarcane lines contain only introduced selectable and/or visual marker genes. All of the introduced genes are derived from plants (rice, sugarcane, barley, bean, thale cress, apple or maize) or the common gut bacterium Escherichia coli. The trial is to take place at up to 3 sites of no more than 2 ha during each of the 3 cropping cycles between February 2007 to November 2010 (ie a total maximum area for the trial of 18 ha). The release may take place in the Queensland local government areas of Bundaberg, Caboolture and/or Cairns. The trial involves early stage ‘proof of concept’ research. The GM sugarcane lines containing only marker genes would be used to compare different genetic modification methods. The other introduced genes are intended to alter plant size and shape, enhance WUE or improve NUE by regulating different biochemical pathways that may result in increased sugar yield. The agronomic performance of GM sugarcane lines containing these genes will be evaluated in the field; some under different irrigation and fertilizer treatments. No GM plant material from the release will be used for human food, animal feed or for the production of other sugarcane commodities. BSES has proposed a number of measures to limit the spread and persistence of the GM sugarcane lines and the introduced genetic materials that were considered during the evaluation of the application. 1 2 WUE is defined as the measure of total yield (sugar) produced per unit of water supplied to the sugarcane crop. NUE is a term used to describe how effectively plants acquire and utilise nitrogen. Executive Summary (February 2007) I DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Risk assessment The hazard identification process considered the circumstances by which people or the environment may be exposed to the GMOs, GM plant materials, GM plant by-products, the introduced genes, or products of the introduced genes. A hazard (source of potential harm) may be an event, substance or organism. A risk is identified when a hazard is considered to have some chance of causing harm. Those events that do not lead to an adverse outcome, or could not reasonably occur, do not advance in the risk assessment process. Sixteen events were identified and assessed whereby the release of the GM sugarcane lines might give rise to harm to people or the environment. These 16 events included consideration of whether, or not, expression of the introduced genes could result in products that are toxic or allergenic to people or other organisms, alter characteristics that may impact on the spread and persistence of the GM plants, or produce unintended changes in their biochemistry or physiology. In addition, consideration was given to the opportunity for gene flow to other organisms, and its effects if this occurred. All events were characterised in relation to both the magnitude and probability of harm in the context of the controls proposed by the applicant to limit the spread and persistence of the GMOs in both time and space. This detailed consideration concluded that none of the sixteen events gave rise to an identified risk that required further assessment. The principle reasons comprise: small scale of the trial is limited in both area and duration suitability of containment and disposal measures to limit the spread and persistence of the GM plants none of the GM plant materials will be used for any other purpose widespread presence of the same or similar proteins and enzymatic products in the environment and lack of evidence of harm from these proteins and their products the lack of known toxicity or allergenicity of the proteins (and enzymatic products) encoded by the introduced genes limited capacity of the GM sugarcane lines to spread and persist outside the areas of the release limited ability and opportunity for the GM sugarcane lines to transfer the introduced genes to commercial sugarcane crops or other sexually compatible species. Therefore, any risks of harm to the health and safety of people, or the environment, from the release of the GM sugarcane lines into the environment are considered to be negligible. Risk management The risk management process builds upon the risk assessment to determine whether measures are required in order to protect people and/or the environment. As none of the 16 events characterised in the risk assessment are considered to give rise to an identified risk that requires further assessment, the level of risk is considered to be negligible. The Regulator’s Risk Analysis Framework defines negligible risks as insubstantial, with no present need to invoke actions for their mitigation. However, containment and disposal measures have been imposed to restrict the release to the locations, size and duration Executive Summary (February 2007) II DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator requested by the applicant, as these were an important part of establishing the context for assessing the risks. The licence conditions require the applicant to limit the duration of the release to between February 2007 and November 2010 on a maximum total area of up to 18 hectares; prevent the use of the GMOs, or materials from the GMOs for any other purposes; maintain physical isolation of the release sites; and conduct post-harvest monitoring to ensure all GM plants are destroyed3. Conclusions of the RARMP The risk assessment concludes that this limited and controlled release of up to 2500 GM sugarcane lines into the areas proposed in Queensland poses negligible risks to the health and safety of people and the environment posed by, or as a result of, gene technology. The risk management plan concludes that these negligible risks do not require specific risk treatment measures. However, licence conditions have been imposed to contain the release to the locations, size and duration requested by the applicant. 3 The licence for DIR 070/2006 is available on the OGTR website (<http://www.ogtr.gov.au/gmorec/ir.htm#table> via the link to DIR 070/2006). Executive Summary (February 2007) III DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator PAGE INTENTIONALLY LEFT BLANK Executive Summary (February 2007) IV DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Table of Contents EXECUTIVE SUMMARY .................................................................................................................................... I INTRODUCTION .................................................................................................................................................... I THE APPLICATION ................................................................................................................................................ I RISK ASSESSMENT ............................................................................................................................................... II RISK MANAGEMENT ............................................................................................................................................ II CONCLUSIONS OF THE RARMP ........................................................................................................................ III TABLE OF CONTENTS ......................................................................................................................................V ABBREVIATIONS ........................................................................................................................................... VII TECHNICAL SUMMARY .................................................................................................................................. 1 INTRODUCTION ................................................................................................................................................... 1 APPLICATION ...................................................................................................................................................... 1 RISK ASSESSMENT ............................................................................................................................................... 3 RISK MANAGEMENT ............................................................................................................................................ 4 LICENCE CONDITIONS TO MANAGE THIS LIMITED AND CONTROLLED RELEASE .................................................... 4 OTHER REGULATORY CONSIDERATIONS .............................................................................................................. 4 IDENTIFICATION OF ISSUES TO BE ADDRESSED FOR FUTURE RELEASES ................................................................ 5 CONCLUSIONS OF THE RARMP .......................................................................................................................... 5 CHAPTER 1 RISK ASSESSMENT CONTEXT .......................................................................................... 7 SECTION 1 SECTION 2 2.1 2.2 2.3 SECTION 3 SECTION 4 4.1 4.2 4.3 4.4 4.5 SECTION 5 5.1 5.2 5.3 5.4 SECTION 6 6.1 6.2 BACKGROUND ............................................................................................................................. 7 THE APPLICATION........................................................................................................................ 8 The proposed locations, size and duration of the trial ..................................................................... 8 The proposed dealings ..................................................................................................................... 8 The proposed measures to limit the spread and persistence of the GMOs ...................................... 9 THE PARENT ORGANISM .............................................................................................................. 9 THE GMOS, NATURE AND EFFECT OF THE GENETIC MODIFICATION ............................................ 9 Introduction to the GMOs ............................................................................................................... 9 Introduction to plant architecture, drought stress and nitrogen use efficiency .............................. 11 The introduced genes and regulatory sequences, and the gene products....................................... 13 Method of genetic modification .................................................................................................... 21 Characterisation of the GMOs ....................................................................................................... 22 THE RECEIVING ENVIRONMENT ................................................................................................. 23 Relevant abiotic factors ................................................................................................................. 23 Relevant agricultural practices ...................................................................................................... 23 Presence of related plants in the receiving environment ............................................................... 24 Presence of the introduced genes or similar genes in the environment ......................................... 25 AUSTRALIAN AND INTERNATIONAL APPROVALS ....................................................................... 25 Australian approvals of the GM sugarcane lines ........................................................................... 25 International approvals .................................................................................................................. 26 CHAPTER 2 RISK ASSESSMENT ............................................................................................................. 27 SECTION 1 SECTION 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 SECTION 3 INTRODUCTION.......................................................................................................................... 27 HAZARD CHARACTERISATION ................................................................................................... 28 Production of a substance toxic to people ..................................................................................... 32 Production of a substance allergenic to people ............................................................................. 34 Production of a substance toxic to organisms other than people ................................................... 36 Spread and persistence of the GM sugarcane in the environment ................................................. 37 Vertical transfer of genes or genetic elements to sexually compatible plants ............................... 44 Horizontal transfer of genes or genetic elements to sexually incompatible organisms. ................ 47 Unintended changes in biochemistry, physiology or ecology ....................................................... 48 Unauthorised activities .................................................................................................................. 50 RISK ESTIMATE PROCESS FOR IDENTIFIED RISKS ........................................................................ 50 CHAPTER 3 RISK MANAGEMENT ......................................................................................................... 51 Table of Contents (February 2007) V DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator SECTION 1 BACKGROUND ........................................................................................................................... 51 SECTION 2 OTHER AUSTRALIAN REGULATORS ........................................................................................... 51 SECTION 3 RISK TREATMENT MEASURES FOR IDENTIFIED RISKS ................................................................. 52 SECTION 4 GENERAL RISK MANAGEMENT ................................................................................................... 52 4.1 Summary of proposed licence conditions ...................................................................................... 52 4.2 Other risk management considerations ......................................................................................... 53 SECTION 5 ISSUES TO BE ADDRESSED FOR FUTURE RELEASES ..................................................................... 54 SECTION 6 CONCLUSIONS OF THE RARMP................................................................................................. 55 SECTION 7 DIR 070/2006 LICENCE............................................................................................................. 55 REFERENCES .................................................................................................................................................... 56 APPENDIX A DEFINITIONS OF TERMS IN THE RISK ANALYSIS FRAMEWORK USED BY THE REGULATOR ........................................................................................................................................... 69 APPENDIX B SUMMARY OF ISSUES RAISED IN SUBMISSIONS RECEIVED FROM PRESCRIBED EXPERTS, AGENCIES AND AUTHORITIES ON APPLICATION DIR 070/2006 ........ 71 APPENDIX C SUMMARY OF ISSUES RAISED IN SUBMISSIONS RECEIVED FROM THE PUBLIC ON APPLICATION DIR 070/2006 ................................................................................................... 72 Table of Contents (February 2007) VI DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Abbreviations ai APVMA ADP ATP AQIS C-terminal DIR DNA dsRNA EMBL FAO FARRP FSANZ g GA GM GMAC GMO GTTAC ha kDa kg km LD50 m mg mRNA μg N/A ng NHMRC NICNAS NUE OECD OGTR PC2 PCR RARMP RNA SDAP T-DNA TGA US FDA USDA WUE Active ingredient Australian Pesticides and Veterinary Medicines Authority Adenosine diphosphate Adenosine triphosphate Australian Quarantine and Inspection Service Carboxy-terminal end of a protein Dealing involving Intentional Release Deoxyribonucleic acid Double stranded RNA European Molecular Biology Laboratory Food and Agricultural Organization of the United Nations Food Allergy Research and Resource Program Food Standards Australia New Zealand (formerly ANZFA) Gram Gibberellic acid Genetically Modified Genetic Manipulation Advisory Committee Genetically Modified Organism Gene Technology Technical Advisory Committee Hectare Kilodalton Kilogram Kilometre Amount of a substance given in a single dose that causes death in 50% of a test population of an organism Metre milligram Messenger ribonucleic acid Microgram Not applicable Nanogram National Health and Medical Research Council National Industrial Chemicals Notification and Assessment Scheme Nitrogen use efficiency Organisation for Economic Co-Operation and Development Office of the Gene Technology Regulator Physical containment level 2 Polymerase chain reaction Risk Assessment and Management Plan Ribonucleic acid Structural database of allergenic proteins Transfer deoxyribonucleic acid Therapeutic Goods Administration United States Food and Drug Administration United States Department of Agriculture Water use efficiency Abbreviations (February 2007) VII DIR 70/2006 – Risk Assessment and Risk Management Plan WHO Office of the Gene Technology Regulator World Health Organisation Abbreviations (February 2007) VIII DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Technical Summary Introduction The Gene Technology Regulator (the Regulator) has decided to issue a licence (DIR 070/2006) to BSES Limited for dealings involving the intentional release of genetically modified (GM) sugarcane lines into the environment, on a limited scale and under controlled conditions. The DIR 070/2006 licence permits the limited and controlled release of up to 2500 GM sugarcane lines. The release will occur at up to 3 sites per cropping cycle between February 2007 to November 2010 in the local government areas of Bundaberg, Caboolture and/or Cairns, Queensland. Each site would be a maximum 2 ha in area, with a total maximum area for the trial of 18 ha. The Gene Technology Act 2000 (the Act), the Gene Technology Regulations 2001 (the Regulations) and corresponding State and Territory law govern the comprehensive and highly consultative process undertaken by the Regulator before making a decision whether to issue a licence to deal with a GMO. The Regulator’s Risk Analysis Framework explains the approach used to evaluate licence applications and to develop the Risk Assessment and Risk Management Plans (RARMPs) that form the basis of her decisions4. This RARMP for DIR 070/2006 has been finalised in accordance with the gene technology legislation. Matters raised in the consultation process regarding risks to the health and safety of people and the environment from the proposed dealings were taken into account by the Regulator in deciding to issue a licence and the licence conditions that have been imposed. Application Title: Applicant: Common name of the parent organism: Scientific name of the parent organism: Modified traits: Identity of the genes responsible for the modified traits: Proposed locations: Proposed release size: Proposed time of release: Limited and controlled release of GM sugarcane with altered plant architecture, enhanced water or improved nitrogen use efficiency* BSES Limited Sugarcane Saccharum spp. hybrid Plant architecture (shoot number, stalk size and height), water use efficiency, nitrogen use efficiency, and marker gene expression (antibiotic resistance and reporter genes) 14 genes for altered plant architecture from the plants Hordeum vulgare subsp. vulgare, Oryza sativa, Phaseolus coccineus and Saccharum spp. 3 genes for enhanced water use efficiency from the bacterium Escherichia coli, and plants Arabidopsis thaliana, Malus x domestica. 1 gene for improved nitrogen use efficiency from Zea mays uidA gene (-glucuronidase reporter gene) from the bacterium E. coli nptII gene (antibiotic resistance selectable marker) from the bacterium E. coli bla gene (antibiotic resistance selectable marker) from the bacterium E. coli. Local government areas of Bundaberg, Caboolture and/or Cairns (Qld) Maximum total area 18 ha, comprising up to 3 sites of no more than 2 ha per cropping cycle February 2007 to November 2010 * The original title of licence application submitted by BSES was GM sugarcane field trial – testing the effect on sugar yield of transformation methods. 4 More information on the assessment of licence applications and copies of the Risk Analysis Framework are available from the Office of the Gene Technology Regulator (OGTR). Free call 1800 181 030 or at <http://www.ogtr.gov.au/ir/process.htm> and <http://www.ogtr.gov.au/pdf/public/raffinal2.2.pdf>, respectively. Technical Summary (February 2007) 1 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator BSES applied for a licence to release up to 2500 GM sugarcane lines into the environment under limited and controlled conditions. Up to 1900 of the GM sugarcane lines have been modified to alter plant architecture (shoot number, stalk size and height), enhance water use efficiency (WUE)5, or improve nitrogen use efficiency (NUE)6. Up to 600 of the GM sugarcane lines contain only introduced selectable antibiotic resistance marker and/or visual marker genes. The trial is intended to take place at up to 3 sites per cropping cycle between February 2007 to November 2010 in the local government areas of Bundaberg, Caboolture and/or Cairns, Queensland. Each site would be a maximum 2 ha in area, with a total maximum area for the trial of 18 ha. The GM sugarcane lines for release were derived by transforming plants of the commercially grown Saccharum spp. hybrid Q117. Up to 1900 of the GM sugarcane lines contain either individual or combinations of 18 different introduced genes intended to improve agronomic characteristics and sucrose yields by either altering plant architecture, or enhancing WUE or improving NUE. The 14 genes for altered plant architecture are derived from the plants Oryza sativa (rice), Saccharum spp. (sugarcane), Hordeum vulgare subsp. vulgare (barley) and Phaseolus coccineus (bean). The 3 genes for enhanced WUE are derived from the plants Arabidopsis thaliana (thale cress), Malus x domestica (apple) and the gut bacterium Escherichia coli. The one gene for enhanced NUE is derived from Zea mays (maize). The introduced genes encode proteins or double-stranded RNAs (dsRNAs) that are intended to alter plant architecture, enhance WUE or improve NUE, by modulating biochemical pathways, either through direct participation (proteins) or regulation of endogenous gene expression (dsRNA) in the sugarcane plants, which in turn may result in increased sugar yield. The GM sugarcane lines modified to alter plant architecture, enhance WUE or improve NUE also contain the E. coli derived nptII marker gene, which confers resistance to some aminoglycoside antibiotics (eg neomycin, paromomycin, geneticin). The nptII gene enabled the identification and selection of GM plant tissues during the initial development of the GM sugarcane lines in the laboratory. All except 100 of the 1900 lines contain the E.coli bla gene (conferring ampicillin resistance). However, this gene is not expressed in the GM sugarcane lines as it is linked to a bacterial promoter that does not function in plants. The bla gene was used to select bacteria carrying the gene construct of interest that were used in the initial plant transformations. In addition to these 1900 GM sugarcane lines, up to 600 more lines contain only the nptII gene (100 lines), or the nptII gene with the bla bacterial marker gene (100 lines), or the nptII gene with the visual reporter gene uidA (400 lines). These lines will be compared during the trial to determine the best transformation technique for generating GM sugarcane lines. The purpose of the trial is to conduct early stage (‘proof of concept’) research to assess the agronomic performance of GM sugarcane lines in the field, including some under different irrigation and fertilizer treatments, and to collect data to assist in optimising the transformation process. The applicant proposed measures to limit the spread and persistence of the GM sugarcane lines in the environment. These were taken into account in establishing the risk assessment context for the release, and their suitability for limiting the release to the locations, size and duration proposed by the applicant was considered as part of the risk assessment process. No 5 6 WUE is defined as the measure of total yield (sugar) produced per unit of water supplied to the sugarcane crop. NUE is a term used to describe how effectively plants acquire and utilise nitrogen. Technical Summary (February 2007) 2 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator material from the GM sugarcane plants will be used for human food, animal feed or other sugarcane products. Risk assessment The risk assessment considered information contained in the application, current scientific knowledge, and issues relating to risks to human health and safety and the environment raised in submissions received from consultation with a wide range of prescribed experts, agencies and authorities on the application (summarised in Appendix B of the RARMP). No new risks to people or the environment were identified from the advice received on the consultation RARMP. However, feedback on some previously raised issues has enabled their clarification in the final RARMP. Two submissions from the public on the application and how they were considered are summarised in Appendix C of the RARMP. No submissions were received from the public on the consultation RARMP. A reference document, The Biology and Ecology of Sugarcane (Saccharum spp. hybrids) in Australia, was produced to inform the risk assessment process for licence applications involving GM sugarcane plants. The document is available from the OGTR or from the website <http://www.ogtr.gov.au>. The hazard identification process considered the circumstances or events by which people or the environment may be adversely affected by exposure to the GMOs, GM plant materials, GM plant by-products, the introduced genes, or products of the introduced genes. A hazard (source of potential harm) may be an event, substance or organism. A risk is identified when a hazard is considered to have some chance of causing harm. Those events that do not lead to an adverse outcome, or could not reasonably occur, do not advance in the risk assessment process. Sixteen events were identified and assessed whereby the release of the GM sugarcane lines might give rise to harm to people or the environment. These 16 events included consideration of whether expression of the introduced genes could result in products that are toxic or allergenic to people or other organisms, alter characteristics that may impact on the spread and persistence of the GM plants, or produce unintended changes in their biochemistry or physiology. In addition, consideration was given to the opportunity for gene flow to other organisms, and its effects if this occurred. All events were characterised in relation to both the magnitude and probability of harm in the context of the controls proposed by the applicant to limit the spread and persistence of the GMOs in both time and space. This detailed consideration concluded that none of the sixteen events gave rise to an identified risk that required further assessment. The principle reasons comprise: small scale of the trial is limited in both area and duration suitability of containment and disposal measures to limit the spread and persistence of the GM plants none of the GM plant materials will be used for any other purpose widespread presence of the same or similar proteins and enzymatic products in the environment and lack of evidence of harm from these proteins and their products the lack of known toxicity or allergenicity of the proteins (and enzymatic products) encoded by the introduced genes Technical Summary (February 2007) 3 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator limited capacity of the GM sugarcane lines to spread and persist outside the areas for release limited ability and opportunity for the GM sugarcane lines to transfer the introduced genes to commercial sugarcane crops or other sexually compatible species. Therefore, as no risks to the health and safety of people, or the environment were identified from the limited and controlled release of the GM sugarcane lines into the environment, the level of risk is considered to be negligible. Risk management A risk management plan builds upon the risk assessment to consider whether any action is required to mitigate the identified risks, and what can be done to protect the health and safety of people and the environment. As none of the 16 events that were characterised in the risk assessment process are considered to give rise to an identified risk that requires further assessment, the level of risk to human health and safety and the environment from the release of GM sugarcane lines is considered to be negligible. The Regulator’s Risk Analysis Framework defines negligible risks as insubstantial with no present need to invoke actions for their mitigation. However, containment measures have been imposed to restrict the release to the locations, size and duration requested by the applicant, as these were important parameters in establishing the context for assessing the risks. Licence conditions to manage this limited and controlled release A number of licence conditions have been imposed to limit and control the release, including requirements to: surround the trial sites by one guard row of non-GM sugarcane and an isolation zone of at least 6 metres locate the trial sites at least 50 m away from natural waterways harvest and process sugarcane from the trial separately from any commercial sugarcane destroy all plant materials not required for experimentation or new plantings following cleaning of sites, monitor for and destroy any GM sugarcane that may grow for at least 12 months until the site is clear of volunteers for a continuous 6 month period. Other regulatory considerations Australia’s gene technology regulatory system operates as part of an integrated legislative framework. The Regulator sought input on the preparation of the RARMP from other agencies that also regulate GMOs or GM products including Food Standards Australia New Zealand (FSANZ), Australian Pesticides and Veterinary Medicines Authority (APVMA), Therapeutic Goods Administration (TGA), National Industrial Chemicals Notification and Assessment Scheme (NICNAS), National Health and Medical Research Council (NHMRC) Technical Summary (February 2007) 4 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator and Australian Quarantine Inspection Service (AQIS). Dealings conducted under a licence issued by the Regulator may also be subject to regulation by one or more of these agencies7. FSANZ is responsible for human food safety assessment, including GM food. As the trial involves early stage research the applicant does not intend any material from the GM sugarcane lines to be used in human food. Accordingly the applicant has not applied to FSANZ to evaluate any of the GM sugarcane lines. FSANZ approval would need to be obtained before they could be used in human food. Identification of issues to be addressed for future releases The risk assessment identified additional information that may be required to assess an application for a larger scale trial, reduced containment conditions or commercial release of any of these GM sugarcane lines. These include: molecular characterisation of GM sugarcane lines selected for possible future releases additional data on the potential toxicity or allergenicity of proteins encoded by the introduced genes for altered plant architecture, enhanced WUE and improved NUE, and of plant materials from the GM sugarcane lines selected for further research biochemical, physiological and agronomic characteristics indicative of weediness in the selected GM sugarcane lines including measurement of tolerance to environmental stresses and reproductive capacity. Conclusions of the RARMP The risk assessment concludes that this proposed limited and controlled release of up to 2500 GM sugarcane lines into the local government areas of Bundaberg, Caboolture and/or Cairns in Queensland poses negligible risks to the health and safety of people and the environment posed by, or as a result of, gene technology. The risk management plan concludes that these negligible risks do not require specific risk treatment measures. However, licence conditions have been imposed to limit the release to the locations, size and duration requested by the applicant, as these were important parameters in establishing the context for assessing the risks. More information on Australia’s integrated regulatory framework for gene technology is contained in the Risk Analysis Framework available from the Office of the Gene Technology Regulator (OGTR). Free call 1800 181 030 or at <http://www.ogtr.gov.au/pdf/public/raffinal2.2.pdf>. 7 Technical Summary (February 2007) 5 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator PAGE INTENTIONALLY LEFT BLANK Technical Summary (February 2007) 6 DIR 70/2006 – Risk Assessment and Risk Management Plan Chapter 1 Section 1 Office of the Gene Technology Regulator Risk assessment context Background 1. This chapter describes the parameters within which risks that may be posed to the health and safety of people and the environment by the proposed release are assessed. These include the scope and boundaries for the evaluation process required by the gene technology legislation8, details of the intended dealings, the GMO(s) and parent organism(s), previous approvals and releases of the same or similar GMOs in Australia or overseas, environmental considerations and relevant agricultural practices. The parameters for the risk assessment context are summarised in Figure 1. Figure 1 Components of the risk context considered during the preparation of the Risk Assessment RISK ASSESSMENT CONTEXT LEGISLATIVE REQUIREMENTS Gene Technology Act and Regulations DEALINGS Activities involving the GMO Location, size and duration of release Proposed containment measures GMO Introduced genes (genotype) Novel traits (phenotype) PARENT ORGANISM RECEIVING ENVIRONMENT Environmental conditions Agronomic practices Sexually compatible relatives Presence of similar genes PREVIOUS RELEASES 2. Sections 49 to 51 of the Gene Technology Act 2000 (the Act) outline the matters which the Regulator must take into account, and who she must consult with, in preparing the RARMPs that form the basis of her decision on licence applications. 3. For this application, establishing the risk assessment context includes consideration of: the locations, size and duration of the trial requested by the applicant proposed dealings containment measures for the GMOs proposed by the applicant characteristics of the parent organism the nature and effect of the genetic modifications the environmental conditions in the location(s) where the release would occur relevant agricultural practices presence of related plants in the environment presence of the introduced or similar genes in the environment 8 The legislative requirements and the approach taken in assessing licence applications are outlined in more detail at <http://www.ogtr.gov.au/ir/process.htm> and in the Risk Analysis Framework (OGTR 2005) <http://www.gov.au/pdf/raffinal2.2pdf>. Chapter 1 - Risk assessment context (February 2007) 7 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator any previous releases of these or other GMOs relevant to this application. 4. Initial consideration of the application under section 49 of the Act determined that public consultation was not required for the preparation of the consultation version of the RARMP. In accordance with section 50 of the Act, the Gene Technology Technical Advisory Committee (GTTAC), State and Territory governments, prescribed Australian Government agencies, the Minister for Environment and Heritage and the local councils where the release may take place were consulted on matters relevant to the preparation of the RARMP. This advice, and where it was taken into account in the RARMP, is summarised in Appendix B. Two submissions from the public were also received. This advice and its consideration is summarised in Appendix C. 5. In accordance with section 52 of the Act, the Regulator notified the public when the consultation version of the RARMP had been prepared and invited written submissions. No submissions were received from the public on the consultation RARMP. Advice on the RARMP was also sought from the same experts, agencies and authorities as before. None of the latter raised any new issues relating to risks to human health and safety and the environment that required further consideration. However, feedback on some previously considered issues has enabled their clarification in the final RARMP. Section 2 The application 6. BSES proposes to release up to 2500 GM sugarcane lines into the environment under limited and controlled conditions. The GM sugarcane lines have been modified to alter plant architecture (shoot number, stalk size and height), enhance water use efficiency (WUE)9 or improve nitrogen use efficiency (NUE)10. 2.1 The proposed locations, size and duration of the trial 7. The trial is proposed to take place at up to 3 sites per cropping cycle from February 2007 to November 2010 in the local government areas of Bundaberg, Caboolture and/or Cairns, Queensland. Each site would be a maximum of 2 ha, with a total maximum area for the trial of 18 ha. 2.2 The proposed dealings 8. The purpose of the trial is to conduct early stage (‘proof of concept’) research to assess the agronomic characteristics of the GM sugarcane lines and to compare different plant transformation procedures used to generate the GM sugarcane lines. 9. The applicant proposes to assess the performance of the GM sugarcane lines in order to determine whether any may warrant further development. Standard sugarcane industry growing practices will be applied, including harvesting the GM sugarcane before flowering to maximise sugar content (flowering reduces potential sugar yields significantly). The WUE and NUE lines will be subject to different irrigation treatments and fertilizer applications (nitrogen), respectively. Phenotypic measurements of the plants would be made periodically during their growth (including stalk number and structure, sugar and fibre content, and leaf morphology) and plant materials would be analysed after harvest (including stalk weight and sugar yield). Some biochemical and molecular analyses of the plant materials would also be made. No products from the release would be used for human food, animal feed or other sugarcane products. 9 WUE can be defined as the measure of total yield (sugar) produced per unit of water supplied to the sugarcane crop. 10 NUE is a term used to describe how effectively plants acquire and utilise nitrogen. Chapter 1 - Risk assessment context (February 2007) 8 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 2.3 The proposed measures to limit the spread and persistence of the GMOs 10. The applicant proposes measures to limit the spread and persistence of the GM sugarcane lines into the environment. These are taken into account in the risk assessment context (this chapter) and their suitability is evaluated in the following chapter for limiting the release to the locations, size and duration proposed by the applicant. 11. The applicant proposes the following containment measures: surround the trial sites by one guard row of non-GM sugarcane and an isolation zone of at least 6 metres locate the trial sites at least 50 m away from natural waterways harvest and process sugarcane from the trial separately from any other commercial sugarcane analyse plant materials at the trial site or in a PC2 laboratory destroy all plant materials not required for experimentation or propagation following cleaning of sites, monitor for and destroy any GM sugarcane that may grow for 12 months and thereafter until the site is free of volunteers for a continuous 6 month period transport of GM plant materials in accordance with OGTR transportation guidelines. Section 3 The parent organism 12. The parent organism is the sugarcane cultivar Q117, which resulted from a cross between cultivars Q77 and Q58-829 (Saccharum spp. hybrids) from a BSES breeding program. Because of domestic quarantine restrictions, this cultivar is only approved for commercial planting and ratooning north of Rockhampton (information supplied by the applicant). More detailed information on sugarcane can be found in the document, The Biology and Ecology of Sugarcane (Saccharum spp. hybrids) in Australia, which was produced to inform the risk assessment process for licence applications involving GM sugarcane plants (OGTR 2002). This document is available at <http://www.ogtr.gov.au>. Section 4 The GMOs, nature and effect of the genetic modification 4.1 Introduction to the GMOs 13. Up to 2500 GM sugarcane lines are proposed for the trial. 14. Up to 1900 GM sugarcane lines with altered plant architecture, enhanced WUE or improved NUE are proposed for release, each line generated using 1 of 19 different transformation vectors (see Table 1). Each transformation vector was used to make up to 100 GM sugarcane lines. Each line contains up to four introduced partial (in both sense and antisense orientations for silencing of endogenous genes) or complete gene sequences for the above traits. Seventeen of the introduced genes are derived from the plants Oryza sativa (rice), Saccharum spp. (sugarcane), Hordeum vulgare subsp. vulgare (barley), Phaseolus coccineus (bean), Arabidopsis thaliana (thale cress), Malus x domestica (apple) and Zea mays (maize). One gene is derived from the common gut bacterium Escherichia coli. Details of the genes are provided in Section 4.3. 15. All of the 1900 GM sugarcane lines with altered plant architecture, enhanced WUE or improved NUE also contain the selectable antibiotic resistance marker gene nptII, which Chapter 1 - Risk assessment context (February 2007) 9 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator encodes neomycin transferase (NPTII), derived from E. coli. The nptII gene confers resistance to some aminoglycoside antibiotics (eg neomycin, paromomycin, geneticin) and enabled the identification and selection of GM plant tissue during the initial development of the GM sugarcane lines in the laboratory. 16. All of the GM sugarcane lines with altered plant architecture, enhanced WUE or improved NUE also contain the E. coli derived bla gene (except those made using the transformation vector 15, see Table 1), which confers ampicillin resistance to bacteria. This bacterial marker gene is not expressed in the GM sugarcane lines as it is linked to a bacterial promoter that does not function in plants. The bla gene was used to select bacteria carrying the gene construct of interest that were used in the initial plant transformation. 17. An additional 600 GM sugarcane lines, each made using 1 of 3 different gene constructs, contain only the nptII gene (100 lines), or the nptII gene with the bla bacterial marker gene (100 lines), or the nptII gene with the plant reporter gene uidA (400 lines). These lines would be analysed during the trial to compare the effectiveness of different transformation techniques used to generate the GM sugarcane lines. 18. Short regulatory sequences (promoters, and sequences for ending transcription) that control expression of the introduced genes are present in all the gene constructs. These are derived from the plants Flaveria trinervia (clustered yellowtops) and Z. mays, and the bacteria Agrobacterium tumefaciens and E. coli. Although A. tumefaciens is a plant pathogen and E. coli is an opportunistic human pathogen, the regulatory sequences comprise only a small part of their respective total genomes, and are not capable of causing disease. 19. All of the introduced genes, except the bla gene, which is under the control of its own bacterial promoter from E. coli, are under the control of the maize ubiquitin1 (ubi1) gene promoter (Christensen et al. 1992). All of the genes, except the bla gene, which again has its own bacterial terminator from E. coli, have the nopaline synthase (nos) gene terminator from A. tumefaciens. Table 1 Details of the GM sugarcane lines modified for altered plant architecture, enhanced WUE or improved NUE Transformation vector*# 1 Trait(s) Altered plant architecture Partial gene sequence (source) None 2 Altered plant architecture None 3 Altered plant architecture None 4 Altered plant architecture None 5 Altered plant architecture None 6 Altered plant architecture None 7 Altered plant architecture None 8 Altered plant architecture None 9 Altered plant architecture None Chapter 1 - Risk assessment context (February 2007) Complete gene sequence (source) PcGA2ox-1 (P. coccineus) HvGA3ox-2 (H. vulgare subsp. vulgare) HvGA20ox-1 (H. vulgare subsp. vulgare) HvGA20ox-2 (H. vulgare subsp. vulgare) OsMAX3 (O. sativa) OsMAX4-1 (O. sativa) OsMAX4-2 (O. sativa) OsTB1 (O. sativa) SoTB1 (Saccharum spp.) 10 DIR 70/2006 – Risk Assessment and Risk Management Plan Transformation vector*# 10 Trait(s) Altered plant architecture 11 Altered plant architecture 12 Altered plant architecture Office of the Gene Technology Regulator Partial gene sequence (source) SoMAX3 (Saccharum spp.) SoTB1 (Saccharum spp.) None Complete gene sequence (source) None None HvGA20ox-2 (H. vulgare subsp. vulgare) SoTB1 (Saccharum spp.) OsMAX3 OsMAX4-1 OsMAX4-2 (O. sativa) 13 Altered plant architecture SoTB1 (Saccharum spp.) 14 Altered plant architecture OsMAX3 OsMAX4-1 OsMAX4-2 (O. sativa) SoTB1 (Saccharum spp.) 15 Enhanced WUE None 16 Enhanced WUE None 17 Enhanced WUE None 18 Enhanced WUE None 19* Improved NUE None MdS6PDH (M x domestica) MdS6PDH (M x domestica) EcTPSP (E. coli) AtMYB2 (A. thaliana) ZmDOF1 (Z. mays) * All lines containing the introduced genes for altered plant architecture, enhanced WUE or improved NUE contain the E. coli nptII and bla antibiotic resistance genes (except the lines made using vector 15). # Vector 15 does not contain the E. coli bla antibiotic resistance gene 4.2 Introduction to plant architecture, drought stress and nitrogen use efficiency Plant architecture and yield 20. Changes in plant architecture have been important in the domestication and improved yields of many plant species such as the single stalk in maize compared to its multiple stalked ancestor teosinte (Galinat 1998). 21. Sugarcane yields may also benefit from changes to plant architecture. This can be achieved by increasing sugar concentration of the cane, increasing the cane yield or both. Cane yield increases can be achieved by increasing the number of stalks produced and/or the weight of the individual stalks through manipulating thickness and/or height. However, an increase in stalk number generally leads to a reduction in stalk weight and diameter (Milligan et al. 1990) leading to limited yield responses (Bell & Garside 2005). 22. Suckering, the late season outgrowth of lateral buds, is another factor affecting sugar yield. Suckers are morphologically different to main stalks and tillers. These differences include a greater stem base diameter and shorter, wider leaves than the primary stalk of a similar age (Bonnett et al. 2001). Suckers can typically reduce sugar yield as their sugar content is much lower than the primary mature cane (Bonnett et al. 2004) and can thus lead to a dilution of yields (information supplied by applicant). Chapter 1 - Risk assessment context (February 2007) 11 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 23. The falling of crops due to stem or root weakness, known as lodging, can decrease yields and this has been investigated through selection of lodging-resistant varieties. However, little effort has been undertaken towards the breeding of lodging-resistant varieties (Singh et al. 2000). 24. Lodging resistance could be achieved by either: increasing stalk number to reduce individual stalk weight and allow increased stalk flexibility in windy conditions; or reducing the stalk number resulting in thicker stems, possibly leading to greater resistance to lodging from high winds placing strains on the base of the plant. 25. A better understanding of the genes controlling plant architecture is critical for developing a purposeful molecular or conventional breeding strategy. Although in sugarcane this is poorly understood, much progress has recently been made in other species including maize, rice and in model dicots, tobacco, garden pea and Arabidopsis. In these species several transcription factors have been identified which regulate plant architecture, including the maize TB1 (Doebley et al. 1997; Takeda et al. 2003), pea RMS (Schmitz & Theres 2005) and Arabidopsis MAX genes (McSteen & Leyser 2005). 26. A transcription factor is any protein required for recognition, by RNA polymerases, of specific regulatory sequences in genes (eg a promoter) (Lewin 1994a). A particular transcription factor may affect the initiation of transcription of many genes, such as those involved in abiotic stress reactions (Shinozaki et al. 2003; Chinnusamy et al. 2004; Chinnusamy et al. 2006). The activity of a regulatory transcription factor may be controlled by its rate of synthesis, covalent modification (eg phosphorylation), binding to a ligand (eg hormone), or inhibitors that can sequestrate or affect the transcription factor’s ability to bind to DNA (Lewin 1994b). Approximately 7.4% (~2000) of the expressed genes in the experimental model plant Arabidopsis encode transcription factors (Iida et al. 2005). 27. Genes involved in the biosynthesis of the plant hormone gibberellin are also known to play a role in plant architecture (Biemelt et al. 2004). Gibberellic acids (GAs) are a large family of diterpenoid compounds of which some are bioactive growth regulators. GAs control seed germination, stem elongation and leaf, trichome, flower and fruit development (reviewed in Davies 2004). Drought stress tolerance (enhanced WUE) and plant responses 28. Drought stress is an abiotic stress; a non-living factor that causes harmful effects to plants. Other types of primary abiotic stresses include salinity, cold, heat and chemical pollution. Plants respond to different abiotic stresses, often through an interconnecting series of signalling and transcription controls that ultimately aim to increase the plant’s ability to tolerate the initial stress through different biochemical and physiological mechanisms. 29. Enhanced WUE is a trait of increasing importance for sugarcane growers. Water allocations in many sugarcane production regions are insufficient to make up the difference between crop water requirement and effective rainfall (Holden 1998). Also, irrigation requirements differ from year to year because of the high variability in rainfall across the areas of sugarcane cultivation. Thus, competition for water, increasing costs, and environmental concerns require that major users of water achieve best-practice in terms of efficiency of water use (Inman-Bamber et al. 2000). 30. At the molecular level there are three broad categories of genes that play a role in a plant’s response to drought stress (Wang et al. 2003; Vinocur & Altman 2005). These include: Chapter 1 - Risk assessment context (February 2007) 12 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator signal sensing, perception and transduction transcriptional control stress tolerance response (via proteins involved in detoxification, osmoprotection, chaperone functions and the control of water and ion movement). 31. Specific receptors are thought to enable plants to directly sense stress (Xiong & Zhu 2001; Zhu 2002). Once sensed, a drought stress signal may be relayed to eventually regulate the transcription of numerous genes involved in the stress response. The transduction of the stress signal may be accomplished through various signalling molecules such as Ca2+, phospholipids, reactive oxygen species, sugars and plant hormones, or via protein messengers such as mitogen activated protein kinases (MAPKs) and serine/threonine phosphatases (Xiong & Zhu 2001; Ramanjulu & Bartels 2002; Zhu 2002; Chaves & Oliveira 2004; Chinnusamy et al. 2004). 32. Drought stress signals activate genes that control the transcription of genes involved in the stress tolerance response mechanisms of cell protection. The majority of these can be categorised as transcription factors (Wang et al. 2003; Shinozaki et al. 2003), but other genes involved in transcription control include those encoding proteins involved in chromatin remodelling (Singh 1998). Certain regulatory genes can be induced by more than one type of abiotic stress (eg drought, cold and salinity) (Seki et al. 2002). Nitrogen use efficiency (NUE) in plants 33. NUE is an important factor in crop plant productivity and as such nitrogen based fertilizers are used extensively in modern agriculture, including sugarcane. Due to the rapid growth and size of sugarcane, current agricultural practices are to apply the fertiliser in a large single quantity early in the crops life cycle. This has implications in nitrogen run-off due to heavy early seasonal rains (information supplied by applicant). The use of fertilizers has an environmental impact through run-off into nearby streams (Addiscott et al. 1991). 34. There are several ways to improve nitrogen use in plants without adding extra nitrogen based fertilizers. These can include: crop rotation practices with nitrogen fixing crops; traditional plant breeding and marker assisted technologies to develop plants with increased NUE; selection of soil microbes that can better fix nitrogen; and genetic modification of the plants for nitrogen use efficiency or to create/enhance their relationship with symbiotic nitrogen fixing bacteria. 35. Several studies have attempted to improve NUE in plants using genetic modification with limited success (Good et al. 2004). These studies have focussed on introducing genes encoding nitrogen transporters, nitrate and nitrite reductases, glutamine synthetase and synthases, aminotransferases and dehydrogenases, and transcription factors. 4.3 The introduced genes and regulatory sequences, and the gene products 36. Genetic material from various sources was introduced into sugarcane. The genetic elements, their sources and function in the source organisms are listed below (see Table 2). 37. The introduced genes that are intended to alter plant architecture, enhance WUE or improve NUE, consist of partial gene sequences that encode dsRNAs (double-stranded RNAs) or complete gene sequences that encode proteins. The introduced genes are intended to modulate biochemical pathways in the GM sugarcane lines, either through encoding enzymes or regulating endogenous gene expression. Chapter 1 - Risk assessment context (February 2007) 13 DIR 70/2006 – Risk Assessment and Risk Management Plan Table 2 Office of the Gene Technology Regulator Source organisms of the introduced genes and regulatory sequences* Genetic Element Source Organism Function in source organism PcGA2ox-1 (complete gene sequence) HvGA3ox-2 (complete gene sequence) Phaseolus coccineus Hordeum vulgare subsp. vulgare H. vulgare subsp. vulgare H. vulgare subsp. vulgare Oryza sativa O. sativa O. sativa O. sativa O. sativa O. sativa O. sativa Saccharum spp. Saccharum spp. Saccharum spp. Malus x domestica Escherichia coli Arabidopsis thaliana Zea mays E. coli E. coli E. coli Z. mays Flaveria trinervia Agrobacterium tumefaciens E. coli E. coli Gibberellin biosynthesis Gibberellin biosynthesis HvGA20ox-1 (complete gene sequence) HvGA20ox-2 (complete gene sequence) OsMAX3 (complete gene sequence) OsMAX3 (partial gene sequence) OsMAX4-1 (complete gene sequence) OsMAX4-1 (partial gene sequence) OsMAX4-2 (complete gene sequence) OsMAX4-2 (partial gene sequence) OsTB1 (complete gene sequence) SoTB1 (complete gene sequence) SoTB1 (partial gene sequence) SoMAX3 (partial gene sequence) MdS6PDH (complete gene sequence) EcTPSP (complete gene sequence) AtMYB2 (complete gene sequence) ZmDOF1 (complete gene sequence) nptII bla uidA ubi1 promoter* pdk intron^ nos terminator* bla promoter bla terminator Gibberellin biosynthesis Gibberellin biosynthesis Transcription factor N/A# Transcription factor N/A# Transcription factor N/A# Transcription factor Transcription factor N/A# N/A# Sorbitol biosynthesis Trehalose biosynthesis Transcription factor Transcription factor Antibiotic resistance Antibiotic resistance Visual marker Transcription initiation of ubi1 gene Intron Transcription termination of nos gene Transcription initiation of bla gene Transcription termination of bla gene *All of the introduced genes, except the bla gene, which is under the control of its own bacterial promoter and terminator from E. coli, are under the control of the maize ubiquitin1 (ubi1) gene promoter from Z. mays and the nopaline synthase (nos) gene terminator from A. tumefaciens (see Section 4.1). ^All of the introduced genes that consist of partial gene sequences encoding dsRNAs also contain the intron sequence of the pyruvate orthophosphate dikinase (pdk) gene from Flaveria trinervia. The intron helps in the formation of a dsRNA hairpin structure necessary for the efficient silencing of the targeted endogenous gene. # Not Applicable. 4.3.1 The introduced genes for altered plant architecture 38. In total, 14 genes or partial gene sequences for altered plant architecture (transformation vectors 1-14, Table 1) have been introduced (up to 4 genes or partial gene sequences per line) into the GM sugarcane lines. 39. Six of the transformation vectors (see Table 1) used to make the GM sugarcane lines contain the complete gene sequences of OsTB1 (O. sativa Teosinte Branching 1) and/or complete or partial gene sequences of SoTB1 (Saccharum spp. Teosinte Branching 1). Both of these gene homologs encode transcription factors that may affect lateral branching and sugar yield in the GM sugarcane lines. The OsTB1 gene reduces lateral branching in its parent species (Takeda et al. 2003; McSteen & Leyser 2005). Both the OsTB1 and SoTB1 genes are homologous to the maize TB1 gene responsible for control of branching in the maize plant (Doebley et al. 1997; Hubbard et al. 2002). 40. Other regulatory genes introduced into the GM sugarcane lines that may alter the plant architecture include genes that affect auxin transport in the plant. The genes, which include the homologs OsMAX3 (O. sativa More Axillary Growth 3), SoMAX3 (Saccharum spp. More Axillary Growth 3), OsMAX4-1 and OsMAX4-2, are present in six of the transformation vectors (Table 1) used to make the GM sugarcane lines. These introduced genes consist of Chapter 1 - Risk assessment context (February 2007) 14 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator complete or partial gene sequences. Auxin plays an important role in the regulation of bud outgrowth and the More Axillary Growth (MAX) 1, 3 and 4 genes of Arabidopsis, and related genes from other plants, have been shown to be responsible for the regulation of shoot branching (Booker et al. 2004; Schwartz et al. 2004; Bainbridge et al. 2005; Bennett et al. 2006) and bud inhibition (Sorefan et al. 2003; Booker et al. 2004; Schwartz et al. 2004; Bainbridge et al. 2005; Snowden et al. 2005; Bennett et al. 2006 and reviewed in McSteen and Leyser, 2005). 41. Some of the GM sugarcane lines contain all three introduced O. sativa MAX genes as complete sequences along with the introduced partial gene sequence of the SoTB1 gene (transformation vector 13, Table 1). GM sugarcane lines containing all three introduced O. sativa MAX as partial sequences along with the introduced complete gene sequence of the SoTB1 gene (transformation vector 14, Table 1) are also proposed for release. 42. Five of the transformation vectors (see Table 1) used to make the GM sugarcane lines contain the complete gene sequences of PcGA2ox-1 (P. coccineus gibberellin 2-oxidase-1), HvGA3ox-2 (H. vulgare subsp. vulgare gibberellin 3-oxidase-2), HvGA20ox-1 and HvGA20ox-2. These gene homologs encode enzymes involved in active gibberellin (GA) (HvGA3ox-2, HvGA20ox-1 and HvGA20ox-2) and inactive GA biosynthesis (PcGA2ox-1) (Hedden & Kamiya 1997) and may affect the height and yield of the GM sugarcane lines. The biologically active GAs includes the compounds GA1 and GA4 and the inactive forms include GA51, GA29, GA34 and GA8 (Hedden & Phillips 2000). 43. One of the transformation vectors used to make the GM sugarcane lines contains both the introduced HvGA20ox-2 and SoTB1 complete gene sequences (transformation vector 12, Table 1). These GM sugarcane lines may have decreased lateral branching, altered plant height and sugar yield. 4.3.2 The introduced genes for enhanced WUE 44. The 3 introduced genes (MdS6PDH, EcTPSP and AtMYB2) intended to enhance WUE (transformation vectors 15-18, Table 1) encode a D-sorbitol-6-phosphate dehydrogenase (S6PDH), a fusion enzyme consisting of a trehalose-6-phosphate synthase and trehalose-6phosphate phosphatase (TPSP), and a myeloblastosis interacting protein 2 (MYB2). The genes are derived from Malus x domestica, E. coli and A. thaliana, respectively. 45. The MdS6PDH and EcTPSP genes encode enzymes involved in sorbitol and trehalose production, respectively. Increased trehalose production in GM rice (Jang et al. 2003) has been shown to increase tolerance to different abiotic stresses, in particular drought stress tolerance (Garg et al. 2002), also known as WUE. Similarly, sorbitol is expected to have an osmoprotective function in the plant as has been seen in marsh plant (Ahmad et al. 1979). Introduction of MdS6PDH into tobacco led to increased boron uptake and increased growth and yield when grown under conditions of limited boron, often seen following drought conditions (Brown et al. 1999; Bellaloui et al. 1999). 46. The EcMYB2 gene encodes the MYB2 transcription factor, which is known to be expressed under salt stress and dehydration (Urao et al. 1993; Abe et al. 2003). 4.3.3 The introduced gene for improved nitrogen use efficiency 47. The introduced gene ZmDOF1 (Z. mays DNA binding with one finger 1) for improved NUE (transformation vector 19, Table 1) encodes the transcription factor DOF1. DOF1 can enhance the expression of many genes simultaneously and has been shown to enhance plant growth when expressed in Arabidopsis plants under low-nitrogen conditions (Yanagisawa et al. 2004). Chapter 1 - Risk assessment context (February 2007) 15 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 48. The DOF protein family of transcription factors share a unique highly conserved DNAbinding domain, a novel zinc finger motif (Yanagisawa 1995; Umemura et al. 2004). DOF proteins have been shown to have diverse functions and associated with a diverse range of promoters (reviewed in (Yanagisawa & Schmidt 1999; Yanagisawa 2002; Yanagisawa 2004). While the DOF binding domain has been shown to function in plant, animal and yeast cells (Yanagisawa 2001), the proteins are plant specific. 49. A study in Arabidopsis (Yanagisawa et al. 2004) showed that expression of the introduced ZmDOF1 gene can improve nitrogen assimilation and enhance growth on a fresh weight basis under low nitrogen input. Previous studies have associated the ZmDOF1 gene with the enhancement of expression of several genes involved in carbon metabolism (Yanagisawa & Sheen 1998; Yanagisawa 2000) in higher plants and micro-organisms, specifically: the C4-type phosphoenol-pyruvate carboxylase (C4PEPC), which catalyses the initial fixation of CO2 in the C4 photosynthetic pathway (Yanagisawa & Sheen 1998) cytosolic orthophosphate dikinase (cyPPDK), which catalyses the production of phosphoenol-pyruvate (PEP) (Edwards et al. 1985), the substrate in the reaction catalysed by PEPC (Chollet et al. 1996) a non-photosynthetic gene encoding PEPC. 4.3.4 Regulatory sequences for expression of the introduced genes 50. Expression of the introduced genes for altered plant architecture, enhanced WUE and improved NUE in GM sugarcane lines is controlled by the ubi1 promoter from maize (Christensen et al. 1992). The ubi1 promoter is thought to lead to constitutive expression of the inserted genes in plants (Christensen et al. 1992). 51. Also required for gene expression in plants is an mRNA termination region, including a polyadenylation signal. The mRNA termination region for the introduced genes for altered plant architecture, enhanced WUE and improved NUE in GM sugarcane lines is the 3’ untranslated region derived from the nopaline synthetase (nos) gene from Agrobacterium tumefaciens (Depicker et al. 1982). Although A. tumefaciens is a plant pathogen, the regulatory sequence comprises only a small part of its total genome and is not capable of causing disease. 52. Five of the introduced gene constructs for altered plant architecture consist of partial gene sequences encoding dsRNAs. These also contain the intron sequence of the pyruvate orthophosphate dikinase (pdk) gene from the plant Flaveria trinervia. The intron helps in the formation of a dsRNA hairpin structure necessary for the efficient silencing of the targeted endogenous gene. 53. The bla gene is under the control of its own bacterial promoter and terminator, both from E. coli. Since the bacterial promoter and terminator sequence do not operate in plants, the bla gene is not expressed in the GM plants. 4.3.5 Toxicity and allergenicity of the proteins (and enzymatic products) encoded by the introduced genes for altered plant architecture 54. All of the introduced genes intended to alter plant architecture were derived from several common crop plants including barley, rice, sugarcane and bean. These source organisms are not known to be toxic to humans or other organisms. 55. Given that expression of the endogenous genes SoTB1 and SoMAX3 is suppressed in sugarcane lines 13 and 14, the amount of TB1 transcription factor or MAX3 enzyme present Chapter 1 - Risk assessment context (February 2007) 16 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator in GM plant tissue is likely to be less than in parent sugarcane. On this basis, if the proteins had any toxicity potential then it should be reduced in these sugarcane lines. 56. Known food allergens generally share a number of characteristics including amino acid sequence homology, a molecular weight (Mr) of 15-70 kDa, are highly expressed, glycosylated, stable [resistant to digestion in the gastrointestinal tract (GIT)] and derived from a food or biological source known to be allergenic (Metcalfe et al. 1996; FAO 2001a). 57. FAO/WHO (FAO 2001a; Codex Alimentarius Commission 2003) guidelines propose that cross-reactivity between an unknown protein and a known allergen has to be considered when there is more than 35% identity in the amino acid sequence over a sliding window of 80 amino acids or a match of six contiguous amino acids. Matches of six amino acids with any allergen have been shown to occur frequently by chance which limits the utility of this criteria for predicting allergenicity (Hileman et al. 2002; Stadler & Stadler 2003; Thomas et al. 2005; Silvanovich et al. 2006). The International Food Biotechnology Council/International Life Sciences Institute have suggested that an optimal length of between eight and twelve amino acids is required for binding to T-cells and that an immunological significant sequence identity requires a match of at least eight contiguous amino acids (Metcalfe et al. 1996). 58. Many protein allergens are N-glycosylated (Herouet et al. 2005) leading to the concern that glycosylation may contribute to the allergenicity (Buchanan 2001). 59. All the introduced genes for altered plant architecture are in the molecular weight range identified as being typical of allergens (15-70 kDa). A motif search (http://www.expasy.org/prosite/) with the amino acid sequences derived from the introduced gene sequences identified that some of the proteins contained putative N-glycosylation sites (Asp-X-Ser/Thr). 60. Amino acid sequence comparisons using SDAP11, Agmobiol12, Food Allergy Research and Resource Program (FARRP) Protein Allergen13, Allergen14, and Allermatch15 databases revealed that none of the sequences of the genes for altered plant architecture contained a region of 80 amino acids with greater than 35% sequence identity to any known allergens. The GA20 OXIDASE-1 sequence from barley showed a match of eight identical continuous amino acids to a known pollen allergen, Amb a 1, from Ambrosia artemisiifolia (short ragweed)(Rafnar et al. 1991). However, this may be a chance occurrence since the related barley GA20 OXIDASE-2 protein did not contain the same match and there were no other regions of similarity between GA20 OXIDASE-1 and Amb a 1. 61. Furthermore, an extensive search of the scientific literature yielded no data or information to suggest that any of the proteins encoded by the introduced genes for altered plant architecture have any toxicity or allergenicity potential to people or other organisms. 4.3.6 Toxicity and allergenicity of the proteins encoded by the introduced genes (and enzymatic products) for enhanced WUE. 62. The MdS6PDH gene encoding for the S6PDH enzyme was isolated from Malus x domestica (apple). Apple is not considered to be toxic or allergenic. The S6PDH enzyme produces sorbitol in apple fruit (information supplied by applicant) and it is not expected that 11 <http://fermi.utmb.edu/SDAP/sdap_who.html <http://ambl.lsc.pku.edu.cn/english/index.phpallergen> 13 <http://www.allergenonline.com> 14 <http://allergen.csl.gov.uk/> 15 <http://allermatch.org/> 12 Chapter 1 - Risk assessment context (February 2007) 17 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator sorbitol will have any allergenic or toxic effects. Sorbitol is approved by Food Standard Australia New Zealand (FSANZ) as a food additive16. 63. The EcTPSP gene encoding for the TPSP protein was derived from E. coli, a bacterium present in the gastrointestinal tract of humans and other animal species. The EcTPSP gene is a fusion gene of two E. coli genes (trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase) for the synthesis of trehalose (Seo et al. 2000), a sugar found naturally in honey, mushrooms, lobster, shrimp and foods produced using baker's and brewer's yeast. Trehalose has been gazetted into the FSANZ code 31 July 2003, A453 Trehalose as a Novel Food. Trehalose has also been evaluated by the US Food and Drug Authority (US FDA) as ‘generally recognised as safe’ (GRAS Notice No. GRN 00004517). 64. The gene coding for the transcription factor, AtMYB2, is derived from A. thaliana, a member of the Brassicaceae family which includes many edible members such as broccoli, cabbage and radish. Many other edible plant species, and possibly all plants, contain MYB genes, such as barley, maize, wheat, rye, rice, soybean, and tomato. Arabidopsis is native to Europe and central Asia but is naturalised worldwide. It is widely used throughout the world for scientific research and there have been no reports of allergenicity or toxicity. It is not commonly eaten, but there are no reports of it causing disease or ill-health in humans. There is no other evidence that this gene exists for any biological role apart from its role in the stress response cycle. 65. MYB2, S6PDH and TPSP are in the molecular weight range identified as being typical of allergens (15-70 kDa). A motif search (http://www.expasy.org/prosite/) with the amino acid sequences identified that MYB2 and S6PDH contain putative N-glycosylation sites (AspX-Ser/Thr). 66. Amino acid sequence comparisons using SDAP9, Agmobiol10, FARRP Protein Allergen11, Allergen12, and Allermatch13 databases revealed no indication that the proteins encoded by the MdS6PDH, EcTPSP and AtMYB2 genes share significant sequence homology with any known allergens. On this basis, the encoded proteins are unlikely to have any allergenic potential in people 67. An extensive search of the scientific literature yielded no data or information to suggest that the proteins encoded by the introduced genes for enhanced WUE have any toxicity or allergenicity potential in people or other organisms. 4.3.7 Toxicity and allergenicity of the introduced gene (ZmDOF1) for improved nitrogen use efficiency 68. The introduced ZmDOF1 gene is derived from the crop plant maize (Z. mays). Maize is a common food and fodder plant and it is not expected that the DOF1 protein encoded by the ZmDOF1 gene would be toxic or allergenic. 69. The DOF1 protein from maize is in the molecular weight range identified as being typical of allergens (15-70 kDa). A motif search (http://www.expasy.org/prosite/) identified one putative N-glycosylation site (Asp-X-Ser/Thr). 70. A comparison of the deduced amino acid sequence of the gene for nitrogen use efficiency to amino acid sequences in the publicly available SDAP9, Agmobiol10, FARRP Protein Allergen11, Allergen12, and Allermatch13 databases revealed no matches of at least 35% identity in a segment of 80 amino acids with any known allergens. One match of eight contiguous identical amino acids to a known allergen was identified with the Gal d 16 17 <http://www.foodstandards.gov.au/thecode/foodstandardscode.cfm#_FSCchapter1> Part 1.3, Standard 1.3.1 <http://www.cfsan.fda.gov/~rdb/opa-gras.html> Chapter 1 - Risk assessment context (February 2007) 18 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator vitellogenin epitope from Gallus domesticus (chicken)(Nardelli et al. 1987). However, this may be a chance occurrence since there were no other regions of similarity between DOF1 and Gal d vitellogenin. 71. Furthermore, an extensive search of the scientific literature yielded no data or information to suggest that DOF1 has any toxicity or allergenicity potential to people or other organisms. 4.3.8 The antibiotic resistance marker gene (nptII) and its encoded protein 72. All of the GM sugarcane lines contain the nptII antibiotic resistance marker gene. The nptII gene was isolated from the bacterial Tn5 transposon (from E. coli) (Beck et al. 1982). It encodes an enzyme, neomycin phosphotransferase type II (NPTII), which confers resistance to some aminoglycoside antibiotics, eg neomycin, paromomycin and geneticin. NPTII uses ATP to phosphorylate those antibiotics, thereby inactivating and preventing them from killing the NPTII-producing cells. The nptII gene functioned as a selectable marker during the laboratory stages of sugarcane plant tissue selection following genetic modification, allowing GM cells to grow in the presence of the antibiotic while inhibiting the growth of non-GM cells. The nptII gene is in common use as a selectable marker in the production of GM plants (Miki & McHugh 2004). 73. Other regulatory agencies, in Australia and in other countries, have previously assessed the nptII gene as safe for use in human food (US FDA 1998; ANZFA 2001a; ANZFA 2001b; ANZFA 2001c; ANZFA 2001d; FSANZ 2003). In addition, a number of genetically modified food crops containing the nptII gene have been approved for commercial release both in Australia (DIRs 012/2002, 021/2002, 022/2002 and 059/2005) and overseas. No adverse effects on humans, animals or the environment have been reported from these releases (US FDA 1998; Flavell et al. 1992; EFB 2001). Regulatory sequences for the expression of the nptII gene 74. The bacterial nptII gene was modified by the addition of regulatory sequences including the ubi1 gene promoter from maize (Christensen et al. 1992) and the nopaline synthase gene (nos) transcription termination region from A. tumefaciens to allow efficient expression in plant cells. 75. A. tumefaciens is a common gram-negative soil bacterium that causes crown gall disease in a wide variety of plants (Van Larebeke et al. 1974). Although A. tumefaciens is a plant pathogen, the regulatory sequence comprises only a small part of its total genome, and is not capable of causing disease. Toxicity of NPTII 76. Protein and DNA sequence comparisons using sequences from four separate databases (Genbank, EMBL, PIR29, Swiss-Prot) indicated that NPTII does not have significant homology to any proteins listed as food toxins in these databases (FDA 1994). 77. Humans (and, by implication, other animals) continually ingest kanamycin-resistant microorganisms, some containing the NPTII enzyme. The diet, especially raw salad, is the major source: estimated conservatively, each human ingests 1.2 x 106 kanamycin-resistant microorganisms daily (Flavell et al. 1992). Large numbers of kanamycin- or neomycinresistant bacteria already inhabit the human digestive system (Levy et al. 1998) with Flavell et al. (1992) estimating about 1012 per person. Kanamycin-resistant bacteria have been isolated from soil, river water and sewage (Smalla et al. 1993). Chapter 1 - Risk assessment context (February 2007) 19 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 78. The insertion of the nptII gene into a wide range of GMOs has not resulted in any adverse effects (Flavell et al. 1992). The nptII gene was introduced into mammalian cell lines with no effects on viability or growth. During gene therapy experiments, mammalian cells expressing the NPTII protein have been infused into cancer patients. Again, no adverse effects have been observed (Flavell et al. 1992). 79. The NPTII protein produced in GM tomatoes has been fed to rodents and reported to be rapidly inactivated and degraded (Calgene Inc. 1990). An acute oral toxicity study in mice, in which the purified NPTII protein was fed at doses of up to 5000 mg/kg of body weight (2500 mg/kg administered twice, four hours apart), did not show any adverse effects (Berberich et al. 1993). A similar study in mice also reported no adverse effects of NPTII at 5000 mg/kg of body weight (Fuchs et al. 1993b). Allergenicity of NPTII 80. The NPTII protein is approximately 29 kDa in size, which is within the typical size range of allergenic proteins. However, it does not possess glycosylation sites, is not stable in the mammalian digestive system and is heat labile, decreasing the probability that it is allergenic (US FDA 1998; Fuchs et al. 1993a; FDA 1994; ANZFA 2001e; ANZFA 2001f). Fuchs et al. reported that no NPTII was detected 10 seconds after addition of simulated gastric fluid as measured by both western blot and enzymatic activity (Fuchs et al. 1993b). Protein sequence comparisons using sequences from four separate protein databases (EMBL, GenBank, PIR29 and Swiss-Prot) indicated that NPTII does not have significant sequence identity to any known protein food allergens (Fuchs & Astwood 1996). 81. The FDA has evaluated data submitted for deliberate releases of GMOs expressing the NPTII protein and concluded that NPTII does not have any of the characteristics associated with allergenic proteins (US FDA 1998). The UK Royal Society have concluded that there is at present no evidence that available GM foods cause allergic reactions, and that the risks posed by GM plants are in principle no greater than those posed by conventional breeding or by plants introduced from other areas of the world (The Royal Society 2002). 4.3.9 β-lactamase gene or ampicillin antibiotic resistance gene (bla or amp) 82. Most of the GM sugarcane lines (up to 2400) contain the bacterial β-lactamase gene (bla; also known as amp) derived from E. coli (Spanu et al. 2002). The bla gene encodes the β-lactamase enzyme, which confers ampicillin resistance. 83. The β-lactamase enzyme is widespread in the environment and in food. Naturally occurring ampicillin-resistant microorganisms have been found in mammalian digestive systems (Spanu et al. 2002). The bla gene was originally isolated from antibiotic resistant strains of E. coli found in hospital patients. 84. The bla gene in the GM sugarcane lines is controlled by bacterial regulatory sequences and therefore will not be expressed. The gene was used in the laboratory prior to the production of the genetically modified plants. 4.3.10 Reporter gene (uidA) and its encoded protein (GUS) 85. Up to 400 of the GM sugarcane lines contain the uidA reporter gene. The uidA gene encodes the enzyme β-glucuronidase (GUS), which is derived from the common gut bacterium E. coli. It is the most widely used reporter gene in GM plants (Miki & McHugh 2004) as it allows GM tissues to be identified using a simple assay. The uidA gene was used as a ‘reporter’ in the laboratory for assessing GM sugarcane plant materials for levels of gene Chapter 1 - Risk assessment context (February 2007) 20 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator expression and can also be used to assess gene expression levels in plants grown under field conditions. 86. The GUS protein is a monomer with a molecular weight of 68 kDa, and the GUS enzyme is active as a tetramer. GUS catalyses the hydrolysis of β-glucuronides and, less efficiently, some β-galacturonides. E. coli lives in the digestive tract of vertebrates, including humans (Jefferson et al. 1986), and the GUS enzyme enables it to metabolise β-glucuronides as a main source of carbon and energy. Regulatory sequences for the expression of the uid gene 87. Expression of the uidA gene in the GM sugarcane lines is controlled by the ubi1 gene promoter from maize (Christensen et al. 1992) and the termination sequence of this gene is the 3′ non-translated region of the nos gene from A. tumefaciens (Rogers et al. 1985). Although A. tumefaciens is a plant pathogen, the regulatory sequence comprises only a small part of its total genome and is not capable of causing disease. Toxicity of GUS 88. Acute oral toxicity studies in mice with purified GUS protein at doses of up to 100 mg/kg did not show any adverse effects (Naylor 1992). Feeding in humans and animals with 1010 GUS-containing E. coli bacteria per ingestion also did not show any toxic or pathogenic reactions (Gilissen et al. 1998). Metabolites of the GUS enzyme are non-toxic (Gilissen et al. 1998). Allergenicity of GUS 89. The GUS protein is approximately 68 kDa in size, which is within the typical size range of allergenic proteins. However, the widespread occurrence of GUS and the constant exposure of humans to the protein without known ill effects indicates that the likelihood that GUS being an allergen is extremely low (Gilissen et al. 1998). In addition, the GUS protein does not possess glycosylation sites and is rapidly denatured in simulated mammalian digestive system (Fuchs & Astwood 1996). The GUS protein from E. coli is rapidly (<15 seconds) degraded in simulated gastric fluid and loses its activity by heating/cooking (Fuchs & Astwood 1996). Protein sequence analysis indicates that GUS does not have significant sequence homology to any known protein food allergens. 4.4 Method of genetic modification 4.4.1 Agrobacterium mediated transformation 90. Up to 400 GM sugarcane lines were generated by Agrobacterium-mediated transformation using standard sugarcane transformation protocols (Arencibia et al. 1998). These 400 GM sugarcane lines only contain the introduced genes nptII and uidA. They do not contain the introduced genes for altered plant architecture, enhanced WUE or improved NUE. Four different strains of A. tumefaciens (Ag10, Ag11, EHA105 and LBA4404) were used in the plant transformation process for the generation of the 400 lines (up to 100 lines per strain). 91. A. tumefaciens is a common gram-negative soil bacterium that causes crown gall disease in a wide variety of plants (Van Larebeke et al. 1974). Plants can be genetically modified by the transfer of DNA (transfer-DNA or T-DNA, located between specific border sequences on a resident plasmid) from A. tumefaciens through the mediation of genes from the virulence region of Ti plasmids. 92. Disarmed Agrobacterium strains have been constructed specifically for plant transformation. The disarmed strains do not contain the genes responsible for the Chapter 1 - Risk assessment context (February 2007) 21 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator overproduction of auxin and cytokinin (iaaM, iaaH and ipt), which are required for tumour induction and rapid callus growth (Klee & Rogers 1989). Agrobacterium plasmid vectors used to transfer T-DNAs contain well characterised DNA segments required for their replication and selection in bacteria, and for transfer of T-DNA from Agrobacterium and its integration into the plant cell genome (Bevan 1984; Wang et al. 1984). 93. Agrobacterium-mediated transformation has been widely used in Australia and overseas for introducing genes, and regulatory sequences for their expression, into plants. Unintended changes in phenotype can occur as a result of transformation, or because of the tissue culture process used after transformation, that are similar to those encountered in conventional breeding and mutation breeding (Cellini et al. 2004; Bradford et al. 2005). Such ‘off types’ are visually identified and eliminated from the breeding program. 94. A disarmed binary plasmid vector was used to introduce the gene construct containing the nptII and uidA genes into sugarcane cultivar (Saccharum hybrid), Q117, using standard Agrobacterium transformation protocols. Following transformation, regeneration of plants was performed in the presence of paromomycin. 95. The 400 GM sugarcane lines were generated from independent transformation events, and therefore the introduced genes are expected to be located at different sites in the sugarcane genome in each line. 4.4.2 Biolistic mediated transformation 96. The remaining 2100 GM sugarcane lines were generated using standard sugarcane biolistic transformation protocols (Bower & Birch 1992; Bower et al. 1996; Birch et al. 2000). This involved coating very small metal (tungsten) particles with the transformation vector containing the introduced genes. The particles were then ‘shot’ into sugarcane embryonic callus. Successfully transformed tissue was selected by its resistance to the antibiotic geneticin, conferred by the introduced nptII gene and regenerated into GM sugarcane plants. 97. All of the GM sugarcane lines were generated from independent transformation events, and therefore the introduced genes are expected to be located at different sites in the sugarcane genome in each line. 4.5 Characterisation of the GMOs 4.5.1 Stability and molecular characterisation 98. The applicant stated that all 2500 sugarcane lines are at an early development stage and have not been tested for genotypic stability. Such data would be required for GM sugarcane lines selected for further development. 99. PCR analyses of the GM sugarcane lines generated by Agrobacterium-mediated and biolistic transformation have confirmed the presence of the introduced genes for altered plant architecture, enhanced WUE or improved NUE. Real Time PCR indicates that the genes are being expressed (information supplied by applicant). Further detailed molecular characterisation may be performed during the trial on selected GM sugarcane lines grown under field conditions. 100. No assessment has been made of the number of copies of the introduced genes inserted into the sugarcane genome. The number of gene copies integrated into a plant genome varies depending on the method of introduction. Copy number of an introduced gene following microprojectile bombardment usually varies from 1 to more than 20 (Pawlowski & Somers 1996), whereas 1-3 copies of introduced genes are commonly seen in transgenic lines obtained through Agrobacterium-mediated transformation (Arencibia et al. 1998). Chapter 1 - Risk assessment context (February 2007) 22 DIR 70/2006 – Risk Assessment and Risk Management Plan 4.5.2 Office of the Gene Technology Regulator Characterisation of the phenotype of the GMOs 101. One of the aims of the proposed trial is to assess whether the GM sugarcane lines demonstrate improved agronomic performance (eg sugar yield), as compared to non-GM sugarcane, under field conditions. 102. The applicant reported that under glasshouse conditions, necrosis is observed on leaves of the GM sugarcane expressing the introduced MdS6PDH gene. The necrosis is more noticeable at the leaf apex where the sorbitol content is the highest. The condition is more severe in the plants producing larger amounts of sorbitol. These GM sugarcane lines are up to 30% shorter compared to non-GM parent. All other aspects of growth seemed to be normal. GM sugarcane lines containing the introduced genes HvGA20ox-1 or 2 appear to produce stalks more rapidly (1-2 months earlier), and GM sugarcane lines containing the introduced gene PcGA2ox-1 grow more slowly and are shorter, than their non-GM sugarcane parent. The GM sugarcane lines containing the other introduced genes appear morphologically similar to the non-GM sugarcane parent (information supplied by applicant). Section 5 The receiving environment 103. The receiving environment forms part of the context in which the risks associated with dealings involving the GMOs are assessed. This includes the size, duration and regions of the dealings, any relevant biotic/abiotic properties of the regions where the release would occur; intended agronomic practices, including those that may be altered in relation to normal practices; other relevant GMOs already released; and any particularly vulnerable or susceptible entities that may be specifically affected by the proposed release (OGTR 2005). 5.1 Relevant abiotic factors 104. The locations, size and duration of the proposed limited and controlled release of the GM sugarcane lines are outlined in Section 2.1 of this Chapter. The release is proposed to take place in the Queensland local government areas of Bundaberg, Caboolture and/or Cairns. These geographically distinct commercial sugarcane growing regions have typical climates for sugarcane growing in Australia (see Table 3) with hot and wet summers, and warm and dry winters. Table 3 Climatic data for sites representative of proposed GM sugarcane trial areas Representative site (within local government area) Average daily max/min temperature (summer) 29.6/21.0ºC Average daily max/min temperature (winter) 22.5/10.7ºC Average monthly rainfall (summer) Average monthly rainfall (winter) Bundaberg airport 151.8 mm 39.8 mm (Bundaberg) Somerset Dam# 30.2/19.0ºC 20.9/7.5ºC 136.1 mm 45.5 mm (Caboolture) Cairns airport* 31.3/23.5ºC 26.0/17.4ºC 340.0 mm 34.4 mm (Cairns) Source: <http://www.bom.gov.au> #The Bureau of Meteorology does not have any averages for the shire of Caboolture. Data from the closely located weather station of Somerset Dam was used instead. * The Summer averages for Cairns were based on January to March and the winter averages were based on July to September. 5.2 Relevant agricultural practices 105. The applicant intends to follow standard agricultural protocols used at BSES research stations to grow the GM sugarcane lines. The sugarcane plants at the trial sites would therefore receive applications of water, fertilizers, herbicides, insecticides and other Chapter 1 - Risk assessment context (February 2007) 23 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator agronomic management practices similar to commercially grown non-GM sugarcane. However, the GM sugarcane lines containing the introduced genes for enhanced WUE may be trialled under optimal and water stress treatments, and the GM sugarcane lines containing the introduced gene for improved NUE may be trialled with varying levels of nitrogen applications. 106. Equipment used on the trial sites would be subject to domestic sugarcane quarantine legislation and regulations (Plant Protection Act 1989 (Qld) and the Plant Protection Regulations 2002 (Qld). Therefore, the applicant has indicated that any equipment used will be routinely cleaned and sterilised. 107. The applicant proposes to use the first season for the planting of up to 2500 individual lines in a non-replicated trial to allow the phenotypic screening of all transgenic lines in the field. A selection of these lines will then be used for generating planting material (setts) for the establishment of a new statistically robust crop trial in the following seasons. 108. The applicant proposes that at the end of each crop cycle all cane will be harvested for plant yield measurements and all harvested material destroyed except for the ratoons or setts that will be used to generate the following year’s crop. 109. The applicant proposes that the final data collection and harvest will occur at the end of each growing season. Commercial sugarcane is routinely harvested before flowering as the process of flowering leads to a reduction in stem sugar content. 5.3 Presence of related plants in the receiving environment 110. All of the proposed trial sites except the Caboolture site have commercial sugarcane (S. officinarum x S. spontaneum) growing within 50 kms of their general vicinity. Sugarcane plants, which may be sexually compatible with the GM sugarcane lines, are maintained on BSES land nearby to all three of the proposed trial sites. 111. The proposed Cairns trial site would be located in the vicinity (but at least 200m away) of S. officinarum and S. spontaneum plants, which form part of a BSES germplasm collection. Both parental species of modern cultivars of sugarcane have also been found naturalised in other areas in the tropics (Hnatiuk 1990). Australia’s Virtual Herbarium (<http://www.cpbr.gov.au/avh.html>) also has records of S. spontaneum in the Northern Territory and Queensland. A few S. officinarum populations have been recorded as escapees from cultivation and subsequently naturalised in subtropical and tropical regions of Australia. 112. Sugarcane is closely related to the genera Erianthus, Narenga, Miscanthus and Sclerostachya. These genera and the Saccharum genera are collectively known as the Saccharum complex. Certain species belonging to different genera within this complex can interbreed naturally (Bull & Glasziou 1979; Grassl 1980; Daniels & Roach 1987). Some accessions of these species are maintained as germplasm for breeding programs in sugarcane experimental stations in Australia. 113. The GM sugarcane may be able cross with other distantly related genera belonging to tribe Andropogoneae. Crossing has been reported between Saccharum spp. and Imperata (blady grass), Sorghum (sorghum), Zea (maize) and Bambusa (bamboo) (Thomas & Venkatraman 1930; Janakiammal 1938; Rao et al. 1967; Nair 1999). However, not all such crosses have been verified, particularly through molecular techniques. 114. Blady grass is common throughout Queensland coastal areas and would probably be in the vicinity of the proposed trial sites (information supplied by applicant). Wild Sorghum species are among the weeds of Australian sugarcane crops (McMahon et al. 2000) and are widespread in Australia (Hnatiuk 1990). However, commercial sorghum and maize crops are Chapter 1 - Risk assessment context (February 2007) 24 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator not cultivated near the proposed trial sites (information supplied by applicant). Bambusa arundinacea is reported in the coastal regions of Queensland, especially at the northern tip of Queensland, and many species of bamboo are grown as garden plants throughout Australia. Bamboo is also present at the BSES Meringa research station, location of one of the proposed trial sites (information supplied by applicant). 5.4 Presence of the introduced genes or similar genes in the environment 115. The majority of the lines containing introduced genes for altered plant architecture, enhanced WUE and improved NUE are derived from either the crop plants O. sativa, Z. mays, Saccharum spp., H. vulgare subsp. vulgare, P. coccineus, M. x domestica, or the well characterised model plant A. thaliana, which is widely used in experimental studies. The introduced genes for altered plant architecture, enhanced WUE and improved NUE (except the EcTPSP gene) are highly homologous and most likely orthologous, to genes in most, if not all, crop and other plants. Therefore, it is expected humans routinely encounter the introduced genes and their gene products, or their homologs, through contact with plants and food. This information forms the baseline data for assessing the risks from exposure to these enzymes as a result of the trial of the GM sugarcane 116. The EcTPSP and nptII genes are derived from E. coli, which is widespread in human and animal digestive systems as well as in the environment (Blattner et al. 1997). As such, it is expected humans routinely encounter the encoded protein through contact with plants and food. 117. The uidA gene, which encodes the GUS enzyme, is also derived from E. coli. GUS enzyme activity has been detected in numerous microbial, plant and animal species (Flavell et al. 1992; Gilissen et al. 1998). The GUS protein used in GM plants is 99.8% homologous to the E. coli GUS protein. GUS is recognised as commonly present on fresh food. The US Environmental Protection Agency (EPA) does not consider the GUS protein to be toxic to mammals and has approved its exemption from the requirement to establish tolerance levels (EPA 2001). 118. The bla (ß-lactamase) gene confers resistance to ß-lactam antibiotics such as ampicillin. It was isolated from the bacterial Tn3 transposon. The bla gene will not be expressed in the GM sugarcane lines because the gene’s bacterial (prokaryotic) promoter does not allow the expression in eukaryotes such as plants. Section 6 Australian and international approvals 6.1 Australian approvals of the GM sugarcane lines 6.1.1 Previous releases approved by GMAC or the Regulator 119. There has been no previous release of these GM sugarcane lines with altered plant architecture, enhanced WUE or improved NUE in Australia. 120. In Australia there have been previous small scale field trials of GM sugarcane lines containing the uidA, nptII (or its homolog gene aphA) or bla genes in trials conducted by both BSES and the University of Queensland (PR-23, PR-23X, PR-68 and PR-68X), BSES (PR-72 and DIR 019/2002) and CSIRO Tropical Agriculture (PR-73 and PR-136). There is small scale field trial of GM sugarcane lines containing the nptII and bla genes being conducted until December 2010 by the University of Queensland (DIR 051/2004). 6.1.2 Approvals by other Australian government agencies 121. The Gene Technology Act 2000 is designed to operate in a cooperative legislative framework with other regulatory authorities that have complementary responsibilities and Chapter 1 - Risk assessment context (February 2007) 25 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator specialist expertise. As well as enhancing coordinated decision making, this arrangement avoids duplication. 122. While the Regulator is responsible for identifying, assessing and managing risks to the health and safety of people and the environment associated with the use of gene technology, other government regulatory requirements may also have to be met in respect of release of GMOs. 123. FSANZ is responsible for human food safety assessment and food labelling, including GM food. The applicant does not intend to use materials from the GM sugarcane lines in human food, accordingly an application to FSANZ has not been submitted. FSANZ approval would need to be obtained before materials from these sugarcane lines could be used in food. 6.2 International approvals 124. There has been no international release of these GM sugarcane lines or other GM plants containing the introduced genes for altered plant architecture, enhanced WUE or improved NUE. However, there have been greater than 20 approvals for field trials in the USA of GM sugarcane lines containing the nptII gene and two approvals for small scale releases of GM sugarcane lines containing the uidA gene. Chapter 1 - Risk assessment context (February 2007) 26 DIR 70/2006 – Risk Assessment and Risk Management Plan Chapter 2 Section 1 Office of the Gene Technology Regulator Risk assessment Introduction 125. Risk assessment is the overall process of identifying the sources of potential harm (hazards) and determining both the seriousness and the likelihood of any adverse outcome that may arise. The risk assessment (summarised in Figure 2) considers risks from the proposed dealings with the GMOs that could result in harm to the health and safety of people or the environment posed by, or as a result of, gene technology. Figure 2 The risk assessment process. RISK ASSESSMENT PROCESS * RISK ASSESSMENT CONTEXT Evaluation of events Consequence assessment IDENTIFIED RISK HAZARD IDENTIFICATION RISK ESTIMATE Likelihood assessment No identified risk * Risk assessment terms are defined in Appendix A. 126. Once the risk assessment context has been established (see Chapter 1) the next step is hazard identification to examine what harm could arise and how it could happen during a release of these GMOs into the environment. 127. It is important to note that the word ‘hazard’ is used in a technical rather than a colloquial sense in this document. The hazard is a source of potential harm. There is no implication that the hazard will necessarily lead to harm. A hazard can be an event, a substance or an organism (OGTR 2005). 128. Hazard identification involves consideration of events (including causal pathways) that may lead to harm. These events are particular sets of circumstances that might occur through interactions between the GMOs and the receiving environment as a result of the proposed dealings. 129. A number of hazard identification techniques are used by the Regulator and staff of the OGTR, including the use of checklists, brainstorming, commonsense, reported international experience and consultation (OGTR 2005). In conjunction with these techniques, hazards identified from previous RARMPs prepared for licence applications of the same and similar GMOs are also considered. 130. The hazard identification process results in the compilation of a list of events. Some of these events lead to more than one adverse outcome and each adverse outcome can result from more than one event. Chapter 2 – Risk assessment (February 2007) 27 DIR 70/2006 – Risk Assessment and Risk Management Plan Section 2 Office of the Gene Technology Regulator Hazard characterisation 131. The list of events compiled during hazard identification are 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. 132. A risk is identified only when there is some chance that harm will occur. Those events that do not lead to an adverse outcome or could not reasonably occur do not represent an identified risk and will not advance in the risk assessment process. Risks associated with the remaining events are assessed further to determine the seriousness of harm (consequence) and chance of harm (likelihood). The identified risks must be posed by or result from gene technology. 133. The criteria used by the Regulator to determine harm are described in Chapter 3 of the Risk Analysis Framework (OGTR 2005). Harm is assessed in comparison to the parent organism and in the context of the proposed dealings and the receiving environment. The risk assessment process focuses on measurable criteria for determining harm. 134. The following factors are taken into account during the analysis of events that may give rise to harm: the proposed dealings, which may include experimentation, development, production, breeding, propagation, use, growth, importation, possession, supply, transport or disposal of the GMOs the size, duration and regions of the release containment measures proposed by the applicant characteristics of the non-GM parent (OGTR 2004) routes of exposure to the GMOs, the introduced gene(s) and its product(s) potential effects of the introduced gene(s) and its product(s) expressed in the GMOs potential exposure to the introduced gene(s) and its product(s) from other sources in the environment properties of the biotic and abiotic environment at the site(s) of release agronomic management practices for the GMOs. 135. There have been no previous releases of these GMOs in Australia. 136. Events that are discussed in detail later in this Section are summarised below in Table 4. Events that share a number of common features are grouped together in broader hazard categories as indicated in the table. Sixteen events were characterised, none of which were considered to lead to an identified risk that required further assessment. 137. The bla gene is controlled by its own bacterial promoter and is unlikely to be expressed in the GM sugarcane. In addition, the prevalence of the bla, nptII and uidA genes in the environment and the lack of evidence for toxicity or allergenicity of the respective encoded proteins are discussed in Chapter 1, Sections 4.3.8 to 4.3.10. Furthermore, the potential effects of the bla and nptII and uidA genes and their products were considered in previous assessments such as DIRs 051/2004, 052/2004, 054/2004, 059/2005, 060/2005 and 066/2006, some of which are for commercial releases. RARMPs for those DIR licences are available from the OGTR or from the website <http://www.ogtr.gov.au>. Risks arising from the introduced bla, nptII or uidA genes in GM sugarcane lines are considered to be negligible. Chapter 2 – Risk assessment (February 2007) 28 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Therefore, the potential effects of the bla, nptII and uidA genes will not be assessed further for this application. Table 4 Hazard category Section 2.1 Production of a substance toxic to people Summary of events that may give rise to an adverse outcome through the expression of the introduced genes for altered plant architecture, enhanced WUE or improved NUE. Event that may give rise to an adverse outcome 1. Ingestion of GM plant materials produced by the sugarcane lines containing the introduced genes. Potential adverse outcome Toxicity for people. Identified risk? No Reason The introduced genes are widespread in the environment because of their presence in many common plants and/or bacteria. There are no reports of toxicity associated with the proteins encoded by the introduced genes. Silencing of genes is unlikely to increase toxicity. None of the GM plant materials from the proposed release would be used as human food. 2. Contact with, or inhalation of, GM plant materials produced by the sugarcane lines containing the introduced genes. Toxicity for people. No The introduced genes are widespread in the environment because of their presence in many common plants and/or bacteria. There are no reports of toxicity associated with the proteins encoded by the introduced genes. Silencing of genes is unlikely to increase toxicity. Contact with, or inhalation of GM plant materials would be limited to workers associated with the proposed field trial. Contact would be further limited by the small size and short duration of the proposed field trial. Section 2.2 3. Ingestion of GM plant Production of a materials, produced by the substance sugarcane lines containing allergenic to people the introduced genes. Allergic reactions in people. No None of the proteins encoded by the introduced genes have been listed in publicly available allergen databases. Silencing of genes is unlikely to increase allergenicity. None of the GM plant materials from the proposed release would be used as human food. 4. Contact with, or inhalation of, GM plant materials, produced by the sugarcane lines containing the introduced genes. Allergic reactions in people. No None of the proteins encoded by the introduced genes have been listed in publicly available allergen databases. Contact with, or inhalation of, GM plant materials would be limited by the use of standard industry cultivation practices and the small size and short duration of the proposed field trial. Silencing of genes is unlikely to increase allergenicity. None of the GM plant materials from the proposed release would be used for the production of human food, animal feed or other sugarcane products. Chapter 2 – Risk assessment (February 2007) 29 DIR 70/2006 – Risk Assessment and Risk Management Plan Hazard category Event that may give rise to an adverse outcome Potential adverse outcome Section 2.3 Production of a substance toxic to organisms other than people 5. Direct or indirect ingestion of GM plant materials, produced by the sugarcane lines containing the introduced genes, by vertebrates, invertebrates and microorganisms Toxicity for vertebrates, invertebrates or microorganisms. Office of the Gene Technology Regulator Identified risk? No Reason Vertebrates, invertebrates and microorganisms are already widely exposed to the same or similar proteins. Exposure of vertebrates, invertebrates and microorganisms to the GM sugarcane lines would be limited by the small size and short duration of the proposed release. None of the GM plant materials from the proposed release would be used as animal feed or other sugarcane products. Section 2.4 Spread and persistence of the GM sugarcane in the environment 6. Expression of the introduced genes improving the survival of the GM sugarcane or volunteers through altered plant architecture, enhanced WUE or improved NUE Weediness. No The main factors limiting the spread and persistence of sugarcane in the area proposed for release are likely to include its low fertility and seed viability, poor ability to establish and thrive without human intervention, pests and diseases, soil type and fertility, and competition from other plants. The release would be of small size and short duration and the applicant proposes measures to limit the spread and persistence of the GM sugarcane lines, including destroying any GM sugarcane volunteers. 7. Expression of the introduced genes improving the survival of the GM sugarcane or volunteers through enhancement of Weediness. No The release would be of small size and short duration and the applicant proposes measures to limit the spread and persistence of the GM sugarcane lines, including destroying GM sugarcane volunteers. Abiotic stress tolerances (other than drought) Biotic stress tolerance 8. Dispersal of GM sugarcane plant materials during transport, research, storage, equipment use, flooding or via animals The main factors limiting the spread and persistence of sugarcane in the area proposed for release are discussed in event 6. Weediness. No The applicant proposes to transport and store all GM plant materials according to OGTR guidelines, to clean and sterilise equipment, and to destroy all harvested plant materials not required for propagation or further research. Research would be carried out in PC2 facilities and any GM plant waste materials would be destroyed. The proposed sites are at least 50 m away from natural waterways and are not prone to flooding. Animals are not known to distribute sugarcane. Chapter 2 – Risk assessment (February 2007) 30 DIR 70/2006 – Risk Assessment and Risk Management Plan Hazard category Event that may give rise to an adverse outcome 9. Exposure of vertebrates (including people), invertebrates and microorganisms to material produced from the GM sugarcane lines or volunteers containing the introduced genes. Section 2.5 10.Expression of the Vertical transfer of introduced genes in other genes or genetic sugarcane or sexually elements to compatible plant species. sexually compatible plants Potential adverse outcome Office of the Gene Technology Regulator Identified risk? Toxicity for, or allergic reactions in, people. Toxicity for other vertebrates, invertebrates and/or micro-organisms. No Weediness. No Reason The potential effects on people are discussed in Events 1-4. The potential effects on organisms other than people are discussed in Event 5. The release would be of small size and short duration and the applicant proposes measures to limit the spread and persistence of the GM sugarcane lines, including destroying GM sugarcane volunteers. The parent sugarcane cultivar rarely flowers and both pollen and seed have low viability. Poor sexual compatibility prevents vertical gene transfer to most sexually compatible plant species. Outcrossing to other sugarcane or sexually compatible plant species would be further limited due to the applicant’s intention to harvest cane before flowering, the small size, short duration and containment measures of the trial. 11.Exposure of vertebrates (including people), invertebrates and microorganisms to material produced by other sugarcane or sexually compatible plant species containing the introduced genes. Toxicity for, or allergic reactions in, people. Toxicity for other vertebrates, invertebrates and/or micro-organisms. No 12.Presence of the introduced regulatory sequences in other sugarcane or sexually compatible plant species as a result of gene transfer. Toxicity for, or allergic reactions in, people. Toxicity for other vertebrates, invertebrates and/or micro-organisms. Weediness. No Section 2.6 13.Presence of the introduced Horizontal transfer genes, or the introduced of genes or genetic regulatory sequences, in elements to other organisms as a result sexually of gene transfer. incompatible organisms Toxicity for, or allergic reactions in, people. Toxicity for other vertebrates, invertebrates and/or micro-organisms. Weediness. No Chapter 2 – Risk assessment (February 2007) The limited potential for gene flow is discussed in Event 10. The potential effects of the introduced genes on people are discussed in Events 1-4. The potential effects of the introduced genes on organisms other than people are discussed in Event 5. The introduced regulatory sequences are not known to behave any differently than endogenous regulatory sequences in plants. Outcrossing to other sugarcane or sexually compatible plant species would be limited due to the small size, short duration and containment measures of the trial. The introduced gene or similar genes and the introduced regulatory sequences are already present in the environment and are available for transfer via demonstrated natural mechanisms. Gene transfer from plants to bacteria has not been demonstrated under natural conditions. 31 DIR 70/2006 – Risk Assessment and Risk Management Plan Hazard category Section 2.7 Unintended changes in biochemistry, physiology or ecology Section 2.8 Unauthorised activities Office of the Gene Technology Regulator Event that may give rise to an adverse outcome Potential adverse outcome Identified risk? 14.Production of innate toxic or allergenic compounds as a result of the expression, or random insertion of the introduced genes into the sugarcane genome during genetic modification. Toxicity for, or allergic reactions in, people. Toxicity for other vertebrates, invertebrates and/or micro-organisms. Weediness. No 15.Altered biochemistry, physiology or ecology of the GM sugarcane lines resulting from expression of the introduced genes. Toxicity for, or allergic reactions in, people. Toxicity for other vertebrates, invertebrates and/or micro-organisms. Weediness. No 16.Use of GMOs outside the proposed licence conditions (non-compliance) Potential adverse outcomes discussed in Events 1-15. No Reason Compositional analysis of the GM sugarcane lines has not been undertaken, as the proposed trial represents early stage research involving phenotypic screening of multiple transformation events and selection of lines for possible further development. The small size, short duration and containment measures of the trial will limit any possible adverse outcomes. Unintended adverse effects, if any, would be limited by the small scale and short duration of the proposed release. Unintended changes typically result in deleterious effects in the plant so are unlikely to proceed in the selection process. The Act provides for substantial penalties for non-compliance and unauthorised dealings with GMOs and also requires consideration of the suitability of the applicant to hold a licence prior to the issuing of a licence by the Regulator. 2.1 Production of a substance toxic to people 138. 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). In order for toxicity to occur, people must be exposed to the substance and at a level necessary to cause an adverse effect on health. The hallmark of toxicity is the concept of a dose-response relationship, that is, the severity of the toxic effect is proportional to dose. Exposure can occur via the oral, dermal or inhalational routes (or a combination of these) and the rate and degree of absorption, tissue distribution, metabolism and elimination of the substance all impact on its toxicity. 139. Toxicity may result from a single exposure (acute toxicity) or due to repeated long-term exposure (chronic toxicity). The adverse health effect(s) may be reversible or irreversible and there is likely to be variability within the population with regard to how people are affected by a substance; some people may be relatively sensitive while others may be refractory. Factors contributing to such variability include genetic polymorphisms, age, gender and pre-existing diseases (Aldridge et al. 2003). 140. There are various in vitro and in vivo experimental testing methods used to assess toxicity, with laboratory animals commonly used as models for people. For chemicals and drugs, a toxicological assessment typically covers acute toxicity, eye and skin irritation, skin sensitisation, repeat-dose toxicity, reproductive toxicity, developmental toxicity, genotoxicity, carcinogenicity and neurotoxicity. Human data from clinical trials, occupational exposure studies and poisoning incidents add further to the toxicological database of a substance. 141. For the risk assessment of naturally-occurring agents (ie those that are endemic in the environment) consideration needs to be given to the background level of exposure to people (in addition to the intrinsic toxicity of the substance). Therefore, the risk will actually be Chapter 2 – Risk assessment (February 2007) 32 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator incremental to this background level of exposure and must take into consideration the range (variability) of the concentration/amount of the substance in the environment. 142. For the purposes of the risk assessment of GMOs, an assessment of toxicity would take into consideration the encoded protein from the introduced gene(s), any end-product(s) produced by the biological activity of the protein(s), the toxicity of the GMO(s) and the source organisms of the genes. Event 1: Ingestion of GM plant materials produced by the sugarcane lines containing the introduced genes 143. The GM sugarcane lines proposed for release differ from non-GM sugarcane in that they contain up to four introduced genes for altered plant architecture, enhanced WUE or improved NUE, and marker and reporter genes. As explained in paragraph 137, the marker and reporter genes bla, nptII and uidA will not be considered further. As the proposal is a ‘proof of concept’ field trial, no toxicity studies have been carried out with the proteins encoded by the introduced genes or the GM sugarcane lines. 144. However, the introduced genes for altered plant architecture, enhanced WUE or improved NUE present in the GM sugarcane lines are the same or similar to those present in many crop plants (sugarcane, maize, barley, apple, soybean and rice), or microorganisms (eg E. coli). People are exposed to the proteins (and enzymatic products) encoded by the introduced genes for altered plant architecture, enhanced WUE or improved NUE, or similar proteins, through normal diet or the environment (see Chapter 1, Section 5.4). There are no reports of toxicity associated with the proteins encoded by the introduced genes (see Chapter 1, Sections 4.3.4 to 4.3.7). Furthermore, some of the introduced genes (containing only partial gene sequences) are intended to silence their endogenous counterparts and therefore the level of these proteins in plant tissues is likely to be lower than in the non-GM sugarcane parent. Therefore if these proteins (or enzymatic products) had any toxicity potential then the respective GM sugarcane lines would be less toxic than the non-GM parent. 145. Food Standards Australia New Zealand (FSANZ) is responsible for human food safety assessment and FSANZ approval would be required before products from the GM sugarcane lines could be used in human food. 146. The applicant does not intend to use GM plant materials (including raw sugarcane, sugar or molasses) from the release in human food or as animal feed, but to destroy any plant materials remaining following the final harvest after collecting samples for analysis. Ingestion of materials from the GM sugarcane lines containing the introduced proteins is not expected to occur from the release. 147. Therefore, no risk is identified. The potential for toxicity for people as a result of ingestion of GM plant materials produced by the sugarcane lines containing the introduced genes will not be assessed further. Event 2: Contact with, or inhalation of, GM plant materials produced by the sugarcane lines containing the introduced genes 148. Dermal and inhalation toxicity studies have not been conducted with the proteins (or their enzymatic products) encoded by the introduced genes for altered plant architecture, enhanced WUE or improved NUE. However, as discussed in Event 1, these or similar proteins (or their enzymatic products) are widespread in the environment, occurring in many common plant and bacterial species, and are not known to be toxic to people. Furthermore, the introduced genes containing partial gene sequences are not expected to increase the Chapter 2 – Risk assessment (February 2007) 33 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator production of toxic products in the GM sugarcane. Therefore, the introduced genes are not expected to increase the toxicity of GM sugarcane plant materials. 149. Dermal and inhalation contact with the proteins (or their enzymatic products) encoded by the introduced genes for altered plant architecture, enhanced WUE or improved NUE may occur when the plant cells have been damaged or broken, or via pollen. Workers may come into contact with the protein (and enzymatic products) encoded by the introduced genes during handling and/or processing the GM sugarcane. However, sugarcane plants possess leaves with sharp edges and irritating hairs which necessitates appropriate protection for the handlers, which would significantly reduce potential for dermal contact. 150. While it is possible that people living nearby could come into contact with GM sugarcane pollen as commercial cultivars rarely flower (Bakker 1999; Berding et al. 2004) the level of exposure is expected to be negligible. Although sugarcane pollen is very small and wind dispersed (OGTR 2004), wind is not expected to disperse any pollen more than one hundred metres (Dr D.M. Hogarth personal communication; Information supplied by the applicant) and there would be no public access to the trial sites. In addition, the applicant intends to follow standard agricultural practice and harvest the GM sugarcane before flowering, to preserve sugar content. This would further reduce the potential for exposure to the GM sugarcane pollen. 151. Since the release is of small size and short duration, the frequency and duration of contact with the proteins (and enzymatic products) encoded by the introduced genes is expected to be limited. In addition, the applicant does not intend to use any sugarcane plant materials from the release in human food, animal feed or for any other sugarcane products. 152. Therefore, no risk is identified and the potential for toxicity for people as a result of contact with, or inhalation of, GM plant materials produced by the sugarcane lines containing the introduced genes will not be assessed further. 2.2 Production of a substance allergenic to people 153. Allergenicity is the potential of a specific substance 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). During the induction or sensitisation phase of an allergic response, a person is exposed for the first time to the substance, which results in a primary immune response and a persistent state of heightened immune responsiveness to the substance. Subsequent challenges (ie exposures) result in a powerful and exaggerated immune response, which can lead to adverse health effects in people. For the purposes of the risk assessment of GM plants, a potential allergen may be a protein or an end-product generated through the biological activity of the protein. 154. Allergic reactions following ingestion or inhalation of a substance are mediated by IgE antibodies (ie they are an immediate-type hypersensitivity reaction). In contrast, allergic reactions following dermal exposure are associated with a cell-mediated immune response (type IV hypersensitivity). However, such a reaction following dermal exposure, particularly to high molecular weight compounds such as proteins, is uncommon as the skin is an effective barrier to penetration (Arts et al. 2006; Holsapple et al. 2006). 155. There continues to be considerable discussion in the scientific literature on the characteristics of protein allergens and whether one can predict allergenic potential based on these characteristics. For the assessment of transgenic food crops, the source of the candidate gene and whether it is derived from a source known to be allergenic is an important characteristic as is its reactivity to sera from patients known to be allergic to the source material (Kimber et al. 1999). Other characteristics commonly used to predict a protein’s Chapter 2 – Risk assessment (February 2007) 34 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator allergenicity include amino acid sequence homology with known human allergens, the stability of the protein, resistance to digestion in the gastrointestinal tract and posttranslational glycosylation (Metcalfe et al. 1996; Huby et al. 2000). Event 3: Ingestion of GM plant materials, produced by the sugarcane lines containing the introduced genes. 156. The proteins encoded by the introduced genes for altered plant architecture, enhanced WUE or improved NUE have not been reported to cause allergic reactions in people. As the proposal is a ‘proof of concept’ field trial, no allergenicity studies have been carried out with the proteins encoded by the introduced genes or the GM sugarcane lines. 157. All except one of the introduced genes are from the plants barley, rice, sugarcane, bean, maize, apple and Arabidopsis, which are not known to cause allergies. The final gene, TPSP is from E.coli, a common gut bacterium. None of the proteins encoded by the introduced genes appear in publicly available allergen databases. 158. However, some of the introduced genes have some of the characteristics of allergens (as discussed in Chapter 1). All of the introduced proteins are in the molecular weight range identified as being typical of allergens (15-70 kDa) and all except the TPSP fusion protein and the GA oxidase proteins possess at least one putative N-glycosylation site (Asp-XSer/Thr) typical of allergens. Two of the proteins also show some amino acid identity with known allergens, although these matches are restricted to short amino acid sequences which are not known to be epitopes. 159. The barley GA20ox-1 protein showed a match of eight identical contiguous amino acids with a pollen allergen from short ragweed (for details see Chapter 1, Section 4.3.2). The short ragweed protein is an inhalant allergen and is not known to cause allergy when ingested. 160. The maize DOF-1 protein sequence showed a match of eight identical contiguous amino acids with a vitellogenin protein from chicken (van het Schip et al. 1987) (for details see Chapter 1, Section 4.3.4). The homology between this protein and the maize DOF-1 protein is restricted to a series of eight contiguous amino acids, with no other sequence identity. In maize the DOF-1 protein is not recognised as being allergenic, suggesting that the short match with the vitellogenin protein may be merely a chance occurrence unrelated to allergenicity. 161. Taken together, the weight of available evidence suggests that it is unlikely that the proteins encoded by the introduced genes would be allergenic to people if they were ingested. Furthermore, some of the introduced genes (containing only partial gene sequences) are intended to silence their endogenous counterparts and therefore the level of protein in plant tissues is likely to be lower than in the non-GM sugarcane parent. Therefore, if these proteins (or enzymatic products) had any allergenic potential then the respective GM sugarcane lines would be less allergenic than the non-GM parent. 162. None of the GM sugarcane materials from the release would be used in human food or animal feed. Thus, the potential for allergic reactions in people resulting from exposure to food is unlikely. Food Standards Australia New Zealand (FSANZ) is responsible for human food safety assessment and FSANZ approval would be required before products from the GM sugarcane lines could be used in human food. 163. Therefore, no risk is identified and the potential for production of a substance allergenic to people will not be assessed further. Chapter 2 – Risk assessment (February 2007) 35 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Event 4: Contact with, or inhalation of, GM plant materials produced by the sugarcane lines containing the introduced genes 164. People working with sugarcane plants would be exposed primarily to the outer waxy cuticle layer at the plant surface, which is essentially free of protein. However, as mentioned in Event 2, dermal and inhalation exposure to proteins (including the proteins encoded by the introduced genes for altered plant architecture, enhanced WUE or improved NUE) or to other cellular components of the sugarcane plants may occur via pollen or if damage to the plants during handling results in rupture of plant cells. This may result in workers at the trial sites being exposed to the proteins (and their enzymatic products). 165. There are no reports of the proteins encoded by the introduced genes for altered plant architecture, enhanced WUE or improved NUE causing allergic reactions in people, however two of the proteins do have some characteristics associated with known allergens (for details see Event 3, and Section 4.3 in Chapter 1). 166. The barley GA20ox-1 protein showed a match of eight identical contiguous amino acids with a pollen allergen (Amb a1) from short ragweed (for details see Chapter 1, Section 4.3.2). Although the ragweed allergen it is a potent inhalant allergen (King et al. 1964), the sequence similarity with barley GA20ox-1 is confined to the eight amino acid match with no additional homology in the remaining protein sequence. 167. Inhalation of pollen by workers could potentially result in allergic reactions if this protein was allergenic and was expressed in the pollen. However, due to the very limited quantities of pollen produced by sugarcane, the applicant’s intention to harvest before flowering and the small size of the trial, it is expected that people would be exposed to very small quantities of pollen. 168. As a result, it is unlikely that contact with GM plant materials would cause allergic reactions in people due to the presence of proteins encoded by the genes for altered plant architecture, enhanced WUE or improved NUE. Furthermore, the introduced genes containing partial gene sequences are not expected to increase the allergenicity of GM sugarcane plant materials. 169. Human contact with the GM plant materials containing the proteins (and enzymatic products) encoded by the introduced genes would be limited because the small scale and short duration of the release and none of the materials would be used in human food, animal feed or in the production of other sugarcane products. 170. Therefore, no risk is identified. The potential for allergic reactions in people, resulting from the use or contact with GM plant materials produced by the sugarcane lines containing the introduced genes, will not be assessed further. 2.3 Production of a substance toxic to organisms other than people 171. A range of organisms may be exposed directly or indirectly to GM plant materials containing the introduced genes for altered plant architecture, enhanced WUE or improved NUE. Organisms may be exposed directly through biotic interactions with GM sugarcane plants (vertebrates, insects, symbiotic microorganisms and/or pathogenic fungi) or through contact with root exudates or dead plant materials (soil biota). Indirect exposure would include organisms that feed on organisms that feed on GM sugarcane or degrade it (vertebrates, insects, fungi, Oomycetes and/or bacteria). Chapter 2 – Risk assessment (February 2007) 36 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Event 5: Direct or indirect ingestion of GM plant materials, produced by the sugarcane lines containing the introduced genes, by vertebrates, invertebrates and microorganisms 172. Vertebrates such as livestock and wildlife (including mammals) may be exposed to the GM sugarcane lines containing the proteins (and enzymatic products) encoded by the introduced genes for altered architecture, enhanced WUE or improved NUE through direct feeding or indirectly through consumption of other organisms which have fed on the GM sugarcane plants. 173. Invertebrates, including beneficial insects, could be exposed directly to the GM sugarcane lines containing the proteins (and enzymatic products) encoded by the introduced genes through feeding on the plants, or via the soil when sugarcane tissues decompose. Exposure of soil invertebrates to the proteins (and enzymatic products) encoded by the introduced genes could also occur as a result of root exudations. Exposure could also occur indirectly, through consumption of other organisms that have fed on the GM sugarcane plants. 174. Microorganisms, particularly soil micro-organisms, will be exposed to the GM sugarcane lines containing the proteins (and enzymatic products) encoded by the introduced genes during growth and decomposition of plant materials, possibly as a result of root exudations. Root exudation has been observed in GM maize (Saxena et al. 1999; Stotzky 2000) and GM cotton (Gupta et al. 2002). Root breakage could also lead to the release of the introduced proteins into the soil. After harvest of GM sugarcane, the remaining plant material may be tilled into the soil, leading to exposure of soil microorganisms to GM plant residues. 175. Contact with or ingestion by vertebrates, invertebrates and microorganisms of GM plant materials containing the proteins (and enzymatic products) encoded by the introduced genes would be limited because of the small scale and short duration of the release. In addition, after harvest the applicant proposed to destroy GM sugarcane materials produced, apart from retaining some plant materials for research purposes and for new plantings within the trial, and none of the materials would be used as animal feed. There are no reports of toxicity associated with the proteins (and enzymatic products) encoded by the introduced genes (see Chapter 1, Sections 4.3.4 to 4.3.7). Furthermore, vertebrates, invertebrates and microorganisms would already be exposed to the introduced proteins (and enzymatic products) or very similar proteins in the environment (see Chapter 1, Section 5.4). As mentioned in Event 1, the introduced genes containing partial gene sequences are not expected to increase the toxicity of GM sugarcane plant materials. 176. Therefore, no risk is identified. The potential for toxicity for organisms other than people as a result of direct or indirect ingestion of the GM plant materials produced by the sugarcane lines containing the introduced genes will not be assessed further. 2.4 Spread and persistence of the GM sugarcane in the environment 177. Information on non-GM sugarcane is included here to establish a baseline for comparison with the GM sugarcane being considered in this risk assessment. Attributes of non-GM sugarcane associated with potential weediness are discussed in the document The Biology and Ecology of Sugarcane (Saccharum spp. hybrids) in Australia (OGTR 2004). This document concludes that non-GM sugarcane is not a problematic weed in Australia and occurs almost exclusively as a managed agricultural crop. In sugarcane districts, volunteer sugarcane plants may occur along roadsides or railways where they may establish after displacement during transport, but there is no indication that these plants become selfperpetuating populations. Sugarcane has been grown for centuries throughout the world without any reports that it is a serious weed. Chapter 2 – Risk assessment (February 2007) 37 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 178. Characteristics of sugarcane that may contribute to its potential to spread and persist include its ability to regenerate by re-sprouting from underground buds or from vegetative cuttings containing viable buds. However, stem cuttings are unlikely to survive very long in the field as they are degraded rapidly by soil micro-organisms in tropical conditions. Normally, stem cuttings need to be dipped in fungicide for survival to germination (FAO 2001b; FAO 2004). 179. Sugarcane is almost exclusively propagated asexually but it may reproduce sexually. However, due to breeding (Berding et al. 2004), climatic conditions (Bakker 1999), and industry agricultural practices, commercial sugarcane cultivars rarely flower. Moreover, if sugarcane does flower it generally exhibits poor fertility (Bakker 1999). In cooler climates such as areas from the Burdekin region and further south, the parent cultivar Q117 has been shown to have very low pollen viability (2-3%)(Bonnett et al. 2007). Furthermore, sugarcane pollen rapidly desiccates and is not viable beyond 35 minutes at 26.5ºC at 65% relative humidity (Venkatraman 1922; Moore 1976). Additionally, sugarcane seed is short lived, losing 90% of its viability in 80 days at 28°C if not treated to enhance its survival (Rao 1980). 180. Modern sugarcane cultivars (Saccharum spp. hybrid) are not invasive in natural undisturbed environments. The inability of sugarcane to establish, spread and persist is likely to be due to factors including competition with other plants, pests and diseases, soil type and fertility, moisture stress, sunlight requirements and low temperatures (Bakker 1999; Hogarth & Allsopp 2000; OGTR 2004). 181. The weed status of sugarcane has also been considered previously in RARMPs produced during the assessment of other GM sugarcane lines (DIRs 019/2002 and 051/2004) and very low risks were identified. Event 6: Expression of the introduced genes improving the survival of the GM sugarcane or volunteers through altered plant architecture, enhanced WUE (drought tolerance) or improved NUE 182. Up to 1400 of the GM sugarcane lines contain introduced gene constructs for altered plant architecture (see Table 1), which if successful could result in plants with different stalk heights, weights and numbers. The GM sugarcane lines may also have increased suckering and resistance to lodging. 183. Many studies in potato, pumpkin, Arabidopsis, aspen, tobacco, rice and lettuce have examined the effects of introducing or silencing genes encoding gibberellin oxidases homologous to those introduced into the proposed GM sugarcane lines (Coles et al. 1999; Carrera et al. 2000; Curtis et al. 2000; Eriksson et al. 2000; Niki et al. 2001; Vidal et al. 2001; Sakamoto et al. 2003; Biemelt et al. 2004; Israelsson et al. 2004; Lee & Zeevaart 2005; Radi et al. 2006). The resulting phenotypes were varied and included increases in plant height, decreased time to flowering, earlier tuber formation, increased lateral root formation and thicker roots, and an increase in fruit and seed numbers. However, some phenotypes were dwarfed, had delayed flowering, were less fertile or sterile, and had smaller roots. The GM sugarcane lines containing the introduced genes HvGA20ox-1 or 2 for altered plant architecture appear to produce stalks more rapidly (1-2 months earlier), and GM sugarcane lines containing the introduced gene PcGA2ox-1 grow more slowly and are shorter, than their non-GM sugarcane parent under glasshouse conditions (see Chapter 1, Section 4.5.2). 184. The other genes for altered plant architecture introduced into the GM sugarcane lines include the MAX and TB1 gene homologs. Studies in Arabidopsis with the endogenous MAX3 and MAX4 genes have shown that when the expression of these genes is suppressed, or is nonexistent, phenotypes included dwarfing, more branching and higher inflorescence numbers Chapter 2 – Risk assessment (February 2007) 38 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator (Seregélyes et al. 2003; Booker et al. 2004; Bainbridge et al. 2005). In petunia, silencing of the endogenous MAX4 gene resulted in plants with a similar phenotype to that reported in the A. thaliana studies but with fewer roots and small flowers and an increased flowering time (Snowden et al. 2005). Over-expression of the endogenous OsTB1 gene in rice led to a reduction in tillers while the converse phenotype resulted from the silencing of the endogenous gene (Takeda et al. 2003). 185. Although alteration of plant architecture by the introduced genes could potentially result in weediness of the GM sugarcane lines, it could also result in GM plants of lower fitness than other commercially available sugarcane varieties because of the potential metabolic/physiological burdens. For example, the sugarcane may have stunted growth, be too tall and fall over, produce less seeds, and have a decreased ability to tolerate competition from other plants. The trial will enable the applicant to assess the effect of the introduced genes on plant physiology and agronomic performance. 186. Up to 300 of the GM sugarcane lines contain introduced gene constructs for enhanced WUE (see Table 1), which if successful would confer enhanced drought stress tolerance. The introduced genes have demonstrated in plants, other than sugarcane, the capacity to produce a water-efficient phenotype (Garg et al. 2002) or are involved in the response to dehydration stress (Urao et al. 1993; Gao et al. 2001). In an environment in which water availability was the main factor limiting the spread and persistence of sugarcane, expression of the genes for WUE could result in weediness of the GM sugarcane lines. However, under glasshouse conditions, necrosis is observed on leaves of the GM sugarcane expressing the introduced MdS6PDH gene for enhanced WUE. The necrosis is more noticeable at the leaf apex where the sorbitol content is the highest. The condition is more severe in the plants producing larger amounts of sorbitol. These GM sugarcane lines are up to 30% shorter compared to non-GM parent (see Chapter 1, Section 4.5.2). Therefore, expression of the MdS6PDH gene for enhanced water use efficiency in the GM sugarcane lines would probably result in less fit plants, which may be more vulnerable to attack by fungal and/or bacterial pathogens, because of an increase in sorbitol content. These necrotic lesions have also been reported on some GM tobacco plants expressing MdS6PDH (Sheveleva et al. 1998). 187. Up to 100 of the GM sugarcane lines contain an introduced DOF1 gene for improved NUE (see Table 1), which if successful would confer improved growth in soil with low nitrogen levels. The ZmDOF1 gene has demonstrated the capacity in the plant A. thaliana to produce a phenotype with improved growth under low nitrogen conditions in the laboratory (Yanagisawa et al. 2004). In an environment in which nitrogen availability was the main factor limiting the spread and persistence of sugarcane, expression of the genes for NUE could result in weediness of the GM sugarcane lines. 188. As discussed above, multiple factors limit the distribution and growth of sugarcane in the areas proposed for release including its 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 (Bakker 1999; Hogarth & Allsopp 2000; OGTR 2004). Alteration of one factor such as WUE or NUE is unlikely to increase the survival of sugarcane enough to lead to an increase in weediness and in some cases the modification may have a negative impact of the plants’ fitness levels. 189. In addition, the release would be of limited size and short duration and the applicant proposed a number of measures to limit the spread and persistence of the GM sugarcane lines (see Chapter 1, Section 2.3). Chapter 2 – Risk assessment (February 2007) 39 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 190. Therefore, no risk is identified and the potential for the spread and persistence of sugarcane outside of the trial site leading to weediness through altered plant architecture, enhanced WUE (drought tolerance) or improved NUE as a result of the expression of the introduced genes will not be assessed further. Uncertainty This is a ‘proof of concept’ field trial and therefore, the effect of the introduced genes for altered plant architecture on the GM sugarcane lines is unknown in the field. Also, the ability of the GM sugarcane plants to withstand drought stress or have improved NUE throughout different stages of their lifecycle, as compared to commercially available sugarcane cultivars, is unknown. Data on the unintentional effects on plant architecture relating to weediness, or enhanced WUE and improved NUE, may be required to assess possible future applications involving larger scale or commercial releases of any of these GM sugarcane lines. However, the data are not required for this release because the trial is limited in locations, size and duration. Event 7: Expression of the introduced genes improving the survival of the GM sugarcane or volunteers through enhancement of abiotic stress tolerances (other than drought stress) or biotic stress tolerances Abiotic stress tolerance 191. The GM sugarcane lines express genes that may alter plant architecture, enhance WUE or improve NUE. It is possible that the introduced genes could potentially confer enhanced tolerances to other abiotic stresses, other than those associated with water or nitrogen deficiencies, which could lead to increased spread and persistence of the GM sugarcane plants. 192. Other enhanced abiotic stress tolerances may improve the GM sugarcane lines’ ability to survive, or have improved performance, under lower and higher temperatures. It is also possible the plants could tolerate higher soil salinity, or have increased seed dormancy, seed or pollen viability, and/or improved seedling germination rates under various abiotic stresses (other than drought stress). For example, the MdS6PDH gene is known to confer enhanced salt stress tolerance to transgenic Japanese persimmon plants (Deguchi et al. 2004) and the introduction of the EcTPSP gene into rice helped the plants withstand salt and low temperature stresses, as well as drought stress (Garg et al. 2002; Jang et al. 2003). In Arabidopsis plants, the expression of AtMYB2 is induced by drought and salt stress, suggesting its involvement in responses to these stresses (Urao et al. 1993). 193. It should be noted that when a gene is expressed in different plant species the same effect(s) on phenotype does not always eventuate (Oh et al. 2005). Therefore, the effect on abiotic stress response as a result of the expression of the introduced genes may not eventuate in the GM sugarcane lines. 194. This is a ‘proof of concept’ field trial and therefore, the ability of the GM sugarcane plants to withstand abiotic stress tolerances throughout different stages of their lifecycle as compared to commercially available sugarcane cultivars is unknown. Biotic stress tolerance 195. The genes for altered plant architecture, enhanced WUE or improved NUE could potentially enhance resistance of the GM sugarcane lines to pests or pathogenic microorganisms. This could lead to spread and persistence of the GM sugarcane lines if pests or diseases were the main limiting factors. Chapter 2 – Risk assessment (February 2007) 40 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 196. Most diseases of sugarcane are controlled by planting resistant clones (Berding et al. 2004). The most economically important disease of sugarcane is probably ratoon stunting disease, caused by a bacterium Clavibacter xyli subsp xyli (McLeod et al. 1999). Other important diseases include orange rust (Puccinia kuehnii), common rust (Puccinia melanocephala), pachymetra root rot (Pachymetra chaunorhiza) and sugarcane smut (Ustilago scitaminea) (Bakker 1999). The major pests of sugarcane are the larvae of cane beetles which destroy the roots of the cane (Berding et al. 2004). Other insect pests such as plant hopper (Perkinsiella saccharicida) do not cause a significant yield loss but do transmit viral diseases such as Fiji virus disease or sugarcane mosaic virus. Sugarcane yields can also be reduced by vertebrate pests such as ground rats (Rattus sordidus), climbing rats (Melomys burtoni) and feral pigs (Sus scrofa) (Agnew 1997). 197. The introduced genes (or orthologs) for altered plant architecture, enhanced WUE or improved NUE are not known to confer enhanced biotic stress tolerance. 198. The high sugar content of sugarcane makes it an ideal host for a number of microorganisms including bacteria and fungi leading to its degradation. It is not expected that the introduced genes will alter this. 199. This is a ‘proof of concept’ field trial and therefore, the ability of the GM sugarcane plants to withstand biotic stress tolerances throughout different stages of their lifecycle as compared to commercially available sugarcane cultivars is unknown. Conclusion 200. As discussed above, multiple factors limit the distribution and growth of sugarcane in the areas proposed for release including its 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 (Bakker 1999; Hogarth & Allsopp 2000; OGTR 2004). 201. In addition, the release would be of limited size and short duration and the applicant proposed a number of measures to limit the spread and persistence of the GM sugarcane lines (see Chapter 1, Section 2.3). 202. Therefore, no risk is identified and the potential for the spread and persistence (weediness) of sugarcane as a result of the expression of the introduced genes for altered plant architecture, enhanced WUE or improved NUE will not be assessed further. Uncertainty 203. Currently, there is no direct evidence that GM sugarcane lines may have enhanced tolerances to a number of abiotic and biotic stresses as compared to their non-GM parent. However, in the highly unlikely instance where the plants may have enhanced tolerances to several environmental stresses, the GM plants will most likely be less fit as compared to other commercially available sugarcane varieties because of the potential metabolic/physiological burdens. For example, the sugarcane may have stunted growth, be too tall and fall over, produce less seeds, and have a decreased ability to tolerate competition from other plants. 204. Data on tolerances to abiotic/biotic factors, additional to that already discussed for enhanced drought tolerance of the GM sugarcane lines may be required to assess possible future applications involving larger scale or commercial releases of any of these GM sugarcane lines. However, the data are not required for this release because the trial is limited in locations, size and duration. Chapter 2 – Risk assessment (February 2007) 41 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Event 8: Dispersal of GM plant materials during transport, research, storage, equipment use, flooding or via animals 205. Commercial sugarcane is propagated vegetatively. A stem cutting called a ‘sett’ contains one or more buds. Usually one bud per cutting is developed into a primary stalk which then gives rise to tillers from underground buds (see more details in Biology and Ecology of Sugarcane (Saccharum spp. hybrids) in Australia (OGTR 2004). 206. It is feasible the GM sugarcane could be dispersed through seeds. However, as discussed in Event 10 seed production is rare and sugarcane seed has low viability (Rao 1980; OGTR 2004), information supplied by the applicant) and does not germinate readily (Breaux & Miller 1987). Therefore, any dispersed seed may not germinate, and if it does, the spread and persistence of any new GM sugarcane plants outside of the trial sites would be limited by multiple factors including its 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 (Bakker 1999; Hogarth & Allsopp 2000; OGTR 2004). In addition, the applicant intends to harvest the majority of the GM sugarcane before flowering, and this would limit seed production. Dispersal by spillage during transport, research, storage or from equipment 207. As discussed in the assessment for DIR 051/2004, viable sugarcane stems could be unintentionally dispersed during transportation. Some sugarcane volunteers have been found growing along roadsides or railways in sugarcane cultivation areas. These volunteers are believed to have originated from stem cuttings displaced or fallen from vehicles. These volunteers generally consist of only a few stools (a group of stems growing from a single original plant base) and do not become self perpetuating or result in further spread. 208. In the course of the dealings the applicant proposed to transport GM sugarcane setts (stem cuttings used for vegetative propagation) to 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. 209. The applicant proposed to transport the GM sugarcane setts to the trial sites according to OGTR transport guidelines (Guidelines for the transport of GMOs, June 2001; Policy on transport and supply of GMOs, July 2005). Therefore, any spillage of setts during transport to and from the release sites would be rare. Moreover, the movement of sugarcane and sugarcane machinery is restricted in Queensland by the Plant Protection Act 1989 (Qld) and the Plant Protection Regulations 2002 (Qld). Any incident involving spillage of GM setts is expected to be readily controlled through cleaning and monitoring of the site of the spill. In addition, the opportunity for any adverse outcome from any such rare occurrence is further diminished by the need for appropriate environmental conditions for survival and persistence of any escaped setts. 210. The applicant proposed to thoroughly clean equipment used in the harvest of the GM sugarcane (the cane may be hand harvested for laboratory samples) to prevent dispersal of GM plant materials to other locations and to meet domestic quarantine requirements. The applicant proposed to destroy all plant materials (including materials from the non-GM guard row) other than materials collected for future research or for new plantings within the trial. The sites would be monitored for volunteers after the final harvest and any volunteer sugarcane plants would be destroyed. Chapter 2 – Risk assessment (February 2007) 42 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 211. The applicant proposed to conduct any lab research in PC2 facilities. Any plant material collected for this purpose and not required for further study will be destroyed in the PC2 facility. Dispersal by flooding of the release site 212. Severe weather conditions (eg flooding) could lead to the dispersal of GM plant materials. The applicant proposed that the trial sites will be at least 50 m away from natural waterways, on land not prone to flooding, and surrounded by a guard row of non-GM sugarcane and a 6 m isolation zone. In case of heavy rain, any GM plant materials on the ground are therefore not expected to be dispersed beyond the area of the release (including the guard row, which would act as a physical barrier once the plants were established). Dispersal of the GM sugarcane would be highly unlikely as sugarcane does not generally propagate vegetatively without human intervention (OGTR 2004). Dispersal by animals 213. As sugarcane can reproduce through vegetative cuttings, it is theoretically possible that pests such as feral pigs or other large animals could disperse viable materials. However, as discussed in the assessment for DIR 051/2004 there are no reports of this occurring. 214. Numerous animal pests of sugarcane, including mammals, birds and insects, are known who could eat GM sugarcane plant materials, including flowers, pollen and seed. Of those, seed has a potential to produce a new GM sugarcane plant if it were still viable after excretion. However, the seed needs appropriate environmental conditions for germination and these are seldom present without human intervention. The applicant proposed to surround GM sugarcane with a row of non-GM sugarcane guard plants, which are expected to minimise dispersal by large mammals. The applicant also intends to harvest the GM sugarcane lines before flowering and this would limit the production of seed. In addition, the animal pests of sugarcane in the release areas are expected to be controlled (as in commercial plantations), further reducing the potential for dispersal by animals. 215. Therefore, no risk is identified and the potential for an adverse outcome as a result of dispersal of GM plant materials during transport, research, storage, equipment use, flooding or via animals will not be assessed further. Event 9: Exposure of vertebrates (including people), invertebrates and microorganisms to material produced by the GM sugarcane lines or volunteers expressing the introduced genes 216. The potential of increased spread and persistence of the GM sugarcane lines in the environment was addressed in Events 6, 7 and 8 no risk was identified. However, in the highly unlikely instance of any of these events occurring, spread and persistence of the GM sugarcane plants in the environment could lead to increased exposure of vertebrates (including people), invertebrates and microorganisms to the proteins (and enzymatic products) encoded by the introduced genes for altered plant architecture, enhanced WUE or improved NUE. 217. An adverse outcome could occur, if the proteins (and enzymatic products) encoded by the introduced genes for altered plant architecture, enhanced WUE or improved NUE were toxic or allergenic for people. The potential for the proteins causing toxic or allergic reactions in people was assessed in Events 1 to 4 and no risk was identified. 218. Organisms other than people may be exposed directly, through feeding on the GM sugarcane plants or indirectly through eating organisms that have fed on or degraded the GM sugarcane plants as a result of spread and persistence of the GM sugarcane in the Chapter 2 – Risk assessment (February 2007) 43 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator environment. These organisms include vertebrates, invertebrates and microorganisms. The toxicity potential of proteins encoded by the genes for altered plant architecture, enhanced WUE and improved NUE to organisms other than people was considered in Event 5 and no risk was identified. 219. In addition, the chain of events that would lead to increased exposure of vertebrates, invertebrates and microorganisms depends on the least likely event to occur, which is an enhanced capacity for spread and persistence in the GM sugarcane lines proposed for release. 220. The release would be of limited size and short duration and the applicant proposed a number of measures to limit the spread and persistence of the GM sugarcane lines in the environment (for details see Chapter 1, Section 2.3). 221. Therefore, no risk is identified and the potential for toxicity or allergic reactions in people or other organisms as a result of spread and persistence of the GM sugarcane lines in the environment will not be assessed further. 2.5 Vertical transfer of genes or genetic elements to sexually compatible plants 222. Transfer of genetic materials to offspring by reproduction, either asexual or sexual (vertical gene transfer) could result in the transfer of the introduced genes altered plant architecture, enhanced WUE or improved NUE, or their associated regulatory elements to other plants. The sexually compatible species present in Australia that could receive genes from the GM sugarcane lines are commercial sugarcane and other Saccharum spp. (S. spontaneum and S. officinarum). Other genera within the tribe Andropogoneae may also be able to receive genes from the GM sugarcane lines, although some level of sexual incompatibility will limit this transfer. 223. Weediness resulting from an increase in the spread and persistence of other sugarcane plants is contingent on both of the following steps: transfer of the introduced gene construct to other sugarcane plants weediness of the recipient plants as a result of expression of the introduced gene. Event 10: Expression of the introduced genes in other sugarcane or sexually compatible plant species Outcrossing to other sugarcane plants 224. All of the proposed trial sites except the Caboolture site have commercial sugarcane (S. officinarum x S. spontaneum) growing within 50 kms of their general vicinity. In one of the areas proposed for release (BSES Meringa research station) Saccharum spp. (commercial sugarcane, S. spontaneum and S. officinarum) are grown as germplasm collections (see Chapter 1, Section 5.3). The genes for altered plant architecture, enhanced WUE or improved NUE could be transferred to these sexually compatible sugarcane plants resulting in an increased potential for weediness. 225. Sugarcane is typically vegetatively propagated but may reproduce sexually. Many sugarcane cultivars do not flower reliably which is reported to interrupt cross-breeding programs (Berding et al. 2004). Observations of the breeding collection at BSES Meringa research station indicated that an average of 38.2% of clones in the breeding collection flowered from 1978 to 1996 and the flowering was variable, ranging from 16% of clones in 1993 to 66% in 1984. Cool or dry climatic conditions negatively impact on the development of the flowers (Bakker 1999). Chapter 2 – Risk assessment (February 2007) 44 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 226. However, even if sugarcane does flower, it generally exhibits poor fertility (Bakker 1999). The non-GM parent cultivar Q117 produces pollen which is only 10-20% viable (McIntyre & Jackson 2001) or less (2-3%) in cooler regions such as Burdekin (Bonnett et al. 2007). As such, Q117 is only used as a female in breeding programs (McIntyre & Jackson 2001); information supplied by the applicant). 227. Under the average temperature and humidity expected for the areas proposed for the release sugarcane pollen would not be expected to travel further than 100 metres (Dr D. M. Hogarth, personal communication; information supplied by applicant). Sugarcane pollen rapidly desiccates and is not viable beyond 35 minutes at 26.5ºC at 65% relative humidity (Venkatraman 1922; Moore 1976). Any seed that may result from a cross would most likely have limited viability and dormancy (OGTR 2004);information supplied by applicant). Furthermore, sugarcane seeds are notoriously hard to germinate and the seedlings are very delicate and vulnerable for the first three to four weeks after germination (Breaux & Miller 1987) and therefore require careful nurturing. 228. Standard cultivation practices for commercial sugarcane cultivation include harvesting prior to flowering to maximise sugar content. Given that this is likely to occur for any nearby commercial crops and the applicant intends to follow the same procedures, the opportunity for gene flow would be limited. Outcrossing to other sexually compatible plants 229. Sugarcane may also be sexually compatible with genera, within the tribe Andropogoneae, outside Saccharum genus (for more detail see Chapter 1, Section 5.3) (OGTR 2004). These genera include Erianthus, Narenga, Miscanthus, Sclerostachya, Imperata (blady grass), Sorghum (sorghum), Zea (maize) and Bambusa (bamboo). 230. Commercial sorghum and maize crops are not cultivated near the proposed trial sites (information supplied by applicant), however, other compatible species may be present in the vicinity of the trial site. 231. Blady grass is common throughout Queensland coastal areas and would probably be in the vicinity of the proposed trial sites (information supplied by applicant). Wild Sorghum species are among the weeds of Australian sugarcane crops (McMahon et al. 2000) and are widespread in Australia (Hnatiuk 1990). 232. Bambusa arundinacea is reported in the coastal regions of Queensland, especially at the northern tip of Queensland, and many species of bamboo are grown as garden plants throughout Australia. Bamboo is also present at the BSES Meringa research station, one of the proposed trial sites (information supplied by applicant). 233. However, 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. For example, out of 42 crosses made between Erianthus arundinaceus and hybrid Saccharum spp., 17 produced seedlings, none of which were genuine hybrids (Piperidis et al. 2000). Crosses using Saccharum officinarum as the female parent produced some hybrids but these were sterile. Saccharum officinarum x Sorghum bicolor crosses have been reported, but these lack vegetative vigour (Nair 1999). 234. Although potentially sexually compatible genera may be growing adjacent to the trial sites, they might not flower at the same time, and outcrossing would be limited due to very low pollen viability, poor sexual compatibility and the containment measures proposed by the applicant (see Chapter 1, Section 2.3). This would make any gene flow highly unlikely. Chapter 2 – Risk assessment (February 2007) 45 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Potential for weediness in plants resulting from outcrossing 235. In the unlikely instance of outcrossing to a commercially grown non-GM sugarcane, the potential for weediness would be the same as for the GM sugarcane lines themselves, which were assessed (Events 6-8, this Chapter) and no risk was identified. The spread and persistence of sugarcane in the areas proposed for release is limited by multiple factors including its 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 (Bakker 1999; Hogarth & Allsopp 2000; OGTR 2004). 236. In the highly unlikely instance of outcrossing to a plant sexually compatible with sugarcane the hybrid will tend to be infertile or exhibit low vigour. This would make enhanced spread and persistence of this plant also highly unlikely. 237. The limited area and duration of this proposed release would further reduce the likelihood of weediness occurring as a result of gene transfer from the GM sugarcane plants to other sugarcane or related plants. Conclusion 238. As discussed above, multiple factors would limit the transfer of genes from the GM sugarcane lines to other sugarcane plants or sexually related species and the potential for weediness of these plants. 239. In addition, the applicant proposed a release of limited size and short duration and a number of measures to limit the spread and persistence of the GM sugarcane lines. These include post harvest monitoring of the trial sites for at least 12 months following the final harvest and the destruction of any volunteers. 240. Therefore, no risk is identified and the potential of expression of the introduced genes for altered plant architecture, enhanced WUE and improved NUE in other sugarcane and sexually compatible plant species leading to weediness will not be assessed further. Event 11: Exposure of vertebrates (including people), invertebrates and microorganisms to material produced by other sugarcane or sexually compatible plant species containing the introduced genes 241. Transfer of the genes for altered plant architecture, enhanced WUE or improved NUE to other sugarcane and sexually compatible plants could lead to increased exposure of vertebrates (including people), invertebrates and microorganisms to the proteins and enzymatic products encoded by the introduced genes. 242. The transfer of the introduced genes to other sugarcane and sexually compatible plants was assessed in Event 10 and no risk was identified. In the highly unlikely event of this occurring the potential adverse outcomes arising from these sexually compatible plants would be highly similar to the GM sugarcane lines for release, which were assessed in Events 1 to 5 and no risk was identified. 243. In addition to the proposed measures to limit the spread and persistence of the GM sugarcane lines, the applicant proposed a release of limited size and short duration. The applicant also proposed to monitor the GM sugarcane trial sites for at least 12 months after the final harvest and destroy any volunteers. 244. Therefore, no risk is identified. The potential for toxicity or allergenicity to vertebrates (including people), invertebrates and microorganisms, through increased exposure to material produced by other sugarcane or sexually compatible plant species containing the introduced genes will not be assessed further. Chapter 2 – Risk assessment (February 2007) 46 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Event 12: Presence of the introduced regulatory sequences in other sugarcane or sexually compatible plant species as a result of gene transfer 245. All of the introduced regulatory sequences operate in the same manner as regulatory elements endogenous to sugarcane plants. The transfer of either endogenous or introduced regulatory sequences could result in unpredictable effects. The impacts from the introduced regulatory elements are equivalent and no greater than the endogenous regulatory elements. 246. The transfer of genetic material to other sugarcane or sexually compatible plant species was assessed in Event 10 and considered highly unlikely. 247. The applicant proposed to monitor the GM sugarcane trial sites for at least 12 months after the final harvest and to destroy any volunteers. 248. Therefore, no risk is identified and the potential for an adverse outcome as a result of vertical gene transfer of introduced regulatory sequences will not be assessed further. 2.6 Horizontal transfer of genes or genetic elements to sexually incompatible organisms. Event 13: Presence of the introduced genes, or the introduced regulatory sequences, in other organisms as a result of gene transfer 249. Transfer of the introduced regulatory sequences and/or the introduced genes for altered plant architecture, enhanced WUE or improved NUE from the GM sugarcane plants to sexually incompatible plants, animals or microorganisms (horizontal gene transfer) could occur only rarely without human intervention. 250. Most gene transfers have been identified through analyses of gene sequences (Worobey & Holmes 1999; Ochman et al. 2000). In general, gene transfers are detected over evolutionary time scales of millions of years (Lawrence 1999). Most gene transfers have been from virus to virus (Lai 1992), or between bacteria (Ochman et al. 2000). In contrast, transfers of plant genetic materials to other microorganisms such as bacteria, viruses or fungi have been exceedingly rare. 251. Transfer of the regulatory sequences to other organisms could alter the expression of endogenous genes in unpredictable ways. However, all of the introduced regulatory sequences operate in the same manner as regulatory elements endogenous to sugarcane plants. The transfer of either endogenous or introduced regulatory sequences could result in adverse unpredictable effects. As there is no difference between those two events, this does not represent a novel adverse outcome as a result of the genetic modification. 252. Horizontal gene transfer has been examined in detail in a number of other RARMPs (most recently DIR 057/2004), which are available from the OGTR website <http://www.ogtr.gov.au> or by contacting the Office. These assessments have concluded that horizontal gene transfer from plants to other sexually incompatible organisms occurs rarely and usually only on evolutionary timescales. Reports of horizontal gene transfer from plants to bacteria occurring during laboratory experiments have not only relied on the use of highly similar sequences to allow homologous recombination to occur, but also on conditions designed to enhance the selective advantage of gene transfer events (Nielsen et al. 2000; Gebhard & Smalla 1998; Mercer et al. 1999; Nielsen 1998; De Vries et al. 2001). Horizontal gene transfer is not expected to produce any adverse outcomes during this limited and controlled release. 253. Therefore, no risk is identified. The potential for an adverse outcome as a result of horizontal gene transfer will not be assessed further. Chapter 2 – Risk assessment (February 2007) 47 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 2.7 Unintended changes in biochemistry, physiology or ecology 254. A single plant gene can have an influence on multiple, sometimes unrelated, plant traits. This phenomenon is known as pleiotropy and all such multiple effects are sometimes unknown. Certain types of genes have a higher likelihood of causing pleiotropic effects. These genes include those that encode proteins that can regulate the transcription of a number of genes (eg transcription factors). 255. Gene technology has the potential to cause unexpected and unintended pleiotropic effects due to the process used to insert new genetic material or by producing a gene product that affects multiple traits. Such 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. 256. Unintended pleiotropic effects might result in adverse outcomes such as toxicity or allergenicity; weediness, pest or disease burden; or reduced nutritional value as compared to the parent organism. However, accumulated experience with genetic modification of plants indicates that the process has little potential for unexpected outcomes that are not detected and eliminated during the routine process of selecting plants that are morphologically similar to the conventional plant except for the new properties (Bradford et al. 2005). Additionally, unintended changes that occur as a result of gene insertions are rarely advantageous to the plant (Kurland et al. 2003). 257. All methods of plant breeding can induce unanticipated changes in plants, including pleiotropic effects (Haslberger 2003). Most often the important nutrients, toxicants, and other components are present in the new plant variety at levels that are within the range expected for commercial varieties. Event 14: Altered levels of innate toxic or allergenic compounds as a result of the expression, or random insertion of the introduced genes into the sugarcane genome during genetic modification 258. 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 2004). 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 and Ecology of Sugarcane (Saccharum spp. hybrids) in Australia (OGTR 2004). 259. There is potential for the GM sugarcane plants for release to produce toxic or allergenic compounds as a result of the expression of the genes for altered plant architecture, enhanced WUE or improved NUE. Additionally, random insertion of the gene construct during the genetic modification process could potentially result in the production of toxic or allergenic compounds in the GM sugarcane lines. Chapter 2 – Risk assessment (February 2007) 48 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 260. Thus far, exposure to plant materials from the GM sugarcane lines has been limited to a few workers maintaining these plants in the glasshouse. The applicant has reported no adverse outcomes from exposure to the GM sugarcane plant materials, suggesting that expression of the introduced genes has not resulted in a change in the level of toxicity or allergenicity associated with the non-GM parent sugarcane. 261. Furthermore, the introduced genes containing partial gene sequences are not expected to increase the toxicity and/or allergenicity of GM sugarcane plant materials. 262. In addition, the release would be of limited size and short duration and the applicant proposed a number of measures to limit the spread and persistence of the GM sugarcane lines. 263. Therefore, no risk is identified and the potential for production of toxic or allergenic compounds as a result of random insertion of the gene construct into the GM sugarcane lines will not be assessed further. Uncertainty 264. Data on the potential toxicity or allergenicity of the proteins encoded by the introduced genes, and plant materials may be required for risk assessments of applications for larger scale or commercial release of these GM sugarcane lines. Further information is not required for assessing the risks of this release because the trial is limited in locations, size and duration and none of the GM sugarcane plant materials are intended for use in human food, animal feed or in the production of other sugarcane products Event 15: Altered biochemistry, physiology or ecology of the GM sugarcane lines resulting from expression of the introduced genes 265. Biochemical, physiological or ecological changes to the GM sugarcane lines for release could occur either as a result of the expression of the introduced genes for altered plant architecture, enhanced WUE or improved NUE, or of the transformation process itself. The applicant has reported that GM sugarcane lines containing the MdS6PDH gene had leaf necrosis (see Chapter 1, Section 4.5.2). The trial will enable the in field agronomic performance of the GM sugarcane lines to be assessed. Unintended changes typically result in deleterious effects in the plant so are unlikely to proceed in the selection process. However, this information is not required for assessing the risks of this release because the trial is limited in locations, size and duration. Furthermore, none of the GM sugarcane plant materials are intended for use in human food, animal feed or in the production of other sugarcane products. 266. Therefore, no risk is identified. The potential for weediness and/or toxicity and/or allergenicity to people and other organisms, as a result of unintended changes in biochemistry, physiology or ecology will not be assessed further. Uncertainty 267. Insertion of new genes and traits by conventional breeding or genetic modification can result in unintended and unexpected changes. Therefore, more data on the potential pleiotropic effects of the genetic modifications on GM sugarcane lines selected for further development, and how these may affect potential weediness, toxicity and allergenicity, may be required to assess any future applications for a larger scale or commercial release of any of these GMOs. Chapter 2 – Risk assessment (February 2007) 49 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 2.8 Unauthorised activities Event 16: Use of GMOs outside the proposed licence conditions (non-compliance) 268. If a licence were to be issued, non-compliance with the conditions of the licence could lead to spread and persistence of the GM sugarcane lines outside of the proposed release areas. The adverse outcomes that this event could cause were assessed in Events 1-15. 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. 269. Therefore, no risk is identified and the potential for an adverse outcome as a result of unauthorised activities will not be assessed further. Section 3 Risk estimate process for identified risks 270. The hazard identification process considered the circumstances by which people or the environment may be exposed to the GMOs, GM plant materials, GM plant by-products, the introduced genes, or products of the introduced genes. 271. Sixteen events were identified and assessed whereby the proposed release of the GM sugarcane lines might give rise to harm to people or the environment. 272. These 16 events included consideration of whether, or not, expression of the introduced genes could result in products that are toxic or allergenic to people or other organisms, produce unintended changes in the biochemistry, physiology or ecology of the GM sugarcane lines, or alter characteristics that may impact on spread and persistence of the GMOs. In addition, consideration was given to the opportunity for gene flow to other organisms, and its effects. 273. All events were characterised in relation to both the magnitude and probability of harm in the context of proposed controls to limit the spread and persistence of the GMOs. Detailed consideration of the sixteen events for this particular field trial demonstrated that none gave rise to an identified risk that required further assessment. The principle reasons include: small scale of the trial is limited in both area and duration suitability of containment and disposal measures proposed by the applicant to limit the spread and persistence of the GM plants none of the GM plant materials will be used for any other purpose widespread presence of the same or similar proteins (and enzymatic products) in the environment and lack of evidence of harm from these proteins and their products the lack of known toxicity or allergenicity of the proteins (and enzymatic products) encoded by the introduced genes, and the unlikely potential for gene suppression to increase toxicity or allergenicity limited capacity of the GM sugarcane lines to spread and persist in the areas proposed for release limited ability and opportunity for the GM sugarcane lines to transfer the introduced genes to commercial sugarcane crops or other sexually compatible species. 274. Therefore, as no risks to the health and safety of people or the environment were identified from the proposed limited and controlled release of GM sugarcane lines, the level of risk is considered to be negligible. Chapter 2 – Risk assessment (February 2007) 50 DIR 70/2006 – Risk Assessment and Risk Management Plan Chapter 3 Office of the Gene Technology Regulator Risk management 275. Risk management includes evaluation of risks identified in Chapter 2 to determine whether or not specific treatments are required to mitigate harm to human health and safety, or the environment, that may arise from the release. Other risk management considerations required under the Act are also addressed in this chapter. Together, these risk management measures are used to inform the decision-making process and determine licence conditions that are imposed by the Regulator under the Act. In addition, the roles and responsibilities of other regulators under Australia’s integrated regulatory framework for gene technology are also explained. Section 1 Background 276. Under section 56 of the Act, the Regulator must not issue a licence unless satisfied that any risks posed by the dealings proposed to be authorised by the licence are able to be managed in a way that protects the health and safety of people and the environment. All licences are required to be subject to three conditions prescribed in the Act. 277. Section 63 requires that each licence holder inform relevant people of their obligations under the licence. Other mandatory statutory conditions contemplate the Regulator maintaining oversight of licensed dealings. For example section 64 requires the licence holder to provide access to premises to OGTR monitors, and section 65 requires the licence holder to report any information about risks or unintended effects of the dealing to the Regulator on becoming aware of them. Matters related to the ongoing suitability of the licence holder are also required to be reported to the Regulator. 278. It is a further requirement that the licence be subject to any conditions imposed by the Regulator. Examples of the matters to which conditions may relate are listed in section 62 of the Act. Licence conditions can be imposed to limit and control the scope of the dealings and the possession, supply, use, transport or disposal of the GMO for the purposes of, or in the course of, a dealing. In addition, the Regulator has extensive powers to monitor compliance with licence conditions under section 152 of the Act. Section 2 Other Australian regulators 279. Australia's gene technology regulatory system operates as part of an integrated legislative framework. Other agencies that also regulate GMOs or GM products include FSANZ, APVMA, Therapeutic Goods Administration (TGA), National Industrial Chemicals Notification and Assessment Scheme (NICNAS), National Health and Medical Research Council (NHMRC) and Australian Quarantine and Inspection Service (AQIS). Dealings conducted under any licence issued by the Regulator may also be subject to regulation by one or more of these agencies18. 280. The Gene Technology Act 2000 requires the Regulator to consult these agencies during the assessment of DIR applications. The Gene Technology (Consequential Amendments) Act 2000 requires the agencies to consult the Regulator for the purpose of making certain decisions regarding their assessments of products that are, or contain a product from, a GMO. 281. FSANZ is responsible for human food safety assessment, including GM food. As the trial involves early stage research the applicant does not intend any material from these GM More information on Australia’s integrated regulatory framework for gene technology is contained in the Risk Analysis Framework available from the Office of the Gene Technology Regulator (OGTR). Free call 1800 181 030 or at <http://www.ogtr.gov.au/pdf/public/raffinal2.2.pdf>. 18 Chapter 3 – Risk management (February 2007) 51 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator sugarcane lines to be used in human food. Accordingly, the applicant has not applied to FSANZ for evaluation of any of the GM sugarcane lines for use in human food. FSANZ approval would need to be obtained before they could be used in this way. 282. No other approvals are required. Section 3 Risk treatment measures for identified risks 283. The risk assessment of events listed in Chapter 2 concluded that there are negligible risks to people and the environment from the proposed trial of GM sugarcane. The Risk Analysis Framework, which guides the risk assessment and risk management process, defines negligible risks as insubstantial with no present need to invoke actions for their mitigation. 284. These events were considered in the context of the scale of the release (up to 3 sites of up to 2 hectares, over the three cropping cycles between February 2007 and November 2010, in the Queensland local government areas of Bundaberg, Caboolture and Cairns), the containment measures and agricultural practices proposed by the applicant and the receiving environment (see Section 5, Chapter 1). Section 4 General risk management 285. Containment measures consistent with the risk assessment context have been imposed to limit the trial to the locations, size and duration requested by the applicant, which are summarised below (Section 4.1). 4.1 Summary of proposed licence conditions 4.1.1 Measures to limit and control the proposed trial 286. A number of licence conditions19 have been imposed to limit the spread and persistence of the GMOs, including requirements to: surround the trial sites by one guard row of non-GM sugarcane and an isolation zone of at least 6 metres locate the trial sites at least 50 m away from natural waterways harvest and process sugarcane from the trial separately from any commercial sugarcane destroy all plant materials not required for experimentation or new plantings following cleaning of sites, monitor for and destroy any GM sugarcane that may grow for at least 12 months until the site is clear of volunteers for a continuous 6 month period. 4.1.2 Measures to control other activities associated with the trial 287. The Regulator has issued guidelines and policies for the transport and supply of GMOs (Guidelines for the transport of GMOs, June 2001; Policy on transport and supply of GMOs, 19 BSES intends to follow standard industry cultivation protocols, including harvesting before flowering to preserve the sugar content of the GM lines. In addition, the applicant proposed the removal of sugarcane flowers at the trial sites prior to anthesis as an additional containment measure to prevent the possibility of gene flow. No risks were identified even if harvesting of the GM sugarcane lines did not occur and flowers were not removed prior to anthesis, primarily because the parent cultivar has low fertility (ie rarely flowers; low pollen production and poor seed viability), is unlikely to flower synchronously with other sexually compatible plants, outcrossing to related species would be limited due to poor sexual compatibility and progeny are likely to have limited viability. In addition, the applicant proposed other suitable measures to contain the release which are required by the licence conditions. Therefore, neither harvesting before flowering nor the removal of sugarcane flowers prior to anthesis have been included as licence conditions. Chapter 3 – Risk management (February 2007) 52 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator July 2005). Licence conditions based on these guidelines and policies have been imposed regarding transportation and storage, and to control possession, use or disposal of the GMOs for the purposes of, or in the course of, the authorised dealings. 4.2 Other risk management considerations 288. All DIR licences issued by the Regulator contain a number of general conditions that relate to general risk management. These include, for example: applicant suitability contingency and compliance plans identification of the persons or classes of persons covered by the licence reporting structures, including a requirement to inform the Regulator if the applicant becomes aware of any additional information about risks to the health and safety of people or the environment monitoring for compliance. 4.2.1 Applicant suitability 289. In making a decision whether or not to issue a licence, the Regulator must have regard to the suitability of the applicant to hold a licence. Under section 58 of the Act matters that the Regulator must take into account include: any relevant convictions of the applicant (both individuals and the body corporate) any revocation or suspension of a relevant licence or permit held by the applicant under a law of the Commonwealth, a State or a foreign country the applicant's history of compliance with previous approved dealings the capacity of the applicant to meet the conditions of the licence. 290. Before making the decision whether or not to issue a licence for this application (DIR 070/2006), the Regulator determined that BSES is suitable to hold a licence. 291. Conditions in the licence include a requirement for the licence holder to inform the Regulator of any circumstances that would affect their suitability or their capacity to meet the conditions of the licence. 292. In addition, any applicant organisation must have access to a properly constituted Institutional Biosafety Committee and be an accredited organisation under the Act. 4.2.2 Contingency and compliance plans 293. The licence requires BSES to submit a plan detailing how it intends to ensure compliance with the licence conditions and document that compliance. This plan is required before the planting of the GM sugarcane lines commences. 294. BSES is also required to submit a contingency plan to the Regulator within 30 days of the issue date of the licence. This plan must detail measures to be undertaken in the event of any unintended presence of the GM sugarcane lines outside of the permitted areas. 295. BSES is also required to provide a method to the Regulator for the reliable detection of the presence of the GMOs and the introduced genetic materials in a recipient organism. This instrument would be required within 30 days of the issue date of the licence. 4.2.3 Identification of the persons or classes of persons covered by the licence Chapter 3 – Risk management (February 2007) 53 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator 296. The persons covered by this licence are the licence holder and employees, agents or contractors of the licence holder and other persons who are, or have been, engaged or otherwise authorised by the licence holder to undertake any activity in connection with the dealings authorised by this licence. 4.2.4 Reporting structures 297. The licence obliges the licence holder to immediately report any of the following to the Regulator: any additional information regarding risks to the health and safety of people or the environment associated with the trial any contraventions of the licence by persons covered by the licence any unintended effects of the trial. 298. The licence holder is also obliged to submit an Annual Report within 90 days of the anniversary of the licence containing any information required by the licence, including the results of inspection activities. 299. A number of written notices are also required under the licence that would assist the OGTR in designing and implementing a monitoring program for all licensed dealings. The notices include: expected and actual dates of planting expected and actual dates of final destroying and cleaning at the end of the trial. 4.2.5 Monitoring for compliance 300. The Act stipulates, as a condition of every licence, that a person who is authorised by the licence to deal with a GMO, and who is required to comply with a condition of the licence, must allow inspectors and other persons authorised by the Regulator to enter premises where a dealing is being undertaken for the purpose of monitoring or auditing the dealing. Post-release monitoring continues until the Regulator is satisfied that all the GMOs resulting from the authorised dealings have been removed from the release sites. 301. If monitoring activities identify changes in the risks associated with the authorised dealings, the Regulator may also vary licence conditions, or if necessary, suspend or cancel the licence. 302. In cases of non-compliance with licence conditions, the Regulator may also instigate an investigation to determine the nature and extent of non-compliance. These include the provision for criminal sanctions of large fines and/or imprisonment for failing to abide by the legislation, conditions of the licence or directions from the Regulator, especially where significant damage to health and safety of people or the environment could result. Section 5 Issues to be addressed for future releases 303. In view of the early stage research involved in the proposed trial, the risk assessment identified additional information that may be required to assess an application for a larger scale trial, reduced containment conditions or a commercial release of any of these GM sugarcane lines. This would include: molecular characterisation of GM sugarcane lines selected for possible future releases Chapter 3 – Risk management (February 2007) 54 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator additional data on the potential toxicity or allergenicity of proteins encoded by the introduced genes for altered plant architecture, enhanced WUE and improved NUE, and of plant materials from the GM sugarcane lines selected for further research altered biochemical, physiological and agronomic characteristics indicative of weediness in the selected GM sugarcane lines including measurement of tolerance to environmental stresses and reproductive capacity. Section 6 Conclusions of the RARMP 304. The risk assessment concludes that this limited and controlled release of up to 2500 GM sugarcane lines on a maximum area of 18 ha over 3 cropping cycles in the Queensland local government areas of Bundaberg, Caboolture and Cairns poses negligible risks to the health and safety of people and the environment. 305. The risk management plan concludes that these negligible risks do not require specific risk treatment measures. However, licence conditions have been imposed to contain the trial to the locations, size and duration requested by the applicant. Section 7 DIR 070/2006 Licence 306. The licence DIR 070/2006 is available on the OGTR website <http://www.ogtr.gov.au/gmorec/ir.htm#table>, following the path to DIR 070/2006. 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References (February 2007) 68 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Appendix A Definitions of terms in the Risk Analysis Framework used by the Regulator (* terms defined as in Australia New Zealand Risk Management Standard AS/NZS 4360:2004) Consequence outcome or impact of an adverse event Marginal: there is minimal negative impact Minor: there is some negative impact Major: the negative impact is severe Event* occurrence of a particular set of circumstances Hazard* source of potential harm Hazard identification the process of analysing hazards and the events that may give rise to harm Intermediate the negative impact is substantial Likelihood chance of something happening Highly unlikely: may occur only in very rare circumstances Unlikely: could occur in some circumstances Likely: could occur in many circumstances Highly likely: is expected to occur in most circumstances Quality control to check, audit, review and evaluate the progress of an activity, process or system on an ongoing basis to identify change from the performance level required or expected and opportunities for improvement Risk the chance of something happening that will have an undesired impact Negligible: risk is insubstantial and there is no present need to invoke actions for mitigation Low: risk is minimal but may invoke actions for mitigation beyond normal practices Moderate: risk is of marked concern requiring mitigation actions demonstrated to be effective High: risk is unacceptable unless actions for mitigation are highly feasible and effective Appendix A (February 2007) 69 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Risk analysis the overall process of risk assessment, risk management and risk communication Risk analysis framework systematic application of legislation, policies, procedures and practices to analyse risks Risk assessment the overall process of hazard identification and risk estimation Risk communication the culture, processes and structures to communicate and consult with stakeholders about risks Risk Context parameters within which risk must be managed, including the scope and boundaries for the risk assessment and risk management process Risk estimate a measure of risk in terms of a combination of consequence and likelihood assessments Risk evaluation the process of determining risks that require treatment Risk management the overall process of risk evaluation, risk treatment and decision making to manage potential adverse impacts Risk management plan integrates risk evaluation and risk treatment with the decision making process Risk treatment* the process of selection and implementation of measures to reduce risk Stakeholders* those people and organisations who may affect, be affected by, or perceive themselves to be affected by a decision, activity or risk States includes all State governments, the Australian Capital Territory and the Northern Territory governments Uncertainty imperfect ability to assign a character state to a thing or process; a form or source of doubt Appendix A (February 2007) 70 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Appendix B Summary of issues raised in submissions received from prescribed experts, agencies and authorities20 on application DIR 070/2006 All issues raised in submissions relating to risks to the health and safety of people and the environment were considered in the context of the currently available scientific evidence that was used in the preparation of the consultation RARMP. Issues raised relating to the Risk Assessment (considered in Ch 2): Enhanced spread and persistence (weediness) of the GM sugarcane (Events 6, 7 and 8). Gene flow to other commercial sugarcane crops, related species or unrelated species (Events 10 to 13). Toxicity and/or allergenicity of the proteins (and enzymatic products) encoded by the introduced genes (Events 1 to 5, 9, and 11 to 15). Dissemination of the GM sugarcane material beyond the intended areas (Events 10 to 13, and 16). Potential pleiotropic effects of the introduced genes (Events 14 and 15). Issues raised relating to the Risk Management Plan (considered in Ch 3 and licence): Containment measures Transport procedures for the GM sugarcane Post-harvest monitoring and practices Disposal of GM plant materials not required for further research or approved plantings. 20 GTTAC, State and Territory governments, Australian Government agencies, the Minister for Environment and Heritage and Local councils where the release may occur. Appendix B (February 2007) 71 DIR 70/2006 – Risk Assessment and Risk Management Plan Office of the Gene Technology Regulator Appendix C Summary of issues raised in submissions received from the public on application DIR 070/2006 Two submissions received from the public are summarised in the table below. All issues relating to risks to the health and safety of people and the environment were considered in the context of the currently available scientific evidence. Issues raised: EN: environmental risks; H: human health and safety. Other abbreviations: OGTR: The Office of the Gene Technology Regulator Submission no. Issue Summary or issues raised Consideration in RARMP Comment 1 None - Noted 2 H, EN Fully supports the proposed application after considering the transgenic phenotypes proposed for release and has confidence in the expertise of BSES staff to comply with OGTR requirements and licence conditions. Such a release could not be controlled. The area could not be de-contaminated, and release to the surrounding environment could not be controlled for very long. Human health and well being are put at risk. Ch 2 Licence conditions have been proposed to contain the small scale release. Ch 2 None of the GM plant material from the proposed trial will be used in human food or animal feed. H Appendix C (February 2007) 72