Dir 111 Limited and controlled release of wheat and barley genetically modified for altered grain composition, nutrient utilisation efficiency, disease resistance or stress tolerance Applicant

Risk management plan

Risk management is used to protect the health and safety of people and to protect the environment by controlling or mitigating risk. The risk management plan evaluates and treats identified risks, evaluates controls and limits proposed by the applicant, and considers general risk management measures. The risk management plan is given effect through the licence conditions.

As none of the seven risk scenarios characterised in the risk assessment give rise to an identified risk that requires further assessment, the level of risk from the proposed dealings is assessed to be negligible. The Regulator's Risk Analysis Framework defines negligible risks as insubstantial, with no present need to invoke actions for their mitigation in the risk management plan. However, conditions have been imposed to restrict the spread and persistence of the GMOs and their genetic material in the environment and to limit the release to the size, location and duration requested by the applicant, as these were important considerations in establishing the context for assessing the risks.

Licence conditions

The Regulator has imposed a number of licence conditions, including requirements to:

limit the release to a total area of up to 2.3 ha per year at one site in the ACT, between May 2012 and June 2017

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  locate the trial site at least 50 m away from natural waterways
  
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  enclose the trial site with a fence capable of excluding livestock, with lockable gates
  
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  establish a 2 m buffer zone and a 10 m monitoring zone around each location, maintained in a manner that does not attract or harbour rodents
  
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  maintain at least 200 m distance between the GMOs and other wheat or barley crops, and destroy other sexually compatible plants found within this area
  
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  harvest the GM wheat and barley plant material separately from other crops
  
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  clean the site, buffer zones and equipment used on the site following harvest
  
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  apply measures to promote germination of any wheat or barley seeds that may be present in the soil after harvest, including irrigation and tillage
  
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  monitor the site for at least 24 months after harvest, and destroy any wheat and barley plants that may grow, until no volunteers are detected for a continuous 6 month period
  
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  transport and store material from the GMO in accordance with Regulator’s guidelines
  
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  not commence nutritional studies involving animals or human volunteers until endorsed by an Animal Ethics Committee or a Human Research Ethics Committee, respectively
  
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  not allow the GM plant materials or products to be used for human food or animal feed, with the exception of the nutritional studies
  
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  destroy all GM plant material not required for further analysis or future trials.
  

Other regulatory considerations

Australia's gene technology regulatory system operates as part of an integrated legislative framework that avoids duplication and enhances coordinated decision making. The Regulator is responsible for assessing risks to the health and safety of people and the environment associated with the use of gene technology. However, dealings conducted under a licence issued by the Regulator may also be subject to regulation by other Australian government agencies that regulate GMOs or GM products, including Food Standards Australia New Zealand (FSANZ), Australian Pesticides and Veterinary Medicines Authority, Therapeutic Goods Administration, National Industrial Chemicals Notification and Assessment Scheme and Australian Quarantine Inspection Service⁶.

APVMA has regulatory responsibility for the supply of agricultural chemicals, including herbicides and insecticidal products, in Australia. The application of these herbicides is subject to regulation by the APVMA. While some GM wheat lines have been modified to be tolerant to glufosinate ammonium containing herbicides, the applicant does not intend to apply these herbicides during the trial.

FSANZ is responsible for human food safety assessment and food labelling, including GM food. The applicant does not intend to commercially use any material from the GM wheat and barley lines in human food, accordingly an application to FSANZ has not been submitted. FSANZ approval would need to be obtained before any material from the GM wheat and barley lines could be sold as food.

In addition, dealings authorised by the Regulator may be subject to the operation of State legislation declaring areas to be GM, GM free, or both, for marketing purposes.

Identification of issues to be addressed for future releases

Additional information has been identified that may be required to assess an application for a large scale or commercial release of these GM wheat and barley lines, or to justify a reduction in containment conditions. This would include:

additional data on the potential toxicity and allergenicity of plant materials from the GM wheat and barley lines

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  additional phenotypic characterisation of the GM wheat and barley lines, particularly with respect to traits that may contribute to weediness, including tolerance to environmental stresses and disease susceptibility
  
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  additional molecular and biochemical characterisation of the GM wheat and barley lines.
  

Suitability of the applicant

The Regulator has assessed the suitability of CSIRO to hold a DIR licence as required by the Act. CSIRO is considered suitable as the Regulator is satisfied that no relevant convictions have been recorded, no licences or permits have been cancelled or suspended under laws relating to the health and safety of people or the environment, and the organisation has the capacity to meet the conditions of the licence.

Conclusions of the RARMP

The risk assessment concluded that this limited and controlled release of up to 292 GM wheat and 41 GM barley lines on a maximum total area of 2.3 ha per year over five years in the ACT, poses negligible risks to the health and safety of people or the environment as a result of gene technology.

The risk management plan concluded that these negligible risks do not require specific risk treatment measures. However, conditions have been imposed to limit the release to the size, location and duration proposed by the applicant, and to require controls in line with those proposed by the applicant as these were important considerations in establishing the context for assessing the risks.

Risk assessment context

1. 
   Background
   

1. 
   This chapter describes the parameters within which potential risks to the health and safety of people or the environment posed by the proposed release are assessed ().
   

Parameters used to establish the risk assessment context

2. 
   The risk assessment context is developed within the framework of the Gene Technology Act 2000 (the Act) and Gene Technology Regulations 2001 (the Regulations, Section 2), the Risk Analysis Framework, and operational policies and guidelines available at the OGTR website <http://www.ogtr.gov.au>.
   
3. 
   In addition, establishing the risk assessment context for this application includes
   consideration of:
   

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  the proposed dealings (8)
  
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  the parent organism ()
  
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  the genetically modified organisms (GMOs), nature and effect of the genetic modification (26)
  
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  the receiving environment (139)
  
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  previous releases of these or other GMOs relevant to this application (165)
  
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  The legislative requirements
  

4. 
   Sections 50, 50A and 51 of the Act outline the matters which the Gene Technology Regulator (the Regulator) must take into account, and with whom he must consult, in preparing the Risk Assessment and Risk Management Plans (RARMPs) that form the basis of his decisions on licence applications. In addition, the Regulations outline matters the Regulator must consider when preparing a RARMP.
   
5. 
   In accordance with section 50A of the Act, the Regulator considered information provided in the application and was satisfied that its principal purpose is to enable the applicant to conduct experiments. In addition, limits on the size, location and duration of the release and controls have been proposed by the applicant to restrict the spread and persistence of the GMOs and their genetic material in the environment. Those limits and controls are such that the Regulator considered it appropriate not to seek the advice referred to in subsection 50(3) of the Act. Therefore, this application is considered to be a limited and controlled release and the Regulator has prepared a RARMP for this application.
   
6. 
   Section 52 of the Act requires the Regulator to seek comment on the RARMP from the States and Territories, the Gene Technology Technical Advisory Committee, Commonwealth authorities or agencies prescribed in the Regulations, the Minister for the Environment, local council(s) where the release is proposed to take place, and the public. The advice from the prescribed experts, agencies and authorities and how it was taken into account is summarised in Appendix A. Three submissions were received from the public and their considerations are summarised in Appendix B.
   
7. 
   Section 52(2)(ba) of the Act requires the Regulator to decide whether one or more of the proposed dealings may pose a ‘significant risk’ to the health and safety of people or to the environment, which then determines the length of the consultation period as specified in section 52(2)(d). The decision is provided in 283 of Chapter 2.
   
8. 
   The proposed dealings
   
9. 
   The Commonwealth Scientific and Industrial Research Organisation (CSIRO) proposes to release up to 292 GM wheat and 41 GM barley lines⁷, that have been genetically modified (GM) for altered grain composition, nutrient utilisation efficiency, disease resistance or stress tolerance, into the environment under limited and controlled conditions.
   
10. 
    The dealings involved in the proposed intentional release would include:
    

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  conducting experiments with the GMOs
  
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  propagating, growing, raising or culturing the GMOs
  
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  breeding the GMOs
  
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  transporting the GMOs
  
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  importing the GMOs
  
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  disposing of the GMOs
  
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  possession, supply or use of the GMOs for the purposes of any of the above.
  

11. 
    These dealings are detailed further throughout the remainder of the current Chapter.
    
12. 
    Some details, including the identities of some of the genes and sequences, associated references and phenotypic data, have been declared or are under consideration as Confidential Commercial Information (CCI) under section 185 of the Act. This information was considered during the preparation of the RARMP and was made available to the prescribed expert groups and authorities that were consulted.
    
13. 
    The proposed activities
    
14. 
    The applicant has stated that the purpose of the trial is to:
    

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  evaluate the agronomic performance of the GMOs under conditions of biotic (exposure to fungal disease) and abiotic (drought/heat) stress
  
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  analyse any changes in grain composition, nutritional characteristics, dough making properties and end product quality
  
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  collect GM material and seeds for subsequent trials.
  

15. 
    Flour derived from the grain of a few GM wheat and barley lines with altered grain composition will be used for a range of carefully controlled, small scale animal nutritional trials, and the same GM wheat lines may be used in nutritional trials with human volunteers. The GM wheat and barley are not permitted to enter the commercial human food or animal feed supply chains.
    
16. 
    Up to 292 GM wheat and 41 GM barley lines are proposed to be released. Some of the lines have been included in previous DIR licences issued to the applicant (see 165 for more details).
    
17. 
    The proposed limits of the dealings (size, location and duration)
    
18. 
    The release is proposed to take place at one site within CSIRO’s Ginninderra Experiment Station in the Australian Capital Territory (ACT), on a maximum area of 2.3 ha per year between May 2012 and June 2017. The applicant may plant at more than one location within the site, if needed.
    
19. 
    Only trained and authorised staff would be permitted access to the proposed location(s).
    
20. 
    The proposed controls to restrict the spread and persistence of the GMOs and their genetic material in the environment
    
21. 
    The applicant has proposed a number of controls to restrict the spread and persistence of the GM wheat and barley lines and the introduced genetic material in the environment (some details are shown in Figure 2), including:
    

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  locating the trial site at least 125 m from the nearest natural waterway
  
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  separating each location where GMOs are grown from other wheat and barley plantings by at least 200 m
  
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  surrounding the GMOs with a 2 m wide buffer zone, a 10 m monitoring zone where growth of related species will be controlled
  
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  restricting animal access by surrounding the trial site with a rabbit and livestock-proof fence and covering with bird netting
  
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  destroying all plant materials not required for testing or future trials
  
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  cleaning of any equipment used prior to removal from the site
  
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  monitoring of the trial site post-harvest for a period of 24 months and destroying any volunteer GMOs before they flower
  
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  transporting and storing all GM material outside the trial site according to the Regulator’s Guidelines for the Transport, Storage and Disposal of GMOs
  
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  not allowing GM plant material to enter the commercial human food or animal feed supply.
  
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  These controls (see also Figure 2), and the limits outlined in Section 17, have been taken into account in establishing the risk assessment context (this chapter), and their suitability for containing the proposed release is evaluated in Chapter 3, Section 1.1.
  

1. 
   Schematic diagram of some of the proposed containment measures (not drawn to scale)
   

The parent organism

22. 
    The parent organisms are bread wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), both of which are exotic to Australia. Commercial wheat and barley cultivation occurs in the wheat belt from south eastern Queensland through New South Wales, Victoria, southern South Australia and southern Western Australia (OGTR 2008b). A small amount of barley is also grown in Tasmania (OGTR 2008a).
    
23. 
    The wheat cultivars used to generate the GM wheat lines are Bobwhite, Frame, Gladius and NB1. The Bobwhite cultivar is not favoured as a commercial bread wheat as it is considered to be of lower quality than most commercial cultivars (Bhalla et al. 2006), but is commonly used in genetic modification work because it is relatively easy to genetically modify and has previously been used in conventional (non-GM) wheat breeding programs. The cultivars Frame and Gladius are commercially cultivated in Australia and have some degree of drought tolerance as they have been bred for Australian conditions. The cultivar NB1 is a breeding line from the United Kindom, which is not commercially grown in Australia.
    
24. 
    The GM barley lines in the proposed release were derived from the barley cultivar Golden Promise. Golden Promise was derived from the Maythorpe cultivar following modification by the use of gamma-ray irradiation. It is a semi-dwarf, malting cultivar that has been found to have greater tolerance to soil salinity than Maythorpe (Forster 2001). While the precise genetic changes are not known, salt tolerance in Golden Promise is a consequence of the plants’ ability to limit the uptake of salt from the soil and results in this cultivar having a higher grain yield than its parental cultivar. Golden Promise is also reported to have some tolerance to drought (Forster 2001) but is not used in commercial plantings.
    
25. 
    Further detailed information about the parent organism is contained in the reference documents The Biology of Triticum aestivum L. em Thell (bread wheat) and The Biology of Hordeum vulgare L. (barley), which were produced to inform the risk assessment process for licence applications involving GM wheat and barley plants (OGTR 2008a; OGTR 2008b). These documents are available at http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/riskassessments-1.
    
26. 
    The GMOs, nature and effect of the genetic modification
    

1. 
   Introduction to the GMOs
   

27. 
    The applicant proposes to release up to 292 GM wheat and 41 GM barley lines, each with one or more genes from among 34 genes of interest derived from wheat and barley. Details of the introduced genes and their anticipated effects are given in Table 1. Table 2 lists the gene combinations used in the GM wheat and barley lines, including the associated regulatory elements (promoters and terminators). Table 2 also identifies those lines that are currently being trialled under other DIR licences.
    

Based on similarities in the introduced genes and modified traits, the GMOs can be classified into six groups belonging to two broad categories:

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  Category 1 consists of 50 wheat lines and one barley line genetically modified for altered grain composition using four partial gene sequences derived from wheat (Groups 1 and 3) and two genes from barley (Group 5).
  
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  Category 2 consists of 242 wheat lines and 40 barley lines genetically modified for improved agronomic performance in drought/heat-prone environments (Groups 2 and 4) and enhanced disease resistance (Group 6) using a total of 28 genes derived from wheat or barley. Among these, 26 genes are expected to enhance carbon assimilation, water use efficiency and photosynthesis (Group 4), one gene is expected to enhance nutrient use efficiency (Group 2) and one gene is responsible for enhanced rust resistance (Group 6).
  

Group 1: up to 23 of the GM wheat lines contain introduced partial sequences of the wheat glucan water dikinase (GWD) gene. GWD is involved in determining grain qualities (starch composition) important for dough making and human nutrition. The gene construct containing partial GWD gene sequences is designed to suppress the expression of the GWD gene through a mechanism known as RNA interference (RNAi; http://www.pi.csiro.au/RNAi; Millar & Waterhouse 2005).

Group 2: up to 95 of the GM wheat and 40 of the GM barley lines contain an introduced alanine aminotransferase (AlaAT) gene from barley that encodes an enzyme involved in nitrogen utilisation. Expression of this gene is expected to result in an increase in plant biomass and yield.

Group 3: 18 of the GM wheat lines contain partial sequences (for RNAi) of the wheat starch branching enzyme IIa (SBE IIa) gene or a combination of partial sequences from the SBE IIa gene and the wheat starch branching enzyme I (SBE I) gene, and one line of GM barley contains two partial sequences (RNAi) of the wheat SBE IIa and SBE IIb genes. Expression of these partial gene sequences leads to altered starch composition by RNAi suppression of the SBE genes in the GM lines.

Group 4: up to 142 of the GM wheat lines contain one or more of 26 genes derived from wheat and barley expected to improve grain weight and yield in heat and drought prone environments through enhanced water use efficiency, photosynthesis and carbon assimilation. The applicant states that some GM plants carrying multiple genes from this group may also be tested in the field trial. These will be generated either by co-bombardment of a mixture of the candidate genes or by conventional crossing of GM lines proposed for release. These GMOs would only be included in the field trial if they pass phenotypic screening at the T₁ stage in controlled environment facilities.

Group 5: nine of the GM wheat lines contain either a barley carbohydrate enzyme A (CME A) gene or a barley carbohydrate enzyme B (CME B) gene, which are expected to alter carbohydrate composition in the endosperm of the wheat grain.

Group 6: up to five GM wheat lines contain the wheat Lr34 gene, which confers partial resistance to leaf rust, stripe rust and powdery mildew.

28. 
    In addition, most of the GM wheat and barley lines also contain one of the three selectable marker genes: bar, hpt and nptII (Table 1). The herbicide resistance gene bar, derived from the bacterium Streptomyces hygroscopicus, encodes the enzyme phosphinothricin acetyl transferase which provides resistance to herbicides containing glufosinate ammonium. The antibiotic resistance selectable marker genes hpt and nptII, derived from the common gut bacterium Escherichia coli, encode the enzymes hygromycin phosphotransferase and neomycin phosphotransferase type II, respectively; the former confers resistance to hygromycin and the latter to kanamycin and related antibiotics.
    
29. 
    Each of the genes or partial gene constructs would be expressed in wheat or barley via one of ten different constitutive (ie widely expressed throughout the plant), tissue specific or inducible promoters derived from wheat, barley, maize and rice as shown in Table 2. The transcription termination regions for the introduced genes are derived from Agrobacterium, rice or wheat. Regulatory elements are discussed further in Section 96 of this Chapter.
    
30. 
    The genes proposed for use in this application and their effects have not been fully characterised in the GM wheat and barley lines. In Table 1 and the following sections, the genes and their encoded products are described in brief to illustrate their potential function within the GM wheat and barley lines.
    

Table . The genes or partial gene sequences introduced into the GM wheat and barley

Gene Number	Gene^a(Source organism^b)	Encoded or predicted protein	Anticipated function (effect) of introduced gene or partial gene
Group 1 - Glucan water dikinase RNAi^c
1	RNAi targeting GWD (Ta)	None	Suppression of GWD gene in the endosperm (altered grain composition)
Group 2 - Alanine aminotransferase
2	AlaAT (Hv)	Alanine aminotransferase	Improved nitrogen use efficiency
Group 3 - Starch branching enzyme RNAi^c
3	RNAi targeting TaSBE IIa^d	None	Suppression of SBE IIa gene and SBE IIb (altered amylose content)
4	RNAi targeting TaSBE IIb	None	Suppression of SBE IIb gene (altered amylose content)
5	RNAi targeting TaSBE I^d	None	Suppression of SBE I gene
Group 4 - Enhanced carbon assimilation, drought and heat tolerance or water use efficiency
6	TaMYBc1	MYB transcription factors	Enhanced carbon assimilation and grain weight
7	TaMYB807 ^e
8	TaMYBc2
9	TaMYB83
10	TaMYB96
11	TaWRKY2	WRKY[Zn] transcription factor
12	TaNF-YAc1^e	NF-Y transcription factor
13	TaB3DBP1	B3 transcription factor
14	TabZIP1	bZIP transcription factors
15	TabZIPc1
16	TaRWD1	RWD transcription factor
17	TaCAT1	Calcium-binding protein
18	TaAuTP1	Auxin carrier
19	TaFBPase^e	Fructose 1,6-bisphosphatase
20	TaIP	Metabolic enzyme	Associated with grain weight and yield
21	TaPSL1	Highly expressed protein with unknown function	Associated with stem water soluble carbohydrates (WSC), grain weight and yield
22	TaPGF1	Peptide growth factor	Enhanced growth, carbon assimilation and grain weight
23	HvEH1	EH-domain protein	Enhanced carbon assimilation and grain weight and yield
24	TaDofsu1^e	Dof transcription factor	Improved biomass production
25	TaNF-YBps1^e	NF-Y transcription factors	Improved photosynthesis
26	TaNF-YCps1^e
27	TaNACdu1^e	NAC transcription factor	Improved water use efficiency
28	TaZFPdu1^e	C2H2 (ZFP) transcription factor	Improved drought tolerance and water use efficiency
29	HvCBF^e	AP2 transcription factor
30	TaHsf1	Heat shock transcription factor	Improved drought and heat tolerance and water use efficiency
31	TaHsf2		Improving heat tolerance
Group 5 - Enhanced fibre content
32	CME A^e (Hv)		Enhanced fibre content
33	CME B^e (Hv)
Group 6 - Enhanced rust resistance
34	Lr34 (Ta)	ABC transporter-like protein	Enhanced rust resistance
Selectable marker
35	nptII (E. coli)	neomycin phosphotransferase type II	Kanamycin (and related antibiotic) resistance
36	hpt (E. coli)	hygromycin phosphotransferase	Hygromycin resistance
37	bar (S. hygroscopicus)	phosphinothricin acetyl transferase	Herbicide resistance
^a The gene names, as supplied by the applicant. ^b Some gene names have two-letter prefixes to denote the species of origin (source organism). Otherwise, this is provided in brackets: Hv=Hordeum vulgare; Ta=Triticum aestivum.^c Only partial gene sequences are used in order to suppress expression of the corresponding endogenous genes. ^d These RNAi contain partial sequences from the SBE IIa and SBE I genes derived from Aegilops tauschii, which is the donor of D genome in wheat. They are therefore identical to TaSBE IIa and TaSBE I, respectively. ^e Identities of these genes are Commercial Confidential Information (CCI). The alternative names, provided by the applicant, are therefore used for these genes in this table and elsewhere in the RARMP.

Table . Gene constructs used to generate the GM wheat and barley lines proposed for release

Gene^a	Construct	Promoter^b	Terminator^c	Parental cultivar	Max no of GM lines	Previous DIR licence
Group 1 - Glucan water dikinase RNAi
1	pBX17GWDcasNOT	Bx17	nos	Bobwhite 26	3	092, 099
	pBX17GWDtwin	Bx17	nos	Bobwhite 26	10	New
	pBX17GWDdrb	Bx17	nos	Bobwhite 26	10
Group 2 - Alanine aminotransferase
2	pARC425	OSAnt1	nos	Bobwhite 26	3	094, 099
	pMDC/POsAnt1 HvAlat Nos	OSAnt1	nos	Bobwhite 26	15
				Frame	2
				Gladius	15	New
				Golden Promise	20
	pARC316	OSAnt1	nos	Gladius	20
	pARC316drb	OSAnt1	nos	Bobwhite 26	20
	pARC425drb	OSAnt1	nos	Bobwhite 26	20
	pMDC-MF1/POsAnt1 HvAlat Nos	OSAnt1	nos	Golden Promise	20
Group 3 - Starch branching enzyme RNAi
3	SBE IIa RNAi	Dx5	nos	NB1	6	093
	BEa3-IR	Dx5	nos	NB1	4	New
	BEa5-IR	Dx5	nos	NB1	4
3 and 5	BEcombo-IR	Dx5	nos	NB1	4
3 and 4	SBE IIa RNAi & SBE IIb RNAi	Dx5	nos	Golden Promise	1	093
Group 4 - Enhanced carbon assimilation, drought and heat tolerance or water use efficiency^d
6	PCR-amplified fragment	Dhn8s or SFT1s	rbcS	Bobwhite 26	6	100
7		SFT1s	rbcS		3
8		SFT1s	rbcS		3
9		Ubi1	rbcS		3
10		Dhn13s	rbcS		3
11		Dhn13s	rbcS		3
12		Dhn8s	rbcS		3
13		Dhn13s	rbcS		3
14		Dhn8s	rbcS		3
15		Dhn8s or SFT1s	rbcS		3
16		Dhn13s	rbcS		3
17		Ubi1	rbcS		3
18		Ubi1	rbcS		3
19		Dhn8s	rbcS		3
20		Ubi1	rbcS		3
21		Dhn8s	rbcS		3
22		Ubi1	rbcS		3
23		Dhn8s	rbcS		3
24		Ubi1	rbcS		3
25		Ubi1	rbcS		3
26		Ubi1	rbcS		3
27		Dhn8s or Dhn4s	rbcS		4
28		HVA1s	rbcS		3
29		Dhn4s	rbcS		3
30		HVA1s	rbcS		3
31		HVA1s	rbcS		3
Any combination of genes 6-31	Multiple PCR-amplified fragments	As above	rbsS		60
Group 5 - Enhanced fibre content
32	pSJ6	Bx17	nos	Bobwhite 26	3	New
33	pSJ33	Bx17	nos	Bobwhite 26	6
Group 6 - Enhanced rust resistance
34	LR34pWGEMN rc	Lr34	Lr34	Bobwhite 26	5	New
^a Use the gene numbers listed in this column to refer to full gene names in Table 1. Most of the GM wheat and barley lines also contain one of three selectable marker genes (gene number 35, 36 and 37 in Table 1). ^b Bx17, endosperm-specific promoter from wheat Bx17 seed storage gene; Dhn4s, drought-inducible promoter from the barley dehydrin 4s gene; Dhn8s and Dhn13s, constitutive promoters from the barley dehydrin 8s and dehydrin 13s genes, respectively; Dx5, endosperm-specific promoter from the wheat High Molecular Weight Glutenin subunit Dx5 gene; HVA1s, drought-inducible promoter from a barley class 3 late embryogenesis-abundant protein gene; Lr34, native promoter from the wheat Lr34 gene; OSAnt1, root-specific promoter from rice antiquitin gene; SFT1s, development-specific promoter from the barley sucrose:fructan 6-fructosyltransferase gene; Ubi1, constitutive promoter from the maize ubiquitin 1 gene. ^c nos, nopaline synthase gene terminator from A. tumefaciens; rbcS, RuBisCo small subunit gene terminator from rice; Lr34, native terminator from the wheat Lr34 gene. ^d Some GM wheat lines in Group 4 may also be cross-bred to produce GM wheat with more than one introduced gene of interest.

The introduced RNAi constructs and their associated effects (Group 1 and 3)

31. 
    In the current application, some wheat and barley lines were genetically modified using RNAi constructs (Groups 1 and 3, Table 1 and Table 2). The RNAi constructs contain fragments, rather than entire coding sequences, of four target genes. Expression of the RNAi constructs is designed to suppress the expression of the target genes. No proteins are encoded by the introduced RNAi constructs.
    
32. 
    RNAi is a mechanism that occurs naturally in plants and other organisms and functions to control the expression of specific genes and remove aberrant RNA molecules (Agrawal et al. 2003). Systemic silencing is generally difficult to achieve; in the case of the GM wheat and barley, organ specific silencing is achieved through using the endosperm specific promoter. In plants, RNAi constructs can also give rise to silencing of closely matching non-target sequences expressed in the same cells. Homology of 95% is generally required for any silencing effect, and increases with greater stretches of homology to the non-target gene. Homology of as little as 20 nucleotides (nt) can give rise to non-target silencing, reviewed by Small (2007). Specific silencing of a single gene of a gene family can be achieved if a specific region of the gene is targeted. Conversely, using highly conserved regions can result in effective silencing of several members of a gene family (Miki et al. 2005).
    
33. 
    When an introduced RNAi construct is expressed, the self complementary parts (sense and anti-sense sequences from the target gene) of the RNA anneal to each other to produce a double stranded RNA, and the intervening intron is spliced out. These duplexed regions are then cut into 21 nucleotide (nt) fragments by the enzyme Dicer. These 21 nt short RNAs (called short interfering RNA, siRNA) guide nuclease complexes to recognise the targeted single-stranded mRNAs for cleavage, leading to the prevention of translation and silencing the targeted gene.
    
34. 
    The Glucan Water Dikinase (GWD) gene
    
35. 
    Gene 1 (Group 1) encodes a siRNA targeting the GWD gene. The GWD gene encodes α-glucan water dikinase (GWD), a starch granule-bound enzyme, which catalyses the ATP dependent phosphorylation of a-glucans. Glucose residues of amylopectin can be phosphorylated on either the C3 or the C6 positions. Experiments with Arabidopsis have shown that GWD phosphorylates the C6 position, and a second enzyme, phosphoglucan water dikinase (PWD), phosphorylates the C3 position (Kotting et al. 2005; Ritte et al. 2006). However, the activity of PWD is dependent on the preceding action of GWD, implying that any defect in the activity of GWD results in a reduction in the phosphorylation of both glucosyl positions. Phosphatase enzymes likely reverse the effects of these kinase enzymes (Hejazi et al. 2010). Phosphorylation of starch is thought to occur during both starch synthesis and starch degradation (Blennow et al. 2002).
    
36. 
    Studies of GWD mutants in Arabidopsis and potato indicate that starch phosphorylation is an important part of starch degradation, as these mutants have a reduced rate of starch degradation, and accumulate excess leaf starch as a result (reviewed by Smith et al. 2005). The level of phosphorylation of starch varies among species, with potato tuber starch being relatively highly phosphorylated (one in 200-300 residues) compared to cereal starches (one in more than 10,000 residues) (reviewed by Mikkelsen et al. 2004). Starch phosphorylation contributes to processing qualities such as pasting, gel strength and stickiness. It is thought that levels of starch phosphorylation may influence the rate of degradation of starch by digestive enzymes (information provided by applicant).
    

Effects of GWD silencing

37. 
    The applicant has provided test results to show that for some GM wheat lines containing a GWD RNAi construct, GWD can no longer be detected. In these lines, the amount of phosphate in the starch is greatly decreased in comparison to the parental wheat cultivar. Except for the decrease in phosphate, no structural or quantitative modifications of the starch composition have been observed.
    
38. 
    The applicant states that the reduction in starch phosphate in the GWD RNAi lines may give rise to altered processing properties, particularly in relation to pasting, for which starch phosphorylation is known to be an important factor (reviewed by Blennow et al. 2002). Nutritional value may be altered in wheat products derived from the GWD RNAi lines, as starch phosphorylation is known to play a role in starch degradation. There is a wide range in levels of starch phosphorylation among plant crops with potatoes considered to produce highly phosphorylated starches and cereals lightly phosphorylated starches (Blennow et al. 2002). The changes observed in the GWD RNAi lines are largely within the range of starch phosphorylation values observed for wheat (Ral et al. 2008).
    
39. 
    Data supplied by the applicant showed that GM wheat lines carrying the GWD RNAi construct display an increase in plant vigour at early growth stages compared to non-GM sibling plants, measured by leaf area (an increase between 40-80%) and dry weight of the above ground tissues (an increase between 20-50%) depending on the line. Mature GM plants exhibit various increases in biomass, grain weight and yield. Field trials for some of the GM wheat lines trialled under licence DIR 092 further confirmed this increase in plant vigour at both early growth and mature stages under field conditions. However, the GM lines produced fewer tillers than the non-GM controls.
    
40. 
    GWD RNAi lines also show an increase in seed production compared to the parental non-GM wheat variety, the result of both an increased number of heads per plant and increased seed weight. However, the increased seed weight is within the natural range observed in 372 diverse wheat lines studied by Bordes et al. (2008).
    
41. 
    The Starch Branching Enzyme (SBE) genes
    
42. 
    Genes 3, 4 and 5 (Group 3) encode siRNAs targeting starch branch enzymes. Starch is an insoluble polymer made up of two D-glucose homopolymers; amylopectin and amylose. Amylopectin is a molecule with a high degree of structural organisation. It is made up of glucose molecules, ranging between 3x10 and 3x10⁵⁶ glucose units, joined via α-1-4 linkages generating linear chains of various lengths. In addition it has α-1-6 branch points, occurring every 24-30 glucose units, catalysed by starch branching enzymes (SBEs) (Myers et al. 2000; James et al. 2003; Van Hung et al. 2006). Amylose is essentially a linear molecule made up of glucose molecules, ranging between 500-6000 glucose units, joined through α-1-4 linkages. It can also contain a small number of α-1-6 branches (Myers et al. 2000; James et al. 2003; Van Hung et al. 2006). The amylopectin component of starch is approximately 70-80% with amylose making up the remaining 20-30%.
    
43. 
    The synthesis of starch in the cereal endosperm involves three main classes of enzymes; the starch branching enzymes (SBE), starch synthases (SS) and starch debranching enzymes. In cereals some isoforms⁸ of these enzymes are unique to the endosperm (Ball et al. 1996; Myers et al. 2000). Mutations in the biosynthesis pathway of either amylose or amylopectin can lead to the production of different ratios of amylose to amylopectin (Ball et al. 1996; Myers et al. 2000; Jobling 2004; Van Hung et al. 2006).
    
44. 
    A change in the amylose and amylopectin composition of starch can also have profound effects on starch physical properties and thus alter flour and dough properties. This can result in flour products with unique qualities that allow for new uses in food and also in non food related applications (Van Hung et al. 2006). For example, low amylose flours are seen as beneficial in the pasta and noodle industry as they result in the production of a higher quality product with better texture. In frozen foods its use is seen as beneficial in improving freeze-thaw stability and preserving flavour (Jobling 2004). More detail on the effects of altered starch composition on flour properties can be found in reviews by Jobling (2004) and Van Hung (2006).
    
45. 
    SBEs catalyse the formation of branches on the linear chain of glucose molecules by generating α-1-6 linkages through cleavage of α-1-4 linkages. Sequences and in vitro activity of SBEs are relatively well characterised, but this knowledge has not resulted in clear understanding of their specific in vivo functions.
    
46. 
    There are two classes of SBEs: SBE I and SBE II (Boyer & Preiss 1978). In vitro studies have shown that SBE II enzymes transfer shorter chains than SBE I and have a higher affinity towards amylopectin. SBE I shows a higher affinity for branching of amylose (Takeda et al. 1993; Tetlow et al. 2004).
    
47. 
    Cereals have two closely related isoforms of SBE II; SBE IIa and SBE IIb, each has a distinct role in endosperm starch synthesis; SBE IIa transfers longer chains than SBE IIb (Boyer & Preiss 1978; Mizuno et al. 1993; Morell et al. 1997; Sun et al. 1998). There is some indication of interactions of SBE with SS as a number of SS mutants show a decrease in the activity of SBE and vice versa (Tetlow et al. 2008).
    
48. 
    Spatial expression of the SBE IIa and SBE IIb genes varies among species. In wheat, rice and maize, SBE IIb appears to be more dominantly expressed in the endosperm. In wheat SBE IIa is expressed mainly within the soluble phase of the endosperm, in maize the SBE IIa form is primarily expressed in the leaves. In barley, SBE IIa is expressed in the endosperm, embryo and vegetative tissues, whereas SBE IIb is expressed only in the endosperm. Both isoforms have similar levels of activity in the barley endosperm (Sun et al. 1998). However, SBE I has been shown to be expressed predominately in seed in maize, rice and wheat (Baba et al. 1991; Kawasaki et al. 1993; Rahman et al. 1999)
    
49. 
    Mutations in the SBE IIa and/or SBE IIb genes result in grains with an increase in the proportion of amylose in starch [see for example Mizuno et al (1993) and Tetlow et al (2004)]. Mutations in SBE IIb can also affect the amount of branching of the residual amylopectin and result in the production of material that is described as being intermediate between amylose and amylopectin (Boyer et al. 1980; Takeda et al. 1993). Mutations in SBE IIa do not appear to alter starch structure (Blauth et al. 2001). In cereals such as maize and rice, the absence or down regulation of SBE IIb in the endosperm leads to higher amylose content in the grain (Garwood et al. 1976; Nishi et al. 2001). In contrast, SBE IIa is a more important isoform in wheat, as silencing of SBE IIa results in dramatic increase of amylose content but silencing of SBE IIb does not alter grain starch composition (Regina et al. 2006).
    
50. 
    In maize, rice and wheat, mutations in the SBE I gene only appear to have minimal impact on the starch content or composition (Blauth et al. 2001; Blauth et al. 2002; Satoh et al. 2003; Regina et al. 2004).
    
51. 
    The wheat TaSBE I and TaSBE IIa genes were isolated from Aegilops tauschii (donor of the D genome in wheat) (Rahman et al. 1999) and the TaSBE IIb gene was isolated from Triticum aestivum (Regina et al. 2005). The naturally occurring TaSBE IIa and TaSBE IIb genes share 86% homology at the protein level and differ mainly at the amino terminus of the protein. The TaSBE IIa sequences from wheat and barley share 98 % sequence homology at the protein level, while the TaSBE IIb sequences from wheat and barley share 95% homology at the protein level (National Center for Biotechnology Information 2001).
    

Effects of SBE silencing

The aim of the genetic modification using the RNAi constructs is to suppress the expression of the corresponding endogenous SBE genes. Suppression of endogenous SBE genes in the GM wheat and barley alters the ratios of amylose to amylopectin produced. By suppressing selected enzymes required for the production of the amylopectin portion of starch, it has been shown that grains with an increase in amylose are produced.

52. 
    In GM wheat and barley lines proposed for release, the RNAi constructs used are under the control of the wheat Dx5 endosperm-specific promoter (Lamacchia et al. 2001). As this promoter is endosperm-specific, the applicant has only characterised the phenotypes of GM wheat and barley lines on seed material, and has not examined changes in other tissues.
    
53. 
    The GM wheat lines 85.2c, 212, YDH7, UW89o.2, X5.3-1 and SSI-853 were generated using the same SBE IIa RNAi construct, which is designed to suppress the expression of the TaSBE IIa gene. The sense and anti-sense sequences are from exons 1-3 of the TaSBE IIa gene (GenBank Accession Number AF338431) and the two sequences are separated by intron 3 from the same gene. The lines 85.2c, 212, YDH7 and SSI-853 have been included in the trials under the licence DIR 093. Expression of this RNAi construct leads to the loss of SBE IIa protein in the transgenic lines, resulting in a reduction in amylopectin biosynthesis and a significant increase in the proportion of amylose in the starch. In these high amylose lines, starch granules are also distorted in appearance.
    
54. 
    The GM wheat line 212 contains the SBE IIa RNAi construct in a SBE I triple null⁹ mutant background (Regina et al. 2004). The SBE I triple null mutant does not show any change in amylose content in wheat (Regina et al. 2004). Characterisation of GM wheat line 212 shows an overall decrease in starch content and a large increase in amylose content compared to the non-GM control.
    
55. 
    The GM wheat line YDH7 contains the SBE IIa RNAi construct in a triple null SS IIa mutant background (Yamamori et al. 2000). Based on other research the triple null mutant line is expected to have increased amylose content (Bird et al. 2007). Mutations in SS II can also lead to a reduction in expression of SBE IIa (Gao et al. 1996). Characterisation of the GM wheat line YDH7 shows a similar overall starch content to the parent line as well as an increased amylose content.
    
56. 
    According to information provided by the applicant, the amylose content varies in GM wheat lines containing different RNAi construct targeting different regions of the SBE genes. The GM wheat lines IIa3’-1 to IIa 3’-4 and IIa5’-1 to IIa5’-4 contain the RNAi constructs BEa3-IR and BEa5-IR respectively designed to alter the expression level of SBE IIa, while the wheat lines Combo-1 to Combo-4 contain the RNAi construct BEcombo-IR designed to suppress the expression levels of SBE I, SBE IIa and SBE IIb genes simultaneously. BEa3-IR and BEa5-IR contain sense and anti-sense sequences from different exons of the TaSBE IIa gene, separated by an intron from the rice tubulin gene (GenBank accession number AF525764). BEcombo-IR contains combined sense and anti-sense sequences from exons of both TaSBE I (GenBank accession number AF525764) and TaSBE IIa genes, separated by the rice tubulin gene intron. The level of amylose content varies in these lines.
    
57. 
    The barley line BC10.5 contains two constructs designed to suppress the expression of both SBE IIa and SBE IIb. This barley line shows altered starch granule morphology and a high amylose phenotype (>70%).
    
58. 
    In GM wheat lines with a construct targeting SBE IIa gene, there is also an associated reduction in the amount of SBE IIb. This effect is not due to DNA sequence similarity resulting in cross-silencing (Regina et al. 2006). In high amylose rice, with a mutation in SBE IIb, a reduction in starch synthase I (SS I) activity was observed, most likely through post transcriptional inhibition of this enzyme. In contrast, transcriptional levels of SS I, SBE IIa and SBE I were not affected (Nishi et al. 2001). The GM wheat and barley lines have not been fully characterised, so the effect of the RNAi constructs on other enzymes involved in starch biosynthesis has not been determined.
    

1. 
   Toxicity/allergenicity associated with the introduced RNAi constructs
   

59. 
    In the GM wheat lines modified for grain starch composition, the use of RNAi has the direct effect of reducing the expression of endogenous transcripts of the target genes, without the expression of novel proteins. The silencing of GWD and SBE genes results in changes to the starch phosphorylation and starch composition of the GM wheat and barley grains, respectively.
    
60. 
    GWD RNAi lines have a decreased level of phosphate in the starch but this is still largely within the range of starch phosphorylation values observed for wheat (Ral et al. 2008).
    
61. 
    The main change displayed by the GM wheat and barley lines containing any SBE RNAi construct is the ratio of amylose to amylopectin in the grain. The grain of non-GM wheat or barley normally contains 25% amylose and 75% amylopectin. The applicant has shown that the SBE RNAi lines all have varying increases in the proportion of amylose. Based on consumption of cereals with similar starch composition, the increased amylose and decreased amylopectin observed in the GM wheat and barley lines are unlikely to be detrimental to human health. A non-GM high amylose barley variety has been previously used in human nutritional studies. The level of amylose in this barley variety was determined to be approximately 70% compared to 25% in the parent variety (Morell et al. 2003). In these nutritional studies, no adverse effects were evident as a result of the high amylose content of the grains.
    
62. 
    Humans are exposed to vast amounts of dietary starch, from a range of sources varying in the parameters changed in the GM lines. For example, conventionally bred high-amylose maize has been incorporated into a wide range of food products since the early 1990s. Also, levels of starch phosphorylation vary greatly between starches from different plant sources. Since no allergic or toxic effects have been reported for human or animal consumption of starch, it is highly unlikely that the changes occurring in the SBE silencing lines would result in altered toxicity or allergenicity of the GM wheat lines compared to parental cultivars.
    
63. 
    No studies on the toxicity or allergenicity of the GM wheat and barley lines and their products have been undertaken to date as the proposed trial is at an early stage. Although the proposed rat and pig studies are not designed as toxicity or allergenicity studies, they may still provide some information about potential adverse effects. Such studies may need to be conducted if approval was sought for the GMOs or their products were to be considered for human consumption in Australia.
    
64. 
    The other introduced genes (Groups 2, 4, 5, 6 and selectable markers) and their encoded proteins
    
65. 
    In addition to the partial gene sequences described above (Section ), 30 genes (Groups 2, 4, 5 and 6, Table 1) are proposed for use in wheat and barley.
    
66. 
    The introduced AlaAT gene, for enhanced nitrogen utilisation efficiency, and the encoded protein
    
67. 
    Nitrogen use efficiency (NUE) is an important factor in crop plant productivity. Nitrogen based fertilizers are used extensively in modern agriculture, including for wheat and barley. Further details of NUE are provided in the RARMP for DIR 094 (available at