Dir 128 Full Risk Assessment and Risk Management Plan (rarmp)

).

Nitrogen use efficiency is an important factor in crop plant productivity, this forming the impetus for research, both using conventional breeding and gene technology, to develop plants that absorb and use this element more efficiently. It can be described as the product of two components: uptake efficicency and utilisation efficiency (Witcombe et al. 2008). Increasing the efficiency of nitrogen use would reduce the need for fertilisers in agriculture and the pollution that excess nitrogen can cause in the environment. Further details of this trait are provided in the RARMP for DIR 094 (available at ).

The other trait that the applicant is attempting to engineer into plants is increased uptake of the micronutrient iron. Deficiencies in micronutrients affects more than 2 billion of the world’s population, with iron deficiency being the most common (Tulchinsky 2010; WHO 2014). Biofortification, the development of new crop varieties with increased levels of micronutrients, by either conventional breeding practices or genetic modification, provides an extremely promising approach to dealing with this problem (Nestel et al. 2006). In particular, it allows the staple food supplies of countries to be used and new germplasm to be spread internationally.

Group 1 – Drought tolerance

The identities of all of the introduced genes linked to drought tolerance have been declared as CCI. The confidential information was made available to the prescribed experts and agencies that were consulted on the RARMP for this application.

In reference to their general function in the cell, these genes are involved in either signalling, transcription, photosynthesis and metabolism, cell specification, proliferation and division, or RNA metabolism. Some involve the expression of microRNAs in plants, thus down-regulating the expression of genes by RNA interference (RNAi).

Group 2 – Salt tolerance

ScNHA1

Some GM wheat and barley lines will contain the ScNHA1 gene encoding a Na⁺, K⁺/H⁺ antiporter (NHA) derived from Saccharomyces cerevisiae (yeast)(Prior et al. 1996). The gene belongs to the CPA2 (cation/proton antiporter) family of Na⁺/H⁺ antiporters in eukaryotes and resides in the subfamily NHA, which includes animal, plant, fungal and bacterial members (Brett et al. 2005).

Deletion of the ScNHA1 gene was found to increase the cytosolic pH of yeast cells suspended in water and acidic buffers, confirming a role of the protein in both regulating the cytoplasmic concentration of Na⁺/K⁺ and buffering the pH of cells (Sychrova et al. 1999). Other studies, investigating the growth of wild-type yeast and strains mutant with respect of this gene in media with varying concentrations of K⁺, have concluded that the protein can affect the main K⁺ influx system in this organism (Banuelos et al. 2002; Banuelos et al. 1998).

PpENA1

Some GM wheat lines and barley lines will contain PpENA1, a gene encoding an ENA-type (exitus natru) Na⁺ pumping ATPase derived from the moss Physcomitrella patens (Benito & Rodriguez-Navarro 2003). Eukaryotes have two types of Na⁺-pump ATPases, the Na⁺/K⁺-ATPases in animals and the ENA-type Na⁺-ATPases in fungi and mosses such as P. patens.

The PpENA1 protein has been shown to be able to complement a salt sensitive yeast strain deficient in Na⁺ and K⁺, as well as to act more generally as a Na⁺ pump in yeast (Benito & Rodriguez-Navarro 2003). Studies of the gene in the moss itself have demonstrated that its transcription is dramatically up-regulated in response to NaCl, and knockout mutants of the gene grow significantly slower than wild-type in media containing NaCl (Lunde et al. 2007).

AtAVP1

Some GM wheat lines and barley lines will contain the Arabidopsis gene AtAVP1, coding for a vacuolar H⁺-pyrophosphatase (H⁺-PPase).

H⁺-PPase proteins are proton pumps that use the energy gained from the breakdown of pyrophosphate to pump protons into the vacuoles of plant cells. This function of the proteins has been exploited in research aimed at enhancing the drought and salt tolerance of plants, the theory being that an increase in the activity of these proteins will generate a higher proton electrochemical gradient, thus energising secondary transporters, especially the Na^+/H⁺ antiporters (Gaxiola et al. 2012; Pasapula et al. 2011).

Overexpression of the AtAVP1 gene in Arabidopsis increased the tolerance of plants to both drought and salt stress (Gaxiola et al. 2001), and similarly the overexpression of a homolog from Thellungiella halophile in maize and cotton improved tolerance to drought in the former and salinity and drought in the latter (Li et al. 2008; Lv et al. 2008; Lv et al. 2009). An unexpected consequence of overexpressing these genes in plants was increased proliferation of roots and shoots. Arabidopsis plants overexpressing AtAVP1 had enhanced leaf areas, root growth and dry weight compared to those observed in wild-type plants, while a loss of function mutant had leaves and roots of reduced size (Li et al. 2005). Overexpression of the Thellungiella halophile gene in cotton was accompanied by increased dry shoot and root masses regardless of whether the plants were grown in the presence or absence of salt (Lv et al. 2008). Another study has shown that maize plants overexpressing the Thellungiella halophile gene were more tolerant to phosphate deficit stress than wild-type plants, this being possibly due to their larger root systems (Pei et al. 2012).

AtCIPK16

Some GM wheat lines and barley lines will contain AtCIPK16, an Arabidopsis gene belonging to a family of calcineurin B-like (CBL) interacting protein kinases (CIPKs) that play a role in regulating plant cell responses to abiotic stress (Luan 2009). Calcium (Ca²⁺) serves as a second messenger during abiotic stress signalling, its release being detected by a number of sensors, including the CBL proteins. These latter proteins recruit the required CIPK to the cell membrane, where the kinase activates transporters and proteins involved in the stress response (Batistic & Kudla 2004; Luan 2009).

There are at least 25 AtCIPK genes in Arabidopsis thaliana, each CIPK interacting with one or more members of the CBL family (Batistic & Kudla 2004). AtCIPK24 (also known as AtSOS2) was detected in a search for genes linked to salt tolerance in Arabidopsis, the mutant sos2 being hypersensitive to NaCl stress. Under salt stress, AtCBL4 (AtSOS3) recruits AtCIPK24 to the plasma membrane, where it activates (via phosphorylation) the Na⁺/H⁺ antiporter AtSOS1 to remove Na⁺ from the cell (Qiu et al. 2003; Qiu et al. 2002). Orthologs of CIPK24 have been identified in many plant species (Martinez-Atienza et al. 2007; Wang et al. 2004; Yu et al. 2007).

Map-based cloning has been used to identify AtCIPK16 as a gene linked to Na⁺ exclusion in Arabidopsis (Roy et al. 2013). Overexpression of the gene in Arabidopsis increased salt tolerance in both hydroponic and soil cultures, while conversely, use of a microRNA to suppress its expression in that plant resulted in elevated levels of Na⁺ in shoots grown in saline media. Overexpression of the gene in barley was also associated with increased salinity tolerance.

Group 3 – Aluminium tolerance

TaALMT1, TaALMT1_minus_insert, ScALMT1.M39.1_wt, ScALMT1.M39.1_plus_insert

Some GM wheat lines and barley lines will contain one of seven genes coding for aluminium activated malate transporters (ALMTs). These genes come from wheat and rye (Secale cereale), some representing in vitro generated variants and mutations (single or double amino acid substitutions) of wild-type sequences. The excretion of malate has been linked to aluminium tolerance in plants, and the addition of malate to solutions containing aluminium can protect plants from toxic levels of this metal by forming a stable harmless complex (Delhaize & Ryan 1995; Delhaize et al. 1993). ALMTs are membrane localised proteins that facilitate the efflux of malate into the apoplast of cells (Sasaki et al. 2004; Yamaguchi et al. 2005), their heterologous expression in a range of organisms (including Xenopus laevis oocytes and the cells of various plants) being associated with an aluminium tolerance phenotype (Delhaize et al. 2004; Sasaki et al. 2004; Yamaguchi et al. 2005). In some cereals, such as rye, the number of ALTM1 genes at a certain chromosomal loci can vary (Collins et al. 2008).

The identities of three of the introduced genes linked to aluminium tolerance have been declared as CCI. The confidential information was made available to the prescribed experts and agencies that were consulted on the RARMP for this application.

HvAACT1

Some GM wheat lines and barley lines will contain the barley gene HvAACT1 (HvMATE), coding for a Multidrug and Toxic Compound Extrusion (MATE) protein. Members of the MATE family of membrane transport proteins occur in both eukaryotes and prokaryotes. They are associated with the transport of a variety of organic molecules, including cationic drugs and plant secondary metabolites such as alkaloids (Omote et al. 2006). HvAACT1 is a citrate transporter which, in the presence of aluminium, facilitates the efflux of citrate from cells, this organic acid (in the manner of malate outlined above) chelating the metal and rendering it harmless.

Group 4 – Nitrogen use efficiency

The identities of all of the introduced genes linked to nitrogen use efficiency have been declared as CCI. The confidential information was made available to the prescribed experts and agencies that were consulted on the RARMP for this application.

In reference to their general function in the cell, these genes are involved in either transcription, primary metabolism, or cell proliferation and division.

Group 5 – Micronutrient uptake

OsNAS2

Some GM wheat lines will contain a rice nicotianamine synthase gene (OsNAS2), the aim being to produce “biofortified” wheat (ie increasing the level of iron in wheat, thus being useful in regions of the world where wheat is a staple crop and iron deficiency amongst people is a problem).

Nicotianamine, a chelator and long distance transporter of transition metals such as iron, is biosynthesised by the trimerisation of S-adenosylmethionine, a reaction catalysed by the NAS enzyme; a by-product of the reaction is S-methyl-5’-thioadenosine. The mugineic acid family of phytosiderophores, molecules also involved in the acquistion of iron from the soil, are produced from nicotianamine by the subsequent activity of other enzymes (Higuchi et al. 1999; von Wiren et al. 2000). Amongst grasses, each species produces its own set of mugineic acids (Kim & Guerinot 2007).

The NAS gene family is largely plant specific, but examples of related genes are known from fungi and archaea (Herbik et al. 1999; Trampczynska et al. 2013). NAS genes have been isolated, or identified via bioinformatics in published genome sequences, from a number of plant species. Among the cereals, these include barley (Herbik et al. 1999; Higuchi et al. 1999), maize (Zhou et al. 2013), and rice (Higuchi et al. 2001).

Overexpression of NAS genes in plants has been shown to increase the levels of both nicotianamine and transition metals in cells. Overexpression of a barley NAS gene in both Arabidopsis and tobacco resulted in plants that showed improved tolerance to nickel, as well as accumulating large quantities of this metal in their shoots (Kim et al. 2005), while overexpression of the same gene in rice showed increased levels of both iron and zinc in seeds (Masuda et al. 2009). The combined expression in rice of an Arabidopsis NAS gene and genes coding for a ferritin and phytase led to a six-fold increase in the iron content of grain (Wirth et al. 2009). In another study in rice, three rice NAS genes were independently overexpresseed, in each case leading to increased levels of both iron and zinc (Johnson et al. 2011).

Selectable marker genes

The vectors used to transform plant tissue contain one or both of two selectable marker genes. These are the neomycin phosphotransferase II (nptII) and hygromycin phosphotransferase (hpt) genes, both from E.coli. The proteins these genes encode inactivate kanamycin (as well as a range of structurally related antibiotics) and hygromycin B, respectively. More information on these genes, and selectable marker genes in general, can be obtained from the OGTR document Marker genes in GM plants, available on the OGTR website.

Toxicity/allergenicity associated with the introduced genes, their encoded proteins and associated products

The introduced genes originate from a range of “higher” plants (Arabidopsis, barley, maize, rice, rye, and wheat), the moss Physcomitrella patens and Saccharomyces cerevisiae (yeast). Other than Arabidopsis thaliana, all of the higher plants are widely consumed by people and animals, and as such people and animals have a long history of exposure to the proteins from these respective plants. P. patens is a common moss found around the peripheries of bodies of water, often exposed in late summer and early autumn. Isolates are available from North America, Europe, Africa, Japan and Australia (Prigge & Bezanilla 2010). There are no reports of this moss producing any toxic or allergenic compounds, but it should be appreciated that it is not part of the human diet. S. cerevisiae is a eukaryotic fungus that is commonly used in baking, brewing and wine making, as well as occurring on fruit and vegetables. As such, it has a long history of use by humans. There are no reports of any isolates of S. cerevisiae producing toxins that negatively affect the health of humans or animals (USEPA 1997).

A comprehensive search of the scientific literature yielded no information to suggest that the genes themselves, their protein products, or any associated products (except iron, see below) were toxic or allergenic to people, or toxic to other organisms. This includes homologues isolated from other species. However, no toxicity/allergenicity tests have been performed on any of the proteins. The protein encoded by one of the genes associated with drought tolerance (declared CCI) has an eight amino acid residue match with a known allergen. Matches of this length occur often when un-related amino acid sequences are compared, and is unlikely to imply that the protein will have allergenic properties (Goodman et al. 2008). Sequence homology searches may establish whether a protein has an allergenic potential, the length of homology being a balance between detecting meaningful information amongst false negative and false positive results (Codex Alimentarius Commission 2003). Further information that has been declared CCI and is pertinent to this gene was made available to the prescribed experts and agencies that were consulted on the RARMP for this application.

Expression of the OsNAS2 gene in wheat is aimed at increasing the level of iron in that plant. Wheat has approximately 30g/g of iron, biofortification targeting a content in this plant of 52g/g (Bouis et al. 2011). Iron is absorbed through the digestive tract and transferred around the body by a bloodstream protein called transferrin (Balmadrid & Bono 2009). This ion plays a number of roles in the body, most prominently being part of the haem group in proteins such as haemoglobin, cytochrome C and catalase. It is hence essential to the transport of oxygen to cells, oxidative phosphorylation, and the decomposition of hydrogen peroxide, respectively. However, excessive iron in the diet quickly leads to saturation of transferrin, resulting in free iron in the blood, something which is directly toxic to organs (Balmadrid & Bono 2009).

Conditions such as thalassemia can be further complicated by iron overload, inducing endocrine diseases, hepatic failure and even death. This may at least in apart be due to inhibition of the expression of hepcidin, an iron regulating peptide (Nemeth 2010; Tanno et al. 2007). Other genetic disorders also lead to excessive iron uptake. For example, hereditary haemochromatosis is characterised by excessive absorption of iron, leading to symptoms such as lethargy, upper abdominal discomfort and loss of libido (Barlow-Stewart et al. 2007).

Characterisation of the GMOs

Stability and molecular characterisation

All the genotypes of the GM plants are stable under glasshouse conditions. The copy number of introduced genes range from one to three in individual GM lines, but the genomic locations are not known for any of the introduced genes.

Phenotypic characterisation

Preliminary phenotypic characterisation of the plants in glasshouse conditions has demonstrated that the introduced genes induce no major visible phenotypes or reduce viability. However, some of the lines have shown delayed flowering, this sometimes occurring over a month later than usually observed. Such delayed maturity may reduce yield in the field, with plants being forced to endure greater exposure to hot and dry conditions.

The receiving environment

The receiving environment includes: any relevant biotic/abiotic properties of the geographic regions where the release would occur; intended agricultural 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 2013).

The factors relevant to the growth, distribution and cultivation of commercial wheat and barley can be found in The Biology of Triticum aestivum L. em Thell (Bread Wheat) (OGTR 2008b) and The Biology of Hordeum vulgare L. (barley) (OGTR 2008a).

The proposed dealings involve two sites in South Australia and three sites in Western Australia.

Site 1, “Glenthorne farm”, is located close to the University of Adelaide’s Waite Campus at O’Halloran Hill. The site is fully fenced and access is managed by a designated laboratory manager.

Site 2, “Karawatha”, is part of a commercial farming operation located in a dryland agricultural area in the Pinery region, approximately 70 km north of Adelaide. Access to the site will be controlled by the owner.

Site 3 is a farm at Kunjin, near Corrigin, Western Australia. The site will be managed by a private company that provides research services.

Sites 4 and 5 are in NGNE facilities, respectively located at Katanning and Merredin, and operated by DAFWA.

Relevant abiotic factors

As noted above, the release is proposed to take place at five sites. Two of these (O-Halloran Hill and Pinery) are located in South Australia, and three (Corrigin, Merredin and Katanning) are located in Western Australia.

• 
  O-Halloran Hill is typical of rain-fed wheat production environments in South Australia, while Pinery is located in a dryland agricultural area that is useful for the assessment of those GM lines that are engineered to show increased drought tolerance.
  
• 
  Corrigin is located in a region of Western Australia that has saline soils and is occasionally subjected to frosts. At present the level of salinity is such that the land is not subject to broad acre cropping. However, these environmental factors make the land useful for the trialling of plants with increased tolerances to salt and frost.
  
• 
  Katanning and Merredin are purpose built facilities, one (Katanning) representing the high rainfall environment used for growing wheat in Western Australia, and the other (Merredin), regions of that state where wheat is grown under low rainfall. The soil of Katanning is largely alkaline and sodic, while that of Merredin is a mixture of yellow sands, gravels, loamy earth and loamy duplex soils, but with also calcareous subsoils.
  

Relevant agricultural practices

It is not anticipated that the agronomic practices for the cultivation of the GM wheat and barley by the applicant will be significantly different from conventional practices for these plants.

GM wheat and barley seeds would be planted in the trial sites in winter or early spring.

The proposed 2 m buffer zone and 10 m monitoring zone surrounding the trial site would be either mowed, herbicide treated or weeded to maintain vegetation at a height of less than 10 cm.

The applicant proposes to harvest all GM wheat and barley at maturity by hand or by machine (such as a custom Plot Harvester). Threshing of GM plants would occur on site, or heads transported to approved facilities for analysis and processing.

Seed that remains after harvest would be either stored in an approved facility for subsequent use or destroyed.

Volunteers would be removed by hand or killed by herbicide application.

Presence of related plants in the receiving environment

The site of O’Halloran Hill is close to urban areas of Adelaide, the nearest commercial production areas for either wheat or barley being approximately 50 km distant. However, the other four sites are located in regions where cereals are grown as crops.

Barley and wheat are not known to hybridise with each other under natural conditions, but do hybridise with a range of other plants present in the Australian environment. This is discussed in the two OGTR documents, The Biology of Triticum aestivum L. em Thell (Bread Wheat) (OGTR 2008b) and The Biology of Hordeum vulgare L. (barley) (OGTR 2008a). A summary of the information contained in these documents is presented below.

Wheat (Triticum aestivum) is sexually compatible with a number of species within the tribe Triticeae that occur in Australia. Of particular importance are durum wheat (Triticum turgidum ssp. Durum), rye (Secale cereale), and Triticale. Hybridisation with durum wheat occurs readily (Wang et al. 2005), whereas that with rye (Dorofeev 1969; Leighty & Sando 1928; Meister 1921) and Triticale (Ammar et al. 2004; Kavanagh et al. 2010) is rarer. It also readily hybridises with Aegilops species, but although some specimens of this genus have been collected in Australia, presumably originating from seed accidently introduced or straying from that brought in for breeding programs (AVH 2012), no Aegilops species is considered to be naturalised.

Australasia possesses four native Triticeae genera – Australopyrum, Stenostachys, Anthosachne (Elymus), and Connorochloa (Barkworth & Jacobs 2011) – as well as a number of introduced species of Triticeae, such as Elytrigia repens (couch grass) and at least four Thinopyrum species (Bell et al. 2010). Thinopyrum ponticum (tall wheatgrass) has been used as a saltland pasture plant in Australia, and in some regions has come to be classified as a weed (Barrett-Lennard 2003; NYNRMP 2011). Although there has been no concerted investigation of natural hybridisation of these native and introduced Triticeae species with wheat, based on experience of hybridising wheat with most other members of the Triticeae, it is likely that it never occurs.

Barley is divided into three gene pools, the basis for this division being primarily the ability to form interspecific hybrids, and the use of data arising from molecular and cytogenetic studies (Zhang et al. 1999). The primary gene pool consists of Hordeum vulgare ssp vulgare (cultivated barley) and H. vulgare ssp spontaneum (the progenitor of cultivated barley), which are fully interfertile. H bulbosum constitutes the secondary pool, while the tertiary pool consists of approximately 30 Hordeum species. Hybridisation of H. vulgare and H. bulbosum usually results in the elimination of the genome of H. bulbosum and the formation of haploids of H. vulgare, but it has proven possible to produce partially fertile hybrids that can be backcrossed to H. vulgare (Pickering & Johnston 2005). Although hybrids can be formed between H. vulgare and members of the tertiary gene pool, due to infertility these have not proven useful in the introgression of germplasm into H. vulgare. However, at least in some cases, colchicine can be used to double chromosome number and lead to the production of hybrid seeds (Islam et al. 2007).

H. bulbosum has been grown as a pasture and forage grass in Australia (Knupffer 2009), and the tertiary pool species H. murinum ssp leporinum (barley grass), H. murinum ssp glaucum (blue barley grass) and H. marinum (sea barley grass) are also widespread in Australian pastures and the environment (Mallett & Orchard 2002; Smith 1968). However, the above described limitations on hybridisation between H. vulgare and plants from the secondary and tertiary gene pools implies there is negligible risk of gene transfer from the GM barley plants to these relatives. Hybridisation between barley and other species is virtually unknown in nature.

As the two NGNE facilities are multi-user facilities, it is possible that other GM and non-GM wheat and barley plants will be grown there in close proximity to those plants that are the subject of this application. Currently, GM wheat and barley plants from DIRs 099 and 112 may be present. Additionally, the other three proposed field trial sites currently have approval for GM wheat and barley from DIR 102 to be grown.

Presence of similar genes and encoded proteins in the environment

All the introduced genes and other genetic elements are from plants and bacteria that are widespread and prevalent in the environment (see ). Most of them are commonly consumed by people or people are naturally exposed to them.

The nptII and hph selectable marker genes are from E.coli, which is widespread in the environment.

Although some of the regulatory sequences are derived from plant pathogens, they comprise only small parts of the total genomes and cannot of themselves cause disease.

Relevant Australian and international approvals

Australian approvals

Previous approval by the Regulator

18. 
    Wheat and/or barley lines possessing some of the introduced genes have previously been approved for limited and controlled release by the Regulator under licence DIR 102:
    

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  AtAVP1
  
• 
  ScNHA1
  
• 
  PpENA1
  
• 
  AtCIPK16
  
• 
  TRANSCRIPTION FACTOR 6
  
• 
  Aminotransferase
  

19. 
    Information on previous DIR licences can be found on the GMO record on the OGTR website. There have been no reports of adverse effects on human health or the environment resulting from any of these releases.
    

Approval by other government agencies

No other approvals relating to human health and safety and environment are currently required from Australian government agencies for this GM wheat and barley trial. However, approvals may be required under some State governments’ legislation unrelated to human health and safety and the environment.

International approvals of GM wheat and barley

20. 
    Field trials of different GM wheat and barley plants have been approved internationally, including in the USA, Canada, Germany, Czech Republic, Denmark, Hungary, Iceland, Italy, Spain, Sweden and the United Kingdom. The traits that have been modified include: novel protein production, disease resistance, insect resistance, altered grain properties and herbicide tolerance⁴.