5.1 Introduction to the GMOs


20. The applicant proposes to release up to 11 varieties of GM cotton containing genes conferring insect resistance and/or herbicide tolerance traits. Some of the GM cotton varieties may also contain an antibiotic resistance selectable marker gene, which was used in the laboratory to select transformed GM plants during early stages of development.

21. The GM cotton varieties also contain short regulatory sequences that control expression of the introduced genes. Regulatory sequences were derived from plants (including thale cress), bacteria (Agrobacterium tumefaciens, Escherichia coli) or viruses.

22. The GM cotton varieties proposed for release include lines containing a single genetic modification (or ‘transformation’) event, as well as varieties containing two or more transformation events which have been combined (or ‘stacked’) by conventional cross-breeding between these lines. All but one of the GM cotton lines containing single transformation events have been previously approved by the Regulator for limited and controlled release in Australia. One GM cotton line, genetically modified for herbicide tolerance, had not been previously assessed by OGTR but has been approved for release by regulatory authorities in other countries.

23. Additional information relevant to the genetic modifications made to the GMOs is covered by CCI. The confidential information was made available to the prescribed experts and agencies that were consulted on the RARMP for this application.

5.2 The introduced genes and their encoded proteins


24. The genes conferring insect resistance traits to the GM cotton varieties were isolated from the common soil bacterium Bacillus thuringensis (Bt). Genes conferring herbicide tolerance were derived from organisms that are widespread in the Australian environment. The antibiotic resistance selectable marker gene was isolated from a commonly occurring bacterium.

25. Additional information relevant to the introduced genes and their encoded proteins is covered by CCI. The confidential information was made available to the prescribed experts and agencies that were consulted on the RARMP for this application.

5.3 Toxicity/allergenicity of the proteins associated with the introduced genes


26. The introduced proteins for insect resistance are derived from Bacillus thuringensis (Bt). Bt is found in soil and plant communities worldwide and strains have been isolated from habitats including soil, insects, stored-product dust and deciduous and coniferous leaves (Schnepf et al. 1998 and references therein). Microbial preparations of Bt have been used for decades as a pesticide, being first commercialised as insecticidal products in France in the late 1930s. Several microbial preparations of Bt, containing similar insecticidal proteins to those described in this application, are used in commercial Bt insecticide sprays in Australia (APVMA). There have also been numerous commercial releases of crops genetically modified to express Bt toxins for insect resistance (Sanchis & Bourguet 2008), both in Australia and overseas. On this basis, people and other organisms have a long history of exposure to Bt insecticidal proteins.

27. The World Health Organisation’s International Programme on Chemical Safety evaluated the environmental safety of use of Bt as a pest control agent and concluded that, because of the specificity of the mode of action of Bt toxins, Bt products are unlikely to pose any hazard to humans, other vertebrates, or the great majority of non-target invertebrates (International Programme on Chemical Safety 1999). In this report it was noted that Bt has not been reported to cause adverse effects on human health when present in drinking water or food. Two human studies found no observable health effect of an oral dose of 1000 mg of Bt spores per day for 3 or 5 days (McClintock et al. 1995; reviewed by Betz et al. 2000).

28. Inhalation and ingestion of Bt is not known to cause allergic reactions (International Programme on Chemical Safety 1999). A survey of farm workers who picked or packed vegetables that had been treated with Bt sprays (Bernstein et al. 1999) indicated that occupational exposure to Bt products could lead to induction of IgE and IgG antibodies. However, there were no reports of occupationally related clinical allergic disease arising from this immunological reaction in any of the workers. The USA Environmental Protection Agency has investigated several claims of dermal allergic reactions attributed to Bt microbial products, and determined that the reactions were not due to Bt itself or any of its insecticidal proteins. The reported reactions were determined to be due to non-insecticidal proteins produced during fermentation or to added formulation ingredients (EPA 2001).

29. Toxicity studies have been conducted on the proteins encoded by each of the introduced genes in the GMOs. All of the proteins were found to be non-toxic towards model vertebrate species.

30. Bioinformatic analysis may assist in the risk assessment process by predicting the allergenic potential of a protein based on similarity to known allergens. The results of such analyses are not definitive and are used to identify those proteins requiring more rigorous testing (Goodman et al. 2008). The applicant compared the predicted amino acid sequences of the proteins encoded by each of the introduced genes to a database of known allergens, and found that none of the proteins to be expressed in the GM cotton varieties has sequence or structure homology to any known allergen.

31. Food Standards Australia New Zealand (FSANZ) has previously assessed all of the GM cotton lines produced by single transformation events that are proposed for release and has approved them as safe for use in food. These approvals would also cover the stacked GM cotton varieties produced by conventional cross-breeding between approved lines.

32. Additional information relevant to toxicity or allergenicity of the GMOs is covered by CCI. The confidential information was made available to the prescribed experts and agencies that were consulted on the RARMP for this application.

5.4 The regulatory sequences


33. Promoters are DNA sequences that are required in order to allow RNA polymerase to bind and initiate correct gene transcription. Also required for gene expression in plants is a transcription termination region, including a polyadenylation signal. Other regulatory sequences, such as introns and protein targeting sequences, may contribute to the expression pattern of a given gene.

34. The introduced regulatory sequences of the GM cotton varieties are derived from plants (including thale cress), common soil and gut bacteria (Agrobacterium tumefaciens and Escherichia coli, respectively) or plant viruses. Although some of the regulatory sequences are derived from plant pathogens (A. tumefaciens, plant viruses), or an opportunistic pathogen of humans and animals (E. coli), the regulatory sequences comprise only a small part of the total genome, and are not in themselves capable of causing disease.

35. Additional information relevant to the introduced regulatory sequences is covered by CCI. The confidential information was made available to the prescribed experts and agencies that were consulted on the RARMP for this application.

5.5 Method of genetic modification


36. Agrobacterium tumefaciens-mediated transformation was used to generate the genetic modifications in the proposed release. A. tumefaciens is a soil bacterium that causes gall formation on a wide range of plant species. The gall is induced by transfer of hormone-producing genes from the bacterial cell into the plant genome. The genes are carried on an extrachromosomal, circular DNA molecule found within the bacterial cell called a Tumour-inducing (Ti) plasmid. During the infection process, only a section of the Ti plasmid known as the Transfer DNA (T-DNA) is transferred to the plant. Molecular biologists have studied the infection and T-DNA transfer process of A. tumefaciens for many years and have used this natural process to facilitate genetic modification of plants. Well-characterised A. tumefaciens Ti plasmids have been produced that lack the genes responsible for tumour formation (disarmed plasmids) and instead enable genes of interest to be inserted between the T-DNA border sequences. When used to infect plants, A. tumefaciens cells carrying such plasmids cannot produce a tumour but will transfer the T-DNA sequence carrying the genes of interest into the plant cell where they stably integrate into the plant genome (Bevan 1984; Klee & Rogers 1989).

37. In addition to transfer of the T-DNA sequence, recent publications have shown that small segments of flanking Ti plasmid sequence and A. tumefaciens chromosomal sequence may be transferred into the plant genome at a low frequency during the transformation process (Smith 1998; Ulker et al. 2008). However, A. tumefaciens-mediated plant transformation has been used extensively in Australia and overseas and is not known to adversely affect human health and safety or the environment.

5.6 Characterisation of the GMOs


38. All of the GM cotton lines containing single transformation events are well characterised, as described below. There is no stability, molecular or phenotypic characterisation data available for some of the stacked GM cotton varieties in which two or more transformation events have been combined by conventional cross-breeding.

5.6.1 Stability and molecular characterisation

39. All plasmid constructs used for generation of the GM cotton lines have been fully sequenced. Southern blot analysis and polymerase chain reaction (PCR) amplification confirmed the presence of the inserted genes in the GM cotton lines and found that no sequences from the A. tumefaciens vector were inserted. Southern blot analysis determined that each line contains either 1 or 2 copies of the inserted genes. The inserted genes have been maintained as dominant Mendelian traits over a number of generations of self-crosses and back-crosses (information supplied by applicant).

40. The exact location of the inserted genes within the cotton genome is not known. A. tumefaciens inserts genetic material into plant genomic DNA via illegitimate recombination, which can potentially result in insertion of the introduced genes anywhere in the host genome.

5.6.2 Characterisation of the phenotype of the GM cotton varieties

41. Some of the introduced genes in the GM cotton varieties are expected to provide resistance against herbivory by target insect species. These GM cotton plants will therefore sustain less pest damage than unsprayed non-GM plants, and may exhibit improved retention of fruiting structures. Some of the introduced genes are expected to provide post-emergence tolerance to specific herbicides. The purpose of the proposed trial is to evaluate the agronomic performance of the GM cotton varieties and to assess the efficacy of the insecticidal proteins against Helicoverpa armigera.

42. The GM cotton lines containing single transformation events are end products of breeding programs in which plant lines with poor agronomic performance were discarded. Field trials of these GM cotton lines in Australia or other countries have not identified secondary effects resulting in agronomic penalties as a result of the genetic modifications. The GM cotton lines have performed in a similar manner to non-GM cotton varieties.