5.1 Introduction to the GMOs
28. The applicant proposes to release up to 190 lines of GM safflower. All lines were produced by Agrobacterium tumefaciens-mediated plant transformation. Information about this transformation method can be found in the risk assessment reference document Methods of plant genetic modification available from the Risk Assessment References page on the OGTR website. Details of the techniques used specifically for transformation of safflower are described elsewhere (Belide et al. 2011).
29. The GM safflower lines contain introduced gene silencing constructs including fragments of either two or three endogenous safflower fatty acid biosynthesis genes. The function of the silencing constructs is to suppress the expression of these target fatty acid biosynthesis genes, and thus alter the oil composition of the GM safflower seeds.
5.2 The introduced genes, encoded proteins and their associated effects
30. The three safflower genes that are targeted for suppression of expression are palmitoyl-ACP thioesterase (FATB), Δ12 desaturase (FAD2) and another fatty acid biosynthesis gene. The identity of the third gene and molecular details of the silencing constructs have been declared CCI. The confidential information was made available to the prescribed experts and agencies that were consulted on the RARMP for this application.
31. Suppression of the target genes is mediated by a natural regulatory mechanism in plants known as ribonucleic acid interference (RNAi) or gene silencing (Baykal & Zhang 2010). Utilising the RNAi pathway, an introduced silencing construct is transcribed into double-stranded RNA, which is processed by endogenous cellular machinery into short interfering RNAs (siRNAs). The siRNAs direct the degradation of messenger RNA (mRNA) molecules with matching sequence after the mRNAs are transcribed from genes and before they are translated into proteins. The efficiency of gene silencing is generally determined by the extent of homology between the silencing construct and the target gene (usually > 95% homology is required) and the length of the homologous region. In plants, introduced silencing constructs have been shown to effectively suppress expression of the target genes but can also give rise to silencing of non-target genes with closely matching sequences.
32. The target gene FATB encodes a carrier protein that mediates export of saturated fatty acids from the plastid, where fatty acid synthesis occurs (Bonaventure et al. 2003). The effect of suppressing expression of FATB is to retain saturated fatty acids in the plastid until they undergo a desaturation reaction (usually to form oleic acid) and can be exported by another carrier protein. This decreases the proportion of saturated fatty acids and increases the proportion of oleic acid in the safflower oil.
33. The target gene FAD2 encodes a desaturase protein that mediates enzymatic conversion of oleic acid to linoleic acid (Harwood 1996). The effect of suppressing expression of FAD2 is to decrease the proportion of linoleic acid and increase the proportion of oleic acid in the safflower oil.
34. The GM safflower lines produce seeds where 90-95% of the total oil content is oleic acid. This high purity oleic oil has potential application as an industrial raw material.
35. All of the GM safflower lines also contain the introduced hph gene which provides resistance to the antibiotic hygromycin B, and is used as a selectable marker during plant transformation. This gene is derived from E. coli and truncated for use in plants. It is expressed under the control of either the enhanced tobacco constitutive ubiquitous promoter (enTCUP) or the 35S constitutive promoter from Cauliflower mosaic virus (CaMV) and the nopaline synthase polyA (nos) terminator from A. tumefaciens.
36. Some of the GM safflower lines also contain the introduced gfp gene, which encodes a green fluorescent protein used to visually identify genetically modified plant cells. This gene is derived from the jellyfish Aequorea victoria. It is expressed under the control of the CaMV 35S promoter and the octopine synthase polyA (ocs) terminator from A. tumefaciens.
37. The hph and gfp marker genes are commonly used in gene technology. Further details about these genes can be found in the risk assessment reference document Marker genes in GM plants available from the Risk Assessment References page on the OGTR website.
5.2.1 Toxicity/allergenicity associated with the introduced safflower genes
38. Insertion of safflower gene fragments as part of gene silencing constructs does not result in expression of a protein, but only in suppression of the expression of endogenous safflower proteins. This is not expected to lead to increased toxicity or allergenicity.
39. The effect of the gene silencing is to increase levels of oleic acid and decrease levels of other fatty acids in GM safflower oil. Oleic acid is a common constituent of food, for example it is the main constituent of olive oil and canola oil, and it is not associated with toxicity or allergenicity.
5.3 Characterisation of the GMOs
5.3.1 Stability and molecular characterisation
40. Transformation of the GM safflower lines was confirmed using both polymerase chain reaction (PCR) assays and analysis of oil content. Lines were self-pollinated and selected through single seed descent for between 2-5 generations. Standard Mendelian inheritance of the introduced genetic material was observed. The copy numbers of the introduced genetic material were determined by Southern blot hybridisation in some lines only. Lines containing either single or multiple insertions are proposed for release. The genomic locations of the introduced genetic material have not been characterised for any of the GM lines.
5.3.2 Phenotypic characterisation
41. The GM safflower lines were grown in greenhouses under controlled conditions. No phenotypic differences between GM plants and non-GM plants from the same genetic background were observed. GM safflower lines had the same growth patterns, morphology and fertility as non-GM comparators.