<p style="text-align: justify;"><b>The Introduced DNA</b></p>

<p style="text-align: justify;">Southern blot analysis of genomic DNA from MON89788 demonstrated that there is a single copy of the T-DNA from PV-GMGOX20 integrated at a single locus in the genome. Hybridisation with different probes confirmed that the integrated T-DNA contains all of the components from the EPSPS expression cassette between the Left Right and Left Borders. Lack of hybridisation with the probes made from the plasmid backbone confirmed the absence of any of these sequences in MON89788. The organization of the genetic elements within the insert of MON89788 was confirmed by DNA sequence analysis, using PCR primers to amplify three overlapping DNA fragments spanning the entire length of the insert. Sequencing of the amplified fragments confirmed that the arrangement of the genetic elements is identical to that in PV-GMGOX20.</p>

<p style="text-align: justify;"><b>Genetic Stability of the Introduced Trait</b></p>

<p style="text-align: justify;">DNA analyses over four generations demonstrated that the <i>cp4 epsps</i> gene was stably inserted. Hybridisation probes spanning the entire T-DNA region were used to ensure that the cassette was stably inherited and the absence of any backbone sequences was confirmed by hybridisation using probes from this region of the plasmid. The glyphosate tolerance trait was also evaluated during the development of MON89788. The R0 transgenic plant was self-pollinated and the R1 population was evaluated for glyphosate tolerance, showing the expected ratio of 3:1 resistant to susceptible plants. Selected R1 plants that survived glyphosate treatment were analysed using quantitative PCR to identify a single plant homozygous for the <i>cp4 epsps</i> gene, which was self pollinated to provide the R2 generation. Testing of this population, and the R3 generation, confirmed that 100% of the plants were tolerant to glyphosate.</p>

<p style="text-align: justify;"><b>Expressed Material</b></p>

<p style="text-align: justify;">Expression of the <i>cp4 epsps</i> gene results is a single 455 amino acid polypeptide of 47.6 kDA, which was characterized using (1) SDS-PAGE, (2) western immunoblot, (3) N-terminal sequence analysis, (4) MALDI-TOF mass spectrometry, (5) enzymatic activity analysis, and (6) glycosylation analysis. The plant expressed protein was compared with the same protein produced in <i>E. coli</i> in order to establish equivalence to existing standards used for safety evaluation of CP4 EPSPS. All of the tests used produced comparable results between the plant expressed and the <i>E. coli</i> expressed proteins, thus establishing the equivalence of the proteins from the two sources.</p>

<p style="text-align: justify;">CP4 EPSPS protein levels in tissues derived from MON89788 were determined by a validated ELISA assay. MON89788 tissues were collected from replicated field trials across five US field locations in 2005 and CP4 EPSPS protein levels from a range of tissues was determined. CP4 EPSPS protein levels in grain averaged 140 &micro;g/g fresh weight and 62 &micro;g/g fresh weight in leaves.</p>
