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

<p style="text-align: justify;">Southern blot analysis and Polymerase Chain Reaction (PCR) amplification of the genomic DNA of MON 71800 wheat demonstrated one site of integration of a single copy of the T-DNA insert of PV-TXGT10. Southern blot analysis also confirmed the insertion of one intact copy of each <i>cp4 epsps</i> gene cassette, including the promoter, terminator and chlorophyll transit peptide sequences. None of the vector backbone sequences were integrated into the genome of MON 71800 wheat.</p>

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

<p style="text-align: justify;">The stability of the inserted DNA was evaluated, across several generations of wheat plants, using Southern blot analysis. The plants tested were progeny from several generations of self-fertilization, as well as from crosses with commercial varieties. The results of the genomic DNA blot analysis confirmed the stable inheritance of the inserted <i>cp4 epsps</i> gene cassettes. The stability of the introduced trait was also demonstrated after 18 generations of selfing of the original homozygous glyphosate-resistant plants. These generations showed no decrease in tolerance to glyphosate.</p>

<p style="text-align: justify;">Mendelian segregation studies for the inheritance of the glyphosate tolerant trait were conducted with heterozygous first generation plants: these were selfed and the progeny were sprayed with glyphosate. The resulting 3:1 ratio of tolerant to sensitive plants was statistically significant and confirmed the inheritance of a single insertion site of the glyphosate tolerant trait.</p>

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

<p style="text-align: justify;">In plant cells, the EPSPS enzyme is transported to the chloroplast by a transit peptide, which then cleaves from the enzyme. The introduced gene sequences in MON 71800 included a gene for a chloroplast transit peptide from <i>Arabidopsis thaliana</i>. Thorough analyses were conducted to investigate whether the same mechanism of binding, transporting, and cleaving of the chloroplast transit peptide to the CP4 EPSPS enzyme exists in MON 71800. The analyses revealed two forms of CP4 EPSPS expressed in MON 71800: one in which the chloroplast transit peptide has completely cleaved from the full length enzyme, and the other in which only part of the transit peptide is bound to the enzyme. Further analysis of Western blots showed that of the total amount of CP4 EPSPS expressed in MON 71800, 80% is in the form where the transit peptide is fully cleaved and 20%, where part of the transit peptide is still bound to the enzyme.</p>

<p style="text-align: justify;">An enzyme-linked immunosorbent assay (ELISA) analysis was used to quantify the levels of the CP4 EPSPS proteins in forage and grain from MON 71800. The mean levels of both CP4 EPSPS proteins, on a fresh weight basis, were 106 &mu;g/g in forage and 13 &mu;g/g in grain. Assuming 14.5% moisture in stored grain, the concentration of both CP4 EPSPS proteins in the grain is approximately 0.0015%, on a dry matter basis.</p>
