<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 ASR368 creeping bentgrass demonstrated that there was one site of integration of a single copy of the DNA insert PV-ASGT08L. The stability of the insertion was assessed over three generations was confirmed using Southern blot analysis and segregation studies.</p>

<p style="text-align: justify;">The <i>cp4 epsps</i> gene was isolated from <i>Agrobacterium tumefaciens</i> strain CP4. The EPSPS protein is a chloroplast-localized enzyme that is transported from the cytosol to the chloroplast by the chlorophyll transit peptide. In ASR368, the inserted <i>ctp2</i> gene codes for a chlorophyll transit peptide that binds to the CP4 EPSPS enzyme to enable transportation to the chloroplast.</p>

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

<p style="text-align: justify;">Mendelian segregation studies were conducted to investigate the stability of inheritance of the glyphosate-tolerance trait. Segregation data were derived from reciprocal crosses between F1 hemizygous plants and nonmodified parental plants. The progeny of the reciprocal crosses segregated in a 1:1 ratio of glyphosate-tolerant to non-tolerant plants. This indicated that the glyphosate-tolerance trait is inherited as a single locus, and is stable across several generations.</p>

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

<p style="text-align: justify;">An enzyme-linked immunosorbent assay (ELISA) analysis and Western blot analysis was used to quantify the levels of the CP4 EPSPS proteins in tissues from ASR368. Samples for analysis were obtained from four field sites. The mean expression level of CP4 EPSPS in ASR368 tissues was 68.6 &mu;g/g fresh weight.</p>
