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

<p style="text-align: justify;">Southern blot analysis of the genomic DNA of events J101 and J163 confirmed the insertion of a single intact copy of the <i>cp4 epsps</i> gene cassette. For each of the transformation events, the <i>cp4 epsps</i> gene cassette was integrated at a different locus. All of the elements of the gene cassette were integrated, including the promoter, enhancer, terminator and chlorophyll transit peptide sequences. None of the vector backbone sequences from the PV-MSHT4 vector were integrated into the genome of either J101 or J163.</p>

<p style="text-align: justify;">The <i>cp4 epsps</i> gene was isolated from <i>Agrobacterium tumefaciens</i> strain CP4. In plants, EPSPS is a chloroplast-localized enzyme that is transported from the cytosol to the chloroplast by the chlorophyll transit peptide. In events J101 and J163, 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 in J101 and J163 alfalfa populations. Segregation data were obtained from five generations of introgression of the trait from events J101 and J163 into unrelated alfalfa germplasm. This was accomplished by initially outcrossing the transformed plants to unrelated elite alfalfa clones. The F1 progeny were subsequently outcrossed to a second set of unrelated elite clones to produce a modified backcross generation. Three additional modified backcross generations were developed with three other sets of unrelated clones. Southern blot analysis and chi-square analysis of the resulting segregation data demonstrated that the glyphosate-tolerance trait in events J101 and J163 was inherited as a single-locus dominant trait, and was stable across five generations.</p>

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

<p style="text-align: justify;">The quantification of the expression levels of CP4 EPSPS in alfalfa populations containing each of J101 and J163 was achieved using enzyme-linked immunosorbent assay (ELISA) analysis. Samples for analysis were obtained from field trials of these varieties grown at six sites in 2001. The mean expression level of CP4 EPSPS in plants containing events J101 was 257 &micro;g/g fresh weight, and 270 &micro;g/g fresh weight in plants containing event J163.</p>
