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

<p style="text-align: justify;">The inserted DNA in event 98140 was characterized via Southern hybridization analysis using hybridization probes derived from plasmid PHP24279. The T-DNA from plasmid PHP24279 contains two expression cassettes: the <i>gat4621</i> cassette comprised of the <i>ubiZM1</i> promoter, <i>ubiZM1</i> intron, the <i>gat4621</i> gene, and the <i>pinII</i> terminator. The <i>zm-hra</i> cassette is comprised of the maize <i>als</i> promoter, <i>zm-hra</i> gene, and <i>pinII</i> terminator. Located between the two cassettes are three copies of the CaMV 35S enhancer element, providing transcription enhancement to both cassettes. Individual plants of the T0S3 generation were analyzed to determine the copy number of each of the genetic elements inserted into event 98140 and to verify that the integrity of the PHP24279 T-DNA was maintained upon integration. The analysis confirmed that event 98140 contained a single, intact, copy of the PHP24279 T-DNA. Southern blot analysis was also used to confirm the absence of plasmid backbone sequences within the event 98140 genome.</p>

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

<p style="text-align: justify;">The stability of the <i>gat4621</i> and <i>zm-hra</i> genes across four generations of event 98140 maize (T0S3, BC0S2, BC1, and BC1S1) was determined from Southern hybridization analysis of <i>Eco</i>RV digests of genomic DNA.</p>

<p style="text-align: justify;">Chi-square analysis of trait inheritance data from four different generations (BC0S1, BC1S1, BC2 and BC3) was also performed to determine the heritability and stability of the <i>gat4621</i> and <i>zm-hra</i> genes in 98140 maize. In order to confirm the expected segregation ratios, polymerase chain reaction (PCR) analysis was performed on leaf punches from seedlings. Qualitative PCR analysis for the <i>gat4621</i> and <i>zm-hra</i> genes was conducted on all plants. The results of this analysis were consistent with the finding of a single locus of insertion of the <i>gat4621</i> and <i>zm-hra</i> genes that segregates in 98140 progeny according to Mendel&rsquo;s laws of genetics. In every case, plants that were positive for the <i>gat4621</i> gene were also positive for the <i>zm-hra</i> gene and vice versa, confirming co-segregation of the two genes as expected.</p>

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

<p style="text-align: justify;">Concentrations of the GAT4621 and ZM-HRA proteins in event 98140 were measured in tissue samples collected from a replicated field study grown at six field locations in North America in 2006. Tissue samples were collected at various developmental growth stages, including V9 (the stage just after glyphosate application), R1 (pollen shed), R4 (forage harvest stage), and R6 (grain harvest stage). Samples of leaf tissue (V9, R1, R4 and R6), root tissue (R1, R4, and R6), pollen (R1), forage (R4), grain (R6), and whole-plant samples (R1 and R6), were tested for GAT4621 and ZM-HRA proteins using quantitative enzyme linked immunosorbent assay (ELISA).</p>

<p style="text-align: justify;">Mean concentrations of GAT4621 protein in various tissues of 98140 maize throughout the growing season ranged from 2.6 ng/mg tissue on a dry weight basis (root at the R6 stage) to 51 ng/mg (leaf at the R1 stage). Mean concentrations of ZM-HRA protein in various tissues of 98140 corn throughout the growing season ranged from below the limit of quantification (pollen at the R1 stage) to 6.7 ng/mg on a dry weight basis (leaf at the V9 stage). As expected, all GAT4621 and ZM-HRA protein concentrations were below the limit of quantification in control corn tissues.</p>
