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

<p style="text-align: justify;">DNA blot (Southern) analyses were used to characterize the introduced DNA in DP356043 soybean confirming that DP356043 contains one insert with a single, intact copy of the transformation fragment PHP20163A containing the <i>gat4601</i> and <i>gm-hra</i> gene cassettes. Southern blot analysis using a plasmid backbone-specific probe confirmed that plasmid backbone sequences, such as those coding for the hygromycin antibiotic resistance gene, were not present in DP356043.</p>

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

<p style="text-align: justify;">The inheritance of the new traits present in DP356043 was studied in five generations (T1, F2, F3, BC1F2, and C2F2). From the Chi-square analysis of the trait inheritance data, it was concluded that the inheritance of the integrated insert with the <i>gat4601</i> and the <i>gm-hra</i> genes is consistent with the Mendelian inheritance pattern of a single locus. The stability of the insert in three generations was assessed using Southern analyses of genomic DNA digested with the restriction enzyme <i>Bgl</i>II. There is a single <i>Bgl</i>II site present in the PHP20163A fragment and this generates a unique, event-specific, DNA hybridization pattern. No changes were detected in the hybridization pattern among the tested generations of the DP356043 soybean (the selfed T4 and T5 generations and the traditionally-bred F3 generation). Given the absence of pattern changes, it was concluded that the insertion of the PHP20163A DNA fragment in 356043 soybean was stable.</p>

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

<p style="text-align: justify;">The levels of the GAT4601 and GM-HRA proteins were assessed in the grain, forage, and root tissue of samples collected from plants grown at six field locations in North America. Three replicated samples per tissue per location were collected for DP356043 and one sample per tissue per location was collected for control soybean. Mean protein levels across six locations for the GAT4601 protein in forage, root, and grain were 1.6, 1.6, and 0.24 nanograms per milligram (ng/mg) of tissue (dry weight), respectively. The mean protein levels for the GM-HRA protein in forage, root, and grain were 27, 3.2, and 0.91 ng/mg of tissue (dry weight), respectively. Neither the GAT4601 nor the GM-HRA proteins were detected in non-transgenic Jack cultivar (control soybean) tissues sampled from the six locations. From this data, it was concluded that expression of the GAT4601 and GM-HRA proteins in 356043 soybean is constitutive. Levels of GAT4601 and GM-HRA proteins in toasted soybean meal and hulls were found to be below the limit of quantitation (LOQ).</p>
