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

<p style="text-align: justify;">Southern blot analysis of genomic DNA isolated from seed of different generations of progeny plants demonstrated that the parental transgenic line, TC1507, contained a single copy of an intact fragment containing both the <i>cry1Fa2</i> and <i>pat</i> gene constructs with their associated noncoding regulatory regions and a second copy of the <i>cry1Fa2</i> coding region lacking the majority of the associated ubiquitin regulatory sequences; and (2) these genetic constructs were stably inherited and cosegregated over four generations of backcrossing.</p>

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

<p style="text-align: justify;">The expression of Cry1F protein in hybrid progeny derived from TC1507 was measured using enzyme linked immunosorbent assay (ELISA). Using a chi square analysis with a 95% confidence interval, a Mendelian ratio of 1:1 was observed for first generation progeny. This pattern of segregation was consistent with the integration of a single functional copy of the <i>cry1Fa2</i> gene in the original transformation event.</p>

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

<p style="text-align: justify;">Levels of expression of Cry1F protein from TC1507 pollen, grain, and grain-derived feeds were measured using quantitative ELISA or Western immunoblotting, and biological activity was determined using insect bioassays with either tobacco budworm or European corn borer larvae.</p>

<p style="text-align: justify;">Cry1F protein was detectable in whole plants (minus the roots) collected at four weeks prior to pollination and following senescence and in leaves, pollen, silk, stalk, and mature grain; whereas PAT was only detectable in leaf tissue. Levels of Cry1F protein expressed in TC1507 ranged from an average of 32 ng/mg in pollen to an average of 110.9 ng/mg total protein in leaf tissue and 89.8 ng/mg total protein in grain samples. Western immunoblotting of sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE) separated proteins in trypsinized plant extracts prepared from TC1507 leaf tissue revealed the presence of a 65 kDa moiety corresponding to the trypsin-resistant core of the Cry1F delta-endotoxin.</p>

<p style="text-align: justify;">The amounts of detectable PAT protein in TC1507 leaf tissue ranged up to 40.8 ng/mg total protein but were undetectable in other tissues, such as pollen, silk, stalk, and grain.</p>

<p style="text-align: justify;">Based on a bioassay with the tobacco budworm (<i>Heliothis virescens</i>), a target species, purified Cry1F proteins incorporated into test soils biodegraded with a half-life of approximately 3.13 days. This half-life is very comparable with the 4-7 days in published reports for other Cry proteins.</p>
