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

<p style="text-align: justify;">The <i>cry1Ab</i> gene, bar gene (used as a selectable marker and for herbicide tolerance) and a selectable marker for ampicillin resistance, beta-lactamase (bla) gene, were introduced into the maize chromosomes of Event 176.</p>

<p style="text-align: justify;">The synthetic <i>cry1Ab</i> gene has approximately 65% homology at the nucleotide level with the native gene. The truncated Cry1Ab protein contains the insecticidal region of the native Cry1Ab as demonstrated by western blot analyses where similar bands were displayed at approximately 65 kDa for Event 176-expressed Cry1Ab and the native protein. Three additional immunoreactive proteins weighing approximately 60, 40 and 36 kDa were detected in the leaves of Event 176, but not in the pollen. It was suggested that these may represent breakdown products resulting from intrinsic proteolysis within the leaf tissue. Southern blot analysis indicated that there may be two or more copies of each plasmid integrated into the genome of Event 176. Southern blot analysis also confirmed the presence of the bar gene in all plant tissues.</p>

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

<p style="text-align: justify;">Segregation and stability data demonstrated that the <i>cry1Ab</i> and <i>bar</i> genes were tightly linked and stably inherited into the genome of Event 176 maize. The production of Cry1Ab and PAT proteins, in leaves and pollen of greenhouse-grown plants was determined to be stable over four successive backcross generations. Segregation analyses indicated that the resistance to ECB and herbicide tolerance traits co-segregate as linked Mendelian traits. A study of 3240 plants indicated that only five plants (0.15%) were identified as being tolerant to glufosinate ammonium but susceptible to damage by ECB larvae.</p>

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

<p style="text-align: justify;">Event 176 was transformed with two synthetic <i>cry1Ab</i> genes; one gene is linked to a specific promoter which confers expression in green tissue, while the other is linked to a pollen-specific promoter, resulting in expression in pollen. Expression of the Cry1Ab protein in green tissues was intended to render the plant resistant to first generation ECB larvae feeding on leaves while expression in pollen was intended to target second generation ECB larvae, which are known to feed on pollen. The glufosinate ammonium tolerance gene (<i>bar</i> gene) is linked to a constitutive promoter active in all plant tissues except pollen.</p>

<p style="text-align: justify;">Cry1Ab production was quantified in leaves, pollen, roots and kernels among three genotypes. Expression in leaves, across genotypes, ranged from 0.596 to 1.159 &micro;g/g (fresh weight) in seedlings, 0.530 to 3.029 &micro;g/g (f.w.) at anthesis, 0.442 to 0.471 &micro;g/g (f.w.) at physiological maturity, and 0.066 to 0.225 &micro;g/g (f.w.) at senescence. Maximum expression in leaves was detected at either the vegetative or anthesis stages, depending on the genotype tested, and expression in all genotypes declined as plants senesced. Expression in pollen, across genotypes, ranged from 1.137 to 2.348 &micro;g/g (f.w.). Expression of Cry1Ab in roots ( 0.008 &micro;g/g f.w.), pith (While Southern blot analysis confirmed the presence of the bar gene in all plant tissues, expressed PAT protein was undetectable in leaves, pollen, roots or kernels of transgenic maize at a detection threshold of 0.2 ppm. The hydroxamic acid, 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-2(4H)-one (DIMBOA) is the only putative endogenous toxin from maize and postulated to play a protective role against specific fungal and bacterial pathogens as well as insect pests. The levels of DIMBOA are normally highest in the leaf tissue of young plants and absent in kernels. The measured concentrations of DIMBOA in leaf tissue from transgenic 176 maize and untransformed control plants grown under identical conditions were statistically identical.</p>

<p style="text-align: justify;">An ampicillin resistance gene (<i>bla</i> gene), regulated by a bacterial promoter, also present in the vectors used to transform Event 176, was not expressed in either leaf tissue or pollen as confirmed by assays and Northern blot analysis. Other regulatory DNA elements inserted to enhance expression levels of <i>cry1Ab</i> did not code for any other protein.</p>
