<p style="text-align: justify;">MON89034 was produced by <i>Agrobacterium</i>-mediated transformation of corn with PVZMIR245, which is a binary vector containing 2 separate T-DNA regions. The first T-DNA, designated as T-DNA I, contained the <i>cry1A.105</i> and the <i>cry2Ab2</i> expression cassettes which express the insecticidal proteins Cry1A.105 and Cry2Ab2. The <i>cry1A.105</i> gene, is a chimeric gene comprising of 4 domains from other <i>cry</i> genes previously used in transgenic plants. The amino acid sequences of Domains I and II are identical with the respective domains from Cry1Ab and Cry1Ac proteins, Domain III is almost identical to the Cry1F protein, and the C-terminal Domain is identical to Cry1Ac protein. Overall, the amino acid sequence homology of Cry1A.105 is 93.6%, 90.0%, and 76.7% respectively with the Cry1Ac, Cry1Ab and Cry1F proteins. The expression cassette for the coding sequence of the Cry1A.105 protein consists of the promoter (<i>P-e35S</i>) and leader for the cauliflower mosaic virus (CaMV) 35S RNA with a duplicated enhancer region. The cassette also contains the 5&#39; untranslated leader of the wheat chlorophyll a/b/ binding protein (<i>L-Cab</i>), the intron from the rice actin gene (I-Ract1), the <i>cry1A.105</i> coding sequence that was optimized for expression in monocots, and the 3&rsquo; nontranslated region of the coding sequence for wheat heat shock protein 17.3 (<i>T-Hsp17</i>), which terminates transcription and provides the signal for mRNA polyadenylation</p>

<p style="text-align: justify;">The Cry2Ab2 protein is a member of the Cry2Ab class of proteins that share &gt;95% amino sequence homology. Like the Cry2Ab2 protein produced in the transgenic cotton event 15985, the Cry2Ab2 protein produced in MON89034 is a slight variant of the wild-type Cry2Ab2 protein isolated from <i>Bacillus thuringiensis</i> subsp. <i>kurstaki</i>. The Cry2Ab2 produced in MON 89034 is identical to the Cry2Ab2 produced in 15985 cotton, and they differ from the wild-type Cry2Ab2 of Bt by only one amino acid. The promoter region of the <i>cry2Ab2</i> gene expression cassette consists of the 35S promoter from figwort mosaic virus (<i>P-FMV</i>) and the first intron from the corn heat shock protein 70 gene (<i>I-Hsp 70</i>). The cassette also contains a <i>cry2Ab2</i> coding sequence with a modified codon usage fused to a chloroplast transit peptide region of corn ribulose 1,5-biphosphate carboxylase small subunit including the first intron (<i>TSSSU-CTP</i>). The 3&rsquo; nontranslated region of the nopaline synthase (<i>T-nos</i>) coding region from <i>Agrobacterium tumefaciens</i> T-DNA terminates transcriptionand directs polyadenylation.</p>

<p style="text-align: justify;">The second T-DNA, designated as T-DNA II, contains the <i>nptII</i> (neomycin phosphotransferase II) expression cassette. The nptII gene cassette that produces the NPTII protein consists of the promoter (<i>P-e35S</i>) from the cauliflower mosaic virus (CaMV) 35S RNA. The cassette also contains the coding sequence for the NPTII protein, followed by the 3&rsquo; nontranslated region of the nopaline synthase (<i>T-nos</i>) sequence from <i>Agrobacterium tumefaciens</i> which terminates the transcription and directs polyadenylation.</p>

<p style="text-align: justify;">During transformation, both T-DNAs were inserted into the genome. The <i>nptII</i> gene confers resistance to Kanamycin and similar antibiotics and was used as the selectable marker for isolation of transformed cells and regeneration of transgenic plants. Once transgenic plants had been regenerated, the selectable marker gene was no longer needed and conventional breeding was used to isolate plants that only contain the <i>cry1A.105</i> and the <i>cry2Ab2</i> expression cassettes (T-DNA I) and did not contain the <i>nptII</i> expression cassette (T-DNA II), thereby, producing marker-free transgenic lines, one of which was selected and designated as MON89034.</p>
