<p style="text-align: justify;"><b>The Introduced DNA</b><br>
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Data from Southern hybridisation analysis and DNA sequencing demonstrate that single copies of both the <i>mcry3A</i> and <i>pmi</i> genes are present in MIR604. Additionally, MIR604 does not contain any of the backbone sequences from the transformation plasmid pZM26. Sequence analysis of the entire T-DNA insert present in MIR604 confirms that the overall integrity of the intended insert and of the functional elements have been maintained. A 43 base-pair truncation at the right border junction of the T-DNA insert and 44 base-pair truncation at the left border junction of the T-DNA insert were identified. Three single nucleotide changes were also identified relative to the intended DNA sequence. One of these changes occurred within a promoter, a regulatory region that does not encode a protein. The remaining two changes occurred within the <i>pmi</i> coding sequence and resulted in the substitution of (1) alanine in place of valine-61 and (2) histidine in place of glutamine-210 in the amino acid sequence of the PMI protein. These substitutions have not resulted in any apparent functional change in PMI as expressed in MIR604.<br>
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<b>Genetic Stability of the Introduced Trait</b><br>
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The hybridization pattern over three generations of MIR604 (BC4, BC5 and BC6) was identical, which demonstrates that the TDNA insert from pZM26 incorporated into MIR604 is stable over several generations. Furthermore, levels of mCry3A and PMI proteins were determined to be stable in MIR604 plants over four successive backcross generations.<br>
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Homozygous plants from the T3 generation were crossed to a non-transgenic inbred to yield the T4 generation, which were selfed in the field and seed was collected in bulk, creating the T5 seed. Individual T5 plants were assayed for the presence of the trait by immuno-detection (ELISA) of mCry3A, as well as by TaqMan® PCR for both <i>mcry3A</i> and <i>pmi</i> genes. The expected Mendelian inheritance ratio of positive and negative plants for a hemizygous trait in these populations is 3:1 and statistical analysis confirmed the expected Mendelian inheritance ratio for both genes.<br>
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<b>Expressed Material</b><br>
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To characterize the range of expression of the mCry3A and PMI proteins in maize (corn) plants derived from MIR604, the concentrations of mCry3A protein and PMI were determined by ELISA in several plant tissues and whole plants at four growth stages (whorl, anthesis, seed maturity and senescence) in two field maize hybrids and one maize inbred.<br>
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Quantifiable levels of mCry3A protein were detected in all MIR604-derived plant tissues analyzed except pollen. Across all growth stages, mean mCry3A levels measured in leaves, roots and whole plants ranged from <i>ca.</i> 3 - 23 µg/g fresh wt, <i>ca.</i> 2 - 14 µg/g fresh wt and <i>ca.</i> 0.9 - 11 µg/g fresh wt, respectively. Mean mCry3A levels measured in kernels from the MIR604 hybrids at senescence (corresponding to the stage closest to grain harvest) were <i>ca.</i> 0.7 µg/g fresh wt. The levels of mCry3A were generally similar between hybrids for each tissue type at each time point. For the inbred line, mCry3A expression was generally higher than in the hybrids in leaves, roots and whole plants at whorl and anthesis stages and in roots at seed maturity.<br>
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PMI protein was detected in most of the MIR604-derived plant tissues analyzed, albeit at low levels. Across all plant stages, mean PMI levels measured in leaves, roots and whole plants ranged from not detectable (ND) to <i>ca</i>. 0.4 µg/g fresh wt., below the LOQ (ca. 0.2 µg/g fresh wt. and below the LOQ (ca 0.3 µg/g fresh wt, respectively. Mean PMI levels measured in kernels from the MIR604 hybrids at senescence (corresponding to the stage closest to grain harvest) were ca. 1.9 – 2.6 µg/g fresh wt. The levels of PMI were generally similar among the inbred and hybrid genotypes for each tissue type at each time point. PMI was not detectable in silage at all three sampling times (day 15, 29 and 75). By comparison, the level of PMI measured in the chopped plant material prior to ensiling was <i>ca</i>. 0.3 µg/g fresh wt.<br>
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