<p style="text-align: justify;">Maize line 3272 was produced by Agrobacterium-mediated transformation of immature embryos derived from the proprietary line A188 of <i>Zea mays</i> (maize) using the transformation vector pNOV7013. The T-DNA segment of this plasmid contained, between the right and left borders, the <i>amy797E</i> and <i>pmi</i> genes and their regulatory elements.</p>

<p style="text-align: justify;">The chimeric <i>amy797E</i> gene was generated to combine the best features of three thermostable amylase enzymes isolated from <i>Thermococcus</i> species bacteria, designated as BD5031, BD5064 and BD5063 (Richardson et al., 2002). Fragments from the three parental genes were combined (in the same relative position) based on the natural sequence homology to create a library of recombinant alpha-amylase genes. The chimeric AMY797E aplha-amylase enzyme (alternatively known as &ldquo;797GL3&rdquo;) was identified by screening these recombined enzymes and is composed of four fragments from BD5031, two fragments from BD5064 and three fragments from BD5063. The <i>amy797E</i> gene includes fusions of a 19 amino acid N-terminal maize gamma-zein signal sequence and a C-terminal endoplasmic reticulum retention signal (Lanahan et al., 2003). The maize gamma-zein signal sequence and the ER retention signal provide for protein targeting to and retention in the endoplasmic reticulum of the cell, respectively. The alpha-amylase coding region of the <i>amy797E</i> gene was synthesized to accommodate the preferred codon usage for maize. The <i>amy797E</i> gene is regulated by the GZein promoter from <i>Zea mays</i> and the 35S terminator from the cauliflower mosaic virus.</p>

<p style="text-align: justify;">The <i>pmi</i> gene represents the <i>manA</i> gene from E. coli and encodes the enzyme phosphomannose isomerase (PMI). It was used as a selectable marker gene during the transformation process. Mannose, a hexose sugar, is taken up by plants and converted to mannose-6-phosphate by hexokinase. This product cannot be further utilised in plants as they lack the PMI enzyme. The accumulation of mannose-6-phosphate inhibits phosphoglucose isomerase, causing a block in glycolysis. It also depletes cells of orthophosphate required for the production of ATP. Therefore, while mannose has no direct toxicity on plant cells, it causes growth inhibition. This does not occur in plants transformed with the <i>pmi</i> gene as they can utilise mannose as a source of carbon. The <i>pmi</i> gene is regulated by the polyubiquitin promoter (ZmUbilnt) from maize and the NOS terminator from <i>Agrobacterium tumefaciens</i>.</p>

<p style="text-align: justify;">Transformed cells were grown on cell culture media containing mannose and tested by PCR for the presence of both the <i>amy797E</i> and <i>pmi</i>i genes and the absence of the spec gene (an antibiotic resistant marker in the plasmid backbone). Regenerated plants meeting these criteria were transferred to the greenhouse for propagation</p>
