<p style="text-align: justify;">Cotton line GHB614 was developed through <i>Agrobacterium</i>-mediated transformation of the cotton variety Coker 312, using the transformation vector pTEM2. Cotton explants were exposed to a culture of disarmed Agrobacterium tumefaciens containing plasmid p-TEM2. After co-culture, the cotton cells were regenerated to whole plants using the appropriate regeneration media with 500 mg/L claforan to eliminate residual <i>Agrobacterium</i>, and then selected with glyphosate.</p>

<p style="text-align: justify;">The shoots that developed were transferred to the greenhouse, further tested for tolerance to glyphosate, and allowed to flower and set seed. The transformation was confirmed by 2mEPSPS enzyme activity assay, by glyphosate application to leaves, and by polymerase chain reaction (PCR) and Southern blot analyses.</p>

<p style="text-align: justify;">The transformation vector used to generate cotton line GHB614, p-TEM2, contains one gene expression cassette within the left and right border segments (T-DNA). The sequence of the 2mepsps gene is derived from the wildtype <i>epsps</i> gene from corn (<i>Zea mays</i>) with two single nucleotide mutations introduced by site directed mutagenesis. A methionine codon has been added to the N-terminal end of the 2mEPSPS protein sequence in order to restore the cleavage site of the optimized plastid transit peptide. The double mutant produces a 47 kDa protein with normal enzyme function and reduced affinity for glyphosate.</p>

<p style="text-align: justify;">The Ph4a748At promoter and h3At intron are regulatory elements used to control expression of the <i>2mepsps</i> gene in cotton and are derived from the histone H4 gene of the plant <i>Arabidopsis thaliana</i>. The use of these elements directs high level constitutive expression, particularly in rapidly growing plant tissues. TPotp C, encodes the optimized transit peptide derived from genes of corn and sunflower and targets the mature protein to the plastids where it is normally located in the cell. The 3&rsquo;histonAt terminator from <i>Arabidopsis thaliana</i> corresponds to the polyadenylation signal which is essential to end transcription of the introduced gene.</p>
