<p style="text-align: justify;">ASR368 creeping bentgrass was produced by microprojectile bombardment of plant cells from the line B99061R. The plasmid vector PV-ASGT08 used for the transformation contained two <i>cp4 epsps</i> gene cassettes coding for glyphosate tolerance. The T-DNA portion of the plasmid (PV-ASGT08L) contained two <i>cp4 epsps</i> gene expression cassettes. Joined with each of the <i>cp4 epsps</i> gene sequences was a chloroplast transit peptide (<i>cpt2</i>) sequence, derived from the <i>Arabidopsis thaliana epsps</i> gene. Two unique promoters were used to regulate the expression of CP4 EPSPS in both vegetative and reproductive tissues. In the first cassette, gene expression was regulated by an enhanced 35S promoter from the cauliflower mosaic virus (CaMV) and the intron of the hsp70 (heat shock protein) gene from <i>Zea mays</i>. The expression of the second <i>cp4 epsps</i> gene was regulated by the 5&rsquo; region of the rice actin1 gene which contains the promoter, transcription start site, and first intron. The transcription termination signal sequences were derived from the 3&rsquo; nontranslated region of the nopaline synthase gene from <i>Agrobacterium tumefaciens</i>.</p>

<p style="text-align: justify;">The PV-ASGT08 vector was replicated in <i>E. coli</i> and digested with the restriction enzyme HindIII to remove the backbone from the DNA segment PV-ASGT08L containing the gene construct. PV-ASGT08L was precipitated onto gold particles and introduced into the line B9961R by microprojectile bombardment. Transformed callus was selected <i>in vitro</i> using culture medium to which glyphosate had been incorporated. Plants were regenerated from tolerant callus using tissue culture techniques. ASR368 was isolated from these regenerated plants.</p>
