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<p style="text-align: justify;">The GA21 maize line was created through biolistic transformation of embryogenic maize cells with DNA-coated gold particles and regeneration of plants by tissue culture on selective medium. The 3.4 kb DNA restriction fragment (<i>Not</i>I digested pDPG434 plasmid DNA) used for transformation contained a copy of the EPSPS encoding gene originally isolated from maize and subsequently modified by <i>in vitro</i> site-directed mutagenesis to be insensitive to inactivation by glyphosate. The expression of the modified (m)-EPSPS encoding gene was controlled in part by the rice actin promoter intron sequences and the NOS 3&#39; termination sequence derived from the Ti plasmid of the plant pathogen <i>Agrobacterium tumefaciens</i>.</p>

<p style="text-align: justify;">The mEPSPS encoding gene was fused to an optimized chloroplast transit peptide (OTP) to allow subcellular targeting of mEPSPS protein into the chloroplast, the site of both the shikimate pathway and glyphosate mode-of-action. The chloroplast transit peptide sequences were derived from ribulose l,5-bisphosphate carboxylase oxygenase (RuBisCo) genes isolated from maize and sunflower.<br />
Because a purified fragment of DNA was used in the transformation, no extraneous bacterial genes, including antibiotic resistance marker genes, were transferred. Genes present in the original pDPG434 plasmid backbone but not in the <i>Not</i>I restriction fragment used for transformation included <i>lac</i>Z, <i>Col</i>E1(ori) and the beta-lactamase encoding <i>bla</i> gene from <i>E. coli</i> plasmid pBR322.</p>
