<p style="text-align: justify;">Maize line MS3 was produced by transforming the inbred line H99 by electroporation of immature embryos in the presence of a preparation of linearized plasmid DNA. The plasmid mixture contained both genes of interest; the male sterility gene (barnase) and the herbicide resistance gene (<i>bar</i>) and additional genetic elements including barstar, <i>bla</i> and <i>cat</i> genes of which none were expressed in the modified maize plant.</p>

<p style="text-align: justify;">The barnase gene encodes an RNAse enzyme that disrupts cell function during anther development and results in male sterility. The expression of the barnase gene was under control of an anther-specific promoter (pTa29), from <i>Nicotiana tabacum</i>, that turns on expression of the gene in the anthers where pollen is produced. The barnase gene was terminated by the 3&#39; -polyadenylation signal of the nopaline synthase (<i>nos</i>) gene isolated from the Ti plasmid of <i>Agrobacterium tumefaciens</i>.</p>

<p style="text-align: justify;">The <i>bar</i> gene encodes the PAT enzyme confering tolerance to glufosinate ammonium herbicide and was used as a selectable marker. Constitutive expression of the <i>bar</i> gene was under control of the cauliflower mosaic virus (CaMV) 35S promoter. Translocation of the introduced PAT enzyme to chloroplasts was accomplished by fusing sequences encoding the chloroplast transit peptide to the 5&#39; terminus of the <i>bar</i> gene. Apart from the sequences encoding for RNAse and PAT, no other plant translatable DNA sequences were introduced into the maize plant genome.</p>

<p style="text-align: justify;">The introduced DNA included the barstar gene from <i>B. amyloliquefaciens</i> that encodes a specific inhibitor of the barnase RNAse. The barstar gene was included to prevent the RNAse from disrupting the development of bacteria in which the introduced DNA was prepared. Also included were two antibiotic resistant genes the beta-lactamase gene (<i>bla</i>) and chloramphenicol acetyl transferase (<i>cat</i>) genes, encoding resistance to the antibiotics ampicillin and chloramphenicol, respectively. These were included as selectable marker genes to identify bacteria transformed with recombinant plasmids. The expression of these three genes (barstar, <i>bla</i>, and <i>cat</i>) was regulated by bacterial promoters that are not active in plants.</p>
