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SAMPLE PETITIONHerbicide-Tolerant Plants

  • IV.

    Donor Genes and Regulatory Sequences

    • A.

      5–Enolpyruvyl Shikimate Phosphate (EPSP) Synthase Gene

The donor organism used to supply the glyphosate tolerance gene, aroA, was the bacterium Salmonella typhimurium. S. typhimurium is a well-characterized enteric bacterium with homology to E. coli (Ochman and Wilson, 1987). Some strains of S. typhimurium are known to cause a disease in susceptible mice and humans, but there is no evidence that strains of the bacterium are plant pests (Le Minor, 198l). Salmonella species may be associated with vegetation as free-living organisms whenever these plants have been contaminated with fertilizers of fecal origin or when they have been irrigated with polluted water. Salmonella organisms do not seem to multiply significantly in the natural environment (outside of digestive tracts), but they can survive several weeks in water and several years in soil if the conditions of temperature, humidity, and pH are favorable (Delage, 1960).

The aroA gene, which encodes the sequence for the enzyme EPSP synthase, has no known inherent plant-pest characteristics nor direct involvement in human and animal disease. EPSP synthase is one of the enzymes in the biosynthetic pathway leading to chorismate, an intermediate in the formation of aromatic amino acids and their derivatives (Pittard, 1987). This pathway is found in most plants, many single-celled organisms, and some lower forms of animals. The aroA gene is constitutively expressed in both E. coli (Tribe et al., 1976) and S. typhimurium (Gollub et al., 1983). The gene was mutated in the bacterium to provide glyphosate resistance by the classical genetic techniques of mutation and selection (Comai et al., 1983). The description of the modified aroA gene, including the method of isolation and complete gene sequence, is contained in two published papers (Comai et al., 1983 and Stalker et al., 1985). The EPSP synthase gene was fused to the CaMV35s promoter and NOS termination/polyadenylation sequences as previously described (White, 1992). Confirmation that this gene was indeed inserted into the cotton chromosome was provided by the following: Southern gel analysis, Mendelian inheritance, measurement of expression levels of


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