Glyphosate is a potent herbicide. It works by competitive inhibition of the enzyme 5-enol-pyruvyl shiki-mate-3-phosphate synthase (EPSPS), which catalyzes an essential step in the aromatic amino acid biosynthetic pathway. We report the genetic engineering of herbicide resistance by stable integration of the petunia EPSPS gene into the tobacco chloroplast genome using the tobacco or universal vector. Southern blot analysis confirms stable integration of the EPSPS gene into all of the chloroplast genomes (5000-10,000 copies per cell) of transgenic plants. Seeds obtained after the first self-cross of transgenic plants germinated and grew normally in the presence of the selectable marker, whereas the control seedlings were bleached. While control plants were extremely sensitive to glyphosate, transgenic plants survived sprays of high concentrations of glyphosate. Chloroplast transformation provides containment of foreign genes because plastid transgenes are not transmitted by pollen. The escape of foreign genes via pollen is a serious environmental concern in nuclear transgenic plants because of the high rates of gene flow from crops to wild weedy relatives. Keywords agricultural biotechnology; transformation; tobacco; glyphosate Glyphosate is a potent, broad spectrum herbicide that is highly effective against a majority of grasses and broad leaf weeds. Glyphosate works by competitive inhibition of the enzyme 5-enol-pyruvyl shikimate-3-phosphate synthase (EPSPS) of the aromatic amino acid biosynthetic pathway. Synthesis of EPSP from shiki-mate-3-phosphate and inorganic phosphate is catalyzed by EPSPS. This particular reaction occurs only in plants and microorganisms, which explains why glyphosate is nontoxic to other living forms. Use of glyphosate is environmentally safe as it is inactivated rapidly in soil, has minimum soil mobility, and degrades to natural products, with little toxicity to non-plant life forms. However, glyphosate lacks selectivity and does not distinguish crops from weeds, thereby restricting its use. EPSPS-based glyphosate resistance has been genetically engineered via the nuclear genome either by the overproduction of the wild-type EPSPS 1 or by the expression of a mutant gene (aroA) encoding glyphosate-resistant EPSPS 2 . *Corresponding author (daniehe@mail.auburn.edu). HHS Public Access Author ManuscriptAuthor Manuscript Author Manuscript Author ManuscriptIn all of the aforementioned examples, without exception, herbicide-resistant genes have been introduced into the nuclear genome. One common concern is the escape of a foreign gene through pollen dispersal from transgenic crop plants engineered for herbicide resistance to their weedy relatives, creating "superweeds." Dispersal of pollen from a central test plot containing transgenic cotton plants to surrounding nontransgenic plants has been observed at varying distances in different directions 3,4 . The escape of foreign genes through pollen is a serious environmental concern, especially in the case of herbicide resistance genes, b...

Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000-to 40,000-fold) and Cry2Aa2-resistant (330-to 393-fold) tobacco budworm Heliothis virescens, cotton bollworm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5,000-10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20-to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field.

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