Containment of herbicide resistance through genetic engineering of the chloroplast genome

Abstract

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...

Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

“…1A). This vector should be able to transform chloroplast genomes of several plant species because the flanking sequences are highly conserved among higher plants 11,12 . This vector contains the 16S ribosomal RNA (rRNA) promoter (Prrn) driving the aadA gene (aminoglycoside 3′-adenylyltransferase) for spectinomycin selection and the three genes of the cry2Aa2 operon.…”

The release of genetically modified crops into the environment

“…Either specific areas of the world should be dedicated for specific productions, or additional molecular mechanisms to prevent gene flow should be put in place. Recent advances in the genetic engineering of chloroplasts offers an approach to further limit gene escape through pollen in species of plants in which chloroplasts are maternally inherited (Daniell et al, 1998). Possibly, the applications of molecular farming may require a revival of the interest in, and acceptance of, other approaches known as genetic use restriction technologies (GURTs), in the popular press better know as 'terminator technology' (Visser et al, 2001).…”

Chloroplast Genetic Engineering

“…In most angiosperms, plastids are maternally inherited, which minimizes the concern of outcrossing of transgenes [12,13] and reduces the potential toxicity of transgenic pollen to non-target insects [1]. Maternal inheritance of transgenes offers containment because of lack of gene flow through pollen [13] and this has been demonstrated in different plant species [7,12,14].…”

“…Maternal inheritance of transgenes offers containment because of lack of gene flow through pollen [13] and this has been demonstrated in different plant species [7,12,14]. The Daniell laboratory developed a chloroplast genome derived cytoplasmic male sterility system in which the tapetal layers are destroyed in developing pollen, making transgenic plants male sterile without affecting other metabolic functions [15].…”

“…Chloroplast transgenic carrot plants have been engineered to withstand salt concentrations only halophytes could tolerate [16]. The Daniell laboratory has conferred several other plant traits, including herbicide [12], insect [17], and disease [18] resistance, drought [10], phytoremediation [19] and production of biopolymers [20].…”