Diane B. Re

Bio: Diane B. Re is an academic researcher from Monsanto. The author has contributed to research in topics: Site-directed mutagenesis & EPSP synthase. The author has an hindex of 6, co-authored 7 publications receiving 1540 citations.

Development, identification, and characterization of a glyphosate-tolerant soybean line

Abstract: Glyphosate (N-phosphonomethyl-glycine) is the active ingredient in the nonselective herbicide Roundup. The sensitivity of crop plants to glyphosate has limited its in-season use as a postemergence herbicide. The extension of the use of Roundup herbicide to allow in-season application in major crops such as soybeans [Glycine max (L.) Merr.] would provide new weed control options for farmers. A glyphosate-tolerant soybean line, 40-3-2, was obtained through expression of the bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, EPSPS) enzyme from Agrobacterium sp. strain CP4. Line 40-3-2 is highly tolerant to glyphosate, showing no visual injury after application of up to 1.68 kg acid equivalent (a.e.) ha -1 of glyphosate under field conditions. Molecular characterization studies determined that the single genetic insert in line 40-3-2 contains only a portion of the cauliflower mosaic virus 35S promoter (P-E35S), the Petunia hybrida EPSPS chloroplast transit peptide (CTP), the CP4 EPSPS gene, and a portion of the 3' nontranslated region of the nopaline synthase gene (NOS 3') terminator. Inheritance studies have shown that the transgene behaves as a single dominant gene and is stable over several generations.

Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer.

Abstract: Transgenic soybean plants have been produced using an Agrobacterium-mediated gene transfer system. This procedure relied on a regeneration protocol in which shoot organogenesis was induced on cotyledons of soybean genotypes selected for susceptibility to Agrobacterium. Cotyledon explants were inoculated with Agrobacterium tumefaciens pTiT37-SE harboring pMON9749 (conferring kanamycin resistance and β-glucuronidase “GUS” activity) or pTiT37-SE∷pMON894 (conferring kanamycin resistance and glyphosate tolerance) and cultured on shoot induction medium containing kanamycin. Plantlets were tested for gene insertion 3–4 months post-inoculation. Approximately 6% of the shoots (8 plants to date) produced on the kanamycin-selected cotyledons were transgenic based on assays for GUS expression, kanamycin resistance or glyphosate tolerance. Progeny from two of these plants demonstrated co-segregation of kanamycin resistance and either GUS expression or glyphosate tolerance in a 3:1 ratio indicating a single insert inherited in a Mendelian fashion.

Stably transformed callus lines from microprojectile bombardment of cell suspension cultures of wheat

Abstract: We have obtained stably transformed callus lines by direct delivery of DNA into plated suspension culture cells of wheat (Triticum aestivum L.) using high velocity microprojectile bombardment. Three different reporter or selectable marker genes, jointly present or on separate plasmids, were introduced: neomycin phospho-transferase (NPTII), β-glucuronidase (GUS) and 5-enolpyruvylshikimate phosphate (EPSP) synthase. Kanamycin was used for the selection of resistant calli, which were screened for GUS expression by a histochemical stain. Southern analysis confirmed that the NPTII, GUS and EPSP synthase genes had been stably integrated in all of the kanamycin resistant and GUS positive lines, and NPTII and EPSP synthase activities were demonstrated in the transformed calli.

Soybean event MON89788 and methods for detection thereof

Abstract: The present invention provides for soybean plant and seed comprising transformation event MON89788 and DNA molecules unique to these events. The invention also provides methods for detecting the presence of these DNA molecules in a sample.

Evolution in Action: Plants Resistant to Herbicides

Abstract: Modern herbicides make major contributions to global food production by easily removing weeds and substituting for destructive soil cultivation. However, persistent herbicide selection of huge weed numbers across vast areas can result in the rapid evolution of herbicide resistance. Herbicides target specific enzymes, and mutations are selected that confer resistance-endowing amino acid substitutions, decreasing herbicide binding. Where herbicides bind within an enzyme catalytic site very few mutations give resistance while conserving enzyme functionality. Where herbicides bind away from a catalytic site many resistance-endowing mutations may evolve. Increasingly, resistance evolves due to mechanisms limiting herbicide reaching target sites. Especially threatening are herbicide-degrading cytochrome P450 enzymes able to detoxify existing, new, and even herbicides yet to be discovered. Global weed species are accumulating resistance mechanisms, displaying multiple resistance across many herbicides and posing a great challenge to herbicide sustainability in world agriculture. Fascinating genetic issues associated with resistance evolution remain to be investigated, especially the possibility of herbicide stress unleashing epigenetic gene expression. Understanding resistance and building sustainable solutions to herbicide resistance evolution are necessary and worthy challenges.

The shikimate pathway.

Abstract: The shikimate pathway links metabolism of carbohydrates to biosynthesis of aromatic compounds. In a sequence of seven metabolic steps, phosphoenolpyruvate and erythrose 4-phosphate are converted to chorismate, the precursor of the aromatic amino acids and many aromatic secondary metabolites. All pathway intermediates can also be considered branch point compounds that may serve as substrates for other metabolic pathways. The shikimate pathway is found only in microorganisms and plants, never in animals. All enzymes of this pathway have been obtained in pure form from prokaryotic and eukaryotic sources and their respective DNAs have been characterized from several organisms. The cDNAs of higher plants encode proteins with amino terminal signal sequences for plastid import, suggesting that plastids are the exclusive locale for chorismate biosynthesis. In microorganisms, the shikimate pathway is regulated by feedback inhibition and by repression of the first enzyme. In higher plants, no physiological feedback inhibitor has been identified, suggesting that pathway regulation may occur exclusively at the genetic level. This difference between microorganisms and plants is reflected in the unusually large variation in the primary structures of the respective first enzymes. Several of the pathway enzymes occur in isoenzymic forms whose expression varies with changing environmental conditions and, within the plant, from organ to organ. The penultimate enzyme of the pathway is the sole target for the herbicide glyphosate. Glyphosate-tolerant transgenic plants are at the core of novel weed control systems for several crop plants.

Cropping systems for managing weeds

Abstract: The invention provides cropping systems for managing weeds in crop environments. The cropping systems comprise, in one embodiment, transgenic plants that display tolerance to an auxin-like herbicide such as dicamba. Method for minimizing the development of herbicide resistant weeds are also provided.

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens.

Abstract: A rapid Agrobacterium tumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. The explants were inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the [beta]-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene. Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium. The inoculated immature embryos or embryogenic calli were selected on G418-containing media. Transgenic plants were regenerated from all three types of explants. The total time required from inoculation to the establishment of plants in soil was 2.5 to 3 months. So far, more than 100 transgenic events have been produced. Almost all transformants were morphologically normal. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to five copies of the transgene were integrated into the wheat genome without rearrangement. Approximately 35% of the transgenic plants received a single copy of the transgenes based on Southern analysis of 26 events. Transgenes in T1 progeny segregated in a Mendelian fashion in most of the transgenic plants.