Stephen C. Weller

Gene amplification in glyphosate tolerant tobacco cells.

Abstract A series of tobacco ( Nicotiana tabacum L.) cell lines have been selected for growth in the presence of normally lethal concentrations of glyphosate, up to 20 mM. Activity of 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), which is inhibited by glyphosate, is elevated in tolerant cells. However, EPSPS isolated from tolerant cells is still inhibited by the herbicide. Increased activity of EPSPS is the result of increased levels of the enzyme in tolerant cells, as determined by immunoblotting, rather than a change in enzyme activity. RNA blots with a petunia EPSPS cDNA demonstrate a correlation between levels of tolerance and steady state level of EPSPS mRNA. Increased expression of EPSPS mRNA results from amplification of at least two genes encoding this enzyme in the tobacco genome. The degree of gene amplification also increases with level of glyphosate tolerance. Selected cells grown in the absence of glyphosate have an elevated EPSPS mRNA abundance and maintain some gene amplification, indicating that selection has resulted in a stable genetic modification.

U.S. Grower Views on Problematic Weeds and Changes in Weed Pressure in Glyphosate-Resistant Corn, Cotton, and Soybean Cropping Systems

Abstract Corn and soybean growers in Illinois, Indiana, Iowa, Mississippi, Nebraska, and North Carolina, as well as cotton growers in Mississippi and North Carolina, were surveyed about their views on changes in problematic weeds and weed pressure in cropping systems based on a glyphosate-resistant (GR) crop. No growers using a GR cropping system for more than 5 yr reported heavy weed pressure. Over all cropping systems investigated (continuous GR soybean, continuous GR cotton, GR corn/GR soybean, GR soybean/non-GR crop, and GR corn/non-GR crop), 0 to 7% of survey respondents reported greater weed pressure after implementing rotations using GR crops, whereas 31 to 57% felt weed pressure was similar and 36 to 70% indicated that weed pressure was less. Pigweed, morningglory, johnsongrass, ragweed, foxtail, and velvetleaf were mentioned as their most problematic weeds, depending on the state and cropping system. Systems using GR crops improved weed management compared with the technologies used before the adoption of GR crops. However, the long-term success of managing problematic weeds in GR cropping systems will require the development of multifaceted integrated weed management programs that include glyphosate as well as other weed management tactics. Nomenclature: Glyphosate; foxtail, Setaria spp.; johnsongrass, Sorghum halepense (L.) Pers.; morningglory, Ipomoea spp.; pigweed, Amaranthus spp.; ragweed, Ambrosia spp.; velvetleaf, Abutilon theophrasti Medik.; corn, Zea mays L.; cotton, Gossypium hirsutum L; soybean, Glycine max (L.) Merr

Transgenic peppermint (Mentha×piperita L.) plants obtained by cocultivation with Agrobacterium tumefaciens

Abstract The first transgenic peppermint (Mentha×piperita L. cultivar Black Mitcham) plants have been obtained by Agrobacterium-mediated transformation by cocultivation with morphogenically responsive leaf explants. Basal leaf explants with petioles, from leaves closest to the apex of in-vitro-culture-maintained shoots (5 cm), exhibited optimal shoot organogenetic responsiveness on medium supplemented with thidiazuron (8.4 µm). Shoot formation occurred at sites of excision on the leaf blade and petiole either directly from cells of the explant or via a primary callus. Analyses of transient GUS activity data indicated that DNA delivery by microprojectile bombardment was more effective than Agrobacterium infection. However, no transgenic plants were obtained from over 22,000 leaf explants after particle bombardment. Cocultivation of leaf explants with Agrobacterium strain EHA 105 and kanamycin selection produced transgenic plants. Greater transient and stable -glucuronidase (GUS) activities were detected in explants or propagules transformed with the construct where gusA was driven by the pBISN1 promoter rather than a CaMV 35S promoter. Eight plants were subsequently regenerated and verified as transgenic based on detection of the nptII transgene by PCR and Southern blot analyses. The Southern analyses indicated that the plants were derived from eight unique transformation events. All transgenic plants appeared morphologically normal. Analyses of GUS activities in leaves sampled from different portions of these transgenic plants, 10 months after transfer to the greenhouse, indicated that six out of the eight original regenerants were uniformly transformed, i.e., did not exhibit chimeric sectors.

A Grower Survey of Herbicide Use Patterns in Glyphosate-Resistant Cropping Systems

Abstract A telephone survey was conducted with growers in Iowa, Illinois, Indiana, Nebraska, Mississippi, and North Carolina to discern the utilization of the glyphosate-resistant (GR) trait in crop rotations, weed pressure, tillage practices, herbicide use, and perception of GR weeds. This paper focuses on survey results regarding herbicide decisions made during the 2005 cropping season. Less than 20% of the respondents made fall herbicide applications. The most frequently used herbicides for fall applications were 2,4-D and glyphosate, and these herbicides were also the most frequently used for preplant burndown weed control in the spring. Atrazine and acetochlor were frequently used in rotations containing GR corn. As expected, crop rotations using a GR crop had a high percentage of respondents that made one to three POST applications of glyphosate per year. GR corn, GR cotton, and non-GR crops had the highest percentage of growers applying non-glyphosate herbicides during the 2005 growing season. A crop rotation containing GR soybean had the greatest negative impact on non-glyphosate use. Overall, glyphosate use has continued to increase, with concomitant decreases in utilization of other herbicides. Nomenclature: 2,4-D; acetochlor; atrazine; glyphosate; corn, Zea mays L.; cotton, Gossipium hirsutum L.; soybean, Glycine max (L.) Merr

Using a Grower Survey to Assess The Benefits and Challenges of Glyphosate-Resistant Cropping Systems for Weed Management in U.S. Corn, Cotton, and Soybean

Abstract Over 175 growers in each of six states (Illinois, Indiana, Iowa, Mississippi, Nebraska, and North Carolina) were surveyed by telephone to assess their perceptions of the benefits of utilizing the glyphosate-resistant (GR) crop trait in corn, cotton, and soybean. The survey was also used to determine the weed management challenges growers were facing after using this trait for a minimum of 4 yr. This survey allowed the development of baseline information on how weed management and crop production practices have changed since the introduction of the trait. It provided useful information on common weed management issues that should be addressed through applied research and extension efforts. The survey also allowed an assessment of the perceived levels of concern among growers about glyphosate resistance in weeds and whether they believed they had experienced glyphosate resistance on their farms. Across the six states surveyed, producers reported 38, 97, and 96% of their corn, cotton, and soybean hectarage planted in a GR cultivar. The most widely adopted GR cropping system was a GR soybean/non-GR crop rotation system; second most common was a GR soybean/GR corn crop rotation system. The non-GR crop component varied widely, with the most common crops being non-GR corn or rice. A large range in farm size for the respondents was observed, with North Carolina having the smallest farms in all three crops. A large majority of corn and soybean growers reported using some type of crop rotation system, whereas very few cotton growers rotated out of cotton. Overall, rotations were much more common in Midwestern states than in Southern states. This is important information as weed scientists assist growers in developing and using best management practices to minimize the development of glyphosate resistance. Nomenclature: Glyphosate; corn, Zea mays L.; cotton, Gossipium hirsutum L.; rice, Oryza sativa L.; soybean, Glycine max (L.) Merr

Influence of Weed Management Practices and Crop Rotation on Glyphosate-Resistant Horseweed (Conyza canadensis) Population Dynamics and Crop Yield-Years III and IV

Abstract Horseweed is an increasingly common and problematic weed in no-till soybean production in the eastern cornbelt due to the frequent occurrence of biotypes resistant to glyphosate. The objective of this study was to determine the influence of crop rotation, winter wheat cover crops (WWCC), residual non-glyphosate herbicides, and preplant application timing on the population dynamics of glyphosate-resistant (GR) horseweed and crop yield. A field study was conducted from 2003 to 2007 in a no-till field located at a site that contained a moderate infestation of GR horseweed (approximately 1 plant m−2). The experiment was a split-plot design with crop rotation (soybean–corn or soybean–soybean) as main plots and management systems as subplots. Management systems were evaluated by quantifying in-field horseweed plant density, seedbank density, and crop yield. Horseweed densities were collected at the time of postemergence applications, 1 mo after postemergence (MAP) applications, and at the time of crop harvest or 4 MAP. Viable seedbank densities were also evaluated from soil samples collected in the fall following seed rain. Soybean–corn crop rotation reduced in-field and seedbank horseweed densities vs. continuous soybean in the third and fourth yr of this experiment. Preplant herbicides applied in the spring were more effective at reducing horseweed plant densities than when applied in the previous fall. Spring-applied, residual herbicide systems were the most effective at reducing season-long in-field horseweed densities and protecting crop yields since the growth habit of horseweed in this region is primarily as a summer annual. Management systems also influenced the GR and glyphosate-susceptible (GS) biotype population structure after 4 yr of management. The most dramatic shift was from the initial GR : GS ratio of 3 : 1 to a ratio of 1 : 6 after 4 yr of residual preplant herbicide use followed by non-glyphosate postemergence herbicides. Nomenclature: Glyphosate; horseweed, Conyza canadensis (L.) Cronq. ERICA.

Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate-tolerant tobacco cells

Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) were obtained from a glyphosate-tolerant tobacco cell line. The cDNAs were 89% identical and the predicted sequences of the mature proteins were greater than 83% identical with EPSPS proteins from other plants. Tobacco EPSPS proteins were more similar to those from tomato and petunia than Arabidopsis. One cDNA clone, EPSPS-1, represented a gene that was amplified in glyphosate-tolerant cells, while the gene for EPSPS-2 was unaltered in these cells. Consequently, EPSPS-1 mRNA was more abundant in tolerant than unselected cells, whereas EPSPS-2 mRNA was at relatively constant levels in these cell lines. Exposure of unselected cells and tobacco leaves to glyphosate produced a transient increase in EPSPS mRNA. However, glyphosate-tolerant cells containing amplified copies of EPSPS genes did not show a similar response following exposure to glyphosate. A significant proportion of the EPSPS gene amplification was maintained when tolerant cells were grown in the absence of glyphosate for eight months. Plants regenerated from these cells also contained amplified EPSPS genes.

A serine-to-threonine substitution in the triazine herbicide-binding protein in potato cells results in atrazine resistance without impairing productivity

A mutation of the psbA gene was identified in photoautotrophic potato (Solanum tuberosum L. cv Superior x U.S. Department of Agriculture line 66–142) cells selected for resistance to 6-chloro-N-ethyl-N[prime]-(1-methylethyl)-1,3,5-triazine-2,4-diamine (atrazine). Photoaffinity labeling with 6-azido-N-ethyl-N[prime]-(1-methylethyl)-1,3,5-triazine-2,4-diamine detected a thylakoid membrane protein with a Mr of 32,000 in susceptible, but not in resistant, cells. This protein was identified as the secondary quinone acceptor of photosystem II (QB) protein. Atrazine resistance in selected cells was attributable to a mutation from AGT (serine) to ACT (threonine) in codon 264 of the psbA gene that encodes the QB protein. Although the mutant cells exhibited extreme levels of resistance to atrazine, no concomitant reductions in photosynthetic electron transport or cell growth rates compared to the unselected cells were detected. This is in contrast with the losses in productivity observed in atrazine-resistant mutants that contain a glycine-264 alteration.

Influence of Weed Management Practices and Crop Rotation on Glyphosate-Resistant Horseweed Population Dynamics and Crop Yield

Abstract Horseweed is an increasingly problematic weed in soybean because of the frequent occurrence of glyphosate-resistant (GR) biotypes. The objective of this study was to determine the influence of crop rotation, winter wheat cover crops (WWCC), residual nonglyphosate herbicides, and preplant herbicide application timing on the population dynamics of GR horseweed and crop yield. A field study was conducted at a site with a moderate infestation of GR horseweed (approximately 1 plant m−2) with crop rotation (soybean–corn or soybean–soybean) as main plots and management systems as subplots. Management systems were evaluated by quantifying horseweed plant density, seedbank density, and crop yield. Crop rotation did not influence in-field horseweed or seedbank densities at any data census timing. Preplant herbicides applied in the spring were more effective at reducing horseweed plant densities than when applied in the previous fall. Spring-applied, residual herbicide systems were the most effective at reducing season long horseweed densities and protecting crop yield because horseweed in this region behaves primarily as a summer annual weed. Horseweed seedbank densities declined rapidly in the soil by an average of 76% for all systems over the first 10 mo before new seed rain. Despite rapid decline in total seedbank density, seed for GR biotypes remained in the seedbank for at least 2 yr. Therefore, to reduce the presence of GR horseweed biotypes in a local no-till weed flora, integrated weed management (IWM) systems should be developed to reduce total horseweed populations based on the knowledge that seed for GR biotypes are as persistent in the seed bank as glyphosate-sensitive (GS) biotypes. Nomenclature: Glyphosate; horseweed, Conyza canadensis L. ERICA; corn, Zea mays L; soybean, Glycine max (L.) Merr; winter wheat, Triticum aestivum L.

Impaired Wound Induction of 3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) Synthase and Altered Stem Development in Transgenic Potato Plants Expressing a DAHP Synthase Antisense Construct

Potato (Solanum tuberosum L.) cells were transformed with an antisense DNA construct encoding part of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15), the first enzyme of the shikimate pathway, to examine the role(s) of this protein in plant growth and development. Chimeric DNA constructs contained the transcript start site, the first exon, and part of the first intron of the shkA gene in antisense or sense orientations under the control of the cauliflower mosaic virus 35S promoter. Some, but not all, of the transgenic plants expressing antisense DAHP synthase RNA showed reduced levels of wound-induced DAHP synthase enzyme activity, polypeptide, and mRNA 12 and 24 h after wounding. No alteration in the wound induction of DAHP synthase gene expression was observed in transgenic potato tubers containing the chimeric sense construct. Reduced steady-state levels of DAHP synthase mRNA were observed in stem and shoot tip tissue. Some plants with the chimeric antisense construct had reduced stem length, stem diameter, and reduced stem lignification.