Paul L. Raymer

Increased fitness of transgenic insecticidal rapeseed under insect selection pressure

Rapeseed Brassica napus L. transgenic for a Bacillus thuringiensis (Bt) transgene was developed and was shown to be insecticidal towards certain caterpillars including the diamondback moth Plutella xylostella L. and the corn earworm Helicoverpa zea Boddie. To simulate an escape of the transgenics from cultivation, a field experiment was performed in which transgenic and nontransgenic rapeseed plants were planted in natural vegetation and cultivated plots and subjected to various selection pressures in the form of herbivory from insects. Only two plants, both transgenic, survived the winter to reproduce in the natural‐vegetation plots which were dominated by grasses such as crabgrass. However, in plots that were initially cultivated then allowed to naturalize, medium to high levels of defoliation decreased survivorship of nontransgenic plants relative to Bt‐transgenic plants and increased differential reproduction in favour of Bt plants. Thus, where suitable habitat is readily available, there is a likelihood of enhanced ecological risk associated with the release of certain transgene/crop combinations such as insecticidal rapeseed. This is the first report of a field study demonstrating the effect of a fitness‐increasing transgene in plants.

Growth, productivity, and competitiveness of introgressed weedy Brassica rapa hybrids selected for the presence of Bt cry1Ac and gfp transgenes

Concerns exist that transgenic crop × weed hybrid populations will be more vigorous and competitive with crops compared with the parental weed species. Hydroponic, glasshouse, and field experiments were performed to evaluate the effects of introgression of Bacillus thuringiensis (Bt) cry1Ac and green fluorescent protein (GFP) transgenes on hybrid productivity and competitiveness in four experimental Brassica rapa × transgenic Brassica napus hybrid generations (F1, BC1F1, BC2F1 and BC2F2). The average vegetative growth and nitrogen (N) use efficiency of transgenic hybrid generations grown under high N hydroponic conditions were lower than that of the weed parent (Brassica rapa, AA, 2n = 20), but similar to the transgenic crop parent, oilseed rape (Brassica napus, AACC, 2n = 38). No generational differences were detected under low N conditions. In two noncompetitive glasshouse experiments, both transgenic and nontransgenic BC2F2 hybrids had on average less vegetative growth and seed production than B. rapa. In two high intraspecific competition field experiments with varied herbivore pressure, BC2F2 hybrids produced less vegetative dry weight than B. rapa. The competitive ability of transgenic and nontransgenic BC2F2 hybrids against a neighbouring crop species were quantified in competition experiments that assayed wheat (Triticum aestivum) yield reductions under agronomic field conditions. The hybrids were the least competitive with wheat compared with parental Brassica competitors, although differences between transgenic and nontransgenic hybrids varied with location. Hybridization, with or without transgene introgression, resulted in less productive and competitive populations.

Genetic load and transgenic mitigating genes in transgenic Brassica rapa (field mustard) × Brassica napus (oilseed rape) hybrid populations.

BackgroundOne theoretical explanation for the relatively poor performance of Brassica rapa (weed) × Brassica napus (crop) transgenic hybrids suggests that hybridization imparts a negative genetic load. Consequently, in hybrids genetic load could overshadow any benefits of fitness enhancing transgenes and become the limiting factor in transgenic hybrid persistence. Two types of genetic load were analyzed in this study: random/linkage-derived genetic load, and directly incorporated genetic load using a transgenic mitigation (TM) strategy. In order to measure the effects of random genetic load, hybrid productivity (seed yield and biomass) was correlated with crop- and weed-specific AFLP genomic markers. This portion of the study was designed to answer whether or not weed × transgenic crop hybrids possessing more crop genes were less competitive than hybrids containing fewer crop genes. The effects of directly incorporated genetic load (TM) were analyzed through transgene persistence data. TM strategies are proposed to decrease transgene persistence if gene flow and subsequent transgene introgression to a wild host were to occur.ResultsIn the absence of interspecific competition, transgenic weed × crop hybrids benefited from having more crop-specific alleles. There was a positive correlation between performance and number of B. napus crop-specific AFLP markers [seed yield vs. marker number (r = 0.54, P = 0.0003) and vegetative dry biomass vs. marker number (r = 0.44, P = 0.005)]. However under interspecific competition with wheat or more weed-like conditions (i.e. representing a situation where hybrid plants emerge as volunteer weeds in subsequent cropping systems), there was a positive correlation between the number of B. rapa weed-specific AFLP markers and seed yield (r = 0.70, P = 0.0001), although no such correlation was detected for vegetative biomass. When genetic load was directly incorporated into the hybrid genome, by inserting a fitness-mitigating dwarfing gene that that is beneficial for crops but deleterious for weeds (a transgene mitigation measure), there was a dramatic decrease in the number of transgenic hybrid progeny persisting in the population.ConclusionThe effects of genetic load of crop and in some situations, weed alleles might be beneficial under certain environmental conditions. However, when genetic load was directly incorporated into transgenic events, e.g., using a TM construct, the number of transgenic hybrids and persistence in weedy genomic backgrounds was significantly decreased.

Chemical and instrumental characterization of maturing kenaf core and bast

Abstract A limiting factor in the production of bast fiber from kenaf is retting, the process by which the fiber is freed from the non-fibrous tissue. An objective of this study was to evaluate the variation in cell wall chemistry with maturity for different cultivars, particularly in relation to lignin and retting. Two cultivars of kenaf (Hibiscus cannabinus), Tainung-1 (T-1) and Everglades-41 (E41), were harvested at 96 and 151 days post planting (DPP), and the top and bottom 15 cm of the stems were excised for analysis. The hand-separated core and bast portions were analyzed for guaiacyl and syringyl groups (indicative of lignin), and arabinose, xylose, mannose, galactose, glucose, and uronic acids. Cell walls from bast were examined for aromatics by ultraviolet (UV) absorption microspectrophotometry. Bottom core contained significantly higher amounts of aromatics. Results suggested that the top bast tissue was not completely lignified by the early harvest. Cellulose deposition, as indicated by the glucose content at 96 DPP, also was not fully complete until the later harvest. UV absorption microspectrophotometry demonstrated that, while the entire bast fiber cell walls were all lignified, the middle lamella had higher absorbance indicative of more aromatic compounds. The λmax at 274–276 nm was consistent with a predominance of syringyl lignin. In a second study, three cultivars of kenaf, Tainung-2 (T-2), E41, and SF459, were harvested at 30, 60, 90, 120, and 180 DPP. The center 15 cm of the stems was excised for analysis. All three cultivars were similar to each other in components within a maturity period. Bast and core fractions were relatively high in lignin even at 30 DPP, and both secondary layers and middle lamellea contained lignin. The core had more lignin than fiber at maturity. Plants increased in lignin to ca 60 DPP and did not increase thereafter, while carbohydrates continued to vary with maturity. Glucose concentrations became stable at about 90 DPP and xylose concentrations remained constant at 60 DPP. Bast fibers had unusually high syringyl:guaiacyl ratios (6.3–9.4). Principal component analysis (PCA) of the mass spectral data of the bast indicated that 30 DPP samples were distinctly different from those harvested at 60–120 DPP, with the 180 DPP sample different from all others. In enzymatic retting studies of Everglades-41 bast from younger plants, i.e. 30–60 DPP were more easily retted than other harvests and mechanical disruption improved retting of more mature bast. Enzymatic retting resulted in separation of fiber bundles, rather than ultimate fibers.

Greenhouse and field evaluations of transgenic canola against diamondback moth, Plutella xylostella, and corn earworm, Helicoverpa zea

Canola (Brassica napus L.) cultivars Oscar and Westar, engineered with a Bacillus thuringiensis (Bt) cryIA(c) gene, were evaluated for resistance to lepidopterous pests, diamondback moth, Plutella xylostella L. (Plutellidae) and corn earworm, Helicoverpa zea (Boddie) (Noctuidae) in greenhouse and field conditions. In greenhouse preference assays conducted at vegetative and flowering plant stages, transgenic plants recorded very low levels of damage. A 100% diamondback moth mortality and ≈90% corn earworm mortality were obtained on transgenic plants in greenhouse antibiosis assays. The surviving corn earworm larvae on transgenic plants had reduced head capsule width and body weight. Mortality of diamondback moth and corn earworm were 100% and ≈95%, respectively, at different growth stages (seedling, vegetative, bolting, and flowering) on the transgenic plants in greenhouse tests. In field tests conducted during 1995–1997, plots were artificially infested with neonates of diamondback moth or corn earworm or left for natural infestation. Transgenic plants in all the treatments were highly resistant to diamondback moth and corn earworm larvae and had very low levels of defoliation. Plots infested with diamondback moth larvae had greater damage in both seasons as compared with corn earworm infested plots and plots under natural infestation. After exposure to defoliators, transgenic plants usually had higher final plant stand and produced more pods and seeds than non‐transgenic plants. Diamondback moth injury caused the most pronounced difference in plant stand and pod and seed number between transgenic and non‐transgenic plants. Our results suggest that transgenic canola could be used for effective management of diamondback moth and corn earworm on canola.

Characterization of directly transformed weedy Brassica rapa and introgressed B. rapa with Bt cry1Ac and gfp genes

Crop to weed transgene flow, which could result in more competitive weed populations, is an agricultural biosafety concern. Crop Brassica napus to weedy Brassica rapa hybridization has been extensively characterized to better understand the transgene flow and its consequences. In this study, weedy accessions of B. rapa were transformed with Bacillus thuringiensis (Bt) cry1Ac- and green fluorescence protein (gfp)-coding transgenes using Agrobacterium to assess ecological performance of the wild biotype relative to introgressed hybrids in which the transgenic parent was the crop. Regenerated transgenic B. rapa events were characterized by progeny analysis, Bt protein enzyme-linked immunosorbent assay (ELISA), Southern blot analysis, and GFP expression assay. GFP expression level and Bt protein concentration were significantly different between independent transgenic B. rapa events. Similar reproductive productivity was observed in comparison between transgenic B. rapa events and B. rapa × B. napus introgressed hybrids in greenhouse and field experiments. In the greenhouse, Bt transgenic plants experienced significantly less herbivory damage from the diamondback moth (Plutella xylostella). No differences were found in the field experiment under ambient, low, herbivore pressure. Directly transformed transgenic B. rapa plants should be a helpful experimental control to better understand crop genetic load in introgressed transgenic weeds.

Sicklepod (Senna obtusifolia) and Red Morningglory (Ipomoea coccinea) Control in Glyphosate-Resistant Soybean with Narrow Rows and Postemergence Herbicide Mixtures1

Field studies were conducted over 2 yr to evaluate weed control, yield, and net returns of glyphosate-resistant soybean using total postemergence (5 wk) (POST) herbicide systems with glyphosate–isopropylamine (Ipa) or glyphosate–trimethylsulfonium (Tms) alone, tank mixed with fomesafen, or in sequential treatments with bentazon, fomesafen, Ipa, or Tms. Soybean early-season injury ranged from 0 to 28% across the test. Although Ipa did not injure soybean, glyphosate–Tms early postemergence (3 wk) (EPOST) injured soybean from 7 to 17% depending on the rate. Glyphosate–Tms mixed with fomesafen EPOST injured soybean from 20 to 28%. Red morningglory control by Ipa and Tms at 0.8 kg ae/ha was no more than 88%. Sequential applications of Tms or Ipa controlled red morningglory 78% or less. Fomesafen improved red morningglory control by Ipa and Tms. Bentazon did not affect the control of red morningglory by these herbicides. Sicklepod, smooth pigweed, and large crabgrass control was 81, 93, and 79%, respectively, or greater for all herbicide treatments. By midseason, narrow-row soybeans had canopied, and competition from weeds was minimal. Overall, the net returns were reflective of soybean yield, and maximum net returns were recorded for treatments with reduced herbicide inputs. Conversely, sequential application of herbicides as EPOST followed by POST treatments resulted in lower net returns because of increased herbicide and application costs. Nomenclature: Bentazon; fomesafen; glyphosate–isopropylamine; glyphosate–trimethylsulfonium; large crabgrass, Digitaria sanguinalis (L.) Scop. #3 DIGSA; red morningglory, Ipomoea coccinea L. # IPOCC; sicklepod, Senna obtusifolia (L.) Irwin and Barnaby # CASOB; smooth pigweed, Amaranthus hybridus L. # AMACH; soybean, Glycine max (L.) Merr. Additional index words: AMACH, Amaranthus hybridus, CASOB, Digitaria sanguinalis, DIGSA, Glycine max, herbicide-resistant crops, IPOCC, Ipomoea coccinea, net returns, Roundup-Ready® soybean, Senna obtusifolia, soybean injury, soybean yield, sulfosate. Abbreviations: EPOST, early postemergence (3 wk); fb, followed by; Ipa, isopropylamine; POST, postemergence (5 wk); Tms, trimethylsulfonium.

A survey of the castor oil content, seed weight and seed-coat colour on the United States Department of Agriculture germplasm collection

Castor (Ricinus communis L.) is an important non-edible oilseed crop that can potentially be used as feedstock for biodiesel production. There are 1033 accessions in the United States Department of Agriculture (USDA) castor germplasm collection. The range of oil content in these accessions has never been surveyed. For exploiting castor as a feedstock for biodiesel production, the entire USDA castor collection was procured from the National Plant Germplasm System (Germplasm Resources Information Network) and the oil content was measured with nuclear magnetic resonance. The variation of oil content among all castor accessions ranged from 37.2 to 60.6% with an average of 48.2%. One hundred seed weights were determined and seed-coat colour was also recorded from each accession. One hundred seed weight ranged from 10.1 to 73.3 g with an average of 28.3 g. There was a significant correlation between seed weight and oil content but the correlation value was low (r ¼ 0.1572, P , 0.0001). Fifty accessions with a wide range of oil content were selected to be field grown for further evaluation. The results obtained from this survey will be useful for castor breeders seeking germplasm accessions with high oil content in the collection.

Characterization of seashore paspalum (Paspalum vaginatum Swartz) germplasm by transferred SSRs from wheat, maize and sorghum

One hundred and thirty SSR markers from wheat, maize and sorghum were screened for the transferability to Paspalum. The transfer rate was 67.5, 49.0 and 66.8% respectively. This would be a very efficient approach for DNA marker development for species which are not well studied molecularly. The polymorphism level for transferred SSR markers was 51.5% within species (Paspalum vaginatum) and 87.1% among Paspalum species. The high level of polymorphism is directly related to the high degree of heterozygosity maintained by its way of reproduction, i.e. self-incompatibility. Forty transferred polymorphic SSR markers were selected and used for characterization and evaluation of seventy-three Paspalum accessions. In total, 209 polymorphic bands were detected from these 40 SSR markers, with an average of five polymorphic bands per marker. The Paspalum accessions clustered into three major groups. Two very similar dendrograms can be generated from either 109 or 209 polymorphic bands. This led us to determine that 18 of the transferred SSR markers were sufficient for genetically differentiating the investigated germplasm accessions. The number of SSR markers required for germplasm characterization and evaluation is discussed. This is the first report of the transfer of SSR markers from major field crops to newly emerged environmental turfgrasses.

Effect of solid state fermentation of peanut shell on its dye adsorption performance

The effect of solid state fermentation of peanut shell to produce beneficial laccase and on its dye adsorption performance was evaluated. The resulting residues from solid fermentation were tested as sorbents (designated as SFs) in comparison to the raw peanut shell (RPS) for their ability to remove crystal violet from water. The fermentation process reduced the adsorption capacity (qm) of SF by about 50%, and changed the sorptive behavior when compared to the RPS. The Langmuir model was more suitable for fitting adsorption by SFs. qm was positively correlated with the surface area of peanut shell, but negatively correlated with acid detergent lignin content. For all the sorbents tested, the process was spontaneous and endothermic, and the adsorption followed both the pseudo 1st and 2nd order kinetic model and the film diffusion model. Dye adsorption efficiency was greater when SFs dispersed solution than when placed in filter packets.