Recovery and evaluation of soybean plants transgenic for aBacillus thuringiensis var.Kurstaki insecticidal gene

Summary Lepidopteran insects are major defoliating pests of soybean in the southeastern United States. Soybean plants transgenic for a nativecryIA(b) gene fromBacillus thuringiensis var.kurstaki HD-1 were obtained. Embryogenic cultures were induced by plating cotyledons on a Murashige and Skoog-based medium supplemented with 40 mg/liter of 2,4-dichlorophenoxyacetic acid (2,4-D). The embryogenic cultures were maintained in liquid medium containing 5 mg/liter 2,4-D. These cultures were subjected to microprojectile bombardment, followed by selection on 50 mg/liter hygromycin. Resistant embryogenic cell lines were transferred to growth regulator-free medium to permit recovery of mature somatic embryos. After a desiccation period, the somatic embryos were returned to growth regulator-free medium for conversion into plants. Southern hybridization analysis verified transformation. Feeding assays of T₁ plants from one cell line deterred feeding, development, and survival of velvetbean caterpillar at a level comparable to that of GatIR81-296, a soybean breeding line with a high level of insect resistance. Reduced feeding on T₁ plants correlated with the presence of the transgene.     

Self-transmissible plasmid in Zymomonas mobilis carrying antibiotic resistance.

The cryptic plasmid pRUT41 from Zymomonas mobilis was examined for its biological properties. This plasmid was found to be conjugally transferred from Z. mobilis CP4 to Escherichia coli BM21 and to carry genes for antibiotic resistance (gentamicin, kanamycin, and streptomycin). Covalently closed circular plasmid DNA was isolated from eight transconjugants of E. coli BM21. These plasmids were identical in mobility on agarose gels and exhibited the same restriction patterns as the native pRUT41 plasmid isolated from Z. mobilis. The plasmid location of the antibiotic resistance genes was further confirmed by transforming E. coli BM21 with isolated pRUT41 plasmid from strain CP4 and with plasmids from the transconjugants of BM21. Resistance to streptomycin, kanamycin, and gentamicin was tightly linked and transferred together in all cases.     

Membrane-initiated steroid signaling (MISS): genomic steroid action starts at the plasma membrane

Plasma membrane (PM) steroid recognition sites are thought to be responsible only for rapid, non-genomic responses without any link to the nuclear receptor-mediated genomic effects of steroids. We focused on a PM "glucocorticoid-importer" (GC-importer) that imports GC into rat liver cells. This site interacts also with particular gestagens (progesterone, P; medroxyprogesterone, MP; ethynodiol, Ethy) and estrogens (ethinylestradiol, EE(2); mestranol), which do not bind to the nuclear GC receptor (GR). To elucidate the role of the GC-importer, we transfected a rat wild-type hepatocyte (CC-1) and a hepatoma cell line, unable to import GC (MH 3924), with a GC<-->GR-responsive luciferase (luc)-reporter gene. Selected steroids were tested for their ability to induce or inhibit luc expression. Corticosterone (B) and dexamethasone (Dex), but also the GC-antagonists cortexolone (Cortex), P and MP, induced luc. Even the PM-impermeable BSA-derivatives of B, Dex and Cortex did so to almost the same extent as the free steroids. MH 3924 cells respond stronger than CC-1 to luc inducing steroids. Luc expression was inhibited by RU 38 486, but also by EE(2) and Ethy. The thiol reactive mesylate-derivatives of B, Dex and Cortex induced to a considerably lesser extent than the free or BSA-steroids. The thiol reagent mersalyl blocks cellular entry of GC and inhibits luc induction in CC-1 cells. Incubation with EE(2) and B of PM-vesicles, isolated from liver cells, resulted in a decrease of the density of two 75 and 52kDa G-proteins reflecting a diminished exchange of GDP by GTP. CONCLUSION: the PM-residing GC-importer, now renamed "Steroid Hormone Recognition and Effector Complex" (SHREC) is an interdependent part of the complete GC signal propagation in which G-proteins are involved. Free SH-groups of SHREC are a prerequisite for genomic GC activity. Specific interactions between SHREC and GC-agonist/-antagonist trigger steroid-dependent signaling. However, import of the ligand into the cell terminates it. Thus, the PM-related non-genomic steroid responses are clearly linked to the GR-related genomic effects.     

Comparative insecticidal properties of two nucleopolyhedrovirus vectors encoding a similar toxin gene chimer

Laboratory, greenhouse and field studies were conducted to characterize the insecticidal properties of genetically altered forms of Autographa californica (Speyer) nucleopolyhedrovirus (AcNPV) and Helicoverpa zea (Boddie) NPV (HzNPV) against selected heliothine species. The altered viruses each contained a chimeric 0.8-kb fragment encoding the insect-specific, sodium channel neurotoxin from the Algerian scorpion Androctonus australis Hector (AaIT, hence recombinant viruses designated Ac-AaIT and Hz-AaIT). Based on LD50 values, results from diet-overlay bioassays showed Ac-AaIT and Hz-AaIT to be equally virulent against larval tobacco budworm, Heliothis virescens (F.), but Hz-AaIT averaged 1,335-fold greater bioactivity than Ac-AaIT against larval cotton bollworm, Helicoverpa zea (Boddie). Hz-AaIT killed larvae of both heliothine species at rates significantly faster than those imparted by HzNPV (viral LT50 values averaged 2.5 and 5.6 d, respectively). In greenhouse studies, foliar sprays of Ac-AaIT and Hz-AaIT were equally effective in controlling H. virescens on cotton; however, Hz-AaIT provided control of H. zea on cotton at a level superior to that of Ac-AaIT. For example, after three weekly sessions of foliar application and H. zea artificial infestation, cotton treated with Ac-AaIT or Hz-AaIT at 10 x 10(11) occulsion bodies (OB)/ha averaged 2.5 and 16.2 nondamaged flower buds per plant, respectively. Another greenhouse study conducted against heliothine species on cotton showed that the quicker killing speed exhibited by Hz-AaIT led to improved plant protection versus HzNPV. Finally, results from three field trials demonstrated that Hz-AaIT at 5-12 x 10(11) OB/ha provided control of the heliothine complex in cotton at levels slightly better than Bacillus thuringiensis, equal to the macrolide, spinosad, and only slightly less than that of selected pyrethroid and carbamate insecticides. Overall, results from these studies indicate that, because of host range differences between the two wild-type viruses, HzNPV is the better vectoring agent (versus AcNPV) for designing recombinant clones as insecticides targeted at the multi-species heliothine complex. Further, these studies suggest that if appropriately tailored for the pest complex, recombinant NPVs may be very effective, insect-specific approaches to managing pests in many cropping scenarios. Possible Hz-AaIT deployment strategies for control of heliothine species on conventional and transgenic cotton varieties are discussed.     

Greenhouse and field evaluations of transgenic canola against diamondback moth, shape Plutella xylostella, and corn earworm, shape 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.     

Development and characterization of an oat TILLING-population and identification of mutations in lignin and β-glucan biosynthesis genes

Background  

Oat, Avena sativa is the sixth most important cereal in the world. Presently oat is mostly used as feed for animals. However, oat also has special properties that make it beneficial for human consumption and has seen a growing importance as a food crop in recent decades. Increased demand for novel oat products has also put pressure on oat breeders to produce new oat varieties with specific properties such as increased or improved β-glucan-, antioxidant- and omega-3 fatty acid levels, as well as modified starch and protein content. To facilitate this development we have produced a TILLING (Targeting Induced Local Lesions IN Genomes) population of the spring oat cultivar SW Belinda.     

Susceptibility of twolined spittlebug (Hemiptera: Cercopidae) life stages to entomophagous arthropods in turfgrass

Prosapia bicincta (Say) (Hemiptera: Cercopidae), the twolined spittlebug, is an economic pest of turfgrass in the southeastern United States. No data concerning natural enemies of P. bicincta in turfgrass have been reported previously. We compared predation of spittlebug eggs, nymphs, and adults in the laboratory by potential generalist predators commonly found in turfgrass: bigeyed bugs Geocoris uliginosus Say and Geocoris punctipes Say; red imported fire ant, Solenopsis invicta Buren; wolf spiders (Lycosa sp. Walckenaer); carabid beetles Harpalaus pensylvanicus DeGeer and Calosoma sayi Dejean; and tiger beetles Megacephala carolina carolina L. Eggs were readily consumed by generalist predators. S. invicta consumed 100% of the eggs offered. H. pensylvanicus and C. sayi were also significant predators of P. bicincta eggs. Nymphs live in spittlemasses that protect them from attack by predators, but exposed nymphs were susceptible to attack when mechanically removed from their spittlemasses. S. invicta and M. carolina carolina caused significant mortality of exposed nymphs. P. bicincta adults are aposematic and have the ability to reflex bleed; however, reflex bleeding did not prevent attack by predators. S. invicta and M. carolina carolina killed 100% of the adult spittlebugs offered in laboratory bioassays. Lycosa sp. are less voracious predators of adults. Sound background knowledge about P. bicincta and its potential natural enemy complex is important for the development and implementation of a detailed, site-specific, biologically based pest management program in turfgrass.     

An axisymmetric single-path model for gas transport in the conducting airways

In conventional one-dimensional single-path models, radially averaged concentration is calculated as a function of time and longitudinal position in the lungs, and coupled convection and diffusion are accounted for with a dispersion coefficient. The axisymmetric single-path model developed in this paper is a two-dimensional model that incorporates convective-diffusion processes in a more fundamental manner by simultaneously solving the Navier-Stokes and continuity equations with the convection-diffusion equation. A single airway path was represented by a series of straight tube segments interconnected by leaky transition regions that provide for flow loss at the airway bifurcations. As a sample application, the model equations were solved by a finite element method to predict the unsteady state dispersion of an inhaled pulse of inert gas along an airway path having dimensions consistent with Weibel's symmetric airway geometry. Assuming steady, incompressible, and laminar flow, a finite element analysis was used to solve for the axisymmetric pressure, velocity and concentration fields. The dispersion calculated from these numerical solutions exhibited good qualitative agreement with the experimental values, but quantitatively was in error by 20%-30% due to the assumption of axial symmetry and the inability of the model to capture the complex recirculatory flows near bifurcations.     

The GRR1 gene of Candida albicans is involved in the negative control of pseudohyphal morphogenesis

The opportunistic fungal pathogen Candida albicans can grow as yeast, pseudohyphae or true hyphae. C. albicans can switch between these morphologies in response to various environmental stimuli and this ability to switch is thought to be an important virulence trait. In Saccharomyces cerevisiae, the Grr1 protein is the substrate recognition component of an SCF ubiquitin ligase that regulates cell cycle progression, cell polarity and nutrient signaling. In this study, we have characterized the GRR1 gene of C. albicans. Deletion of GRR1 from the C. albicans genome results in a highly filamentous, pseudohyphal morphology under conditions that normally promote the yeast form of growth. Under hypha-inducing conditions, most cells lacking GRR1 retain a pseudohyphal morphology, but some cells appear to switch to hyphal-like growth and express the hypha-specific genes HWP1 and ECE1. The C. albicans GRR1 gene also complements the elongated cell morphology phenotype of an S. cerevisiae grr1Delta mutant, indicating that C. albicans GRR1 encodes a true orthologue of S. cerevisaie Grr1. These results support the hypothesis that the Grr1 protein of C. albicans, presumably as the F-box subunit of an SCF ubiquitin ligase, has an essential role in preventing the switch from the yeast cell morphology to a pseudohyphal morphology.     

Genetic diversity and population structure in the Brazilian Cattleya labiata (Orchidaceae) using RAPD and ISSR markers

Brazilian orchids are currently threatened with extinction due to habitat loss and, because of their high ornamental value, intense collecting pressure. Genetic diversity can play a key role in the survival of endangered orchid species. Here we provide the first data on genetic diversity and structure of wild populations in the genus Cattleya, in particular C.?labiata, using random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers. We studied 130 individuals, 117 belonging to Cattleya?labiata and 13 from 10 other species in the same genus. Data generated from 12 ISSR and 12 RAPD primers were used to determine genetic variability via a model-based Bayesian procedure (Structure) and molecular variance analysis. In addition, Shannon index, genetic diversity and Jaccard coefficients were also estimated. The marker data indicated that C. labiata has a high level of polymorphism, and five reconstructed populations were identified by Structure. The unweighted pair group method with arithmetic mean dendrogram did not group the samples by origin, which was also confirmed by Bayesian analysis, demonstrating the complex genetic structure of C.?labiata. Other Cattleya species showed no relationship with any C.?labiata sample. This genetic characterization of Cattleya from northeast Brazil contributes to knowledge of the genetic structure of the species and can be used to define strategies for conservation and breeding programmes.