Effect of various irradiation treatments of plant protoplasts on the transformation rates after direct gene transfer

Summary In P. hybrida and B. nigra an enhancement of transformation rates (direct gene transfer) of about six to seven-fold was obtained after irradiation of protoplasts with 12.5 Gy (X-ray). The effect of protoplast irradiation was similar in experiments where protoplasts were irradiated 1h before transformation (X-ray/DNA) or 1h after completion of the transformation procedure (DNA/X-ray). Increased X-ray doses up to 62.5 Gy resulted in further enhancement of percentages of transformed colonies, indicating a correlation between relative transformation frequencies (RTF) and the doses applied. Estimation of degradation rates of plasmid sequences in plant protoplasts yielded a reduction of plasmid concentration to 50% 8–12 h after transformation. In 1-day-old protoplasts, the level of plasmid fragments dropped to 0%–10% compared to 1h after transformation. The results demonstrate that the integration rates of plasmid sequences into the plant genome may in part be governed by DNA repair mechanisms. This could be an explanation for the observed genotypic dependence of transformation rates in different plant species and plant genotypes. Gene copy number reconstructions revealed enhanced integration rates of plasmid sequences in transformed colonies derived from irradiated protoplasts.     

The power of interval mapping of quantitative trait loci, using selected sib pairs.

The interval-mapping procedure of Fulker and Cardon for analysis of a quantitative-trait loci (QTL) is extended for application to selected samples of sib pairs. Phenotypic selection of sib pairs, which is known to yield striking increases in power when a single marker is used, provides further increases in power when the interval-mapping approach is used. The greatest benefits of the combined approach are apparent with coarse maps, where QTLs of relatively modest (15%-20%) heritability can be detected with widely spaced markers (40-60 cM apart) in reasonably sized sibling samples. Useful information concerning QTL location is afforded by interval mapping in both selected and unselected samples.     

A sib-pair approach to interval mapping of quantitative trait loci.

An interval mapping procedure based on the sib-pair method of Haseman and Elston is developed, and simulation studies are carried out to explore its properties. The procedure is analogous to other interval mapping procedures used with experimental material, such as plants and animals, and yields very similar results in terms of the location and effect size of a quantitative trait locus (QTL). The procedure offers an advantage over the conventional Haseman and Elston approach, in terms of power, and provides useful information concerning the location of a QTL. Because of its simplicity, the method readily lends itself to the analysis of selected samples for increased power and the evaluation of multilocus models of complex phenotypes.     

Why is CpG suppressed in the genomes of virtually all small eukaryotic viruses but not in those of large eukaryotic viruses?

Dinucleotide over- and underrepresentation is evaluated in all available completely sequenced DNA or RNA viral genomes, ranging in size from 3 to 250 kb (available RNA viruses fall into the small-virus category). The dinucleotide CpG is statistically underrepresented (suppressed) in all but four of the small viruses (more than 75 with lengths of < 30 kb) but has normal relative abundances in most large viruses (> or = 30 kb). Most retrotransposons in eukaryotic species also show low CpG relative abundances. Interpretations, especially in some cases of DNA viruses or viruses with a DNA intermediate, might relate to methylation effects and modes of viral integration and excision. Other possible contributing factors relate to dinucleotide stacking energies, special mutation mechanisms, and evolutionary events.     

Soil boron and selenium removal by three plant species

High concentrations of boron (B) and selenium (Se) naturally found in the environment are detrimental to sustainable agriculture in the western USA. Greenhouse pot experiments were conducted to study B and Se uptake in three different plant species; Brassica juncea (L.) Czern (wild brown mustard), Festuca arundinacea Schreb. L. (tall fescue), and Brassica napus (canola) were grown in soil containing naturally occurring concentrations of 3.00 mg extractable B kg^–1 and 1.17 mg total Se kg^–1 soil. During the growing season, four intermediate harvests were performed on wild mustard and tall fescue. Final harvest I consisted of harvesting wild mustard, canola, and clipping tall fescue. Final harvest II consisted of harvesting wild mustard, which had been planted in soil in which wild mustard was previously grown, and harvesting previously clipped tall fescue. The greatest total amount of above ground biomass and below surface biomass was produced by tall fescue. Plants were separated into shoots and roots, weighted, and plant tissues were analyzed for total B and Se. The highest concentrations of tissue B were recovered in shoots of wild mustard and canola at final harvest I, while roots from tall fescue contained the highest concentrations of B irrespective of the harvest. Tissue Se concentrations were similar in all plants species. Soils were analyzed for residual B and Se. Extractable soil B concentrations at harvest times were lowered no less than 32% and total Se no less than 24% for all three species. The planting of wild mustard, canola, or tall fescue can reduce water-extractable B and total Se in the soil.     

Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco

A transposon tagging system for heterologous hosts, based on the maize En/Spm transposable element, was developed in transgenic tobacco. In this system, the two En-encoded trans-acting factors necessary for excision are expressed by fusing their cDNAs to the CaMV 35S promoter. The dSpm receptor component is inserted in the 5-untranslated leader of the bar gene. Germinal revertants can therefore be selected by seed germination on L-PPT-containing medium or by spraying seedlings with the herbicide Basta. Using this bar-based excision reporter construct, an average frequency of germinal excision of 10.1% was estimated for dSpm-S, an En/Spm native internal deletion derivative. Insertion of En-foreign sequences in a receptor, such as a DHFR selectable marker gene in dSpm-DHFR, does not abolish its capacity to transpose. However, dSpm-DHFR has a lower frequency of somatic and germinal excision than dSpm-S. Revertants carrying a transposed dSpm-DHFR element can be selected with methotrexate. Germinal excision is frequently associated with reinsertion but, as in maize, dSpm has a tendency to integrate at chromosomal locations linked to the donor site. Concerning the timing of excision, independent germinal transpositions are often found within a single seed capsule. All activity parameters analysed suggest that transposon tagging with this system in heterologous hosts should be feasible.     

Influence of rhizodeposition under elevated CO2 on plant nutrition and soil organic matter

Atmospheric CO₂ concentrations can influence ecosystem carbon storage through net primary production (NPP), soil carbon storage, or both. In assessing the potential for carbon storage in terrestrial ecosystems under elevated CO₂, both NPP and processing of soil organic matter (SOM), as well as the multiple links between them, must be examined. Within this context, both the quantity and quality of carbon flux from roots to soil are important, since roots produce specialized compounds that enhance nutrient acquisition (affecting NPP), and since the flux of organic compounds from roots to soil fuels soil microbial activity (affecting processing of SOM).From the perspective of root physiology, a technique is described which uses genetically engineered bacteria to detect the distribution and amount of flux of particular compounds from single roots to non-sterile soils. Other experiments from several labs are noted which explore effects of elevated CO₂ on root acid phosphatase, phosphomonoesterase, and citrate production, all associated with phosphorus nutrition. From a soil perspective, effects of elevated CO₂ on the processing of SOM developed under a C4 grassland but planted with C3 California grassland species were examined under low (unamended) and high (amended with 20 g m^–2 NPK) nutrients; measurements of soil atmosphere 13C combined with soil respiration rates show that during vegetative growth in February, elevated CO₂ decreased respiration of carbon from C4 SOM in high nutrient soils but not in unamended soils.This emphasis on the impacts of carbon loss from roots on both NPP and SOM processing will be essential to understanding terrestrial ecosystem carbon storage under changing atmospheric CO₂ concentrations.Abbreviations SOM soil organic matter - NPP net primary productivity - NEP net ecosystem productivity - PNPP p-nitrophenyl phosphate     

Seasonal timing of inundation affects riparian plant growth and flowering: implications for riparian vegetation composition

Changes to the timing of peak river flows caused by flow regulation affect riparian vegetation composition, but the mechanisms driving such vegetation changes are not well understood. We investigated experimentally the effects of timing of inundation on riparian plant growth and flowering. We collected 168 sods from 14 sites across five lowland rivers in south-eastern Australia. Plant cover and flowering within the sods were surveyed each season for a year. During this period, sods were inundated for 6 weeks in either early spring or in summer. Terrestrial plant taxa (which included most exotic species) senesced in response to inundation, regardless of its timing. In contrast, native amphibious species (particularly amphibious forbs) responded favourably to inundation in spring, but were unaffected by inundation in summer. Native and exotic emergent macrophytes responded favourably to inundation regardless of timing, and flowered frequently in both the spring- and the summer-inundation treatments. In contrast, many native annuals flowered only in the spring-inundation treatment, while more exotic grasses flowered in the summer-inundation treatment. In temperate climates, inundation in early spring followed by non-flooded conditions is likely to be important for promoting the growth of amphibious forbs and the recruitment and flowering of riparian annuals. Without inundation in spring, many terrestrial exotic weeds may flourish and set seed prior to any subsequent inundation (e.g. in summer). We contend that natural seasonal timing (i.e. winter-early spring) of flow peaks is important for the maintenance of native riverbank vegetation and reducing the extent of terrestrial exotic species within the riparian zone.     

Angiovision: Aortic stent-graft placement by augmented angionavigation

The endovascular treatment of abdominal aortic aneurysm (EVAR - EndoVascular Aneurism Repair) has become an alternative therapy to conventional open surgery. The objective of Angiovision project was to contribute to the improvement of EVAR procedures in terms of accuracy and customization of the interventional strategy. Our approach was mainly based on the use of patient-specific data, computer-aided endovascular navigation and numerical simulation. We proposed a new FEM-based patient-specific simulation solution to estimate the deformations caused by the introduction of stiff endovascular devices. We developed and implemented an original endovascular navigation system to augment the intraoperative fluoroscopy with the visualization of deformed preoperative aorto-iliac structure, and related information concerning vessel wall (calcifications), as well as stent-graft virtual deployment. The proposed solution, that requires only lightweight computer equipment, can be used in a standard operating room in order to optimize device placement (from a single angle of incidence). Evaluation is currently under way.