Genetic Transformation of Common Wheat (Triticum aestivum L.) Using Biolistics.

The following protocol describes the genetic transformation of wheat using the BioRad PDS/1000-He particle delivery system. Immature embryos are isolated 12-16 days post-anthesis, the embryonic axis is removed, and the immature scutella are precultured for 1-2 days prior to particle bombardment. Gold particles are coated with plasmid DNA containing the gene(s) of interest plus a selectable marker gene, in this instance bar (bialaphos resistance), and are fired into the cells to deliver the DNA. Subsequent tissue culture and regeneration steps allow recovery of plantlets, assisted by the inclusion of PPT (phosphinothricin tripeptide), the active ingredient of glufosinate-ammonium containing herbicides, to help select transformants. This updated method introduces selection earlier in the regeneration process which provides a shortened protocol while maintaining high transformation efficiencies.

Organochlorine pesticides in the soils from Benevento provincial territory, southern Italy: Spatial distribution, air-soil exchange, and implications for environmental health.

This study comprehensively interprets the contamination status of organochlorine pesticides (OCPs) in the soils from Benevento provincial territory, southern Italy, and its implications for environmental health by means of a systematic grid sampling method and geostatistics. The total concentrations of OCPs in the soils ranged from 0.058 to 16.9 ng/g, with a geometric mean (GM) of 0.72 ng/g and an arithmetic mean (AM) of 1.71 ng/g. The levels of OCPs were dominated by p,p'-DDE, p,p'-DDD, HCB, contributing together to 73.5% of the total OCPs. The higher levels of HCB, DDTs, and HCHs found in southwestern, central and east Benevento provincial territory, all occurring adjacent to landfill sites. The residues of OCPs in soil are largely ascribed to their historical use. The OCP inventories in soils of Benevento provincial territory ranged from 0.13 to 4.84 metric tons, with GM = 0.42 metric tons and AM = 0.44 metric tons. The soil is likely to be a sink for DDTs under the influence of regional air transport from pollution hotspots and has the potential to release other chemicals with a high vapor pressure, e.g., HCB, HCHs, and α-Endosulfan. And the mean level of the air-soil exchange flux of HCB, HCHs, and DDTs is estimated to be -1.59, -0.72, and 0.10 ng/m2/day respectively. The potential ecological and human health risks caused by OCPs in the soils are deemed essentially negligible in Benevento provincial territory.

Residues of hexachlorobenzene and chlorinated cyclodiene pesticides in the soils of the Campanian Plain, southern Italy.

A systematic grid sampling method and geostatistics were employed to investigate the spatial distribution, inventory, and potential ecological and human health risks of the residues of hexachlorobenzene (HCB) and chlorinated cyclodiene pesticides in soils of the Campanian Plain, Italy, and explore their relationship with the soils properties. The geometric mean (Gmean) concentrations of HCB and cyclodiene compounds followed the order CHLs (heptachlor, heptachlor epoxide, trans-chlordane, and cis-chlordane) > DRINs (aldrin, dieldrin, and endrin) > SULPHs (α-endosulfan, ß-endosulfan, and endosulfan sulfate) > HCB. The residual levels of most cyclodienes in agricultural soils were generally higher than those of corresponding counterparts in the other land uses. Significant differences in the concentration of HCB and cyclodienes in the soils across the region are observed, and the Acerra-Marigliano conurbation (AMC) and Sarno River Basin (SRB) areas exhibit particularly high residual concentrations. Some legacy cyclodienes in the Campanian Plain may be attributed to a secondary distribution. The Gmean inventory of HCB, SULPHs, CHLs, and DRINs in the soil is estimated to be 0.081, 0.41, 0.36, and 0.41 metric tons, respectively. The non-cancer and cancer risks of HCB and cyclodienes for exposed populations are deemed essentially negligible, however, endosulfan poses significant ecological risks to some terrestrial species.

The status of organochlorine pesticide contamination in the soils of the Campanian Plain, southern Italy, and correlations with soil properties and cancer risk.

The distribution, inventory, and potential risk of organochlorine pesticides (OCPs), including Hexachlorocyclohexanes (HCHs) and Dichlorodiphenyltrichloroethanes (DDTs), and their correlation with soil properties and anthropogenic factors were investigated in soils of the Campanian Plain. The total concentrations of HCHs and DDTs ranged from 0.03 to 17.3 ng/g (geometric mean: GM = 0.05 ng/g), and 0.08-1231 ng/g (GM = 14.4 ng/g), respectively. In general, the concentration of OCPs in farmland and orchards was higher than on land used for non-agricultural purposes. There are significant differences in the concentration of OCPs in the soils across the region, more specifically, the Acerra-Marigliano conurbation (AMC) and Sarno River Basin (SRB) are recognized as severely OCP-contaminated areas. The recent application of technical HCHs and DDTs in large quantities appears unlikely in light of the ratio of α-HCH/ß-HCH and p,p'-DDT/p,p'-DDE, and the prohibition of the use of these chemicals in Italy nearly forty years ago. The clear correlation between the concentration of DDTs and organic carbon suggests a typical secondary distribution pattern. The mass inventory of OCPs in soils of the Campanian Plain is estimated to have a GM of 17.3 metric tons. There is no clear evidence linking the impact of geographical distribution of OCPs on the incidence of cancer, and the 95% confidence interval of total incremental lifetime cancer risk (TILCR) data falls below the internationally accepted benchmark value of 1 × 10(-5).

The first crop plant genetically engineered to release an insect pheromone for defence.

Insect pheromones offer potential for managing pests of crop plants. Volatility and instability are problems for deployment in agriculture but could be solved by expressing genes for the biosynthesis of pheromones in the crop plants. This has now been achieved by genetically engineering a hexaploid variety of wheat to release (E)-ß-farnesene (Eßf), the alarm pheromone for many pest aphids, using a synthetic gene based on a sequence from peppermint with a plastid targeting amino acid sequence, with or without a gene for biosynthesis of the precursor farnesyl diphosphate. Pure Eßf was produced in stably transformed wheat lines with no other detectable phenotype but requiring targeting of the gene produced to the plastid. In laboratory behavioural assays, three species of cereal aphids were repelled and foraging was increased for a parasitic natural enemy. Although these studies show considerable potential for aphid control, field trials employing the single and double constructs showed no reduction in aphids or increase in parasitism. Insect numbers were low and climatic conditions erratic suggesting the need for further trials or a closer imitation, in the plant, of alarm pheromone release.

Cystinuria-a urologist's perspective.

Cystinuria is a genetic disease that leads to frequent formation of stones. In patients with recurrent stone formation, particularly patients <30 years old or those who have siblings with stone disease, urologists should maintain a high index of suspicion of the diagnosis of cystinuria. Patients with cystinuria require frequent follow-up and a multidisciplinary approach to diagnosis, prevention and management. Patients have reported success in preventing stone episodes by maintaining dietary changes using a tailored review from a specialist dietician. For patients who do not respond to conservative lifestyle measures, medical therapy to alkalinize urine and thiol-binding drugs can help. A pre-emptive approach to the surgical management of cystine stones is recommended by treating smaller stones with minimally invasive techniques before they enlarge to a size that makes management difficult. However, a multimodal approach can be required for larger complex stones. Current cystinuria research is focused on methods of monitoring disease activity, novel drug therapies and genotype-phenotype studies. The future of research is collaboration at a national and international level, facilitated by groups such as the Rare Kidney Stone Consortium and the UK Registry of Rare Kidney Diseases.

Genetic transformation of wheat via Agrobacterium-mediated DNA delivery.

The method described involves an initial incubation of wheat immature embryos in a liquid culture of Agrobacterium tumefaciens. The Agrobacterium strain is engineered to contain a binary vector with a gene of interest and a selectable marker gene placed between the T-DNA borders; the T-DNA is the region transferred to the plant cells, thus harnessing the bacterium's natural ability to deliver specific DNA into host cells. Following the initial inoculation with the Agrobacterium, the embryos are co-cultivated for several days after which the Agrobacterium is selectively destroyed using an antibiotic. Tissue culture of the embryos on plant media with a correct balance of hormones allows embryogenic callus formation followed by regeneration of plantlets, and in the later stages of tissue culture a selectable marker (herbicide) is included to minimize the incidence of non-transformed plants. This protocol has been used successfully to generate transformed plants of a wide range of wheat varieties, both spring and winter bread wheats (T. aestivum L.) and durum wheats (T. turgidum L.).

Increasing the amylose content of durum wheat through silencing of the SBEIIa genes.

BACKGROUND: High amylose starch has attracted particular interest because of its correlation with the amount of Resistant Starch (RS) in food. RS plays a role similar to fibre with beneficial effects for human health, providing protection from several diseases such as colon cancer, diabetes, obesity, osteoporosis and cardiovascular diseases. Amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. In particular, the inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content. In this work, genes encoding starch branching enzymes of class II (SBEIIa) were silenced using the RNA interference (RNAi) technique in two cultivars of durum wheat, using two different methods of transformation (biolistic and Agrobacterium). Expression of RNAi transcripts was targeted to the seed endosperm using a tissue-specific promoter. RESULTS: Amylose content was markedly increased in the durum wheat transgenic lines exhibiting SBEIIa gene silencing. Moreover the starch granules in these lines were deformed, possessing an irregular and deflated shape and being smaller than those present in the untransformed controls. Two novel granule bound proteins, identified by SDS-PAGE in SBEIIa RNAi lines, were investigated by mass spectrometry and shown to have strong homologies to the waxy proteins. RVA analysis showed new pasting properties associated with high amylose lines in comparison with untransformed controls. Finally, pleiotropic effects on other starch genes were found by semi-quantitative and Real-Time reverse transcription-polymerase chain reaction (RT-PCR). CONCLUSION: We have found that the silencing of SBEIIa genes in durum wheat causes obvious alterations in granule morphology and starch composition, leading to high amylose wheat. Results obtained with two different methods of transformation and in two durum wheat cultivars were comparable.

Transient transformation of plants.

Transient expression in plants is a valuable tool for many aspects of functional genomics and promoter testing. It can be used both to over-express and to silence candidate genes. It is also scaleable and provides a viable alternative to microbial fermentation and animal cell culture for the production of recombinant proteins. It does not depend on chromosomal integration of heterologous DNA so is a relatively facile procedure and can lead to high levels of transgene expression. Recombinant DNA can be introduced into plant cells via physical methods, via Agrobacterium or via viral vectors.

Agrobacterium-mediated transformation of bread and durum wheat using freshly isolated immature embryos.

Agrobacterium-mediated transformation of wheat is becoming a viable alternative to the more established biolistic protocols. It offers advantages in terms of simple, low-copy-number integrations and can be applied with similar efficiencies to specific durum wheat and spring and winter bread wheat types varieties.

Efficient and rapid Agrobacterium-mediated genetic transformation of durum wheat (Triticum turgidum L. var. durum) using additional virulence genes.

Genetic transformation of wheat, using biolistics or Agrobacterium, underpins a range of specific research methods for identifying genes and studying their function in planta. Transgenic approaches to study and modify traits in durum wheat have lagged behind those for bread wheat. Here we report the use of Agrobacterium strain AGL1, with additional vir genes housed in a helper plasmid, to transform and regenerate the durum wheat variety Ofanto. The use of the basic pSoup helper plasmid with no additional vir genes failed to generate transformants, whereas the presence of either virG542 or the 15 kb Komari fragment containing virB, virC and virG542 produced transformation efficiencies of between 0.6 and 9.7%. Of the 42 transgenic plants made, all but one (which set very few seeds) appeared morphologically normal and produced between 100 and 300 viable seeds. The transgene copy number and the segregation ratios were found to be very similar to those previously reported for bread wheat. We believe that this is the first report describing successful genetic transformation of tetraploid durum wheat (Triticum turgidum L. var. durum) mediated by Agrobacterium tumefaciens using immature embryos as the explant.

Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat.

Since the first report of wheat transformation by Agrobacterium tumefaciens in 1997, various factors that influence T-DNA delivery and regeneration in tissue culture have been further investigated and modified. This paper reviews the current methodology literature describing Agrobacterium transformation of wheat and provides a complete protocol that we have developed and used to produce over one hundred transgenic lines in both spring and winter wheat varieties.