Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated ...(Cas)-guide RNA technology are reported in maize. DNA vectors expressing maize codon-optimizedStreptococcus pyogenesCas9 endonuclease and single guide RNAs were cointroduced with or without DNA repair templates into maize immature embryos by biolistic transformation targeting five different genomic regions: upstream of the liguleless1 (LIG1) gene, male fertility genes (Ms26andMs45), and acetolactate synthase (ALS) genes (ALS1andALS2). Mutations were subsequently identified at all sites targeted, and plants containing biallelic multiplex mutations atLIG1,Ms26, andMs45were recovered. Biolistic delivery of guide RNAs (as RNA molecules) directly into immature embryo cells containing preintegrated Cas9 also resulted in targeted mutations. Editing theALS2gene using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded chlorsulfuron-resistant plants. Double-strand breaks generated by RNA-guided Cas9 endonuclease also stimulated insertion of a trait gene at a site nearLIG1by homology-directed repair. Progeny showed expected Mendelian segregation of mutations, edits, and targeted gene insertions. The examples reported in this study demonstrate the utility of Cas9-guide RNA technology as a plant genome editing tool to enhance plant breeding and crop research needed to meet growing agriculture demands of the future.
Recently discovered bacteria and archaea adaptive immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) endonuclease has been ...explored in targeted genome editing in different species.Streptococcus pyogenesCas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis, gene integration, and gene editing in soybean (Glycine max). Two genomic sites,DD20andDD43on chromosome 4, were mutagenized with frequencies of 59% and 76%, respectively. Sequencing randomly selected transgenic events confirmed that the genome modifications were specific to the Cas9-gRNA cleavage sites and consisted of small deletions or insertions. Targeted gene integrations through homology-directed recombination were detected by border-specific polymerase chain reaction analysis for both sites at callus stage, and oneDD43homology-directed recombination event was transmitted to T1 generation. T1 progenies of the integration event segregated according to Mendelian laws and clean homozygous T1 plants with the donor gene precisely inserted at theDD43target site were obtained. The Cas9-gRNA system was also successfully applied to make a directed P178S mutation of acetolactate synthase1 gene through in planta gene editing.
It has recently been reported that ceramics can be sintered in a few seconds with the aid of an electric field (“flash sintering”). This investigation tests the possibility that the accelerated ...sintering is a consequence of the rapid heating rate involved rather than a direct effect of the electric field on mass transport. The sintering of 3YSZ powder compacts at a temperature of ∼1300°C was compared (i) in flash sintering, (ii) with rapid heating rates produced without the application of an electric field, and (iii) with conventional heating rates. The results show that rapid heating can accelerate sintering by over 2 orders of magnitude compared with heating to the same temperature at conventional rates, even without the application of an electric field. It is concluded that the rapid densification in flash sintering of 3YSZ is at least partly a consequence of the rapid heating involved. Possible explanations are discussed.
The paper presents an overview of the results of diagnostic and monitoring activities carried out in the last years through satellite radar interferometry (2011–2016) and in situ measurements ...(2017–2019) in the historical city of Gubbio, Italy. The study is aimed at contributing to understanding the potential of remote sensing technologies in measuring complex deformation phenomena in historic buildings, such as those caused by soil-structural interaction and earthquakes, considering the monumental Consoli Palace and the Town Walls as case studies. The research activities have been carried out within the European HERACLES project, funded in the framework of Horizon (2020) and aimed at proposing novel diagnostic monitoring solutions for enhancing heritage resilience against various types of hazards. Particular attention is focused on the cross-correlation of the outputs of satellite radar interferometry and in-situ structural monitoring data aimed at achieving engineering meaningful results supporting decision making and proactive interventions.
•Structural monitoring of CH constructions through InSAR and in-situ data is presented.•Remote sensing and in-situ measurements show consistent seasonal displacement trends.•InSAR technique proves promising in detecting permanent earthquake-induced deformations.•Integration between InSAR and in-situ monitoring is identified as a fruitful research direction.
Summary
The liguleless locus (liguleless1) was chosen for demonstration of targeted mutagenesis in maize using an engineered endonuclease derived from the I‐CreI homing endonuclease. A single‐chain ...endonuclease, comprising a pair of I‐CreI monomers fused into a single polypeptide, was designed to recognize a target sequence adjacent to the LIGULELESS1 (LG1) gene promoter. The endonuclease gene was delivered to maize cells by Agrobacterium‐mediated transformation of immature embryos, and transgenic T0 plants were screened for mutations introduced at the liguleless1 locus. We found mutations at the target locus in 3% of the T0 plants, each of which was regenerated from independently selected callus. Plants that were monoallelic, biallelic and chimeric for mutations at the liguleless1 locus were found. Relatively short deletions (shortest 2 bp, longest 220 bp) were most frequently identified at the expected cut site, although short insertions were also detected at this site. We show that rational re‐design of an endonuclease can produce a functional enzyme capable of introducing double‐strand breaks at selected chromosomal loci. In combination with DNA repair mechanisms, the system produces targeted mutations with sufficient frequency that dedicated selection for such mutations is not required. Re‐designed homing endonucleases are a useful molecular tool for introducing targeted mutations in a living organism, specifically a maize plant.
In this paper, after a review of the evolution of the literature on climate change economics in agriculture, I present some evidence of the impact of different moments of the distribution of rainfall ...on farmers risk aversion. It is found that while more rainfall is negatively associated with the probability of observing risk aversion, rainfall variability is positively correlated. This result highlights an important behavioural dimension of climatic factors.
In resource dependent rural areas of developing countries, common property resource management has been considered as one of the most viable options for combining poverty reduction, enhancement of ...local level economic development and biodiversity conservation. The past decade has witnessed an increasing emphasis on community-based forest management, with transference of forest management responsibility into the hands of local communities. However, although community forestry (CF) has succeeded in halting resource degradation and conservation of biodiversity, the equity aspect of CF not been fully examined. Nepal is a good location for a case study to examine this question, as community forest programs have been in place longer than in many other countries. This study analyzes the relationship between key household characteristics and common property resources used in order to assess whether poorer households are able to gain greater access to community forests as a result of institutional change. Both quantitative and qualitative analyses suggest that forest product collection from community forests is dependent on various socio-economic variables. In general it appears that land and livestock holdings, caste, education of family members and household economic status exert a strong influence on appropriating benefits from the commons. Based on this analysis, it can be concluded that, at least for some key products, poorer households are currently facing more restricted access to community forests than ‘less poor’ or relatively better off households.
Recombinase-mediated DNA cassette exchange (RMCE) has been successfully used to insert transgenes at previously characterized genomic sites in plants. Following the same strategy, groups of ...transgenes can be stacked to the same site through multiple rounds of RMCE. A gene-silencing cassette, designed to simultaneously silence soybean (Glycine max) genes fatty acid ω-6 desaturase 2 (FAD2) and acyl-acyl carrier protein thioesterase 2 (FATB) to improve oleic acid content, was first inserted by RMCE at a precharacterized genomic site in soybean. Selected transgenic events were subsequently retransformed with the second DNA construct containing a Yarrowia lipolytica diacylglycerol acyltransferase gene (DGAT1) to increase oil content by the enhancement of triacylglycerol biosynthesis and three other genes, a Corynebacterium glutamicum dihydrodipicolinate synthetase gene (DHPS), a barley (Hordeum vulgare) high-lysine protein gene (BHL8), and a truncated soybean cysteine synthase gene (CGS), to improve the contents of the essential amino acids lysine and methionine. Molecular characterization confirmed that the second RMCE successfully stacked the four overexpression cassettes to the previously integrated FAD2-FATB gene-silencing cassette. Phenotypic analyses indicated that all the transgenes expressed expected phenotypes.
An analysis was made of the transport and fate of the organophosphate pesticide chlorpyrifos in productive soils from the Alto Valle of the Río Negro in Argentine Patagonia. The climate of the region ...is arid, so traditional fruit production is under flood irrigation. The soils in the floodplain are predominantly Aridisols with textures ranging from sandy loam to clay loam.
The calibration was performed with water table data and chlorpyrifos concentration in the soil horizons. Field experiments made with Brilliant Blue FCF at the profile scale enabled the parametrisation of the dual-permeability model MACRO. The model calibration was evaluated by a comparison of observed and simulated data and statistics.
The simulation of the groundwater table depth was satisfactory and the chlorpyrifos leaching revealed a different pattern in the two soil types studied. The sandy loam texture soil produced more percolation of irrigation water, but the clay loam soil produced greater leaching of chlorpyrifos under similar application conditions, presumably due to preferential flow under non-equilibrium conditions.
Productive management alternatives to reduce leaching into the underlying unconfined aquifer were simulated. Among these, the incorporation of organic matter was the best alternative.
•Field experiment with dye was used to describe macropores and heterogeneities in soil.•Chlorpyrifos leaching in clay loam soil was greater due to preferential transport.•Through MACRO model the best alternative to reduce chlorpyrifos leaching was chosen.
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