Induction of transgenic hairy roots in soybean genotypes by Agrobacterium rhizogenes‑mediated transformation

The objective of this work was to perform the screening of soybean genotypes as to their ability to respond to the induction of hairy roots by Agrobacterium rhizogenes‑mediated transformation. Four Brazilian soybean cultivars (BRSMG 68 Vencedora, BRS 137, Embrapa 48, and MG/BR 46 Conquista) and two North American ones adapted to Brazilian cropping conditions (Bragg and IAS‑5) were screened for their capacity to respond to A. rhizogenes in protocols for in vitro hairy root culture and ex vitro composite plant production. Four‑day‑old seedlings with uniform size were injected with A. rhizogenes harboring the plasmid p35S‑GFP. Seedlings expressing green fluorescent protein (GFP) in at least one hairy root were used to determine the transformation frequency. Using an axenic in vitro protocol, excised cotyledons from four‑day‑old seedlings were infected with A. rhizogenes harboring the pCAMBIA1301 plasmid, containing the gusA reporter gene. The transformation frequency and the number of days for hairy root emergence after bacterial infection (DAI) were evaluated. The transformation frequency and DAI varied according to the genotype. Cultivars MG/BR 46 Conquista and BRSMG 68 Vencedora are more susceptible to A. rhizogenes and can be recommended for transformation experiments.

Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class. (C) 2011 Elsevier Inc. All rights reserved.

GFP-tagged CFTR transgene is functional in the G551D cystic fibrosis mouse colon

Trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is central to its function, with the most common mutation, DeltaF508, resulting in abnormal processing and trafficking. Therefore, there is a significant need to develop tools, which enable the trafficking of CFTR to be studied in vitro and in vivo. In previous studies it has been demonstrated that fusion of the green fluorescent protein (GFP) to the N-terminus of CFTR does lead to functional expression of CFTR chloride channels in epithelial cell lines. The aim of the present study was to examine whether it is possible to express GFP-tagged CFTR as a transgene in colonic and airway epithelial cells of cystic fibrosis (CF) mice and to correct the CF defect. Using the epithelial-specific human cytokeratin promoter K18, we generated bitransgenic mice cftr(G551D/G551D) K18-GFP-CFTR+/-, designated GFP mice. Transcripts for GFP-CFTR could be detected in bitransgenic mice by use of RT-PCR techniques. Expression of GFP-CFTR protein was detected specifically in the colonic epithelium by both direct GFP fluorescence and the use of an anti-GFP antibody. Ussing chamber studies showed that the ion transport defect in colon and airways observed in cftr(G551D/G551D) mice was partially corrected in the bitransgenic animals. Thus, K18-GFP-CFTR is functionally expressed in transgenic mice, which will be a valuable tool in studies on CFTR synthesis, processing and ion transport in native epithelial tissues.

Malignant Transformation Of An Odontogenic Cyst In A Period Of 10 Years.

Primary intraosseous carcinoma of the jaws (PIOSCC) might arise from odontogenic epithelium, more commonly from a previous odontogenic cyst. The aim of this case is to illustrate that the clinician should consider that an apparent benign dentigerous cyst can suffer malignant transformation and that all material removed from a patient must be evaluated histologically. A 44-year-old man presented in a routine periapical X-ray an impacted lower left third molar with radiolucency over its crown. Ten years later, the patient complained of pain in the same region and the tooth was extracted. After one month, the patient still complained of pain and suffered a fracture of the mandible. A biopsy was performed and carcinoma was diagnosed. The patient was treated surgically with adjuvant radio- and chemotherapy and after 8 years, he is well without signs of recurrences. This report describes a central mandibular carcinoma probably developed from a previous dentigerous cyst.

The Rg1 allele as a valuable tool for genetic transformation of the tomato 'Micro-Tom' model system

Background: The cultivar Micro-Tom (MT) is regarded as a model system for tomato genetics due to its short life cycle and miniature size. However, efforts to improve tomato genetic transformation have led to protocols dependent on the costly hormone zeatin, combined with an excessive number of steps. Results: Here we report the development of a MT near-isogenic genotype harboring the allele Rg1 (MT-Rg1), which greatly improves tomato in vitro regeneration. Regeneration was further improved in MT by including a two-day incubation of cotyledonary explants onto medium containing 0.4 mu M 1-naphthaleneacetic acid (NAA) before cytokinin treatment. Both strategies allowed the use of 5 mu M 6-benzylaminopurine (BAP), a cytokinin 100 times less expensive than zeatin. The use of MT-Rg1 and NAA pre-incubation, followed by BAP regeneration, resulted in high transformation frequencies (near 40%), in a shorter protocol with fewer steps, spanning approximately 40 days from Agrobacterium infection to transgenic plant acclimatization. Conclusions: The genetic resource and the protocol presented here represent invaluable tools for routine gene expression manipulation and high throughput functional genomics by insertional mutagenesis in tomato.

Phase transformation and residual stress probed by Raman spectroscopy in diamond-turned single crystal silicon

Single-point diamond turning of monocrystalline semiconductors is an important field of research within brittle materials machining. Monocrystalline silicon samples with a (100) orientation have been diamond turned under different cutting conditions (feed rate and depth of cut). Micro-Raman spectroscopy and atomic force microscopy have been used to assess structural alterations and surface finish of the samples diamond turned under ductile and brittle modes. It was found that silicon undergoes a phase transformation when machined in the ductile mode. This phase transformation is evidenced by the creation of an amorphous surface layer after machining which has been probed by Raman scattering. Compressive residual stresses are estimated for the machined surface and it is observed that they decrease with an increase in the feed rate and depth of cut. This behaviour has been attributed to the formation of subsurface cracks when the feed rate is higher than or equal to 2.5 mu m/rev. The surface roughness was observed to vary with the feed rate and the depth of cut. An increase in the surface roughness was influenced by microcrack formation when the feed rate reached 5.0 mu m/rev. Furthermore, a high-pressure phase transformation induced by the tool/material interaction and responsible for the ductile response of this typical brittle material is discussed based upon the presented Raman spectra. The application of this machining technology finds use for a wide range of high quality components, for example the creation of a micrometre-range channel for microfluidic devices as well as microlenses used in the infrared spectrum range.

Anticariogenic Properties of ent-Pimarane Diterpenes Obtained by Microbial Transformation

In the present work, the anticariogenic activities of three pimarane-type diterpenes obtained by fungal biotransformation were investigated. Among these metabolites, ent-8(14), 15-pimaradien-19-ol was the most active compound, displaying very promising MIC values (ranging from 1.5 to 4.0 mu g mL(-1)) against the main microorganisms responsible for dental caries: Streptococcus salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis, and Lactobacillus casei. Time kill assays performed with ent-8(14), 15-pimaradien-19-ol against the primary causative agent S. mutans revealed that this compound only avoids growth of the inoculum in the first 12 h (bacteriostatic effect). However, its bactericidal effect is clearly noted thereafter (between 12 and 24 h). The curve profile obtained by combining ent-8(14), 15-pimaradien-19-ol and chlorhexidine revealed a significant reduction in the time necessary for killing S. mutans compared with each of these two chemicals alone. However, no synergistic effect was observed using the same combination in the checkerboard assays against this microorganism. In conclusion, our results point out that ent-8(14), 15-pimaradien-19-ol is an important metabolite in the search for new effective anticariogenic agents.

Relevance of extractives and wood transformation products on the biodegradation of Pinus taeda by Ceriporiopsis subvermispora

Ceriporiopsis subvermispora is a promising white-rot fungus for biopulping. However, the underlying biochemistry involved in lignin removal and insignificant cellulose degradation by this species is not completely understood. This paper addresses this topic focusing on the involvement of ethanol-soluble extractives and wood transformation products in the biodegradation process. Cultures containing ethanol-extracted or in natura wood chips presented similar levels of extracellular enzymes and degradation of wood components. Fe3+-reducing compounds present in undecayed Pinus taeda were rapidly diminished by fungal degradation. Lignin-degradation products released during biodegradation restored part of the Fe3+-reducing activity. However, Fe3+ reduction was ineffective in presence of 0.5 mM oxalate at pH 4.5. Fungal consumption of Fe3+-reducing compounds and secretion of oxalic acid minimized the significance of Fenton`s reaction in the initial stages of wood biotreatment. This would explain limited polysaccharide degradation by the fungus that also lacks a complete set of hydrolytic enzymes. Scientific relevance of the paper: Ceriporiopsis subvermispora is a white-rot fungus suitable for biopulping processes because it degrades lignin selectively and causes significant structural changes on the wood components during the earlier decay stages. However, the intricate mechanism to explain lignin transformation and insignificant cellulose degradation by this species remains poorly understood. Some recent evidences pointed out for lipid peroxidation reactions as all initiating process explaining lignin degradation. On the other hand, alkylitaconic acids produced by the fungus via transformations of fatty acids occurring in wood showed to prevent polysaccharide degradation in Fenton reactions. In this context, one may conclude that the involvement of native wood substances or their transformation products in the overall wood biodegradation process induced by C subvermispora is still a matter of discussion. While free and esterified fatty acids present in wood extractives may be involved in the biosynthesis of alkylitaconic acids and in lipid peroxidation reactions, some extractives and lignin degradation products can reduce Fe3+, providing Fe2+ species needed to form OH radical via Fenton`s reaction. The present study focuses on this topic by evaluating the relevance of ethanol-soluble extractives and wood transformation products on the biodegradation of P. taeda by C subvermispora. For this, solid-state cultures containing ethanol-extracted and in natura wood chips were evaluated in details for up to 4 weeks. (C) 2007 Elsevier Ltd. All rights reserved.

A Levinson algorithm based on an isometric transformation of Durbin`s

Starting from the Durbin algorithm in polynomial space with an inner product defined by the signal autocorrelation matrix, an isometric transformation is defined that maps this vector space into another one where the Levinson algorithm is performed. Alternatively, for iterative algorithms such as discrete all-pole (DAP), an efficient implementation of a Gohberg-Semencul (GS) relation is developed for the inversion of the autocorrelation matrix which considers its centrosymmetry. In the solution of the autocorrelation equations, the Levinson algorithm is found to be less complex operationally than the procedures based on GS inversion for up to a minimum of five iterations at various linear prediction (LP) orders.

Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic beta-Glucosidase

By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP`s fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable beta-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.

Genetic transformation of Citrus sinensis cv. Hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker

Transgenic Citrus sinensis (L.) Osb. cv. Hamlin plants expressing the hrpN gene were obtained by Agrobacterium tumefaciens (Smith and Towns) Conn-mediated transformation. hrpN encodes a harpin protein, which elicits the hypersensitive response and systemic acquired resistance in plants. The gene construct consisted of gst1, a pathogen-inducible promoter, a signal peptide for protein secretion to the apoplast, the selection genes nptI1 or aacC1 and the Nos terminator. The function of gst1 in citrus was evaluated in transgenic C. sinensis cv. Valencia harboring the reporter gene uidA (gus) driven by this promoter. Histochemical analysis for gus revealed that gst1 is activated in citrus leaves by both wounding and inoculation with Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al. Genetic transformation was confirmed by Southern blot hybridization in eight cv. Hamlin acclimatized plants. RT-PCR confirmed hrpN gene expression in seven cv. Hamlin transgenic lines before pathogen inoculation. Some hrpN transgenic lines showed severe leaf curling and abnormal growth. Six hrpN transgenic lines were propagated and evaluated for susceptibility to X axonopodis pv. citri. RT-PCR confirmed gene expression in all six hrpN transgenic lines after pathogen inoculation. Several of the hrpN transgenic lines showed reduction in susceptibility to citrus canker as compared with non-transgenic plants. One hrpN transgenic line exhibited normal vegetative development and displayed very high resistance to the pathogen, estimated as up to 79% reduction in disease severity. This is the first report of genetic transformation of citrus using a pathogen-inducible promoter and the hrpN gene. Further evaluations of the transgenic plants under field conditions are planned. Nevertheless, the evidence to date suggests that the hrpN gene reduces the susceptibility of citrus plants to the canker disease. (C) 2009 Elsevier B.V. All rights reserved.

Endophytic population of Pantoea agglomerans in citrus plants and development of a cloning vector for endophytes

Harmless bacteria inhabiting inner plant tissues are termed endophytes. Population fluctuations in the endophytic bacterium Pantoea agglomerans associated with two species of field cultured citrus plants were monitored over a two-year period. The results demonstrated that populations of P. agglomerans fluctuated in Citrus reticulata but not C. sinensis. A cryptic plasmid pPA3.0 (2.9 kb) was identified in 35 out of 44 endophytic isolates of P. agglomerans and was subsequently sequenced. The origins of replication were identified and nine out of 18 open reading frames (ORFs) revealed homology with described proteins. Notably, two ORFs were related to cellular transport systems and plasmid maintenance. Plasmid pPA3.0 was cloned and the gfp gene inserted to generate the pPAGFP vector. The vector was introduced into P. agglomerans isolates and revealed stability was dependent on the isolate genotype, ninety-percent stability values were reached after 60 hours of bacterial cultivation in most evaluated isolates. In order to definitively establish P. agglomerans as an endophyte, the non-transformed bacterium was reintroduced into in vitro cultivated seedlings and the density of inner tissue colonization in inoculated plants was estimated by bacterium re-isolation, while the tissue niches preferred by the bacterium were investigated by scanning electronic microscopy (SEM). Cells from P. agglomerans (strain ARB18) at similar densities were re-isolated from roots, stems and leaves and colonization of parenchyma and xylem tissues were observed. Data suggested that P. agglomerans is a ubiquitous citrus endophyte harboring cryptic plasmids. These characteristics suggest the potential to use the bacterium as a vehicle to introduce new genes in host plants via endophytic bacterial transformation.

Genetic Transformation of Passionflower and Evaluation of R(1) and R(2) Generations for Resistance to Cowpea aphid borne mosaic virus

We report on the production and evaluation of passionflower transgenic lines for resistance to Cowpea aphid borne mosaic virus (CABMV). Genetic transformation was done using Agrobacterium tumefaciens and transgene integration was confirmed by Southern blot analyses, resulting in nine transgenic lines for `IAC 275` and three for `IAC 277`. Transgenic lines were clonally propagated and evaluated for resistance to CABMV After the third inoculation, under higher inoculum pressure, only propagated plants of the transgenic line T16 remained asymptomatic, indicating a high resistance to infection with CABMV. This transgenic line was self-pollinated and the RI generation was evaluated together with the RI generation of another resistant transgenic line (T2) identified previously. Plants were inoculated with CABMV by means of viruliferous Myzus nicotianae. All 524 T2R(1) plants became infected, whereas 13 of 279 T16R(1) remained asymptomatic after four successive inoculations. A TI6R(2) generation was obtained and plants were inoculated with CABMV mechanically or by aphids. After successive inoculations, 118 of 258 plants were symptomless, suggesting that the resistance to CABMV was maintained in the plant genome as the homozygous condition was achieved. Five selected resistant TI6R(2) plants which contained the capsid protein gene are being crossed for further analyses.