Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with A Gene (CsTL1) Encoding a Chestnut Thaumatin-Like Protein.

We present a reproducible procedure for transforming somatic embryos of cork oak with the CsTL1 gene that codes for a thaumatin-like protein, in order to confer tolerance to Phytophthora cinnamomi. Different concentrations/combinations of the antibiotics carbenicillin and cefotaxime, as bacteriostatic agents, and kanamycin, as a selective agent, were tested. A lethal dose of 125 mg/L kanamycin was employed to select transgenic somatic embryos, and carbenicillin was used as a bacteriostatic agent at a concentration of 300 mg/L, which does not inhibit somatic embryo proliferation. The transformation efficiency was clearly genotype-dependent and was higher for the TGR3 genotype (17%) than for ALM80 (4.5%) and ALM6 (2%). Insertion of the transgenes in genomic DNA was confirmed by PCR analysis, whereas expression of the CsTL1 gene was evaluated by semi-quantitative real-time PCR (qPCR) analysis. A vitrification treatment successfully cryopreserved the transgenic lines generated. The antifungal activity of the thaumatin-like protein expressed by the gene CsTL1 was evaluated in an in vitro bioassay with the oomycete P. cinnamomi. Of the eight transgenic lines analyzed, seven survived for between one or two times longer than non-transgenic plantlets. Expression of the CsTL1 gene and plantlet survival days were correlated, and survival was generally greater in plantlets that strongly expressed the CsTL1 gene.

Genetic Transformation of Quercus ilex Somatic Embryos with a Gnk2-like Protein That Reveals a Putative Anti-Oomycete Action.

Holm oak is a key tree species in Mediterranean ecosystems, whose populations have been increasingly threatened by oak decline syndrome, a disease caused by the combined action of Phytophthora cinnamomi and abiotic stresses. The aim of the present study was to produce holm oak plants that overexpress the Ginkbilobin-2 homologous domain gene (Cast_Gnk2-like) that it is known to possess antifungal properties. Proembryogenic masses (PEMs) isolated from four embryogenic lines (Q8, E2, Q10-16 and E00) were used as target explants. PEMs were co-cultured for 5 days with Agrobacterium EHA105pGnk2 and then cultured on selective medium containing kanamycin (kan) and carbenicillin. After 14 weeks on selective medium, the transformation events were observed in somatic embryos of lines Q8 and E2 and a total of 4 transgenic lines were achieved. The presence of the Cast_Gnk2-like gene on transgenic embryos was verified by PCR, and the number of transgene copies and gene expression was estimated by qPCR. Transgenic plants were obtained from all transgenic lines after cold storage of the somatic embryos for 2 months and subsequent transfer to germination medium. In an in vitro tolerance assay with the pathogen P. cinnamomi, we observed that transgenic plants were able to survive longer than wild type.

Holm Oak Somatic Embryogenesis: Current Status and Future Perspectives.

Quercus ilex (holm oak) is one of the most representative trees in the Mediterranean basin, but now the sustainability of its ecosystems is at serious risk due to the lack of natural regeneration and to the presence of a severe disease called oak decline that has caused the death of thousands of trees. The application of biotechnological tools, such as somatic embryogenesis, allows programs of genetic improvement of the species to be speeded up and helps in the conservation of its ecosystems. Somatic embryogenesis is currently considered one of the main biotechnological techniques that has demonstrated significant benefits when has applied to forest tree species, providing significant advantages such as mass propagation, genetic transformation, application of synthetic seed, and cryopreservation of elite genotypes. In this report, the state of the art of somatic embryogenesis in holm oak is reviewed. Factors affecting the induction (plant donor age, type of explant, or plant growth regulators) and maintenance and proliferation of the embryogenic cultures are summarized. Advances on the conversion of somatic embryos into plants and on the acclimatization of these plantlets, as well as the results obtained on the application of the genetic transformation and the cryopreservation procedures to holm oak embryogenic cultures, are also presented.

Initiation of leaf somatic embryogenesis involves high pectin esterification, auxin accumulation and DNA demethylation in Quercus alba.

Somatic embryogenesis is considered a convenient tool for investigating the regulating mechanisms of embryo formation; it is also a feasible system for in vitro regeneration procedures, with many advantages in woody species. Nevertheless, trees have shown recalcitrance to somatic embryogenesis, and its efficiency remains very low in many cases. Consequently, despite the clear potential of somatic embryogenesis in tree breeding programs, its application is limited since factors responsible for embryogenesis initiation have not yet been completely elucidated. In the present work, we investigated key cellular factors involved in the change of developmental program during leaf somatic embryogenesis initiation of white oak (Quercus alba), aiming to identify early markers of the process. The results revealed that pectin esterification, auxin accumulation and DNA demethylation were induced during embryogenesis initiation and differentially found in embryogenic cells, while they were not present in leaf cells before induction or in non-embryogenic cells after embryogenesis initiation. These three factors constitute early markers of leaf embryogenesis and represent processes that could be interconnected and involved in the regulation of cell reprogramming and embryogenesis initiation. These findings provide new insights into the mechanisms underlying plant cell reprogramming, totipotency and embryogenic competence acquisition, especially in tree species for which information is scarce, thus opening up the possibility of efficient manipulation of somatic embryogenesis induction.

5-azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation, but prevents subsequent embryo development in rapeseed and barley.

Microspores are reprogrammed by stress in vitro toward embryogenesis. This process is an important tool in breeding to obtain double-haploid plants. DNA methylation is a major epigenetic modification that changes in differentiation and proliferation. We have shown changes in global DNA methylation during microspore reprogramming. 5-Azacytidine (AzaC) cannot be methylated and leads to DNA hypomethylation. AzaC is a useful demethylating agent to study DNA dynamics, with a potential application in microspore embryogenesis. This work analyzes the effects of short and long AzaC treatments on microspore embryogenesis initiation and progression in two species, the dicot Brassica napus and the monocot Hordeum vulgare. This involved the quantitative analyses of proembryo and embryo production, the quantification of DNA methylation, 5-methyl-deoxy-cytidine (5mdC) immunofluorescence and confocal microscopy, and the analysis of chromatin organization (condensation/decondensation) by light and electron microscopy. Four days of AzaC treatments (2.5 µM) increased embryo induction, response associated with a decrease of DNA methylation, modified 5mdC, and heterochromatin patterns compared to untreated embryos. By contrast, longer AzaC treatments diminished embryo production. Similar effects were found in both species, indicating that DNA demethylation promotes microspore reprogramming, totipotency acquisition, and embryogenesis initiation, while embryo differentiation requires de novo DNA methylation and is prevented by AzaC. This suggests a role for DNA methylation in the repression of microspore reprogramming and possibly totipotency acquisition. Results provide new insights into the role of epigenetic modifications in microspore embryogenesis and suggest a potential benefit of inhibitors, such as AzaC, to improve the process efficiency in biotechnology and breeding programs.

Social robots in advanced dementia.

AIMS: Pilot studies applying a humanoid robot (NAO), a pet robot (PARO) and a real animal (DOG) in therapy sessions of patients with dementia in a nursing home and a day care center. METHODS: In the nursing home, patients were assigned by living units, based on dementia severity, to one of the three parallel therapeutic arms to compare: CONTROL, PARO and NAO (Phase 1) and CONTROL, PARO, and DOG (Phase 2). In the day care center, all patients received therapy with NAO (Phase 1) and PARO (Phase 2). Therapy sessions were held 2 days per week during 3 months. Evaluation, at baseline and follow-up, was carried out by blind raters using: the Global Deterioration Scale (GDS), the Severe Mini Mental State Examination (sMMSE), the Mini Mental State Examination (MMSE), the Neuropsychiatric Inventory (NPI), the Apathy Scale for Institutionalized Patients with Dementia Nursing Home version (APADEM-NH), the Apathy Inventory (AI) and the Quality of Life Scale (QUALID). Statistical analysis included descriptive statistics and non-parametric tests performed by a blinded investigator. RESULTS: In the nursing home, 101 patients (Phase 1) and 110 patients (Phase 2) were included. There were no significant differences at baseline. The relevant changes at follow-up were: (Phase 1) patients in the robot groups showed an improvement in apathy; patients in NAO group showed a decline in cognition as measured by the MMSE scores, but not the sMMSE; the robot groups showed no significant changes between them; (Phase 2) QUALID scores increased in the PARO group. In the day care center, 20 patients (Phase 1) and 17 patients (Phase 2) were included. The main findings were: (Phase 1) improvement in the NPI irritability and the NPI total score; (Phase 2) no differences were observed at follow-up.

Motor effects of REAC in advanced Alzheimer's disease: results from a pilot trial.

We conducted a pilot, randomized, controlled trial to mainly investigate the feasibility, safety, and short-term motor effects of brain stimulation with radio electric asymmetric conveyer (REAC) technology in patients with advanced Alzheimer's disease (AD) who also experience some gait dysfunction. Neuropostural optimization (NPO) or sham protocol was administered to 31 nursing home patients (mean [SD] age 84.7 [7.0], 77.4% female, 6.5% moderate dementia, 51.6% moderately severe dementia, and 41.9% severe dementia). Motor, cognitive, functional, and behavioral measures were conducted at baseline (T1), immediately after treatment (T2), and 1-3 weeks after treatment (T3). There was transitory dysfunction in axial movements at T2 in the experimental group with no other differences between the experimental group and the control group in the planned analyses. However, after reanalysis of data based on outcome, improvement in capacity of walking was observed at T3 in the experimental group (p < 0.05). NPO administration was comfortable and safe. These results warrant further research with NPO and other REAC protocols to improve motor deterioration in AD.

Concepto de corrupción y su representación en los estudiantes universitarios / The concept of corruption and its representation among university students

En este trabajo se fundamenta epistemológica y psicológicamente en un modo de conceptuar la corrupción. Se plantea que en la sociedad contemporánea, en particular en países como Argentina, se pueden distinguir dos grandes formas de corrupción que, aunque representan momentos históricos distintos, tienen plena vigencia y, además, una fuerte dinámica de entrelazamiento. Una de esas formas de corrupción es propia de las sociedades de mercado desarrolladas (capitalismo) mientras que la otra es inherente a las sociedades donde priman formas de poder precapitalista. Se considera que una claridad conceptual es decisiva para percibir no sólo la magnitud sino también la cadena causal a que da lugar o en la cual se halla implicado ese fenómeno psico-político. Aquí se presentan y analizan los datos obtenidos en un relevamiento empírico efectuado en septiembre-octubre de 2003 con estudiantes universitarios. El objetivo del mismo era registrar las conceptualizaciones que éstos presentaban acerca de la corrupción y compararlas con las construidas desde nuestro marco teórico. También se analiza la estimación que los entrevistados realizan acerca del grado de vigencia de la corrupción en las instituciones más notorias del país. En este último aspecto, los resultados muestran una ligera variación respecto de los datos obtenidos en las encuestas del año 2001 y 2002. A la par, en la población, se mantiene un nivel de conceptualización y de explicación insuficientemente objetivo de la noción estudiada.