Potato snakin-1 gene enhances tolerance to Rhizoctonia solani and Sclerotinia sclerotiorum in transgenic lettuce plants.

Snakin-1 is a cysteine-rich antimicrobial peptide (AMP) isolated from potato tubers, with broad-spectrum activity. It belongs to the Snakin/GASA family, whose members have been studied because of their diverse roles in important plant processes, including defense. To analyze if this defensive function may lead to disease tolerance in lettuce, one of the most worldwide consumed leafy vegetable, we characterized three homozygous transgenic lines overexpressing Snakin-1. They were biologically assessed by the inoculation with the fungal pathogens Rhizoctonia solani and Sclerotinia sclerotiorum both in vitro and in planta at the greenhouse. When in vitro assays were performed with R. solani on Petri dishes containing crude plant extracts it was confirmed that the expressed Snakin-1 protein has antimicrobial activity. Furthermore, transgenic lines showed a better response than wild type in in vivo challenges against R. solani both in chamber and in greenhouse. In addition, two of these lines showed significant in vivo protection against the pathogen S. sclerotiorum in challenge assays on adult plants. Our results show that Snakin-1 is an interesting candidate gene for the selection/breeding of lettuce plants with increased fungal tolerance.

Competencia profesional como indicador de calidad en el programa de bajo peso al nacer / Professional competence as indicator of quality in the low birth weight program

Se realizó una investigación en sistemas y servicios de salud, desde septiembre de 2015 hasta marzo de 2016, con vistas a evaluar la calidad de la atención prenatal para la prevención del bajo peso al nacer, a través de la competencia profesional de 43 médicos vinculados a la labor asistencial en consultorios del Policlínico Docente Ramón López Peña de Santiago de Cuba. Se utilizó la metodología propuesta por la Escuela Nacional de Salud Pública y se preestablecieron criterios, indicadores y estándares de evaluación. La calidad del programa evaluado no fue la mejor, pues se detectaron dificultades relacionadas con la conducta a seguir en situaciones de riesgo, asociadas frecuentemente con el embarazo. Se hace necesario evaluar otras dimensiones de la calidad en este programa, así como la atención a otros grupos poblacionales como las mujeres en edad fértil, con riesgo de tener hijos con bajo peso, y capacitar a los profesionales en los aspectos con dificultades

An investigation in health systems and services, was carried out from September, 2015 to March, 2016, aimed at evaluating the quality of prenatal care for the low birth weight prevention, through the professional competence of 43 doctors linked to the assistance work in doctor offices from Ramón López Peña Teaching Polyclinic in Santiago de Cuba. The proposed methodology was used by the National School of Public Health and approaches, indicators and evaluation standards were preestablished. The quality of the evaluated program was not the best, because some difficulties related to the follow-up protocols in risk situations were detected, frequently associated with pregnancy. It is necessary to evaluate other dimensions of this program quality, as well as care to other populational groups as fertility age women, with risk of having low weight children, and to qualify the professionals in the aspects with difficulties

Simplified methodology for large scale isolation of homozygous transgenic lines of lettuce

Background: Lettuce is a globally important leafy vegetable and a model plant for biotechnology due to its adaptability to tissue culture and stable genetic transformation. Lettuce is also crucial for functional genomics research in the Asteraceae which includes species of great agronomical importance. The development of transgenic events implies the production of a large number of shoots that must be differentiated between transgenic and non-transgenic through the activity of the selective agent, being kanamycin the most popular. Results: In this work we adjusted the selection conditions of transgenic seedlings to avoid any escapes, finding that threshold concentration of kanamycin was 75 mg/L. To monitor the selection system, we studied the morphological response of transgenic and non-transgenic seedlings in presence of kanamycin to look for a visual morphological marker. Several traits like shoot length, primary root length, number of leaves, fresh weight, and appearance of the aerial part and development of lateral roots were affected in non-transgenic seedlings after 30 d of culture in selective media. However, only lateral root development showed an early, qualitative and reliable association with nptII presence, as corroborated by PCR detection. Applied in successive transgenic progenies, this method of selection combined with morphological follow-up allowed selecting the homozygous presence of nptII gene in 100% of the analyzed plants from T2 to T5. Conclusions: This protocol allows a simplified scaling-up of the production of multiple homozygous transgenic progeny lines in the early generations avoiding expensive and time-consuming molecular assays.

Sunflower (Helianthus annuus L.).

Sunflower (Helianthus annuus L.) is still considered as a recalcitrant species to in vitro culture and transformation in spite of the publication of different protocols. Here we describe a routine transformation system of this crop which requires mature HA89 genotype seeds and Agrobacterium tumefaciens EHA105 strain for gene delivery, being both easily available. Selection of transformed shoots depends on root development in kanamycin-selective media, instead of shoot color, avoiding selection of escapes. The establishment of this protocol proved successful for the incorporation of both reporter and agronomic important genes and also for the evaluation of the specific expression patterns of different promoters in transgenic sunflower plants. Stable expression of the incorporated transgenes was confirmed by RT-PCR and GUS reporter gene visualization. Stable inheritance of transgenes was successfully followed until T2 generation in several independent lines.

Rooting in Km selective media as efficient in vitro selection method for sunflower genetic transformation

Despite of numerous publications in sunflower genetic transformation, there is no efficient or reproducible protocol with low number of escapes. The latter would indicate that the selection method is not effective. In this work we used Km as selective agent, Agrobacterium tumefaciens EHA105 strain and a vector with the nptII gene under the nos promoter and uidA gene under 35S promoter. The response of agroinfected (A) and control (C) explants during the in vitro culture was studied and in both cases in presence or absence of Km in order to assign a differential morphologic response between transformed and non-transformed plants. The characteristics analyzed were: height, colour/aspect of the plantlets, in vitro rooting and in vitro bud-flower development. Selection was applied from the third regeneration media. Among the A plantlets two were capable of rooting, being positive by PCR, whereas the C were unable to root in presence of Km. One of them gave 6 seeds and in these plants, it was determined the presence of the transgene by PCR and GUS staining. This work shows that in Km selection, colour/aspect of shoots is not useful as selection criteria whereas rooting is an effective selection method in which no escapes were obtained.