Excision of a selectable marker gene in transgenic banana using a Cre/lox system controlled by an embryo specific promoter.

Antibiotic and herbicide resistance genes have been used in transgene technology as powerful selection tools. Nonetheless, once transgenic events have been obtained their presence is no longer needed and can even be undesirable. In this work, we have developed a system to excise the selectable marker and the cre recombinase genes from transgenic banana cv. 'Grande Naine' (Musa AAA). To achieve this, the embryo specific REG-2 promoter was isolated from rice and its expression pattern in banana cell clumps, somatic embryos and regenerated plantlets was characterized by using a pREG2::uidA fusion construct. Subsequently, the REG-2 promoter was placed upstream of the cre gene, conferring Cre functionality in somatic embryos and recombination of lox sites resulting in excision of the selectable marker and cre genes. PCR analysis revealed that 41.7 % of the analysed transgenic plants were completely marker free, results that were thereafter confirmed by Southern blot hybridization. These results demonstrate the feasibility of using developmentally controlled promoters to mediate marker excision in banana. This system does not require any extra handling compared to the conventional transformation procedure and might be useful in other species regenerating through somatic embryogenesis.

La formación del capital humano de Atención Estomatológica en Villa Clara / The formation of human resources in the stomatologic attention in Villa Clara

El presente trabajo resultó de una investigación descriptiva con enfoque histórico-lógico llevada a cabo en la Facultad de Tecnología de la Salud "Julio Trigo López" de Villa Clara, desde el curso 1985-1986 hasta 2009-2010, consistió en una revisión documental sobre la historia, formación y evolución del personal técnico de apoyo al estomatólogo. Se emplearon métodos del nivel teórico: histórico-lógico, analítico-sintético e inductivo-deductivo y del empírico: análisis documental para consultar los planes de estudio, la fundamentación de la carrera y el registro nominal de graduados de forma escalonada (formación de la asistente dental, el técnico medio y el licenciado en atención estomatológica) con el objetivo de describir la historia de esta especialidad en la provincia y determinar su número de egresados hasta la fecha. El estudio permitió conocer las diferentes etapas por las que ha transitado este proceso a partir de sus transformaciones curriculares, y valorar el salto cualitativo que generó el diseño progresivo de su formación.

The current work was based on a previous descriptive research work that had a historical-logical approach which was carried out in Villa Clara Health Technology Faculty from the academic year1985-1986 to 2009-2010; it was a documental review about the history, formation and evolution of the technical personnel which bach up odontologists in their work. Theoretical methods were used such as: Historical-logical, anatytical-synthetic and inductive-deductive. Empirical methods were also used: Documental analysis so as to consult the study plans, the foundations of the career and the register of graduated students, (the formation of dental assistants, dental technicians and the Bachelor in Stomatologic Attention) with the objective to describe the history of the specialty in the province and to determine the number of graduated students up to the current date. The study allowed to know the different stages of this process as well as the curricular transformations and to assess the qualitative advancing steps which generated its progressive formation.

La formación del capital humano de Atención Estomatológica en Villa Clara / The formation of human resources in the stomatologic attention in Villa Clara

El presente trabajo resultó de una investigación descriptiva con enfoque histórico-lógico llevada a cabo en la Facultad de Tecnología de la Salud “Julio Trigo López” de Villa Clara, desde el curso 1985-1986 hasta 2009-2010, consistió en una revisión documental sobre la historia, formación y evolución del personal técnico de apoyo al estomatólogo. Se emplearon métodos del nivel teórico: histórico-lógico, analítico-sintético e inductivo-deductivo y del empírico: análisis documental para consultar los planes de estudio, la fundamentación de la carrera y el registro nominal de graduados de forma escalonada (formación de la asistente dental, el técnico medio y el licenciado en atención estomatológica) con el objetivo de describir la historia de esta especialidad en la provincia y determinar su número de egresados hasta la fecha. El estudio permitió conocer las diferentes etapas por las que ha transitado este proceso a partir de sus transformaciones curriculares, y valorar el salto cualitativo que generó el diseño progresivo de su formación(AU)

Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system.

Selectable marker genes are indispensable for efficient production of transgenic events, but are no longer needed after the selection process and may cause public concern and technological problems. Although several gene excision systems exist, few have been optimized for vegetatively propagated crops. Using a Cre-loxP auto-excision strategy, we obtained transgenic banana plants cv. Grande Naine (Musa AAA) devoid of the marker gene used for selection. We used T-DNA vectors with the cre recombinase gene under control of a heat shock promoter and selectable marker gene cassettes placed between two loxP sites in direct orientation, and a gene of interest inserted outside of the loxP sites. Heat shock promoters pGmHSP17.6-L and pHSP18.2, from soybean and Arabidopsis respectively, were tested. A transient heat shock treatment of primary transgenic embryos was sufficient for inducing cre and excising cre and the marker genes. Excision efficiency, as determined by PCR and Southern hybridization was 59.7 and 40.0% for the GmHSP17.6-L and HSP18.2 promoters, respectively. Spontaneous excision was not observed in 50 plants derived from untreated transgenic embryos. To our knowledge this is the first report describing an efficient marker gene removal system for banana. The method described is simple and might be generally applicable for the production of marker-free transgenic plants of many crop species.

Analysis of expressed sequence tags derived from a compatible Mycosphaerella fijiensis-banana interaction.

Mycosphaerella fijiensis, a hemibiotrophic fungus, is the causal agent of black leaf streak disease, the most serious foliar disease of bananas and plantains. To analyze the compatible interaction of M. fijiensis with Musa spp., a suppression subtractive hybridization (SSH) cDNA library was constructed to identify transcripts induced at late stages of infection in the host and the pathogen. In addition, a full-length cDNA library was created from the same mRNA starting material as the SSH library. The SSH procedure was effective in identifying specific genes predicted to be involved in plant-fungal interactions and new information was obtained mainly about genes and pathways activated in the plant. Several plant genes predicted to be involved in the synthesis of phenylpropanoids and detoxification compounds were identified, as well as pathogenesis-related proteins that could be involved in the plant response against M. fijiensis infection. At late stages of infection, jasmonic acid and ethylene signaling transduction pathways appear to be active, which corresponds with the necrotrophic life style of M. fijiensis. Quantitative PCR experiments revealed that antifungal genes encoding PR proteins and GDSL-like lipase are only transiently induced 30 days post inoculation (dpi), indicating that the fungus is probably actively repressing plant defense. The only fungal gene found was induced 37 dpi and encodes UDP-glucose pyrophosphorylase, an enzyme involved in the biosynthesis of trehalose. Trehalose biosynthesis was probably induced in response to prior activation of plant antifungal genes and may act as an osmoprotectant against membrane damage.

An efficient method for the extraction of high-quality fungal total RNA to study the Mycosphaerella fijiensis-Musa spp. Interaction.

Efficient RNA isolation is a prerequisite for gene expression studies and it has an increasingly important role in the study of plant-fungal pathogen interactions. However, RNA isolation is difficult in filamentous fungi. These organisms are notorious for their rigid cell walls and the presence of high levels of carbohydrates, excreted from the fungal cells during submerged growth, which interferes with the extraction procedures. Although many commercial kits are already available for RNA isolation, they do not provide, in most cases, enough amount of pure RNA to be used in upstream applications. In the present work, we propose an easy and efficient protocol for isolating total RNA from the filamentous fungus Mycosphaerella fijiensis, the most important foliar pathogen of Musa spp. varieties worldwide. In addition, we applied the proposed protocol to the isolation of total RNA from banana leaves infected with the pathogen. Our methodology was developed based on the SDS method with modifications including a carbohydrate precipitation step. The protocol resulted in high-quality total RNA, from fungal mycelium grown in PDB medium and infected banana leaves, suitable for further molecular studies. The proposed methodology is also applicable to the ascomycete fungus Passalora fulva (syn. Cladosporum fulvum).