Rafael Rojas-Herrera

Embryo production through somatic embryogenesis can be used to study cell differentiation in plants

Somatic embryogenesis is the process by which somatic cells, under induction conditions, generate embryogenic cells, which go through a series of morphological and biochemical changes that result in the formation of a somatic embryo. Somatic embryogenesis differs from zygotic embryogenesis in that it is observable, its various culture conditions can be controlled, and a lack of material is not a limiting factor for experimentation. These characteristics have converted somatic embryogenesis into a model system for the study of morphological, physiological, molecular and biochemical events occurring during the onset and development of embryogenesis in higher plants; it also has potential biotechnological applications. The focus of this review is on embryo development through somatic embryogenesis and especially the factors affecting cell and embryo differentiation.

New insights into somatic embryogenesis: leafy cotyledon1, baby boom1 and WUSCHEL-related homeobox4 are epigenetically regulated in Coffea canephora.

Plant cells have the capacity to generate a new plant without egg fertilization by a process known as somatic embryogenesis (SE), in which differentiated somatic cells can form somatic embryos able to generate a functional plant. Although there have been advances in understanding the genetic basis of SE, the epigenetic mechanism that regulates this process is still unknown. Here, we show that the embryogenic development of Coffea canephora proceeds through a crosstalk between DNA methylation and histone modifications during the earliest embryogenic stages of SE. We found that low levels of DNA methylation, histone H3 lysine 9 dimethylation (H3K9me2) and H3K27me3 change according to embryo development. Moreover, the expression of LEAFY COTYLEDON1 (LEC1) and BABY BOOM1 (BBM1) are only observed after SE induction, whereas WUSCHEL-RELATED HOMEOBOX4 (WOX4) decreases its expression during embryo maturation. Using a pharmacological approach, it was found that 5-Azacytidine strongly inhibits the embryogenic response by decreasing both DNA methylation and gene expression of LEC1 and BBM1. Therefore, in order to know whether these genes were epigenetically regulated, we used Chromatin Immunoprecipitation (ChIP) assays. It was found that WOX4 is regulated by the repressive mark H3K9me2, while LEC1 and BBM1 are epigenetically regulated by H3K27me3. We conclude that epigenetic regulation plays an important role during somatic embryogenic development, and a molecular mechanism for SE is proposed.

A Simple Silica-based Method for Metagenomic DNA Extraction from Soil and Sediments

A new method is described for extraction of metagenomic DNA from soil and sediments which is based on DNA adsorption to silica without the use of phenol, ethanol precipitation or a cesium chloride gradient. High-quality DNA was obtained, and PCR inhibition was overcome by adding bovine serum albumin and adjusting magnesium concentration. By using PCR-DGGE with Firmicutes and lactic acid bacteria-specific primers the extracted metagenomic DNA was shown to contain a mixture of bacterial genomes. This method can be used for screening bacterial diversity in soil and sediment samples.

Differential gene expression in embryogenic and non-embryogenic clusters from cell suspension cultures ofCoffea arabica

Summary Somatic embryogenesis (SE) is a very useful system for studying the differentiation process in plants and involves gene regulation at several levels. During SE induction in Coffea arabica cv. Catura Rojo two types of cell clusters, embryogenic (EC) and non-embryogenic (NEC), were observed. The goal of this work was to compare the most relevant characteristics between EC and NEC for a better understanding of the mechanism driving SE. Morphohistological observations indicated a correlation between the morphological features of clusters and their embryogenic competence. On the other hand, no variation at the DNA level, studied by AFLP, were found to explain the disparity in embryogenic competence of clusters, but gene expression, observed by RNA differential display, and SDS-PAGE showed differences that can explain that disparity. Our results lead us to propose that differential gene expression can modulate the embryogenic capacity of coffee cells and that the number of genes turned off in somatic cells to allow for the change from a somatic to an embryogenic state, is higher than those genes that are turned on.

Isolation and identification of lactic acid bacteria from sediments of a coastal marsh using a differential selective medium

Aims: To identify lactic acid bacteria (LAB) colonies isolated from sediments of a coastal marsh by the reduction of 2,3,5‐triphenyltetrazolium chloride (TTC) in MRS medium.

Modification of the embryogenic response of Coffea arabica by the nitrogen source

Somatic embryogenesis was induced in coffee from in vitro cultured plants as starting material and the faster response obtained allowed lines from selected plants to be generated more quickly. In contrast to other systems, where embryos take 2 or 3 months to develop, globular embryos were obtained after 3 weeks. The optimum nitrogen concentrations for embryogenesis were between 3.75 and 15 mM nitrogen with a nitrate/ammonium molar ratio of 2:1 or 1:2.

Differential Gene Expression During Somatic Embryogenesis in Coffea arabica L., Revealed by RT-PCR Differential Display

Molecular and biochemical studies of somatic embryogenesis may help to shed light on the mechanisms governing this phenomenon. In this article, a differential display analysis approach was employed to investigate the changes taking place during the induction of somatic embryogenesis in leaf explants and suspension cultures of coffee. Cloned fragments show homologies to several proteins reported in databases, but only one has previously been described as regulated during somatic embryogenesis. By a reverse dot blot modification, the expression pattern of such fragments was evaluated.

Stem Cells from Dental Pulp: What Epigenetics Can Do with Your Tooth

Adult stem cells have attracted scientific attention because they are able to self-renew and differentiate into several specialized cell types. In this context, human dental tissue-derived mesenchymal stem cells (hDT-MSCs) have emerged as a possible solution for repairing or regenerating damaged tissues. These cells can be isolated from primary teeth that are naturally replaced, third molars, or other dental tissues and exhibit self-renewal, a high proliferative rate and a great multilineage potential. However, the cellular and molecular mechanisms that determine lineage specification are still largely unknown. It is known that a change in cell fate requires the deletion of existing transcriptional programs, followed by the establishment of a new developmental program to give rise to a new cell lineage. Increasing evidence indicates that chromatin structure conformation can influence cell fate. In this way, reversible chemical modifications at the DNA or histone level, and combinations thereof can activate or inactivate cell-type-specific gene sequences, giving rise to an alternative cell fates. On the other hand, miRNAs are starting to emerge as a possible player in establishing particular somatic lineages. In this review, we discuss two new and promising research fields in medicine and biology, epigenetics and stem cells, by summarizing the properties of hDT-MSCs and highlighting the recent findings on epigenetic contributions to the regulation of cellular differentiation.

A method for isolating RNA from metabolically active bacterial flora associated with octopus

Aim:  Development of a method for the isolation and purification of metagenomic RNA (mgRNA) from the ectopic bacterial flora of octopus.

Effects of two intake levels of Leucaena leucocephala on rumen function of sheep

Ruminant production systems based on grass pastures often produce only poor animal performance as expressed by growth or reproductive rates. The nutrient imbalance affecting rumen function is due to low energy and protein intake. The incorporation of leguminous forages such as Leucaena leucocephala, in the diet of ruminants, can stimulate rumen function by providing protein-rich forage (Barros-Rodriguez et al. 2012). This increases the availability of compounds such as ammonia, amino acids and peptides as well as branched short-chain fatty acids, which are produced as a result of degradation of proteins. These substances promote fiber breakdown by acting as ruminal growth activators for rumen bacteria, especially cellulolytic bacteria (Hoover and Stokes 1991). This study aimed to evaluate the effects of 2 intake levels of L. leucocephala (leucaena) on rumen function of sheep fed Pennisetum purpureum.