[show abstract][hide abstract] ABSTRACT: 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.
PLoS ONE 01/2013; 8(8):e72160. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Colletotrichum capsici is an important fungal species that causes anthracnose in many genera of plants causing severe economic losses worldwide. A primer set was designed based on the sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a conventional PCR assay. The primer set (CcapF/CcapR) amplified a single product of 394 bp with DNA extracted from 20 Mexican isolates of C. capsici. The specificity of primers was confirmed by the absence of amplified product with DNA of four other Colletotrichum species and eleven different fungal genera. This primer set is capable of amplifying only C. capsici from different contaminated tissues or fungal structures, thereby facilitating rapid diagnoses as there is no need to isolate and cultivate the fungus in order to identify it. The sensitivity of detection with this PCR method was 10 pg of genomic DNA from the pathogen. This is the first report of a C. capsici-specific primer set. It allows rapid pathogen detection and provides growers with a powerful tool for a rational selection of fungicides to control anthracnose in different crops and in the post-harvest stage.
[show abstract][hide abstract] ABSTRACT: Genetic studies and pathogen detection in plants using molecular methods require the isolation of DNA from a large number of samples in a short time span. A rapid and versatile protocol for extracting high-quality DNA from different plant species is described. This method yields from 1 to 2 mg of DNA per gram of tissue. The absorbance ratios (A260/A280) obtained ranged from 1.6 to 2.0. A minimal presence of contaminating metabolites (as polymerase chain reaction [PCR] inhibitors) in samples and a considerable savings in reagents are characteristics of this protocol, as well as the low cost of the analysis per sample. The quality of the DNA was suitable for PCR amplification.
[show abstract][hide abstract] ABSTRACT: Histocytological analysis carried out on leaf explants of Coffea arabica undergoing somatic embryogenesis revealed that, using a culture method involving a single Gelrite-containing semisolid medium, the entire region surrounding the edge of the plant-derived leaf explants showed the differentiation of organized structures with little or no callusing. Histological examination of embryogenesis without callus formation (direct somatic embryogenesis) revealed that at approximately 1 week after the explant had been placed in culture, the development of the embryo began in the form of a small, isodiametric, densely cytoplasmic cell that underwent a series of organized divisions. In embryogenesis from callus (indirect somatic embryogenesis), however, the embryogenic cell was observed within the first week. Our histological observations indicate that both direct and indirect somatic embryos of coffee that form on explanted leaf segments and callus, respectively, have a unicellular origin.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Plant Physiology 01/2002; 159(11):1267-1270. · 2.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: A gene fragment, termed AR-52, was cloned by differential display analysis during the induction of somatic embryogenesis in foliar explants of Coffea arabica. It is homologous to several class III chitinases and Northern blot shows that it is up regulated during somatic embryogenesis and scarcely expressed in embryos at any developmental stage. AR-52 is also induced by wounding the leaves of in vitro cultured plantlets but not induced in a non-embryogenic suspension culture. Chitinase zymography revealed the existence of at least three-chitinolitic bands that are also regulated during somatic embryogenesis and wounding.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
Plant Cell Tissue and Organ Culture 86(3):285-301. · 3.63 Impact Factor
[show abstract][hide abstract] ABSTRACT: Genetic improvement of coffee, an important commercial crop, through classical breeding is slow and cumbersome. Biotechnology
offers alternative strategies for generating new and improved coffee varieties, including those resistances to environmental
extremes, pests, and diseases, low in caffeine, and with uniform fruit maturation. Large improvements in somatic embryogenesis,
development of haploids, and scale-up of micropropagation have been accomplished in the last 5yr. The recent identification
of expressed sequence tags (EST) that are differentially expressed during the infestation of coffee plants by coffee leaf
miners and the isolation and cloning of the promoter for the N-methyltransferase gene associated with caffeine production open up the possibility of producing varieties of coffee with
new traits. This review provides a summary of in vitro biological advances and directions as to how they could be applied to improve the production and quality of coffee.