Francisco Quiroz-Figueroa

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.

Expression of WUSCHEL in Coffea canephora causes ectopic morphogenesis and increases somatic embryogenesis

Cell differentiation depends on the proper and sequential expression of key genes required for morphogenesis. Several aspects of control are required for this which include: chromatin modifications, DNA methylation, correct amount of particular transcription factors, proper nuclear arrangement, etc. During the last few years the homeobox transcription factor WUSCHEL (WUS) has been shown to cause dedifferentiation when expressed on somatic cells followed by a production of new stem cells that can lead to somatic embryogenesis or organogenesis. We found that expression of WUS in coffee plants can induce calli formation as well as a 400% increase somatic embryo production. The results show that transgenic expression of the transcription factor WUS can be useful to increase somatic embryogenesis in heterologous systems. However, a critical developmental stage and additional hormonal requirements are required for the induction of embryogenesis by WUS in Coffea canephora.

Culture-induced variation in plants of Coffea arabica cv. Caturra rojo, regenerated by direct and indirect somatic embryogenesis

Amplified fragment-length polymorphism (AFLP) was used to evaluate the stability of DNA in regenerated plantlets of Coffea arabica obtained by direct (DSE) and indirect somatic embryogenesis (ISE). Cluster analysis using the unweighted pair-group method (UPGMA), showed no specific grouping pattern related to the type of embryogenesis. These results suggest that the somatic embryogenesis (SE) process has a mechanism for the selection of normal and competent cells. Bulked DNA from regenerated plants obtained by DSE and ISE, and from the mother plants, was used to characterize specific AFLP fragments associated with each SE process. Twenty-three primer combinations were tested. A total of 1446 bands were analyzed, with 11.4% being polymorphic and 84% being specific for regenerated plants. Furthermore, specific bands were detected for DSE, ISE, and the mother plants. These results indicate that the SE process induces rearrangements at the DNA level and demonstrates discrepancies between the mechanisms involved in each SE process. Coffea arabica breeding programs that involve DSE and ISE can use AFLP as an additional tool for assessing DNA stability.

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.

Direct somatic embryogenesis in Coffea canephora.

Somatic embryogenesis (SE) provides a useful model to study embryo development in plants. In contrast to zygotic embryogenesis, SE can easily be observed, the culture conditions can be controlled, and large quantities of embryos can be easily obtained. In Coffea spp several model systems have been reported for in vitro SE induction. SE for coffee was first reported in Coffea canephora. Several systems have been developed since then, including SE from callus cultures derived from leaf explants; a two-phase experimental protocol for SE from leaves of Coffea arabica; and from leaf explants of Arabusta or C. arabica using a medium with cytokinins. Here we report a protocol using young leaves from in vitro seedling pre-conditioned with growth regulators. This is a simplified method to obtain a faster and more efficient protocol to produce direct somatic embryos in C. canephora.

Plant Tissue Culture

Plant tissue culture (PTC) is a set of techniques for the aseptic culture of cells, tissues, organs and their components under defined physical and chemical conditions in vitro and controlled environment (Fig. 50.1). PTC technology also explores conditions that promote cell division and genetic re-program-ming in in vitro conditions and it is considered an important tool in both basic and applied studies, as well as in commercial application

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.

Accumulation of High Levels of ABA Regulates the Pleiotropic Response of the nhr1 Arabidopsis Mutant

Plants have evolved a variety of mechanisms for responding to environmental cues, which allows them to survive in the presence of limited resources or environmental stresses. One of the most significant growth adaptations plants have attained is tropism, a growth response that involves bending of plant organs toward or away from a stimulus. Roots exhibit hydrotropism in response to moisture gradients, which is thought to be critical in acquiring water and establishing their stand in the soil. However, the mechanism underlying hydrotropism remains unsolved. Here, we report that the no hydrotropic response (nhr1) mutant of Arabidopsis, which is impaired in hydrotropism, is tolerant to drought. The no hydrotropic response phenotype of nhr1 was repressed by AbamineSG, an inhibitor of abscisic acid (ABA) biosynthesis, indicating that ABA negatively regulates hydrotropism. Furthermore, the content of ABA was higher in nhr1 compared to those of wild type (wt). However, the higher ABA levels in nhr1 plants were not due to higher transcript levels of 9-cis-epoxycarotenoid dioxygenase (NCED3), since these were diminished compared to those of wt. Our results indicated that the root hydrotropic response of the nhr1 mutant is modulated by ABA and that the higher ABA levels of the mutant might confer it drought resistance.