Diego Ismael Rocha

Somatic embryogenesis of a wild passion fruit species Passiflora cincinnata Masters: histocytological and histochemical evidences.

The characterization of cellular changes that occur during somatic embryogenesis is essential for understanding the factors involved in the transition of somatic cells into embryogenically competent cells and determination of cells and/or tissues involved. The present study describes the anatomical and ultrastructural events that lead to the formation of somatic embryos in the model system of the wild passion fruit (Passiflora cincinnata). Mature zygotic embryos were inoculated in Murashige and Skoog induction media supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Zygotic embryo explants at different development stages were collected and processed by conventional methods for studies using light, scanning, and transmission electron microscopy (TEM). Histochemical tests were used to examine the mobilization of reserves. The differentiation of the somatic embryos began in the abaxial side of the cotyledon region. Protuberances were formed from the meristematic proliferation of the epidermal and mesophyll cells. These cells had large nuclei, dense cytoplasm with a predominance of mitochondria, and a few reserve compounds. The protuberances extended throughout the abaxial surface of the cotyledons. The ongoing differentiation of peripheral cells of these structures led to the formation of proembryogenic zones, which, in turn, dedifferentiated into somatic embryos of multicellular origin. In the initial stages of embryogenesis, the epidermal and mesophyll cells showed starch grains and less lipids and protein reserves than the starting explant. These results provide detailed information on anatomical and ultrastructural changes involved in the acquisition of embryogenic competence and embryo differentiation that has been lacking so far in Passiflora.

Morphoanatomy and development of leaf secretory structures in Passiflora amethystina Mikan (Passifloraceae)

Extrafloral nectaries (EFNs) are commonly found in Passiflora L. Reports have been made on the occurrence of resin-producing structures morphologically similar to EFNs in the genus. The objective of this study was to characterise the morphoanatomy and development of leaf secretory structures in Passiflora amethystina and to use chemical and histochemical tests to detect the presence of sugars in the exudates. Samples of leaf blade and petioles in different developmental stages were collected and subjected to usual techniques using light and scanning electron microscopy. Secretion samples were analysed by high performance liquid chromatography. The concentration of total sugars in the secretion amounted to 39.67% for blade EFNs and 52.82% for petiolar EFNs. EFNs consist of a secretory, uni- or bistratified palisade epidermis, arising from the protoderm by means of anticlinal and periclinal divisions, glandular parenchyma originated from the ground meristem, and xylem and phloem elements formed from the procambium. Exudate accumulated in a subcuticular space formed outside the epidermal cells from where it was then released. Histochemical tests showed a positive reaction for neutral polysaccharides. The results confirm that the leaf secretory structures are indeed extrafloral nectaries, and these findings constitute important information for studies on the taxonomy and ecology of this species.

Cellular and molecular changes associated with competence acquisition during passion fruit somatic embryogenesis: ultrastructural characterization and analysis of SERK gene expression

The integration of cellular and molecular data is essential for understanding the mechanisms involved in the acquisition of competence by plant somatic cells and the cytological changes that underlie this process. In the present study, we investigated the dynamics and fate of Passiflora edulis Sims cotyledon explants that were committed to somatic embryogenesis by characterizing the associated ultrastructural events and analysing the expression of a putative P. edulis ortholog of the Somatic Embryogenesis Receptor-like Kinase (SERK) gene. Embryogenic calli were obtained from zygotic embryo explants cultured on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Callus formation was initiated by the division of cells derived from the protodermal and subprotodermal cells on the abaxial side of the cotyledons. The isodiametric protodermal cells of the cotyledon explants adopted a columnar shape and became meristematic at the onset of PeSERK expression, which was not initially detected in explant cells. Therefore, we propose that these changes represent the first observable steps towards the acquisition of a competent state within this regeneration system. PeSERK expression was limited to the early stages of somatic embryogenesis; the expression of this gene was confined to proembryogenic zones and was absent in the embryos after the globular stage. Our data also demonstrated that the dynamics of the mobilization of reserve compounds correlated with the differentiation of the embryogenic callus.

Morpho-histological, histochemical, and molecular evidences related to cellular reprogramming during somatic embryogenesis of the model grass Brachypodium distachyon

The wild grass species Brachypodium distachyon (L.) has been proposed as a new model for temperate grasses. Among the biotechnological tools already developed for the species, an efficient induction protocol of somatic embryogenesis (SE) using immature zygotic embryos has provided the basis for genetic transformation studies. However, a systematic work to better understanding the basic cellular and molecular mechanisms that underlie the SE process of this grass species is still missing. Here, we present new insights at the morpho-histological, histochemical, and molecular aspects of B. distachyon SE pathway. Somatic embryos arose from embryogenic callus formed by cells derived from the protodermal-dividing cells of the scutellum. These protodermal cells showed typical meristematic features and high protein accumulation which were interpreted as the first observable steps towards the acquisition of a competent state. Starch content decreased along embryogenic callus differentiation supporting the idea that carbohydrate reserves are essential to morphogenetic processes. Interestingly, starch accumulation was also observed at late stages of SE process. Searches in databanks revealed three sequences available annotated as BdSERK, being two copies corresponding to SERK1 and one showing greater identity to SERK2. In silico analysis confirmed the presence of characteristic domains in a B. distachyon Somatic Embryogenesis Receptor Kinase genes candidates (BdSERKs), which suggests SERK functions are conserved in B. distachyon. In situ hybridization demonstrated the presence of transcripts of BdSERK1 in all development since globular until scutellar stages. The results reported in this study convey important information about the morphogenetic events in the embryogenic pathway which has been lacking in B. distachyon. This study also demonstrates that B. distachyon provides a useful model system for investigating the genetic regulation of SE in grass species.

Molecular overview on plant somatic embryogenesis

The developmental pathway leading to plant somatic embryogenesis (SE) is true demonstration of totipotency of plant cells. During this process, somatic cells, under appropriate conditions, divide and differentiate into embryos. This developmental pathway plays an important role as an efficient means for plant regeneration and large-scale propagation. It includes a profound reprogramming of gene expression leading to changes in cell division and differentiation patterns, becoming a suitable platform to study the morpho-physiological and molecular aspects involved in plant cell differentiation and embryo development. Plant growth regulators such as auxin, as well as stress factors and DNA methylation, are key components to induce entry into SE pathways. Proteome and transcriptome analysis allowed isolation and characterization of embryogenic-specific gene markers involved in promoting vegetative-to-embryogenic transition as well as in maturation of somatic embryos contributing to the understanding of complex relationships between inductive conditions and somatic embryo formation. This review describes current advances made, mainly at the molecular level, in discovery of the main factors involved in the induction and maturation of somatic embryos providing a basic background for understanding genetic reprogramming that is at the heart of this process. We paid special attention to extracellular protein markers during SE as well as to auxin, abscisic acid and ethylene response genes, transcriptor factors and proteins involved in embryogenic competence acquisition.

Histology and Histochemistry of Somatic Embryogenesis

The seminal reports of somatic embryogenesis in the umbellifers Oenanthe aquatica by Harry Waris in 1957 (Krikorian and Simola, Physiol Plant 105:348–355 (1999)) and carrot (Steward et al., Am J Bot 45:693–703 (1958)) paved the way for current studies on the mechanisms involved in the transition of somatic cells to the embryogenic state for many species (Feher et al., Plant Cell Tiss Org 74:201–228, 2003; Elhiti and Stasolla, Plant embryo culture: methods and protocols, Humana Press, New York, 2011; Feher, Biochim Biophys Acta 1849:385–402, 2015). Somatic embryogenesis has been a focal point of research in plant development. This process relies on somatic cell totipotency (i.e., the capacity to regenerate the entire plant from single somatic cells), and it has been long used in biotechnological breeding techniques as an efficient system for regenerating plants in a large-scale basis. Also, because it is a unique system which includes a large number of events—such as physiological reprogramming of explants as well as changes in the gene expression and cell division patterns, and in cell fate (Feher, Acta Biol Szeged 52:53–56, 2008; Rose et al., Plant developmental biology-biotechnological perspectives. Springer, Heidelberg, 2010)—somatic embryogenesis has also become an appropriate method for studying the morphophysiological and molecular aspects of cell differentiation. The comprehension of the developmental events during the induction phase as well as the development of somatic embryos is essential to regulate each stage of the somatic embryogenesis developmental program efficiently. Additionally, it may be useful for the development of efficient protocols for somatic embryogenesis induction and validation in genetic transformation systems (Feher et al., Plant Cell Tiss Org 74:201–228, 2003; Yang and Zhang, Crit Rev Plant Sci 29:36–57, 2010; Rocha and Dornelas, CAB Rev 8:1–17, 2013; Mahdavi-Darvari et al., Plant Cell Tiss Org 120:407–422, 2015). Anatomical and ultrastructural studies have contributed to the better understanding of the basic cellular mechanisms involved in the acquisition of competence and histodifferentiation of somatic embryos (Canhoto et al., Ann Bot 78:513–521, 1996; Verdeil et al., Trends Plant Sci 12:245–252, 2001; Moura et al., Plant Cell Tiss Org 95:175–184, 2008; Moura et al., Sci Agric 67:399–407, 2010 ; Almeida et al., Plant Cell Rep 31:1495–1515, 2012; Rocha et al., Protoplasma 249:747–758, 2012; Rocha et al., Plant Cell Tiss Org 120:1087–1098, 2015; Rocha et al., Protoplasma 111:69–78, 2016). In addition, histochemical methods have enabled the monitoring of the mobilization and synthesis of reserve compounds during embryogenic development. This way, the dynamic and fate of cells committed to the somatic embryogenesis can be supported by microscopy techniques. The formation of an embryogenic callus and the subsequent differentiation of somatic embryos can be analyzed over time, and the cytological changes that have occurred during these processes can also be of great value, by associating the observed cytological changes with the expression patterns of several genes from the initial explant through competence acquisition to the formation of somatic embryos. Somatic embryogenesis has been intensively studied over the past decades. A range of descriptive studies using light and electron microscopy has provided a detailed characterization of histocytological events underlying the progression from somatic cells to the formation of embryos. Here, we review recent studies that have advanced our understanding of the anatomical and ultrastructural changes that characterize the somatic embryogenesis developmental pathway.

Somatic Embryogenesis in Annatto (Bixa orellana L.)

Our research group has pioneered the work on somatic embryogenesis of Bixa orellana (annatto), and since then we have directed efforts in understanding several aspects of this morphogenic pathway in annatto. Here, we present a synthetic description of such works, emphasizing anatomical analyzes and the characterization of the cellular alterations that occur in the process, and the association of the SERK gene expression and somatic embryogenesis. These results are unprecedented and contribute to a better understanding of the processes involving somatic embryogenesis in the species. Advances in this area will facilitate the improvement of the mass propagation, genetic manipulation of the carotenoid biosynthetic pathway, and the overall breeding perspectives of the genus.

Novel functions of the Arabidopsis transcription factor TCP5 in petal development and ethylene biosynthesis

Summary The flowers of most dicotyledons have petals that, together with the sepals, initially protect the reproductive organs. Later during development petals are required to open the flower and to attract pollinators. This diverse set of functions demands tight temporal and spatial regulation of petal development. We studied the functioning of the Arabidopsis thaliana TCP5‐like transcription factors (TFs) in petals. Overexpression of TCP5 in petal epidermal cells results in smaller petals, whereas tcp5 tcp13 tcp17 triple knockout lines have wider petals with an increased surface area. Comprehensive expression studies revealed effects of TCP5‐like TFs on the expression of genes related to the cell cycle, growth regulation and organ growth. Additionally, the ethylene biosynthesis genes 1‐amino‐cyclopropane‐1‐carboxylate (ACC) synthase 2 (ACS2) and ACC oxidase 2 (ACO2) and several ETHYLENE RESPONSE FACTORS (ERFs) are found to be differentially expressed in TCP5 mutant and overexpression lines. Chromatin immunoprecipitation–quantitative PCR showed direct binding of TCP5 to the ACS2 locus in vivo. Ethylene is known to influence cell elongation, and the petal phenotype of the tcp5 tcp13 tcp17 mutant could be complemented by treatment of the plants with an ethylene pathway inhibitor. Taken together, this reveals a novel role for TCP5‐like TFs in the regulation of ethylene‐mediated petal development and growth.

Protocol for Somatic Embryogenesis in Passiflora cincinnata Mast. (Passifloraceae)

The process of somatic embryogenesis has become an essential asset, as it enables plant regeneration and large-scale propagation. Our research team has pioneered a reproducible protocol for somatic embryogenesis using mature zygotic embryo of P. cincinnata. Here, we describe in details the protocol for P. cincinnata, in which the explants were exposed to medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Due to the efficacy and reproducibility of this regeneration protocol, new perspectives arise as the protocol can be extended to other Passifloraceae species that arouse agronomic, ornamental and commercial interest. Moreover, this is an alternative for genetic transformation protocols, based up to now on the organogenic system.

Cellular and Morpho-histological Foundations of In Vitro Plant Regeneration

In vitro plant regeneration systems have turned into invaluable tools to plant biotechnology. Despite being poorly understood, the molecular mechanisms underlying the control of both morphogenetic pathways, de novo organogenesis and somatic embryogenesis, have been supported by recent findings involving proteome-, metabolome-, and transcriptome-based profiles. Notwithstanding, the integration of molecular data with structural aspects has been an important strategy of study attempting to elucidate the basis of the cell competence acquisition to further follow commitment and determination to specific a particular in vitro regeneration pathway. In that sense, morpho-histological tools have allowed to recognize cellular markers and patterns of gene expression at cellular level and this way have collaborated in the identification of the cell types with high regenerative capacity. This chapter ties together up those fundamental and important microscopy techniques that help to elucidate that regeneration occurs, most of the time, from epidermis or subepidermal cells and from the procambial cells (pericycle and vascular parenchyma). Important findings are discussed toward ultrastructural differences observed in the nuclear organization among pluripotent and totipotent cells, implying that regeneration occurs from two cellular mechanisms based on cellular reprogramming or reactivation.