Lorena Melo Vieira

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.

Microenxertia ex vitro para eliminação do vírus CABMV em maracujá-azedo

O objetivo deste trabalho foi avaliar diferentes meios de cultura, utilizados sobre o ponto da enxertia, na microenxertia ex vitro para a eliminacao do Cowpea aphid-borne mosaic virus (CABMV), em plantas de maracuja-azedo (Passiflora edulis Sims f. flavicarpa Deg.). Apices caulinares, provenientes de plantas infectadas, foram microenxertados em plântulas obtidas pela germinacao de sementes em substrato comercial esterilizado. Foram conduzidos experimentos com a microenxertia realizada no hipocotilo e no epicotilo, e foram utilizados cinco meios de cultura, que diferiam na concentracao de fitorreguladores, aplicados no local da enxertia. O indice medio de microenxertos com folha expandida foi de 27,22 e 32,22%, quando a microenxertia foi realizada no hipocotilo e no epicotilo, respectivamente. Na microenxertia realizada no hipocotilo, nao houve efeito da aplicacao de meios de cultura. Na microenxertia realizada no epicotilo, o meio MS acrescido de 0,1 mg L-1 de AIB e 1 mg L-1 de BAP proporcionou 53,3% de microenxertos com folha expandida, numero superior aos demais tratamentos e maior desenvolvimento das brotacoes. A indexacao realizada pelo teste ELISA indireto, 80 a 100 dias apos a microenxertia, mostrou que 93% das plantas testadas nao apresentavam virus detectavel.

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.

Microenxertia interespecífica ex vitro em maracujazeiros

This study aimed at evaluating the effect of Passiflora species used as rootstocks on the success and development of ex vitro interspecific micrografting, and at anatomically characterizing the region where it is done. Stem apices of Passiflora edulis f. flavicarpa were grafted onto the hypocotyl of P. alata, P. cincinnata, P. edulis, and P. setacea seedlings. The micrografting development, the occurrence of adventitious shoots in rootstocks and the percentage of micrografts with expanded leaves were evaluated. The rootstocks were subjected to anatomical and histochemical evaluation. Micrograft development and adventitious shoot number were higher when P. edulis rootstocks were used, and lower for rootstocks of P. setacea and P. alata. P. edulis, P. cincinnata, P. alata, and P. setacea rootstocks developed 30.3, 10, 1.6, and 0% of micrografts with expanded leaves, respectively. The histological evaluation revealed increased thickness of the cortical region in rootstocks of P. edulis, while in P. setacea a lower number of cortical layers, increased phloem fibers, and increased alkaloids were observed, characteristics that negatively influenced the micrografting outcome.

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.