title: Morpho-histological, histochemical, and molecular evidences related to cellular reprogramming during somatic embryogenesis of the model grass Brachypodium distachyon.
abstract: 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.
authors.affiliation: Laboratório de Cultura de Tecidos/BIOAGRO, Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Avenida P. H. Rolfs s/n, 36570-900, Viçosa, MG, Brazil.
authors.forename: Evelyn Jardim
authors.initials: EJ
authors.lastname: Oliveira
authors.affiliation: Laboratório de Cultura de Tecidos/BIOAGRO, Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Avenida P. H. Rolfs s/n, 36570-900, Viçosa, MG, Brazil.
authors.forename: Andréa Dias
authors.initials: AD
authors.lastname: Koehler
authors.affiliation: Instituto de Biociências, Universidade Federal de Goiás, Regional Jataí, BR 364, km 195, 75801-615, Jataí, GO, Brazil.
authors.forename: Diego Ismael
authors.initials: DI
authors.lastname: Rocha
authors.affiliation: Laboratório de Cultura de Tecidos/BIOAGRO, Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Avenida P. H. Rolfs s/n, 36570-900, Viçosa, MG, Brazil.
authors.forename: Lorena Melo
authors.initials: LM
authors.lastname: Vieira
authors.affiliation: Laboratório de Cultura de Tecidos/BIOAGRO, Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Avenida P. H. Rolfs s/n, 36570-900, Viçosa, MG, Brazil.
authors.forename: Marcos Vinícius Marques
authors.initials: MVM
authors.lastname: Pinheiro
authors.affiliation: Laboratório de Cultura de Tecidos/BIOAGRO, Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Avenida P. H. Rolfs s/n, 36570-900, Viçosa, MG, Brazil.
authors.forename: Elyabe Monteiro
authors.initials: EM
authors.lastname: de Matos
authors.affiliation: Laboratório de Cultura de Tecidos/BIOAGRO, Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Avenida P. H. Rolfs s/n, 36570-900, Viçosa, MG, Brazil.
authors.forename: Ana Claudia Ferreira
authors.initials: ACF
authors.lastname: da Cruz
authors.affiliation: Laboratório de Cultura de Tecidos/BIOAGRO, Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Avenida P. H. Rolfs s/n, 36570-900, Viçosa, MG, Brazil.
authors.forename: Thais Cristina Ribeiro
authors.initials: TCR
authors.lastname: da Silva
authors.affiliation: Departamento de Fitopatologia e Nematologia, Universidade de São Paulo/ESALQ, Av. Pádua Dias, 13418-900, Piracicaba, SP, Brazil.
authors.forename: Francisco André Ossamu
authors.initials: FAO
authors.lastname: Tanaka
authors.affiliation: Laboratorio de Genética Molecular do Desenvolvimento Vegetal (LGMDV), Universidade de São Paulo/ESALQ, Av. Pádua Dias, 13418-900, Piracicaba, SP, Brazil. ftsnogue@usp.br.
authors.forename: Fabio Tebaldi Silveira
authors.initials: FTS
authors.lastname: Nogueira
authors.affiliation: Laboratório de Cultura de Tecidos/BIOAGRO, Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Avenida P. H. Rolfs s/n, 36570-900, Viçosa, MG, Brazil. wcotoni@gmail.com.
authors.forename: Wagner Campos
authors.initials: WC
authors.lastname: Otoni
mesh_terms: D058431:Brachypodium / Q000196:embryology* / Q000378:metabolism; D065150:Cellular Reprogramming / Q000235:genetics / Q000502:physiology*; D018745:Genome, Plant / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D010940:Plant Proteins
keywords: Cellular competency; Grass; Histology; In situ hybridization; SERK genes; Somatic embryogenesis