SÜREÇ ANALİZİ; Deneyimim

INVESTIGAÇÕES MORFOLÓGICAS, HISTOLÓGICAS E HISTOQUÍMICAS.

RESUMO

Este estudo fornece a primeira descrição de alterações morfológicas, histológicas e histoquímicas associadas ao processo de embriogenese somática em B. distachyon linhagem BD21 usando técnicas de microscopia de luz e eletrônica. A regeneração in vitro de plântulas derivadas de embriões somáticos ocorreu por embriogênese somática através da via indireta. Os embriões somáticos tiveram uma origem multicelular e originaram-se de calo embriogênico formado a partir de células da epiderme na região do nó escutelar que se estenderam para a periferia do IZE. A ordem de acúmulo de reservas nos embriões somáticos foi semelhante a dos embriões zigóticos. Nas culturas embriogênicas, proteínas e lipídios de armazenamento foram mobilizados nos primeiros 2 dias em meio de indução (DAC). O teor de amido aumentou nos primeiros 2 DAC e diminuiu em seguida em número de grânulos que se tornaram maiores e apareceram principalmente nas células vacuoladas adjacentes às massas pró- embriogênicas. Pequenos grânulos de amido começaram a acumular nos pró-embrióides após 4 DAC e tornaram-se maior e mais abundantes em células do escutelo aos 12 DAC. A diferenciação do embrião somático seguiu a mesma sequência de desenvolvimento verificado em outros membros da família Poaceae, ou seja, a passagem pelos estádios globular, escutelar e coleoptilar. Estes resultados fornecem informações importantes para a compreensão dos processos de desenvolvimento, e os mecanismos que conduzem à diferenciação celular e da transição das células somáticas para o estágio embriogênico que ainda não foram relatados em B. distachyon.

Palavras-chave: Brachypodium distachyon, embriogênese somática, histologia, histoquímica, mobilização de reservas.

23 INTRODUCTION

Over the past decade, Brachypodium distachyon has been proposed as a model species for temperate grasses and cereals (Draper et al., 2001; Vogel and Bragg, 2009).

B. distachyon is an ideal system for functional genomic studies, because of its easy

growth requirements, small stature, and rapid life cycle, small genome and self- pollination (Opanowicz et al., 2008). In addition, important genomic resources have been developed for using B. distachyon as a model for grass crops: transformation protocols, large expressed sequence tag (EST) databases, tools for forward and reverse genetic screens, highly refined cytogenetic probes, germplasm collections and, recently, a complete genome sequence has been generated (Vain, 2011; Brkljacic et al., 2011).

Functional genomics studies require from any model plant efficient transformation and regeneration systems. Efficient tissue culture protocols have been established for B. distachyon (Ye and Tao, 2008), but there have been no reports on the literature of basic histological and histochemical studies of the events taking place in the explant cells during the regeneration process. This fundamental knowledge for the understanding of the developmental processes occurring during plant growth and development as well as the mechanisms that lead to cell differentiation and passage from the somatic to the competent stage to form organs or embryos is lacking in B.

distachyon.

Somatic embryogenesis (SE) is the process by which somatic cells differentiate into somatic embryos (von Arnold, 2002). SE plays a very important role in in vitro plant regeneration of various cereal and grass species (Ozias-Akins and Vasil, 1982; Vasil et al., 1985; Brisibe et al., 1993; Taylor and Vasil, 1996; Mariani et al., 1998; Wrobel et al., 2011). When integrated with conventional breeding programs and molecular and genetic engineering techniques, SE provides a valuable tool to enhance genetic improvement of crop species (Quiroz-Figueroa et al., 2006). However, genetic engineering or mutagenesis techniques cannot be successfully achieved if the processes underlying morphogenesis are not well understood (Fortes and Pais, 2000).

The transition and induction of embryogenic competence is the most important, step during SE, but, in spite of the accumulation of experimental data, the key events underlying the transition of differentiated somatic cells to the totipotent and embryogenic cell state is still not elucidated (Fehér et al., 2003). During this step, competent cells are those which are in a transitional state and which still require some

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stimuli to become embryogenic (Namasivayam, 2007). It is not clear how the embryogenic cells originate within the explants and what mechanisms control this process. Cells will change fate and the direction of differentiation by erasing the genetic developmental program and starting a new one. It is unknown how the explant cells do so. Studies indicate that changes in the developmental program occur through the physical isolation of a cell or a group of cells from the surroundings. It has been shown that there are some features of the transition from the somatic to the embryogenic state on the cellular and histological level which allows the recognition of this developmental stage (Kurczynska et al., 2012).

In most embryogenic systems described until now, embryogenic cells show characteristics common to meristematic cells, including a high nucleus:cytoplasm ratio, a dense cytoplasm, and small fragmented vacuoles (Williams and Maheswaran 1986; Fehér et al., 2003). However, meristematic cells have spherically shaped nuclei with several small nucleoli and most of the chromatin exists as heterochromatin, whereas embryogenic cells have irregularly shaped nuclei with invaginations of the nuclear membrane, contain one large nucleolus and thick cell walls (Verdeil et al., 2007).

The morphological, histological and cytological analysis of SE is also an object of studies leading to an understanding of the basis of the totipotency, differentiation, dedifferentiation, transdifferentiation and changes in cell fate and can help in the understanding of the developmental processes taking place during plant growth and development (Quiroz-Figueroa et al., 2006; Sugimoto et al., 2011).

Descriptions of the changes occurring during the transition of somatic cells into embryogenically competent cells and histodifferentiation of somatic embryos were reported for Acrocomia aculeata (Moura et al., 2010), Hordeum vulgari (Wrobel et al., 2011), Musa spp. (Pan et al., 2011) and Passiflora (Rocha et al.,2012). Histochemical tests and ultrastructure analysis have also been used to monitor the synthesis and mobilization of reserves during the embryogenic process (Taylor and Vasil, 1996; Moura et al., 2010; Rocha et al., 2012).

Somatic embryogenesis of Brachypodium distachyon has not been characterized. Particularly, early stages of embryogenic callus development have not been examined by light and scanning electron microscopy to confirm somatic embryogenesis and to understand the mechanisms underlying the changes occurring in the transition from the somatic to the embryogenic stage. Therefore, the objectives of this study were to characterize the morphological and anatomical changes involved in somatic embryo

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formation and monitor, using histochemical methods, reserve mobilization during the induction of somatic embryogenesis from immature zygotic embryos (IZE) of B.

distachyon community standard line Bd21.

MATERIALS AND METHODS