Somatic embryogenesis in Brachypodium distachyon (L.) Beauv. (Poaceae): morphoanatomical and histochemical characterization and analysis of SERK gene expression

Brachypodium distachyon (L.) P. Beauv. (Poaceae: Poideae) tem se destacado como planta modelo para gramíneas de clima temperado e espécies usadas para a produção de biocombustíveis. A linhagem Bd21 de Brachypodium distachyon tem um genoma completamente seqüenciado e montado, além de protocolos de genômica e de transformação bem estabelecidos com base em calos embriogênicos. No entanto, as informações sobre a origem e as alterações celulares que ocorrem durante a diferenciação de embriões somáticos nos estágios iniciais não foi documentada para B. distachyon. Também não há relatos sobre o uso de abordagens moleculares para investigar o processo de embriogênese somática nesta espécie. Portanto, os objetivos deste trabalho foram: (1) caracterizar as alterações morfológicas, anatômicas e histoquímicas que ocorrem durante a indução de embriogênese somática a partir de embriões zigóticos imaturos (EZI) da B. distachyon linhagem de referência Bd21 usando microscopia de luz e de varredura em associação com testes histoquímicos e, (2), realizar a clonagem e caracterização de genes SERK (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE) e analisar sua expressão na indução de embriogênese somática utilizando hibridização in situ para monitorar os processos morfogenéticos in vitro. Culturas embriogênicas de B. distachyon (BD21) foram estabelecidas usando EZI 15 dias após a antese, em meio Murashige e Skoog (1962) contendo ácido 2,4- diclorofenoxiacético. A regeneração in vitro de plântulas derivadas de embriões somáticos ocorreu pela 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 EZI. A ordem de acumulação de reservas nos embriões somáticos foi semelhante a dos embriões zigóticos. Nas culturas embriogênicas, proteínas e lipídios foram utilizados nos primeiros 2 dias em meio de indução. O teor de amido aumentou nos primeiros 2 dias em meio de indução 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-embrioides após 4 dias em meio de indução e tornaram- se maior e mais abundantes em células do escutelo aos 12 dias em meio de indução. 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. O gene SERK tem sido usado como um marcador para as células competentes na embriogênese somática de várias espécies. Neste estudo, utilizando iniciadores degenerados, uma sequência específica homóloga de um fragmento do gene SERK foi amplificada de B. distachyon Bd21. A análise da sequência do fragmento de SERK (766 bp) revelou altos níveis de similaridade com genes SERK relatados em outras espécies. A análise de hibridização in situ mostrou que o gene SERK estava presente nos tecidos embriogênicos de B. distachyon antes do desenvolvimento de embriões somáticos e continuou sendo expresso nos estágios globular e escutelar. Estes resultados sugerem que a expressão do gene SERK de B. distachyon pode estar associada com a indução da embriogênese somática. Este estudo faz a primeira descrição de mudanças morfoanatômicas e histoquímicas durante o processo de embriogênese somática em B. distachyon linhagem Bd21. Este é também o primeiro relato sobre a clonagem e expressão de um gene SERK para a espécie e sugere que este gene pode servir como um marcador molecular para monitorar a transição de células somáticas em células competentes e embriogênicas também em B. distachyon.Brachypodium distachyon (L.) P. Beauv. (Poaceae: Poideae) has been proposed as a new model for temperate and biofuel grasses. Brachypodium distachyon inbred line Bd21 has a fully sequenced and assembled genome, a series of genomics resources, and well-established somatic embryogenesis-based transformation protocols. However, information about origin and cellular changes occurring during the early differentiation of somatic embryos has not been documented for B. distachyon. There are also no reports on the use of molecular approaches to investigate somatic embryogenesis in B. distachyon. Therefore, the objectives of this study were (1) to characterize the morphological, anatomical and histochemical changes occurring during the induction of somatic embryogenesis from immature zygotic embryos (IZE) of B. distachyon community standard line Bd21 using light and scanning electron microscopy in association with histochemical tests and, (2), to carry out the cloning and characterization of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes and analysis of its expression in somatic embryogenesis induction using in situ hybridization for monitoring the morphogenetic processes in vitro. Somatic embryogenic cultures of B. distachyon (Bd21) were established following culture of IZE, 15 days post anthesis, on Murashige and Skoog (1962) medium containing 2,4- dichlorophenoxyacetic acid. In vitro regeneration of plantlets derived from somatic embryos occurred by the indirect somatic embryogenesis pathway. Somatic embryos had a multicellular origin and originated from embryogenic callus formed from cells of the epidermis in the region of the scutellar node and extended to the periphery of IZE. The order in accumulation of storage reserves in somatic embryos was similar to that of zygotic embryos. In the embryogenic cultures, storage proteins and lipids were used up in the first 2 days after culture (DAC). The levels of starch increased in the first 2 DAC and then decreased in number of granules that became larger and appeared mainly in the vacuolated cells subtending the proembryonic masses. Small starch granules started accumulating in proembryoids after 4 DAC and became larger and abundant in scutellar cells 12 DAC. Somatic embryo differentiation in B. distachyon proceeded through globular, scutellar and coleoptilar stages, following the morphological pattern of development of that reported in other members of the Poaceae. The SERK gene has been used successfully as a marker for competent cells in somatic embryogenesis of several species. In this study, using degenerate primers, it was possible to amplify a specific homologous sequence of a SERK gene fragment from B. distachyon Bd21. Sequence analysis of the SERK fragment (766 bp) revealed high levels of similarity to other reported SERKs. In situ hybridization analysis showed that the SERK gene was present in embryogenic tissues of B. distachyon before somatic embryo development and continued expressing through globular and scutellar stages. These results suggest that the expression of the B. distachyon SERK gene was associated with induction of somatic embryogenesis. This study provides the first description of morphoanatomical and histochemical changes underlying the embryogenic process in B. distachyon reference line Bd21. It is also the first report on the cloning and expression of a SERK gene for the species, suggesting that it could serve as a molecular marker to monitor the transition of IZE cells into competent and embryogenic cells also in B. distachyon line Bd21.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Somatic embryogenesis from mature zygotic embryos of commercial passionfruit (Passiflora edulis Sims) genotypes

Mature zygotic embryos of three genotypes of Passiflora edulis Sims, including ‘FB-100’, ‘FB-200’, and ‘FB-300’ were incubated on a Murashige and Skoog (MS) (1962) medium supplemented with different concentrations (18.1–114.8 μM) of 2,4-diclorophenoxyacetic acid (2,4-D) and 4.4 μM of 6-benzyladenine (BA). MS basal medium and MS with BA induced germination of P. edulis embryos. The highest frequencies of embryogenic calli were observed when explants were incubated on MS medium supplemented with 72.4 μM 2,4-D and 4.4 μM BA for ‘FB-200’, which showed the highest potential for embryogenic callus formation. Cytological and histological analyses of pro-embryogenic callus revealed two distinct cell types: thin-walled, small, isodiametric cells with large nuclei and dense cytoplasm, typical of intense metabolic activity; and elongated and vacuolated cells, with small nuclei and less dense cytoplasm. Differentiation of somatic embryos was promoted on MS medium supplemented with activated charcoal and indole-3-acetyl-l-aspartic acid (IAA-Asp) either with or without 2,4-D. However, no conversion of somatic embryos into plantlets was observed

Somatic embryogenesis in anthurium (Anthurium andraeanum cv. Eidibel) as affected by different explants - doi: 10.4025/actasciagron.v36i1.16557

This study establishes a protocol for the induction of somatic embryogenesis in Anthurium andraeanum cv. Eidibel. The experiment was arranged in a completely randomized 5 x 5 x 5 factorial design using five explant types (whole leaves, half leaves; petiole; nodal segments and root segments) from in vitro plantlets; five auxins: indole-3-acetic acid (IAA), α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 4-amino-3,5,6-trichloropicolinic acid (Picloram); at five concentrations (0, 2.5, 5, 7.5 and 10 µM), with five replications using five Petri dishes. The cultures were maintained in a growth room at 25 ± 2ºC in the dark. The explant type was investigated for the induction of somatic embryogenesis in anthurium cv. Eidibel, and nodal segments were shown to be the most suitable explant for this process. After 60 days in culture, the highest number of embryogenic calli was recorded for the nodal segments cultured in NAA (5, 7.5 and 10 µM), 2,4-D (10 µM) and Picloram (7.5 and 10 µM). The histological analysis confirmed the presence of embryos with established polarization, procambium, ground meristem and protoderm in the nodal segments cultured in Pierik medium containing 10 µM NAA. After the conversion of the somatic embryos into plantlets, these plantlets were acclimatized transferred to in vivo conditions and grown into normal plants.  

Somatic embryogenesis in anthurium (Anthurium andraeanum cv. Eidibel) as affected by different explants

This study establishes a protocol for the induction of somatic embryogenesis in Anthurium andraeanum cv. Eidibel. The experiment was arranged in a completely randomized 5 x 5 x 5 factorial design using five explant types (whole leaves, half leaves; petiole; nodal segments and root segments) from in vitro plantlets; five auxins: indole-3-acetic acid (IAA), α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 4-amino-3,5,6-trichloropicolinic acid (Picloram); at five concentrations (0, 2.5, 5, 7.5 and 10 μM), with five replications using five Petri dishes. The cultures were maintained in a growth room at 25 ± 2oC in the dark. The explant type was investigated for the induction of somatic embryogenesis in anthurium cv. Eidibel, and nodal segments were shown to be the most suitable explant for this process. After 60 days in culture, the highest number of embryogenic calli was recorded for the nodal segments cultured in NAA (5, 7.5 and 10 μM), 2,4-D (10 μM) and Picloram (7.5 and 10 μM). The histological analysis confirmed the presence of embryos with established polarization, procambium, ground meristem and protoderm in the nodal segments cultured in Pierik medium containing 10 μM NAA. After the conversion of the somatic embryos into plantlets, these plantlets were acclimatized transferred to in vivo conditions and grown into normal plants.O objetivo foi estabelecer um protocolo para indução de embriogênese somática em Anthurium andraeanum cv. Eidibel. O experimento foi arranjado em delineamento inteiramente casualizado, com esquema fatorial 5 x 5 x 5, cinco explantes (folha inteira e seccionada ao meio; pecíolo; segmento nodal e de raiz); cinco auxinas (AIA, ANA, AIB, 2,4-D e Picloram) em cinco concentrações (0,0; 2,5; 5,0; 7,5 e 10 μM) e cinco repetições, consistindo de cinco placas de Petri, mantidas em sala de crescimento a 25 ± 2 oC, no escuro. Após 60 dias de cultivo, a maior produção de calos embriogênicos foi registrada nos segmentos nodais cultivados na presença de ANA (5; 7,5 e 10 μM), 2,4-D (10 μM) ou Picloram (7,5 e 10 μM). As análises histológicas confirmaram a presença de embriões com polaridade definida, procâmbio, meristema fundamental e protoderme em segmentos nodais cultivados em meio Pierik com 10 μM ANA. Depois da conversão dos embriões somáticos, as plantas foram aclimatizadas e transferidas para as condições in vivo, com crescimento normal das mesmas

Somatic embryogenesis inanthurium (Anthurium andraeanum cv. Eidibel) as affected by different explants

This study establishes a protocol for the induction of somatic embryogenesis in Anthurium andraeanum cv. Eidibel. The experiment was arranged in a completely randomized 5 x 5 x 5 factorial design using five explant types (whole leaves, half leaves; petiole; nodal segments and root segments) from in vitro plantlets; five auxins: indole-3-acetic acid (IAA), α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 4-amino-3,5,6-trichloropicolinic acid (Picloram); at five concentrations (0, 2.5, 5, 7.5 and 10 µM), with five replications using five Petri dishes. The cultures were maintained in a growth room at 25 ± 2ºC in the dark. The explant type was investigated for the induction of somatic embryogenesis in anthurium cv. Eidibel, and nodal segments were shown to be the most suitable explant for this process. After 60 days in culture, the highest number of embryogenic calli was recorded for the nodal segments cultured in NAA (5, 7.5 and 10 µM), 2,4-D (10 µM) and Piclora

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

NEOTROPICAL XENARTHRANS: a data set of occurrence of xenarthran species in the Neotropics

Xenarthrans—anteaters, sloths, and armadillos—have essential functions for ecosystem maintenance, such as insect control and nutrient cycling, playing key roles as ecosystem engineers. Because of habitat loss and fragmentation, hunting pressure, and conflicts with domestic dogs, these species have been threatened locally, regionally, or even across their full distribution ranges. The Neotropics harbor 21 species of armadillos, 10 anteaters, and 6 sloths. Our data set includes the families Chlamyphoridae (13), Dasypodidae (7), Myrmecophagidae (3), Bradypodidae (4), and Megalonychidae (2). We have no occurrence data on Dasypus pilosus (Dasypodidae). Regarding Cyclopedidae, until recently, only one species was recognized, but new genetic studies have revealed that the group is represented by seven species. In this data paper, we compiled a total of 42,528 records of 31 species, represented by occurrence and quantitative data, totaling 24,847 unique georeferenced records. The geographic range is from the southern United States, Mexico, and Caribbean countries at the northern portion of the Neotropics, to the austral distribution in Argentina, Paraguay, Chile, and Uruguay. Regarding anteaters, Myrmecophaga tridactyla has the most records (n = 5,941), and Cyclopes sp. have the fewest (n = 240). The armadillo species with the most data is Dasypus novemcinctus (n = 11,588), and the fewest data are recorded for Calyptophractus retusus (n = 33). With regard to sloth species, Bradypus variegatus has the most records (n = 962), and Bradypus pygmaeus has the fewest (n = 12). Our main objective with Neotropical Xenarthrans is to make occurrence and quantitative data available to facilitate more ecological research, particularly if we integrate the xenarthran data with other data sets of Neotropical Series that will become available very soon (i.e., Neotropical Carnivores, Neotropical Invasive Mammals, and Neotropical Hunters and Dogs). Therefore, studies on trophic cascades, hunting pressure, habitat loss, fragmentation effects, species invasion, and climate change effects will be possible with the Neotropical Xenarthrans data set. Please cite this data paper when using its data in publications. We also request that researchers and teachers inform us of how they are using these data