miRVine: a microRNA expression atlas of grapevine based on small RNA sequencing

miRNAs are the most abundant class of small non-coding RNAs, and they are involved in post-transcriptional regulations, playing a crucial role in the refinement of genetic programming during plant development. Here we present a comprehensive picture of miRNA regulation in Vitis vinifera L. plant during its complete life cycle. Furthering our knowledge about the post-transcriptional regulation of plant development is fundamental to understand the biology of such an important crop

Produção de sementes sintéticas de maracujazeiro silvestre com potencial ornamental

Passiflora cincinnata Mast. é uma espécie silvestre de maracujazeiro de ampla distribuição. Possui hábito trepador e crescimento vigoroso, com flores muito vistosas e perfumadas. O trabalho teve como objetivo a produção de sementes sintéticas da espécie P. cincinnata, utilizando-se de embriões somáticos e zigóticos em diferentes formas de cultivo. Os embriões somáticos em estágio de desenvolvimento pré cotiledonar e cotiledonar foram obtidos a partir de embriões zigóticos cultivados em meio de MS na presença de 18,1 μM de 2,4-Ácido-dichlorophenoxiacetico (2,4-D) e 4,5 μM Benziladenine (BA). Embriões zigóticos e embriões somáticos foram encapsulados em matriz de alginato de sódio a 2,5% (p v -1 ) e complexados em solução estéril de CaCl 2 .2H 2 O a 0,1M. Embriões somáticos e zigóticos foram encapsulados na Matriz (I) com alginato de sódio, Matriz (II) com alginato de sódio + endosperma artificial e Matriz (III) - alginato de sódio + endosperma artificial e suplementado com 0,15% (p v -1 ) de carvão ativado. Embriões zigóticos cultivados em frascos encapsulados na Matriz (I) 79% germinaram, 76% Matriz (II) e na Matriz (III) 86%. Os embriões somáticos cotiledonares encapsulados nos três tipos de matrizes responderam com maior percentual de germinação quando cultivados em plugs de celulose com mais de 50% de embriões convertidos. Os embriões somáticos pre cotiledonares encapsulados nos três tipos de matrizes e nas diferentes formas de cultivo não responderam. No cultivo ex vitro nos dois tipos de substratos PlantMax e Florialite o número de embriões convertidos foi baixo, sendo o melhor resultado com 12,67 % no Florialite e encapsulados na Matriz (I). Palavras-Chave: Passiflora cincinnata, alginato de sódio, sementes artificiais, cultivo in vitroPassiflora cincinnata is a wild species of passion fruit with a wide geographical distribution. It has vigorous growth, climbing habit and very showy and fragrant flowers. The aim of the present investigation was to obtain synthetic seeds from encapsulated zygotic and somatic embryos of P. cincinnata, cultivated under different conditions. Precotyledonary and cotyledonary stage embryos were obtained from zygotic embryos cultivated on MS medium supplemented with 18.1 μM of 2,4-Acid-dichlorophenoxyacetic (2,4-D) and 4.5 μM of Benzyladenine (BA). Zygotic embryos and somatic embryos stages were encapsulated using sodium alginate (2.5% w v-1) and CaCl2.2H2O (1 mM) as complexing agent. The zygotic and somatic embryos were encapsulated in a matrix containing (I) sodium alginate, (II) sodium alginate + artificial endosperm and (III) sodium alginate + artificial endosperm supplemented with activated charcoal (0.15% w/v). Zygotic embryos encapsulated in the matrix (I), matrix (II) and matrix (III) and cultivated in flasks, germinated at rates of 79%, 76% and 86% respectively. The cotyledonary somatic embryos encapsulated in the 3 different matrices showed better germination rates when cultivated on cellulose plugs, with more than 50% of embryos converted into plants. Precotyledonary somatic embryos did not germinated regardless the matrix and cultivation. When cultivating the alginate beads ex vitro, both substrate Plantmax and Florialite showed low number of germinated embryos, and the best result (12.67%) were obtained using Florialite and embryos encapsulated in the matrix (I)

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.25

A novel regeneration system for a wild passion fruit species (Passiflora cincinnata Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 mu M benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 mu M 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog`s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.FAPEMIG[CAG 1527/2005]Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)CAPESConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNP

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

Repetitive somatic embryogenesis from wild passion fruit (Passiflora cincinnata Mast.) anthers

Induction of somatic embryogenesis from in vitro-cultivated anthers represents a recent but poorly understood regeneration pathway for passion fruit species. Here, we aimed to develop an efficient system to produce and proliferate somatic embryos from cultivated anthers of Passiflora cincinnata. The floral buds were categorized into five different developmental stages (DS1 to DS5) according to their length and diameter. Their anthers were then cultured in induction medium at various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 μM 6-benzyladenine. The control contained no plant growth regulators. Somatic embryogenesis started from the diploid sporophytic tissues of the anthers and continued indirectly through the formation of yellow and friable embryogenic calluses at 9.1 to 27.1 μM 2,4-D. Embryogenic calluses and primary and secondary embryos were significantly more numerous only when anthers at the DS2 stage were cultivated with 18.1 μM 2,4-D and 4.5 μM 6-benzyladenine. Secondary diploid somatic embryos formed on the surface of primary embryos via direct and repetitive embryogenesis, as well as directly from the hypocotyl of regenerated P. cincinnata emblings. The capacity to induce repetitive somatic embryogenesis represents a promising tool for Passiflora micropropagation

High responsiveness in de novo shoot organogenesis induction of Passiflora cristalina (Passifloraceae), a wild Amazonian passion fruit species

The aim of the present study was to establish a regeneration system via de novo organogenesis from different types of non-meristematic explants of Passiflora cristalina. Leaf, hypocotyl, root segments, cotyledons, and endosperm of P. cristalina seeds were inoculated in Murashige and Skoog (MS)-basal medium, supplemented with different concentrations of 6-Benzyladenine (BA), Thidiazuron (TDZ), or Kinetin (KIN). BA was found to be the most efficient cytokinin in induction of de novo organogenesis from most the explants used in the study. The highest frequencies of adventitious bud formation in the hypocotyl and cotyledon explants were observed in medium supplemented with 1.0 mg L^−1 BA. For leaf and endosperm segments, the best concentration was 2.0 mg L^−1 BA; while for root segments, the highest mean values were observed with 1.0 mg L^−1 KIN. The different morphogenetic responses obtained from each explant source were characterized using light microscopy. P. cristalina revealed a remarkable organogenic potential, with superior production of adventitious shoots compared with the other Passiflora species evaluated elsewhere. These results will be helpful to establish a reproducible and reliable micropropagation protocol, as well as to implement conservationist and biotechnological-based genetic breeding strategies for this wild Passiflora species

Novel and efficient transformation of wild passion fruit (Passiflora cincinnata Mast.) using sonication-assisted Agrobacterium-mediated transformation

Passiflora cincinnata stands out among Passifloraceae because of its medicinal properties and its resistance to pathogens, which promotes its use as a rootstock for other Passiflora species. A valuable strategy for obtaining disease-resistant passion fruit plants is represented by genetic transformation; however, this requires efficient regeneration. Here, we aimed to establish an efficient protocol for generating transgenic passion fruit plants using sonication-assisted Agrobacterium-mediated transformation of somatic embryos. The latter were obtained from anthers, sonicated, and exposed to an Agrobacterium suspension for 15 or 30 s. As a comparison, non-sonicated embryos were also exposed to the same bacterial treatment, whereas non-infected embryos were used as controls. Plant identity was confirmed by PCR, qPCR, and histochemical assay. Transgenic plants were obtained at higher rates in the treatments applying sonication. Overall, 171 plantlets were regenerated, 38 of which showed stable uidA reporter gene expression. Of these 38 transgenic plants, 22 (57.89%) and 13 (34.21%) were obtained by sonication-assisted Agrobacterium-mediated transformation for 30 s and 15 s, respectively, whereas the remaining 3 (7.89%) were exposed for 30 s but without prior sonication. Our results indicate that sonication-assisted Agrobacterium-mediated transformation for 30 s enhanced transformation efficiency in P. cincinnata. We believe that this system will allow for more efficient production of transgenic passion fruit plants