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 lM benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 lM 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.
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 lM) of 2,4-diclorophenoxyacetic acid (2,4-D) and 4.4 lM 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 lM 2,4-D and 4.4 lM 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.
BackgroundmiRNAs 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.ResultsWe analyzed 70 small RNA libraries, prepared from berries, inflorescences, tendrils, buds, carpels, stamens and other samples at different developmental stages. One-hundred and ten known and 175 novel miRNAs have been identified and a wide grapevine expression atlas has been described. The distribution of miRNA abundance reveals that 22 novel miRNAs are specific to stamen, and two of them are, interestingly, involved in ethylene biosynthesis, while only few miRNAs are highly specific to other organs. Thirty-eight miRNAs are present in all our samples, suggesting a role in key regulatory circuit. On the basis of miRNAs abundance and distribution across samples and on the estimated correlation, we suggest that miRNA expression define organ identity. We performed target prediction analysis and focused on miRNA expression analysis in berries and inflorescence during their development, providing an initial functional description of the identified miRNAs.ConclusionsOur findings represent a very extensive miRNA expression atlas in grapevine, allowing the definition of how the spatio-temporal distribution of miRNAs defines organ identity. We describe miRNAs abundance in specific tissues not previously described in grapevine and contribute to future targeted functional analyses. Finally, we present a deep characterization of miRNA involvement in berry and inflorescence development, suggesting a role for miRNA-driven hormonal regulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1610-5) contains supplementary material, which is available to authorized users.
Despite their diversity and ecological importance, many areas of the SAR-Stramenopila, Alveolata, and Rhizaria-clade are poorly understood as the majority (90%) of SAR species lack molecular data and only 5% of species are from well-sampled families. Here, we review and summarize the state of knowledge about the three major clades of SAR, describing the diversity within each clade and identifying synapomorphies when possible. We also assess the "dark area" of SAR: the morphologically described species that are missing molecular data. The majority of molecular data for SAR lineages are characterized from marine samples and vertebrate hosts, highlighting the need for additional research effort in areas such as freshwater and terrestrial habitats and "non-vertebrate" hosts. We also describe the paucity of data on the biogeography of SAR species, and point to opportunities to illuminate diversity in this major eukaryotic clade. See also the video abstract here: https://youtu.be/_VUXqaX19Rw.
In this study, flow cytometric analysis was used to evaluate the genetic stability of Passiflora cincinnata Mast. plants regenerated via primary and secondary somatic embryogenesis. Embryogenic calli obtained from culturing zygotic embryos on Murashige and Skoog (MS) medium containing 18.1 lM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 lM benzyladenine (BA) were transferred to differentiation medium. Torpedo and cotyledonary embryos were obtained. These primary embryos were maintained on differentiation medium to generate secondary embryos. Conversion of primary and secondary embryos yielded 305 and 138 normal plants, respectively. Almost 90% of plantlets survived following acclimatization. Flow cytometric analysis revealed that seed-derived plants had on average 3.01 pg nuclear DNA (2C), and all plants, except for a single plant regenerated via primary embryogenesis, maintained their ploidy. This single plant contained more than twice the average DNA content: 6.21 pg (4C). Epidermal stomata of leaves of the tetraploid plant were larger but lower in density than those of diploid plants, indicating that stomatal characteristics are useful in distinguishing between diploid and tetraploid plants of passion fruit. In summary, the procedure we employed to regenerated P. cincinnata plants via somatic embryogenesis generated mostly genetically true-to-type plants.
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