Denise Maria Trombert Oliveira

Large Plant Samples: How to Process for GMA Embedding?

It is often necessary to process large plant samples for light microscopy studies, but due to structural characteristics of plant tissues, especially intercellular spaces, large vacuoles, and phenolic substances, results are often unsatisfactory. When large samples are embedded in glycol methacrylate (GMA), their core may not polymerize, remaining soft and moist and making it difficult to cut microtome sections. This situation has been erroneously interpreted as the result of poor infiltration, when the soft core of these samples is actually the result of incomplete polymerization. While GMA is in fact present inside samples, unsatisfactory polymerization results from rapid external polymerization that does not allow sufficient hardener to reach the sample core, while the relatively large volume of GMA inside the tissue block also dilutes the hardener. In this chapter we propose a new method for processing large plant specimens that avoids these problems by: (1) slowing the polymerization process through cooling in order to permit the penetration of hardener into the sample core and (2) increasing the hardener:GMA ratio to aid polymerization of the sample core.

Cypsela or achene? Refining terminology by considering anatomical and historical factors

The worry about the indiscriminate use of the terms cypsela and achene for the fruits of Asteraceae has been frequently detached by specialists in this family. The present work was developed aiming to verify the existence of arguments to justify the adoption of a term against the other. After historical and anatomical analysis, we concluded that there is technical basis to consider cypsela and achene as different types of fruits. For Asteraceae, the correct is to call cypsela; achenes are only derived from superior ovaries, as in Plumbaginaceae.

Evolution of physiological dormancy multiple times in Melastomataceae from Neotropical montane vegetation

We investigated seed dormancy among species of Melastomataceae from Neotropical montane vegetation of Brazil. Four out of 50 studied species had dormant seeds: Miconia corallina (Miconieae), Tibouchina cardinalis (Melastomeae), Comolia sertularia (Melastomeae) and Chaetostoma armatum (Microlicieae). For these four species, germinability of seeds collected in different years was always ,10% and the percentages of embryoless seeds and non-viable embryos were both insufficient to explain low or null germinability. This is the first unequivocal report of seed dormancy in tropical Melastomataceae. The production of seeds with permeable seed coats and fully developed, differentiated embryos indicates the occurrence of physiological dormancy. The reconstructed phylogenetic tree of the 50 species suggests that physiological dormancy evolved multiple times during the evolutionary history of Melastomataceae in this vegetation. Physiological dormancy evolved in species and populations associated with xeric microhabitats, where seeds are dispersed in unfavourable conditions for establishment. Therefore, droughtinduced mortality may have been a strong selective pressure favouring the evolution of physiological dormancy in Melastomataceae. We argue that dormancy may have been independently selected in other lineages of Cerrado plants colonizing xeric microhabitats and dispersing seeds at the end of the rainy season. The contributions of our data to the understanding of seed dormancy in tropical montane vegetation are discussed.

Anatomy and ontogeny of the pericarp of Pterodon emarginatus Vogel (Fabaceae, Faboideae), with emphasis on secretory ducts

Discrepant and incomplete interpretations of fruits of Pterodon have been published, especially on the structural interpretation of the pericarp portion that remain attached to the seed upon dispersal. The present work clarified these doubts and analyzed ultrastructural aspects of the Pterodon emarginatus diaspores using light and transmission electron microscopes. Cell divisions are prevalent among the initial phases of development, and the subadaxial and adaxial meristems form the fibrous inner mesocarp and the endocarp composed of multi-seriate epidermis, respectively. At the median mesocarp, numerous secretory ducts differentiate between the lateral bundles, by lytic process. After lysis of the central cells and the formation of the lumen, the ducts show unistratified secretory epithelium with dense cells; oil droplets are observed on the secretory epithelium and the subadjacent tissues. At maturity, the uniseriate exocarp and the outer mesocarp slough off in an irregular fashion, leaving the diaspore composed of a papery and brittle wing linked to a seed chamber that includes the median mesocarp composed of lignified cells, bordering vascular bundles and many secretory ducts whose epithelial cells develop large vacuoles that accumulate oleoresins. The Pterodon emarginatus fruit is a cryptosamara.

Structure and ontogeny of the pericarp of six Eupatorieae (Asteraceae) with ecological and taxonomic considerations

The ontogeny of cypselae and their accessory parts were examined using light and scanning electron microscopy for the species Campuloclinium macrocephalum, Chromolaena stachyophylla, Mikania micrantha, Praxelis pauciflora, Symphyopappus reticulatus, and Vittetia orbiculata, some of these being segregated from the genus Eupatorium. A layer of phytomelanin observed in the fruit appears to be secreted by the outer mesocarp into the schizogenous spaces between the outer and inner mesocarp; its thickness was observed to vary among the different species examined. The bristles of the pappus are vascularized, except in M micrantha, and have cells that are superficially projected and arranged acropetally; in S. reticulatus some of the projections are retrorse and a fracture line on the floral disk that is only seen in this species may indicate a double dispersal process. Numerous differences observed among the cypselae examined here reinforce earlier segregations of the genus Eupatorium sensu lato.

Morfoanatomia e ontogênese do fruto e semente de Vernonia platensis (Spreng.) Less. (Asteraceae)

The Asteraceae comprises approximately 23,000 species and Vernonieae is best represented in Brazil; Vernonia is the largest genus in this tribe. Due to occurrence in extensive areas of Cerrado, Vernonia platensis was selected for this work that aims to describe the morphology, anatomy and development of the pericarp and seed of this species, comparing the results with the literature. The collected material was processed by conventional techniques. The ovary is inferior, bicarpellate, syncarpous, unilocular with a single anatropous ovule, unitegmic, tenuinucelate, attached in a basal placenta. The ovary wall is homogenous with dense cells peripherally. The integument has three zones, with a conspicuous endothelium. At maturity, most layers collapse, only the outer mesocarp fibers remaining; the double pappus persists and has lignified cells. The mature seed has a testa composed of collapsed cells; the endosperm is cellular, residual at maturity, and the embryo has a thick, short, and axial embryo axis. Completely developed seeds were found in only 40% of the mature cypselae evaluated. Our observations corroborate previous research with Asteraceae; we call attention to the nutritive role of the seed coat in seed development, and the low production of seeds in V. platensis.

The ribs of Eupatorieae (Asteraceae): of wide taxonomic value or reliable characters only among certain groups?

Rib numbers have traditionally been used in identification keys of Eupatorieae. Some species, however, demonstrate variations in this character much greater than those registered in the literature, making the use of this trait controversial. The large variations seen among some species of Eupatorieae examined in this work were found to be related to the fact that rib number is more closely associated with the pressure exerted by neighboring flowers on the capitulum than with genetic factors. According to our results, ribs are not a reliable taxonomic character for broad use in Eupatorieae, being especially inappropriate for Chromolaena stachyophylla, Praxelis pauciflora, and Vittetia orbiculata. The association between the occurrence of vascular bundles and ribs is also discussed, and the variability seen in some Eupatorieae is analyzed. Our data indicate that the formation of the ribs on the cypselae is determined more by the position occupied by the flower on the inflorescence than by the occurrence of vascular bundles.

The role of fibres and the hypodermis in Compositae melanin secretion.

Melanins are dark, insoluble pigments that are resistant to concentrated acids and bleaching by oxidising agents. Phytomelanin (or phytomelan) is present in the seed coat of some Asparagales and in the fruits of some Compositae. In Compositae fruits, melanin is deposited in the schizogenous spaces between the hypodermis and underlying fibrous layer. Phytomelanin in Compositae is poorly understood, and there are only speculations regarding the cells that produce the pigment and the cellular processes involved in the secretion and polymerisation of phytomelanin. This report describes the cellular processes involved in the secretion of phytomelanin in the pericarp of Praxelis diffusa, a species with a structure typical of the family. The ovaries and fruits at different stages were fixed and processed according to the standard methods of studies of light microscopy and transmission electron microscopy. Hypodermal cells have abundant rough endoplasmic reticulum and mitochondria, and the nuclei have chromatin that is less dense than other cells. These characteristics are typical of cells that synthesise protein/amino acids and suggest no carbohydrate secretion. The fibres, however, have a dense cytoplasm rich in the Golgi bodies that are associated with vesicles and smooth endoplasmic reticulum, common characteristics of carbohydrate secretory cells. Our results indicate that the hypodermal cells are not responsible for the secretion of phytomelanin, as previously described in the literature; in contrast, this function is assigned to the adjacent fibres, which have an organisation typical of cells that secrete carbohydrates.

A new seed coat water-impermeability mechanism in Chaetostoma armatum (Melastomataceae): evolutionary and biogeographical implications of physiophysical dormancy

Determining the phylogenetic and biogeographic distribution of physical dormancy remains a major challenge in germination ecology. Here, our goal was to describe a novel water-impermeable seed coat mechanism causing physical dormancy (PY) in the seeds of Chaetostoma armatum (Melastomataceae). Although seed coat permeability tests indicated a significant increase in seed weight after soaking in distilled water, anatomical and dye-tracking analyses showed that both water and dyes penetrated the seed coat but not the embryo, which remained in a dry state. The water and dye penetrated the lumen of the exotestal cells, which have a thin outer periclinal face and thickened secondary walls with U-shaped phenolic compounds. Because of this structure, water and dye do not penetrate the inner periclinal face of the exotestal cells, indicating PY. Puncturing the seeds increased germination more than tenfold compared to that of the control, but GA 3 did not increase germination further. A significant fraction of the seeds did not germinate after puncturing, indicating that embryos are also physiologically dormant (PD). This paper constitutes the first report of the water-impermeable seed coat in the Myrtales and the first report of physiophysical (PD+PY) dormancy in a shrub from a tropical montane area.

Pericarp structure in Banisteriopsis C.B.Rob. and Diplopterys A.Juss. (Malpighiaceae): new data supporting generic segregation

Pericarp structure in Banisteriopsis C.B.Rob. and Diplopterys A.Juss. (Malpighiaceae): new data supporting generic segregation). Molecular studies have indicated that some species of Banisteriopsis would be more appropriately placed in Diplopterys. Both of these genera are neotropical members of the Malpighiaceae, a family that is widely diverse in South America, especially in the Brazilian savanna (cerrado). Studies concerning the anatomy of the reproductive organs of Malpighiaceae from the cerrado are very rare. Thus, the present work describes the morphology, anatomy, and ontogeny of the pericarp of B. campestris, B. oxyclada, B. stellaris, and D. pubipetala, comparing them and identify- ing characteristics that could support the segregation of genera. The fruits of Banisteriopsis studied here demonstrate structures very similar among themselves, while D. pubipetala shows numerous differences. The fruits of all species are schizocarpic and formed by three samara with dorsal wings, while the fruits of D. pubipetala also have lateral winglets on the seed chamber. The mesocarp of the Banisteriopsis species has groups of thick and elongated cells, while these cells form a continuous band in D. pubipetala; the endocarp of D. pubipetala proliferates markedly, forming an aerenchyma layer not seen in Banisteriopsis. Besides the homogeneous structure of the pericarp of the Banisteriopsis species, all referred distinctive characteristics support the maintenance of D. pubipetala in a different genus.