Hormonal control of inflorescence development in plantlets of calla lily (Zantedeschia spp.) grown in vitro

Hormonal control of flower induction and inflorescence development in vitro was investigated in photoperiodically day-neutral calla lily (Zantedeschia spp., colored cultivars). The effects of gibberellins (GAs, 5.8–2900 M) and the cytokinin benzyl adenine (BA, 0.4–13.3 M) on inflorescence development were studied in plantlets regenerated in tissue culture. Plantlets were dipped in GA and BA solutions prior to replanting in new media. GA was mandatory for the shift from the vegetative to the reproductive stage. GA3, GA1 and GA4 had the same florigenic effect. Inflorescence development in the apical bud was observed after 30–50 days in GA-treated plantlets grown in vitro and resembled the pattern occurring under natural conditions. The transition from the vegetative to the reproductive phase was characterized by a swollen, dome-shaped apex that transformed into a smooth elongated apex surrounded by the spathe primordium, at the tip of the elongating peduncle primordium. Floret primordia developed in inflorescences at a more advanced stage. The female florets located at the base of the primordial spadix, could be clearly distinguished from male florets located above them. BA did not have an effect on flower induction but, in the presence of GA, BA at concentrations up to 4.4 M enhanced inflorescence differentiation. The results indicate that inflorescence development in Zantedeschia plantlets in tissue culture can serve as a potential model to study the role of GAs and other factors in the flowering process of day-neutral plants that do not require external signals for flower induction.     

The hypothesis of sympatric speciation as the dominant generator of endemism in a global hotspot of biodiversity

Allopatric or sympatric speciation influence the degree to which closely related species coexist in different manners, altering the patterns of phylogenetic structure and turnover among and between communities. The objective of this study was to examine whether phylogenetic community structure and turnover in the Brazilian Atlantic Forest permit conclusions about the dominant process for the formation of extant angiosperm richness of tree species. Therefore, we analyzed phylogenetic community structure (MPD, MNTD) as well as taxonomic (Jaccard similarity) and phylogenetic turnover (betaMPD, betaMNTD) among and between 49 tree communities distributed among three different habitat types. Mean annual precipitation and mean annual temperature in each survey area were estimated. Phylogenetic community structure does not differ between habitat types, although MPD reduces with mean annual temperature. Jaccard similarity decreases and betaMNTD increases with spatial distance and environmental differences between study sites. Spatial distance explains the largest portions of variance in the data, indicating dispersal limitation and the spatial aggregation of recently formed taxa, as betaMNTD is related to more recent evolutionary events. betaMPD, that is related to deep evolutionary splits, shows no spatial or environmental pattern, indicating that older clades are equally distributed across the Brazilian Atlantic Forest. While similarity pattern indicates dispersal limitations, the spatial turnover of betaMNTD is consistent with a high degree of sympatric speciation generating extant diversity and endemism in the Brazilian Atlantic Forest. More comprehensive approaches are necessary to reduce spatial sampling bias, uncertainties regarding angiosperm diversification patterns and confirm sympatric speciation as the dominant generator for the formation of extant species diversity in the Brazilian Atlantic Forest.     

Amyrin esters induce cell death by apoptosis in HL-60 leukemia cells

Four derivatives of an α,β-amyrin mixture were synthesized by acylation with appropriate anhydrides. The structures of the compounds were confirmed by means of IR and (1)H and (13)C NMR. The compounds were screened for cytotoxic activity using four human tumor cell lines (HL-60, MDAMB-435, SF-295 and HCT-8) and normal peripheral blood mononuclear cells (PBMC). 3-O-Carboxymaleinate of α,β-amyrin (3a/3b) were found to be the only active compounds of the series (high cytotoxicity), showing IC(50) values ranging from 1.8 to 3μM. In PBMC, 3a/3b were not toxic, suggesting selectivity for tumor cells. To better understand the mechanism of action involved in the cytotoxicity of 3a/3b, HL-60 cells treated with 3a/3b were examined for morphological changes, DNA fragmentation, cell cycle perturbation, externalization of phosphatidylserine and activation of caspases 3/7, with doxorubicin serving as the positive control. The results indicate that the cytotoxicity of 3a/3b involves the induction of cell death by apoptosis.     

Simple bioreactors for mass propagation of plants

Bioreactors provide a rapid and efficient plant propagation system for many agricultural and forestry species, utilizing liquid media to avoid intensive manual handling. Large-scale liquid cultures have been used for micropropagation through organogenesis or somatic embryogenesis pathways. Various types of bioreactors with gas-sparged mixing are suitable for the production of clusters of buds, meristems or protocorms. A simple glass bubble-column bioreactor for the proliferation of ornamental and vegetable crop species resulted in biomass increase of 3 to 6-fold in 3–4 weeks. An internal loop bioreactor was used for asparagus, celery and cucumber embryogenic cultures. However, as the biomass increased, the mixing and circulation were not optimal and growth was reduced. A disposable pre-sterilized plastic bioreactor (2–5-l volume) was used for the proliferation of meristematic clusters of several ornamental, vegetable and woody plant species. The plastic bioreactor induced minimal shearing and foaming, resulting in an increase in biomass as compared to the glass bubble-column bioreactor. A major issue related to the use of liquid media in bioreactors is hyperhydricity, that is, morphogenic malformation. Liquid cultures impose stress signals that are expressed in developmental aberrations. Submerged tissues exhibit oxidative stress, with elevated concentrations of reactive oxygen species associated with a change in antioxidant enzyme activity. These changes affect the anatomy and physiology of the plants and their survival. Malformation was controlled by adding growth retardants to decrease rapid proliferation. Growth retardants ancymidol or paclobutrazol reduced water uptake during cell proliferation, decreased vacuolation and intercellular spaces, shortened the stems and inhibited leaf expansion, inducing the formation of clusters. Using a two-stage bioreactor process, the medium was changed in the second stage to a medium lacking growth retardants to induce development of the meristematic clusters into buds or somatic embryos. Cluster biomass increased 10–15-fold during a period of 25–30 days depending on the species. Potato bud clusters cultured in 1.5 1 of medium in a 2-l capacity bioreactor, increased during 10–30 days. Poplar in vitro roots regenerated buds in the presence of thidiazuron (TDZ); the biomass increased 12-fold in 30 days. Bioreactor-regenerated clusters were separated with a manual cutter, producing small propagule units that formed shoots and initiated roots. Clusters of buds or meristematic nodules with reduced shoots, as well as arrested leaf growth, had less distortion and were optimal for automated cutting and dispensing. In tuber-, bulb- and corm-producing plants, growth retardants and elevated sucrose concentrations in the media were found to enhance storage organ formation, providing a better propagule for transplanting or storage. Bioreactor-cultures have several advantages compared with agar-based cultures, with a better control of the contact of the plant tissue with the culture medium, and optimal nutrient and growth regulator supply, as well as aeration and medium circulation, the filtration of the medium and the scaling-up of the cultures. Micropropagation in bioreactors for optimal plant production will depend on a better understanding of plant responses to signals from the microenvironment and on specific culture manipulation to control the morphogenesis of plants in liquid cultures.     

Oxanine DNA glycosylase activity from Mammalian alkyladenine glycosylase

Oxanine (Oxa) is a deaminated base lesion derived from guanine in which the N(1)-nitrogen is substituted by oxygen. This work reports the mutagenicity of oxanine as well as oxanine DNA glycosylase (ODG) activities in mammalian systems. Using human DNA polymerase beta, deoxyoxanosine triphosphate is only incorporated opposite cytosine (Cyt). When an oxanine base is in a DNA template, Cyt is efficiently incorporated opposite the template oxanine; however, adenine and thymine are also incorporated opposite Oxa with an efficiency approximately 80% of a Cyt/Oxa (C/O) base pair. Guanine is incorporated opposite Oxa with the least efficiency, 16% compared with cytosine. ODG activity was detected in several mammalian cell extracts. Among the known human DNA glycosylases tested, human alkyladenine glycosylase (AAG) shows ODG activity, whereas hOGG1, hNEIL1, or hNEIL2 did not. ODG activity was detected in spleen cell extracts of wild type age-matched mice, but little activity was observed in that of Aag knock-out mice, confirming that the ODG activity is intrinsic to AAG. Human AAG can excise Oxa from all four Oxa-containing double-stranded base pairs, Cyt/Oxa, Thy/Oxa, Ade/Oxa, and Gua/Oxa, with no preference to base pairing. Surprisingly, AAG can remove Oxa from single-stranded Oxa-containing DNA as well. Indeed, AAG can also remove 1,N(6)-ethenoadenine from single-stranded DNA. This study extends the deaminated base glycosylase activities of AAG to oxanine; thus, AAG is a mammalian enzyme that can act on all three purine deamination bases, hypoxanthine, xanthine, and oxanine.     

Leaf anatomy as an additional taxonomy tool for 16 species of Malpighiaceae found in the Cerrado area (Brazil)

This work describes the leaf anatomy of 16 species belonging to three genera of the Malpighiaceae family found in the Cerrado (Minas Gerais State, Brazil). The scope of this study was to support the generic delimitation by contributing to the identification of the species and constructing a dichotomous identification key that includes anatomical characters. The taxonomic characters that were considered to be the most important and used in the identification key for the studied Malpighiaceae species were as follows: the presence and location of glands; presence of phloem in the medullary region of the midrib; mesophyll type; presence and type of trichomes; and presence, quantity, and disposition of accessory bundles in the petiole. It was also possible to indicate promising characters for future taxonomic and phylogenetic studies in the Malpighiaceae family, especially for the Banisteriopsis, Byrsonima, and Heteropterys genera.     

Anatomy of the extrafloral nectaries in species of Chamaecrista section Absus subsection Baseophyllum (Leguminosae,Caesalpinioideae)

In this paper the ontogenesis and histochemistry of the petiolar glands found on the petiole/rachis of the eight Chamaecrista species of the section Absus, subsection Baseophyllum (Leguminosae, Caesalpinioideae) are studied by using light microscopy techniques, aiming to characterise these structures and to provide taxonomic characters which may be useful in phylogenetic approaches. Strips for glucose identification reacted positively with the exudates of the glands, confirming the presence of nectar in the secretion, characterising these glands as extrafloral nectaries (EFN). Histochemical tests also detected the presence of neutral and acid muco-polysaccharides, pectins, mucilages, total proteins, and phenolic compounds in the EFNs. The EFNs arise from a group of meristem cells (protodermis, ground meristem and procambium) in the petiole/rachis. All EFNs of the investigated taxa share some morpho-anatomical characters, so that their peculiarities are too weak to be used alone in the identification of particular species. Rather their similarities may be used to include these species into a single group, supporting the hypothesis of monophyly of the subsection Baseophyllum.     

Toxoplasma gondii antigens: recovery analysis of tachyzoites cultivated in Vero cell maintained in serum free medium

Vero cells have been used successfully in Toxoplasma gondii maintenance. Medium supplementation for culture cells with fetal bovine serum is necessary for cellular growth. However, serum in these cultures presents disadvantages, such as the potential to induce hypersensitivity, variability of serum batches, possible presence of contaminants, and the high cost of good quality serum. Culture media formulated without any animal derived components, designed for serum-free growth of cell lines have been used successfully for different virus replication. The advantages of protozoan parasite growth in cell line cultures using serum-free medium remain poorly studied. Thus, this study was designed to determine whether T. gondii tachyzoites grown in Vero cell cultures in serum-free medium, after many passages, are able to maintain the same antigenic proprieties as those maintained in experimental mice. The standardization of Vero cell culture in serum-free medium for in vitro T. gondii tachyzoite production was performed establishing the optimal initial cell concentration for the confluent monolayer formation, which was 1×10(6) Vero cell culture as initial inoculum. The total confluent monolayer formatted after 96 h and the best amount of harvested tachyzoites was 2.1×10(7) using parasite inoculum of 1.5×10(6) after 7 days post-infection. The infectivity of tachyzoites released from Vero cells maintained in serum-free medium was evaluated using groups of Swiss mice infected with cell-culture tachyzoites. The parasite concentrations were similar to those for mice infected with tachyzoites collected from other infected mice. The data from both in vivo and in vitro experiments showed that in at least 30 culture cell passages, the parasites maintained the same infectivity as maintained in vivo. Another question was to know whether in the several continued passages, immunogenic progressive loss could occur. The nucleotide sequences studied were the same between the different passages, which could mean no change in their viability in the lysate antigen. Thus, the antigen production by cell culture has clear ethical and cost-saving advantages. Moreover, the use of culture media formulated without any human or animal derived components, designed for serum-free growth of cell lines, successfully produced tachyzoites especially for antigen production.