In Vitro Propagation of "Salsaparrilha" [Herreria salsaparrilha Mart. (Asparagaceae)]: An Antisyphilitic Medicinal Species.

An efficient procedure for in vitro propagation of Herreria salsaparrilha Martius was established from single-node explants (fourth and fifth nodes from apex to the base) derived from donor plants maintained under shading-house conditions. After surface sterilization, explants are inoculated in test tubes containing 15 mL of Murashige and Skoog (MS) medium without growth regulators. Cultures are maintained under 35 µmol m-2 s-1 irradiance, a 16/8-h light/dark light regime, at 26 ± 2 °C. The subcultures are carried out under the same conditions, adding 6-benzyladenine 1.0 mg/L and Phytagel® 2.8 g/L. Shoots are elongated and rooted by transferring individual shoots to half-strength MS medium without growth regulators. After 25-30 days, elongated rooted shoots are transferred to plastic pots containing 25-30 mL of sterile distilled water, covered with a transparent plastic bag, and kept under the same growth room conditions for 2 days. Plants are transferred to cups containing autoclaved and washed sand and kept in a shading house (50% light interception) for acclimatization. True-to-type adult plants were successfully recovered under ex vitro conditions.

Water stress modulates terpene biosynthesis and morphophysiology at different ploidal levels in Lippia alba (Mill.) N. E. Brown (Verbenaceae).

Monoterpenes are the main component in essential oils of Lippia alba. In this species, the chemical composition of essential oils varies with genome size: citral (geraniol and neral) is dominant in diploids and tetraploids, and linalool in triploids. Because environmental stress impacts various metabolic pathways, we hypothesized that stress responses in L. alba could alter the relationship between genome size and essential oil composition. Water stress affects the flowering, production, and reproduction of plants. Here, we evaluated the effect of water stress on morphophysiology, essential oil production, and the expression of genes related to monoterpene synthesis in diploid, triploid, and tetraploid accessions of L. alba cultivated in vitro for 40 days. First, using transcriptome data, we performed de novo gene assembly and identified orthologous genes using phylogenetic and clustering-based approaches. The expression of candidate genes related to terpene biosynthesis was estimated by real-time quantitative PCR. Next, we assessed the expression of these genes under water stress conditions, whereby 1% PEG-4000 was added to MS medium. Water stress modulated L. alba morphophysiology at all ploidal levels. Gene expression and essential oil production were affected in triploid accessions. Polyploid accessions showed greater growth and metabolic tolerance under stress compared to diploids. These results confirm the complex regulation of metabolic pathways such as the production of essential oils in polyploid genomes. In addition, they highlight aspects of genotype and environment interactions, which may be important for the conservation of tropical biodiversity.

Functional Fruit Trees from the Atlantic and Amazon Forests: Selection of Potential Chestnut Trees Rich in Antioxidants, Nutrients, and Fatty Acids.

The Amazon rainforest and the biodiversity hotspot of the Atlantic Forest are home to fruit trees that produce functional foods, which are still underutilized. The present study aimed to select potential functional nut donor trees from two Brazilian chestnuts, by evaluating the nutritional and antioxidant composition of the nuts and the fatty acid profile of the oil. The nutritional characteristics, antioxidants, oil fatty acid profile, and X-ray densitometry of the nuts were evaluated, as well as the characterization of leaf and soil nutrients for each parent tree. The nut oil was evaluated through Brix (%), mass (g), yield (%), and the fatty acid profile. For L. pisonis, the most nutritious nuts were produced by L. pisonis tree 4 (N > P > K > Mg > Ca > Zn > Fe) and L. pisonis tree 6 (P > Ca > Mg > Mn > Zn > Cu > Fe), and for the species L. lanceolata, L. lanceolata tree 6 (N > P > Ca > Mg > Zn > Fe > Cu) and L. lanceolata tree 2 (P > K > Mg > Zn > Cu). In L. pisonis, the highest production of anthocyanins, DPPH, total phenolics, and flavonoids was obtained from the nuts of L. pisonis tree 4 as well as for L. lanceolata, from L. lanceolata tree 1, except for flavonoids. The Brix of the oil from the nuts of both species showed no difference between the trees and the fatty acid profile with a similar amount between saturated (48-65%) and unsaturated (34-57%) fatty acids. Both species have nuts rich in nutrients and antioxidant compounds and can be considered unconventional functional foods. The data collected in the present study confirm that the nuts of these species can replace other foods as a source of selenium.

Pfaffia glomerata hydroalcoholic extract stimulates penile tissue in adult Swiss mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Roots of Pfaffia glomerata are used in traditional medicine as aphrodisiacs and sexual stimulants. AIM OF THE STUDY: The aim of this study was to evaluate the action of the hydroalcoholic extract from the roots of Pfaffia glomerata on the Leydig cells, cavernous bodies and other penile constituents, as well as on serum testosterone and 17ß-estradiol levels of adult mice. MATERIALS AND METHODS: Mature male Swiss mice were divided into 6 groups: control (water), sildenafil citrate, 3 groups receiving daily doses of P. glomerata extract (100, 200 and 400 mg/kg) and one group receiving intermittent doses of P. glomerata (200 mg/kg/3-3d). RESULTS: The proportions of blood vessels, lymphatic space and estradiol levels were increased. On the other hand, reduction of testosterone levels due to Leydig cells death was observed. As for penile parameters, volumetric proportions of cavernous bodies, collagen and nitric oxide were increased, while smooth muscle content was decreased. CONCLUSIONS: Despite that the long term intake of P. glomerata extract was related to a stimulant action, reduction on Leydig cell viability induced decreased testosterone production.

Accessions of Brazilian ginseng (Pfaffia glomerata) with contrasting anthocyanin content behave differently in growth, antioxidative defense, and 20-hydroxyecdysone levels under UV-B radiation.

Ultraviolet-B (UV-B) radiation is an elicitor of secondary metabolites in plant tissue culture, but the effects on 20-hydroxyecdysone (20E) are still unclear. The 20E may show biotechnological, pharmacological, medical, and agrochemical applicability. Here, we use Pfaffia glomerata, a medically important species, to understand the impacts of UV-B radiation on their physiological performance, the expression of key genes involved in the 20E biosynthesis, and the 20E content. Two accessions (A22 and A43) of plants 20 days old grown in vitro were exposed to 0 (control), 2 (6.84 kJ m-2), and 4 (13.84 kJ m-2) h UV-B radiation for 20 consecutive days. Our data showed that UV-B reduced glucose concentration in A22 and A43 under 4 h of exposure (29 and 30%, respectively), while sucrose concentration increased (32 and 57%, respectively). UV-B also differentially impacted the accessions (A22 and A43), where the A22 under 4 h of UV-B had reduced total dry weight (8%) and electron transport rate (31%); in contrast, A43 did not change. Also, only A22 had increased POD activity under 4 h of UV-B (66%), as well as increased gene expression of the 20E pathway and the 20E content under 2 and 4 h of UV-B in leaves (28 and 21%, respectively) and roots (16 and 13%, respectively). This differential performance to UV-B can be explained by the contrasting anthocyanin contents. Notably, A43 displayed 56% more anthocyanin to the former, a possible defense against UV-B. In conclusion, UV-B radiation is a potential elicitor for increasing 20E content in P. glomerata grown in vitro.

Genetic, evolutionary and phylogenetic aspects of the plastome of annatto (Bixa orellana L.), the Amazonian commercial species of natural dyes.

MAIN CONCLUSION: The plastome of B. orellana reveals specific evolutionary features, unique RNA editing sites, molecular markers and the position of Bixaceae within Malvales. Annatto (Bixa orellana L.) is a native species of tropical Americas with center of origin in Brazilian Amazonia. Its seeds accumulate the apocarotenoids, bixin and norbixin, which are only found in high content in this species. The seeds of B. orellana are commercially valued by the food industry because its dyes replace synthetic ones from the market due to potential carcinogenic risks. The increasing consumption of B. orellana seeds for dye extraction makes necessary the increase of productivity, which is possible accessing the genetic basis and searching for elite genotypes. The identification and characterization of molecular markers are essential to analyse the genetic diversity of natural populations and to establish suitable strategies for conservation, domestication, germplasm characterization and genetic breeding. Therefore, we sequenced and characterized in detail the plastome of B. orellana. The plastome of B. orellana is a circular DNA molecule of 159,708 bp with a typical quadripartite structure and 112 unique genes. Additionally, a total of 312 SSR loci were identified in the plastome of B. orellana. Moreover, we predicted in 23 genes a total of 57 RNA-editing sites of which 11 are unique for B. orellana. Furthermore, our plastid phylogenomic analyses, using the plastome sequences available in the plastid database belonging to species of order Malvales, indicate a closed relationship between Bixaceae and Malvaceae, which formed a sister group to Thymelaeaceae. Finally, our study provided useful data to be employed in several genetic and biotechnological approaches in B. orellana and related species of the family Bixaceae.

Translationally controlled tumour protein (TCTP) from tomato and Nicotiana benthamiana is necessary for successful infection by a potyvirus.

Translationally controlled tumour protein (TCTP) is a ubiquitously distributed protein in eukaryotes, involved in the regulation of several processes, including cell cycle progression, cell growth, stress protection, apoptosis and maintenance of genomic integrity. Its expression is induced during the early stages of tomato (Solanum lycopersicum) infection by the potyvirus Pepper yellow mosaic virus (PepYMV, a close relative of Potato virus Y). Tomato TCTP is a protein of 168 amino acids, which contains all the conserved domains of the TCTP family. To study the effects of TCTP silencing in PepYMV infection, Nicotiana benthamiana plants were silenced by virus-induced gene silencing (VIGS) and transgenic tomato plants silenced for TCTP were obtained. In the early stages of infection, both tomato and N. benthamiana silenced plants accumulated less virus than control plants. Transgenic tomato plants showed a drastic reduction in symptoms and no viral accumulation at 14 days post-inoculation. Subcellular localization of TCTP was determined in healthy and systemically infected N. benthamiana leaves. TCTP was observed in both the nuclei and cytoplasm of non-infected cells, but only in the cytoplasm of infected cells. Our results indicate that TCTP is a growth regulator necessary for successful PepYMV infection and that its localization is altered by the virus, probably to favour the establishment of virus infection. A network with putative interactions that may occur between TCTP and Arabidopsis thaliana proteins was built. This network brings together experimental data of interactions that occur in other eukaryotes and helps us to discuss the possibilities of TCTP involvement in viral infection.

Flask sealing on in vitro seed germination and morphogenesis of two types of ornamental pepper explants / Tipo de vedação na germinação e na morfogênese in vitro de dois tipos de explante de pimenteira ornamental

ABSTRACT: The influence of flask sealing and explant source on the in vitro morphogenesis of pepper, were evaluated in Capsicum annuum. Seeds were sterilized and inoculated in Murashige and Skoog media supplemented with vitamins, myo-inositol, sucrose, and agar. Gas exchange was evaluated in the germination stage by comparing 3 flask-sealing systems: rigid polypropylene lids (PLs) without vents, PLs with 1 vent, and PLs with 2 vents covered with membranes. In the regeneration stage, cotyledon and hypocotyl segments were transferred to the organogenesis-inducing media, being the different sealing types also tested in a factorial scheme. Photosynthetic pigments, morphological and hystological analyses were conducted for each treatment. Plants maintained in glass flasks capped with vented lids showed more vigorous growth and differentiated anatomical structures. These treatments resulted in taller plants, higher numbers and more expanded leaves, higher fresh and dry weights, and an increase in photosynthetic pigments. Cultivation of C. annuum in flasks with reduced gas exchange was more effective on callus induction. During the regeneration stage, hypocotyls were more effective than cotyledons. Sealing type influenced the morphogenic responses of pepper, demonstrating that an increase in gas exchange has a positive effect on biomass production and acclimatization of the plantlets.

RESUMO: Objetivando avaliar a influência de trocas gasosas e da fonte de explante na morfogênese in vitro de pimenteira ornamental, sementes de Capsicum annuum foram desinfestadas e inoculadas em meio de Murashige e Skoog suplementado com vitaminas, mio-inositol, sacarose e ágar. O efeito das trocas gasosas foi avaliado na fase de germinação, utilizando três tipos de vedações nos frascos: tampas de polipropileno rígido (TPR) sem membrana (controle), TPR com um orifício (10mm de diâmetro) coberto com membrana e TPR com dois orifícios cobertos com membranas permeáveis à gases. Na fase de regeneração, segmentos de hipocótilo e cotilédones foram transferidos para o meio de indução de organogênese e os diferentes tipos de vedação foram novamente testados em esquema fatorial. Análises anatômicas de pigmentos fotossintéticos e de características morfológicas foram realizadas. Plântulas cultivadas em frascos com tampas com membranas apresentaram estruturas anatômicas diferenciadas e mais vigorosas. Esses tratamentos resultaram em plântulas maiores, com folhas mais expandidas, maior número de folhas, maior massa fresca e seca e maior quantidade de pigmentos fotossintéticos. O cultivo de C. annuum em frascos com menores trocas gasosas foi mais eficaz na indução de calos. Durante o estágio de regeneração, hipocótilos foram mais eficazes do que cotilédones. O tipo de vedação influenciou nas respostas morfogênicas em pimenteira, demonstrando que o aumento nas trocas gasosas tem um efeito positivo sobre a produção de biomassa e aclimatização das plântulas.

Morphogenesis in vitro of chrysanthemum (Dendranthema grandiflora tzelev) from stem and pedicel explants / Morfogênese in vitro de crisântemo (Dendranthema grandiflora tzelev) a partir de explantes de caule e pedicelo

Visando a adequação de um protocolo de morfogênese in vitro para plantas de crisântemo (Dendranthema grandiflora Tzelev) analisou-se três fatores que afetam a frequência da regeneração. Foram avaliadas três concentrações de ácido indolil-acético (AIA) e 6- Benzilaminopurina (BAP), a fonte de explante (caule e pedicelo) e a orientação de inoculação do explante no meio de cultivo. Ao final de 15-30 dias de cultivo, observou-se que a fonte de explante, o tipo de explante e a interação explante e meio de cultivo afetaram significativamente a frequência de regeneração (FR) e o número de ramos por explantes (NRE) em crisântemo. Os explantes de pedicelo apresentaram maiores frequências de regeneração (15-20 dias) em meio de cultivo suplementado com 0,5 mg L-1 de BAP e 2,0 mg L-1 de AIA e os explantes caulinares (30 dias) em meio com 2,0 mg L-1 de BAP e 0,5 mg L-1 de AIA. Respostas diferenciais em relação à morfogênese foram observadas quando as duas posições do pedicelo foram inoculadas no meio de cultivo, a região seccionada em contato com o meio de cultivo suplementado com 0,5 mg L-1 de BAP e 2,0 mg L-1 de AIA proporcionou maior frequência de brotos. Foram obtidas plantas com flores 45-60 dias após a transferência para a casa de vegetação. As flores apresentavam os mesmos padrões e características das flores das plantas doadoras de explantes. Podese concluir que a o tipo de explante, sua orientação em relação ao meio de cultura bem como as combinações de reguladores de crescimento são fatores-chave envolvidos no sucesso da regeneração de plantas de crisântemo in vitro.

Aiming the optimization of a regeneration protocol for chrysanthemum plants (Dendranthema grandiflora), experiments of regeneration in vitro were accomplished, stem explants and pedicel inoculated in several shoots induction mediuns. The stem explants and of pedicels they were sectioned longitudinally and inoculated with the face opposed to the section in the medium of cultivation with the basic salts MS, with different concentrations and combinations of growth regulators BAP and AIA. At the end of 15-30 days of cultivation, significant differences were observed in the regeneration frequency, number of shoots for explante and in the length of the shoots among the appraised explantes. Explants of pedicel presented potential the best to the explants stem. The pedicel explants presented larger regeneration frequencies in medium of cultivation contends basic salts of MS, with 0,5 mg L-1 of BAP and 2,0 mg L-1 of AIA and the explantes of stem in medium with basic salts of MS + 2,0 mg L-1 of BAP and 0,5 mg L-1 of AIA. They were obtained plants with flowers 45-60 days after the transfer to the vegetation house. In function of the obtained results, it can be concluded that the choice of the explant type and the medium of cultivation are factor-key involved in the success of the regeneration of plants for posterior use in experiments of genetic transformation.

Azadirachtin biosynthesis induction in Azadirachta indica A. Juss cotyledonary calli with elicitor agents

The use of cell and plant tissues culture techniques to produce economically important active metabolites has been growing. Among these substances, azadirachtin (AZA), produced by the neem tree (Azadirachta indica), has received considerable attention due to its bioinsecticide action. The main goal of this work was to analyze the AZA levels in neem cotyledonary calli. The calli were grown in agitated Woody Plant Medium (WPM) liquid medium, supplemented with glucose (Gl), hydrolyzed casein (HC) and methyl jasmonate (MeJ) as elicitor agent. An interaction was observed between these substances, depending on in vitro cultivation time with orbital agitation. The highest concentrations (average of 0.2470 µg g-1) of AZA were produced in the first and second weeks of culture when the cell mass was grown in a medium with 2% Gl v/v, 500 mg L-1 HC and 100 µM of MeJ. This corresponded to approximately 57% of the AZA content stored in the donor plants seeds, used as a source of explants to induce in vitro callus formation. It was concluded that the nutrition, as well as the concentration of MeJ as signal transduction of secondary metabolism in neem cells, might influence the AZA content produced in vitro.