Ovarian Tissue Culture to Preserve Fertility in Transgender Male Patients after Hormonal Treatment / Cultura de tecido ovariano para preservação da fertilidade em pacientes transexuais masculinos após tratamento hormonal

Abstract Objective To evaluate the reproductive and histological characteristics of fresh cultured ovarian tissue from transgender male patients. Methods An in vitro pilot study in which samples were collected during sex reassignment surgery for transgender male patients. The ovarian cortex was cut into fragments of 2 mm, 3mm, and 4 mm, and placed in a 96-well plate suitable for cultivation at days 0, 2, 4, 6, and 8, when the histology was analyzed. Results Stromal hyperplasia was observed in all samples, and it was not associated with the obtainment of primordial or primary follicles. Peripheral reduction in cell count was also a recurrent finding. Primordial and primary follicles were identified with a heterogeneous pattern in fragments from the same patient and from different patients, and follicles in more advanced stages of development (secondary and antral) were not found. There was an association between the diameter of the ovarian fragments and the identification of primary follicles (p=0.036). The number of days in culture was associated with histological signs of tissue damaging in the fragments (p=0.002). The total number of follicles identified in the samples with 2mm in diameter was significantly lower than in those that measured 4mm in diameter (p=0.031). Conclusion A diameter of 4mm is suitable for ovarian tissue culture with the benefit of ease of handling. Even after prolonged exposure to testosterone, the ovarian fragments presented primordial and primary follicles, maintaining viability throughout the days they were exposed to the culture. Freezing the ovarian cortex of transgender patients who will undergo surgery for gender reassignment would be an interesting option, in the future, for the preservation of fertility.

Resumo Objetivo Avaliar as características reprodutivas e histológicas de tecido ovariano cultivado a fresco de pacientes transexuais masculinos. Métodos Estudo experimental in vitro e piloto, no qual amostras foram coletadas durante a cirurgia de redesignação de sexo para pacientes transexuais masculinos. O córtex ovariano foi cortado em fragmentos de 2mm, 3mm, e 4mm, e colocado em placa de 96 poços própria para cultivo nos dias 0, 2, 4, 6 e 8, quando a histologia foi analisada. Resultados Hiperplasia estromal foi observada em todas as amostras, e não esteve associada à obtenção de folículos primordiais ou primários. A redução periférica no número de células também foi um achado recorrente. Folículos primordiais e primários foramidentificados com padrão heterogêneo emfragmentos domesmo paciente e em fragmentos de pacientes diferentes, não sendo encontrados folículos em estágios mais avançados de desenvolvimento (secundários e antrais). Houve associação entre o diâmetro dos fragmentos ovarianos e a identificação dos folículos primários (p=0,036). O número de dias de cultura esteve associado a sinais histológicos de lesão tecidual nos fragmentos (p=0,002). O número total de folículos identificados nas amostras de 2mm de diâmetro foi significativamente menor do que nas de 4mm de diâmetro (p=0,031). Conclusão O diâmetro de 4mm parece ser mais adequado para a cultura de tecido ovariano com a vantagem de fácil manejo. Mesmo após exposição prolongada à testosterona, os fragmentos ovarianos apresentavam folículos primordiais e primários, e manteve a viabilidade ao longo dos dias de exposição à cultura. No futuro, o congelamento da cortical do ovário de pacientes transgêneros que se submeterão à cirurgia de redesignação sexual poderia ser uma opção interessante para a preservação da fertilidade.

In vitro establishment and multiplication of Echynochloa polystachya

The objective of this work was to carry out the in vitro establishment of Echynochloa polystachya aiming at obtaining a micropropagation protocol for works involving the selection of superior genotypes and the cultivation of the species. E. polystachya stems were collected in the municipality of Manaus-AM. Explants were inoculated in test tubes containing Murashige and Skoog (MS) medium. Thirty days after in vitro establishment, the rate of sprouting and contamination were evaluated. Experiments were also carried out to assess the effects of sucrose and 6-benzylaminopurine (BAP) concentrations on the tillering rate of explants. It was found that during the successive subcultures there was a decrease in internodes and the consequent loss of vigor. There were responses in the multiplication rate at concentrations starting from 45 g L-1 sucrose. In addition, BAP and sucrose interfered the development and in vitro multiplication. Sucrose in conjunction with BAP was harmful and shortened internodes. The physiological state of the explants for the species under study was intrinsically linked to the concentrations of sucrose used for the culture medium and the concentrations of BAP. However, the sucrose and BAP concentrations suggested for in vitro cultivation of E. Polystachya must be adjusted during successive subcultures. Absence of contamination in the in vitro establishment occurred at concentrations 15, 30 and 60 g L-1 sucrose. The combination of 1.5 mg L-1 BAP and 30 g L-1 sucrose promoted greater induction of sprouts. In addition, the in vitro rooting of E. polystachya was 45%.

Presencia y Expresión del Receptor ACE2 (Target de SARS-CoV-2) en Tejidos Humanos y Cavidad Oral. Posibles Rutas de Infección en Órganos Orales / Presence and Expression of ACE2 Receptor (Target of SARS-CoV-2) in Human Tissues and Oral Cavity. Possible Routes Infection in Oral Organs

RESUMEN: Un nuevo coronavirus (SARS-CoV-2) ha sido reconocido como el agente etiológico de una misteriosa neumonía originada en Wuhan, China. La OMS ha nombrado a la nueva enfermedad como COVID-19 y, además, la ha declarado pandemia. Taxonómicamente, SARS-CoV-2 pertenece al género de los betacoronavirus junto con SARS-CoV y MERS-CoV. SARS-CoV-2 utiliza la enzima convertidora de la angiotensina 2 (ACE2) como el receptor objetivo para el ingreso en una célula huésped. La expresión de ACE2 en células de tejidos humanos podría indicar un potencial riesgo de reconocimiento por parte del virus y, por ende, ser susceptibles a la infección. Mediante algunas técnicas de laboratorio y de bioinformática, se ha visto una alta presencia de ACE2 en células epiteliales alveolares tipo II de pulmón y en enterocitos del intestino delgado. En la cavidad oral, se ha podido identificar la presencia de ACE2, principalmente, en células epiteliale s de glándulas salivales y células epiteliales de la lengua. Además, se ha reportado la manifestación de algunos síntomas, como sequedad bucal y ambligeustia, los que podrían estar relacionadas con una infección de SARS-CoV-2 en estos órganos. Sin embargo, son necesarios mayores estudios que evidencien esta situación.

ABSTRACT: A novel coronavirus (SARS-CoV-2) has been recognized as a etiologic agent of a mysterious pneumonia originating in Wuhan, China. WHO has named the new disease as COVID-19 and, in addition, has declared it a pandemic. Taxonomically, SARS-CoV-2 belongs to the betacoronavirus genus along with SARS-CoV and MERS-CoV. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the target receptor for entry into a host cell. The expression of ACE2 in cells of human tissues could indicate a potential risk of recognition by the virus and, therefore, be susceptible to infection. Through some laboratory and bioinformatics techniques, high presence of ACE2 has been seen in type II alveolar epithelial cells of the lung and enterocytes of the small intestine. In oral cavity, mainly presence of ACE2 has been identified in epithelial cells of salivary glands and epithelial cells of tongue. In addition, manifestation of some symptoms, such as dry mouth and amblygeustia, have been reported, which could be related to a SARS-CoV-2 infection in these organs. However, further studies are needed to prove this situation.

Effect of light and sucrose on photoautotrophic and photomixotrophic micropropagation of Physalis angulata / Efeito da luz e sacarose na micropropagação fotoautotrófica e fotomixotrófica de Physalis angulata

The biotechnological interest in genus Physalis has increased in the last decades, however, there are still few micropropagation studies of this genus. The objective of this study was to evaluate P. angulata photoautotrophic and photomixotrophic micropropagation with gas exchange under seven light spectra and five concentrations of sucrose. Lighting were yellow, blue, white, red, green, red + blue LEDs and natural light filtered by mesh. Sucrose concentrations were 0, 7.5, 15, 22.5 and 30 g.L-1. Phytotechnical, anatomical features and photopigment contents were evaluated through stem and root segment length, leaf number, leaf area, chlorophyll a and b contents, carotenoids, adaxial epidermis, palisadic and spongy parenchyma and abaxial epidermis. The data were compared by Scott-Knott's mean test and principal components analysis using the R software. Comparing the variables within lighting types, it was observed that only the screen treatment, screen-filtered natural illumination, obtained assessment in all variables. Comparing the levels of sucrose, it was observed that the treatment 15 g.L-1 sucrose obtained the highest number of averages with maximum evaluation. It was concluded that the natural light filtered by screen with 50% of shading allowed the photoautotrophic micropropagation of P. angulata. Better development results were observed in photomixotrophic micropropagation with 15 g.L-1 of sucrose.

O interesse biotecnológico em Physalis aumentou nas últimas décadas, porém, ainda existem poucos trabalhos de micropropagação desse gênero. Objetivou-se avaliar sua micropropagação fotoautotrófica e fotomixotrófica com troca gasosa sob sete tipos de iluminação e cinco concentrações de sacarose. Foram utilizados LEDs amarelo, azul, branco, vermelho, verde, vermelho + azul e luz natural filtrada por malha. As concentrações de sacarose foram 0, 7,5, 15, 22,5 e 30 g.L-1. Características fitotécnicas, anatômicas e teor de fotopigmentos foram avaliados através de comprimento de segmento de caule e raíz, número de folhas, área foliar, teores de clorofilas a e b, carotenoides, epiderme adaxial, parênquimas paliçádico e esponjoso e epiderme abaxial. Os dados foram comparados por teste de média Scott-Knott e análise de componentes principais utilizando-se o software R. Comparando-se as variáveis dentro de tipos de iluminação, observou-se que apenas o tratamento screen, iluminação natural filtrada por malha, obteve avaliação máxima em todas as variáveis. Comparando-se os níveis de sucrose, observou-se que o tratamento 15 g.L-1 sacarose obteve o maior número de médias com avaliação máxima. Concluiu-se que a luz natural filtrada por tela com 50% de sombreamento permitiu a micropropagação fotoautotrófica de P. angulata. Observou-se melhores resultados de desenvolvimento na micropropagação fotomixotrófica com 15 g.L-1 de sacarose.

Polyploidy induction in Physalis alkekengi / Indução de poliploidia em Physalis alkekengi

Physalis alkekengi is an ornamental plant that can also be used as a medicinal plant due to its anti-inflammatory, bactericidal, antitumor and fungicidal properties. Polyploidization can be an important tool in the genetic improvement of this species. The objective this work was to obtain tetraploids in vitro and to evaluate the phytotechnical traits of P. alkekengi. For this, nodal segments of P. alkekengi var. Franchettii were inoculated into petri dishes containing 100 ml of MS medium supplemented with colchicine at concentrations 0; 0.04; 0.08; 0.12; and 0.16% and kept in the dark for 24 and 48h. After the respective treatment periods with colchicine the segments were inoculated into test tubes. The tetraploids were identified by flow cytometry and classical cytogenetics. In vitro seedlings were measured: root length, nodal segment length, leaflet number and total leaf area. In the acclimatization phase, the area of the second leaf and total leaf, petiole radius, stem length, fruit weight with calyx, without calyx, fruit diameter, number of seeds and brix of the pulp were evaluated. Chlorophyll a, chlorophyll b, total chlorophyll, total carotenoid, total chlorophyll / total carotenoid ratio and chlorophyll a / b ratio were also estimated. The treatment that most produced tetraploid seedlings was with 0.08% colchicine per 24h. No significant difference was observed in 7 (seven) variables, these being all variables of photopigments, stem diameter (steam) and brix. In general, diploid (2x) plants were better in 9 (nine) while tetraploid seedlings were better in 6 (six) of the phytotechnical variables. It was concluded that the MS medium supplemented with 0.08% colchicine for 24 h allowed P. alkekengi tetraploides to be obtained with better phytotechnical qualities.

Physalis alkekengi é uma planta ornamental que também pode ser usada como planta medicinal devido às suas propriedades anti-inflamatórias, bactericidas, antitumorais e fungicidas. A poliploidização pode ser uma ferramenta importante para o melhoramento genético dessa espécie. O objetivo deste trabalho foi obter tetraplóides in vitro e avaliar as características fitotécnicas de P. alkekengi. Para isso, segmentos nodais de P. alkekengi var. Franchettii foram inoculados em placas de Petri contendo 100 ml de meio MS suplementado com colchicina nas concentrações 0; 0,04; 0,08; 0,12; e 0,16% e mantido no escuro por 24 e 48h. Após os respectivos períodos de tratamento com colchicina, os segmentos foram inoculados em tubos de ensaio. Os tetraplóides foram identificados por citometria de fluxo e citogenética clássica. As plântulas in vitro foram medidas: comprimento da raiz, comprimento do segmento nodal, número de folhetos e área foliar total. Na fase de aclimatação foram avaliadas a área da segunda folha e área foliar total, raio do pecíolo, comprimento do caule, peso do fruto com cálice, sem cálice, diâmetro do fruto, número de sementes e brix da polpa. Também foram estimadas clorofila a, clorofila b, clorofila total, carotenóides totais, razão clorofila total / carotenóide total e razão clorofila a / b. O tratamento que mais produziu mudas tetraplóides foi com colchicina a 0,08% por 24 horas. Não foi observada diferença significativa em 7 (sete) variáveis, sendo todas variáveis de fotopigmentos, diâmetro do caule (vapor) e brix. Em geral, as plantas diplóides (2x) foram melhores em 9 (nove) variáveis fitotécnicas, enquanto as mudas tetraplóides foram melhores em 6 (seis). Concluiu-se que o meio MS suplementado com colchicina a 0,08% por 24 h permitiu obter tetraploides de P. alkekengi com melhores qualidades fitotécnicas.

Toxicity evaluation of water extract of tissue-cultured Taraxacum formosanum by acute, subacute administration, and Ames test

BACKGROUND: Taraxacum species (commonly known as dandelion) used as herbal medicine have been reported to exhibit an antiproliferative effect on hepatoma cells and antitumor activity in non-small-cell lung cancer cells. Although several investigations have demonstrated the safety of Taraxacum officinale, the safety of tissue-cultured plants of T. formosanum has not been assessed so far. Therefore, the present study examines the safety of the water extract of the entire plant of tissue cultured T. formosanum based on acute and subacute toxicity tests in rats, as well as the Ames tests. RESULTS: No death or toxicity symptoms were observed in the acute and subacute tests. The results of the acute test revealed that the LD50 (50% of lethal dose) value of the T. formosanum water extract for rats exceeded 5 g/kg bw. No abnormal changes in the body weight, weekly food consumption, organ weight, or hematological, biochemical, and morphological parameters were observed in the subacute toxicity test. Thus, the no observed adverse effect level (NOAEL) of T. formosanum water extract was estimated to be higher than 2.0 g/kg. Finally, the results of the Ames test revealed that T. formosanum water extract was not genotoxic at any tested concentration to any of five Salmonella strains. CONCLUSIONS: The water extract of tissue-cultured T. formosanum was non-toxic to rats in acute and subacute tests and exhibited no genotoxicity to five Salmonella strains.

A comparison of callus induction in 4 Garcinia species

Background: This research is intended to determine suitable types and concentrations of plant growth regulators (PGRs) to induce callus on stem and leaf sections of 4 species of the genus Garcinia, namely, Garcinia mangostana, Garcinia schomburgkiana, Garcinia cowa, and Garcinia celebica. The base medium was MS medium containing 30 g l -1 sucrose, 0.5 g l-1 polyvinylpyrrolidone (PVP), and 7 g l-1 agar, and for the different treatments, PGRs were added to the medium as follows: thidiazuron (TDZ) at concentrations of 0, 0.1, 0.5, 1, and 2 mg l-1; 6-(3- hydroxybenzylamino) purine (meta-topolin) at concentrations of 0, 0.5, 2.5, and 5 mg l-1; 4-amino-3,5,6- trichloro-2-pyridinecarboxylic acid (picloram) at concentrations of 0, 0.5, 2.5, and 5 mg l-1; and 2,4- dichlorophenoxyacetic acid (2,4-D) at concentrations of 0, 0.5, 1, 2, and 4 mg l-1. The occurrence of callus was observed after 4 weeks. Results: A maximum of 100% and 93% of G. mangostana leaf explants formed callus in the 0.5 mg l-1 and 1 mg l-1 TDZ treatments, respectively, while 100% of G. schomburgkiana stem explants formed callus in the 1 mg l-1 TDZ treatment and 89% of G. schomburgkiana leaf explants formed callus in the 0.5 mg l-1 picloram treatment. The highest callus induction rate for G. cowa was 62% in the 1 mg l-1 TDZ treatment and for G. celebica was 56% in the 0.5 mg l-1•mT-1 treatment. Conclusions: For all 4 species, the greatest amount of large nodular callus was observed in the TDZ treatments. White, friable callus was observed on most of the 2,4-D and picloram treatment groups. Most meta-topolin treatments resulted in minimal callus formation.

Influence of Silver Nitrate on Somatic Embryogenesis Induction in Arabica Coffee (Coffea arabica L)

Abstract The influence of silver nitrate (AgNO3), benzyladenine (BAP), and indole-3-acetic acid (IAA) on low frequency somatic embryogenesis (LFSE) induction in Caturra and Catuaí arabica coffee was evaluated. For the Caturra cultivar, the production of somatic embryos was significantly increased by adding AgNO3 to the semisolid culture medium. The highest average number of somatic embryos for this cultivar was obtained using 6.6 μM BAP, 2.85 μM IAA, and 40 μM AgNO3. In contrast, for the Catuaí cultivar, the highest average number of somatic embryos was obtained using semisolid medium supplemented with 8.8 μM BAP, and 2.85 μM IAA. Using these protocols, somatic embryos were directly induced using leaf sections of in vitro plants of both coffee cultivars within 8 weeks. The somatic embryos developed into rooted plants with a 100% survival rate upon transfer to the greenhouse.

Kir2.1 Channel Regulation of Glycinergic Transmission Selectively Contributes to Dynamic Mechanical Allodynia in a Mouse Model of Spared Nerve Injury / 神经科学通报·英文版

Neuropathic pain is a chronic debilitating symptom characterized by spontaneous pain and mechanical allodynia. It occurs in distinct forms, including brush-evoked dynamic and filament-evoked punctate mechanical allodynia. Potassium channel 2.1 (Kir2.1), which exhibits strong inward rectification, is and regulates the activity of lamina I projection neurons. However, the relationship between Kir2.1 channels and mechanical allodynia is still unclear. In this study, we first found that pretreatment with ML133, a selective Kir2.1 inhibitor, by intrathecal administration, preferentially inhibited dynamic, but not punctate, allodynia in mice with spared nerve injury (SNI). Intrathecal injection of low doses of strychnine, a glycine receptor inhibitor, selectively induced dynamic, but not punctate allodynia, not only in naïve but also in ML133-pretreated mice. In contrast, bicuculline, a GABA receptor antagonist, induced only punctate, but not dynamic, allodynia. These results indicated the involvement of glycinergic transmission in the development of dynamic allodynia. We further found that SNI significantly suppressed the frequency, but not the amplitude, of the glycinergic spontaneous inhibitory postsynaptic currents (gly-sIPSCs) in neurons on the lamina II-III border of the spinal dorsal horn, and pretreatment with ML133 prevented the SNI-induced gly-sIPSC reduction. Furthermore, 5 days after SNI, ML133, either by intrathecal administration or acute bath perfusion, and strychnine sensitively reversed the SNI-induced dynamic, but not punctate, allodynia and the gly-sIPSC reduction in lamina IIi neurons, respectively. In conclusion, our results suggest that blockade of Kir2.1 channels in the spinal dorsal horn selectively inhibits dynamic, but not punctate, mechanical allodynia by enhancing glycinergic inhibitory transmission.

Human Brain Slice Culture: A Useful Tool to Study Brain Disorders and Potential Therapeutic Compounds / 神经科学通报·英文版

Investigating the pathophysiological mechanisms underlying brain disorders is a priority if novel therapeutic strategies are to be developed. In vivo studies of animal models and in vitro studies of cell lines/primary cell cultures may provide useful tools to study certain aspects of brain disorders. However, discrepancies among these studies or unsuccessful translation from animal/cell studies to human/clinical studies often occur, because these models generally represent only some symptoms of a neuropsychiatric disorder rather than the complete disorder. Human brain slice cultures from postmortem tissue or resected tissue from operations have shown that, in vitro, neurons and glia can stay alive for long periods of time, while their morphological and physiological characteristics, and their ability to respond to experimental manipulations are maintained. Human brain slices can thus provide a close representation of neuronal networks in vivo, be a valuable tool for investigation of the basis of neuropsychiatric disorders, and provide a platform for the evaluation of novel pharmacological treatments of human brain diseases. A brain bank needs to provide the necessary infrastructure to bring together donors, hospitals, and researchers who want to investigate human brain slices in cultures of clinically and neuropathologically well-documented material.

Study on tissue culture system of Polygonatum cyrtonema / 中国中药杂志

In order to accelerate the breeding of the excellent seedlings of Polygonatum cyrtonema,tissue culture system of P. cyrtonema was established through the comprehensive regulation of key factors( leaf age,leaf location,basic media and plant growth regulators) and cytological basis of callus formation and differentiation was analyzed through paraffin section. The results showed that the 30-day-old leaf base explanton medium MS+6-BA 1. 50 mg·L~(-1)+2,4-D 0. 20 mg·L~(-1) had the highest induction rate( 80. 00%). The callus was initiated from cells on leaf base epidermis and near cortex,formed by the differentiation of middle vascular bundle cells. The optimal medium for adventitious bud differentiation was MS+ 6-BA 4. 00 mg·L~(-1)+ 2,4-D 0. 20 mg·L~(-1) with the differentiation rate of90. 33%,and the average number of buds was 5. 16. The adventitious buds had two origin types: exogenous and endogenous origin,formed by callus proximal cells and callus internal meristemoid. The adventitious bud proliferation medium was screened by orthogonal design,which determined the optimum combination was MS+ 6-BA 2. 00 mg·L~(-1)+NAA 0. 10 mg·L~(-1) and MS+ 6-BA 2. 00 mg·L~(-1)+NAA 0. 20 mg·L~(-1). The tubers with three leaves were cut and inoculated in the medium 1/2 MS+IBA 2. 00 mg·L~(-1),showing the highest rooting rate of 94. 00%. The rooting seedlings transplanted into the peat-vermiculite( 1 ∶ 1) matrix grew healthy and the survival rate was over 85. 00%. This research provided a novel solution for large-scale cultivation of P. cyrtonema seedling.

Effects of exogenous IBA and fungal elicitor on growth of in vitro roots culture of Dysosma versipellis and production of podophyllotoxin / 中国中药杂志

Using the White as basic medium, the effects of the exogenous IBA and endophytic fungal elicitor on the growth of in vitro roots cultures of Dysosma versipellis and production of podophyllotoxin were investigated in this study. The results showed that the IBA and the endophytic fungus Zasmidium syzygii elicitor could increase the content of podophyllotoxin of in vitro roots of D. versipellis after 3 weeks. The White medium added with 3 mg·L~(-1) IBA induced the highest increase of podophyllotoxin(1 830.86 μg·g~(-1)), which was 2.07 folds greater than the control, and followed by 1.5 mg·L~(-1) IBA, fungal elicitor, 1 mg·L~(-1) IBA, 0.5 mg·L~(-1) IBA and 4.5 mg·L~(-1) IBA, which was 1.82, 1.71, 1.63, 1.43 and 1.1 folds greater than the control, respectively. The results also showed that the growth of roots was certain positively correlated with the change of IBA concentration. Therefore, 3 mg·L~(-1) IBA was the most suitable for the production of podophyllotoxin in the in vitro roots of D. versipellis, and the stimulating effect of Z. syzygii fungal elicitor was between 1.5 mg·L~(-1) and 1 mg·L~(-1) IBA, which was a potential natural elicitor to induce the accumulation of podophyllotoxin in future production.

Simulating stress condition through compaction in banana plant cultivated In vitro / Mulação de estresse por compactação em bananeiras cultivadas In vitro

Banana cultivation is an agricultural activity practiced in different regions and constantly subjected to abiotic stresses that limit its productivity. To treat the effect of these stresses research can be undertake by simulating them in vitro since it limit the effect of the external factor in the experiment. Therefore, the objective of this study was to simulate the compaction in banana plant cultivated in vitro using Phytagel. To realize the experiment, we used MS culture medium with two distinct consistency added in the testing tubes. In the bottom part was added 10 mL of culture medium with consistency of jellification: 1.8, 2.8, 3.8, 4.8 and 5.8 g L-1 of Phytagel. Over this culture medium was added 5mL of half MS with consistency standard 1.8 g L-1 of Phytagel. Posteriorly, the planting materials from cultivar Grand Naine, BRS Vitória and BRS Princesa were inoculated in the growth medium. After 30 days of culture the plant materials were submitted for agronomic and physiological evaluation. The result showed non-significant difference among the compaction factors on physiological parameters however variety Grand Naine presents better performance in this character. Besides variety Grand Naine increased the rate of photosynthesis under compaction. This behaviour probably occurred due to overcoming the effect of stress by the variety. Therefore, it can be concluded that the cultivar Grand Naine is superior to cultivars BRS Vitória and BRS Princesa since it produces superior performance on rate of photosynthesis, stomatal conductance, and transpiration under simulated compaction conditions that favor the accumulation of plant biomass.

A bananicultura é uma atividade agrícola difundida em diferentes áreas e constantemente sujeita a estresses abióticos, os quais limitam sua produtividade. Para sanar o efeito dos estresses abióticos pesquisas podem ser simuladas in vitro, pois devido à característica de ambiente controlado pode proporcionar efeito puro do fator de estresse. Sendo assim, o objetivo do presente trabalho foi simular a compactação em bananeiras cultivadas in vitro mediante o uso de Phytagel. Para instalação do experimento utilizou-se o meio de cultura MS em duas consistências distintas, adicionadas a tubos de ensaios. Na parte inferior desses, adicionou-se 10 mL do meio de cultura com distintas consistências de geleificação: 1,8; 2,8; 3,8; 4,8 e 5,8 g L-1 de Phytagel. Sobre este meio de cultura foi adicionado mais 5 mL de meio MS, mas na consistência padrão de 1,8 g L-1 de Phytagel. Posteriormente foram inoculados propágulos das cultivares Grand Naine, BRS Vitória e BRS Princesa. Aos 30 dias de cultivo o material vegetal foi submetido a avaliações fitotécnicas e fisiológicas. Observa-se que as variáveis fitotécnicas não foram significativas para o fator compactação e que a cultivar Grand Naine obteve maior desempenho nessa característica. Observa-se também que a cultivar Grand Naine aumenta sua taxa fotossintética mediante a compactação. Esse comportamento provavelmente ocorre visando a superação desse estresse e explica porque as variáveis fitotecnicas não são comprometidas. Portanto, conclui-se que a cultivar Grand Naine é superior as cultivares BRS Vitória e BRS Princesa, porque mediante a simulação de compactação apresenta superioridade em taxa fotossintética, condutância estomática e transpiração, favorecendo o acumulo de biomassa vegetal.

Application of tissue culture techniques in potato / Aplicação da cultura de tecidos vegetais em batata

Potato is the world's most important non-cereal food crop, and therefore, it is considered one of the major food sources for humankind. Its conventional propagation is asexual, by using the tuber, which allows the accumulation and dissemination of pathogens to new cultivation areas. This fact not only impairs the yield of this solanaceous plant, but also threatens the maintenance of genotypes for commercial or breeding purposes. Due to the impossibility of using botanical seed, conservation and exchange of germplasm of this species by means of conventional methods are not feasible. In all potato-producing regions, the demand for high-quality tubers has been paramount to ensure crops production. Thus, biotechnological techniques based on tissue culture are very important. Plant tissue culture offers alternative methods of propagation by in vitro techniques that provide production and multiplication of material with high sanity. Thus, this literature review summarizes the history and current situation of tissue culture techniques applied to potato crop. Besides clonal multiplication, this biotechnological tool makes available initial indexed material to breeding programs and certified seed potato, and facilitates the exchange and conservation of germplasm. For all these reasons, the use of these techniques in potato production chain directly benefits producers by providing high-quality propagules.

A batata é a cultura não-cereal mais importante do mundo e, portanto, uma das principais fontes de alimento para a humanidade. Sua multiplicação convencional é assexuada utilizando o próprio tubérculo, o que permite o acúmulo e a difusão de patógenos para novas áreas de cultivo, comprometendo a produtividade desta solanácea e ameaçando a manutenção de genótipos de interesse comercial ou para fins de melhoramento. Devido à inviabilidade de utilização das sementes botânicas, a conservação e o intercâmbio de germoplasma dessa espécie por meio de métodos convencionais torna-se inviável. Em todas as regiões produtoras de batata, a demanda por tubérculos de alta qualidade tem sido primordial para garantir a produção das lavouras. Dessa forma, técnicas biotecnológicas baseadas na cultura de tecidos são de suma importância. A cultura de tecidos vegetais oferece métodos alternativos de propagação através das técnicas in vitro que proporcionam a produção e multiplicação de material com alta sanidade. Dessa maneira, esta revisão visa sumarizar o histórico e panorama atual das aplicações da cultura de tecidos em batata. Além da multiplicação clonal, essa ferramenta biotecnológica fornece material inicial indexado para programas de melhoramento e de produção certificada de batata-semente e facilita o intercâmbio e a conservação de germoplasma. Por tudo isso, o emprego destas técnicas na cadeia produtiva da batata proporciona benefícios diretos aos produtores, uma vez que fornece material propagativo com elevada qualidade genética e fitossanitária.

Development of a direct transformation method by GFP screening and in vitro whole plant regeneration of Capsicum frutescens L

Background: Capsicum is a genus of an important spice crop that belongs to the chili lineage. However, many Capsicum species (family Solanaceae) are known to be recalcitrant to genetic transformation and in vitro regeneration, thus hampering the effort in using Capsicum species for detailed biological investigation. In this study, we have developed an optimized protocol for the direct transformation of Capsicum frutescens L. cv. Hot Lava using a biolistic particle delivery system. In addition, a procedure for in vitro whole plant regeneration from the hypocotyl explants of C. frutescens was established. Results: In this study on the biolistic system, explant target distance, bombardment helium (He) pressure, and the size of the microcarrier were the key parameters to be investigated. The optimized parameters based on the screening of GFP expression were determined to have a target distance of 6 cm, helium pressure of 1350 psi, and gold particle (microcarrier) size of 1.6 µm. The greatest number of shoots was obtained from hypocotyls as explants using Murashige and Skoog medium supplemented with 5.0-mg/L 6-benzylaminopurine and 0.1-mg/L 1-naphthaleneacetic acid. On an average, five shoots per explant were formed, and of them, one shoot managed to form the root and developed into a whole plant. Conclusions: We obtained an optimized protocol for the biolistic transformation of chili and in vitro regeneration of chili plantlets. The establishment of the protocols will provide a platform for molecular breeding and biological studies of chili plants.

Influence of N-P-K fertilization at the acclimatization stage on micropropagated seedlings of Tillandsia bulbosa Hook / Influência da adubação com N-P-K na fase de aclimatização em mudas micropropagadas de Tillandsia bulbosa Hook

Bromeliads are known worldwide for their ornamental potential. In Brazil, species of the genus Tillandsia occur in the Atlantic rainforest, Amazon rainforest, and rocky fields. This work aimed to evaluate the influence of nitrogen, phosphorus, and potassium fertilization on micropropagated seedlings of Tillandsia bulbosa, at the acclimatization stage, and their leaf anatomy. The experiment was carried out in a completely randomized design, in a 4x4+1 factorial scheme, using coconut coir: earthworm humus: sand mixture as substrate (2:1:1). Urea, single superphosphate, and potassium chloride were used as nitrogen, phosphorus and potassium sources, respectively, at proportions of 50, 100, 200, and 400% of the dose recommended. The doses were distributed in four applications, testing total application at planting (1); ½ application at planting and ½ at 80 DAP (2); application at planting, at 50 DAP, and at 100 DAP (3); and » application at planting, » at 30 DAP, » at 60 DAP, and » at 120 DAP (4); and a control (without fertilization). Leaves anatomy was analyzed at 180 days after planting. Fertilization did not significantly influence the development of seedlings during acclimatization. The doses of 50, 100, and 200% provided thicker parenchyma of chlorophyll and aquifer and leaf blade. Tillandsia bulbosa can be acclimatized without fertilizer application.

As bromélias são conhecidas mundialmente por seu potencial ornamental. No Brasil, as espécies de Tillandsia podem ser encontradas na Mata Atlântica, Floresta Amazônica e campos rupestres. O objetivo deste trabalho foi verificar a influência de adubação com nitrogênio, fósforo e potássio em plântulas micropropagadas de Tillandsia bulbosa, na fase de aclimatização e na sua anatomia foliar. O experimento foi implantado em delineamento inteiramente casualizado, em esquema fatorial 4x4+1, utilizando como substrato a mistura de pó de coco: húmus de minhoca: areia (2:1:1). Ureia, super fosfato simples e cloreto de potássio foram utilizados como fontes de nitrogênio, fósforo e potássio, nas proporções de 50, 100, 200 e 400% da dose recomendada. As doses foram distribuídas em quatro aplicações, testando aplicação total no plantio (1); ½ no plantio e ½ aos 80 DAP (2); no plantio, aos 50 DAP e aos 100 DAP (3) e » no plantio, » aos 30 DAP, » aos 60 DAP e » aos 120 DAP (4), e uma testemunha (sem adubação). Foi realizado o estudo anatômico das folhas aos 180 dias. A adubação não influenciou significativamente o desenvolvimento das plântulas durante a aclimatização. As doses de 50, 100 e 200% proporcionaram maior espessura de parênquimas clorofiliano e aquífero e do limbo foliar. A aclimatização de Tillandsia bulbosa pode ser realizada sem a necessidade de adubação.

Proteic and phenolics compounds contents in Bacupari callus cultured with glutamine and nitrogen sources / Conteúdo de proteínas e compostos fenólicos em calos de Bacupari cultivados com glutamina e fontes de nitrogênio

Abstract In this study was evaluated the influence of glutamine supplementation on the endogenous content of amino acids, proteins, total phenolics, flavonoids and proanthocyanidins in Bacupari callus. The explants were inoculated in MS medium, MS with half concentration of the nitrogen salts (MS½) and nitrogen-free MS, supplemented with glutamine (5, 10, 30 and 60mM) named as Gln5, Gln10, Gln30 and Gln60. Amino acids and proteins were analyzed after 20, 80 and 140 days and the secondary metabolites on the 140th day. There was no difference in the amino acids on the 20th day. On the 80th day the treatments MS and MS½ presented the lowest levels. On the 140th day MS and MS½ presented the lowest amino acid concentration and Gln10 the highest. Concerning proteins, there was difference only on the 140th day, being the highest concentrations observed in Gln5, and the lowest in MS½ treatment. Total phenolics content was higher in the treatment Gln60 and lowest in MS. Treatments Gln5, Gln10, Gln30 and MS½ were statistically equal. For flavonoids, the highest values occurred in the treatments Gln30, Gln60 and MS½ and the lowest in Gln5, Gln10 and MS. Similarly, for the proanthocyanidins the highest concentrations were observed in treatment Gln60 and the lowest in Gln5 and MS. In conclusion, the treatment with 60mM of glutamine favors the protein accumulation and production of secondary metabolites in Bacupari callus.

Resumo Nesse estudo foi avaliado o efeito da suplementação com glutamina no conteúdo endógeno de aminoácidos, proteínas, fenólicos totais, flavonoides e proantocianidinas em calos de Bacupari. Os explantes foram inoculados em meio MS, meio MS com metade da concentração de dos sais de nitrogênio (MS½) e meio MS sem nitrogênio suplementado com glutamina (5, 10, 30 e 60mM) denominados como Gln5, Gln10, Gln30 e Gln60. Os aminoácidos e as proteínas foram analisados após 20, 80 e 140 dias e os metabólitos secundários no 140° dia. Não houve diferença nos aminoácidos no 20° dia. No 80° dia os tratamentos MS e MS½ apresentaram os menores níveis. No 140° dia, MS e MS½ apresentaram as menores concentrações de aminoácidos e o Gln10 as maiores. A respeito das proteínas, houve diferença apenas no 140° dia, sendo as maiores concentrações observadas nos tratamentos Gln, e as menores no MS½. O conteúdo de fenólicos totais foi maior no tratamento Gln60 e menor no MS. Os tratamentos Gln5, Gln10, Gln30 e MS½ foram estatisticamente iguais. Para os flavonóides, os maiores valores ocorreram nos tratamentos Gln30, Gln60 e MS½ e os menores no Gln5, Gln10 e MS. Da mesma forma, para as proantocianidinas, as maiores concentrações foram observadas no tratamento Gln60 os menores no Gln5 e MS. Em conclusão, o tratamento com 60 mM de glutamina favorece o acúmulo de proteínas e a produção de metabólitos secundários em calos de Bacupari.

Cyproheptadine Regulates Pyramidal Neuron Excitability in Mouse Medial Prefrontal Cortex / 神经科学通报·英文版

Cyproheptadine (CPH), a first-generation antihistamine, enhances the delayed rectifier outward K current (I) in mouse cortical neurons through a sigma-1 receptor-mediated protein kinase A pathway. In this study, we aimed to determine the effects of CPH on neuronal excitability in current-clamped pyramidal neurons in mouse medial prefrontal cortex slices. CPH (10 µmol/L) significantly reduced the current density required to generate action potentials (APs) and increased the instantaneous frequency evoked by a depolarizing current. CPH also depolarized the resting membrane potential (RMP), decreased the delay time to elicit an AP, and reduced the spike threshold potential. This effect of CPH was mimicked by a sigma-1 receptor agonist and eliminated by an antagonist. Application of tetraethylammonium (TEA) to block I channels hyperpolarized the RMP and reduced the instantaneous frequency of APs. TEA eliminated the effects of CPH on AP frequency and delay time, but had no effect on spike threshold or RMP. The current-voltage relationship showed that CPH increased the membrane depolarization in response to positive current pulses and hyperpolarization in response to negative current pulses, suggesting that other types of membrane ion channels might also be affected by CPH. These results suggest that CPH increases the excitability of medial prefrontal cortex neurons by regulating TEA-sensitive I channels as well as other TEA-insensitive K channels, probably I and inward-rectifier Kir channels. This effect of CPH may explain its apparent clinical efficacy as an antidepressant and antipsychotic.

Advances in plant stem cell culture / 生物工程学报

Plant stem cells are the cells that are located in meristems and are kept in a state of undifferentiation. Plant stem cell possesses lower vacuolization, higher mitochondrial activity, more genetic stability and stronger self-renewal capacity compared with calli. Plant stem cell culture has a wide application in pharmaceutical, functional food as well as cosmetic industries. Here we describe the procedure of induction, isolation and identification of plant stem cells, to provide a reference for further research in this field.

Follicle Viability after Vitrification of Bovine Ovarian Tissue / Viabilidade folicular após a vitrificação de tecido ovariano bovino

Abstract Purpose The present study aimed to evaluate the impact of vitrification on the viability of follicles using a three-dimensional (3D) in vitro culture. Methods Bovine ovarian tissue samples (n = 5) obtained from slaughterhouses were utilized. The cortex was cut into small fragments of 2 x 3 x 0.5 mm using a tissue slicer. From these fragments, secondary follicles were first isolated by mechanical and enzymatic methods, then encapsulated in alginate gel and individually cultured for 20 days. Additional fragments of the same ovarian tissue were vitrified in a solution containing 25% glycerol and 25% ethylene glycol. After warming, the follicles underwent the same follicular isolation process that was performed for the fresh follicles. Results A total of 61 follicles were isolated, 51 from fresh ovarian tissue, and 10 from vitrified tissue. After the culture, the vitrified and fresh follicles showed 20% and 43.1% survival rates respectively (p = 0.290),with no significant differences. At the end of the culture, therewere no significant differences in follicular diameter between the vitrified (422.93 ± 85.05 μm) and fresh (412.99 ± 102.55 μm) groups (p = 0.725). Fresh follicles showed higher mean rate of antrum formation when compared with vitrified follicles (47.1% and 20.0% respectively), but without significant difference (p = 0.167). Conclusions The follicles were able to develop, grow and form antrum in the 3D system after vitrification, despite the lower results obtained with the fresh tissue.

Resumo Objetivo O presente estudo teve como objetivo avaliar o impacto da vitrificação na viabilidade dos folículos utilizando a cultura in vitro tridimensional (3D). Métodos Foi utilizado tecido ovariano bovino (n = 5) obtido de abatedouros. O córtex foi cortado em pequenos fragmentos de 2 x 3 x 0,5 mm, utilizando o tissue slicer e a partir destes fragmentos foram isolados folículos secundários por meio de método enzimático e mecânico, encapsulados em gel de alginato e cultivados individualmente durante 20 dias. Outros fragmentos do mesmo tecido ovariano foram vitrificados em solução contendo 25% de glicerol e 25% de etilenoglicol. Após aquecimento, os folículos passaram pelo mesmo processo de isolamento folicular realizado a fresco. Resultados Foram isolados 61 folículos, sendo 51 originários de tecido ovariano a fresco, e 10 de tecido vitrificado. Após a cultura, os folículos vitrificados apresentaram taxa de sobrevida de 20%, e o grupo a fresco apresentou taxa de 43,1% (p = 0,290). O diâmetro folicular ao final da cultura também não apresentou diferença significativa entre o grupo vitrificado (422,93 ± 85,05 μm) e a fresco (412,99 ± 102,55 μm) (p = 0,725). Os folículos a fresco apresentarammaior taxa média de formação de antro do que os folículos vitrificados (47,1% e 20,0%, respectivamente), mas sem diferença significativa (p = 0,167). Conclusões Os folículos foram capazes de se desenvolver, crescer e formar antro em sistema 3D após a vitrificação.