Evaluation Effectiveness of in Vitro Cultivation of Bezoar on Mouse Model Combining Disease with Syndrome of Coronavirus Pneumonia with Yidu Xifei Syndrome / 中国实验方剂学杂志

Objective:To determine the therapeutic effect of <italic>in vitro</italic> cultivation of bezoar on a mouse model adding disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome. Method:BALB/c mice were randomly divided into six groups according to their weight grade： normal group， HCoV-229E infection group， cold and damp group， a mouse model combining disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome， and high and low dose group of <italic>in vitro</italic> cultivation of bezoar. The combination model of human coronavirus pneumonia with Yidu Xifei syndrome mice was established by the method of cold dampness condition stimulation+coronavirus HCoV-229E infection. <italic>In vitro</italic> cultivation of bezoar （0.128，0.064 g·kg^-1） was administrated by gavage for 3 days from the day of infection. The observation indexes included： general state observation of mice， inhibition rate of lung index and lung index of mice. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the viral load in the lung tissues of mice. Serum levels of motilin（MTL）， gastrin （GAS）， and cytokines interleukin（IL）-10，IL-6， tumor necrosis factor-<italic>α</italic>（TNF-<italic>α</italic>）and interferon-<italic>γ</italic>（IFN-<italic>γ</italic>） in lung tissue of mice were determined by enzyme-linked immunosorbent assay（ELISA）. The percentages of CD4⁺ T lymphocytes，CD8⁺ T lymphocytes and B lymphocytes in the blood of mice were determined by flow cytometry. Result:The high and low dose group of <italic>in vitro</italic> cultivation of bezoar can significantly improve the general condition of model mice. Compared with blank group， model group mice lung index increased significantly （<italic>P</italic><0.01）， nucleic acids significantly increased expression of lung tissue in mice （<italic>P</italic><0.01）， significantly higher serum MTL content in mice， GAS content significantly decreased （<italic>P</italic><0.05，<italic>P</italic><0.01）， lung tissue cells in the immune factor TNF-<italic>α</italic>， IL-10 and IL-6 were significantly increased （<italic>P</italic><0.01）， peripheral blood lymphocyte CD4⁺ T cells in mice， The percentages of CD8⁺ T cells and B cells were significantly decreased （<italic>P</italic><0.01）. Compared with model group， <italic>in vitro</italic> cultivation bezoar mice lung index of high and low dose group were significantly lower （<italic>P</italic><0.01）， the lung tissue of mice express nucleic acid decreased significantly （<italic>P</italic><0.01）， MTL content decreased significantly （<italic>P</italic><0.01）， the lung tissue of mice in the IL-6， IL-10， the TNF-<italic>α</italic>， IFN-<italic>γ</italic> levels were significantly lower （<italic>P</italic><0.01）， <italic>in vitro</italic> cultivation bezoar high dose group can significantly increase the CD4⁺ T cell percentage （<italic>P</italic><0.05）， <italic>in vitro</italic> cultivation bezoar can to a certain extent reduce model mice lung inflammatory exudation， pulmonary interstitial edema， as well as blood stasis symptoms. Conclusion:<italic>In vitro</italic> cultivation of bezoar has a significant therapeutic effect on a mice model adding disease with syndrome of coronavirus pneumonia with Yidu Xifei syndrome. It can be treated by reducing the lung index of the model mice， improving the pathological damage of the lung tissue， adjusting the immune effective and inhibiting the clearing of inflammatory factors， and to provide a laboratory basis for clinical medication.

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.

Chemical sterilization of culture medium for in vitro multiplication of Cochlospermum regium / Esterilização química do meio de cultura para a multiplicação in vitro de Cochlospermum regium

ABSTRACT: Cochlospermum regium roots are used in popular medicine and its extract has diverse phytochemical molecules some with antimicrobial activity, consequently exposing this specie to genetic erosion risks. Thus, the objective of this study was to develop an in vitro multiplication protocol using chemical sterilization of culture medium. Therefore, explants obtained from apical buds of C. regium seedlings were inoculated into with 0.05mg L-1 NAA and 1mg L-1 BAP sterilized by chemical agent sodium hypochlorite (NaOCl) at 0.001%, 0.003% and 0.005% of active chlorine (Cl). Autoclaved culture medium was used as control. Result showed that the contamination by bacterial at 91 days of cultivation was significantly (P<0.05) controlled by autoclaving, 0.001% and 0.005% Cl. Moreover, the callus induction in the culture medium with 0.001% and 0.005% Cl was, respectively, 30% and 20% major than autoclaving sterilization. There was not significant (P<0.05) in the percentage of shoot induction among the sterilization preparations methods, and 65% of the explants survived in the presence of culture medium with 0.005% Cl. Histological analyses indicated that the Cl did not have any deleterious effects on morphogenic events. These results indicated that the chemical sterilization using 0.001% - 0.005% Cl controlled the fungal and bacterial multiplication in the culture medium and no affected the C. regium explants development, becoming it an alternative to autoclaving method.

RESUMO: As raízes de Cochlospermum regium são usadas na medicina popular, pois seu extrato apresenta diversas moléculas fitoquímicas, algumas com atividade antimicrobiana, que, consequentemente, expõe esta espécie ao risco de erosão genética. Assim, o objetivo deste estudo foi elaborar um protocolo de multiplicação in vitro para explantes de C. regium usando esterilização química do meio de cultura. Gemas apicais obtidas de plântulas de C. regium germinadas in vitro foram inoculadas em meio de cultivo MS suplementado com 0,05mg L-1 de ANA e 1mg L-1 de BAP e esterilizado pela adição do agente químico hipoclorito de sódio (NaOCl) nas concentrações de cloro ativo de 0,001%, 0,003% e 0,005%. O meio autoclavado foi utilizado como controle. Os resultados mostraram que a contaminação por bactérias aos 91 dias de cultivo foi significativamente (P<0,05) controlada pela autoclavagem, 0,001% e 0,005% de cloro ativo. Além disso, a indução de calos no meio de cultura com 0,001% e 0,005% de cloro ativo foi, respectivamente, 30% e 20% maior do que na esterilização por autoclavagem. Não houve diferença significativa (P<0,05) da porcentagem de indução de brotos entre os métodos de esterilização e 65% dos explantes sobreviveram na presença de 0,005% de cloro ativo. Análises histológicas indicaram que o cloro ativo não afetou os eventos morfogênicos. Os resultados indicaram que a esterilização química por meio do uso de 0,001% - 0,005% de cloro ativo auxiliou no controle da proliferação de bactérias e fungos no meio de cultura e não afetou o desenvolvimento dos explantes de C. regium, tornando-a uma alternativa em relação a autoclavagem.

Influence of growth regulators on distribution of trichomes and the production of volatiles in micropropagated plants of Plectranthus ornatus

ABSTRACT The profile of volatile organic compounds, the glandular and non-glandular trichomes of Plectranthus ornatus, obtained by in vitro cultivation, was evaluated in plants grown in Murashide and Skoog medium supplemented with benylaminopurine at 4.5, 9.0, and 18.0 µM + naphthaleneacetic acid at 5.37 µM, kinetin at 4.7, 9.3 and 18.5 µM + naphthaleneacetic acid (5.37 µM) or Murashide and Skoog 0 medium (as a control). Scanning Electron Microscopy was performed on samples of the third leaf node of the 90 days old plants obtained from treatment with 4.5 or 9.0 µM benylaminopurine, and 4.7 or 9.3 µM kinetin. Headspace Solid Phase Micro-Extraction of the 30, 60 and 90 days old in vitro plants permitted to determinate by GC/MS the composition comprised of 62 compounds. The data were analyzed using Principal Component Analysis and Hierarchical Clustering Analysis and, the major constituents of these oils after treatment and aging were monoterpenes and sesquiterpenes. Morphoanatomical analysis of trichomes, by Scanning Electron Microscopy, enabled the identification of non-glandular trichomes and four types of glandular trichomes, which comprised capitate and peltate glandular trichomes that were distributed on both sides of the leaf. We observed that the regulators influenced qualitative and quantitative profiles of the volatile organic compounds and the number and distribution of hairs on the leaf surface.

INDUÇÃO E HISTOLOGIA DE EMBRIÕES SOMÁTICOS PRIMÁRIOS E SECUNDÁRIOS DO HÍBRIDO Phalaenopsis CLASSIC SPOTTED PINK (ORQUIDACEAE) / Induction and Histology of Primary and Secondary Somatic Embryos of Phalaenopsis Hybrid Classic Spotted Pink (Orquidaceae) / Inducción e histología de embriones somáticos primarios y secundarios del híbrido Classic Spotted Pink (Orquidaceae)

O presente trabalho teve como objetivos induzir a formação de embriões somáticos in vitro no híbrido Phalaenopsis Classic Spotted Pink, utilizando diferentes meios nutritivos e avaliar a morfologia interna desses embriões por meio de análises histológicas e histoquímicas. Folhas jovens de plantas cultivadas in vitro foram utilizadas como explantes para indução de embriões somáticos em diferentes meios nutritivos: New Dogashima Medium, contendo ANA (0,537μM) e BAP (4,440μM), acrescido de phytagel e com pH 5,8 (NDM) e o Murashige & Skoog com a metade da concentração dos sais, acrescido de ANA (0,537μM) e TDZ (13,621μM), gelificado com gelrite e o pH 5,2 (½ MS). Embriões somáticos primários foram obtidos aos 90 dias de cultivo no meio ½MS e foram transferidos para o mesmo meio para obtenção de embriões secundários. Os embriões somáticos primários e secundários foram subcultivados para meio MS com metade da concentração de sais, sem fitoregulador submetidos a fotoperíodo de 16 horas, o qual estimulou a produção de clorofila tanto nos embriões primários como secundários, promovendo o desenvolvimento desses em protocormos e posteriormente em plantas. As análises histológicas demonstraram que os embriões somáticos foram formados diretamente das camadas epidérmicas dos explantes, sem passar pela fase de calo, caracterizando embriogênese somática direta. Os métodos histoquímicos utilizados possibilitaram evidenciar a deposição de amido e lipídeos nas células embriogênicas em decorrência de mecanismos fisiológicos, permitindo o desenvolvimento dos embriões primários e secundários em plantas. Portanto, o meio ½ MS acrescido de ANA (0,537μM) e TDZ (13,621μM), gelificado com gelrite e o pH 5,2 promoveu a obtenção de embriões primários e secundários com capacidade para regenerar plantas apresentando características morfológicas semelhantes a planta matriz.

The present work had as objectives to induce the formation of somatic embryos in vitro on Phalaenopsis hybrid Classic Spotted Pink, using different nutrient medium and assess the internal morphology of these embryos by means of histological and histochemical analysis. Young leaves of plants grown in vitro were used as explants for induction of somatic embryos in different nutrient medium: New Dogashima Medium, containing ANA (0.537 μM) and BAP (4.440 μM) plus phytagel and with pH 5.8 (NDM) and the Murashige & Skoog with half the concentration of salts, plus NNA (0.537 μM) and TDZ (13.621 μM), jellied with gelrite and pH 5.2 (0.5 MS). Primary somatic embryos were obtained to 90 days of cultivation in half MS and have been transferred to the same means for obtaining of secondary embryos. The primary and secondary somatic embryos were subcultived for MS with half the concentration of salts, without fitoregulator subjected to photoperiod of 16 hours, which stimulated the production of chlorophyll in primary embryos as secondary, promoting the development of those in protocorms and later in plants. The histological analysis showed that the somatic embryos were formed directly from the epidermal layers of the explants, without going through the phase of callus, featuring direct somatic embryogenesis. The histochemical methods used made it possible to highlight the deposition of starch and lipids in cells embriogenics as a result of physiological mechanisms, enabling the development of primary and secondary embryos in plants. Therefore, the medium 0.5 MS Plus ANA (0.537 μM) and TDZ (13.621 μM), jellied with gelrite and pH 5.2 promoted to obtain primary and secondary embryos with ability to regenerate plants showing morphological similar the mother plant.

El presente trabajo tuvo como objetivos inducir la formación de embriones somáticos in vitro en el híbrido Phalaenopsis Classic Spotted Pink, utilizando diferentes medios nutritivos, y evaluar la morfología interna de estos embriones mediante análisis histológico e histoquímico. Hojas jóvenes de plantas cultivadas in vitro se utilizaron como explantes para la inducción de embriones somáticos en diferentes medios nutritivos: New Dogashima Medium, contenido de ANA (0.537 mM) y BAP (4.440 μM) además de phytagel y con pH 5.8 (NDM) y el Murashige Skoog con la mitad de la concentración de sales, además de ANA (0.537 μM) y TDZ (13.621 μM), gelificado gelrite y pH 5.2 (½ MS). Se obtuvieron embriones somáticos primarios a los 90 días de cultivo en el medio ½ MS y a estos se les transfirió al mismo medio (½ MS) para la obtención de embriones secundarios. Los embriones somáticos primarios y secundarios fueron subcultivados para MS con la mitad de la concentración de sales, sin reguladores de crecimiento y sometidos a fotoperiodo de 16 horas, lo que estimuló la producción de clorofila tanto en los embriones primarios como en los secundarios, promoviendo el desarrollo de los protocormos y más tarde en las plantas. Los análisis histológicos demostraron que los embriones somáticos fueron formados directamente en las capas epidérmicas de los explantes, sin pasar por la fase de callo, vía embriogénesis somática directa. Los métodos histoquímicos hicieron posible destacar la deposición de almidón y lípidos en las células embriogénicas como resultado de mecanismos fisiológicos, que permiten el desarrollo de los embriones primarios y secundarios en las plantas. Por lo tanto, el medio ½ MS contenido de ANA (0.537 μM) y TDZ (13.621 μM), con gelrite y pH 5.2 permitió obtener embriones primarios y secundarios con capacidad para regenerar plantas con caracteres morfológicos similares a los dela planta matriz.

Light in intermediate acclimatization of in vitro germinated seedlings of Dendrobium phalaenopsis Deang Suree / Luz na aclimatização intermediária de mudas germinadas in vitro de Dendrobium phalaenopsis Deang Suree

The success in micropropagation of Dendrobium phalaenopsis Deang Suree is high, but when transplanted into the greenhouse, their survival is minimal. To increase survival in production in the present study it was evaluated the effect of intermediate acclimatization for 30 days in a grow room utilizing the following luminosity conditions: 1- white fluorescent light (B) (18.9µmol m-2 s-1); 2- white fluorescent light + red fluorescent light (GRO-LUX(r)) (BV) (14.85µmol m-2 s-1); 3- red fluorescent light (GRO-LUX(r)) (V) (9.45µmol m-2 s-1) and the control plants were accommodated directly in a greenhouse (162.0µmol m-2 s-1). After this the leaves were characterized anatomically and the plants transferred to the control greenhouse. It was evaluated survival percentage and final number of roots, and calculated the relations between the final and initial values of fresh weight, number of leaves, length and diameter of the largest pseudo bulb, number of pseudo bulbs and longest root length. Only plants submitted to red light, were statistically better than the control in relation to the survival percentage and in relation to fresh weight, while the control showed a higher number of roots that plants acclimatized in this luminosity conditions. Intermediate acclimatization, using red light or red + white light, is recommended for D. phalaenopsis Deang Suree.

O sucesso na micropropagação de Dendrobium phalaenopsis Deang Suree é alto, porém, quando transplantado para o viveiro, sua sobrevivência é mínima. Com o intuito de aumentar a sobrevivência na produção, no presente trabalho, avaliou-se o efeito da aclimatização intermediária, por 30 dias em sala de crescimento, sob as seguintes condições de luminosidade: 1- luz fluorescente branca (18,9µmol m-2 s-1); 2- luz fluorescente branca + luz fluorescente vermelha (14,85µmol m-2 s-1); e 3- luz fluorescente vermelha (9,45µmol m-2 s-1), o controle foi acondicionado diretamente em viveiro coberto (162,0µmol m-2 s-1). Na sequência, as folhas foram caracterizadas anatomicamente e as plantas foram transferidas para o viveiro que continha o controle. Foi avaliada a porcentagem de sobrevivência e o número final de raízes, sendo calculadas também as relações entre os valores finais e iniciais de massa fresca, número de folhas, comprimento e diâmetro do maior pseudobulbo, número de pseudobulbos e comprimento da maior raiz das plantas aclimatizadas. Apenas as plantas submetidas à luz vermelha foram estatisticamente superiores ao controle, em relação à porcentagem de sobrevivência e relação de massa fresca, enquanto que o controle apresentou maior número de raízes que plantas aclimatizadas nessa condição de luminosidade. Aclimatização intermediária, utilizando luz vermelha ou a luz vermelha + branca, é recomendada para D. phalaenopsis Deang Suree.

Multiplicação de abacaxizeiros ornamentais em diferentes concentrações de bap e períodos de avaliação do desenvolvimento in vitro / Multiplication of ornamental pineapple in different concentrations of bap and periods evaluation of development in vitro

O mercado mundial de flores tem apresentando grande destaque econômico nos últimos anos. Apesar do estabelecimento das técnicas de micropropagação, melhorias no protocolo ainda são necessárias. Neste sentido, objetivou-se com o presente trabalho avaliar a multiplicação in vitro das espécies de abacaxi ornamental Ananas comosus var. bracteatus e A. comosus var. erectifolius em diferentes períodos de avaliação e concentrações de BAP. Foram utilizados como explantes gemas axilares de abacaxizeiro ornamental. Utilizou-se delineamento experimental inteiramente casualizado, composto de 16 tratamentos com 5 repetições e fatorial 2x4x2, sendo um explante por tubo de ensaio. Para o número de folhas produzidas, a variedade erectifolius foi mais eficiente que a bracteatus; na avaliação realizada aos 60 dias, o número de folhas foi 2 vezes superior aos 30 dias na concentração de 1 mg.L-1 de BAP. A variedade erectifolius apresentou folhas mais compridas na concentração de 1 mg.L-1 de BAP. O comprimento das folhas diminuiu com o aumento da concentração de BAP. O número de raízes produzidas não foi influenciado pela variedade, nem pelo período de avaliação. A variedade erectifolius apresentou maior comprimento de raiz principal que a bracteatus na ausência de BAP aos 60 dias. A concentração de 3 mg.L-1 de BAP foi mais eficiente na formação de brotos. A variedade bracteatus aos 60 dias apresentou a maior formação de brotos em abacaxizeiro ornamental.

The world market for flowers is showing great economic prominence in recent years. Despite the establishment of micropropagation techniques, improvements are still needed in the protocol. In this sense, the aim of the present study was to evaluate the in vitro multiplication of species of ornamental pineapple Ananas comosus var. bracteatus and A. comosus var. erectifolius at different evaluation periods and concentrations of BAP. Were used as explants axillary buds of ornamental pineapple. We used a completely randomized design, consisting of 16 treatments with 5 replicates and 2x4x2 factorial design, with one explant per tube. For leaf number, variety erectifolius was more efficient than bracteatus; evaluation performed 60 days, the number of leaves was two times higher than at 30 days at the concentration of 1 mg l-1 BAP. The variety erectifolius presented leaves longer in the concentration of 1 mg L-1 BAP. The length of the leaves decreased with increasing concentration of BAP. The number of roots produced was not influenced by the variety, or the evaluation period. The variety erectifolius showed greater root length main bracteatus that in the absence of BAP at 60 days. The concentration of 3 mg L-1 BAP was more effective in the formation of shoots. The variety bracteatus at 60 days showed the highest shoot formation in ornamental pineapple.

Growth curve and development of the internal calli structure of Eucalyptus camaldulensis Dehn

The objective of this work was to elucidate the growth curve of Eucalyptus camaldulensis Dehn. calli analyzing their anatomical modifications. A sigmoid aspect of the growth curve of the calli fresh matter was observed, with five different phases (lag, exponential, linear, deceleration and decline). In the lag phase, the highest growth percentage 87%, was observed, which reduced during the evaluation period to 17% in the linear phase. As for the anatomical analyses, cellular multiplications was observed during the lag and exponential phases and increase in cell size during the linear phase, promoting the calli volume growth and the establishment of the globular conformation.

Germinação assimbiótica e desenvolvimento de Dendrobium nobile Lindl. sob efeito de reguladores vegetais no tratamento pré-germinativo / Asymbiotic germination and development of Dendrobium nobile Lindl. under the effect of plant growth regulators in pre-germinative treatment

O presente trabalho teve como objetivo estudar a influência dos reguladores vegetais BAP e GA3 como tratamentos pré-germinativos no processo de germinação e desenvolvimento inicial de plântulas de Dendrobium nobile, espécie importante pelas propriedades farmacológicas como anti-oxidante, vasodilatadora e até mesmo anti-cancerígena, além do valor ornamental. Os tratamentos pré-germinativos consistiram de BAP e GA3, separadamente, nas concentrações de 0,0; 1,0; 2,0 e 5,0 mg L-1. Após seis meses da semeadura in vitro e manutenção em câmara de germinação e de crescimento com temperatura e foto-período controlados (12 horas e 23ºC ± 2), foram avaliados os parâmetros número de sementes germinadas, porcentagem de germinação, massa fresca e altura das plântulas, diâmetro e número de pseudobulbos, número de folhas, número de raízes, e o comprimento da maior raiz. O delineamento experimental foi inteiramente casualizado. Todas as variáveis foram submetidas à análise de variância e de regressão, quando significativas. As sementes de D. nobile germinaram melhor na ausência de reguladores vegetais e os tratamentos com BAP ou GA3 na embebição das sementes pouco beneficiaram o desenvolvimento in vitro de D. nobile.

The present study aimed to investigate the influence of plant growth regulators BAP and GA3 as pre-germinative treatment in the process of germination and initial development of seedlings of Dendrobium nobile, a species important for its pharmacological properties like antioxidant, vasodilator and even anticancer, besides its ornamental value. Pre-germinative treatments consisted of BAP and GA3, separately, at the concentrations of 0.0; 1.0; 2.0 and 5.0 mg L-1. At six months after in vitro sowing and maintenance in a germination and growth chamber with controlled temperature and photoperiod (12 hours and 23ºC ± 2), the following parameters were evaluated: number of germinated seeds, percentage of germination, fresh mass and height of seedlings, number and diameter of pseudo-bulbs, number of leaves, number of roots, and length of the largest root. The experimental design was completely randomized. All variables underwent analysis of variance and regression analysis when significant. D. nobile seeds presented better germination in the absence of plant growth regulators and the treatments with BAP or GA3 in seed imbibition little benefited D. nobile in vitro development.

Aplicações da cultura de tecidos vegetais em fruteiras do Cerrado / Applications of tissue culture techniques in Brazilian Cerrado fruit trees

Atualmente, percebe-se uma preocupação em relação às plantas do cerrado, com grande enfoque nas fruteiras em função de suas características e usos. Apesar de ser uma área ainda pouco explorada, é crescente o número de estudos dessas espécies nativas, dentre eles, os que abrangem as técnicas de cultura de tecidos. Isso se deve uma vez que essa ferramenta biotecnológica permite a propagação de espécies com dificuldade de germinação, minimiza o problema de sementes recalcitrantes, promove a produção de mudas em larga escala, complementa bancos de germoplasma e facilita as trocas de materiais genéticos. Dessa maneira, esta revisão visa a sumarizar o histórico e panorama atual das aplicações da cultura de tecidos em fruteiras do cerrado, proporcionando sustentação para novos estudos.

Currently, it's been given a huge concern to the cerrado plants, focusing on fruit trees due to their characteristics and uses. Despite being a fairly unexplored area, the number of studies on these native species has increased, especially those involving tissue culture techniques. That's because this biotechnological tool provides the propagation of species with germination difficulty, reduces problems of recalcitrant seeds, promotes large scale seedling production, complements germplasm banks and facilitates the exchange of genetic materials. Therefore, this review summarizes the history and current situation of tissue culture techniques applied to Brazilian Cerrado fruit trees, providing support to further studies.

Micropropagação de Dioscorea multiflora Grised / Micropropagation of Dioscorea multiflora Grised

Dioscorea multiflora uma planta nativa do Sul do Brasil produz a diosgenina como metabólito secundário majoritário, uma substância potencialmente usada pela indústria farmacêutica para a produção de cortisona e substâncias com ação contraceptiva. Objetivou-se, neste trabalho otimizar o protocolo de micropropagação de D. multiflora, visando a produção de mudas em escala comercial. Segmentos nodais subcultivados em meio MS sólido foram transferidos para multiplicação em meio MS suplementado com BAP (0,01; 0,1; 0,5; 1,0 e 3,0 mg L-1)e meio MS suplementado com 0,1 mg L-1 ou 0,5 mg L-1 de BAP acrescido de diferentes concentrações de sacarose (2, 4, 6, 8 e 10 por cento). Para o enraizamento, as brotações foram cultivadas em meio MS suplementado com AIB (0,1; 0,5; 1,0 e 3,0 mg L-1) e meio MS suplementado com ANA (0,1; 0,5; 1,0 e 3,0 mg L-1). Os experimentos in vitro foram instalados em delineamento experimental inteiramente casualizado e cada tratamento constituiu-se de 3 repetições e 10 cubetas/parcela. Plântulas com e sem raízes foram aclimatizadas em casa de vegetação. Melhores resultados de multiplicação e enraizamento foram obtidos em meio MS + 0,1 mg L-1 de BAP (80 por cento) e em meio MS + 1,0 mg L-1 de AIB (42,6 por cento), respectivamente. Não houve diferença quanto à porcentagem de sobrevivência das plântulas in vitro e ex vitro durante a aclimatização (75 por cento). O protocolo de micropropagação para D. Multiflora é efetivo e pode ser usado para a produção em escala comercial.

Dioscorea multiflora is a plant native to southern Brazil that produces diosgenin as a major secondary metabolite, a substance which is used by the pharmaceutical industry for the production of cortisone and substances with contraceptive action. The objective of this work was to optimize the micropropagation protocol of D. multiflora, for the production of seedlings on a commercial scale. Nodal segments subcultured in solid MS medium were transferred for multiplication to MS medium supplemented with BAP (0.01, 0.1, 0.5, 1.0 and 3.0 mg L-1) and MS medium supplemented with 0.1 mg L-1 or 0.5 mg L-1 BAP plus different concentrations of sucrose (2, 4, 6, 8 and 10 percent). For rooting, the shoots were cultured on MS medium supplemented with IBA (0.1, 0.5, 1.0 and 3.0 mg L-1) and MS medium supplemented with NAA (0.1, 0.5, 1.0 and 3.0 mg L-1). A completely randomized design was used with treatment consisting of 3 replicates with 10 buckets per plot. Seedlings with and without roots were acclimatized in a greenhouse. The best results of multiplication and rooting were obtained in MS medium + 0.1 mg L-1 BAP (80 percent) and in MS medium + 1.0 mg L-1 IBA (42.6 percent), respectively. There was no difference in the survival percentage of seedlings in vitro and during ex vitro acclimatization (75 percent). The micropropagation protocol for production of D. multiflora is effective and can be used for commercial production.

Protoplast production from napier grass and pearl millet triploid hybrids / Obtenção de protoplastos de híbridos triplóides entre o capim-elefante e milheto

The objective of this work was to obtain protoplasts from napier grass and pearl millet triploid hybrids as a basis for future studies on chromosomal duplication. Explants were taken from mesophyll of in vitro- and in vivo-cultured plants or from calli of two triploid hybrids (H1 and H2), which were treated with enzymatic solutions containing different concentrations of cellulase R-10 (0.5, 1.0, 1.5 and 2.0 percent) with an additional 0.2 percent macerozyme and 0.1 percent driselase or 1.0 percent pectolyase Y-23 and 0.5 percent hemicellulase. Enzymatic digestion was monitored once every hour for five hours. Protoplasts were obtained from in vitro and in vivo leaflets of both triploid hybrids, and in vitro leaflets were the best explant sources. The quantity of produced protoplasts varied according to the hybrid, the enzymatic solution and the treatment time.

Objetivou-se, neste trabalho, a obtenção de protoplastos de híbridos triplóides entre o capim-elefante e o milheto como base para futuros trabalhos de duplicação cromossômica. Foram utilizados explantes de mesofilo de plantas cultivadas in vitro e in vivo, ou de calos de dois híbridos triplóides (H1 e H2), os quais foram tratados com soluções enzimáticas em diferentes concentrações da enzima celulase R-10 (0,5; 1,0; 1,5 e 2,0 por cento), acrescidas de 0,2 por cento macerozyme e 0,1 por cento driselase ou 1,0 por cento pectolyase Y-23 e 0,5 por cento hemicelulase. A digestão enzimática foi monitorada a cada hora durante 5 horas. Obtiveram-se protoplastos a partir de folhas in vitro e in vivo dos dois híbridos triplóides, sendo as folhas in vitro as melhores fontes de explante. A quantidade de protoplastos variou em função do híbrido, da solução enzimática e do tempo de tratamento.

In Vitro Cultivation of Cymatocarpus solearis (Brachycoeliidae) Metacercariae to Obtain the Adult Stage without the Marine Turtle Definitive Host

In vitro cultivation of trematodes would assist studies on the basic biology of the parasites and their hosts. This is the first study to use the yolk of unfertilized chicken eggs as a simple and successful method of ovocultivation and the first time to obtain the adult-stage of the trematode Cymatocarpus solearis Braun, 1899 (Digenea: Brachycoeliidae). Chicken eggs were inoculated with metacercariae from the muscle of the spiny lobster, Panulirus argus (Latreille, 1804). The metacercariae were excysted and incubated for 576 hr (24 days) at 38degrees C to obtain the adult stage. Eggs in utero were normal in shape and light brown color. The metacercariae developed into mature parasites that have been identified as the adult-stage found in marine turtles. The adult lobsters collected in Quintana Roo State, Mexico, showed the prevalence of 49.4% and the mean intensity of 26.0 per host (n = 87). A statistical study was performed to determine that no parasitic preference was detected for male versus female parasitized lobsters. Morphometric measurements of the adult-stage of C. solearis obtained in our study have been deposited in the National Helminths Collection of the Institute of Biology of the National Autonomous University of Mexico. This study is significant because it is the first time that a digenean of the family Brachycoeliidae has been demonstrated to develop in vitro from metacercariae into adults capable of producing eggs using the yolk of unfertilized chicken eggs. Secondly, this technique allows to obtain the adult stage of C. solearis without the presence of its marine turtle host, allows us to describe the mature parasites, and thus contribute to our understanding of the biology of C. solearis.

Plasmodium falciparum Cultivation Using the Petri Dish: Revisiting the Effect of the 'Age' of Erythrocytes and the Interval of Medium Change

Differences in the characteristics of the culture conditions can influence the multiplication rate of Plasmodium falciparum. The Petri dish method is one of the most popular methods of cultivating this parasite. In many previous studies, ideal culture conditions of the Petri dish method were achieved by using erythrocytes collected from blood that had been stored for at least 2 weeks, with daily changes of the medium. In the present study, we studied the multiplication rate of P. falciparum in cultures containing erythrocytes of various ages together with changing the medium at various intervals of time. Our results strongly suggest that the rate of in vitro multiplication of P. falciparum was higher in freshly collected erythrocytes than in aged erythrocytes regardless of the anticoagulant and that when the parasitemia is lower than 8% with a hematocrit of 5%, the medium change interval can be as long as 48 hr without a great reduction in the rate of multiplication.

OBSERVATION OF ULTRASTRUCTURE AND DRUG SENSITIVITY OF IN VITRO CULTURED EXOERYTHROCYTIC FORM OF PLASMODIUM BERGHEI / 中国寄生虫学与寄生虫病杂志

The infrastructure of in vitro cultured exoerythrocytic stage (EE) of Plasmodium ber-ghei (P.b.)was observed under transmission electron microscope (TEM).The drug sensitivity of EE was also measured in vitro.The EE was cultured in monolayer host cell,fixed and embeded in situ.The ultrathin sections were prepared and examined by routine methods.The TEM pictures showed that the fine structure of in vitro cultured EE was similar with that of EE grown in rat hepatocytes in vivo,as described by Meis et al.The parasite was found within a parasitophorous vacuole,with nucleus,mitochondria,endoplasmic reticuia and Golgi apparatus were observed inside the parasite.After 24h cultivation,the EE was incubated for 48h in medium containing a serial concentrations of primaquine or chlo-roquine,then examined under light microscope.The percentage of abnormal parasites was calculated.The preliminary results showed that the sensitivity of cultured P.b.EE to primaquine and chloroquine was significantly different.At the same concentration of 1?10-5 mol/L,the percentage of abnormal parasites was 38.5?3.9% and 5.7?1.9% ,respectively These results demonstrated that the in vitro cultivation system of P.b.EE would have potential utility in antimalarial drug research.(Figs.1-6)