Toxic, cytotoxic and genotoxic potential of synthetics food flavorings / Potencial tóxico, citotóxico y genotóxico de saborizantes sintéticos de alimentos

Food flavorings in general are few studied for the toxicological aspect. This condition justifies toxicity, cytotoxicity and genotoxicity assessments of the substances. In the present study, the toxicity of banana, cherry and hazelnut flavorings was evaluated in meristematic cells of roots of Allium cepa, in pure form (as marketed) and in the concentrations of 12.5; 25 and 50%, after 24 and 48 hours of exposure. Toxic potential of these food additives was also evaluated against Artemia salina nauplii at concentrations of 0.78; 1.56; 3.12; 6.25; 12.5; 25 and 50%, after 24 hours of exposure. The three additives, in all treatments and times of analysis considered, caused significant inhibition of cell division in A. cepa, however did not cause cellular alterations to the evaluated meristems. These food flavorings also caused significant mortality to micro crustaceans with LC50<100 μg/mL. From this, under the conditions of mentioned analyzes, cherry, banana and hazelnut flavorings induced significant toxicity and cytotoxicity to the bioassays used.

En general, los aspectos toxicológicos de los saborizantes de los alimentos son poco estudiados. Esta condición justifica las evaluaciones de toxicidad, citotoxicidad y genotoxicidad de estas sustancias. En el presente estudio, se evaluó la toxicidad de los aromas de plátano, cereza y avellana en células meristemáticas de raíces de Allium cepa, en forma pura (según comercializa) y en concentraciones de 12.5; 25 y 50%, después de 24 y 48 horas de exposición. El potencial tóxico de estos aditivos alimentarios también se evaluó frente a nauplios de Artemia salina a concentraciones de 0,78; 1,56; 3.12; 6.25; 12.5; 25 y 50%, después de 24 horas de exposición. Los tres aditivos, en todos los tratamientos y tiempos de análisis considerados, causaron inhibición significativa de la división celular en A. cepa, sin embargo, no causaron alteraciones celulares a los meristemos evaluados. Estos saborizantes alimentarios también causaron una mortalidad significativa a microcrustáceos con LC50 <100 μg/ mL. A partir de esto, bajo las condiciones de los análisis descriptos, los aromatizantes de cereza, plátano y avellana indujeron toxicidad significativa y citotoxicidad para los bioensayos utilizados.

Toxicity of the goji berry fruit associated with artificial excipients and dried without additives / Toxicidade do fruto goji berry associado a excipientes artificiais e desidratado sem aditivos

The aim of this study was to evaluate the cytotoxic and genotoxic potential of goji berry fruit-based pharmaceutical powders obtained from three pharmaceutical laboratories. The product A was tested at concentrations of 0.012; 0.025 and 0.05 g mL-1, and B and C at concentrations 0.02; 0.04 and 0.08 g mL-1. It was also evaluated the tea of the dried goji berry fruit (non-additives) in the concentrations 0.035; 0.07 and 0.14 g mL-1 for comparison to the results obtained with powdered goji berry. Tea concentrations in the two exposure times did not cause inhibition of cell division nor cellular alterations to meristem tissues. For the industrialized goji products, all concentrations analyzed caused significant antiproliferative effect to the tissues evaluated at the shortest time of analysis. There were no significant cellular changes in tissues exposed to industrialized goji. Therefore, under the conditions of analysis, goji berry powder, at the three concentrations evaluated, was cytotoxic to root meristems.

Objetivou-se na presente pesquisa avaliar, em células meristemáticas de raízes de A. cepa, nos tempos de exposição 24 e 48 horas, o potencial citotóxico e genotóxico de produtos farmacêuticos do fruto goji berry em pó, provenientes de três laboratórios farmacêuticos. O produto A foi avaliado nas concentrações 0,012; 0,025 e 0,05 g mL-1, e B e C nas concentrações 0,02; 0,04 e 0,08 g mL-1. Avaliou-se também o chá do fruto seco de goji (não aditivado), nas concentrações 0,035; 0,07 e 0,14 g mL-1, para comparação com os resultados obtidos do fruto em pó. Verificou-se que o chá nas concentrações avaliadas, nos dois tempos de exposição estabelecidos, não ocasionaram inibição da divisão celular e nem alterações celulares aos meristemas de raízes. Para os goji industrializados, todas as concentrações analisadas causaram efeito antiproliferativo significativo aos tecidos avaliados logo no menor tempo de análise considerado. Nenhum dos produtos industrializados causou número significativo de alterações aos meristemas analisados. Assim, os goji em pó foram citotóxicos ao bioensaio utilizado por terem acarretado relevante instabilidade genética aos meristemas de raízes.

Antiproliferative and genotoxic effects of nature identical and artificial synthetic food additives of aroma and flavor / Efeito antiproliferativo e genotóxico de aditivos alimentares de aroma e sabor sintéticos, dos tipos idêntico ao natural e artificial

Abstract This study aimed to analyze the antiproliferative and genotoxic potential of synthetic food flavorings, nature identical passion fruit and artificial vanilla. This assessment used root meristem cells of Allium cepa L., in exposure times of 24 and 48 hours and using doses of 0.2; 0.4 and 0.6 mL. Roots were fixed in Carnoy’s solution, hydrolyzed in hydrochloric acid, stained with acetic orcein and analyzed with optical microscope at 400× magnification, 5,000 cells for each treatment. For data analysis, it was used Chi-square test at 5%. Doses of 0.2 mL at ET 48 h; 0.4 and 0.6 mL at ET 24 and 48 h of passion fruit flavor, and the three doses of the vanilla flavor at ET 24 and 48 h significantly reduced the cell division rate in the meristems of roots, proving to be cytotoxic. Doses of 0.2; 0.4 and 0.6 mL of the passion fruit additive, and the three doses of vanilla tested, in the two exposure times, induced mitotic spindle changes and micronuclei formation in the cells of the test organism used, proving to be genotoxic. Therefore, under the studied conditions, flavoring solutions of vanilla and passion fruit, marketed nationally and internationally, significantly altered the functioning of the cell cycle in root meristem cells of A. cepa.

Resumo Neste trabalho teve-se por objetivo analisar o potencial antiproliferativo e genotóxico de aromatizantes alimentares sintéticos, idêntico ao natural de Maracujá, e artificial de Baunilha. Esta avaliação foi realizada por meio das células meristemáticas de raízes de Allium cepa L., nos tempos de exposição de 24 e 48 horas e nas doses de 0,2; 0,4 e 0,6 ml. As raízes foram fixadas em solução de Carnoy, hidrolisadas em ácido clorídrico e coradas com orceína acética. Analisou-se, em microscópio óptico em aumento de 400×, 5.000 células por grupo tratamento, e utilizou-se o teste estatístico Qui-quadrado a 5% para análise dos dados. Verificou-se que as doses de 0,2 ml, no TE 48 h; 0,4 e 0,6 ml, nos TE 24 e 48 h, do aromatizante de Maracujá, e as três doses analisadas, nos TE 24 e 48 h, do aditivo de Baunilha reduziram significativamente o índice de divisão celular dos meristemas de raízes, mostrando-se citotóxicas. As doses 0,2; 0,4 e 0,6 ml do aditivo de Maracujá, e a de 0,6 ml do aromatizante de Baunilha, nos dois tempos de exposição considerados, induziram alterações de fuso mitótico e micronúcleos as células do organismo de prova utilizado, mostrando-se genotóxicas. Portanto, nas condições analisadas, as soluções aromatizantes de Baunilha e Maracujá, comercializadas nacional e internacionalmente, alteraram significativamente o funcionamento do ciclo celular das células meristemáticas de raízes de A. cepa.

Meristem culture and subsequent micropropagation of Chilean strawberry (Fragaria chiloensis (L) Duch. )

BACKGROUND: Vegetative propagation of Fragaria sp. is traditionally carried out using stolons. This system of propagation, in addition to being slow, can spread plant diseases, particularly serious being viral. In vitro culture of meristems and the establishment of micropropagation protocols are important tools for solving these problems. In recent years, considerable effort has been made to develop in vitro propagation of the commercial strawberry in order to produce virus-free plants of high quality. These previous results can serve as the basis for developing in vitro-based propagation technologies in the less studied species Fragaria chiloensis. RESULTS: In this context, we studied the cultivation of meristems and establishment of a micropropagation protocol for F. chiloensis. The addition of polyvinylpyrrolidone (PVP) improved the meristem regeneration efficiency of F. chiloensis accessions. Similarly, the use of 6-benzylaminopurine (BAP) in the culture media increased the average rate of multiplication to 3-6 shoots per plant. In addition, the use of 6-benzylaminopurine (BAP), had low levels (near zero) of explant losses due to oxidation. However, plant height as well as number of leaves and roots were higher in media without growth regulators, with average values of 0.5 cm, 9 leaves and 4 roots per plant. CONCLUSIONS: For the first time in Chilean strawberry, meristem culture demonstrated to be an efficient tool for eliminating virus from infected plants, giving the possibility to produce disease free propagation material. Also, the addition of PVP into the basal MS medium improved the efficiency of plant recovery from isolated meristems. Farmers can now access to high quality plant material produced by biotech tools which will improve their technological practices.

Antimitotic and antimutagenic action of the Hymenaea stigonocarpa bark on dividing cells / Ação antimitótica e antimutagênica do ritidoma de Hymenaea stigonocarpa Mart ex Hayne na divisão celular

Abstract The objective of this study was to evaluate the action of Hymenaea stigonocarpa bark hydroalcoholic extract against a mutagenic compound using A. cepa meristematic root cells as a test system. The treatment groups were: Negative Control (NC) – distilled water; Positive Control (PC) – paracetamol at a concentration of 0.008 mg/mL, Jatoba Control (JC) – aqueous fraction jatobá-do-cerrado at 0.5 or 1.0 or 1.5 mg/mL, and Simultaneous Treatment (ST) - jatobá-do-cerrado aqueous fraction at a concentration of 0.5 or 1.0 or 1.5 mg/mL associated with paracetamol solution at a concentration of 0.008 mg/mL. All groups were analyzed at 24 and 48 h. Five onion bulbs (five replications) were used for each treatment group. The root tips were fixed in Carnoy and slides prepared by the crush technique. Cells were analyzed throughout the cell cycle, totaling 5,000 for each treatment group at each exposure time. Mitotic indices were subjected to statistical analysis using the chi-square test (p<0.05). From the results it was found that the ST group, at the three concentrations, significantly potentiated the antiproliferative effect of the test system cells when compared to PC, NC and TJ at the three concentrations. Furthermore, the three ST concentrations significantly reduced the number of cell aberrations when compared to the number of aberrant cells obtained for the PC, demonstrating antimutagenic action on the A. cepa test system cells.

Resumo O objetivo do presente trabalho foi avaliar a ação do extrato hidroalcólico do ritidoma de Hymenaea stigonocarpa frente a um composto mutagênico, utilizando como sistema teste as células meristemáticas de raízes de A. cepa. Os grupos tratamentos avaliados foram: Controle Negativo (CN) – água destilada; Controle Positivo (CP) – paracetamol na concentração de 0,008 mg/mL, Controle Jatobá (CJ) – fração aquosa de jatobá-do-cerrado na concentração de 0,5 ou 1,0 ou 1,5 mg/mL, e Tratamento Simultâneo (TS) – fração aquosa de jatobá-do-cerrado na concentração de 0,5 ou 1,0 ou 1,5 mg/mL associada a solução de paracetamol na concentração de 0,008 mg/mL. Todos os grupos foram analisados nos tempos de 24 e 48 h. Para cada grupo tratamento cinco bulbos de cebolas (cinco repetições) foram utilizados. As radículas foram fixadas em Carnoy e as lâminas preparadas pela técnica de esmagamento. Analisaram-se células em todo ciclo celular, totalizando 5.000 para cada grupo tratamento em cada tempo de exposição. Os índices mitóticos obtidos foram submetidos à análise estatística do Qui-quadrado (p<0,05). A partir dos resultados verificou-se que o grupo TS, nas três concentrações, potencializou o efeito antiproliferativo significativo as células do sistema teste quando comparado ao CP, CN e TJ nas três concentrações. Ainda, o TS nas três concentrações reduziu de forma significativa o número de aberrações celulares quando comparado com o número de células aberrantes obtidas para o CP, demonstrando ação antimutagênica as células do sistema teste A. cepa.

Cytotoxicity of Cheese and Cheddar Cheese food flavorings on Allim cepa L root meristems / Citotoxicidade de aromatizantes alimentares de Queijo e Queijo Cheddar sob meristemas de raízes de Allim cepa L

Abstract Despite their great importance for the food industry, flavorings, in general, raise a number of questions regarding their cytotoxicity, mutagenicity and carcinogenicity, since, in the literature, there are few studies found evaluating the toxicity on the systemic and cellular level, of these chemical compounds. The root meristems of Allium cepa (onion) are widely used for the assessment of toxicity of chemical compounds of interest. Thus, this study aimed to evaluate, in A. cepa meristematic cells, individually and in combination at the cellular level, the toxicity of synthetic Cheese and Cheddar Cheese food flavorings, identical to the natural, at doses of 1.0 and 2.0 mL, at exposure times of 24 and 48 hours. In combination we used 0.5 mL of Cheese flavor associated with 0.5 mL of Cheddar flavor; and 1.0 mL of Cheese flavor associated with 1.0 mL of Cheddar flavor, at exposure times of 24 and 48 hours. For these evaluations, we used groups of five onion bulbs, which were first embedded in distilled water and then transferred to their respective doses. The root tips were collected and fixed in acetic acid (3:1) for 24 hours. The slides were prepared by crushing and were stained with 2% acetic orcein. Cells were analyzed throughout the cell cycle, totaling 5,000 for each control and exposure time. The mitotic indices calculated and cellular aberrations observed were subjected to statistical analysis using the chi-square test (p <0.05). No chromosomal abnormalities nor those of mitotic spindle were observed for the treatments performed. The results, both individually and in combination, showed that the flavorings under study significantly reduced the cell division rate of the test system cells used. Therefore, under the conditions studied, the two flavorings were cytotoxic.

Resumo Apesar da grande importância para a indústria alimentícia, os aromatizantes, em geral, suscitam uma série de dúvidas quanto a sua citotoxicidade, mutagenicidade e carcinogenicidade, visto que, na literatura, poucos são os trabalhos encontrados avaliando a toxicidade, em nível sistêmico e celular, destes compostos químicos. Os meristemas de raízes de Allium cepa (cebola) são muito utilizados para a avaliação da toxicidade de compostos químicos de interesse. Desta forma, este trabalho teve por objetivo avaliar em células meristemáticas de A. cepa, de forma individual, a toxicidade em nível celular de aromatizantes alimentares sintéticos, idênticos aos naturais, de sabores Queijo e Queijo Cheddar, nas doses de 1,0 e 2,0 mL, nos tempos de exposição de 24 e 48 horas; e de forma associada, onde se utilizou 0,5 mL do aromatizante sabor Queijo associado a 0,5 mL do aromatizante sabor Queijo Cheddar; e 1,0 mL do aromatizante sabor Queijo associado a 1,0 mL do aromatizante sabor Queijo Cheddar, nos tempos de exposição de 24 e 48 horas. Para estas avaliações utilizou-se grupos de cinco bulbos de cebolas, que primeiramente foram enraizados em água destilada, e em seguida transferidos para as suas respectivas doses. As radículas foram coletadas e fixadas em ácido acético (3:1) por 24 horas. As lâminas foram preparadas pela técnica de esmagamento e coradas com orceína acética a 2%. Analisaram-se células em todo ciclo celular, totalizando 5.000 para cada controle e tempo de exposição. Os índices mitóticos calculados e as aberrações celulares observadas foram submetidos à análise estatística do Qui-quadrado (p<0,05). Não foram observadas alterações cromossômicas e anomalias de fuso mitótico para nenhum dos tratamentos realizados. Os resultados obtidos, tanto individualmente como de forma associada, mostraram que os aromatizantes em estudos reduziram de forma significativa os índices de divisões celulares das células do sistema teste utilizado. Portanto, nas condições analisadas, os dois aromatizantes foram citotóxicos.

Evaluation of cytotoxicity and genotoxicity of Hancornia speciosa latex in Allium cepa root model / Avaliação da citotoxicidade e genotoxicidade do látex de Hancornia speciosa usando o modelo da raiz de Allium cepa

Abstract The latex obtained from Hancornia speciosa Gomes (Mangabeira tree) is widely used in traditional medicine to treat a variety of diseases, including diarrhea, ulcer, gastritis, tuberculosis, acne and warts. In this study, the cytotoxicity and genotoxicity effects of H. speciosa latex on the root meristem cells of Allium cepa were examined. Onion bulbs were exposed to different concentrations of latex and then submitted to microscopic analysis using Giemsa stain. Water was used as a negative control and sodium azide as a positive control. The results showed that, under the testing conditions, the mitotic index (MI) of the onion roots submitted to latex treatment did not differ significantly from the negative control, which suggests that the latex is not cytotoxic. Low incidence of chromosome aberrations in the cells treated with H. speciosa latex was also observed, indicating that the latex does not have genotoxic effect either. The MI and the chromosome aberration frequency responded to the latex concentration, requiring more studies to evaluate the dosage effect on genotoxicity. The results indicate that in tested concentrations H. speciosa latex is probably not harmful to human health and may be potentially used in medicine.

Resumo O látex obtido de Hancornia speciosa é amplamente utilizado na medicina popular para tratar uma variedade de doenças, tais como: diarreia, úlcera, gastrite, tuberculose, acne e verrugas. Nesse estudo, foram avaliados os efeitos citotóxicos e genotóxicos do látex de H. speciosa sobre as células meristemáticas das raízes de Allium cepa. Os bulbos das cebolas foram expostos a diferentes concentrações de látex e depois submetidos à analise microscópica usando o corante Giemsa. A água foi usada como controle negativo e a ázida sódica como controle positivo. Os resultados mostraram que o índice mitótico (IM) das raízes de cebola submetidas ao tratamento com látex, nas condições testadas, não diferiram significativamente do controle negativo, e sugerem que o látex não é citotóxico. Também foi observada uma baixa incidência de aberrações cromossômicas nas células tratadas com látex de H. speciosa, o que sugere que o látex também não possui efeito genotóxico. O IM e a frequência de aberrações cromossômicas foram dependentes da concentração de látex. Outros estudos devem ser realizados para avaliar o efeito da dose na genotoxidade. Os resultados indicam que o látex de mangabeira, nas concentrações testadas, provavelmente não é danoso para saúde humana e pode ter potencial para ser usado na medicina.

Efeito de metabólitos produzidos por Trichoderma spp. sobre o índice mitótico em células das pontas de raízes de Allium cepa / Effect of metabolites produced by Trichoderma spp. on mitotic index in cells of Allium cepa root tips

Microrganismos do solo produzem metabólitos importantes para a produção de compostos biologicamente ativos. Dentre estes microrganismos, as espécies de trichoderma (Trichoderma spp.) possuem a capacidade de produzir metabólitos. A promoção do desenvolvimento de plantas por trichoderma pode estar relacionada, entre outros fatores, ao estimulo à multiplicação celular, através da produção de hormônios, entre outros. O método de avaliação do índice mitótico em raízes de Allium cepa é validado pelo Programa Internacional de Segurança Química (IPCS, OMS) e o Programa Ambiental das Nações Unidas (UNEP) como um eficiente teste para análise e monitoramento in situ. O objetivo do estudo foi selecionar cepas e produtos biológicos comerciais de trichoderma, utilizados no controle biológico de fitopatógenos que promovam o aumento do índice mitótico em células de pontas de raízes pelo sistema teste de A. cepa. Foram utilizadas cepas de Trichoderma harzianum (2B2, 2B22, 2B12); Trichoderma viride (TSM1, TSM2, C1) e os produtos biológicos comerciais de trichoderma, Agrotrich® e Trichodermil®. Os bulbos de Allium cepa foram arranjados em dez tratamentos (quatro repetições por tratamento), oito com metabólitos de cepas de Trichoderma spp. e dois controles na ausência de metabólitos. A contagem das células ocorreu na região meristemática, onde foram contadas 500 células por bulbo em cada uma das lâminas. Foram observadas as células em interfase, prófase, metáfase, anáfase e telófase, com auxílio de microscópio ótico com a objetiva de 40X. Foram contadas as células de cada uma das fases do ciclo celular estudadas, após calculou-se os índices mitóticos. A análise estatística dos dados foi realizada pelo teste c 2 (Qui-quadrado), com probabilidade de 5%, pelo programa estatístico BioEstat 4.0. Os tratamentos T1, T3 e T4 (metabólitos das cepas 2B2, 2B12 e C1 de trichoderma, respectivamente) apresentaram os maiores números de células para as fases prófase, metáfase, anáfase e telófase e alcançaram os maiores IM (c 2= 5.45 a= 0.05), (c 2= 5.0 a= 0.05) e (c 2= 3.92 a= 0.05), respectivamente. O tratamento controle T10 (meio líquido BD), apresentou o maior número de células em interfase, e o menor nas fases da divisão celular. O teste com raízes de Allium cepa possibilita selecionar cepas de trichoderma que induzem o aumento do índice mitótico em pontas de raízes pela ação de metabólitos e apresenta variabilidade entre as cepas estudadas.

Soil microorganisms produce metabolites important for the production of biologically active compounds. In relation to these organisms, species of trichoderma (Trichoderma spp.) have the ability to produce metabolites. Promoting the development of plants by trichoderma may be related, among other factors, to stimulate cell proliferation through the production of hormones, among others. The method of evaluation of mitotic index in Allium cepa roots is validated by the International Programme on Chemical Safety (IPCS, WHO) and United Nations Environment Programme (UNEP) as an effective test for in situ analysis and monitoring. The aim of the study was to select strains and commercial biological products trichoderma used in biological control of plant pathogens that promote increased mitotic index in cells of the root tips of A. cepa test system. Strains of Trichoderma harzianum (2B2, 2B22, 2B12) and Trichoderma viride (TSM1, TSM2, C1) and the biological products commercials Agrotrich® and Trichodermil® of trichoderma were used. The bulbs of Allium cepa were arranged in ten treatment (four replicates per treatment), eight with metabolite of Trichoderma spp. and two controls in the absence of metabolites. Cell counts occurred on the meristematic region where 500 cells were counted for each bulb on the blades. Cells were observed in interphase, prophase, metaphase, anaphase and telophase, with the aid of an optical microscope with a 40X objective. The cells were counted in each phase of the cell cycle analyzed and after it was calculated mitotic rates. Statistical analysis of data was performed by c 2 test (chi-square), with a probability of 5%, the statistical program BioEstat 4.0. Treatments T1, T3 and T4 (metabolites of isolates 2B2, 2B12 and C1 of trichoderma, respectively) showed higher cell numbers for the phases prophase, metaphase, anaphase and telophase and reached the highest IM (c 2= 5.45 a= 0.05), (c 2= 5.0 a= 0.05) e (c 2= 3.92 a= 0.05, respectively. The T10 control (liquid medium BD), had the highest number of interphase cells, and the lower phases of cell division. The test with Allium cepa roots enables selecting trichoderma isolates that induce increased mitotic index in root tips by the action of metabolites and shows variability among the isolates studied.

Origin of the subepidermal tissue in Piper L. leaves / Origem do tecido subepidérmico em folhas de Piper L.

Studies on the anatomy of Piper leaves demonstrate the presence of a subepidermal tissue distinct from the adjacent epidermis, which cells show thin walls and hyaline contents. Some authors consider such cells a hypodermal tissue, while others refer to them as components of a multiple epidermis. In this study, the nature of this subepidermal tissue was investigated through the analysis of leaf ontogeny in three Piper species. The analysis showed that the referred tissue originates from the ground meristem and, thus, should be considered a hypodermis. The studied species suggests that the role of the hypodermis would be to protect the photosynthetic apparatus from excess light, regulating the intensity of light reaching the chlorophyll parenchyma.

Os estudos de anatomia foliar de espécies de Piper revelam a presença de um tecido subepidérmico distinto da epiderme e cujas células apresentam paredes finas e conteúdo hialino. Estas células são referenciadas por alguns autores como um tecido hipodérmico e por outros, como sendo constituintes de uma epiderme múltipla. Nesse estudo verificou-se a natureza deste tecido subepidérmico a partir da análise da ontogênese foliar de três espécies de Piper. A análise revelou que o referido tecido tem origem do meristema fundamental e, portanto, deve ser denominado de hipoderme. Para as espécies avaliadas, sugere-se que a hipoderme teria função de, proteger o aparato fotossintético do excesso de luminosidade, regulando a intensidade luminosa que atinge o parênquima clorofiliano.

Multiplicação in vitro de segmentos apicais caulinares de segurelha (Satureja hortensis L. ) / In vitro multiplication of apical shoot segments of Summer Savory (Satureja hortensis L. )

O presente estudo teve por objetivo avaliar o efeito de BAP, na presença e ausência de ANA, sobre a multiplicação in vitro de segmentos apicais caulinares de Satureja hortensis. Os explantes foram isolados de plântulas germinadas in vitro e cultivados em meio nutritivo MS. O delineamento experimental utilizado foi o inteiramente casualizado, em esquema fatorial 2x5, correspondendo às concentrações de ANA (0 e 1 µM) e de BAP (0; 5; 10; 15 e 20 µM), com seis repetições, cada uma composta por três explantes. Para a porcentagem de explantes com brotações houve efeito significativo para o fator BAP, aumentando à medida que cresce a concentração da citocinina. A variável "número de brotos por explante" apresentou interação entre os fatores, havendo a maior formação de brotos na presença de ANA na faixa de 10 - 15 µM de BAP. Na presença da auxina, o maior valor ocorreu com 15 µM de BAP tendendo a diminuir independente da presença de ANA. O enraizamento dos segmentos apicais foi maior na presença de ANA e ausência de BAP, diminuindo com o aumento da citocinina. O número de folhas foi influenciado pela maior concentração de BAP sendo que a 15 µM foi observado o maior número, tendendo à queda com aumento da concentração. A concentração de 15 µM de BAP, independente de ANA, proporcionou os melhores resultados na multiplicação da espécie produzindo aumento de brotações e número de folhas, à exceção do enraizamento que foi influenciado pela auxina.

This study aimed to evaluate the effect of BAP in the presence and absence of NAA, on the in vitro multiplication of shoot apical segments of Satureja hortensis. The explants were isolated from seedlings germinated in vitro and cultured in nutrient medium MS. The experimental design was completely randomized in a 2x5 factorial arrangement, corresponding to the concentrations of NAA (0 and 1 µM) and BAP (0, 5, 10, 15 and 20 µM), with six replicates, each consisting of three explants. For the percentage of explants with shoots, there was no significant effect for the factor BAP, increasing as the concentration of BAP grows. The variable number of shoots per explant showed interaction between factors, with the highest shoot formation in the presence of NAA and BAP in the range of 10 -15 µM. In the presence of auxin, the highest value occurred with 15 µM BAP, tending to decline regardless of the presence of NAA. The rooting of the apical segments was higher in the presence of NAA and absence of BAP, decreasing with increasing cytokinin. Leaf number was influenced by the higher concentration of BAP, and the amount of 15 µMhadthe largest number, tending to decrease with increasing concentration. The concentration of 15 µM BAP, regardless of NAA, provided better results in the multiplication of the species, producing increased number of shoots and leaves, except for rooting, whichwas influenced by auxin.

Diferentes meios de cultura e antioxidantes no estabelecimento in vitro do Jacarandá da Bahia / Different culture media and antioxidants in vitro establishment of Jacaranda da Bahia

O Jacarandá da Bahia é uma espécie florestal nativa pertencente a família Leguminosaea Papilionoideae com ocorrência desde o sul da Bahia até o estado de São Paulo. O extrativismo e a pecuária contribuíram para a sua quase extinção e apesar disso, pouco tem sido feito para a multiplicação desta espécie. A micropropagação é uma alternativa para obtenção de grande quantidade de mudas sadias em curto espaço de tempo. Foram utilizados meristemas apicais e gemas axilares de plântulas de Jacarandá da Bahia, cultivadas em condições assépticas nos tratamentos compostos por meios de cultura (MS e WPM), concentrações de BAP (4, 9 e 18 µM) e com a ausência ou presença de agente antioxidante (PVP ou carvão ativado), totalizando 18 tratamentos, cada um com 10 repetições. As avaliações foram realizadas com 4 e 8 semanas de cultivo, considerando a porcentagem de explantes oxidados. Os meios de cultura de antioxidantes testados não diferiram estatisticamente entre si, porém tratamentos com BAP na concentração de 18 µM foram os que apresentaram maior oxidação.

Dalbergia nigra is a native forest species belonging the Papilionoideae-Leguminosaea family occurring from southern Bahia to the state of Sao Paulo. The extraction and livestock contributed to its near extinction and yet little has been done to the multiplication of this species. The micropropagation techniques are an alternative in order to achieve a large amount of seedlings in a short time and under conditions plant without diseases. Used apical and lateral meristems of Dalbergia nigra seedlings, grown in aseptic conditions in treatments consisting of different culture media (MS and WPM), BAP concentrations (4, 9 and 18 µM) and the absence or presence of antioxidant agent (PVP or activated charcoal), all crossed each other, totaling 18 treatments, each with 10 repetitions. Evaluations were performed at 4 and 8 weeks of cultivation, considering the percentage of browning explants. The culture media of antioxidants tested did not differ statistically, but BAP at a concentration of 18 µM were those with the highest oxidation.

Genotoxicity and toxicity evaluations of ECF cellulose bleaching effluents using the Allium cepa L. Test / Genotoxicidade e toxicidade em Allium cepa L.: avaliação da qualidade do efluente no processo de branqueamento da celulose

Toxicity and genotoxicity tests were performed on root cells of Allium cepa in order to evaluate wastewater quality following an ECF cellulose bleaching process. The results revealed a toxic effect of the effluent, with inhibition of meristem growth and generally lower values of metaphase, anaphase and telophase indices at pH 10.5 than pH 7 for all effluent concentrations. The genotoxicity effect was different from the toxic effect given that the micronucleus and the chromosomal aberration tests in anaphase-telophase cells were low over all ranges of the studied effluent concentrations.

Os testes de toxicidade e genotoxicidade foram realizados em células da raiz de Allium cepa, a fim de avaliar a qualidade do efluente na sequência de um processo de branqueamento de celulose ECF. Os resultados revelaram um efeito tóxico do efluente, com inibição de crescimento do meristema e valores geralmente baixos de metáfase, anáfase e índices de telófase, a pH 10,5 e pH 7, para todas as concentrações do efluente. O efeito de genotoxicidade foi diferente do efeito tóxico, uma vez que o micronúcleo e os testes de aberrações cromossômicas em anáfase-telófase das células foram baixos em todas as gamas de concentrações do efluente estudado.

Elimination of TSWV from impatiens hawkerii Bull. and regeneration of virus-free plant

The possibility for obtaining virus free plants from Impatiens hawkerii Bull. shoots infected with Tomato spotted wilt virus (TSWV) through meristem-tip culture was examined. TSWV presence in I. hawkerii plants was detected by DAS-ELISA and RT-PCR and identification of the virus was confirmed by sequencing one of the chosen isolate (GenBank Accesion CQ132190). Meristem-tip explants (0.3-1.5 mm) from virus-infected shoots are cultured on MS media supplemented with different concentrations of the cytokinins, CPPU or TDZ (0.01-1.0 uM), respectively. Using this system, a large number of in vitro shoots could be produced from a single explant. Also, cytokinins showed a stimulatory effect on the length, fresh and dry weights of the newly formed shoots. Plant pigments content in I. hawkerii shoots increased significantly in the presence of cytokinins. Rooting of shoots was spontaneous on the same media. Rooted plantlets were transferred to soil where 97 percent successfully acclimatized. By DAS-ELISA and RT-PCR, 80 percent of the in vitro plantlets were shown to be a virus-free. Considering these, the present protocol seems to be an efficient method for in vitro generation of virus-free I. hawkerii plantlets by meristem tip cultures.

Cytogenotoxicity of Cymbopogon citratus (DC) Stapf (lemon grass) aqueous extracts in vegetal test systems

The lemon grass, Cymbopogon citratus (DC) Stapf, is an important species of Poaceae family commonly used in the folk medicine in many countries. The aim of this study was to investigate the cytotoxic and genotoxic effects of aqueous extracts from C. citratus leaves on Lactuca sativa (lettuce) root tip meristem cells by cytogenetic studies that have never been done before for lemon grass extracts. For this, lettuce seeds were treated for 72h with different concentrations of lemon grass aqueous extracts (5; 10; 20 and 30 mg/mL). The percentage of germination, root development and cellular behavior were analyzed, and the results showed that the highest concentration of aqueous extracts reduced the mitotic index, the seed germination and the root development of lettuce. The extracts have also induced chromosome aberrations and cellular death in the roots cells of L. sativa.

O capim-limão, Cymbopogon citratus (DC) Stapf, é uma importante espécie da família Poaceae com uma comum utilização na medicina popular em vários países. O objetivo deste estudo foi investigar os efeitos citotóxicos e genotóxicos do extrato aquoso das folhas de C. citratus em células meristemáticas de Lactuca sativa (alface) por meio de estudos citogenéticos, uma vez que estudos desta natureza não existem para extratos aquosos de capim-limão. Para isso, sementes de alface foram tratadas por 72h com diferentes concentrações de extratos aquosos feitos das folhas de capim-limão (5, 10, 20 e 30 mg/mL). O percentual de germinação, desenvolvimento radicular e o comportamento celular foram avaliados e os resultados mostraram que as concentrações mais elevadas dos extratos aquosos reduziram o índice mitótico, o percentual de germinação das sementes e desenvolvimento radicular da alface. Os extratos também induziram aberrações cromossômicas e morte celular nas células das raízes de L. sativa.

Effect of morphological heterogeneity of somatic embryos of Melia azedarach on conversion into plants

Embryogenic cultures were initiated from immature Melia azedarach (Meliaceae) zigotic embryos. Explants were induced on Murashige and Skoog (1962) medium with 4.54 microM thidiazuron or 0.45 microM dichlorophenoxyacetic acid. After 6 weeks of culture on induction medium, somatic embryos were categorized in four morphological classes based on the presence of single or fused embryos and if they remained united or not to the original explant; that were evaluated histologically. The somatic embryos of every category were transferred, in groups or individually, on a 1/4 MS medium. Bipolar embryos, the more typically normal ones, had well defined shoot and root apical meristems and produced single plants; subcultured individually their conversion was 28%, and subcultured in groups the conversion declined to 6.8%. Fused embryos subcultured in groups had only a 2.1% conversion and produced plants with fused stems. None conversion rate in the others classes was associated to poorly developed shoot and root meristematic areas or with their absence. The converted plants were acclimatized and transferred, in a mist, to soil, with an independent of the class 95% survival rate.

Cytogenetical and ultrastructural effects of copper on root meristem cells of Allium sativum L

Different copper concentrations, as well as different exposure times, were applied to investigate both cytogenetical and ultrastructural alterations in garlic (Allium sativum L.) meristem cells. Results showed that the mitotic index decreased progressively when either copper concentration or exposure time increased. C-mitosis, anaphase bridges, chromosome stickiness and broken nuclei were observed in the copper treated root tip cells. Some particulates containing the argyrophilic NOR-associated proteins were distributed in the nucleus of the root-tip cells and the amount of this particulate material progressively increased with increasing exposure time. Finally, the nucleolar material was extruded from the nucleus into the cytoplasm. Also, increased dictyosome vesicles in number, formation of cytoplasmic vesicles containing electron dense granules, altered mitochondrial shape, disruption of nuclear membranes, condensation of chromatin material, disintegration of organelles were observed. The mechanisms of detoxification and tolerance of copper are briefly discussed.

Determination of vanadium accumulation in onion root cells (Allium cepa L) and its correlation with toxicity

The vanadium is a metal that presents great interest from the toxicological point of view, because of the numerous alterations that can take place in different biological systems. This work evaluated the capacity of vanadium accumulation and its correlation with genotoxic effects in root cells of Allium cepa L. The bulbs were cultivated in renovated filtered water each 24 h, at a temperature of 25 +/- 0.5 degrees C, in darkness and constant aeration. Treatments were carried out under the same experimental conditions, using water solutions of vanadium of 25, 50, 75 and 100 microg/g for 0, 12, 24, 48 and 72 h. A control was carried out where metal solution was substituted by distilled water. After the treatment, the meristems were fixed with alcohol--acetic acid (3:1) and stained according to the technique of Feulge n. The capacity of accumulation was determined by GFAAS. The analysis of the results revealed an accumulation of the metal for all times and concentrations. No correlation was presented among vanadium accumulation, growth and mitotic index; however, positive correlation was given with the induction of chromosomic aberrations. In conclusion, vanadium is able to induce cytotoxic effect in the exposed roots, but only genotoxic effect was correlated with metal accumulation.

Evaluation of cytotoxic potential of latex of Calotropis procera and Podophyllotoxin in Allum cepa root model

In the present study we have utilized the Allium cepa root tip meristem model to evaluate the cytotoxic and anti-mitotic activities of latex of Calotropis procera (DL) and podophyllotoxin. Standard cyto-toxic drug cyclophosphamide and non-cytotoxic drugs cyproheptadine and aspirin served as controls. Like cyclophosphamide, both DL and podophyllotoxin significantly inhibited the growth of roots and mitotic activity in a dose-dependent manner. However, podophyllotoxin was more potent in this regard and produced root decay. Cyproheptadine and aspirin, on the other hand, showed a marginal effect on the root growth and mitotic activity at much higher concentrations

The G2 checkpoint activated by DNA damage does not prevent genome instability in plant cells

Root growth, G2 length, and the frequency of aberrant mitoses and apoptotic nuclei were recorded after a single X-ray irradiation, ranging from 2.5 to 40 Gy, in Allium cepa L. root meristematic cells. After 72 h of recovery, root growth was reduced in a dose-dependent manner from 10 to 40 Gy, but not at 2.5 or 5 Gy doses. Flow cytometry plus TUNEL (TdT-mediated dUTP nick end labeling) showed that activation of apoptosis occurred only after 20 and 40 Gy of X-rays. Nevertheless, irrespective of the radiation dose, conventional flow cytometry showed that cells accumulated in G2 (4C DNA content). Simultaneously, the mitotic index fell, though a mitotic wave appeared later. Cell accumulation in G2 was transient and partially reversed by caffeine, thus it was checkpoint-dependent. Strikingly, the additional G2 time provided by this checkpoint was never long enough to complete DNA repair. Then, in all cases, some G2 cells with still-unrepaired DNA underwent checkpoint adaptation, i.e., they entered into the late mitotic wave with chromatid breaks. These cells and those produced by the breakage of chromosomal bridges in anaphase will reach the G1 of the next cell cycle unrepaired, ensuring the appearance of genome instability.

Meristematic activity of the Endodermis and the Pericycle in the primary thickening in monocotyledons: considerations on the "PTM"

A proposta deste trabalho é mostrar uma nova interpretação do meristema de espessamento primário em monocotiledôneas. Anatomia dos órgãos vegetativos das seguintes espécies foi examinada: Cephalostemon riedelianus (Rapataceae), Cyperus papyrus (Cyperaceae), Lagenocarpus rigidus, L. Junciformis (Cyperaceae), Echinodorus paniculatus (Alismataceae) and Zingiberofficinale (Zingiberaceae). A atividade meristemática da endoderme foi observada nas raizes de todas as espécies, no caule de Cyperus, Cephalostemum e Lagenocarpus rigidus, e no traço foliar de Cyperus e folha de Echinodorus. Considerando a continuidade dos tecidos através da raiz, caule e folha, as autoras concluem que no caule o periciclo permanece ativo durante a vida da planta, como um gerador de tecidos vasculares. O "Meristema de Espessamento Primário" é o periciclo em fase meristemática, juntamente com a endoderme e suas derivadas (ou apenas o periciclo). Próximo ao ápice caulinar, esses tecidos se assemelham a um único meristema, dando origem ao córtex interno e aos tecidos vasculares.