Synthesis and Phytocytogenotoxic Activity of N-Phenyl-2-phenoxyacetamides Derived from Thymol.

The excessive use of herbicides has caused a series of problems related to human health, environmental pollution, and an increase in the resistance of plants to commercial herbicides. As an alternative, natural compounds and their semisynthetic derivatives have been widely studied to obtain environmentally friendly and more effective herbicides than the usual ones. In view of these factors, the aim of this work was to synthesize new molecules with herbicidal potential using thymol as a starting material, a natural phenol that has a pronounced phytotoxic effect. Novel N-phenyl-2-thymoxyacetamides were synthesized and characterized by MS and by 1H and 13C NMR. All prepared molecules were subjected to phytotoxic and cytotoxic activity assays using Lactuca sativa L. and Sorghum bicolor L. as model plants. Molecules containing chlorine in the para position of the thymoxy group exhibited phytotoxic and cytogenotoxic effects superior to those of the commercial herbicides 2,4-D and glyphosate.

Phytogenotoxicity of thymol and semisynthetic thymoxyacetic acid in pre/post emergence of model plants and weeds.

Herbicides are commonly used to control weed. However, some plants are resistant to such products. To identify less harmful herbicides, it is crucial to search for different mechanisms of action. Thymol is an easily acquired allelopathic compound, capable of producing its respective semisynthetic derivative, thymoxyacetic acid. The aim of this study was to determine the effects of thymol and thymoxyacetic acid molecules as bioherbicides in greenhouse at the concentration of 3 mmol L-1 in pre- and postemergence applications in five species: Amaranthus viridis L., Cucumis sativus L., Lactuca sativa L., Eleusine indica L., and Sorghum bicolor L. The initial seedling development and DNA changes were analyzed. These molecules were contrasting with the solvent, in the negative control, and with the glyphosate, in the positive control, promoting phytogenotoxic activities. The toxic effect of thymoxyacetic acid was more effective in preemergence and thymol's in postemergence. We also observed a reduction in the germination speed index and root growth with a negative correlation to the increase in potassium leaching. Damage to the root and shoot of the seedlings was verified at the DNA level, and the phytotoxicity of the plants treated with the herbicide glyphosate was similar to the plants treated with the natural molecules tested. The bioherbicidal effect of thymol and thymoxyacetic acid exacerbates the reduction of the environmental impact caused by the disordered and increased use of residual pesticides.

Short-term changes related to autotetraploidy in essential oil composition of Eucalyptus benthamii Maiden & Cambage and its applications in different bioassays.

Some forest trees have been polyploidized to improve their traits and to supply new germplasms for breeding programs. As trees have a long juvenile stage, the early characterization of the chromosome set doubling effects is crucial for previous selection. Thus, we aimed to characterize the chemical variability of essential oils from diploid and autotetraploid germplasms (autotetraploid A and B) of Eucalyptus benthamii, as well as to evaluate their larvicidal and allelopathic effects. Autotetraploid A showed a higher essential oil yield than diploid and autotetraploid B, which did not differ quantitatively. Aromadendrene, viridiflorol and α-pinene were the major compounds in the diploid essential oil. In contrast, compounds were present in autotetraploids, such as 1,8-cineole, limonene, α-terpineol, and α-terpinyl-acetate. Essential oils from the diploid at 50-200 ppm were twice as larvicidal than those from autotetraploids against Aedes aegypti larvae. Considering the phytotoxicity bioassays using Lactuca sativa, essential oils from both ploidy levels affected root growth. Moreover, the essential oils inhibited shoot growth at all concentrations tested (187.5; 375; 750; 1500; and 3000 ppm). Autotetraploid A and B had the same effect on shoot growth as glyphosate. The essential oils had no cytogenotoxic effect on root meristematic cells of L. sativa, whereas phytotoxic potential was identified mainly in shoot growth. This work demonstrated a dramatic change in secondary metabolism (terpene composition) related to an increase in the ploidy level in Eucalyptus germplasms. In addition, we report the novelty of the chemical composition of essential oils among germplasms and their potential use as larvicidal and post-emergence weed control agents.

Phytocytotoxicity of volatile constituents of essential oils from Sparattanthelium Mart. species (Hernandiaceae).

The intensive application of agrochemicals in crops has negatively impacted the environment and other organisms. The use of naturally occurring compounds may be an alternative to mitigate these effects. Plants are secondary metabolite reservoirs and may present allelopathic activity, which is potentially interesting to be used in bioherbicide formulations. In this context, the present work aimed to evaluate the phytotoxic and cytotoxic effects of essential oils extracted from leaves of Sparattanthelium botocudorum and Sparattanthelium tupiniquinorum in bioassays with the plant models Lactuca sativa L. and Sorghum bicolor L. Moench. The essential oils were applied at concentrations of 3,000, 1,500, 750, 375 and 187.5 ppm. Chemical characterization of the oils was performed, and their impact on the percentage of germinated seeds, initial development of L. sativa and S. bicolor seedlings, and changes in the mitotic cycle of meristematic cells from L. sativa roots was evaluated. The major compound of the essential oils was germacrene D, followed by bicyclogermacrene, ß-elemene and germacrene A. The phytotoxicity assay showed that the essential oils of both species reduced the root and shoot growth in L. sativa and decreased the germination and shoot growth in S. bicolor. Inhibition was dependent on the tested oil concentration. In the cytotoxicity assay, a decrease in mitotic index and chromosomal and nuclear alterations were observed, which resulted from aneugenic and clastogenic action.

Phytochemical analysis and effect of the essential oil of Psidium L. species on the initial development and mitotic activity of plants.

The use of allelopathic compounds is an alternative for weeds control, since they present low toxicity when compared with the synthetic herbicides, that may cause several damages, as the contamination of the environment. Our objective was to determine the chemical composition and allelopathic properties of the essential oils of Psidium cattleianum, P. myrtoides, P. friedrichsthalianum, and P. gaudichaudianum on the germination and root growth of Lactuca sativa and Sorghum bicolor, and to evaluate their action on the cell cycle of root meristematic cells of L. sativa. The main compound found in all the studied species was (E)-caryophyllene (P. cattleianum-23.4 %; P. myrtoides-19.3%; P. friedrichsthalianum-24.6% and P. gaudichaudianum-17.0%). The different essential oils were tested at different concentrations on L. sativa and S. bicolor, reducing germination, germination speed index, and root and shoot growth of lettuce and sorghum seedlings. The cytotoxicity and aneugenic potential of these oils were evidenced by the reduction of the mitotic index and increase of the frequency of chromosomal alterations in L. sativa. The essential oils of the species of Psidium studied have potential to be used in weeds control.

Cytogenetic bioassays as tools to distinguish between toxic and non-toxic varieties of Jatropha curcas (Euphorbiaceae)

Abstract The tropical and subtropical naturalized physic nut (Jatropha curcas L.), has been explored for biodiesel production in recent times. The oil is extracted from the seeds and, for the production to be feasible, utilization of the residual seed cake is crucial. Although the cake could be employed as a protein source in animal feed, it is rich in phorbol ester, which is toxic for animals. Therefore, breeding programs have been working to reduce or eliminate the phorbol ester content in physic nut. In this context, the present work aimed to evaluate the physic nut oil of toxic and non-toxic varieties (containing known or undetectable amounts of phorbol ester, respectively) with regards to phytotoxicity in a model experiment with Lactuca sativa L. For this, the percentage of germinated seeds was evaluated after 8, 16, 24, 36 and 48 hours of exposure to the treatments with toxic and non-toxic oil at concentrations of 22.5 %, 45 % and 67.5 % of emulsion (physic nut oil energetically mixed with distilled water). Root growth was determined after 48 hours of exposure and the germination speed index was obtained. The different stages of mitotic division as well as possible chromosomal and nuclear alterations were also recorded. The mitotic index was calculated as the number of dividing cells, as a fraction of the total number of cells, and the frequency of chromosome and nuclear alterations, expressed as the percentage of number of alterations divided by the total number of cells. Both varieties exhibited phytotoxicity, inducing significant reductions in percentage of germinated seeds (reduction of 98 %), germination speed index (reduction of 24.44) and root growth (reduction of 8.54 mm). In microscopic analysis, a mitodepressive effect was observed for both oils at the three concentrations used when compared to the negative control; however, it was possible to distinguish between the toxic and the non-toxic varieties based on the more expressive reduction of division promoted by the first, 2.19 %. Significant increments in the frequency of mitotic cells showing chromosome alterations as well, as the presence of condensed nuclei, were observed in the treated cells. However, these parameters were not significantly different from the control in the cells treated with both physic nut oils. In conclusion, the evaluation of root growth and cell division in the plant model L. sativa, can be proposed as an alternative to animal tests to distinguish the varieties with high and low phorbol ester concentration, thus contributing to the detection of toxicity in varieties used in breeding programs.

Resumen Jatropha curcas L., naturalizado tropical y subtropical, ha sido explorado para la producción de biodiesel. El aceite se extrae de las semillas y, para que la producción sea factible, la utilización de la torta de semillas residual es crucial. Aunque la torta se puede emplear como una fuente de proteína en la alimentación animal, es rica en éster de forbol, que es tóxico para los animales. Por lo tanto, los programas de mejoramiento han procurado reducir o eliminar el contenido de éster de forbol de J. curcas. En este contexto, el presente trabajo tuvo como objetivo evaluar el aceite de J. curcas de las variedades tóxicas y no tóxicas (con cantidades conocidas o indetectables de éster de forbol, respectivamente) con respecto a la fitotoxicidad en el modelo Lactuca sativa L. El porcentaje de semillas germinadas se evaluó después de 8, 16, 24, 36 y 48 horas de tratamiento. El crecimiento de la raíz se determinó después de 48 horas de exposición y se obtuvo el índice de velocidad de germinación. Se registraron las diferentes etapas de la división mitótica así como posibles alteraciones cromosómicas y nucleares. El índice mitótico se calculó como el número de células en división como una fracción del número total de células y la frecuencia de las alteraciones cromosómicas y nucleares, expresada como el porcentaje del número de alteraciones dividido entre el número total de células. Ambas variedades exhibieron fitotoxicidad, induciendo reducciones significativas en el porcentaje de semillas germinadas (Reducción del 98 %), índice de velocidad de germinación (Reducción de 24.44) y crecimiento de raíces (Reducción de 8.54 mm). En el análisis microscópico, se observó un efecto mitodepresivo para ambos aceites. Sin embargo, fue posible distinguir entre las variedades tóxicas y las no tóxicas basándose en la reducción más expresiva de la división promovida por la primera, 2.19 %. Se observaron incrementos significativos en la frecuencia de células mitóticas que mostraban alteraciones cromosómicas, así como la presencia de núcleos condensados en las células tratadas. Sin embargo, estos parámetros no fueron significativamente diferentes del control en las células tratadas con ambos aceites de J. curcas. En conclusión, la evaluación del crecimiento de las raíces y la división celular en el modelo L. sativa se puede proponer como una alternativa a las pruebas en animales para distinguir las variedades con concentraciones altas y bajas de éster de forbol, contribuyendo así a la detección de toxicidad en variedades utilizadas en programas de mejoramiento genético.

The contribution of cytogenetics and flow cytometry for understanding the karyotype evolution in three Dorstenia (Linnaeus, 1753) species (Moraceae).

Chromosome morphometry and nuclear DNA content are useful data for cytotaxonomy and for understanding the evolutionary history of different taxa. However, the chromosome number is the only karyotype aspect reported for the species of Dorstenia so far. In this study, the nuclear genome size of Dorstenia arifolia (Lamarck, 1786), Dorstenia bonijesu (Carauta & C. Valente, 1983) and Dorstenia elata (Hooker, 1840) was evaluated and their karyotype morphometry accomplished, with the aim of verifying the potential of those parameters to understand evolutionary issues. Mean nuclear 2C value ranged from 2C = 3.49 picograms (pg) for Dorstenia elata to 2C = 5.47 pg for Dorstenia arifolia, a variation of ± 1.98 pg. Even though showing a marked difference in 2C value, the three species exhibited the same 2n = 32. Corroborating the flow cytometry data, differences in chromosome morphology were found among the karyotypes of the species investigated. Based on this and the only phylogeny proposed for Dorstenia thus far, structural rearrangements are related to the karyotype variations among the three species. Besides, the karyological analysis suggests a polyploid origin of the Dorstenia species studied here.

C-value reassessment of plant standards: an image cytometry approach.

Image cytometry (ICM) has been used to measure DNA 2C-values by evaluating the optical density of Feulgen-stained nuclei. This optical measurement is carried out using three basic tools: microscopy, digital video camera, and image analysis software. Because ICM has been applied to plants, some authors have remarked that studies should be performed before this technique can be accepted as an accurate method for determination of plant genome size. Based on this, the 2C-value of eight plants, which are widely used as standards in DNA quantifications, was reassessed in a cascade-like manner, from A. thaliana through R. sativus, S. lycopersicum, Glycine max, Z. mays, P. sativum, V. faba, to A. cepa. The mean 2C-values of all plants were statistically compared to the values reported by other authors using flow cytometry and/or ICM. These analyses demonstrated that ICM is an accurate and reliable method for 2C-value measurement, representing an attractive alternative to flow cytometry. Statistical comparison of the results also indicated Glycine max 'Polanka' as the most adequate primary standard. However, distinct authors have been advised that 2C DNA content of the reference standard should be close to that of the sample. As three further approaches also revisited the 2C-value of these eight plants, we have thus proposed a mean 2C-value for each eight species.

Revisiting the DNA C-values of the genome size-standards used in plant flow cytometry to choose the "best primary standards".

Flow cytometry (FCM) techniques have enabled characterization of the genome size for various plant species. In order to measure the nuclear genome size of a species, reference standards with well-established DNA content are necessary. However, different 2C-values have been described for the same species used as reference standard. This fact has brought about inaccurate genome measurements, making relevant the establishment of optimal DNA reference standards for plant cytometric analyses. Our work revisited the genome size of Arabidopsis thaliana and other seven plant standards, which were denominated "Dolezel's standard set" and have been widely used in plant DNA measurements. These eight plant standards were reassessed for a comparative measurement of their DNA content values, using each plant species as primary standard in a cascade-like manner, from A. thaliana to Allium cepa. The genome size values obtained here were compared to those reported in the literature by statistical analyses. As a result, Raphanus sativus and Drosophila melanogaster were considered the most inadequate primary standards, whereas A. thaliana, Solanum lycopersicum and Pisum sativum were found to be the most suitable.