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Nanotechnology has brought about significant progress through the use of goods based on nanomaterials. However, concerns remain about the accumulation of these materials in the environment and their potential toxicity to living organisms. Plants have the ability to take in nanomaterials (NMs), which can cause changes in their physiology and morphology. On the other hand, nanoparticles (NPs) have been used to increase plant development and control pests in agriculture by including them into agrochemicals. The challenges of the interaction, internalization, and accumulation of NMs within plant tissues are enormous, mainly because of the various characteristics of NMs and the absence of reliable analytical tools. As our knowledge of the interactions between NMs and plant cells expands, we are able to create novel NMs that are tailored, targeted, and designed to be safe, thus minimizing the environmental consequences of nanomaterials. This review provides a thorough examination and comparison of the main microscopy techniques, spectroscopic methods, and far-field super-resolution methodologies used to examine nanomaterials within the cell walls of plants.

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This chapter describes a step-by-step protocol for rapid serological quantification of global DNA methylation by enzyme-linked immunosorbent assay (ELISA) in plant tissue culture specimens. As a case study model, we used the coconut palm (Cocos nucifera), from which plumules were subjected to somatic embryogenesis followed by embryogenic calli multiplication. DNA methylation is one of the most common epigenetic markers in the regulation of gene expression. DNA methylation is generally associated with non-expressed genes, that is, gene silencing under certain conditions, and the degree of DNA methylation can be used as a marker of various physiological processes, both in plants and in animal cells. Methylation consists of adding a methyl radical to carbon 5 of the DNA cytosine base. Herein, the global DNA methylation was quantified by ELISA with antibodies against methylated cytosines using a commercial kit (Zymo-Research™). The method allowed the detection of methylation in total DNA extracts from coconut palm embryogenic calli (arising from somatic embryogenesis) cultivated in liquid or solid media by using antibodies against methylated cytosines and enzymatic development with a colorimetric substrate. Control samples of commercially provided Escherichia coli bacterial DNA with previously known methylation percentages were included in the ELISA test to construct an experimental methylation standard curve. The logarithmic regression of this E. coli standard curve allowed methylation quantification in coconut palm samples. The present ELISA methodology, applied to coconut palm tissue culture specimens, is promising for use in other plant species and botanical families. This chapter is presented in a suitable format for use as a step-by-step laboratory procedure manual, with theoretical introduction information, which makes it easy to apply the protocol in samples of any biological nature to evaluate DNA global methylation associated with any physiological process.

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Sugarcane is used to produce sugar, ethanol, and other by-products, so it is considered one of the most important crops worldwide. Using temporary immersion systems for sugarcane micropropagation represents an alternative to reduce the labor force, increase plant development, and improve plant quality. Temporary immersion systems are semi-automated bioreactors designed for the large-scale propagation of tissues, embryos, and organs. These are temporarily exposed in a liquid culture medium under a specific time and immersion frequency. Using this protocol and a temporary immersion bioreactor, it is possible to achieve multiplication and rooting. The use of temporary immersion bioreactors improves the multiplication rate and the rooting of sugarcane, reducing the culture time, labor force, and reagents needed while maintaining high survival rates during acclimatization.

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The Guadalupe cypress (Cupressus guadalupensis S. Watson) is an endangered species included in the list of the NOM-059-SEMARNAT-2010. The presence of wild goats in the habitat has been the greatest threat to the propagation and survival of this species. Therefore, there is a need to generate propagation protocols that facilitate the regeneration of the species. Plant tissue culture offers various possibilities that can facilitate the regeneration of species under some risk. Temporary immersion systems have proven to be an option with various advantages in plant tissue culture, such as increasing the number of seedlings generated and reducing production times, compared to semisolid media. The objective of this chapter is to describe a protocol to propagate Guadalupe cypress tissues in a RITA® temporary immersion system.

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BACKGROUND: The current techniques for determining carbon and nitrogen content to provide information about the nutritional status of plants are time-consuming and expensive. For this reason, the objective of this study was to develop an analytical method for the direct and simultaneous determination of nitrogen and carbon elemental content in soybean leaves using near-infrared spectroscopy and compare the performance of conventional (1100-2500 nm spectral range) and portable equipment (1100-1700 nm spectral range). Partial least-squares regression models were developed using 27 soybean leaf samples collected during the 2021 harvest and applied for the simultaneous determination of carbon and nitrogen in 13 samples collected during the 2022 harvest. RESULTS: The root-mean-square error of prediction values for nitrogen and carbon were low (2.42 g kg-1 and 4.37 g kg-1 respectively) for the benchtop method yielded low but higher for the portable method (3.82 g kg-1 and 10.7 g kg-1 respectively). The benchtop method did not show significant differences when compared with the reference method for determining nitrogen and carbon. In contrast, the portable methodology showed potential as a screening method for determining nitrogen levels, particularly in fieldwork. CONCLUSION: The methodologies evaluated in this study were implemented and evaluated under real crop monitoring conditions, using independent sets of calibration and prediction samples. Their utilization enables the acquisition of cost-effective, safe analytical data aligning with the principles of green analytical chemistry. © 2023 Society of Chemical Industry.

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Plant growth regulators (PGR) are essential for somatic embryogenesis (SE) in different species, and Coffea canephora is no exception. In our study model, previously, we have been able to elucidate the participation of various genes involved in SE by using different strategies; however, until now, we have not used a proteomic approach. This research seeks to contribute to understanding the primary cellular pathways involved in developing SE in C. canephora. The process of our model consists of two stages: (1) preconditioning in MS medium with auxin (NAA) and cytokinin (KIN), and (2) induction in Yasuda liquid medium added with cytokinin (BA). Therefore, in this study, we analyzed different days of the SE induction process using shotgun label-free proteomics. An amount of 1630 proteins was found among different sampling days of the process, of which the majority were accumulated during the induction stage. We found that some of the most enriched pathways during this process were the biosynthesis of amino acids and secondary metabolites. Eighteen proteins were found related to auxin homeostasis and two to cytokinin metabolism, such as ABC, BIG, ILR, LOG, and ARR. Ten proteins and transcription factors related to SE were also identified, like SERK1, SKP1, nuclear transcription factor Y, MADS-box, and calreticulin, and 19 related to other processes of plant development, among which the 14-3-3 and PP2A proteins stand out. This is the first report on the proteomic approach to elucidate the mechanisms that operate during the induction of SE in C. canephora. So, our findings provide the groundwork for future, more in-depth research. Data are available via ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD047172.

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Candidatus Liberibacter spp is the most prevalent microorganism in the citrus plant, associated with Citrus Huanglongbing (HLB), which is transmitted by the psyllid vector. In Colombia, the vector Diaphorina citri Kugayama has been reported in different regions, but "Ca. Liberibacter asiaticus" (CLas) has only been detected in insect vectors, not in citrus host plants. To identify the presence and quantify the pathogen in citrus tissues, we employed a combined strategy that involved three techniques based on polymerase chain reaction (PCR). First, we used endpoint PCR with specific primers for CLas (OI1-OI2c) to confirm the infection. Second, we used qPCR with specific primers CIT295a - CIT298 designed on 16S rDNA gene regions to quantify the pathogen load. Finally, we employed droplet digital PCR (ddPCR) to determine the copy number of the pathogen in citrus tissues using the ß-subunit of ribonucleotide reductase (RNR) gene (nrdB) that is specific to CLas. We identified the presence of CLas in citrus plants for the first time in Colombia and quantified its titer in the plant tissue. We employed ddPCR and qPCR to provide crucial information for the country's disease management, control strategies, and general crop health.

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INTRODUCTION: Cecropia angustifolia Trécul. is a native Andean plant containing high levels of pentacyclic triterpenes (PTs), including several isobaric molecules that serve as chemical markers. Preclinical studies suggest that PTs positively modulate metabolic and vascular diseases. However, their low oral absorption reduces their bioactive effects. OBJECTIVE: The objective of this study was (1) to improve the absorption of PTs from C. angustifolia and (2) to establish a platform to produce biomass or botanical reference material using a strategy for their accumulation. METHODS: MALDI-TOF and UPLC-MS were used to characterize and quantify PTs in different matrices. An in vitro platform for PT production was established. Chemical profiles of triterpenes were also evaluated from wild and in vitro herbal material using TLC coupled with mass spectrometry. RESULTS: To overcome the low absorption of PTs, a premier raw material was used, which increased their bioavailability to 9.2%. Active ingredients in herbal material can vary, and there is an urgent need for standardized extracts using pharmacokinetics as an effective tool to reveal the dynamics of active ingredients in vivo. A temporary immersion system was produced as a promising platform with a total PT accumulation exceeding 50% of the content in the dry fraction, indicating it is a feasible mechanism to produce biomass or botanical reference material. CONCLUSIONS: Plant tissue culture is a promising eco-friendly technology for phytochemical production and a modern strategy to protect biodiversity in natural assets. Alternative and modern, yet environmentally friendly production methods are needed to meet the large demand for herbal products.

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Salt stress is one of the most severe abiotic stresses affecting plant growth and development. The application of silicon (Si) is an alternative that can increase the tolerance of plants to various types of biotic and abiotic stresses. The objective was to evaluate salt stress's effect in vitro and Si's mitigation potential on Aechmea blanchetiana plants. For this purpose, plants already established in vitro were transferred to a culture medium with 0 or 14 µM of Si (CaSiO3). After growth for 30 days, a stationary liquid medium containing different concentrations of NaCl (0, 100, 200, or 300 µM) was added to the flasks. Anatomical and physiological analyses were performed after growth for 45 days. The plants cultivated with excess NaCl presented reduced root diameter and effective photochemical quantum yield of photosystem II (PSII) (ΦPSII) and increased non-photochemical dissipation of fluorescence (qN). Plants that grew with the presence of Si also had greater content of photosynthetic pigments and activity of the enzymes of the antioxidant system, as well as higher values of maximum quantum yield of PSII (FV/FM), photochemical dissipation coefficient of fluorescence (qP) and fresh weight bioaccumulation of roots and shoots. The anatomical, physiological and biochemical responses, and growth induced by Si mitigated the effect of salt stress on the A. blanchetiana plants cultivated in vitro, which can be partly explained by the tolerance of this species to grow in sandbank (Restinga) areas.

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Effective protocols for in vitro rooting for woody fruit trees are still a challenge for in vitro seedling production, especially when there is a need to insert new cultivars or rootstocks. These protocols are essential to accelerate studies in plant breeding programs and for seedling distribution. This study evaluated the use of 6-Benzylaminopurine (IBA) in in vitro rooting of Pyruscomunnis rootstocks, clones 'OHxF87' and Pyrodwarf. Explant exposure times (0, 24, 48, 72, and 96 hours) to 20 mg L-1 IBA were tested for in vitro rooting. The exposure to IBA resulted in rooting rates above 80%, surpassing some results reported in the literature. The 24-hour treatment provided 81,81% survival, leading to an average growth of five roots with 19 mm length, for 'OHxF87' rootstock. The same exposure time resulted in the highest survival rate (75%) and the highest mean root number, seven roots per plant with 10 mm length, for 'PDW' rootstock. Root formation did not occur in the absence of synthetic auxin. Therefore, it can be concluded that a 24-hour exposure at 20 mg L-1 IBA was sufficient to promote in vitro rooting in 'OHxF87' and Pyrodwarf rootstocks'.

Protocolos eficazes de enraizamento in vitro de frutíferas lenhosas ainda são um desafio para produção de mudas in vitro, especialmente quando há necessidade de inserção de novas cultivares ou porta-enxerto. Esses protocolos são essenciais para acelerar estudos nos programas de melhoramento genético e também para distribuição posterior das mudas. Nesse sentido, o objetivo deste estudo foi avaliar a utilização da 6-Benzilaminopurina no enraizamento in vitro de porta-enxerto Pirus comunnis, clones 'OHxF87' e Pyrodwarf. Para o enraizamento, foi testado o tempo de exposição dos explantes ao AIB. Para tanto, foram utilizados 20 mg L-1 do fitohormônio nas horas 0, 24, 48, 72 e 96 horas. A exposição ao AIB resultou em taxas de enraizamento acima de 80%, superando alguns resultados encontrados na literatura. Para o porta-enxerto 'OHxF87', o tratamento de 24 horas proporcionou 81,81% de sobrevivência, promovendo em média cinco raízes com comprimento de 19 mm. O mesmo tratamento para o porta-enxerto 'PDW' resultou na maior taxa de sobrevivência (75%), bem como no maior número médio de raízes, sete raízes por planta, com comprimento de 10 mm. Na ausência de auxina sintética, a formação de raízes não ocorreu. Assim sendo, podemos concluir que o tempo de exposição de 24 horas a 20 mg L-1 de IBA foi suficiente para promover o enraizamento de porta-enxertos Pyrus communis de 'OHxF87' e 'PDW'.

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Hippeastrum hybridum is an important bulbous flower plant in world floriculture, which are propagated conventionally by the technique known as double or twin scales to obtain plants with clonal origin. However, this technique promotes the propagation of systemic diseases, particularly mosaic-inducing viruses. The aim of this paper was to evaluate the somatic embryogenesis (SE) from tepals as an alternative to provide a technique for SE induction and to obtaining virus-free plantlets of Hippeastrum from infected plants. The concentrations of 2,4-Dichlorofenoxiacetic Acid (2,4-D) and thidiazuron (TDZ) was evaluated in SE induction pathway. The monitoring of viruses during the assays with tepals was performed by Reverse Transcription-Polymerase Chain Reaction. SE induction was obtained, for the first time, in tepal segments from flower buds of Hippeastrum. The 2,4-D was the main factor for embryogenic callus induction, and TDZ increased the SE induction rate. However, conversion of somatic embryos into plantlets were only developed in free-2,4-D media, replaced by 1.0 mg L-1 6-Benziladenine. Out of five virus species monitored during the experiment, Cucumber mosaic virus was detected in tepals and Hippeastrum mosaic virus in leaves of donor plants. The SE-derived plantlets that germinated in vitro were acclimatized and tested negative for all viruses assayed.

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Plants that produce secondary metabolites with allelopathic activity or phytotoxicity can be biotechnologically important, serving as sources of allelochemicals, and thus contributing to the agroindustrial sector. Vismia japurensis (Hypericaceae) is an Amazonian species that grows in clumps called vismiais, from which most other plants are absent. Accordingly, the objective of this study was to identify possible phytotoxicity effects of hexane and methanol extracts of Vismia japurensis leaves and branches in vivo and from seedlings grown in vitro on Lactuca sativa. In addition, fresh and dry leaves were assayed by the sandwich method in order to determine their ability to release allelochemicals. The hexanic extract from in vitro seedlings reduced germination by 10%, while the methanol extract produced a 16% reduction in germination speed. Root growth of Lactuca sativa was inhibited by 64.7% when subjected to hexane leaf extract, by 39.3% under the influence of hexane branch extract, and by 96.09% for in vitro seedling hexanic extract. When analysed by thin layer chromatography and 1H nuclear magnetic resonance, extracts showed evidence of terpenes, anthraquinones and flavonoids, with greater intensity of signals in the aromatic region of in vitro seedling hexanic extract. Clearly, Vismia japurensis has a high biotechnological potential in terms of the production of substances of low polarity with capacity to interfere in plant development.

Plantas que produzem metabólitos secundários com atividade alelopática ou fitotóxica podem ser biotecnologicamente importantes, servindo como fontes de aleloquímicos e, assim, contribuindo para o setor agroindustrial. Vismia japurensis (Hypericaceae) é uma espécie amazônica que cresce em grupos, formando vismiais. Assim, o objetivo deste estudo foi identificar possíveis efeitos fitotóxicos de extratos hexânicos e metanólicos de folhas e ramos de Vismia japurensis in vivo e de plântulas cultivadas in vitro sobre Lactuca sativa. Além disso, folhas frescas e secas foram analisadas pelo método sanduíche, a fim de determinar sua capacidade de liberação de aleloquímicos. O extrato hexânico de plântulas in vitro reduziu a germinação em 10% e o extrato metanólico promoveu uma redução de 16% na velocidade de germinação. O crescimento radicular de Lactuca sativa foi inibido em 64,7% quando submetido ao extrato hexânico das folhas, em 39,3% sob influência do extrato hexânico dos galhos e em 96,09% para o extrato de hexânico das plântulas in vitro. Quando analisados por cromatografia em camada delgada e ressonância magnética nuclear de 1H, os extratos mostraram evidências de terpenos, antraquinonas e flavonoides, com maior intensidade de sinais na região aromática do extrato hexânico das plântulas in vitro. Assim, Vismia japurensis possui elevado potencial biotecnológico em termos de produção de substâncias de baixa polaridade com capacidade de interferência no desenvolvimento de plantas.

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Abstract Plants that produce secondary metabolites with allelopathic activity or phytotoxicity can be biotechnologically important, serving as sources of allelochemicals, and thus contributing to the agroindustrial sector. Vismia japurensis (Hypericaceae) is an Amazonian species that grows in clumps called vismiais, from which most other plants are absent. Accordingly, the objective of this study was to identify possible phytotoxicity effects of hexane and methanol extracts of Vismia japurensis leaves and branches in vivo and from seedlings grown in vitro on Lactuca sativa. In addition, fresh and dry leaves were assayed by the sandwich method in order to determine their ability to release allelochemicals. The hexanic extract from in vitro seedlings reduced germination by 10%, while the methanol extract produced a 16% reduction in germination speed. Root growth of Lactuca sativa was inhibited by 64.7% when subjected to hexane leaf extract, by 39.3% under the influence of hexane branch extract, and by 96.09% for in vitro seedling hexanic extract. When analysed by thin layer chromatography and 1H nuclear magnetic resonance, extracts showed evidence of terpenes, anthraquinones and flavonoids, with greater intensity of signals in the aromatic region of in vitro seedling hexanic extract. Clearly, Vismia japurensis has a high biotechnological potential in terms of the production of substances of low polarity with capacity to interfere in plant development.

Resumo Plantas que produzem metabólitos secundários com atividade alelopática ou fitotóxica podem ser biotecnologicamente importantes, servindo como fontes de aleloquímicos e, assim, contribuindo para o setor agroindustrial. Vismia japurensis (Hypericaceae) é uma espécie amazônica que cresce em grupos, formando vismiais. Assim, o objetivo deste estudo foi identificar possíveis efeitos fitotóxicos de extratos hexânicos e metanólicos de folhas e ramos de Vismia japurensis in vivo e de plântulas cultivadas in vitro sobre Lactuca sativa. Além disso, folhas frescas e secas foram analisadas pelo método sanduíche, a fim de determinar sua capacidade de liberação de aleloquímicos. O extrato hexânico de plântulas in vitro reduziu a germinação em 10% e o extrato metanólico promoveu uma redução de 16% na velocidade de germinação. O crescimento radicular de Lactuca sativa foi inibido em 64,7% quando submetido ao extrato hexânico das folhas, em 39,3% sob influência do extrato hexânico dos galhos e em 96,09% para o extrato de hexânico das plântulas in vitro. Quando analisados por cromatografia em camada delgada e ressonância magnética nuclear de 1H, os extratos mostraram evidências de terpenos, antraquinonas e flavonoides, com maior intensidade de sinais na região aromática do extrato hexânico das plântulas in vitro. Assim, Vismia japurensis possui elevado potencial biotecnológico em termos de produção de substâncias de baixa polaridade com capacidade de interferência no desenvolvimento de plantas.

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Plants that produce secondary metabolites with allelopathic activity or phytotoxicity can be biotechnologically important, serving as sources of allelochemicals, and thus contributing to the agroindustrial sector. Vismia japurensis (Hypericaceae) is an Amazonian species that grows in clumps called vismiais, from which most other plants are absent. Accordingly, the objective of this study was to identify possible phytotoxicity effects of hexane and methanol extracts of Vismia japurensis leaves and branches in vivo and from seedlings grown in vitro on Lactuca sativa. In addition, fresh and dry leaves were assayed by the sandwich method in order to determine their ability to release allelochemicals. The hexanic extract from in vitro seedlings reduced germination by 10%, while the methanol extract produced a 16% reduction in germination speed. Root growth of Lactuca sativa was inhibited by 64.7% when subjected to hexane leaf extract, by 39.3% under the influence of hexane branch extract, and by 96.09% for in vitro seedling hexanic extract. When analysed by thin layer chromatography and 1H nuclear magnetic resonance, extracts showed evidence of terpenes, anthraquinones and flavonoids, with greater intensity of signals in the aromatic region of in vitro seedling hexanic extract. Clearly, Vismia japurensis has a high biotechnological potential in terms of the production of substances of low polarity with capacity to interfere in plant development.

Plantas que produzem metabólitos secundários com atividade alelopática ou fitotóxica podem ser biotecnologicamente importantes, servindo como fontes de aleloquímicos e, assim, contribuindo para o setor agroindustrial. Vismia japurensis (Hypericaceae) é uma espécie amazônica que cresce em grupos, formando vismiais. Assim, o objetivo deste estudo foi identificar possíveis efeitos fitotóxicos de extratos hexânicos e metanólicos de folhas e ramos de Vismia japurensis in vivo e de plântulas cultivadas in vitro sobre Lactuca sativa. Além disso, folhas frescas e secas foram analisadas pelo método sanduíche, a fim de determinar sua capacidade de liberação de aleloquímicos. O extrato hexânico de plântulas in vitro reduziu a germinação em 10% e o extrato metanólico promoveu uma redução de 16% na velocidade de germinação. O crescimento radicular de Lactuca sativa foi inibido em 64,7% quando submetido ao extrato hexânico das folhas, em 39,3% sob influência do extrato hexânico dos galhos e em 96,09% para o extrato de hexânico das plântulas in vitro. Quando analisados por cromatografia em camada delgada e ressonância magnética nuclear de 1H, os extratos mostraram evidências de terpenos, antraquinonas e flavonoides, com maior intensidade de sinais na região aromática do extrato hexânico das plântulas in vitro. Assim, Vismia japurensis possui elevado potencial biotecnológico em termos de produção de substâncias de baixa polaridade com capacidade de interferência no desenvolvimento de plantas.

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Plants that produce secondary metabolites with allelopathic activity or phytotoxicity can be biotechnologically important, serving as sources of allelochemicals, and thus contributing to the agroindustrial sector. Vismia japurensis (Hypericaceae) is an Amazonian species that grows in clumps called vismiais, from which most other plants are absent. Accordingly, the objective of this study was to identify possible phytotoxicity effects of hexane and methanol extracts of Vismia japurensis leaves and branches in vivo and from seedlings grown in vitro on Lactuca sativa. In addition, fresh and dry leaves were assayed by the sandwich method in order to determine their ability to release allelochemicals. The hexanic extract from in vitro seedlings reduced germination by 10%, while the methanol extract produced a 16% reduction in germination speed. Root growth of Lactuca sativa was inhibited by 64.7% when subjected to hexane leaf extract, by 39.3% under the influence of hexane branch extract, and by 96.09% for in vitro seedling hexanic extract. When analysed by thin layer chromatography and 1H nuclear magnetic resonance, extracts showed evidence of terpenes, anthraquinones and flavonoids, with greater intensity of signals in the aromatic region of in vitro seedling hexanic extract. Clearly, Vismia japurensis has a high biotechnological potential in terms of the production of substances of low polarity with capacity to interfere in plant development.(AU)

Plantas que produzem metabólitos secundários com atividade alelopática ou fitotóxica podem ser biotecnologicamente importantes, servindo como fontes de aleloquímicos e, assim, contribuindo para o setor agroindustrial. Vismia japurensis (Hypericaceae) é uma espécie amazônica que cresce em grupos, formando vismiais. Assim, o objetivo deste estudo foi identificar possíveis efeitos fitotóxicos de extratos hexânicos e metanólicos de folhas e ramos de Vismia japurensis in vivo e de plântulas cultivadas in vitro sobre Lactuca sativa. Além disso, folhas frescas e secas foram analisadas pelo método sanduíche, a fim de determinar sua capacidade de liberação de aleloquímicos. O extrato hexânico de plântulas in vitro reduziu a germinação em 10% e o extrato metanólico promoveu uma redução de 16% na velocidade de germinação. O crescimento radicular de Lactuca sativa foi inibido em 64,7% quando submetido ao extrato hexânico das folhas, em 39,3% sob influência do extrato hexânico dos galhos e em 96,09% para o extrato de hexânico das plântulas in vitro. Quando analisados por cromatografia em camada delgada e ressonância magnética nuclear de 1H, os extratos mostraram evidências de terpenos, antraquinonas e flavonoides, com maior intensidade de sinais na região aromática do extrato hexânico das plântulas in vitro. Assim, Vismia japurensis possui elevado potencial biotecnológico em termos de produção de substâncias de baixa polaridade com capacidade de interferência no desenvolvimento de plantas.(AU)

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Coffea arabica is one of the most important crops worldwide. In vitro culture is an alternative for achieving Coffea regeneration, propagation, conservation, genetic improvement, and genome editing. The aim of this work was to identify proteins involved in auxin homeostasis by isobaric tandem mass tag (TMT) and the synchronous precursor selection (SPS)-based MS3 technology on the Orbitrap Fusion™ Tribrid mass spectrometer™ in three types of biological materials corresponding to C. arabica: plantlet leaves, calli, and suspension cultures. Proteins included in the ß-oxidation of indole butyric acid and in the signaling, transport, and conjugation of indole-3-acetic acid were identified, such as the indole butyric response (IBR), the auxin binding protein (ABP), the ATP-binding cassette transporters (ABC), the Gretchen-Hagen 3 proteins (GH3), and the indole-3-acetic-leucine-resistant proteins (ILR). A more significant accumulation of proteins involved in auxin homeostasis was found in the suspension cultures vs. the plantlet, followed by callus vs. plantlet and suspension culture vs. callus, suggesting important roles of these proteins in the cell differentiation process.

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Indirect somatic embryogenesis (ISE) establishment for Coffea species started in the 1970s. Since then, intraspecific variations in the morphogenic pathway have been reported, even in the common environmental condition in vitro. Several authors have suggested that these variations are the result of genetic, epigenetic, and/or physiological events, highlighting the need for investigations to know the causes. Along these lines, this study aimed to investigate and describe, for the first time, the global 5-methylcytosine and physiological changes that occur in the cells of the aggregate suspensions of Coffea canephora during proliferation and somatic embryo regeneration steps. The cell proliferation step was characterized by increase in cell mass in all subcultures; relatively low mean values of global 5-methylcytosine (5-mC%), abscisic acid (ABA), and indole-3-acetic acid (IAA); high mean value of 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor); and increase followed by decrease in spermidine (Spd, a polyamine) level. Therefore, these epigenetic and physiologic aspects promoted the cell proliferation, which is fundamental for ISE. In turn, the somatic embryo regeneration was correlated with global 5-mC% and physiological changes. The competence acquisition, determination, and cell differentiation steps were marked by increases in mean values of 5-mC%, IAA and ABA, and decreases in ACC and Spd, evincing that these changes are the triggers for regeneration and maturation of somatic embryos. Therefore, dynamic and coordinated epigenetic and physiologic changes occur in the cells of the aggregate suspensions during the C. canephora ISE in liquid system.

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The main challenge of plant chemical diversity exploration is how to develop tools to study exhaustively plant tissues. Their sustainable sourcing is a limitation as bioguided strategies and dereplication need quite large amounts of plant material. We examine if alternative solutions could overcome these difficulties by obtaining a secure, sustainable, and scalable source of tissues able to biosynthesize an array of metabolites. As this approach would be as independent of the botanical origin as possible, we chose eight plant species from different families. We applied a four steps culture establishment procedure, monitoring targeted compounds through mass spectrometry-based analytical methods. We also characterized the capacities of leaf explants in culture to produce diverse secondary metabolites. In vitro cultures were successfully established for six species with leaf explants still producing a diversity of compounds after the culture establishment procedure. Furthermore, explants from leaves of axenic plantlets were also analyzed. The detection of marker compounds was confirmed after six days in culture for all tested species. Our results show that the first stage of this approach aiming at easing exploration of plant chemodiversity was completed, and leaf tissues could offer an interesting alternative providing a constant source of natural compounds.

Assuntos

RESUMO

RESUMEN La albahaca (Ocimum basilicum L.) es un cultivo promisorio de reciente explotación y expansión en Colombia; sin embargo, esta tendencia se ve limitada por el desconocimiento de la nutrición con elementos, como N y K. Con la aplicación de dosis óptimas de N y K, los productores pueden incrementar la productividad y calidad de este sistema productivo, atrayendo nuevos productores. El objetivo de este estudio fue evaluar el efecto de la aplicación de nitrógeno y potasio sobre el contenido y relación de los macronutrientes en el tejido vegetal de albahaca. Las dosis de N y K fueron combinadas con base en la matriz Box-Berard aumentada (3):2k+2k+2k+1+1, constituyendo un total de 14 tratamientos, dispuestos bajo un diseño completamente al azar, con tres repeticiones. Se estudió la masa seca (MS) y las relaciones N/K, N/P, N/S, N/Ca, K/Ca, K/Mg, K/P y K/S, las cuales, fueron analizas con pruebas de medias y la metodología de superficie de respuesta. La albahaca desarrolló una mayor MS cuando la relación foliar de N y K con el resto de los nutrientes fue alta, a excepción de la relación de estos (N/K), la cual, fue estrecha. En este sentido, la MS fue 116,72% superior para las relaciones asociadas con nitrógeno y 120,84% para las de potasio, con respecto a las dosis mínimas de N y K. En albahaca, la concentración foliar de N y K debe predominar sobre el resto de macroelementos, condición obtenida con dosis de 100-190kg ha-1 de N y de 125-237,5kg ha-1 de K.

ABSTRACT Basil (Ocimum basilicum L.) is a promising crop of recent exploitation and expansion in Colombia, however, this trend is limited by ignorance of nutrition requirements of elements such as N and K. With the application of optimal doses of N and K, producers can increase the productivity and quality of this production system, attracting new producers. The objective of this study was to evaluate the effect of the application of nitrogen and potassium on the content and ratio of macronutrients in basil plant tissue. The doses of N and K were combined based on the increased Box-Berard matrix (3): 2k+2k+2k+1+1, constituting a total of 14 treatments, arranged under a completely randomized design with three repetitions. The dry mass (DM) and the relationships N/K, N/P, N/S, N/Ca, K/Ca, K/Mg, K/P and K/S were studied, and analyzed with tests of means and the response surface methodology. Basil developed a higher DM when the leaf ratio of N and K with the rest of the nutrients was high, except for the ratio of these (N/K), which was close. In this sense, the DM was 116.72% higher for the ratios associated with nitrogen and 120.84% for those of potassium, with respect to the minimum doses of N and K. In basil, the foliar concentration of N and K should predominate over the rest of macroelements, a condition obtained with doses of 100-190kg ha-1 of N and 125-237.5kg ha-1 of K.

RESUMO

RESUMEN La planta Lippia origanoides ha sido ampliamente estudiada debido al efecto antimicrobiano y antifúngico que poseen sus extractos y aceites esenciales, los cuales han sido probados contra un gran número de microorganismos patógenos. Sin embargo, es escasa la literatura que registra la diversidad de bacterias endófitas asociadas a esta especie de plantas. El objetivo del trabajo fue evaluar in vitro la capacidad de promoción de crecimiento vegetal de bacterias endófitas de Lippia origanoides en el municipio de Sincelejo -Sucre, Colombia. En este estudio se aislaron bacterias endófitas en medio de cultivo agar R2A a partir de diferentes tejidos, se evaluó la densidad poblacional (UFC/g de tejido) por conteo en superficie y la promoción de crecimiento vegetal de forma cualitativa en medios selectivos específicos. Se observaron diferencias significativas para la densidad poblacional de bacterias endófitas respecto al tipo de tejido, con mayores valores en la raíz (2,0 x 1010/g raíz), seguido del tallo (1,3 x 1010/g tallo) y hojas (9,2 x 109/g hoja). Se obtuvieron un total de 20 bacterias endófitas, los cuales dos mostraron capacidad solubilizadora de fosfato, fijación biológica de nitrógeno, producción de sideróforos y ACC desaminasa. Los morfotipos TLO5 y RLO4 fueron identificados molecularmente como Bacillus cereus, mostrando buenos resultados de promoción de crecimiento vegetal.

ABSTRACT The Lippia origanoides plant has been widely studied due to the antimicrobial and antifungal effect of its extracts and essential oils, which have been tested against a large number of pathogenic microorganisms. However, there is little literature that records the diversity of endophytic bacteria associated with this plant species. The objective of the work was to evaluate in vitro the plant growth promotion capacity of endophytic Lippia origanoides bacteria in the municipality of Sincelejo-Sucre, Colombia. In this study, endophytic bacteria were isolated in R2A agar culture medium from different tissues, population density (CFU / g of tissue) was evaluated by surface counting and the promotion of plant growth qualitatively in specific selective media. Significant differences were observed for the population density of endophytic bacteria regarding tissue type, with higher values in the root (2.0 x 1010 / g root), followed by the stem (1.3 x 1010 / g stem) and leaves (9.2 x 109 / g sheet). A total of 20 endophytic bacteria were obtained, which two showed phosphate solubilizing capacity, biological nitrogen fixation, production of siderophores and ACC deaminase. The TLO5 and RLO4 morphotypes were molecularly identified as Bacillus cereus, showing good results in promoting plant growth.