Tonoplast and Peroxisome Targeting of γ-tocopherol N-methyltransferase Homologs Involved in the Synthesis of Monoterpene Indole Alkaloids.

Many plant species from the Apocynaceae, Loganiaceae and Rubiaceae families evolved a specialized metabolism leading to the synthesis of a broad palette of monoterpene indole alkaloids (MIAs). These compounds are believed to constitute a cornerstone of the plant chemical arsenal but above all several MIAs display pharmacological properties that have been exploited for decades by humans to treat various diseases. It is established that MIAs are produced in planta due to complex biosynthetic pathways engaging a multitude of specialized enzymes but also a complex tissue and subcellular organization. In this context, N-methyltransferases (NMTs) represent an important family of enzymes indispensable for MIA biosynthesis but their characterization has always remained challenging. In particular, little is known about the subcellular localization of NMTs in MIA-producing plants. Here, we performed an extensive analysis on the subcellular localization of NMTs from four distinct medicinal plants but also experimentally validated that two putative NMTs from Catharanthus roseus exhibit NMT activity. Apart from providing unprecedented data regarding the targeting of these enzymes in planta, our results point out an additional layer of complexity to the subcellular organization of the MIA biosynthetic pathway by introducing tonoplast and peroxisome as new actors of the final steps of MIA biosynthesis.

Vacuole-Targeted Proteins: Ins and Outs of Subcellular Localization Studies.

Accurate and efficient demonstrations of protein localizations to the vacuole or tonoplast remain strict prerequisites to decipher the role of vacuoles in the whole plant cell biology and notably in defence processes. In this chapter, we describe a reliable procedure of protein subcellular localization study through transient transformations of Catharanthus roseus or onion cells and expression of fusions with fluorescent proteins allowing minimizing artefacts of targeting.

Virus-induced gene silencing in Rauwolfia species.

Elucidation of the monoterpene indole alkaloid biosynthesis has recently progressed in Apocynaceae through the concomitant development of transcriptomic analyses and reverse genetic approaches performed by virus-induced gene silencing (VIGS). While most of these tools have been primarily adapted for the Madagascar periwinkle (Catharanthus roseus), the VIGS procedure has scarcely been used on other Apocynaceae species. For instance, Rauwolfia sp. constitutes a unique source of specific and valuable monoterpene indole alkaloids such as the hypertensive reserpine but are also well recognized models for studying alkaloid metabolism, and as such would benefit from an efficient VIGS procedure. By taking advantage of a recent modification in the inoculation method of the Tobacco rattle virus vectors via particle bombardment, we demonstrated that the biolistic-mediated VIGS approach can be readily used to silence genes in both Rauwolfia tetraphylla and Rauwolfia serpentina. After establishing the bombardment conditions minimizing injuries to the transformed plantlets, gene downregulation efficiency was evaluated at approximately a 70% expression decrease in both species by silencing the phytoene desaturase encoding gene. Such a gene silencing approach will thus constitute a critical tool to identify and characterize genes involved in alkaloid biosynthesis in both of these prominent Rauwolfia species.

Avances en la propagación vía embriogénesis somática de Psychotria ipecacuanha (Brot. ) Stokes, planta medicinal en peligro crítico / Advances in vitro propagation via somatic embryogenesis of Psychotria ipecacuanha (Brot. ) Stokes, medicinal plant critically endangered

Psychotria ipecacuanha (Brot.) Stokes (Rubiaceae Juss), es una especie vegetal con reconocidas propiedades medicinales. Esta especie se encuentra en peligro crítico de extinción, debido a la sobreexplotación de las poblaciones naturales. Conociendo además las dificultades para su propagación por medio de semillas (debido a la baja tasa de germinación y elevada muerte prematura de las plántulas) y por vía vegetativa (lento crecimiento), el presente trabajo tuvo como objetivo evaluar el potencial de propagación vía embriogénesis somática directa. Segmentos de hojas jóvenes de plantas mantenidas en casa malla fueron desinfectados y sembrados en el medio de cultivo MS (Murashige y Skoog) suplementado con diferentes concentraciones y combinaciones de reguladores de crecimiento. Las combinaciones IBA y BAP a 1 y 2 mg/L y 2 y 1 mg/L, respectivamente; mostraron ser efectivas en la formación de embriones somáticos en esta especie. La procedencia de la planta donadora parece tener influencia en la sensibilidad del tejido foliar a la respuesta. Este es el primer reporte de embriogénesis somática directa para esta especie y el primer reporte de cultivo in vitro de poblaciones colombianas.

Psychotria ipecacuanha (Brot.) Stokes (Rubiaceae Juss), is a specie with known medicinal properties. This species is critically endangered due to overexploitation of natural populations.Besides knowing the difficulties in propagation by seed (due to the low rate of germination and high seedling premature death) and by vegetative (slow growth), the present study evaluated the potential for propagation by using direct somatic embryogenesis.Young leaves segments from plants cultivated in a greenhouse were disinfected and planted in MS medium (Murashige y Skoog), supplemented with different concentrations of growth regulators. IBA and BAP combinations at 1 and 2 mg / L and 2 and 1 mg / L, respectively, shown to be effective in the formation of somatic embryos in this species. The origin of the donor plant seems to influence foliar tissue sensitivity to the answer. This is the first report of direct somatic embryogenesis for this species and the first report of in vitro culture of Colombian populations.

Cultivos celulares de Choibá Dipteryx oleifera Benth / Cell cultures of Choiba Dipteryx oleifera Benth

Choibá (Dipteryx oleifera Benth) es un árbol de la familia Fabaceae (Papilionoideae), con una distribución geográfica reportada desde Nicaragua hasta Colombia a una altura de hasta 1000 msnm. Crece en bosque húmedo, muy húmedo o premontano húmedo. Esta especie es considerada vulnerable debido a la sobreexplotación de su madera, ya que es un árbol altamente apetecido por esta y por sus frutos. Su almendra almacena una buena cantidad de aceites con potencial para la industria alimentaria, lo que podría resultar en una nueva fuente alimenticia, por lo cual el cultivo in vitro de vegetales con el propósito de producir compuestos de interés, marca un punto de partida para reducir el uso del suelo y lograr componentes bioactivos bajo condiciones controladas. En este trabajo, como una primera etapa experimental, se evaluó el crecimiento celular en suspensiones, a partir de callo inducido en explantes de cotiledón; se ensayaron 6 tratamientos diferentes, la mitad de estos con MS como medio basal y la otra mitad con B5, cada uno de los dos grupos con un control y la combinación hormonal de 2.5 mg/L de 2,4-D y 1 mg/L de BAP o kinetina, suplementado con adenina, biotina, glutamina y ácido pantoténico y 30 g/L de sacarosa, bajo completa oscuridad. Se encontró que dos tratamientos con MS en combinación con 2.5 mg/L de 2,4-D y 1 mg/L de kinetina o BAP fueron los mejores.

Choibá (Dipteryx olifera) is a tree of the Fabaceae family, with a geographical distribution reported from Nicaragua to Colombia, nearly 1.000 msnm in a tropical rain forest. This species is a highly desired tree for its timber and fruits, the kernel store a lot of important oils for the food industry, resulting in a new possible food source, so we are making in vitro cultivation of vegetables with the purpose of producing compounds of interest and that mark a starting point on the reduction of land use and it achieves bioactive components under controlled conditions. In this work, as a first experimental step was evaluated the tissue cell growth in suspension, using fragments of cotyledon and testing 6 different treatments, the half of those with MS as basal medium and the other half with B5, each of the two groups with one control and hormone combination of 2.5 mg/L 2,4-D and 1 mg/L BAP or kinetin, supplemented with adenine, biotin, pantothenic acid and glutamine and 30 g l sucrose under complete darkness. It was found that two treatments with MS in combination with 2.5 mg/L 2,4-D and 1 mg/L kinetin or BAP were the best.

Evaluation of a method using high performance liquid chromatography with ultraviolet detection for the determination of statins in macromycetes of the genus Pleurotus cultivated by fermentation processes.

The applicability of high-performance liquid chromatography with ultraviolet light (HPLC-UV) for the determination of the presence of statins in macromycetes of the genus Pleurotus was analyzed. The fungi were obtained by liquid-state fermentation (LSF) using unconventional sources of carbon as substrates and solid-state fermentation (SSF) employing agro industrial wastes. Five statins were used as standards: lovastatin and simvastatin in the lactone form (LOVL and SIML), their corresponding hydro-acidic forms (LOVH and SIMH) and pravastatin (PRA). The following measures were evaluated: the linearity, accuracy and precision, detection limit (DL) and quantification limit (QL). The results demonstrated HPLC-UV to be an effective tool for detecting the presence of statins in extracts of LSF and SSF products. Likewise, it was hypothesized that the strains that were used for the study do not produce statins. This finding highlights the importance of continuing to evaluate other strains of the same genus by using techniques such as HPLC to first separate sufficient quantities of the compounds that were detected using the standard technique but that did not match the retention time (tR) of any of the standards used.

Lipid profile of in vitro oil produced through cell culture of Jatropha curcas.

Recent increases in energy demands as a consequence of population growth and industrialization, and pollution caused during the extraction and combustion of fossil fuel sources have driven the development of new energy sources that do not cause pollution and are inexpensive and renewable. Consequently, it is necessary to develop alternative ways of generating biofuels that put less pressure on agricultural lands and water supplies, and ensure ecosystems conservation. In order to achieve the proposed goals related to energetic coverage and independence, several approaches have been developed, including biodiesel production using vegetal oils as feedstock. The aim of the current research project was to apply a nonconventional bioprocess for in vitro biomass and oil production of Jatropha curcas, for assessing different J. curcas varieties, where seed tissue was isolated and used for callus induction. Once friable callus was obtained, cell suspension cultures were established. The cell viability, fatty acid content, and characteristics were used to select the most promising cell line according to its fatty acid profile and ability to grow and develop under in vitro conditions. Oil produced by cell suspension culture of the Jatropha varieties studied was extracted and characterized by GC/MS. Differences encountered among Jatropha varieties were related to their fatty acid profiles, oil content (% on dry basis), and cell viability measurements (%).

Production of biomass, polysaccharides, and ganoderic acid using non-conventional carbon sources under submerged culture of the Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt.:Fr.)P. Karst. (higher Basidiomycetes).

The effect of different non-conventional carbon sources was studied in the submerged culture of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum, for simultaneous production of mycelial biomass, bioactive ganoderic acid, and polysaccharides, in less time, using non-conventional carbon sources to minimize the high costs of current culture media. The optimal medium composition was defined as (g/L): 50 of barley flour, 0.2 of KH2PO4, 0.1 of MgSO4ⁱ7H2O, and 1 NH4Cl. Cultivated under this complex culture medium, the mycelial biomass production was 23.49 ± 0.37 g/L; the extracellular polysaccharides production was 2.72 ± 0.11 g/L; the intracellular polysaccharides production was 2.22 ± 0.06 g/L; the ganoderic acids production was 299.67 ± 11.63 mg/L. One liter of culture medium developed in this project was priced at USD $ 0.11 if barley flour is used as carbon source or $ 0.13 with oat flour in order to get a good amount of products of interest.

Propagación in vitro de Phalaenopsis (Orchidaceae) a partir de protocormos, mediante el sistema de inmersión temporal RITA / In vitro propagation of Phalaenopsis (Orchidaceae) from protocorms, using the temporary inmersion system RITA

Protocormos in vitro de Phalaenopsis de tres meses de edad se transfirieron a contenedores RITA® con el fin depropagarlos masivamente. Los factores evaluados fueron la concentración de sacarosa en el medio y la frecuencia de inmersión. Se dispusieron cinco pares de contenedores RITA® con medio de cultivo líquido a concentraciones de sacarosa de 0, 15, 30, 45 y 60 g/L. El medio utilizado fue el MS a la mitad de la concentración de las sales, suplementado con vitaminas y tidiazuron (5 mg/L). El experimento se realizó en dos etapas, cada una con duración de dos meses. La primera etapa con una frecuencia de inmersión de cuatro horas y la segunda con una frecuencia de inmersión de ocho horas, ambas con un tiempo de inmersión de un minuto. Los resultados mostraron que la mejor respuesta proliferativa, con 8,2 protocormos adventicios por protocormo por mes, se obtuvo en el medio con 15 g/L de sacarosa y un tiempo de inmersión de un minutocada cuatro horas.

In order to massively propagate Phalaenopsis orchids, three months old in vitro protocorms were transferred to RITA® vessels. The evaluation factors were the sucrose concentration in the culture medium and the frequency immersion. There was a set of five pairs of RITA® vessels with liquid culture medium containing sucrose at 0, 15, 30, 45, 60 g/L. A half-strength MS medium plus vitamins, supplemented with vitamins and thidiazuron (5 mg/L) was used. The experiment had two stages, each lasting two months. Each stage had a one minute immersion. The first stage had a four hour immersion frequency and the second one had an eight hour immersion frequency. Results showed that the best proliferating response was reached in a 15 g/L of sucrose medium with one minute of immersion time every four hours, resulting in 8,2 adventitious protocorms per protocorm per month.