Estudios termodinámicos de los procesos de vitrificación en la crioconservación de germoplasma vegetal=Thermodynamic studies of vitrification processes in plant germplasm cryopreservation

En la actualidad, las técnicas de crioconservación poseen una importancia creciente para el almacenamiento a largo plazo de germoplasma vegetal. En las dos últimas décadas, estos métodos experimentaron un gran desarrollo y se han elaborado protocolos adecuados a diferentes sistemas vegetales, utilizando diversas estrategias como la vitrificación, la encapsulación-desecación con cuentas de alginato y el método de “droplet”-vitrificación. La presente tesis doctoral tiene como objetivo aumentar el conocimiento sobre los procesos implicados en los distintos pasos de un protocolo de crioconservación, en relación con el estado del agua presente en los tejidos y sus cambios, abordado mediante diversas técnicas biofísicas, principalmente calorimetría diferencial de barrido (DSC) y microscopía electrónica de barrido a baja temperatura (crio-SEM). En un primer estudio sobre estos métodos de crioconservación, se describen las fases de enfriamiento hasta la temperatura del nitrógeno líquido y de calentamiento hasta temperatura ambiente, al final del periodo de almacenamiento, que son críticas para la supervivencia del material crioconservado. Tanto enfriamiento como calentamiento deben ser realizados lo más rápidamente posible pues, aunque los bajos contenidos en agua logrados en etapas previas de los protocolos reducen significativamente las probabilidades de formación de hielo, éstas no son del todo nulas. En ese contexto, se analiza también la influencia de las velocidades de enfriamiento y calentamiento de las soluciones de crioconservación de plantas en sus parámetros termofísicos referente a la vitrificación, en relación su composición y concentración de compuestos. Estas soluciones son empleadas en la mayor parte de los protocolos actualmente utilizados para la crioconservación de material vegetal. Además, se estudia la influencia de otros factores que pueden determinar la estabilidad del material vitrificado, tales como en envejecimiento del vidrio. Se ha llevado a cabo una investigación experimental en el empleo del crio-SEM como una herramienta para visualizar el estado vítreo de las células y tejidos sometidos a los procesos de crioconservación. Se ha comparado con la más conocida técnica de calorimetría diferencial de barrido, obteniéndose resultados muy concordantes y complementarios. Se exploró también por estas técnicas el efecto sobre tejidos vegetales de la adaptación a bajas temperaturas y de la deshidratación inducida por los diferentes tratamientos utilizados en los protocolos. Este estudio permite observar la evolución biofísica de los sistemas en el proceso de crioconservación. Por último, se estudió la aplicación de películas de quitosano en las cuentas de alginato utilizadas en el protocolo de encapsulación. No se observaron cambios significativos en su comportamiento frente a la deshidratación, en sus parámetros calorimétricos y en la superficie de las cuentas. Su aplicación puede conferir propiedades adicionales prometedoras. ABSTRACT Currently, cryopreservation techniques have a growing importance for long term plant germplasm storage. These methods have undergone great progress during the last two decades, and adequate protocols for different plant systems have been developed, making use of diverse strategies, such as vitrification, encapsulation-dehydration with alginate beads and the dropletvitrification method. This PhD thesis has the goal of increasing the knowledge on the processes underlying the different steps of cryopreservation protocols, in relation with the state of water on tissues and its changes, approached through diverse biophysical techniques, especially differential scanning calorimetry (DSC) and low-temperature scanning electron microscopy (cryo-SEM). The processes of cooling to liquid nitrogen temperature and warming to room temperature, at the end of the storage period, critical for the survival of the cryopreserved material, are described in a first study on these cryopreservation methods. Both cooling and warming must be carried out as quickly as possible because, although the low water content achieved during previous protocol steps significantly reduces ice formation probability, it does not completely disappear. Within this context, the influence of plant vitrification solutions cooling and warming rate on their vitrification related thermophysical parameters is also analyzed, in relation to its composition and component concentration. These solutions are used in most of the currently employed plant material cryopreservation protocols. Additionally, the influence of other factors determining the stability of vitrified material is studied, such as glass aging. An experimental research work has been carried out on the use of cryo-SEM as a tool for visualizing the glassy state in cells and tissues, submitted to cryopreservation processes. It has been compared with the better known differential scanning calorimetry technique, and results in good agreement and complementary have been obtained. The effect on plant tissues of adaptation to low temperature and of the dehydration induced by the different treatments used in the protocols was explored also by these techniques. This study allows observation of the system biophysical evolution in the cryopreservation process. Lastly, the potential use of an additional chitosan film over the alginate beads used in encapsulation protocols was examined. No significant changes could be observed in its dehydration and calorimetric behavior, as well as in its surface aspect; its application for conferring additional properties to gel beads is promising

Glassy State and Cryopreservation of Mint Shoot Tips

Vitrification refers to the physical process by which a liquid supercools to very low tem- peratures and finally solidifies into a metastable glass, without undergoing crystallization at a practical cooling rate. Thus, vitrification is an effective freeze-avoidance mechanism and living tissue cryopreservation is, in most cases, relying on it. As a glass is exceedingly vis- cous and stops all chemical reactions that require molecular diffusion, its formation leads to metabolic inactivity and stability over time. To investigate glassy state in cryopreserved plant material, mint shoot tips were submitted to the different stages of a frequently used cryopreservation protocol (droplet-vitrification) and evaluated for water content reduction and sucrose content, as determined by ion chromatography, frozen water fraction and glass transitions occurrence by differential scanning calorimetry, and investigated by low-tempera- ture scanning electron microscopy, as a way to ascertain if their cellular content was vitri- fied. Results show how tissues at intermediate treatment steps develop ice crystals during liquid nitrogen cooling, while specimens whose treatment was completed become vitrified, with no evidence of ice formation. The agreement between calorimetric and microscopic observations was perfect. Besides finding a higher sucrose concentration in tissues at the more advanced protocol steps, this level was also higher in plants precultured at 25/21?C than in plants cultivated at 25?C

Fast measurement of cooling and heating rates in standard cryopreservation protocols based on vitrification

Estudio de la cinética de la crioconservación de tejidos vegetale

Betalains and phenolic compounds of leaves and stems of Alternanthera brasiliana and Alternanthera tenella

Betacyanins and phenolic compounds from acetonitrile:acidified water extracts of Alternanthera brasiliana and Alternanthera tenella were characterized and quantified using a high-performance liquid chromatography system coupled with diode array and electrospray mass spectrometry detection. Four betacyanins (amaranthine, isoamaranthine, betanin and isobetanin) were tentatively identified and quantified. Twenty eight phenolic compounds of four different families (hydroxybenzoic and hydroxycinnamic acids, flavones and flavonols) were separated and characterized on the basis of their accurate MS and MS/MS information out of which ten compounds were confirmed by authentic standards. These plant species could be considered as an especially rich source of natural bioactive compounds and potential food colorants. A. brasiliana showed the highest betacyanin and polyphenols content (89 μg/g and 35,243 μg/g, respectively). Among polyphenols, flavonols were the more abundant (kaempferol-glucoside, kaempferol-rutinoside and kaempferol-rhamnosyl-rhamnosyl-glycoside). Meanwhile, A. tenella showed a different polyphenols profile with flavones as major compounds (glucopyranosil-vitexin and vitexin). As a novelty, pentosyl-vitexin and pentosyl-isovitexin were detected for the first time in Alternanthera plants. Both A. brasiliana and A. tenella leaves showed high total polyphenol content and in vitro antioxidant activity (FRAP). These results provide an analytical base concerning the phenolic and betalains composition and the antioxidant properties of two members of the promising Alternanthera gender, for subsequent applications, such as functional food ingredients.Fil: Deladino, Lorena. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Alvarez, I.. Consejo Superior de Investigaciones Científicas. Instituto de Ciencia y Tecnologia de Alimentos y Nutrición; EspañaFil: De Ancos, B.. Consejo Superior de Investigaciones Científicas. Instituto de Ciencia y Tecnologia de Alimentos y Nutrición; EspañaFil: Sánchez Moreno, C.. Consejo Superior de Investigaciones Científicas. Instituto de Ciencia y Tecnologia de Alimentos y Nutrición; EspañaFil: Molina García, A. D.. Consejo Superior de Investigaciones Científicas. Instituto de Ciencia y Tecnologia de Alimentos y Nutrición; EspañaFil: Schneider Teixeira, Aline. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentina. Consejo Superior de Investigaciones Científicas. Instituto de Ciencia y Tecnologia de Alimentos y Nutrición; Españ

Interactions between water and triacylglycerols may explain faster aging rates in stored germplasm at low temperatures

The purpose of this research is to explore the extent and significance of possible interacting factors on the viability of stored germplasm. Our work begins with characterizing the kinetics of TAG and water phase changes in peanut (Arachis hypogaea) and Papaya (Carica papaya) seeds equilibrated to different water contents and stored at temperatures between -5 and -80°C. Water and TAG phase was measured using a Perkin Elmer Differential Scanning Calorimeter. Cytoplasm ultra-structure was visualized without chemical fixatives using low temperature scanning electron microscopy (cryo-SEM) performed with a Zeiss DSN 960 scanning microscope equipped with a Cryotrans CT-1500 cold plate (Oxford, UK)

Análise da coluna d'água na região de perfuração de poços petrolíferos em águas profundas

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Evaluación de la actividad antioxidante y polifenoles totales en Avena sativa proveniente de la ciudad de Arequipa

The Avena sativa, a food that besides offering the basic requirements in the diet provides additional benefits towards the health, preparing diseases chronically degenerative as: obesity, diabetes, cardiovascular and cancer. The aim of this work was to determine the profile of polyphenols of the ethanolic extract of oats by means of HPLC and to evaluate the antirust activity by means of the tests beta-carotene/linoleic acid, test FRAP, inhibition of the radical DPPH• and the total present polyphenols for the test Folin-Ciocalteau. The results provide a preliminary basis for further exploitation of this plant and its chemical components and application development.La Avena sativa, un alimento que además de brindar los requerimientos básicos en la dieta provee beneficios adicionales hacia la salud, previniendo enfermedades crónico degenerativas como: obesidad, diabetes, cardiovasculares y cáncer. El objetivo de este trabajo fue determinar el perfil de polifenoles del extracto etanólico de avena mediante HPLC y evaluar la actividad antioxidante mediante los ensayos beta-caroteno/ácido linoleico, ensayo FRAP, e inhibición del radical DPPH• y los polifenoles totales presentes por el ensayo Folin-Ciocalteau. Los resultados proporcionan una base preliminar para una mayor exploración de esta planta y sus componentes químicos y desarrollo de una aplicación

Invariance of the Glass Transition Temperature of Plant Vitrification Solutions with Cooling Rate

The solutions studied were Plant Vitrification Solutions 1, 2 and 3: (PVS1: Uragami et al. 1989, Plant Cell Rep. 8, 418; PVS2: Sakai et al. 1990, Plant Cell Rep. 9, 30; PVS3: Nishizawa et al. 1993, Plant Sci. 91, 67). Cooling was performed using the calorimeter control (5, 10 and 20°C min-1), or for higher rates, by quenching the closed pan with PVS in LN, either naked (faster - 5580°C min-1) or introduced in cryovials (reduced rate 360°C min-1). Quenched pans were then transferred to the sample chamber, pre-cooled to -196°C. Glass transition temperature was observed by DSC with a TA 2920 instrument, upon warming pans with solution samples from -145°C to room temperature, at standard warming rate10°C min-1

Measurement of cooling and warming rates in vitrification-based plant cryopreservation protocols

Cryopreservation protocols include the use of additives and pre-treatments aimed to reduce the probability of ice nucleation at all temperatures, mainly through micro-viscosity increase. Still, there is a risk of ice formation in the temperature region comprised between the equilibrium freezing (Tf) and the glass transition (TG) temperatures. Consequently, fast cooling and warming, especially in this region, is a must to avoid ice-derived damage. Vitrification and droplet-vitrification techniques, frequently used cryopreservation protocols based in fast cooling, were studied, alongside with the corresponding warming procedures. A very fast data acquisition system, able to read very low temperatures, down to that of liquid nitrogen, was employed. Cooling rates, measured between -20 and -120 ºC, ranged from ca. 5 ºC s-1 to 400 ºC s-1, while warming rates spanned from ca. 2 ºC s-1 to 280 ºC s-1, for the different protocols and conditions studied. A wider measuring window (0 ºC to -150 ºC) produced lower rates for all cases. The cooling and warming rates were also related to the survival observed after the different procedures. Those protocols with the faster rates yielded the highest survival percentages.Fil: Schneider Teixeira, Aline. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigaciones En Criotecnología de Alimentos (i); Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentina. Instituto de Ciencia y Tecnología de Alimentos y Nutrición; EspañaFil: Gonzalez Benito, M. Elena. Universidad Politecnica de Madrid; EspañaFil: Molina García, Antonio D.. Consejo Superior de Investigaciones Científicas. Instituto de Ciencia y Tecnologia de Alimentos y Nutrición; Españ

Major Phenolics in Yerba Mate Extracts (<i>Ilex paraguariensis</i>) and Their Contribution to the Total Antioxidant Capacity

Yerba mate (Ilex paraguariensis) is an indigenous crop which is highly consumed as an infusion in the South American subtropical forest. It has a high concentration of antioxidant substances, providing health benefits and helping to prevent diseases. The objectives of this work were to characterize an aqueous yerba mate extract by spectrophotometric and chromatographic (HPLC) methods and to study the effect of the freeze-drying process on the polyphenols profile and antioxidant activity, determined by a novel method. An aqueous extract was obtained and lyophilized to obtain a yerba mate powder with antioxidant properties. The extracts showed a high polyphenol content, determined by Folin-Ciocalteau and HPLC, and a high antioxidant activity towards the DPPH· radical and after the recently developed method of photochemiluminescence. A linear correlation was found between Folin-Ciocalteau and DPPH methods for lyophilized samples. HPLC analysis allowed determining antioxidant components like rutin, caffeine and chlorogenic acid. Lyophilization caused a decrease in total polyphenol content and antiradical activity of the extracts and this fact was mainly attributed to changes in the chlorogenic acid related compounds and rutin structures, after their photochemiluminescence data. The photoluminiscent method proves to be an advantageous approach for antioxidant capacity determination.Centro de Investigación y Desarrollo en Criotecnología de AlimentosConsejo Nacional de Investigaciones Científicas y Técnica