ES2694706B2 - PROCEDURE TO INCREASE THE PRODUCTION OF GLUCOSINOLATES IN CELL CULTURES - Google Patents
PROCEDURE TO INCREASE THE PRODUCTION OF GLUCOSINOLATES IN CELL CULTURES Download PDFInfo
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- ES2694706B2 ES2694706B2 ES201730836A ES201730836A ES2694706B2 ES 2694706 B2 ES2694706 B2 ES 2694706B2 ES 201730836 A ES201730836 A ES 201730836A ES 201730836 A ES201730836 A ES 201730836A ES 2694706 B2 ES2694706 B2 ES 2694706B2
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- culture medium
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- glucosinolates
- concentration
- plant cells
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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Description
DESCRIPCIÓNDESCRIPTION
Procedimiento para incrementar la producción de glucosinolatos en cultivos celularesProcedure to increase the production of glucosinolates in cell cultures
La presente invención se encuentra dentro del campo de la biotecnología y la farmacia, y se refiere a un procedimiento para incrementar la producción de glucosinolatos a partir de cultivos de células vegetales mediante la adición al medio de cultivo de coronatina (un análogo estructural y funcional del precursor octadecanoide del jasmonato de metilo) o jasmonato de metilo y, opcionalmente, ciclodextrinas.The present invention is within the field of biotechnology and pharmacy, and relates to a process to increase the production of glucosinolates from plant cell cultures by adding to the culture medium coronatin (a structural and functional analog of the octadecanoid precursor of methyl jasmonate) or methyl jasmonate and, optionally, cyclodextrins.
ANTECEDENTES DE LA INVENCIÓNBACKGROUND OF THE INVENTION
El brócoli (Brassica olerácea var. italica) también conocido como brécol, o bróculi, es una planta comestible que pertenece a la familia de las brasicáceas o crucíferas, al igual que la col, las coles de Bruselas, la coliflor, el rábano, el repollo y la rúcula.Broccoli ( Brassica olerácea var. Italica) also known as broccoli, or broccoli, is an edible plant that belongs to the brassicaceae or cruciferous family, like cabbage, Brussels sprouts, cauliflower, radish, cabbage and arugula.
Las plantas pertenecientes a la familia Brassicaceae (crucíferas) se caracterizan por poseer una gran cantidad de compuestos fitoquímicos, apareciendo la mayor concentración de estos en un estado fisiológico juvenil (en forma de brote), a diferencia del estado fisiológico adulto con menor contenido fitoquímico, debido a la dilución de estos compuestos provocada por el crecimiento de los diferentes tejidos. Dentro de esta familia de plantas, el brócoli (Brassica oleracea var. italica) se caracteriza por ser una hortaliza con efectos beneficiosos para la salud, cuya producción mundial (junto con la coliflor) ha ido incrementando en las últimas décadas alcanzando una producción mundial de 24.175.000 de toneladas.Plants belonging to the Brassicaceae family (cruciferous) are characterized by having a large amount of phytochemical compounds, the highest concentration of these appearing in a juvenile physiological state (in the form of a sprout), unlike the adult physiological state with less phytochemical content. due to the dilution of these compounds caused by the growth of different tissues. Within this family of plants, broccoli ( Brassica oleracea var. Italica) is characterized by being a vegetable with beneficial effects on health, whose world production (together with cauliflower) has been increasing in recent decades, reaching a world production of 24,175,000 tons.
Durante las últimas décadas, se ha prestado especial atención a los compuestos bioactivos naturales con potencial para el tratamiento y la prevención de enfermedades humanas. Los compuestos bioactivos presentes en frutas y hortalizas, son productos del metabolismo secundario de los vegetales, que se encuentran en cantidades pequeñas en las plantas, con respecto a otros macronutrientes, pero que contribuyen significativamente a la regulación de los mecanismos de protección frente a situaciones de estrés y tienen propiedades biológicas de interés para la prevención de algunas enfermedades en los humanos que los consumen. Además, contribuyen al mantenimiento de los tejidos corporales, aumentan la resistencia a las infecciones, regulan el correcto desarrollo del sistema nervioso e intervienen en el crecimiento, y resultan beneficiosos para la síntesis de enzimas en el hígado (Hooper, L., & Cassidy, A. (2006). A review of the health care potential of bioactive compounds. Journal of the Science of Food and Agriculture, 86(12), 1805-1813) .During the last decades, special attention has been paid to natural bioactive compounds with potential for the treatment and prevention of human diseases. The bioactive compounds present in fruits and vegetables are products of the secondary metabolism of vegetables, which are found in small amounts in plants, with respect to other macronutrients, but which contribute significantly to the regulation of protection mechanisms against situations of stress and have biological properties of interest for the prevention of some diseases in humans who consume them. In addition, they contribute to maintenance of body tissues, increase resistance to infections, regulate the correct development of the nervous system and intervene in growth, and are beneficial for the synthesis of enzymes in the liver (Hooper, L., & Cassidy, A. (2006 A review of the health care potential of bioactive compounds. Journal of the Science of Food and Agriculture, 86 (12), 1805-1813).
Entre los compuestos bioactivos del brócoli, cabe destacar los glucosinolatos y los compuestos fenólicos (flavonoides y ácidos hidroxicinámicos), además de otros nutrientes como carotenoides, esteroles, vitamina C, contenido en fibra y elementos minerales esenciales para la salud. Los glucosinolatos, también llamados tioglucósidos, son metabolitos secundarios aniónicos nitrógeno-azufrados que se encuentran casi exclusivamente en plantas de la familia Brassicaceae (cruciferas), entre ellas el brócoli, donde alcanzan la mayor concentración, es decir, son los compuestos bioactivos por excelencia del brócoli. El esqueleto básico de los glucosinolatos (P-D-tioglugósido-N-hidroxisulfato) consiste en un grupo p-D-tioglucosa, una oxima sulfonada y una cadena lateral derivada de los aminoácidos metionina, fenilalanina, o triptófano con una cadena lateral variable. La estructura química de los glucosinolatos y su contenido pueden variar entre especies y entre variedades dentro de la misma especie. En la actualidad se conocen más de 120 tipos de glucosinolatos en las cruciferas, siendo los más abundantes en brócoli la glucorafanina (80%), la glucobrasicina (8%), la 4-metoxi-glucobrasicina (4%) y la 1-metoxi-glucobrasicina (4%). Entre los compuestos de degradación de los glucosinolatos se encuentran los isotiocianatos comúnmente denominados "aceites de mostaza”, siendo el sulforafano el principal isotiocianato encontrado en el brócoli.Among the bioactive compounds in broccoli, it is worth highlighting glucosinolates and phenolic compounds (flavonoids and hydroxycinnamic acids), as well as other nutrients such as carotenoids, sterols, vitamin C, fiber content and mineral elements essential for health. Glucosinolates, also called thioglucosides, are nitrogen-sulfur anionic secondary metabolites that are found almost exclusively in plants of the Brassicaceae family (crucifers), including broccoli, where they reach the highest concentration, that is, they are the bioactive compounds par excellence of the broccoli. The basic skeleton of glucosinolates (PD-thioglucose-N-hydroxysulfate) consists of a pD-thioglucose group, a sulfonated oxime, and a side chain derived from the amino acids methionine, phenylalanine, or tryptophan with a variable side chain. The chemical structure of glucosinolates and their content can vary between species and between varieties within the same species. Currently more than 120 types of glucosinolates are known in crucifers, the most abundant in broccoli being glucoraphanin (80%), glucobrassicin (8%), 4-methoxy-glucobrassicin (4%) and 1-methoxy -glucobrassicin (4%). Among the degradation compounds of glucosinolates are the isothiocyanates commonly called "mustard oils", with sulforaphane being the main isothiocyanate found in broccoli.
Los glucosinolatos son compuestos beneficiosos para la salud humana (ya que en algunos casos pueden ofrecer protección frente a algunos tipos de cáncer como el de pulmón, mama, colon y próstata Jeffery EH, Araya M. 2009. Physiological effects of broccoli consumption. Phytochemistry Reviews 8 (1): 283-298.). Sin embargo, estos metabolitos no son bioactivos en el animal que los consume hasta que han sido hidrolizados a isotiocianatos por la enzima mirosinasa. De hecho, los isotiocianatos son agentes preventivos del cáncer debido a su capacidad para inducir enzimas de fase II de desintoxicación tales como las quinonareductasas y las glutation-S-transferasas. El sulforafano, que es el producto de hidrólisis del glucosinolato glucorafanina es un mono-inductor muy potente del metabolismo de la fase II. Induce la apoptosis e inhibe el crecimiento tumoral durante las fases de iniciación, promoción y progresión (Mi LX, Wang XT, Govind S, Hood BL, Veenstra TD, Conrads TP, Saha DT, Goldman R, Chung FL. 2007. The role of protein binding in induction of apoptosis by phenethylisothiocyanate and sulforaphane in human non-small lung cancer cells. Cancer Research 67: 6409-6416). También, recientemente se ha descrito el tratamiento con sulforafano en varios tipos de autismo con resultados positivos, observándose una mejoría significativa en pacientes que consumían brócoli de manera regular frente a aquellos que no lo consumían (Singh K, L. Connors S, A. Macklin E, D. Smith K, W. Fahey J, Talalay P, W. Zimmerman A. 2014. Sulforaphane treatment of autism spectrum disorder (ASD). PNAS. vol. 111 no.43, 15550-15555).Glucosinolates are beneficial compounds for human health (since in some cases they can offer protection against some types of cancer such as lung, breast, colon and prostate cancer Jeffery EH, Araya M. 2009. Physiological effects of broccoli consumption. Phytochemistry Reviews 8 (1): 283-298.). However, these metabolites are not bioactive in the consuming animal until they have been hydrolyzed to isothiocyanates by the enzyme myrosinase. In fact, isothiocyanates are cancer preventive agents due to their ability to induce phase II detoxification enzymes such as quinonereductases and glutathione S-transferases. Sulforaphane, which is the hydrolysis product of glucoraphanin glucosinolate, is a very potent mono-inducer of phase II metabolism. Induces apoptosis and inhibits tumor growth during the initiation, promotion and progression phases (Mi LX, Wang XT, Govind S, Hood BL, Veenstra TD, Conrads TP, Saha DT, Goldman R, Chung FL. 2007. The role of protein binding in induction of apoptosis by phenethylisothiocyanate and sulforaphane in human non-small lung cancer cells. Cancer Research 67: 6409-6416). Also, recently sulforaphane treatment has been described in various types of autism with positive results, observing a significant improvement in patients who consumed broccoli regularly compared to those who did not consume it (Singh K, L. Connors S, A. Macklin E, D. Smith K, W. Fahey J, Talalay P, W. Zimmerman A. 2014. Sulforaphane treatment of autism spectrum disorder (ASD). PNAS. Vol. 111 no.43, 15550-15555).
Los glucosinolatos son metabolitos secundarios que van a ser sintetizados y almacenados por las células vegetales para responder a diferentes estreses a los que se ven sometidas, de modo que su producción en células vegetales es aumentada por moléculas señalizadoras o elicitores que actúan como mensajeros químicos en la planta para contrarrestar el estrés biótico o abiótico al que se ve sometido la planta. El cultivo de células vegetales constituye una prometedora alternativa para la producción de compuestos naturales difíciles o poco rentables de preparar por síntesis química o que suponen graves agresiones al medio ambiente al recurrir a la fuente natural.Glucosinolates are secondary metabolites that will be synthesized and stored by plant cells to respond to different stresses to which they are subjected, so that their production in plant cells is increased by signaling molecules or elicitors that act as chemical messengers in the plant to counteract the biotic or abiotic stress to which the plant is subjected. The cultivation of plant cells is a promising alternative for the production of natural compounds that are difficult or not very profitable to prepare by chemical synthesis or that pose serious damage to the environment when resorting to a natural source.
En este sentido, el cultivo in vitro de células vegetales ha abierto nuevos caminos como fuente renovable de compuestos bioactivos de gran valor añadido debido a las ventajas que presenta su utilización:In this sense, the in vitro culture of plant cells has opened new paths as a renewable source of bioactive compounds of great added value due to the advantages of their use:
- son independientes de factores geográficos, estacionales y ambientales.- are independent of geographical, seasonal and environmental factors.
- constituyen sistemas de producción estables ya que aseguran la obtención continua de compuestos con calidad y productividad uniformes- they constitute stable production systems as they ensure the continuous obtaining of compounds with uniform quality and productivity
- los requerimientos de espacio para el desarrollo de la producción son reducidos. - el proceso de purificación de los compuestos de interés es más fácil y se optimiza cuando éste se libera al medio de cultivo y se puede realizar a gran escala.- the space requirements for the development of the production are reduced. - the process of purification of the compounds of interest is easier and is optimized when it is released into the culture medium and can be carried out on a large scale.
- permite obtener nuevos compuestos que no son sintetizados por las plantas de forma natural.- allows to obtain new compounds that are not synthesized by plants naturally.
Hasta la fecha, no existen estudios previos donde se utilicen los cultivos celulares de brócoli para la producción de metabolitos de interés. Sin embargo, la adición exógena de diversos elicitores (ácido salicílico o jasmonato de metilo (MJ)) a pellas de brócoli se ha usado con anterioridad para mejorar su composición fitoquímica. En este sentido Baenas et al., 2014 (Baenas, N.; García-Viguera, C.; Moreno, D.A. Biotic elicitors effectively increase the glucosinolates content in brassicaceae sprouts. J. Agric. Food Chem. 2014, 62, 1881-1889) mostraron que MJ provocó un aumento de la cantidad total de glucosinolatos en brotes de Brassica olerácea var. italica, correspondiendo la mayor producción a los glucosinolatos indólicos y glucorafanina, mientras que ácido salicílico produjo un menor aumento de la cantidad de glucosinolatos totales en estos brotes. Además, se ha descrito que una concentración de 250 pM de MJ aplicada a pellas de brócoli produjo un aumento tanto de glucosinolatos como de sus productos de degradación entre un 10 y un 300% (Ku KM, Jeffery EH, Juvik JA (2013) Influence of seasonal variation and methyl jasmonate mediated induction of glucosinolate biosynthesis on quinone reductase activity in broccoli florets. J Agric Food Chem 61: 9623-9631). Sin embargo, concentraciones menores de MJ (5-100 pM) no parecieron tener efecto en el contenido de glucosinolatos, a excepción de un incremento del 10% de indol-glucosinolatos cuando se aplicaba MJ a una concentración de 10 pM (Pérez-Balibrea S, Moreno DA, García-Viguera C (2011) Improving the phytochemical composition of broccoli sprouts by elicitation. Food Chem 129: 35-44).To date, there are no previous studies where broccoli cell cultures are used for the production of metabolites of interest. However, the exogenous addition Various elicitors (salicylic acid or methyl jasmonate (MJ)) to broccoli pellets have been used previously to improve their phytochemical composition. In this sense, Baenas et al., 2014 (Baenas, N .; García-Viguera, C .; Moreno, DA Biotic elicitors effectively increase the glucosinolates content in brassicaceae sprouts. J. Agric. Food Chem. 2014, 62, 1881-1889 ) showed that MJ caused an increase in the total amount of glucosinolates in shoots of Brassica olerácea var. italica, with the highest production corresponding to indole glucosinolates and glucoraphanin, while salicylic acid produced a lower increase in the amount of total glucosinolates in these shoots. In addition, it has been described that a concentration of 250 pM of MJ applied to broccoli pellets produced an increase in both glucosinolates and their degradation products between 10 and 300% (Ku KM, Jeffery EH, Juvik JA (2013) Influence of seasonal variation and methyl jasmonate mediated induction of glucosinolate biosynthesis on quinone reductase activity in broccoli florets. J Agric Food Chem 61: 9623-9631). However, lower concentrations of MJ (5-100 pM) did not seem to have an effect on the glucosinolate content, except for a 10% increase in indole-glucosinolates when MJ was applied at a concentration of 10 pM (Pérez-Balibrea S , Moreno DA, García-Viguera C (2011) Improving the phytochemical composition of broccoli sprouts by elicitation. Food Chem 129: 35-44).
Por otra parte, Alvárez et al. (2008) (Alvarez, S., He, Y., Chen, S. (2008). Comparative Investigations of the Glucosinolate-Myrosinase System in Arabidopsis Suspension Cells and Hypocotyls. Plant Cell Physiol. 49(3), 324-333) mostraron que suspensiones celulares de Arabidopsis thaliana derivadas de hipocótilo sintetizaron 7 glucosinolatos diferentes, entre los que se encontraron glucosinolatos indólicos o glucorafanina, algunos de los cuales estuvieron presentes en mayor cantidad que en el tejido de vegetal que dio origen a la suspensión celular. En conjunción a esto, este grupo mostró que la elicitación del cultivo celular con MJ provocó un considerable aumento de la producción de glucosinolatos, demostrando también un aumento de la actividad mirosinasa en suspensiones celulares A. thaliana tratadas con MJ.On the other hand, Alvárez et al. (2008) (Alvarez, S., He, Y., Chen, S. (2008). Comparative Investigations of the Glucosinolate-Myrosinase System in Arabidopsis Suspension Cells and Hypocotyls. Plant Cell Physiol. 49 (3), 324-333) showed that hypocotyl-derived Arabidopsis thaliana cell suspensions synthesized 7 different glucosinolates, among which were indole glucosinolates or glucoraphanin, some of which were present in greater amounts than in the plant tissue that gave rise to the cell suspension. In conjunction with this, this group showed that MJ cell culture elicitation caused a considerable increase in glucosinolate production, also demonstrating an increase in myrosinase activity in MJ-treated A. thaliana cell suspensions.
Sin embargo, en ningún caso de describe el uso de coronatina o MJ como estimulador de la producción de glucosinolatos en cultivos celulares de brócoli. En ese sentido, se ha observado que la coronatina, una toxina producida por Pseudomonas syringae (Weiler, E. W., Kutchan, T. M., Gorba, T., Brodschelm, W., Niesel, U., & Bublitz, F. (1994). The Pseudomonas phytotoxin coronatine mimics octadecanoid signalling molecules of higher plants. FEBS letters, 345(1), 9-13), es un análogo estructural de la forma activa el jasmonato de metilo (JA-Ile) y ha resultado ser un potente elicitor para la producción de taxanos y trans-resveratrol en cultivos celulares de Taxus sp y Vitis vinífera, respectivamente (Onrubia, M., Moyano, E., Bonfill, M., Cusidó, R. M., Goossens, A., & Palazón, J. (2013). Coronatine, a more powerful elicitor for inducing taxane biosynthesis in Taxus media cell cultures than methyl jasmonate. Journal of plant physiology, 170(2), 211-219; Almagro, L., Belchí-Navarro, S., Martínez-Márquez, A., Bru, R., & Pedreño, M. A. (2015). Enhanced extracellular production of transresveratrol in Vitis vinifera suspension cultured cells by using cyclodextrins and coronatine. Plant Physiology and Biochemistry, 97, 361-367).However, in no case is the use of coronatin or MJ described as a stimulator of glucosinolate production in broccoli cell cultures. In this sense, it has been observed that coronatin, a toxin produced by Pseudomonas syringae (Weiler, EW, Kutchan, TM, Gorba, T., Brodschelm, W., Niesel, U., & Bublitz, F. (1994). The Pseudomonas phytotoxin coronatine mimics octadecanoid signaling molecules of higher plants. FEBS letters, 345 ( 1), 9-13), is a structural analog of the active form methyl jasmonate (JA-Ile) and has proven to be a powerful elicitor for the production of taxanes and trans-resveratrol in cell cultures of Taxus sp and Vitis vinífera, respectively (Onrubia, M., Moyano, E., Bonfill, M., Cusidó, RM, Goossens, A., & Palazón, J. (2013). Coronatine, a more powerful elicitor for inducing taxane biosynthesis in Taxus media cell cultures than methyl jasmonate. Journal of plant physiology, 170 (2), 211-219; Almagro, L., Belchí-Navarro, S., Martínez-Márquez, A., Bru, R., & Pedreño , MA (2015). Enhanced extracellular production of transresveratrol in Vitis vinifera suspension cultured cells by using cyclodextrins and coronatine. Plant Physiology and Biochemistry, 97, 361-367).
DESCRIPCIÓN DE LA INVENCIÓNDESCRIPTION OF THE INVENTION
El incremento de la síntesis de glucosinolatos se ejemplifica en cultivos celulares de Brassica oleracea variedad italica cv Chronos, una de las especies productoras de glucosinolatos más importantes. Los autores de la presente invención han observado que estableciendo cultivos de células vegetales y adicionando al medio de cultivo coronatina o jasmonato de metilo, se incrementa el rendimiento de producción y extracción de glucosinolatos.The increase in glucosinolate synthesis is exemplified in cell cultures of Brassica oleracea variety italica cv Chronos, one of the most important glucosinolate-producing species. The authors of the present invention have observed that by establishing plant cell cultures and adding coronatin or methyl jasmonate to the culture medium, the yield of glucosinolate production and extraction is increased.
Jasmonato de metilo CoronatinaMethyl Jasmonate Coronatin
Por tanto, un primer aspecto de la presente invención es el incremento de la producción de glucosinolatos, que comprende:Therefore, a first aspect of the present invention is the increase in the production of glucosinolates, which comprises:
a. la adición de coronatina o jasmonato de metilo a un medio de cultivo,to. the addition of coronatin or methyl jasmonate to a culture medium,
b. poner en contacto células vegetales potencialmente productoras de glucosinolatos con el medio de cultivo de (a), b. contact potentially glucosinolate-producing plant cells with the culture medium of (a),
c. la incubación de las células vegetales del paso b) en el medio de cultivo del paso a), d. la separación de los glucosinolatos obtenidos tras el paso c) del cultivo celular.c. incubation of the plant cells from step b) in the culture medium from step a), d. the separation of the glucosinolates obtained after step c) of the cell culture.
Se entiende por células vegetales potencialmente productoras de glucosinolatos, cualquier línea celular capaz de producir glucosinolatos, bien de forma natural o tras modificación genética.Potentially glucosinolate-producing plant cells are understood to be any cell line capable of producing glucosinolates, either naturally or after genetic modification.
Cualquier tejido o célula vegetal capaz de producir glucosinolatos puede ser empleado en la invención. Esto incluye aquellas células procedentes de un organismo que, aunque no posean de forma natural la capacidad de sintetizar glucosinolatos, ha adquirido dicha capacidad mediante procesos de manipulación genética.Any plant tissue or cell capable of producing glucosinolates can be used in the invention. This includes those cells from an organism that, although they do not naturally possess the ability to synthesize glucosinolates, has acquired this ability through genetic manipulation processes.
En una realización preferida de la invención, son tejidos o células vegetales procedentes de plantas de especies del orden Brassicales. En otra realización más preferida pertenecen a la familia Brassicaceae. En otra realización aún más preferida, pertenecen al género Brassica, en otra realización particular de la invención, pertenecen a la especie Brassica oleracea L. var. italica cv Chronos.In a preferred embodiment of the invention, they are plant tissues or cells from plants of species of the order Brassicales. In another more preferred embodiment they belong to the Brassicaceae family. In another even more preferred embodiment, they belong to the genus Brassica , in another particular embodiment of the invention, they belong to the species Brassica oleracea L. var. italica cv Chronos.
También se entienden como células vegetales potencialmente productoras de glucosinolatos aquellas que provienen de vitroplantas, órganos o tejidos de dichas vitroplantas, preferiblemente hojas de vitroplantas.Potentially glucosinolate-producing plant cells are also understood to be those that come from vitroplants, organs or tissues of said vitroplants, preferably vitroplant leaves.
El término” planta” u "organismo” incluye partes, tejidos, células o protoplastos procedentes de la planta o del organismo, cultivos de células, cultivos de tejidos, callos, óvulos, embriones y semillas procedentes en última instancia de la planta o el organismo.The term "plant" or "organism" includes parts, tissues, cells or protoplasts derived from the plant or the organism, cell cultures, tissue cultures, callus, ovules, embryos and seeds ultimately derived from the plant or the organism. .
Taxonómicamente Brassica oleracea L. var. italica cv Chronos se define como un organismo celular, que pertenece al supereino Eukaryota, reino Viridiplantae, phylum Streptophyta, orden Brassicales, familia Brassicaceae, tribu Brassiceae, género Brassica.Taxonomically Brassica oleracea L. var. italica cv Chronos is defined as a cellular organism, which belongs to the superkingdom Eukaryota, kingdom Viridiplantae, phylum Streptophyta, order Brassicales, family Brassicaceae, tribe Brassiceae, genus Brassica .
El término "cultivo de células” en esta memoria, hace referencia a un cultivo de células aisladas del mismo o diferente tipo de tejido, o una colección de tales células organizadas en partes de una planta o en tejidos (cultivos tisulares). Tipos de cultivos de este tipo son, por ejemplo, cultivos de protoplastos, callos (grupos de células vegetales indiferenciadas capaces de regenerar una planta completa) y células vegetales que están aisladas de plantas o partes de las plantas, tales como embriones, protoplastos, células meristemáticas, polen, hojas, raíces, anteras, pistilos, flores, semillas, vainas o vástagos de las plantas.The term "cell culture" herein refers to a culture of cells isolated from the same or different type of tissue, or a collection of such cells organized in parts of a plant or in tissues (tissue cultures). Types of cultures of this type are, for example, cultures of protoplasts, calluses (groups of undifferentiated plant cells capable of regenerating a whole plant) and plant cells that are isolated from plants or parts of plants, such as embryos, protoplasts, meristematic cells, pollen , leaves, roots, anthers, pistils, flowers, seeds, pods or shoots of plants.
En otra realización preferida de la presente invención, la concentración de jasmonato de metilo es de entre 5 y 500 micromoles/por litro de medio de cultivo. Más preferiblemente la concentración de jasmonato de metilo es de entre 25 y 150 micromoles/L medio de cultivo y aún más preferiblemente de entre 75 y 125 micromoles/L medio de cultivo.In another preferred embodiment of the present invention, the concentration of methyl jasmonate is between 5 and 500 micromoles / per liter of culture medium. More preferably the concentration of methyl jasmonate is between 25 and 150 micromoles / L culture medium and even more preferably between 75 and 125 micromoles / L culture medium.
En otra realización preferida de la presente invención, la concentración de coronatina es de entre 0.1 y 100 micromoles/por litro de medio de cultivo. Más preferiblemente la concentración de coronatina es de entre 0.5 y 50 micromoles/L medio de cultivo y aún más preferiblemente de entre 0.75 y 10 micromoles/L medio de cultivo.In another preferred embodiment of the present invention, the coronatin concentration is between 0.1 and 100 micromoles / per liter of culture medium. More preferably the concentration of coronatin is between 0.5 and 50 micromoles / L culture medium and even more preferably between 0.75 and 10 micromoles / L culture medium.
Opcionalmente, en la etapa a) se pueden adicionar, además del coronatina o jasmonato de metilo, ciclodextrinas al medio de cultivo.Optionally, in step a), in addition to coronatin or methyl jasmonate, cyclodextrins can be added to the culture medium.
Las ciclodextrinas son oligosacáridos cíclicos y se obtienen a partir de la degradación del almidón. A veces también son llamadas cicloamilosas. Presentan un exterior hidrofílico y una cavidad interior hidrofóbica donde pueden atrapar moléculas orgánicas no polares. Para cambiar las propiedades físicas y químicas de las ciclodextrinas se han desarrollado diversos derivados. Unos de los más utilizados son los derivados parcialmente metilados que tienen una solubilidad en agua hasta 150 veces superior a la del producto de partida.Cyclodextrins are cyclic oligosaccharides and are obtained from the degradation of starch. They are also sometimes called cycloamylose. They have a hydrophilic exterior and a hydrophobic interior cavity where they can trap nonpolar organic molecules. Various derivatives have been developed to change the physical and chemical properties of cyclodextrins. Some of the most used are partially methylated derivatives that have a solubility in water up to 150 times higher than that of the starting product.
Así, en otra realización preferida de la invención, las ciclodextrinas se eligen del grupo que comprende ciclodextrina metilada aleatoriamente (CDMA).Thus, in another preferred embodiment of the invention, the cyclodextrins are chosen from the group comprising randomly methylated cyclodextrin (CDMA).
Preferiblemente, el grado de sustitución por metilos por unidad de glucosa (anhidra) de la CDMA es de entre 1 y 3. En particular, el grado de sustitución por metilos por unidad de glucosa (anhidra) de la CDMA es de 2. En otra realización preferida de este aspecto de la invención un maltooligosacárido cíclico constituido por 7 unidades de D-glucosa unidas por enlaces glucosídicos de tipo a (1^ 4) (P-ciclodextrinas).Preferably, the degree of methyl substitution per glucose unit (anhydrous) of CDMA is between 1 and 3. In particular, the degree of methyl substitution per glucose unit (anhydrous) of CDMA is 2. In another preferred embodiment of this aspect of the invention a cyclic maltooligosaccharide made up of 7 D-glucose units linked by glucosidic bonds of type a (1 ^ 4) (P-cyclodextrins).
En otra realización preferida de este aspecto de la invención la concentración de ciclodextrinas es de entre 6,5 y 130 g/L medio de cultivo. Más preferiblemente la concentración de ciclodextrinas es de entre 10 y 100 g/L medio de cultivo y aún más preferiblemente es de entre 50 y 75 g/L medio de cultivo.In another preferred embodiment of this aspect of the invention the concentration of cyclodextrins is between 6.5 and 130 g / L culture medium. More preferably the concentration of cyclodextrins is between 10 and 100 g / L culture medium and even more preferably it is between 50 and 75 g / L culture medium.
En otra realización preferida de la invención, la concentración celular es de entre 10 g peso fresco (pf)/l, 1g peso seco (ps)/l y 300 gpf/l, 15 gps/l. Más preferiblemente, la concentración celular es de entre 100 gpf/l, 5 gps/l y 250g pf/l, 10 gps/l.In another preferred embodiment of the invention, the cell concentration is between 10 g fresh weight (mp) / l, 1g dry weight (ps) / l and 300 gpf / l, 15 gps / l. More preferably, the cell concentration is between 100 gpf / l, 5 gps / l and 250g pf / l, 10 gps / l.
Otro aspecto de la invención se refiere al uso de una composición, de ahora en adelante composición de la invención, que comprende coronatina o jasmonato de metilo para incrementar la producción de glucosinolatos en células vegetales con el potencial de producir glucosinolatos.Another aspect of the invention refers to the use of a composition, hereinafter composition of the invention, comprising coronatin or methyl jasmonate to increase the production of glucosinolates in plant cells with the potential to produce glucosinolates.
En una realización preferida, la composición de la invención comprende, además de coronatina o jasmonato de metilo, ciclodextrinas.In a preferred embodiment, the composition of the invention comprises, in addition to coronatin or methyl jasmonate, cyclodextrins.
Otro aspecto de la invención se refiere al uso de un medio de cultivo, de ahora en adelante medio de cultivo de la invención, que comprende coronatina o jasmonato de metilo para incrementar la producción de glucosinolatos y en células vegetales con el potencial de producir glucosinolatos.Another aspect of the invention relates to the use of a culture medium, hereinafter the culture medium of the invention, comprising coronatin or methyl jasmonate to increase the production of glucosinolates and in plant cells with the potential to produce glucosinolates.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y figuras se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. Throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
BREVE DESCRIPCIÓN DE LAS FIGURASBRIEF DESCRIPTION OF THE FIGURES
FIG. 1: Gráfica que muestra la producción total de glucosinolatos en cultivos celulares de B. olerácea var. italica cv Chronos en respuesta a los diferentes elicitores tras 168 h de incubación. FIG. 1 : Graph showing the total production of glucosinolates in cell cultures of B. olerácea var. italica cv Chronos in response to the different elicitors after 168 h of incubation.
FIG. 2: Gráfica que muestra la producción de 4-hidroxi-glucobrasicina, glucobrasicina, 4-metoxi-glucobrasicina y neoglucobrasicina por suspensiones celulares de B. olerácea var. italica cv Chronos en respuesta a diferentes elicitores tras 168 h de incubación. FIG. 2 : Graph showing the production of 4-hydroxy-glucobrassicin, glucobrassicin, 4-methoxy-glucobrassicin and neoglucobrassicin by cell suspensions of B. olerácea var. italica cv Chronos in response to different elicitors after 168 h of incubation.
EJEMPLOSEXAMPLES
A continuación se ilustrará la invención mediante unos ensayos realizados por los inventores, que pone de manifiesto el efecto del uso de coronatina o jasmonato de metilo y opcionalmente, ciclodextrinas en la producción de glucosinolatos mediante la utilización de suspensiones celulares de B. olerácea L. var italica. cv Chronos.The invention will now be illustrated by means of tests carried out by the inventors, which show the effect of the use of coronatin or methyl jasmonate and optionally, cyclodextrins in the production of glucosinolates by using cell suspensions of B. olerácea L. var italica. cv Chronos.
Preparación y mantenimiento de material vegetalPreparation and maintenance of plant material
Línea celular: Brassica olerácea L. var. italica cv ChronosCell line: Brassica olerácea L. var. italica cv Chronos
Para la inducción de callos de brócoli se utilizaron vitroplantas que se obtuvieron por germinación in vitro de semillas.For the induction of broccoli calluses, vitroplants were used that were obtained by in vitro germination of seeds.
Germinación in vitro de semillas de brócoli In vitro germination of broccoli seeds
El objetivo fundamental de la germinación de semillas in vitro es la obtención de plantas estériles para su utilización en la inducción de líneas celulares de los diferentes órganos de la planta: raíz, tallo y hoja.The fundamental objective of in vitro seed germination is to obtain sterile plants for use in the induction of cell lines of the different organs of the plant: root, stem and leaf.
Para obtener las vitroplantas de brócoli se utilizaron semillas que se sometieron a un proceso de desinfección con etanol al 70% durante 1 minuto y a continuación, se sumergieron durante 15-20 minutos en una disolución de hipoclorito cálcico al 7% que contenía Tween 20 al 0,1%. Transcurrido este tiempo, las semillas se lavaron tres veces con agua destilada estéril trabajando a partir de este lavado y en las etapas siguientes en la cabina de flujo laminar.To obtain the broccoli vitroplants, seeds were used that were subjected to a disinfection process with 70% ethanol for 1 minute and then immersed for 15-20 minutes in a 7% calcium hypochlorite solution containing 0 Tween 20. ,1%. After this time, the seeds were washed three times with sterile distilled water working from this wash and in the following stages in the laminar flow cabinet.
Las semillas desinfectadas se transfirieron a tubos que contenían el medio de cultivo basado en el descrito por Murashige y Skoog (1962) (Murashige, T., &Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia plantarum, 15(3), 473-497), suplementado con pantotenato cálcico (1mg/l), mioinositol (100 mg/l), biotina (0,01 mg/l), piridoxina (1 mg/l), tiamina (1 mg/l), ácido nicotínico (1 mg/l) e hidrolizado de caseína (250 mg/L). Como fuente carbonada se utilizó sacarosa (30 g/l). Este medio se ajusta a pH 6,0 y se esteriliza mediante la aplicación de calor húmedo (autoclave) durante 20 minutos a 1.2 atmósferas de sobrepresión, adquiriendo la consistencia sólida mediante la adición de agar purificado (6,2 g/l) cuando el medio se enfría.The disinfected seeds were transferred to tubes containing the culture medium based on that described by Murashige and Skoog (1962) (Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia plantarum, 15 ( 3), 473-497), supplemented with calcium pantothenate (1mg / l), myoinositol (100 mg / l), biotin (0.01 mg / l), pyridoxine (1 mg / l), thiamine (1 mg / l), nicotinic acid (1 mg / l) and casein hydrolyzate (250 mg / l L). Sucrose (30 g / l) was used as a carbon source. This medium is adjusted to pH 6.0 and sterilized by applying humid heat (autoclave) for 20 minutes at 1.2 atmospheres of overpressure, acquiring the solid consistency by adding purified agar (6.2 g / l) when the medium cools down.
Obtención de callos y mantenimiento de suspensiones celulares de brócoli Los hipocótilos de las vitroplantas se utilizaron como fuente de explanto. Las placas Petri con los explantos se mantuvieron bajo un fotoperiodo de 16 horas de luz, 8 horas de oscuridad a una irradiancia de 17,4 w/m2 a 25 °C, observándose al cabo de 2-3 semanas la aparición de microcallos que se transfirieron a matraces de 250 mL de capacidad que contenían 100 ml del medio de cultivo anteriormente descrito suplementado con ácido naftalenacético (1 mg/l) y benciladenina (10 mg/l). Obtaining callus and maintaining broccoli cell suspensions The hypocotyls of the vitroplants were used as a source of explant. The Petri dishes with the explants were kept under a photoperiod of 16 hours of light, 8 hours of darkness at an irradiance of 17.4 w / m2 at 25 ° C, observing after 2-3 weeks the appearance of microcallos that were transferred to 250 mL flasks containing 100 ml of the above-described culture medium supplemented with naphthaleneacetic acid (1 mg / l) and benzyladenine (10 mg / l).
Las suspensiones celulares se iniciaron, a partir de callos friables, en matraces de 250 ml de capacidad que contenían 80 ml de medio cultivo sin agar. Las células en medio líquido se mantuvieron en un agitador orbital a 100 rpm, en las mismas condiciones de luz y Ta y se subcultivaron cada 17 días.Cell suspensions were started from friable calli in 250 ml flasks containing 80 ml of agar-free culture medium. The cells in liquid medium were kept on an orbital shaker at 100 rpm, under the same conditions of light and Ta and were subcultured every 17 days.
Elicitación de las célulasCell elicitation
En cada experiencia de elicitación se tomaron, en condiciones de esterilidad, entre 90 y 240 g de peso fresco de células por litro que habían sido previamente lavadas con medio fresco y filtradas. Utilizando esta densidad celular, las células se repartieron en matraces que contenían el medio fresco suplementadoIn each elicitation experiment, between 90 and 240 g of fresh weight of cells per liter that had been previously washed with fresh medium and filtered were taken under sterile conditions. Using this cell density, cells were partitioned into flasks containing supplemented fresh medium.
- solo con jasmonato de metilo (en adelante, MJ) a una concentración 100 micromolar. - solo con coronatina (en adelante, Cor) a una concentración 1 micromolar- only with methyl jasmonate (hereinafter, MJ) at a concentration of 100 micromolar. - only with coronatin (hereinafter Cor) at a concentration of 1 micromolar
- sólo con una B-ciclodextrina metilada aleatoriamente con un grado de sustitución por metilos de entre 1,6 y 1,9 (en adelante, CDMA) a una concentración de 62,4 g/l.- only with a randomly methylated B-cyclodextrin with a degree of methyl substitution between 1.6 and 1.9 (hereinafter CDMA) at a concentration of 62.4 g / l.
- con ciclodextrinas anteriormente mencionadas (CDMA) a una concentración 62,5 g/l y con jasmonato de metilo (MJ) a una concentración 100 micromolar.- with previously mentioned cyclodextrins (CDMA) at a concentration of 62.5 g / l and with methyl jasmonate (MJ) at a concentration of 100 micromolar.
- con ciclodextrinas anteriormente mencionadas (CDMA) a una concentración 62,5 g/l y con coronatina (Cor) a una concentración 1 micromolar.- with previously mentioned cyclodextrins (CDMA) at a concentration of 62.5 g / l and with coronatin (Cor) at a concentration of 1 micromolar.
El jasmonato de metilo se esteriliza aparte del medio por filtración, disuelto en etanol, y posteriormente se mezcla con el resto del medio estéril. La concentración final de etanol en el medio de cultivo es de 0,2% en volumen. The methyl jasmonate is sterilized apart from the medium by filtration, dissolved in ethanol, and subsequently mixed with the rest of the sterile medium. The final concentration of ethanol in the culture medium is 0.2% by volume.
Los matraces se incubaron en las mismas condiciones descritas anteriormente para su mantenimiento en medio líquido durante 168 horas.The flasks were incubated under the same conditions described above for maintenance in liquid medium for 168 hours.
Muestreo y preparación de muestras para análisisSampling and sample preparation for analysis
Los cultivos incubados con elicitores se recogieron cada cierto tiempo para su análisis. Las células fueron separadas del medio por filtración realizando un ligero vacío, y se utilizaron para la extracción de los compuestos y posterior análisis por HPLC acoplado a un espectrómetro de masas.The cultures incubated with elicitors were collected from time to time for analysis. The cells were separated from the medium by filtration using a slight vacuum, and were used for the extraction of the compounds and subsequent analysis by HPLC coupled to a mass spectrometer.
Análisis de glucosinolatosGlucosinolate Analysis
Una vez realizados los experimentos de elicitación, la extracción de los compuestos presentes en las células de brócoli, se realizó añadiendo 4,5 mL de metanol al 70% a 0,2 g de material vegetal previamente liofilizado. A continuación, las muestras se incubaron en un baño a 73°C durante 20 minutos. Durante el tiempo de incubación las muestras se agitan vigorosamente cada 5 minutos, con el fin de favorecer la eficiencia del proceso de extracción. Transcurrido ese tiempo las muestras se dejaron enfriar en hielo. Posteriormente las muestras se centrifugaron 15 minutos a 10000 rpm y 4°C. Se guardó el sobrenadante y el precipitado se resuspendió en 3 ml de metanol al 70%, para después agitar y volver a centrifugar 15 min, a 10000 rpm y 4°C. Tras la centrifugación, se mezclaron ambos sobrenadantes y se concentraron en un rotavapor. El residuo seco se resuspendió en 2 ml de agua ultrapura y se filtró a través de membranas inorgánicas de 0,22 pm de poro para su análisis por HPLC-MS.Once the elicitation experiments had been carried out, the extraction of the compounds present in the broccoli cells was carried out by adding 4.5 mL of 70% methanol to 0.2 g of previously lyophilized plant material. The samples were then incubated in a 73 ° C bath for 20 minutes. During the incubation time, the samples are vigorously shaken every 5 minutes, in order to favor the efficiency of the extraction process. After that time the samples were allowed to cool on ice. Afterwards, the samples were centrifuged for 15 minutes at 10,000 rpm and 4 ° C. The supernatant was saved and the precipitate was resuspended in 3 ml of 70% methanol, then stirred and centrifuged again for 15 min, at 10,000 rpm and 4 ° C. After centrifugation, both supernatants were mixed and concentrated on a rotary evaporator. The dry residue was resuspended in 2 ml of ultrapure water and filtered through 0.22 pm pore inorganic membranes for analysis by HPLC-MS.
La identificación y cuantificación de los glucosinolatos se realizó en un cromatógrafo (Agilent Series 1200, Agilent Technologies) acoplado a un detector espectrómetro de masas triple cuadrupolo (MS/MS), con fuente de ionización por electrospray (ESI) operando en modo negativo. Para ello, se utilizó nitrógeno como gas nebulizador a una presión de 60 psi y ajustándose el flujo a 13 L/ min. La temperatura y el voltaje del capilar se mantuvieron a 350 °C y 4 kV, respectivamente. La separación cromatográfica se realizó en una columna Luna C18 (250 mm * 46 mm, tamaño de partícula de 5 m; con precolumna C18-ODS (4 * 30 mm).The identification and quantification of glucosinolates was carried out in a chromatograph (Agilent Series 1200, Agilent Technologies) coupled to a triple quadrupole mass spectrometer (MS / MS) detector, with an electrospray ionization source (ESI) operating in negative mode. For this, nitrogen was used as the nebulizer gas at a pressure of 60 psi and the flow was adjusted to 13 L / min. Capillary temperature and voltage were maintained at 350 ° C and 4 kV, respectively. Chromatographic separation was performed on a Luna C18 column (250mm * 46mm, 5m particle size; with C18-ODS guard column (4 * 30mm).
El tiempo de retención de los diferentes glucosinolatos y la respuesta del detector a la concentración de los mismos (curva de calibrado para cuantificación) se determinó usando patrones externos. The retention time of the different glucosinolates and the response of the detector to their concentration (calibration curve for quantification) was determined using external standards.
EJEMPLO 1: Elicitación de células de brócoli (Brassica olerácea L. variedad italica cv Chronos) con coronatina (Cor), jasmonato de metilo (MJ) y/o ciclodextrinas (CDMA)EXAMPLE 1: Elicitation of broccoli cells ( Brassica olerácea L. variety italica cv Chronos) with coronatin (Cor), methyl jasmonate (MJ) and / or cyclodextrins (CDMA)
Las suspensiones de Brassica olerácea L. variedad italica cv Chronos se trataron con: - controles sin CDMA, MJ ni CorSuspensions of Brassica olerácea L. variety italica cv Chronos were treated with: - controls without CDMA, MJ or Cor
- con MJ (100 micromoles/l)- with MJ (100 micromoles / l)
- con Cor (1 micromol/l)- with Cor (1 micromol / l)
- con CDMA (62,5 g/l)- with CDMA (62.5 g / l)
- con CDMA (62,5 g/l) y MJ (100 micromoles/l)- with CDMA (62.5 g / l) and MJ (100 micromoles / l)
- con CDMA (62,5 g/l) y Cor (1 micromol/l)- with CDMA (62.5 g / l) and Cor (1 micromol / l)
Los experimentos se llevaron a cabo por triplicado en matraces de 250 ml de capacidad conteniendo 100 ml de medio de cultivo. Al cabo de 168 horas, se recolectaron las células, se pesaron y se determinó la cantidad total de glucosinolatos en el interior celular. El resultado del experimento se muestra en la Tablas A y B y Figura 1 y 2.The experiments were carried out in triplicate in 250 ml flasks containing 100 ml of culture medium. After 168 hours, the cells were harvested, weighed and the total amount of glucosinolates in the cell interior was determined. The result of the experiment is shown in Tables A and B and Figure 1 and 2.
Cuando el cultivo se elicitó solo con MJ o Cor, se observó una acumulación de glucosinolatos por unidad de biomasa superior a cualquiera de los tratamientos realizados.When the culture was elicited only with MJ or Cor, an accumulation of glucosinolates per unit of biomass was observed higher than any of the treatments carried out.
Por otro lado se observó que no existían diferencias significativas con los distintos tratamientos tras 168 horas de elicitación en la producción de glucosinolatos.On the other hand, it was observed that there were no significant differences with the different treatments after 168 hours of elicitation in the production of glucosinolates.
Además, la producción de glucobrasicina y 4-metoxi-glucobrasicina representó más del 85% del total de los glucosinolatos producidos alcanzando valores máximos de 996.68 ± 46.30 y 865.70 ± 39.16 pg/g ps, respectivamente en el tratamiento sólo con MJ siendo los valores alcanzados tras el tratamiento con coronatina de 1185.96 ± 111.06 y 843.35 ± 101.28 pg/g ps de glucobrasicina y 4-metoxi-glucobrasicina, respectivamenteFurthermore, the production of glucobrassicin and 4-methoxy-glucobrassicin represented more than 85% of the total glucosinolates produced, reaching maximum values of 996.68 ± 46.30 and 865.70 ± 39.16 pg / g ps, respectively in the treatment only with MJ, the values reached being after coronatin treatment of 1185.96 ± 111.06 and 843.35 ± 101.28 pg / g ps of glucobrassicin and 4-methoxy-glucobrassicin, respectively
TABLA A: Producción de glucosinolatos en pg/gps de B. olerácea var italica cv Chronos tras 168 horas de incubación. TABLE A: Production of glucosinolates in pg / gps of B. olerácea var italica cv Chronos after 168 hours of incubation.
Densidad celular: 12 g ps/l Cell density: 12 g ps / l
Control: medio de cultivo sin elicitoresControl: culture medium without elicitors
MJ: tratamiento con 100 micromoles/lMJ: treatment with 100 micromoles / l
Cor: tratamiento con 1 micromol/lCor: treatment with 1 micromole / l
CDMA: tratamiento con 62,5 g/lCDMA: treatment with 62.5 g / l
CDMA+MJ: tratamiento con 62,5 g/l de CDMA combinado con 100 micromoles/l de MJ CDMA+Cor: tratamiento con 62,5 g/l de CDMA combinado con 1 micromol/l de CorCDMA + MJ: treatment with 62.5 g / l of CDMA combined with 100 micromoles / l of MJ CDMA + Cor: treatment with 62.5 g / l of CDMA combined with 1 micromol / l of Cor
TABLA B: Producción de glucobrasicina, 4-metoxi-glucobrasicina, 4-hidroxiglucobrasicina y neoglucobrasicina en pg/g PS de B. olerácea var italica cv Chronos tras 168 horas de incubación. TABLE B: Production of glucobrassicin, 4-methoxyglucobrassicin, 4-hydroxyglucobrassicin and neoglucobrassicin in pg / g PS of B. olerácea var italica cv Chronos after 168 hours of incubation.
Densidad celular: 12 g ps/lCell density: 12 g ps / l
Control: medio de cultivo sin elicitoresControl: culture medium without elicitors
MJ: tratamiento con 100 micromoles/lMJ: treatment with 100 micromoles / l
Cor: tratamiento con 1 micromol/lCor: treatment with 1 micromole / l
CDMA: tratamiento con 62,5 g/lCDMA: treatment with 62.5 g / l
CDMA+MJ: tratamiento con 62,5 g/l de CDMA combinado con 100 micromoles/l de MJ CDMA+Cor: tratamiento con 62,5 g/l de CDMA combinado con 1micromol/l de Cor
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