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    • 专利摘要:
    Procedure to increase the production of glucosinolates in cell cultures. The present invention relates to a process for increasing the production of glucosinolates in cell cultures by adding to the culture medium coronatin (structural and functional analogue of the octadecanoid precursor of methyl jasmonate) or methyl jasmonate. It also relates to the use of a culture medium comprising coronatin or methyl jasmonate to increase the production of glucosinolates in plant cells potentially producing them. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2694706A1
    申请号:ES201730836
    申请日:2017-06-23
    公开日:2018-12-26
    发明作者:Ana Belén SABATER JARA；Pedro Joaquín SÁNCHEZ PUJANTE；María BORJA MARTÍNEZ；María Ángeles PEDREÑO GARCÍA
    申请人:Agricola Santa Eulalia S L；Agricola Santa Eulalia Sl；Universidad de Murcia；
    IPC主号:
    专利说明:

    Procedure to increase the production of glucosinolates in cell cultures
    The present invention is within the field of biotechnology and pharmacy, and relates to a method for increasing the production of glucosinolates from plant cell cultures by adding to the culture medium coronatin (a structural and functional analog of the invention). octadecanoid precursor of methyl jasmonate) or
    10 methyl jasmonate and, optionally, cyclodextrins.
    BACKGROUND OF THE INVENTION
    Broccoli (Brassica oleracea var. Italica) also known as broccoli, or broccoli, is an edible plant that belongs to the family of Brassicaceae or cruciferous, as well as cabbage, Brussels sprouts, cauliflower, radish, the cabbage and the arugula.
    The plants belonging to the family Brassicaceae (crucifers) are characterized by having a large number of phytochemical compounds, with the largest
    20 concentration of these in a juvenile physiological state (in the form of an outbreak), unlike the adult physiological state with lower phytochemical content, due to the dilution of these compounds caused by the growth of different tissues. Within this family of plants, broccoli (Brassica oleracea varo italica) is characterized for being a vegetable with beneficial effects for health, whose
    25 world production (together with cauliflower) has been increasing in the last decades reaching a world production of 24,175,000 tons.
    During the last decades, special attention has been given to natural bioactive compounds with potential for the treatment and prevention of
    30 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 prevention
    35 of some diseases in humans that consume them. In addition, they contribute to the 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 5 Science of Food and Agriculture, 86 (12), 1805-1813).
    Among the bioactive compounds of broccoli, include glucosinolates and phenolic compounds (flavonoids and hydroxycinnamic acids), as well as other nutrients such as carotenoids, sterols, vitamin E, fiber content and essential minerals for health. Glucosinolates, also called thioglucosides, are nitrogen-sulfur anionic secondary metabolites that are found almost exclusively in plants of the family Brassicaceae (cruciferous), including broccoli, where they reach the highest concentration, that is, they are the bioactive compounds par excellence of the broccoli The basic skeleton of the 15 glucosinolates (j3-D-thiogluC; osido-N-hydroxysulfate) consists of a j3-D-thioglucose group, a sulphonated 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 may vary between species and between varieties within the same species. Currently more than 120 types of glucosinolates are known
    20 in the cruciferous, being the most abundant in broccoli glucoraphanin (80%), glucobrassicin (8%), 4-methoxy-glucobrassicin (4%) and 1-methoxy-glucobrassicin (4%). Among the degradation compounds of glucosinolates are the isothiocyanates commonly referred to as "mustard oils", with sulforaphane being the main isothiocyanate found in broccoli.
    25 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 Jeffery EH, Araya M. 2009. Physiological effects of broccoli. Reviews 8 (1): 283-298.). However, these
    30 meta bolites are not bioactive in the animal that consumes them until they have been hydrolysed 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 quinonareductases and glutathione-Stransferases. Sulforaphane, which is the hydrolysis product of glucosinolate
    35 glucoraphanin is a very potent mono-inducer of the metabolism of phase 11. Induces
    apoptosis and inhibits tumor growth during initiation, promotion and progression phases (My LX, Wang XT, Govind S, Hood BL, Veenstra TD, Conrads TP, Saha OT, 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 5 Cancer Research 67: 6409-6416). Also, recently treatment with sulforaphane has been described in several types of autism with positive results, showing a significant improvement in patients who consumed broccoli on a regular basis compared to those who did not consume it (Singh K, L. Connors S, A. Macklin E, O. Smith K, W. Fahey J, Talalay P, W. Zimmerman A. 2014. Sulforaphane treatment of
    10 autism spectrum disorder (ASO). PNAS. vol. 111 no. 43, 15550-15555).
    The glucosinolates are secondary meta bolites that are going to be synthesized and stored by the plant cells to respond to different stresses to which they are subjected, so that their production in plant cells is increased by
    15 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 difficult or unprofitable natural compounds to be prepared by chemical synthesis or that pose serious aggressions to the environment when resorting to the source
    20 natural.
    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 that its use presents:
    25 -are independent of geographic, seasonal and environmental factors. -constitute stable production systems since they ensure the continuous obtaining of compounds with uniform quality and productivity -the space requirements for the development of production are reduced. -the process of purification of the compounds of interest is easier and is optimized
    30 when it is released into the culture medium and can be performed on a large scale. -permits to obtain new compounds that are not synthesized by plants naturally.
    To date, there are no previous studies where cell cultures of broccoli are used for the production of metabolites of interest. However, exogenous addition
    of various elicitors (salicylic acid or methyl jasmonate (MJ) to broccoli pellets has been used previously to improve its 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 5 Chem. 2014, 62, 1881-1889) showed that MJ caused an increase in the total amount of glucosinolates in shoots of Brassica oleracea varo italica, the highest production corresponded to the indole glucosinolates and glucoraphanin, while salicylic acid produced a smaller increase in the amount of total glucosinolates in these shoots, and it has been described that a concentration of 250 μl of MJ applied to 10 pellets of broccoli produced an increase of both glucosinolates and their degradation products between 10 and 300% (Ku KM, Jeffery EH, Juvik JA (2013) Influence of seasonal variation and methyl jasmonate media ted 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 IJM) did not appear
    15 to have an effect on the content of glucosinolates, with the exception of an increase of 10% of indole-glucosinolates when MJ was applied at a concentration of 10 IJM (PérezBalibrea S, Moreno DA, Garcia-Viguera C (2011) Improving the phytochemical composition of broccoli sprouts by elicitation, Food Chem 129: 35--44)
    20 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 cell suspensions of Arabidopsis thaliana derived from hypocotyl synthesized 7 different glucosinolates, among which indole glucosinolates or glucoraphanin were found,
    25 some of which were present in greater quantity than in the vegetable tissue that gave rise to the cell suspension. In conjunction with this, this group showed that the elicitation of cell culture with MJ caused a considerable increase in glucosinolate production, also demonstrating an increase in myrosinase activity in A. thaliana cell suspensions treated with MJ.
    30 However, in no case does it describe the use of coronatin or MJ as a stimulator of glucosinolate production in broccoli cell cultures. In that sense, it has been observed that coronatin, a toxin produced by Pseudomonas syringae (Weiler, E.W., Kutchan, T.M., Garba, T., Brodschelm, W., Niesel, U., & Bublilz, F.
    35 (1994). The Pseudomonas phytotoxin coronatine mimics octadecanoid signalling
    molecules of higher plants. FEBS leffers, 345 (1), 9-13), is a structural analogue of the active form methyl jasmonate (JA-lIe) and has proved to be a potent elicitor for the production of taxanes and trans-resveratrol in cell cultures of Taxus sp and Vitis vinifera, respectively (Onrubia, M., Moyana, E., Bonfill, M., Cusidó, RM, 5 Goossens, A., & Palazón, J. (2013) Coronatine, a more powerful elicitor for inducing taxane biosynthesis in Taxus media cell cultures than methyl jasmonate Journal of Plan! Physiology, 170 (2), 211-219; Almagro, L., Belchi-Navarro, S., Martinez-Marquez, A., Bru, R., & Pedreño, MA (2015) Enhanced extracellular production of transresveratrol in Vitis vinifera suspension cultured cells by using cyclodextrins and
    10 coronatine. Plant Physiology and Biochemistry, 97, 361-367).
    DESCRIPTION OF THE INVENTION The increase in glucosinolate synthesis is exemplified in cell cultures of Brassica o / eracea variety italica cv Chronos, one of the species producing
    15 most important glucosinolates. The authors of the present invention have observed that by establishing plant cell cultures and adding to the culture medium coronatin or methyl jasmonate, the production yield is increased and
    twenty extraction of glucosinolates or .... ~
    25
    30 Methyl Jasmonate Thus, a first aspect of the present production of glucosinolates, comprising:
    or
    H
    Coronatina invention is the increase of
    to. the addition of coronatin or methyl jasmonate to a culture medium,
    b. contacting plant cells potentially producing glucosinolates with the culture medium of (a),
    c. Incubation of the plant cells of step b) in the culture medium of step a),
    d. the separation of the glucosinolates obtained after step c) of the cell culture.
    It is understood by plant cells potentially producing glucosinolates,5 any cell line capable of producing glucosinolates, either naturally or aftergenetic modification.
    Any tissue or plant 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, have acquired this capacity through genetic manipulation processes.
    In a preferred embodiment of the invention, they are plant tissues or cells from plants of species of the Brassicales order. In another embodiment more
    15 preferred 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.
    Also understood as plant cells potentially producing glucosinolates are those that come from vitroplants, organs or tissues of said vitroplants, preferably leaves from vitroplants.
    The term "plant" or "organism" includes parts, tissues, cells or protoplasts from the plant or organism, cell cultures, tissue cultures, corns, ovules, embryos and seeds ultimately derived from the plant or the body. organism.
    Taxonomically Brassica oleracea L. var. italica cv Chronos is defined as a cellular organism, belonging to the supereno Eukaryota, kingdom Viridiplantae, phylum 30 Streptophyta, order Brassicales, family Brassicaceae, tribe Brassiceae, genus Brassica.
    The term "cell culture" herein refers to a culture of cells isolated from the same or different tissue type, or a collection of such cells organized in parts of a plant or in tissues (tissue cultures). Types of crops
    of this type are, for example, protoplast cultures, callus (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,
    5 flowers, seeds, pods or stems of plants.
    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
    10 micromoles / L culture medium and even more preferably between 75 and 125 micromoles / L culture medium.
    In another preferred embodiment of the present invention, the concentration of coronatin 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.
    Optionally, in step a), in addition to the coronatin or methyl jasmonate, cyclodextrins can be added to the culture medium.
    Cyclodextrins are cyclic oligosaccharides and are obtained from the degradation of starch. Sometimes they are also called cycloamymal. They present a hydrophilic exterior and a hydrophobic interior cavity where they can trap non-polar organic molecules. To change the physical and chemical properties of
    25 cyclodextrins have been developed various derivatives. Some of the most commonly used are partially methylated derivatives which have a solubility in water up to 150 times higher than that of the starting product.
    Thus, in another preferred embodiment of the invention, the cyclodextrins are selected from group 30 comprising randomly methylated cyclodextrin (CDMA).
    Preferably, the degree of substitution by methyl (per anhydrous) glucose unit of the CDMA is between 1 and 3. In particular, the degree of substitution by methyls per glucose unit (anhydrous) of the CDMA is 2. In another preferred embodiment of this aspect of the invention a cyclic maltooligosaccharide consisting of 7 units of D
    glucose bound by glycosidic bonds of type 0 (1 _4) (~ -cyclodextrins).
    In another preferred embodiment of this aspect of the invention the concentration of cyclodextrins is between 6.5 and 130 gIL culture medium. More preferably the concentration of cyclodextrins is between 10 and 100 gIL culture medium and even more preferably is between 50 and 75 gIL culture medium.
    In another preferred embodiment of the invention, the cell concentration is between 10 g fresh weight (pf) / I, 19 dry weight (ps) / I and 300 gpf / I, 15 gps / l. More preferably, the cell concentration is between 100 gpf / l, 5 gps / l and 250 g ptll, 10 gps / l.
    Another aspect of the invention relates to the use of a composition, hereinafter the composition of the invention, comprising coronatin or methyl jasmonate to increase the production of glucosinolates in plant cells with the potential to produce glucosinolates.
    In a preferred embodiment, the composition of the invention comprises, in addition to coronatin or methyl jasmonate, cyclodextrins.
    Another aspect of the invention relates to the use of a culture medium, hereinafter 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.
    Throughout the description and the claims the word "comprises" and its variants do not intend 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 partly from the description and partly 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.
    BRIEF DESCRIPTION OF THE FIGURES FIG. 1: Graph showing the total production of glucosinolates in cell cultures
    of B. oleracea var. italica cv Chronos in response to the different elicitors after 168 h of incubation.
    5 FIG. 2: Graph showing the production of 4-hydroxy-glucobrassicin, glucobrassicin,4-methoxy-glucobrassicin and neoglucobrasicin by cell suspensions of B.oleracea varo italica cv Chronos in response to different elicitors after 168 h ofincubation.
    10 EXAMPLES
    The invention will now be illustrated by means of tests carried out by the inventors, which reveals the effect of the use of coronatin or methyl jasmonate and, optionally, cyclodextrins in the production of glucosinolates by means of the
    15 use of cell suspensions of B. oleracea L. var italica. cv Chronos.
    Preparation and maintenance of plant material Cell line: Brassica o / eracea L. var. italica cv Chronos For the induction of broccoli tripe, vitroplants that were obtained by
    20 in vitro germination of seeds.
    In vitro germination of broccoli seeds The fundamental objective of germination of seeds in vitre is the obtaining of sterile plants for use in the induction of cell lines of the
    25 different organs of the plant: root, stem and leaf. 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 solution of 7% calcium hypochlorite containing Tween 20 at 0 ,one%. After this time, the seeds were washed three times
    30 times with sterile distilled water working from this wash and in the following stages in the laminar flow cabinet. 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.
    35 Physiologia plantarum, 15 (3), 473-497), supplemented with calcium pantothenate (1 mg / 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). Sucrose (30 gIl) was used as the carbon source. This medium is adjusted to pH 6.0 and sterilized by applying moist heat (autoclave) for 20 minutes at 1.2 atmospheres of
    5 overpressure, acquiring solid consistency by adding purified agar (6.2 gIl) when the medium is cooled.
    Obtaining Calluses and Maintaining Cellular Suspensions of Broccoli The hypocotyls of vitroplants were used as a source of explant. The plates
    10 Petri 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 oC, and after 2-3 weeks the appearance of microcallos was observed, which were transferred to 250 mL capacity flasks containing 100 mL of the above-described culture medium supplemented with naphthaleneacetic acid (1 mg / L) and benzyladenine (10 mg / L).
    15 Cell suspensions were started, from friable calluses, in 250 ml flasks containing 80 ml of culture medium without agar. The cells in liquid medium were kept in an orbital shaker at 100 rpm, in the same light conditions and Ta and subcultured every 17 days.
    20 Elicitation of the cells In each elicitation experiment, between 90 and 240 g of fresh weight of cells per liter, which had been previously washed with fresh medium and filtered, were taken under sterile conditions. Using this cell density, the cells were divided into flasks containing the supplemented fresh medium
    25 - only with methyl jasmonate (hereinafter, MJ) at a 100 micromolar concentration. -only with coronatin (hereafter, Cor) at a concentration of 1 micromolar -only with one r ..- cyclodextrin methylated randomly with a degree of substitution by methyls between 1.6 and 1.9 (hereinafter, COMA) at a concentration of 62.4 gIl. -with aforementioned cyclodextrins (COMA) at a concentration 62.5 gIl
    30 And with methyl jasmonate (MJ) at a 100 micromolar concentration. -with previously mentioned cyclodextrins (COMA) at a concentration 62.5 gIl Y with coronatin (Cor) at a concentration of 1 micromolar. 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
    The ethanol in the culture medium is 0.2% by volume.
    The flasks were incubated in the same conditions described above for maintenance in liquid medium for 168 hours.
    Sampling and preparation of samples for analysis
    5 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.
    10 Analysis of glucosinolates Once the elicitation experiments were 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. Next, the samples were incubated in a bath at 73 ° C for 20 minutes. During the incubation time the
    15 samples are shaken vigorously every 5 minutes, in order to favor the efficiency of the extraction process. After that time the samples were allowed to cool on ice. Subsequently, the samples were centrifuged 15 minutes at 10,000 rpm and 4 ° C. The supernatant was stored and the precipitate was resuspended in 3 ml of 70% methanol, and then stirred and re-centrifuged 15 min at 10,000 rpm and 4 ° C. Behind the
    After centrifugation, both supernatants were mixed and concentrated in a rotary evaporator. The dried residue was resuspended in 2 ml of ultra pure water and filtered through inorganic membranes of 0.22 iJm pore for analysis by HPLC-MS. The identification and quantification of glucosinolates was performed in a chromatograph (Agilent Series 1200, Agilent Technologies) coupled to a detector spectrometer of
    25 triple quadrupole masses (MS / MS), with electrospray ionization source (ESI) operating in negative mode. For this, nitrogen was used as a nebulizer gas at a pressure of 60 psi and the flow was adjusted to 13 LI min. Temperature and capillary voltage were maintained at 350 oC and 4 kV, respectively. The chromatographic separation was carried out on a Luna C18 column (250 mm) (46 mm,
    30 particle of 5 m; with precolumn C18-0DS (4) (30 mm).
    The retention time of the different glucosinolates and the response of the detector to the concentration thereof (calibration curve for quantification) was determined using external standards.
    EXAMPLE 1: Elicitation of broccoli cells (Brassica o / eracea L. variety italica cv Chronos) with coronatin (Cor), methyl jasmonate (MJ) and / or cyclodextrins (COMA)
    5 Suspensions of Brassica o / eracea L. variety italica cv Chronos were treated with:controls without CDMA, MJ or Corwith MJ (100 micromoles / l)with Cor (1 micromol / l)
    with CDMA (62.5 gil) 10 with CDMA (62.5 gil) YMJ (100 mieromoles / l) with CDMA (62.5 gil) Y Cor (1 mieromol / l)
    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 was determined
    15 inside the cell. The result of the experiment is shown in Tables A and B and Figures 1 and 2.
    When the culture was elicited only with MJ or Cor, an accumulation of glucosinolates per unit of biomass superior to any of the treatments 20 was observed.
    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.
    25 In addition, the production of glucobrassicin and 4-methoxy-glucobrassicin accounted for more than 85% of the total glucosinolates produced reaching maximum values of
    996.68 ± 46.30 and 865.70 ± 39.16 1-19 / g ps, respectively in the treatment with MJ only, being the values reached after the treatment with coronatin of 1185.96 ±
    111 .06 and 843.35 ± 101.28 1-19 / g ps of glucobrassicin and 4-methoxy-glucobrassicin, 30 respectively
    TABLE A: Production of glucosinolates in 1-19 / gps of B. o / eracea var italica cv Chronos after 168 hours of incubation.
    35 Cell density: 12 9 psl l Control: culture medium without MJ elicitors: treatment with 100 micromoles / l Cor: treatment with 1 micromol / l COMA: treatment with 62,5 gi l COMA + MJ: treatment with 62,5 gil of COMA combined with 100 micromoles / l of MJ COMA + Cor: treatment with 62.5 gi l of COMA combined with 1 micromol / l of Cor
    Total glucosinolates
    ~ g / g PS
    Control 571 .51 ± 118.24
    MJ 1950.82 ± 515.36
    Cor 2653.34 ± 608.92
    COMA 522.59 ± 75.46
    COMA + MJ 462.03 ± 50.71
    CDMA + Cor 1767.82 ± 85.48
    10 TABLE B: Production of glucobrassicin, 4-methoxy-glucobrassicin, 4-hydroxyglucobrasicin and neoglucobrassicin at 1-19 μg PS of B. oJeracea var italica cv Chronos after 168 hours of incubation.
    15 Cell density: 12 g psll Control: culture medium without elicitors MJ: treatment with 100 micromoles / l Cor: treatment with 1 micromol / l COMA: treatment with 62, S gIl
    20 COMA + MJ: treatment with 62.5 g of COMA combined with 100 micromoles / l of MJ COMA + Cor: treatment with 62.5 g of COMA combined with 1 micromol / l of Cor
    4-Hydroxy ~ g / gPS Glucobrassicin Control 63.39 ± 0.14 MJ 16.67 ± 2.10 Cor 13.92 ± 2.64 COMA 36.36 ± 5.62 COMA + MJ 10.88 ± 7.97 COMA + Cor 53.26 ± 11 .16 Glucobrassicin 247.31 ± 1 6.27 996.67 ± 46.30 11 85.96 ± 111 .07 186.54 ± 38.53 81.15 ± 21.39 560.79 ± 56.694-Methoxy-Glucobrassicin 240.87 ± 92.61 865.69 ± 39.16 843.34 ± 101.28 294.24 ± 27.44 354.12 ± 15. 18 1008.70 ± 4.64Neoglucobrasicin 19.93 ± 1.54 71.768 ± 32.46 610. 11 ± 53.93 5.44 ± 3.87 15.87 ± 6.17 145.05 ± 12.99
    权利要求:
    Claims (20)
    [1]
    1. Procedure to increase the production of glucosinolates in cell cultures, comprising:
    to. the addition of coronatin or methyl jasmonate to a culture medium,
    b. contacting plant cells potentially producing glucosinolates with the culture medium of step (a),
    c. incubating the cells of step (b) in the culture medium of step (a), and
    d. the separation of the glucosinolates obtained after step (c) of the cell culture.
    [2]
    two. Process according to the preceding claim, wherein the plant cells potentially producing glucosinolates of step (b) are plant cells from plants of the order Brassicales.
    [3]
    3. Process according to any of claims 1-2, wherein the plant cells potentially producing glucosinolates are plant cells from plants that are selected from the Brassicaceae family.
    [4]
    Four. Process according to any of claims 1-3, wherein the plant cells potentially producing glucosinolates are plant cells from plants of the Brassica genus.
    [5]
    5. Process according to any of claims 1-4, wherein the plant cells potentially producing glucosinolates are plant cells from B. oleracea var italica.
    [6]
    6 Process according to any of claims 1-5, wherein the plant cells potentially producing glucosinolates come from vitroplants.
    [7]
    7 The method according to claim 6, wherein the plant cells potentially producing glucosinolates come from leaves of vitroplants.
    [8]
    8 Process according to any of claims 1-7, wherein step a) comprises the addition of methyl jasmonate to the culture medium and where the
    The concentration of methyl jasmonate in the culture medium of step (a) is between 5 and 500 micromoles / liter.
    [9]
    9. Process according to claim 8, wherein the concentration of methyl methyl jasmonate in the culture medium of step (a) is between 25 and 150 micromoles / liter.
    [10]
    10. Process according to claim 9, wherein the concentration of methyl jasmonate in the culture medium of step (a) is between 75 and 125 micromoles! liter.
    10 11. The process according to any of claims 1-7, wherein step a) comprises the addition of coronatin to the culture medium and wherein the concentration of coronatin in the culture medium of step (a) is between 0.1 and 100 micromoles! liter.
    [12]
    12. Method according to claim 11, wherein the concentration of coronatin in the culture medium of step (a) is between 0.5 and 50 micromoles / liter.
    [13]
    13. Method according to claim 12, wherein the concentration of coronatin in the culture medium of step (a) is between 0.75 and 10 micromoles! liter
    14. Process according to any of claims 1 to 13 wherein cyclodextrins are further added to the culture medium in step (a).
    [15]
    15. Process according to claim 14, wherein the cyclodextrins are selected from the group comprising randomly methylated cyclodextrin.
    [16]
    16. The process according to claim 15, wherein the randomly methylated cyclodextrin has a degree of substitution by methyls between 1 and 3.
    [17]
    17. Process according to claim 16 wherein the methylated cyclodextrin is randomly dimethylated.
    [18]
    18 Process according to any of claims 14 to 17 wherein the cyclodextrin is a 3-cyclodextrin.
    [19]
    19 Process according to any of claims 14 to 18 wherein the concentration of cyclodextrins in the culture medium of step (a) is between 6.5 gIl and
    130 gi l.
    20. Method according to any of claims 14 to 19 wherein the concentration of cyclodextrins in the culture medium of step (a) is between 10 gIl and
    100 gi l.
    [21 ]
    twenty-one . Process according to any of claims 14 to 20 wherein the concentration of cyclodextrins in the culture medium of step (a) is between 50 gIl and
    75 gil.
    [22]
    22. Use of a culture medium comprising coronatin or methyl jasmonate for
    increase the production of glucosinolates in plant cells potentially producing them.
    [23]
    23. Use of a composition according to claim 22 wherein the composition or culture medium further comprises cyclodextrins.
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    同族专利:
    公开号 | 公开日
    ES2694706B2|2021-06-22|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    EP3707993A1|2019-03-12|2020-09-16|Perpetuum CropScience BVBA|Producing isothiocyanates from callus suspension cultures|
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