Production of thapsigargin by thapsigaga cell suspension culture

Abstract

The present invention relates to a method of producing sesquiterpene lactones of the thapsigargin family, said method comprising the steps of: (a) culturing plant cells of Thapsia in a suspension cell culture in a nutrient medium, wherein said cells produce one or more sesquiterpene lactones of the thapsigargin family; and (b) recovering the one or more sesquiterpene lactones of the thapsigargin family produced in (a). The present invention also relates to a suspension cell culture comprising plant cells of Thapsia, wherein said plant cells are capable of producing one or more sesquiterpene lactones of the thapsigargin family, and to a plant cell biomass comprising plant cells of Thapsia obtained from said suspension cell culture.

Description

Invention Field

[0001] This invention relates to a method for producing sesquiterpene lactones of the thapsigargin family, the method comprising the steps of: (a) culturing plant cells of the genus *Thapsia* in a nutrient medium in a suspension cell culture, wherein the cells produce one or more sesquiterpene lactones of the thapsigargin family; and (b) recovering the one or more sesquiterpene lactones of the thapsigargin family produced in (a). The invention also relates to suspension cell cultures comprising *Thapsia* plant cells capable of producing one or more sesquiterpene lactones of the thapsigargin family, and to plant cell biomass comprising *Thapsia* plant cells obtained from said suspension cell cultures.

[0002] This specification references numerous documents, including patent applications and manufacturer manuals. The disclosures of these documents (not considered to be related to the patentability of this invention) are incorporated herein by reference in their entirety. More specifically, all documents are incorporated herein by reference to the extent that each document specifically and individually indicates its inclusion herein. Background of the Invention

[0004] Plants contain a wide range of compounds, including pharmaceuticals. In many cases, the medicinal activity of these secondary metabolites has been studied. The most prominent example is the anticancer compound paclitaxel, expressed by members of the genus *Taxus*. For many years, products containing paclitaxel as a pharmaceutically active ingredient have been commercially available. Paclitaxel has been shown to effectively kill highly proliferating human cells—rapid cell growth is characteristic of many tumors.

[0005] The activity of naturally occurring carotenoids (belonging to the sesquiterpene lactone class) in inducing cell death has been investigated. Members of the carotenoid family of sesquiterpene lactones have been found to act as apoptosis inducers in a proliferation-independent manner. A unique member of the carotenoid family, carotenoid (…), has been discovered… Figure 1 Carotenoids are specific inhibitors of the sarcoplasmic reticulum calcium transport ATPase (SERCA) family, thus demonstrating potential applications in cancer treatment. To target carotenoids to cancer cells, such as prostate cancer cells, they can be conjugated to peptides that act as selective substrates for cancer cell-specific proteolytic enzymes, such as prostate-specific antigen (PSA) [SB Christensen et al. 2009, Anti-Cancer Agents in Medicinal Chemistry; 9: 276-294]. Such so-called prodrugs containing carotenoids as cytotoxic compounds are currently under clinical development.

[0006] Members of the carotenoid family are typically isolated from plants in the genus *Thapsia*, such as *Thapsia garganica*, *Thapsia transtagana*, and *Thapsia villosa*, or any of the *Thapsia* species mentioned in Table 2. The distribution of carotenoids varies among different members of the *Thapsia* genus. The most prominent compound—carotenoids—was initially isolated from *Thapsia*, but has also been identified in other members of the *Thapsia* genus.

[0007] Currently, the plant *Carotene toxicaria* is the only commercial source of the compound carotene because its complex chemical structure prevents its economically viable chemical synthesis. In vitro culture for the production of valuable plant-derived secondary metabolites has long been considered an alternative source. The measurability of in vitro culture allows for the commercial supply of a given compound in high quantities and sustainably, independent of the natural environment.

[0008] et al. [Smitt et al. 1993; The Plant Cell Reports: 12, 517-520] investigated the in vitro culture of carotenoid species as an alternative source for the production of carotenoids. When they studied differentiated in vitro plant materials (i.e., green cotyledonous embryos, shoots, and roots) cultured on solid media, the authors identified carotenoids pentoxides, nortrifolinolide and trifolinolide, in these plant materials. However, carotenoids hexaoxides were not identified. See also Smitt et al. 1996 [Smitt, UW, AK, and Nyman, U; XXIV Thapsia garganica L.: Invitro culture, somatic embryogenesis, and the production of Thapsigargins; In: Biotechnology in Agriculture and Forestry, Vol 37; Medicinal and Aromatic Plants IX (ed.By YPS Bajaj); Springer-Verlag Berlin Heidelberg 1996].

[0009] Furthermore, when using other in vitro materials cultured in solid or suspension media, such as callus, protozoa, or globular embryos, these authors failed to identify sesquiterpene lactones of the toxic carotenoid family.

[0010] Therefore, although much effort has been made in terms of methodology to establish plant cells for the production of carotenoids, there is currently no economical and simple method for producing these compounds from plant cell cultures, especially suitable for large-scale cultivation. Therefore, there is a need to provide such methods and such cell cultures.

[0011] This requirement is achieved through the embodiments characterized in the claims. Invention Overview

[0013] This invention provides various methods for producing sesquiterpene lactones of the carotenoid family from plant cell cultures. One objective of this invention is to obtain a commercially viable quantity of the final product from suspension cultures.

[0014] The present invention thus provides a method for producing one or more sesquiterpene lactones of the toxic carotenoid family from suspension cell cultures, wherein plant cells of the genus *Carotenoidea* are cultured in a nutrient medium to form a cell culture that produces one or more toxic carotenoids, and one or more toxic carotenoids are recovered.

[0015] Preferably, the plant cells are *Thapsia garganica* cells. More preferably, the plant cells are selected from the group consisting of cells from: *Thapsia garganica*, *Thapsia villosa*, *Thapsia transtagana*, *Thapsia gymnesica*, *Thapsia maxima*, *Thapsia decussate*, *Thapsia laciniata*, including subspecies / varieties of *Thapsia garganica* ssp. *decussata* var. *angusta*, *Thapsia villosa* var. *dissecta*, *Thapsia villosa* var. *microcarpa*, and *Thapsia villosa* var. *stenoptera*. More preferably, the cells are undifferentiated cells, differentiated cells, meristematic cells, or mixtures thereof (but not embryogenic cells).

[0016] More preferably, the plant cells are cultured in a growth medium. More preferably, the growth medium is capable of inducing at least 50% growth within one week. Alternatively, or additionally, the plant cells are cultured on a production medium. More preferably, the plant cells are cultured on a growth medium and then on a production medium. Preferably, the growth and production media are different. More preferably, the growth medium does not contain the plant growth regulator 2,4-D and / or the production medium does not contain the plant growth regulator 2,4-D. Brief description of the attached diagram

[0018] The attached image shows:

[0019] Figure 1 The general core structure of hexaoxide sesquiterpene lactones of the carotenoid family is shown in Table 1 for details of substituents R1 and R2.

[0020] Figure 2 : Molecular structure of carotenoids.

[0021] Figure 3 UPLC-MS / MS spectra of toxic carotene biomass samples derived from non-embryonic suspension cell cultures and toxic carotene standards were analyzed by UPLC / MS. In the non-embryonic suspension cell culture samples, the precursor ion of toxic carotene (m / z = 668; M+NH4) was detected above the noise level. + The two seed ions (the first (standard) and the third (sample) curves m / z = 491 and the second (standard) and the fourth (sample) curves m / z = 551). Invention Details

[0023] The present invention relates to a method for producing sesquiterpene lactones of the toxic carotenoid family, the method comprising the steps of: (a) culturing plant cells of the genus *Carotenoidea* in a nutrient medium in a suspension cell culture, wherein the cells produce one or more sesquiterpene lactones of the toxic carotenoid family; and (b) recovering the one or more sesquiterpene lactones of the toxic carotenoid family produced in (a).

[0024] The term “sesquiterpene lactones of the thapsigargin family” is used interchangeably with the term “thapsigargins” herein and refers to any member of the guaianolides superfamily, which includes the 2β,3α,7β,8α,10β,11α-hexaoxygenated-6β,12-guaianolide nucleus, i.e., hexaoxygenated thapsigargins. Figure 1 ), or 3α,7β,8α,10β,11α-pentaoxygenated-6β,12-guaianolide nucleus, also known as pentaoxygenated thapsigargins. The characteristic feature of sesquiterpene lactones in the thapsigargin family is the 7β-hydroxy group that leads to trans-annelation of the lactone ring. Non-restrictive members of the thapsigargin family include hexaoxygenated thapsigargins with structural differences in the acyl groups attached to O(2) and O(8), such as thapsigargins (…). Figure 2 ), thapsigargicin, thapsitranstagin, thapsivillosin A, thapsivillosin B, thapsivillosin C, thapsivillosin D, thapsivillosin E, thapsivillosin G, thapsivillosin H, thapsivillosin I, thapsivillosin J and thapsivillosin K; and pentoxide thapsigargicin with different structures of the acyl group attached to O(8), such as trilobolide, nortrilobolide and thapsivillosin F [Christensen et al. 1997; 129-167; in Progress in the Chemistry of Organic Natural Compounds; Ed. Herz, Kirby, Moore, Steglich and Tamm; Springer-Verlag Wien; ISBN 3-211-82850-8].

[0025] Name (Carotene hexaoxide) CAS Reg.No. / (Replacement Pattern) Toxic carotene <![CDATA[67526-95-8 / (R 1 =Oct / R 2 =But)]]> Toxic carrots <![CDATA[67526-94-7 / (R 1 =Hex / R 2 =But)]]> Thapsitranstagin <![CDATA[81127-21-1 / (R 1 =iVal / R 2 =2-MeBut)]]> Long-haired hairs, carotene A <![CDATA[81127-16-4 / (R 1 =The / R 2 (Sen)]]> Long-haired hairs, carotene B <![CDATA[81127-17-5 / (R 1 =The / R 2 (2-MeBut)]]> Long-haired hairs, carotene C <![CDATA[- / (R 1 =Oct / R 2 =2-MeBut)]]> Long-haired hairs, carotene D <![CDATA[- / (R 1 =6-MeOct / R 2 =Sen)]]> Long-haired hairs, carotene E <![CDATA[81127-19-7 / (R 1 =6-MeOct / R 2 =2-MeBut)]]> Long-haired hairs, carotene G <![CDATA[- / (R 1 =6-MeHep / R 2 =2-MeBut)<!-- 3 --> ]]> Long-haired hairs, carotene H <![CDATA[- / (R 1 or R 2 =Ang or Sen)]]> Long-haired hairs, carotene I <![CDATA[94567-55-2 / (R 1 =The / R 2 (But)]]> Long-haired hairs, carotene J <![CDATA[- / (R 1 =iVal / R 2 =But)]]> Long-haired hairs, carotene K <![CDATA[- / (R 1 =Sen / R 2 =2-MeBut)]]> Name (Toxic Carotene Pentoxide) CAS Reg.No. / Replacement Pattern Trifolin lactone <![CDATA[50657-07-3 / (R 1 =Deoxygenation / R 2 =2-MeBut)]]> nortrifolin lactone <![CDATA[136051-63-3 / (R 1 =Deoxygenation / R 2 =But)]]> Long-haired hairs, carotene F <![CDATA[- / (R 1 =Deoxygenation / R 2 =Sen)]]>

[0026] Preferably, the sesquiterpene lactone of the carotenoid family is a sesquiterpene lactone of the carotenoid family that can be produced from carotenoid cells. More preferably, the sesquiterpene lactone of the carotenoid family that can be produced from carotenoid cells is the compound carotenoid.

[0027] Preferably, the sesquiterpene lactones of the carotenoid family have therapeutic activity, or they can be modified to produce bioactive compounds. Bioactive compounds are characterized in that they comprise a sesquiterpene lactone of the carotenoid family (fused to a targeting peptide) with therapeutic activity, as described, for example, in [US 7,468,345]. In a preferred embodiment, the sesquiterpene lactone of the carotenoid family is itself therapeutically active and, by fusing to a targeting peptide, can target a specific environment in the human or animal body in which the targeting peptide is specifically cleaved (e.g., but not limited to proteases).

[0028] As used herein, the term encompasses, indicating that additional steps and / or components may be included in addition to the specifically mentioned steps and / or components. However, this term also covers the claim that the subject matter consists precisely of the mentioned steps and / or components.

[0029] According to the present invention, cells that produce sesquiterpene lactones of the toxic carotenoid family are cultured to produce one or more sesquiterpene lactones of the toxic carotenoid family (as defined above). The term "cells producing sesquiterpene lactones of the toxic carotenoid family" refers to any cell capable of producing one or more sesquiterpene lactones of the toxic carotenoid family under at least one set of culture conditions. According to the present invention, the "cells producing sesquiterpene lactones of the toxic carotenoid family" are cells capable of producing a detectable amount of one or more sesquiterpene lactones of the toxic carotenoid family under the culture conditions defined in the method of the present invention.

[0030] According to the method of the present invention, the cell culture comprises cells of the genus *Thapsia*. Plants of the genus *Thapsia* include, but are not limited to: *Thapsia*, *Thapsia longhair*, *Thapsia transtagana*, *Thapsia gymnesica*, *Thapsia maxima*, *Thapsia decussate*, *Thapsia laciniata*, including the subspecies / varieties *Thapsia decussata* subspecies *angusta*, *Thapsia dissecta*, *Thapsia microcarpa*, and *Thapsia stenoptera*, as shown in Table 2 below. In a preferred embodiment of the method of the present invention, the plant cells of the culture are selected from the group consisting of: *Thapsia* cells, *Thapsia gymnesica* cells, and *Thapsia longhair* cells. In a more preferred embodiment of the method of the present invention, the cell culture comprises *Thapsia* cells.

[0031] genus Exemplary species Exemplary subspecies / variants Poisonous carrots Poisonous carrot (T. garganica) T. villosa (long-haired poisonous carrot) T.transtagana T. gymnesica T.maxima T. decussata T.laciniata Poisonous carrots Decussata subspecies angusta variety Long-haired poisonous carrot Dissecta variant Microcarpa variant Stenoptera variant

[0032] For example, cells in the same suspension cell culture can be derived from one or more species, subspecies, varieties, or plants. Preferably, the cells are derived from one species, more preferably from one subspecies, even more preferably from one variety, and most preferably from one plant.

[0033] The plant tissue used to initiate the suspension cell culture can be any plant tissue, preferably selected based on the following: for example, the ability to produce one or more sesquiterpene lactones of the carotenoid family. Non-limiting tissues for initiating the suspension cell culture include, for example, roots, leaves, stems, meristems, or cells (derived from previously formed callus, preferably from fragile callus material). The plant cells can be cells directly derived from the plant or cryopreserved cells.

[0034] The method includes the step of culturing a suspension culture in a nutrient medium. The method may also include more than one step of culturing a suspension culture in a nutrient medium.

[0035] According to the present invention, a nutrient medium is used. The term "nutrient medium," as used herein, refers to a medium suitable for culturing the plant cell callus and / or suspension cultures. The term "nutrient medium" encompasses both "growth medium" and "production medium." The term "growth medium" refers to a nutrient medium that promotes the growth of the cultured cells. In a preferred embodiment, the growth medium provides at least 50% growth within one week. Preferably, but not exclusively, the growth is determined based on dry weight. More preferably, the growth is determined based on fresh weight. According to the present invention, a "production medium" is a nutrient medium that promotes the production of one or more sesquiterpene lactones of the carotenoid family. It should be understood that growth can also be carried out in a production medium and production can also be carried out in a growth medium, whereby the growth and production media can be the same. However, more preferably, a production medium is selected that promotes a greater degree of production of the target compound than the growth medium used.

[0036] It is well known in the art that a balanced or relatively low carbon-to-inorganic ratio (such as nitrogen and phosphorus) generally favors cell growth, while a relatively high carbon-to-inorganic ratio limits cell growth. Therefore, the production medium can utilize growth-limiting conditions, such as a high carbon-to-inorganic ratio, to promote the production of sesquiterpene lactones of the carotenoid family rather than cell growth [see, for example, Majerus F. & Pareilleux A., “Alkaloid accumulation in Ca-alginate entrapped cells of Catharanthus roseus: Using alimiting growth medium,” Plant Cell Reports (1986) 5: 302-305].

[0037] Nutrient media in the art are well established and can be based on, for example, Murashige and Skoog Basal Salts (MS), Schenk and Hildebrandt Basal Salts (SH), or compositions of compounds according to Gamborg (B5).

[0038] Exemplary production media (PM) include, but are not limited to, salt-based media (e.g., MS or SH) with added macronutrients, micronutrients, vitamins, and a carbon source (preferably sucrose).

[0039] The preferred amount of sucrose used is between 0.01% and 10%, more preferably between 0.05% and 8%, even more preferably between 0.5% and 6%, such as between 1% and 4%, and most preferably about 3%. The sucrose can be obtained from, for example, Sigma Aldrich, Carl Roth, and / or VWR. The preferred amount of nitrogen used is between 0.1% and 10%, more preferably between 0.5% and 5%, such as between 1% and 3%, and most preferably 2.63%. Nitrogen can be obtained from nitrates, ammonium, and amino acids.

[0040] Exemplary growth media include, but are not limited to, salt-based media (e.g., MS, SH, or B5) with an added carbon source (preferably sucrose).

[0041] The preferred amount of sucrose to be used is between 0.01% and 6%, more preferably between 0.1% and 5%, such as between 1% and 3%, and most preferably about 2%. The sucrose can be obtained from, for example, Sigma Aldrich, Carl Roth, and / or VWR. The preferred amount of nitrogen to be used is between 0.1% and 10%, more preferably between 0.5% and 5%, such as between 1% and 3%, and most preferably 2.63%. Nitrogen can be obtained from nitrates, ammonium, and amino acids.

[0042] Therefore, the preferred growth medium contains a base (e.g., MS, SH, or B5) plus 2% sucrose.

[0043] Suspension cultures producing sesquiterpene lactones of the carotenoid family can achieve rapid growth rates and high cell densities when using suitable nutrients and reaction conditions. Those skilled in the art can readily combine, modify, and manipulate culture medium conditions to achieve optimal performance (which varies between cell lines) with reference to the guidance provided herein and known in the art.

[0044] The production of secondary metabolites in plant suspension cultures is known to be affected by plant growth regulators. Similarly, in media used to produce sesquiterpene lactones of the carotenoid family, omitting or reducing the content of plant growth regulators can improve production. Improving the production of sesquiterpene lactones of the carotenoid family through the selection of plant growth regulators can be experimentally verified by those skilled in the art without excessive labor.

[0045] The productivity of carotenoids is not determined by a single productivity-limiting step, but rather by the complex interactions between multiple limiting factors. Mitigation of any limiting factor will enhance carotenoid production, but the extent of this enhancement depends on specific culture conditions, which determine the relative limiting effects of other steps in carotenoid production once that particular limitation is mitigated. Culture conditions influencing the interactions between various limiting factors include: the genetic composition of the cells; the composition of the culture medium; and temperature.

[0046] Plant growth regulators include a wide variety of substances readily known from general botanical literature. Among them, growth regulators particularly suitable for suspension cultures of poisonous carrots include, but are not limited to, growth hormones and / or cytokinin-like compounds, such as indoleacetic acid, indolebutyric acid, naphthaleneacetic acid (NAA), chlorhexidine, dicamba, benzoic acid purine, kinetin, zeatin, phenylthiazolidinone, and indoleacetic acid. Amino acids include any natural or synthetic amino acids utilized by cells, such as glutamine, glutamic acid, and aspartic acid. These can be used alone or in any combination. Preferably, a combination of dicamba, benzoic acid purine, and phenylthiazolidinone is used, as shown in the appended examples.

[0047] Since the effects of the plant growth regulator 2,4-dichlorophenoxyacetic acid on embryogenesis in toxic carrot suspension cultures are known, a nutrient medium free of 2,4-dichlorophenoxyacetic acid is preferred in order to maintain the plant cells in a non-embryogenic state.

[0048] It is also desirable to reduce or avoid oxidative browning in cell cultures. Therefore, anti-browning agents can be added to inhibit the oxidation of phenolic exudates. Preferably, the suspension culture is composed of undifferentiated cells, which are less susceptible to browning than embryonic cells.

[0049] Recommended concentration ranges for these culture medium components are given below; however, for specific cell lines and / or specific culture conditions, routine optimization may prove preferred outside these ranges. All concentrations refer to the average initial values ​​in the extracellular medium after addition. Concentrations in the added solution, and therefore local concentrations in contact with cells, can be higher than shown. Formulations containing cell material can be added based on the specific component concentrations or as a portion of the culture volume.

[0050] Plant growth regulators can be used at concentrations from about 0.001 μmol / L to about 2 mmol / L, preferably from about 0.01 μmol / L to about 1 mmol / L.

[0051] Amino acids can be used at concentrations from about 1 μmol / L to about 20 mmol / L, preferably from about 10 μmol / L to about 10 mmol / L.

[0052] Using these recommended concentrations as guidelines, routine optimization can identify which components or combinations of components, and which concentrations (including those not recommended above), are useful for maximizing the production of sesquiterpene lactones from the carotenoid family.

[0053] In a preferred embodiment, cells are first cultured in a growth medium, followed by a culture in a production medium different from the growth medium. The growth medium also includes an inorganic or organic nitrogen source, such as amino acids. When cells are transferred from the growth medium to the production medium, the production medium preferably has a higher level of carbon source and / or C:N ratio, for example, a higher concentration of sugar. The production medium also preferably contains an inorganic or organic nitrogen source, such as amino acids. After the cells and culture medium have initially come into contact, other components of the nutrient medium can be introduced into the culture. In one embodiment, these components, such as additional sugars, are supplied intermittently or continuously as a feed stream, as needed.

[0054] It should be understood that the method of the present invention may include one or more steps of culturing cells in a growth medium and / or one or more steps of culturing cells in a production medium.

[0055] Typical cell culture conditions are well known in the art, and the desired effects, such as growth or production, are known to be achievable by manipulating the culture medium conditions described above (by adding, removing, or altering the concentration of one or more nutrients or other substances). In addition to modifying the culture medium composition, other reaction conditions can also be modified to obtain the desired results, for example, by manipulating conditions including but not limited to temperature and pH, or by manipulating any combination of these conditions.

[0056] For example, the temperature range can be adjusted. Preferred temperatures include approximately 0°C to approximately 40°C, more preferably approximately 15°C to approximately 35°C, and even more preferably approximately 20°C to approximately 30°C. More preferably, the cell culture conditions include shaking conditions, preferably at approximately 40 rpm to approximately 350 rpm, more preferably approximately 80 rpm to approximately 200 rpm, such as, for example, between approximately 100 rpm and approximately 150 rpm, more preferably approximately 130 rpm, and most preferably a shaking frequency of 130 rpm, in the dark at a temperature of approximately 23°C to 27°C, preferably approximately 25°C. Additionally, it is preferable to subculture the cell culture every 14 days.

[0057] As used herein, the term "approximately" covers the explicitly stated value and any small deviations therefrom. In other words, a shaking speed of "approximately 130 rpm" includes, but is not necessarily precisely the stated 130 rpm, but can have deviations of several rpm, and thus includes, for example, 131 rpm, 132 rpm, 129 rpm, or 128 rpm. Those skilled in the art will understand that values ​​are relative and do not need to be perfectly accurate as long as they roughly correspond to the stated value. Therefore, the term "approximately" covers deviations from the stated value of, for example, 15%, more preferably, 10%, and most preferably, 5%.

[0058] Beyond the above, those skilled in the art can easily adjust general processing procedures without much effort. The operational mode of plant cell culture refers to the addition or removal of nutrients, cells, and products over time [Payne, G., et al., 1991; "Plant Cell and Tissue Culture in Liquid System", 62-66 & 298-297 (Hanser)]. [Publishers]. Components supplied to cells can be provided in many different ways. Components can be added at specific stages of growth, such as delayed growth, exponential growth, or stationary growth phases. All components can be provided immediately and then subsequently at appropriate time intervals, with the product being recoverable. In other cases, not all components can be provided all at once. Instead, one or more of them can be provided at different times during culture. Furthermore, addition can be discontinuous or phased depending on the time of initial contact, and the duration of such provision varies for different components. Components can be provided in multiple portions. One or more components can be supplied as portions of a solution, contacting the cell culture or portions thereof separately. Portions of suspension cultures can be removed at any time or periodically for cryopreservation, further cell passage, production, and / or recovery. Such cell-containing portions can be further exposed to nutrients or other components as desired. Exemplary subculture procedures are described herein.

[0059] In one embodiment, culture medium containing nutrients or other components may be added to replenish the removed portion or all. Such removed material may be added back to the original culture; for example, cells and culture medium may be removed, a portion of which may be used for product recovery, while the remaining cells or culture medium may be returned. The supply rate of components in the culture or the levels of various components in the culture may be controlled to advantageously produce and recover the product. Separate portions of the culture may be exposed to components in any of the aforementioned modes and subsequently combined in proportions determined to be advantageous for production. The cell content in the culture may also be adjusted to advantageously produce the product or passage cells. Adjustments to the cell content may be advantageously combined with strategies for contacting nutrients or other components.

[0060] Supplementing cells undergoing active biosynthesis with fresh culture medium can also enhance production by providing depleted basic nutrients. For example, Miyasaka et al. were able to stimulate stable-phase cells of Salvia miltiorrhiza to produce the diterpenoid metabolites cryptotanshinone and ferruginol simply by adding sucrose to the culture medium [[Miyasaka et al., "Regulation of Ferruginol and Cryptotanshinone Biosynthesis in Cell Suspension Cultures of Salvia miltiorrhiza", Phytochemistry 25: 637-640 (1986)]. It is presumed that biosynthesis has ceased due to carbon confinement during the stable-phase growth period.

[0061] Using a periodic culture medium replacement protocol in this culture method can provide similar benefits.

[0062] The amount of culture medium replaced, the frequency of replacement, and the composition of the supplemented culture medium can be varied according to various embodiments of the invention. The ability to stimulate biosynthesis through culture medium replacement is of great importance for the design and operation of efficient commercial processes in continuous, semi-continuous, or batch feeding modes. In a "batch feeding" operation, specific culture medium components, such as nutrients, are supplied periodically or continuously. In a preferred embodiment, a large portion (but not all) of the contents of the batch culture is replaced with fresh culture medium for continuous cell growth and production; this processing mode is similar to a "repeated removal and addition" operation and is termed a "semi-continuous process." In another preferred embodiment, the process is "continuous," i.e., a continuous supply of fresh culture medium and continuous or repeated removal of the effluent culture medium.

[0063] The term "suspension culture," as used herein, refers to a culture of cells (preferably structurally undifferentiated cells) dispersed in a liquid nutrient medium. Due to this culture technique, the cells do not adhere to a solid support or culture vessel. It is readily understood that suspension cultures contain cells at various stages of aggregation. A range of aggregate sizes can be encountered in suspensions, ranging from tens of micrometers in diameter (a single cell or several aggregates) to aggregates many millimeters in diameter, composed of thousands of cells. The method of this invention covers suspension cultures containing such aggregates.

[0064] To transfer cells to a suspension culture, for example, by removing them from callus and transferring them to a sterile culture vessel containing nutrient medium, suspension culture can be initiated, for example, using a nutrient medium (preferably without a gelling agent) that has previously produced, for example, fragile callus. However, it should be understood that the optimal medium for suspension cultures may differ from the optimal conditions for callus of the same cell line. Those skilled in the art can determine a suitable medium without excessive effort.

[0065] Alternatively, or otherwise, the plant cell culture may also be derived from a cryopreserved cell collection.

[0066] Once initiated, the suspension culture can be further cultured by: (i) substantially separating the cells from the culture medium (usually by filtration) and then introducing a portion into a nutrient-containing medium; or (ii) transferring a certain volume of culture medium (cells and culture medium) into a nutrient-containing medium; or (iii) precipitating the cells and then removing any portion of the existing culture medium and reintroducing a nutrient-containing medium. When the cells and culture medium are transferred by volumetric measurement, the volume transferred to the final volume can preferably be from about 1% to substantially all of the volume, more preferably from about 5% to about 50%, and even more preferably from 10% to about 20%. In the case of transferring all volumes, fresh nutrients can be supplied in a concentrated form, resulting in only a small volume increase. The culture can thus be divided into fractions, which can be used individually to continue cell growth to produce carotenoids or both. Each fraction can (but is not required to) be cultured under the same conditions as each other or under the same conditions as the original culture. The duration of growth can be extended by replenishing partially depleted nutrient-containing culture medium.

[0067] Cells grown according to the method of the present invention produce one or more sesquiterpene lactones of the toxic carotenoid family (as defined above).

[0068] As used herein, the term "one or more," for example in the term "one or more sesquiterpene lactones of the toxic carotenoid family," refers to a specific one, but also to more than one, such as, for example, two, three, four, five, six, seven, etc. Furthermore, the term "one or more" does not define the exact number of molecules of a single type, but rather the number of different molecules within the mentioned category. For example, the term "one or more sesquiterpene lactones of the toxic carotenoid family" specifically refers to one sesquiterpene lactone, such as the preferred sesquiterpene lactone toxic carotenoid, but also to more than one sesquiterpene lactone from the stated family, such as, for example, two, three, four, five, six, seven, etc.

[0069] In the second step of the method of the present invention, sesquiterpene lactones of the carotenoid family produced by the cultured cells are recovered. The carotenoids can be recovered from the entire culture, or from any part of the culture, and they can be recovered at any time during the culture or after the culture is completed. It should be understood that all carotenoids produced can be recovered, or, preferably, one or more carotenoids of interest can be recovered, such as, for example, only carotenoids hexaoxide, only carotenoids pentoxide, or even only a single compound carotenoid.

[0070] Cell material can be lyophilized prior to the extraction process. Other methods known in the art can be used to prepare cell material or suspension material for use in a suitable extraction method. Sesquiterpene lactones of the carotenoid family can be recovered by any method known in the art, including but not limited to extraction using non-aqueous polar solvents, extraction using acidic media, extraction using alkaline media, and recovery by adsorption through resin (wherein the resin is inside or outside the culture vessel).

[0071] The methods for separating the resulting molecules are well known in the art and include, but are not limited to, method steps such as extraction from lyophilized cell or aqueous cell suspensions using organic solvents such as, for example, ethyl acetate, acetone, or toluene, followed by chromatographic separation using, for example, liquid chromatography (LC), reversed-phase LC, or liquid / liquid chromatography. The only sesquiterpene lactone to be recovered is carotenoid, preferably recovered polarly using the purification sequence described, for example, in Ollivier A et al. 2013 [Ollivier, A. 2013; J Chromatogr B Analyt Technol Biomed Life Sci.; 926: 6-20] or Rasmussen et al. 1978 [Rasmussen U. et al. 1978; Acta Pharm Suec.; 15(2); 133-40].

[0072] Preferably, the recovery is carried out as described in the appended examples, i.e., by freeze-drying the biomass obtained from the cell culture, homogenizing the freeze-dried biomass with a solution in a mill, centrifuging to obtain a crude extract, and purifying the components of the crude extract as described in Ollivier A et al. 2013 [Ollivier, A. 2013; J Chromatogr B Analyt Technol Biomed Life Sci.; 926: 6-20].

[0073] According to the present invention, a novel method for producing sesquiterpene lactones of the toxic carotenoid family is provided. Large-scale production of toxic carotenoids is made possible by using cells grown in suspension culture, for example, through semi-continuous or continuous cell culture techniques. To the best of the inventors' knowledge, there is currently no such plant cell culture route for the large-scale production of toxic carotenoids. The lack of such methods threatens the survival of plants in the genus *Carotenoides*, which is currently the only viable source for the large-scale isolation of toxic carotenoids [SB Christensen et al. 2009, Anti-Cancer Agents in Medicinal Chemistry; 9: 276-294]. This is because synthetic methods for producing these compounds are economically unfeasible, as synthesis requires at least 42 steps.

[0074] In a preferred embodiment of the method of the present invention, one or more sesquiterpene lactones of the carotenoid family recovered in step (b) comprise a 2β,3α,7β,8α,10β,11α-hexaoxide-6β,12-guaiacol lactone core.

[0075] As detailed above, the sesquiterpene lactones produced by various plant cells can be the same, that is, they represent only one molecular type of sesquiterpene lactone, or they can be a mixture of different molecular types of sesquiterpene lactones. Also as detailed above, all of these sesquiterpene lactones of the toxic carotenoid family produced can be recovered, or only preferred mixtures can be recovered from this group.

[0076] Therefore, according to this preferred embodiment, it covers: (i) a sesquiterpene lactone of the carotenoid family recovered in step (b) of the method according to the invention comprising a 2β,3α,7β,8α,10β,11α-hexaoxidized -6β,12-guaiacol lactone core; (ii) a variety (but not all) sesquiterpene lactones of the carotenoid family recovered in step (b) of the method according to the invention comprising a 2β,3α,7β,8α,10β,11α-hexaoxidized -6β,12-guaiacol lactone core; or (iii) all sesquiterpene lactones of the carotenoid family recovered in step (b) of the method according to the invention comprising a 2β,3α,7β,8α,10β,11α-hexaoxidized -6β,12-guaiacol lactone core.

[0077] In a more preferred embodiment of the method of the present invention, the sesquiterpene lactone of the toxic carotenoid family containing a 2β,3α,7β,8α,10β,11α-hexaoxide-6β,12-guaiacol lactone core is a toxic carotenoid.

[0078] Carotene is the most commonly used sesquiterpene lactone in the carotene family, and its structure is... Figure 1 As shown in the figure. Preferably, the toxic carotenoid is a sesquiterpene lactone of the only toxic carotenoid family that can be recovered from plant cells by the method according to the invention.

[0079] In another preferred embodiment of the method of the present invention, one or more sesquiterpene lactones of the carotenoid family recovered in step (b) comprise a 3α,7β,8α,10β,11α-pentoxide-6β,12-guaiacol lactone core.

[0080] The same considerations for hexaoxide-containing carotenoids also apply to pentoxide-containing carotenoids, i.e., according to this preferred embodiment, it covers: (i) a sesquiterpene lactone of the carotenoid family recovered in step (b) of the method according to the invention, comprising a 3α,7β,8α,10β,11α-pentoxide-6β,12-guaiacol lactone core; (ii) a variety (but not all) of the carotenoid family of sesquiterpene lactones recovered in step (b) of the method according to the invention, comprising a 3α,7β,8α,10β,11α-pentoxide-6β,12-guaiacol lactone core; or (iii) all of the carotenoid family of sesquiterpene lactones recovered in step (b) of the method according to the invention, comprising a 3α,7β,8α,10β,11α-pentoxide-6β,12-guaiacol lactone core.

[0081] As can be seen from the above, a mixture of carotenoid hexaoxide and carotenoid pentoxide can also be recovered.

[0082] In another preferred embodiment of the method of the present invention, the plant cells are non-embryonic cells.

[0083] According to the present invention, the term "non-embryonic cell" refers to undifferentiated, dedifferentiated, and meristematic cells. Non-embryonic cells can be distinguished from embryogenic cells by the presence of vacuoles, which are highly cytoplasmic and non-vacuolated.

[0084] Measures and methods for obtaining non-embryogenic cells for use in the methods of the present invention are well known in the art. Non-limiting methods for obtaining non-embryogenic cells include, for example, their derivation from fragile callus tissue. As an example, a non-embryogenic suspension culture can be prepared using an inoculum from about 0.01 to about 10 g / 25 ml of such fragile callus tissue for use in the methods according to the invention. According to the invention, the plant cells remain non-embryogenic during suspension culture.

[0085] The term "fragile callus material" refers to a substantially undifferentiated cluster of cells cultured on a solidified culture medium. Methods for forming fragile callus material and for callus passage are well known in the art. Non-limiting methods for obtaining callus include, for example, surface sterilization of plant-derived material, such as thorough washing using clean water, disinfectants like hypochlorite, wetting agents like Tween or Triton, antibodies, and / or antifungal agents. Callus formation can begin from any surviving plant part (also referred to herein as "plant explant"), preferably from the root tissue of a plant. The plant-derived material can be a whole part of a plant or a portion thereof. Typically, the plant-derived material is placed on a solidified culture medium surface and incubated under sterile conditions for approximately 1–12 weeks until a large number of undifferentiated cells (the fragile callus material) grow adjacent to the plant-derived material. After establishing the fragile callus material, the cells are cultured in a nutrient medium according to the method of the invention.

[0086] Culture conditions for passage of fragile callus, including culture medium composition, pH range, carbon source, nitrogen source, macro- and micro-salts, vitamins, and growth regulators, are well known in the art and have been described, for example, in WO 97 / 44476 (incorporated herein). In a preferred embodiment, passage of fragile callus includes the use of a gelling agent. Gelling agents include, for example, agar, hydrogel, gelatin, and... Charcoal can be used to remove waste and unwanted organic compounds. In another preferred embodiment, the passage of fragile callus includes initiating fragile callus material on a medium free of 2,4-dichlorophenoxyacetic acid. In another preferred embodiment, the passage of fragile callus includes culturing fragile callus on a medium free of 2,4-dichlorophenoxyacetic acid. For all embodiments of the invention, the medium used for the initiation and passage of fragile callus can be, for example, a solid medium or a semi-solid medium. Preferably, for all embodiments of the invention, the medium used for the initiation and passage of fragile callus is a solid medium.

[0087] Subculture techniques known in the art can be used to periodically transfer fractional portions of fragile callus tissue to a fresh source of nutrients. Preferably, the frequency of callus transfer is between 4 and 6 weeks.

[0088] As described above, this invention provides for the first time a suspension cell culture suitable for the large-scale production of carotenoids. While attempts to produce carotenoids from cell cultures have been described in the prior art (see...),... (As cited above, 1993), but they did not provide a method for producing a favorable form of toxic carotenoids from suspension cultures. Unbound by theory, the inventors observed that the cells used in the prior art are derived from callus tissue induced in a manner that leads to embryogenesis in the plant material, which negatively impacts the production of toxic carotenoids using cells derived from said plant material.

[0089] According to the above, in a more preferred embodiment, the method further includes performing an additional step (a-0) before step (a), which includes culturing the poisonous carrot plant explants on a culture medium to obtain fragile callus material. In all embodiments of the present invention, the culture medium used to start the fragile callus can be, for example, a solid or semi-solid culture medium. Preferably, in all embodiments of the present invention, the culture medium used to start the fragile callus is a solid culture medium.

[0090] The definitions and preferred implementation schemes provided above are applicable after necessary modifications.

[0091] In a particularly preferred embodiment, the present invention provides a method for producing sesquiterpene lactones of the toxic carotenoid family, the method comprising the following steps:

[0092] (a) Plant cells of the genus *Carotenoidea* are cultured in a nutrient medium in a suspension cell culture, wherein the cells produce one or more sesquiterpene lactones of the carotenoid family; wherein the cell culture conditions include shaking, preferably at 130 rpm, in the dark at a temperature of about 23°C to 27°C, preferably about 25°C, and wherein the cell culture is periodically subcultured, preferably every 14 days; and

[0093] (b) Recover sesquiterpene lactones of the toxic carotenoid family produced in (a).

[0094] In another preferred embodiment of the method of the present invention, the nutrient culture medium does not contain 2,4-dichlorophenoxyacetic acid.

[0095] 2,4-Dichlorophenoxyacetic acid (also referred to herein as 2,4-D) is a synthetic growth hormone (i.e., a plant hormone) that is commonly used in plant research as a supplement to plant cell culture media, where it is used, for example, as a growth regulator for the induction and maintenance of callus material, and for the culture of suspensions until they become embryos.

[0096] To avoid embryogenesis, according to this preferred embodiment, the suspension culture is cultured in a nutrient medium free of 2,4-dichlorophenoxyacetic acid. In another preferred embodiment, the suspension culture is maintained in a nutrient medium free of 2,4-dichlorophenoxyacetic acid. According to the invention, the suspension culture is maintained in a way that the cells are viable but do not grow significantly. In another preferred embodiment, the suspension culture is passaged in a nutrient medium free of 2,4-dichlorophenoxyacetic acid.

[0097] As described above, attempts to produce toxic carotenoids from cell cultures have been described in the prior art (see [link to article]). (As cited above, 1993), but no production of carotenoids from suspension cultures was shown. Again, without being limited by theory, the inventors observed that the cells used in the prior art are derived from culture techniques utilizing the plant hormone 2,4-D, which induces embryogenesis in plant material. This would negatively impact the production of carotenoids from cells derived from said plant material. The positive role of 2,4-D in initiating embryogenesis has been discussed and acknowledged. The addition of 2,4-D to the culture medium and the embryogenesis initiated therefrom can explain why these authors only identified carotenoids pentoxide and these only in the further differentiated embryonic stages, namely the cotyledonary stage, shoots, and roots (grown on solid media).

[0098] Therefore, in a particularly preferred embodiment, the present invention provides a method for producing sesquiterpene lactones of the toxic carotenoid family, the method comprising the following steps:

[0099] (a) Culturing non-embryogenic *Carotenoidea* plant cells in suspension cell culture in a nutrient medium free of 2,4-dichlorophenoxyacetic acid, wherein said cells produce one or more sesquiterpene lactones of the carotenoid family; and

[0100] (b) Recover sesquiterpene lactones of the toxic carotenoid family produced in (a).

[0101] In a most preferred embodiment, the present invention provides a method for producing sesquiterpene lactones of the toxic carotenoid family, the method comprising the following steps:

[0102] (a) Plant cells of the genus *Carotenoidea* are cultured in suspension cell culture in a nutrient medium free of 2,4-dichlorophenoxyacetic acid, wherein the cells produce one or more sesquiterpene lactones of the carotenoid family; wherein the cell culture conditions include shaking, preferably at 130 rpm, in the dark at a temperature of about 23°C to 27°C, preferably about 25°C, and wherein the cell culture is periodically subcultured, preferably every 14 days; and

[0103] (b) Recover sesquiterpene lactones of the toxic carotenoid family produced in (a).

[0104] In another particularly preferred embodiment, the present invention provides a method for producing sesquiterpene lactones of the toxic carotenoid family, the method comprising the following steps:

[0105] (a-0) Poisonous carrot plant explants were cultured on a culture medium to obtain fragile callus material;

[0106] (a) The *Carotenoides* plant cells obtained in step (a-0) are cultured in suspension cell culture in a nutrient medium free of 2,4-dichlorophenoxyacetic acid, wherein the cells produce one or more sesquiterpene lactones of the carotenoid family; and

[0107] (b) Recover sesquiterpene lactones of the toxic carotenoid family produced in (a).

[0108] In a more preferred embodiment, the present invention provides a method for producing sesquiterpene lactones of the toxic carotenoid family, the method comprising the following steps:

[0109] (a-0) Poisonous carrot plant explants were cultured on a medium free of 2,4-dichlorophenoxyacetic acid to obtain fragile callus material;

[0110] (a) The *Carotenoides* plant cells obtained in step (a-0) are cultured in a nutrient medium in a suspension cell culture, wherein the cells produce one or more sesquiterpene lactones of the carotenoid family; and

[0111] (b) Recover sesquiterpene lactones of the toxic carotenoid family produced in (a).

[0112] In a more preferred embodiment, the present invention provides a method for producing sesquiterpene lactones of the toxic carotenoid family, the method comprising the following steps:

[0113] (a-0) Poisonous carrot plant explants were cultured on a medium free of 2,4-dichlorophenoxyacetic acid to obtain fragile callus material;

[0114] (a) The *Carotenoides* plant cells obtained in step (a-0) are cultured in suspension cell culture in a nutrient medium free of 2,4-dichlorophenoxyacetic acid, wherein the cells produce one or more sesquiterpene lactones of the carotenoid family; and

[0115] (b) Recover sesquiterpene lactones of the toxic carotenoid family produced in (a).

[0116] In a most preferred embodiment, the present invention provides a method for producing sesquiterpene lactones of the toxic carotenoid family, the method comprising the following steps:

[0117] (a-0) Poisonous carrot plant explants were cultured on a medium free of 2,4-dichlorophenoxyacetic acid to obtain fragile callus material;

[0118] (a) The *Carotenoides* plant cells obtained in step (a-0) are cultured in suspension cell culture in a nutrient medium free of 2,4-dichlorophenoxyacetic acid, wherein the cells produce one or more sesquiterpene lactones of the carotenoid family; wherein the cell culture conditions include shaking, preferably at 130 rpm, in the dark at a temperature of approximately 23°C to 27°C, preferably approximately 25°C, and wherein the cell culture is periodically subcultured, preferably every 14 days; and

[0119] (b) Recover sesquiterpene lactones of the toxic carotenoid family produced in (a).

[0120] The present invention also relates to suspension cell cultures comprising plant cells of the genus *Carotenoidea*, wherein said plant cells are capable of producing one or more sesquiterpene lactones of the carotenoid family. Preferably, these suspension cell cultures are cultured / produced / producible / provided according to any of the embodiments described above.

[0121] The definitions and preferred embodiments of the method of the present invention provided above, after necessary modifications, are also applicable to this type of suspended cell culture of the present invention.

[0122] To the best of the inventors' knowledge, there is no prior art for suspension cell culture of *Carotenoidea* plants capable of producing sesquiterpene lactones of the carotenoid family. Based on the method of this invention, a suspension cell culture can be provided for the first time, and it is suitable for the large-scale production of one or more carotenoids.

[0123] The present invention also relates to plant cell biomass comprising plant cells of the genus *Carotenoides*, wherein the plant cells of the genus *Carotenoides* are derived from or may be derived from the suspension cell culture of the present invention.

[0124] Furthermore, all the definitions and preferred embodiments of the method of the present invention provided above, after necessary modifications, are also applicable to the plant cell biomass of the present invention.

[0125] The term "biomass," as used herein, refers to the biological material that constitutes the cell mass of a suspension culture. In other words, biomass is obtained when the cells of a suspension cell culture are separated from the liquid culture medium. Preferably, the biomass used according to the invention comprises one or more sesquiterpene lactones from the carotenoid family.

[0126] The biomass of this invention represents a valuable source of carotenoids. The biomass can be freshly obtained or it can be pre-stored (e.g., in the form of frozen samples or lyophilized powder).

[0127] It should be understood that all method steps described herein are performed in the order described, i.e., step (a) is performed before step (b). When an additional step (a-0) is included, this step is performed first before step (a). In other words, the claimed method includes (or consists of) the first step (a) and the subsequent step (b), or the claimed method includes (or consists of) the first step (a-0), the subsequent step (a), and the third step (b) after step (a).

[0128] Unless otherwise expressly stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of any conflict, the patent specification, including its definitions, shall prevail.

[0129] For the embodiments characterized in this specification (in particular the claims), each embodiment mentioned in a dependent claim is to be combined with each embodiment in each claim (independent or dependent) to which that dependent claim is to be attached. For example, in a situation where: independent claim 1 refers to three options A, B, and C; dependent claim 2 refers to three options D, E, and F; and claim 3 is dependent on claims 1 and 2 and refers to three options G, H, and I, it should be understood that: the specification unequivocally discloses embodiments corresponding to the following combinations: A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless otherwise expressly stated.

[0130] Similarly, and where multiple options are not mentioned in the independent and / or dependent claims, it should be understood that if a dependent claim refers to multiple prior claims, any combination of the subject matter covered therein is considered to be clearly disclosed. For example, in a situation where independent claim 1, dependent claim 2 refers to claim 1, and dependent claim 3 refers to claims 2 and 1, then the combination of the subject matter of claims 3 and 1 is clearly and unambiguously disclosed, as is the combination of the subject matter of claims 3, 2, and 1. In the case where there is a further dependent claim 4 and it refers to any one of claims 1-3, then the following combinations of the subject matter of the claims are clearly and unambiguously disclosed: claims 4 and 1, claims 4, 2 and 1, claims 4 and 31, and claims 4, 3, 2, and 1.

[0131] The above considerations, with necessary modifications, also apply to all appended claims. For the purpose of providing a non-limiting example, the combination of claims 9, 8, and 1 is clearly and unambiguously disclosed according to the structure of the claims. The same applies to, for example, combinations of claims 9, 8, 7, and 1.

[0132] The foregoing detailed description has illustrated the invention with reference to specific exemplary embodiments. However, it should be understood that various modifications and alterations may be made without departing from the scope of the invention as set forth in the appended claims. The detailed description and accompanying drawings should be considered merely illustrative and not restrictive, and all such modifications or alterations (if any) should fall within the scope of the invention described herein.

[0133] More specifically, illustrative exemplary embodiments of the invention have been described herein, but the invention is not limited to these embodiments, but includes all embodiments having modifications, omissions, combinations (e.g., combinations across aspects of various embodiments), adaptations, and / or alterations that would be understood by those skilled in the art based on the detailed description above. The limitations in the claims are to be understood broadly based on the language therein, and not to be limited to the instances described in the detailed description above or during the implementation of this application; the instances should be understood as non-exclusive. Example

[0134] The present invention will be illustrated by the following examples:

[0135] Example 1: Surface disinfection of intact plant materials

[0136] Thoroughly wash the roots of *Thapsia garganica* with tap water. Cut the roots into small cubes of approximately 3x3cm (plant explants). Wash the plant explants thoroughly in detergent and running water for approximately 10 to 15 minutes. Surface sterilization of the explants is performed aseptically by immersing them in a 70% isopropanol (IPA) (v / v) solution containing 2-3 drops of Tween 20 for 1 minute (with gentle agitation during this time). Afterward, treat the plant explants in a NaOCl solution (2.8g / 100ml sodium hypochlorite) for 15 to 30 minutes. Subsequently, rinse the explants simply 3 to 4 times with sterile distilled water to remove all traces of disinfectant. After surface sterilization, keep the explants in covered petri dishes in a laminar flow hood until ready for processing to prevent dehydration. Before placing the explants on a solid culture medium, the incisions of the explants are removed with a sterile scalpel and the explants are cut into smaller pieces of appropriate size (0.5-1 cm).

[0137] Example 2: Induction of callus formation in embryos on a solid culture medium containing 2,4-D

[0138] Plant explants were placed on solid modified Musharige and Skoog basal media (half-strength MS basal salts, 3% sucrose, 0.5 mg / L BAP, and 0.5 mg / L 2,4-D). The cultures were then incubated in the dark at 25 ± 2 °C. Primary callus material was obtained after 6–8 weeks. Explants cultured on MS medium supplemented with 2,4-D transformed into embryogenic callus, which further developed into somatic embryos at different stages with increasing culture time. The frequency of callus formation from transferred embryos depended on the growth rate and ranged from 4 to 8 weeks.

[0139] Example 3: Induction of fragile callus on solid culture medium without 2,4-D

[0140] Plant explants were placed on two different solid modified Gamborg basal media (full strength B5 basal salt, 2% sucrose, 5 mM chlorhexidine, and 0.01 mM BAP or 1 mg / L dicamba and 0.22 mg / L TDZ). The cultures were then incubated in the dark at 25 ± 2 °C. Primary callus material was obtained after 6–8 weeks. Fragile callus material was obtained from explants cultured on the two modified B5 media (without 2,4-D) after 4–6 weeks. The frequency of transfer of fragile callus depended on the growth rate and ranged from 4–6 weeks.

[0141] Example 4: Initiation of a suspension culture of poisonous carrots

[0142] To initiate suspension culture, fragile callus material (approximately 40-60 g / L) was transferred to liquid B5 medium without 2,4-D as described above. The suspension culture grew as large cell aggregates and was cultured in 250 ml Erlenmeyer flasks (50 ml of medium, on a rotary shaker at 130 rpm in the dark). The culture temperature was 25 ± 2 °C. Further medium modifications, such as a higher initial sucrose concentration or a higher nitrate-to-ammonia ratio, can be applied to obtain better suspension cell lines.

[0143] Example 5: Maintenance of Toxic Carrot Suspension Culture

[0144] To maintain the suspension, vacuum-filtered biomass (40-60 g / L) was transferred to 50 ml of fresh, 2,4-D-free liquid B5 medium as described above. The cultures were maintained in 250 ml Erlenmeyer flasks on a rotating shaker (130 rpm, in the dark) and subcultured every 14 days. The incubation temperature was 25 ± 2 °C. All established cultures were maintained for at least 3 months.

[0145] Example 6: Extraction of biomass from poisonous carrots

[0146] Following sampling, the biomass of the poisonous carrot (from callus material in suspension or vacuum-filtered biomass) was directly transferred to liquid nitrogen to avoid intracellular cell metabolism. To prepare the extract, the lyophilized biomass sample was weighed and 15 times its volume of solvent was added. EtOAc, acetone, or ethanol were used as solvents. The cells were homogenized in a grinder for 90 seconds, and then the mixture was centrifuged for 10 minutes (14,000 rpm) to obtain a crude extract for further analysis.

[0147] Example 7: Extraction of Suspension

[0148] The suspension culture of poisonous carrots was lyophilized and then weighed. The lyophilized material was resuspended in 15 times its volume of solvent. EtOAc, acetone, or ethanol were used as solvents. This mixture was homogenized in a grinder for 90 seconds and then centrifuged for 10 minutes (14,000 rpm) to obtain a crude extract for further analysis.

[0149] Example 8: Analysis of sesquiterpene lactones of the carotenoid family

[0150] The following methods are used to detect sesquiterpene lactones of the carotenoid family:

[0151]

[0152] Applying these conditions to cell extracts, for example, as described in Example 7, by comparing with reference materials (see...) Figure 3 (Standards) compared to samples (see) Figure 3 Toxic carotenoids were clearly identified in the sample. For both the standard and the sample, it was clearly observed that the parent ion at m / z = 668 (M+NH4)+ split into daughter ions at m / z = 491 (upper curve in the standard and sample) and m / z = 551 (lower curve in the standard and sample).

[0153] Example 9: Isolation of carotenoids and other sesquiterpene lactones from the carotenoid family

[0154] Carotenoids can be isolated from PCF cell suspensions using typical methods known in the art. Initial extraction of freeze-dried cells or aqueous cell suspensions using organic solvents such as ethyl acetate, acetone, or toluene is followed by chromatographic separation using, for example, a normal or reversed stable phase. For examples of potential purification sequences, see: Ollivier A, Grougnet R, Cachet X, Meriane D, Ardisson J, Boutefnouchet S, Deguin B.J Chromatogr B Analyt Technol Biomed Life Sci. 2013;926;16-20 or Rasmussen U, Christensen S, Sandberg F. Acta Pharm Suec. 1978; 15(2); 133-40.

Claims

1. A method for producing sesquiterpene lactones of the toxic carotenoid family, the method comprising the following steps: (a) Plant cells of the genus *Thapsia* are cultured in a nutrient medium in a suspension cell culture, wherein said cells produce one or more sesquiterpene lactones of the toxic carotenoid family; and (b) Recover one or more sesquiterpene lactones of the toxic carotenoid family produced in (a). The plant cells mentioned above are non-embryonic cells. The sesquiterpene lactones of the carotenoid family mentioned above are carotenoids, and The nutrient medium described herein does not contain 2,4-dichlorophenoxyacetic acid. The structure of the carotenoid is shown below.

2. The method of claim 1, wherein the plant cells are selected from: poison carrot (Thapsia garganica) cells, Thapsia gymnesica cells, and long-haired poison carrot (Thapsia villosa) cells.

3. The method of claim 1 or 2, further comprising an additional step prior to step (a): (a-0) Explants of the genus *Caragana* were cultured on a culture medium to obtain fragile callus material.

4. The method of any one of claims 1-3, wherein the nutrient medium in step (a) is a growth medium capable of inducing at least 50% growth within one week.

5. A suspension cell culture containing plant cells of the genus *Carotenoidea*, wherein the plant cells are capable of producing one or more sesquiterpene lactones of the carotenoid family, and wherein the plant cells are non-embryogenic cells, and wherein the sesquiterpene lactones of the carotenoid family are carotenoids, the structure of which is shown below.

6. A plant cell biomass comprising plant cells of the genus *Caragana* obtained from the suspension cell culture of claim 5.

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