EP3125675A2 - Pfropfenströmungsherstellungsverfahren für pflanzenwurzeln - Google Patents

Pfropfenströmungsherstellungsverfahren für pflanzenwurzeln

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Publication number
EP3125675A2
EP3125675A2 EP15735834.2A EP15735834A EP3125675A2 EP 3125675 A2 EP3125675 A2 EP 3125675A2 EP 15735834 A EP15735834 A EP 15735834A EP 3125675 A2 EP3125675 A2 EP 3125675A2
Authority
EP
European Patent Office
Prior art keywords
support
roots
opening
root
culture
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP15735834.2A
Other languages
English (en)
French (fr)
Inventor
Pierre-Antoine Mariage
Thierry MARIQUE
Mario GODOY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Green2chem SA
Original Assignee
Green2chem SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Green2chem SA filed Critical Green2chem SA
Publication of EP3125675A2 publication Critical patent/EP3125675A2/de
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G31/00—Soilless cultivation, e.g. hydroponics
    • A01G31/02—Special apparatus therefor
    • A01G31/04—Hydroponic culture on conveyors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G31/00—Soilless cultivation, e.g. hydroponics
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
    • A01G24/50—Growth substrates; Culture media; Apparatus or methods therefor contained within a flexible envelope
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
    • Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures

Definitions

  • the field of the invention is that of processes for producing roots of plants above ground by scrolling a culture medium and devices implementing said methods. More specifically, the invention also relates to a support for producing roots of plants above ground.
  • Medicinal plants are the main source of medicines for the majority of the world's population and account for 40% of drugs marketed in Europe and the United States. At present, these compounds are extracted from wild plants or cultivated by extraction with solvents (ethanol, hexane, acetone, etc.) and are marketed in the pharmaceutical, cosmetic, nutraceutical, dye and food sectors. However, the production of these compounds from conventionally grown or wild harvested plants is not sufficient to meet the growing market demand. In addition, harvesting wild plants is in many cases a significant threat to species as well as their ecosystem, which is in contradiction with consumer demand for more sustainability.
  • solvents ethanol, hexane, acetone, etc.
  • bioactive secondary metabolites is in many cases too metabolically complex to be carried out in bacterial or yeast microorganisms that do not have adequate genetic material. Culture of unicellular plant cells in a bioreactor is rarely satisfactory. Indeed, cell lines are generally genetically unstable, fragile, demanding complex nutrients and the production of secondary metabolites under these conditions is unstable. Moreover, many bioactive molecules require, to be produced, interactions between several plant cell lines that can only be found in differentiated tissues such as plant roots.
  • Plant roots are a potential source of bioactive molecules that include a disconcerting diversity of metabolites and proteins, and therefore, are considered the site of the sole secondary metabolism of the whole plant.
  • Recent advances in in vitro root culture have resulted in the production of many secondary metabolites from either adventitious and phytohormone-derived root lines to grow, or transformed with Rhizobium Rhizogenes and able to grow in higher media.
  • the roots of plants can indeed be cultivated in vitro in a solid or liquid support without aerial organs (stems and leaves) provided they are brought into contact with a carbon source (glucose, etc.) and growth hormones specific to root multiplication. These hormones are very expensive, which prevents the development of the culture of this type of roots on an industrial scale.
  • An example of this type of liquid bioreactor root culture is given in Korean Patent WO2008100016.
  • In vitro root culture can also be carried out without hormones using roots transformed by bacteria such as Agrobacterium Rhizogenes.
  • Roots transformed in this way are particularly easy to sterilely cultivate in an artificial medium in a liquid, solid or aerial medium (with feeding of the medium in the form of fog), and have a high genetic and biochemical stability. This type of root is commonly called “hairy roots” or “hair roots”.
  • an object of the invention in at least one of its embodiments, is to provide a continuous flow piston process for producing roots of plants above ground by scrolling a culture medium.
  • Another object of the invention in at least one of its embodiments, is to provide a device implementing a continuous flow piston process for producing roots of plants above ground.
  • the invention in at least one of its embodiments, is also intended to provide an above-ground plant roots production support suitable for use in a continuous piston-flow process.
  • the invention relates to a continuous piston-flow process for producing roots of plants above ground by moving a culture support, said support serving as containment chamber.
  • such a continuous piston flow process comprises the following successive steps: ⁇ deployment of the culture support for production of plant roots, said support being provided with at least a first isolation means for compartmentalizing the support and to isolate the different compartments thus created from each other, Seeding within said support of a root inoculum through at least a first opening located, in the direction of travel of the support, downstream of said at least first isolation means,
  • Opening of at least one second isolation means making it possible to compartmentalize the support and to isolate the different compartments thus created from each other, situated, in the direction of the scrolling of the support, downstream from said at least one first opening ,
  • Root harvesting the speed of travel of said support allowing an increase in root mass by a factor of at least 4 between the inoculum seeding step and the harvest.
  • the general principle of the invention is based on the fact that a root inoculum is introduced into a culture medium by at least a first opening located between a first means and a second insulation means held in the closed position, said culture support forming a containment vessel or bioreactor. Then, the roots are fed by nebulization of a culture medium through at least a second opening located between the first means and the second insulating means, the culture support forming a containment enclosure or bioreactor being maintained preferentially under overpressure.
  • Said second isolation means being tilted in the open position in order to avoid any tearing due to excessively high pressure or when the two insulation means are kept in the closed position, said support being provided with at least one additional opening allowing avoid any excess pressure tear important such as a vent or a valve and / or allowing the evacuation of liquid.
  • the roots are fed into the support by nebulising a culture medium through the at least one second opening, thereafter stopping the supply by nebulization and the support is advanced, this succession of operation being reproduced as the progress of the support.
  • the containment vessel or bioreactor is thus closed at both ends and has one or more openings for eliminating excess air or liquid and preferably maintained at overpressure, preferably using a mist-laden air.
  • the support forming the confinement enclosure or bioreactor is set in motion by a displacement device by carrying within it the roots, preferably of the hairy or adventitious type, being grown by any techniques known to those skilled in the art. art such as a treadmill.
  • the roots develop within the support as the deployment and thus the advancement of said support.
  • the support is finally closed with a third means of isolation and the roots are harvested with the bioreactor, the latter can be used as packaging or be harvested directly.
  • the bioreactor or containment chamber is renewed as and when to replace the part that has moved and allow the introduction of a new inoculum.
  • the inventors have also determined that feeding the roots by nebulization of a culture medium makes it possible to supply nutrients to a containment vessel or bioreactor having a length of more than 10 m while maintaining a productivity greater than 500 g / m. 2 / day root dry matter, preferably greater than 1 kg / m 2 / day.
  • the invention is based on a completely new and inventive approach of seeding a support parade by an inoculum of roots, said support serving as containment chamber or bioreactor, the supply of said inoculum being carried out by nebulization of a culture medium. Because of the scrolling, the beginning of the confinement enclosure or bioreactor is renewed progressively so as to replace the part of the containment or bioreactor having moved, roots being introduced in the form of a inoculum for growing in said reactor and / or producing metabolites of interest therein.
  • the support serving as containment vessel or bioreactor is composed of a transparent envelope, and more preferably of a flexible transparent envelope consisting of polymer such as polyvinyl chloride (PVC), polyamide, polyethylene, polypropylene, polyethylene terephthalate, polylactic acid (PLA), poly-succinate.
  • PVC polyvinyl chloride
  • PLA polylactic acid
  • metabolites of interest is intended to denote an organic compound that is intermediate or derived from metabolism, generally in the form of small molecules or monomers.
  • continuous piston flow process is meant a process in which a bioreactor is filled at one end continuously and / or with successive charges at time intervals suitably distributed over time and drained at the other end. end continuously and / or by successive discharging at time intervals suitably distributed in time so that at a given distance from the first end of the bioreactor, the state of the roots is identical
  • silling a "support” or “support parade” is meant to indicate that the support is moving relative to where the seeding is performed.
  • deployment of the support it is meant to position the support before the place where seeding is performed. Said deployment corresponds to unfolding or unfolding of the support, preferably an unwinding of the support.
  • insulation means means a mechanical insulation means such as clip, clip.
  • inoculum is intended to denote a sample containing whole plant roots or in pieces, intended to be introduced into a medium favorable to its multiplication, in order to produce a greater amount.
  • root harvesting is meant the actual root collection or the recovery of the support comprising the roots, said support being closed at its ends by the insulation means thus forming a sealed pocket.
  • the at least first opening allowing the inoculum to be seeded with a root inoculum corresponds to the at least one second opening allowing the nebulization feed of a culture medium, thus the seeding of the medium.
  • support and the nebulized feed of a culture medium are made by the same opening.
  • said at least one first opening and at least one second opening are merged.
  • the at least first opening and / or the at least second opening are located on the upper part of the support.
  • the opening will not be encumbered by the roots and / or the liquid formed by the condensed mist.
  • a feed from above also allows a better distribution of particles of nebulized medium in the sky of the enclosure formed by the support above the growing roots, the particles undergoing the force of gravity.
  • the first isolation means of the deployment step corresponds to the second isolation means of the seeding step when the cycle of these steps is repeated.
  • the culture support is in the form of a strip comprising at least one lower sheet and at least one upper sheet interconnected by at least one securing means.
  • solidifying means any means for securing the upper and lower sheets between said means being selected from the group consisting of heat sealing, sewing, gluing, preferably the sheets are secured at their lateral ends or edges by gluing or heat-sealing to form a tube during the overpressure of the support after seeding.
  • the containment vessel or bioreactor formed by the support is physically separated from the external environment and allows root growth in a controlled and sanitized and / or sterile environment.
  • the culture support comprises at least one permeable sheet secured to at least a first lower sheet and at least a first upper sheet and situated between them, said support being provided with at least a vent, at least one drain and at least one opening for seeding and / or allowing the nebulization feed of a culture medium.
  • such a support has the advantage of allowing the roots to grow around the at least one permeable sheet by using it as a support and by avoiding settling under the effect of their own weight.
  • the roots can maintain a sufficiently low root bed density and sufficiently high root spacing that will allow the aerosol particles to penetrate and feed the heart of the root bed.
  • This type of support with at least one bonded permeable sheet is simple to manufacture, has a small steric hindrance and is therefore also simple to sterilize.
  • the method according to the invention is such that the step of deploying the culture medium corresponds to a running of said support.
  • the non-deployed support has a small steric hindrance which facilitates its storage as well as possible sterilization step for example by irradiation and / or pasteurization.
  • the process according to the invention is such that the root inoculum comprises pieces of roots having a size of at least 50 mm and at most
  • the pieces of roots have both a size compatible with rapid growth and a sufficient number per mass unit to form after their growth a homogeneous root mat inside the support.
  • the process according to the invention is such that the roots are of the hair type.
  • the roots grown in the process do not require the addition of growth hormone in the culture medium.
  • the process according to the invention is such that the aerosol particles constituting the nebulization feed have an average diameter less than or equal to 4 ⁇ and preferably less than or equal to 2 ⁇ m.
  • the aerosol particles will have a lower mass, a higher specific surface per unit mass and therefore will have less tendency to sediment within the support and therefore can be transported over greater distances.
  • their small size will allow them to penetrate deeper into the root bed and feed the roots in the center of the bed more efficiently compared to aerosol particles with an average diameter greater than 4 ⁇ .
  • Example 3 below shows that the growth of Salvia miltiorrhiza roots is directly affected by the average size of the aerosol particles.
  • the process according to the invention is such that the seeding of said support is carried out in a white chamber.
  • the various manipulations such as inoculation and the connections between the nebulization chamber and the second opening, can be performed under aseptic conditions and limit the risk of contamination of the controlled environment inside the support.
  • the method according to the invention is such that it comprises a step of stimulating root growth by illumination and / or modification of the composition of the culture medium.
  • it is possible to stimulate the production of metabolites of interest as bioactive compounds by induced light stress to increase root growth and / or production of active compounds.
  • These light stresses can be achieved either at wavelengths visible by conventional lighting or specialized horticulture, or at specific wavelengths with lighting with 380-500 nm LEDs, preferably between 400-450 nm, or again by lighting at UV wavelengths, preferably between 270-290 nm.
  • the advantage of the technology that is the subject of the invention is that the lighting can be arranged at any time during the growth of the roots. It is thus possible, for example, to stimulate the production of bioactive compounds only at an advanced stage of growth so as to prevent these bioactive compounds in excessive concentration from inhibiting root growth.
  • the method according to the invention is such that it comprises a root elicitation step by illumination and / or modification of the composition of the culture medium and / or acoustic stimulation.
  • a root elicitation step by illumination and / or modification of the composition of the culture medium and / or acoustic stimulation.
  • the method according to the invention is such that the stimulation and / or elicitation steps are performed on all or part of the support.
  • the method according to the invention is such that the cultivated root is selected from the group consisting of the root of the species Arabidopsis thaliana, Arctium majus, Armoracia rusticana, Artemisia annua, Astragalus membranaceous, Atropa belladonna, Azadirachta indica, Berberis vulgaris, Calystegia sepium, Catharanthus roseus and Trichophyllus, Cinchosa pubescens, Cistanche tubulosa, Datura stramonium, Derris trifolia, Dioscorea vollosa, Echinacea angustifolia, Pallida and purpurea, Eleutherococcus senticosus, Fallopia multiflora, Gloriosa superba, Glycyrrhiza glabra and Uralensis, Harpagophytum Procumbens, Hydrastis canadensis, Hyoscyamus mutic
  • the process according to the invention is such that the root elicitation step for producing a tanshinone-type metabolite of interest is carried out by illumination by means of illumination. wavelengths between 380 and 500 nm, said elicitation preferably being carried out on roots of salvia miltiorrhiza type.
  • the method according to the invention is such that the elicitation step and / or the step of stimulating root growth is carried out in the presence of at least one compound selected from the group of compounds consisting of sodium acetate, jasmonic acid, methyl jasmonate, yeast extract, salicylic acid, fatty acids, C12 fatty acids, ethylene, nitrous oxide and carbon dioxide.
  • Example 6 below shows the increased production of Tanshinone with an elicitation of 100 ppm of yeast extract.
  • C12 fatty acids is intended to denote a fatty acid containing 12 carbon atoms.
  • the invention also relates to a device for semi-continuous production of roots of plants above ground by scrolling a culture support, said support serving as confinement chamber or bioreactor, adapted to implement the method according to the invention, said device comprising:
  • the support being provided with insulating means to compartmentalize the support and isolate the various compartments thus created each other, at least one vent and at least a drain and at least one opening allowing the seeding and / or allowing the nebulization feed of a culture medium located between two successive isolation means.
  • the support forming the confinement enclosure or bioreactor has shrinkage of the circumference when the latter is fed to the culture medium, these shrinkage being obtained preferentially with the aid of external clamp.
  • media feed unit any system that makes it possible to limit the steric bulk of the storage of the support, such as folding-stacking, winding in a reel, preferably the feed unit. present in the form of a coil.
  • support scrolling mechanism any system for moving the support such as a treadmill, suspension on moving cables, tray equipped with rollers, preferably a treadmill on which the support is deposited.
  • nebulizing chamber is meant more particularly to designate a closed chamber within which culture medium is introduced for example by means of a peristaltic pump or any other techniques known to man of the the art and nebulized in the chamber either with a diffuser fed with pressurized medium, or with an ultrasonic nebulizer or with any other techniques known to those skilled in the art to form liquid particles of average diameter less than or equal to 50 ⁇ , preferably less than or equal to 10 ⁇ , more preferably less than or equal to 5 ⁇ , most preferably less than or equal to 4 ⁇ and most preferably less than or equal to 2 ⁇ .
  • a compressed air inlet on the fogging chamber makes it possible to carry the haze formed at a flow rate sufficient to supply, through one or more connections, the confinement enclosure or mist reactor over a distance greater than 5 m, and preferentially greater than 10 m.
  • nebulization chamber fed with a carrier gas is meant a chamber provided with a feed in culture medium in mist form, the culture medium in fogged form being either inserted into the chamber by injection nozzles, is formed within the chamber using ultrasonic nebulizer and mixed with a gas stream to carry the aerosol particles formed throughout the support.
  • connection between the nebulizing chamber and the support is meant to designate one or more pipes with or without valves and connections for supplying the flow of gas carrying the aerosol particles between the mist chamber and a nozzle. or more openings of the support.
  • the device according to the invention is such that it comprises a white room for seeding.
  • white room is meant a room or space where the concentration of microorganisms is controlled to minimize the introduction, generation, retention of these microorganisms within the support as by filtration air and / or air treatment with radiation and / or chemical and / or thermal treatment of the air.
  • the white chamber will be kept under pressure by injection of filtered air in order to keep the concentration of microorganisms at lower levels as exposed by the ISO 14644-1 Class 3 standard.
  • FIG. 1 schematically describes the successive steps of a method according to the invention
  • FIG. 2 illustrates a device able to implement a method according to the invention
  • FIG. 3 shows in graphical form a production of Salvia Milthiorriza hair roots in a bioreactor using a method according to the invention
  • FIG. 4 shows in graphical form a production of Salvia Milthiorriza hairy roots in a bioreactor as a function of the frequency of the fogger by implementing a process according to the invention
  • FIG. 5 graphically presents a production of Panax Ginseng hairy roots in a bioreactor using a method according to the invention.
  • FIG. 1 shows an embodiment of a continuous piston-flow process for producing roots of plants above ground by moving a culture support, said support acting as confinement enclosure. Said method comprises the following successive steps:
  • the method according to the invention may comprise an additional step of stimulating root growth by illumination and / or by modifying the composition of the culture medium and / or a root elicitation step by illumination and / or modification. the composition of the culture medium and / or by stimulation acoustically, said additional step being inserted between steps 4 and 5 or being confused with step 4.
  • a culture support reel for producing plant roots said support being in the form of a strip comprising two sheets joined at their ends or edges and being provided with a first means of insulation in the form of forceps or closing clip for bag,
  • the support is transported by a treadmill, the root inoculum introduced into the support develops there simultaneously with the transport of this until it is harvested at the exit.
  • the support constituting the containment enclosure or reactor is renewed progressively so as to replace the part s' moved and roots are introduced to grow and / or produce metabolites of interest.
  • the nebulization chamber consists of a closed chamber inside which culture medium is introduced for example using a peristaltic pump or any other techniques known to those skilled in the art and nebulized in the chamber is using a diffuser fed with pressurized medium, an ultrasonic nebulizer or any other technique known to those skilled in the art to form liquid particles of average diameter less than or equal to 50 ⁇ , preferably less than or equal to 10 ⁇ , more preferably less than or equal to 5 ⁇ m, most preferably less than or equal to 4 ⁇ and most preferably less than or equal to 2 ⁇ m average diameter.
  • a compressed air inlet on the fogging chamber enables the haze formed to be carried at a sufficient flow rate to supply, through one or more connections, the external mist tunnel over a distance greater than 5 m and preferably greater than 5 m. 10 m.
  • the support is preferentially transparent, and more preferentially transparent and flexible, and consists of a polymer such as polyvinyl chloride (PVC), polyethylene, polypropylene, polyethylene terephthalate, polylactic acid (PLA), poly succinate consisting of a polymer such as poly-vinyl chloride, polyethylene, polypropylene, polyethylene terephthalate, poly-lactic acid, poly-succinate.
  • PVC polyvinyl chloride
  • PLA polylactic acid
  • PDA poly succinate consisting of a polymer such as poly-vinyl chloride, polyethylene, polypropylene, polyethylene terephthalate, poly-lactic acid, poly-succinate.
  • the support closed at both ends, has one or more openings for
  • the roots are harvested by isolating the part of the support containing them from the rest of the support and detaching this part of the support. This operation can be done by any technique known to those skilled in the art and preferably by crushing the walls of the support with 2 clamps and cutting the portion between the two clamps so as to limit the contacts between the two. inside the support and its external environment. When the support is set in motion, said support is continuously renewed and inoculated with hairy roots.
  • the roots can be illuminated at a photon intensity of 150 / ⁇ mole / sec / m 2 at wavelengths of 600 nm +/- 20 nm to stimulate their growth.
  • Panax Ginseng described in Examples 7 and 8 below or the species Arabidopsis thaliana, Arctium majus, Armoracia rusticana, Artemisia annua, Astragalus membranaceous, Atropa belladonna, Azadirachta indica, Berberis vulgaris, Calystegia sepium, Catharanthus roseus and Trichophyllus, Cinchosa pubescens, Cistanche tubulosa, Datura stramonium, Derris trifolia, Dioscorea vollosa, Echinacea angustifolia, pallida and purpurea, Eleutherococcus senticosus, Fallopia multiflora, Gloriosa superba, Glycyrrhiza glabra and uralensis, Harpagophytum procumbens, Hydrastis canadensis, Hyoscyamus muticus
  • This tunnel is composed of 15 compartments of 1 m 3 communicating with each other by narrowing of the circumference of the tunnel using external metal clamp,
  • a controlled atmosphere sterile zone at the inlet of the system for attaching the inlet and outlet connections aseptically to the opening of the bag and inoculating the bag with roots (22).
  • This sterile zone consists of a CLEAN AIR TECHNIEK W DLF 660EC laminar flow hood adapted to the walls.
  • a sterile compressed air supply that ensures positive pressure inside the plastic tunnel and provides the oxygen needed for root growth (23).
  • This sterile air supply is through a Pall Advanta 10 "filter and a CoenCo deoil air compressor (at a flow rate of 3 m 3 / h) through the nebulizer unit.
  • a nebulization culture medium feed (24) stainless steel tank equipped with a level detector and a Beijing Ultrasonic 2Mhz ultrasonic atomization unit.
  • the haze whose particles have an average size of 2 ⁇ ⁇ is transferred thanks to the flow of air over the entire length of the tunnel.
  • the nebulizer tank is fed at a rate of 81 / h by a Masterflex peristaltic pump.
  • the leaves are wounded with a sterile scalpel, then infected with Rhizobium rhizogenes strain LMG-152, which has an optical density of between 0.7 and 1.
  • the leaves are then laid for one week on the "Murashige and Skoog” (MS) medium (Sigma-Aldrich ref: M5519), in the dark and at 24 ° C.
  • the medium is composed of 4.4 g / l of MS powder, 20 g / l of sucrose, 7 g / l of agar and no phytohormones. After incubation, the leaves are transferred to a liquid "MS" medium (same composition as the solid but without agar) with 250 mg / L Ampicillin and 250mg / L Cefotaxime for 4-5 days with agitation to get rid of the bacteria.
  • MS liquid "MS" medium (same composition as the solid but without agar) with 250 mg / L Ampicillin and 250mg / L Cefotaxime for 4-5 days with agitation to get rid of the bacteria.
  • Example 2 Production of Salvia Milthiorrhiza Roots in the Misted Reactor An example of such a continuous bioreactor which meets the description of the invention is given in this example.
  • the autoclaved medium MS composed of the ingredients mentioned in the following table, is introduced into the fogger using a peristaltic pump at a flow rate of 8 l / h.
  • the compartment n is isolated from the n + 1 compartment and the upstream bag roll by 2 separation clamps. Every 24 hours, the treadmill is started, and the bag is unrolled to the exit of the barren chamber of lm. The compartment n then becomes the compartment n + 1 and the empty bag under the hood becomes the compartment n.
  • Roots cut into 50-100 mm pieces of Salvia Miltiorrhiza transformed with Agrobacterium Rhizogenes obtained in Example 1 to grow in the absence of aerial parts and growth hormones are inoculated by the upper opening of the first compartment (n) of the Waterproof and sterile PVC plastic tunnel with a spatially homogeneous distribution of 10 kg of roots per square meter.
  • This compartment is then connected to the compartment " n + 1 by removing the separation clamp while keeping the separation clamp with the bag roll upstream of the system .
  • the arrival of the air / mist mixture is then disconnected from the compartment n + 1 and reconnected to compartment n.
  • Figure 2 shows the masses of roots harvested over a month in the reactor. In total 1743 kg of fresh roots were produced by this technology in one month.
  • the root bed at the outlet of the reactor has an average thickness of 21.4 cm for a bulk density of 27%.
  • the roots are then harvested by cutting the bag and dried in a microwave drying tunnel of the brand Amisy AMSD-FN12 with a capacity of 100 kg / h up to a dry matter concentration of 85%.
  • Example 4 To determine the impact of fog particle size on nutrient supply to the roots and the transport of these nutrients along the tunnel, we modified the experiment in Example 4 by changing the frequency of day 1 the generator of 2 Mhz at 1, 6 Mhz or 1 Mhz which corresponds to an increase in the size of the fog particles from 2 ⁇ to 4 ⁇ and 10 / im.
  • Tanshinone A was analyzed according to the protocol described in Gupta et al (2011) on a Waters C18 column (5 ⁇ , 4.6 ⁇ 250 mm) with a mixture of acetonitrile: water (58:42) at 1 ml min-1 at room temperature and detection at 245 nm.
  • EXAMPLE 5 Elicitation Using a 600 nm Illumination of Salvia Mitiorrhiza's Hairy Root Growth
  • VEGELED 600 nm 9W from Colasse SA VEGELED 600 nm 9W from Colasse SA
  • These lamps are lit 16 hours a day providing a brightness of about 150 ⁇ mol / s / m2.
  • EXAMPLE 6 Chemical elicitation using yeast extract from the production of Tanshinone by Salvia Miltiorrhiza roots
  • the seeds were then soaked 1 min. in ethanol and 3 minutes in a 1 g / l HgCl 2 solution at room temperature.
  • the seeds whose surface has thus been sterilized are soaked in a sterile solution of 500 ppm Chloramphenicol, Neomycin 500 ppm and tetracycline 500 ppm for one hour before being deposited on an Agar-Mushashino gel purchased from Sigma-Aldrich in a box of Petri.
  • the roots and leaves of the plants obtained by germination of these seeds and culture for 30 days are cut into pieces of 2-3 cm and injected with a pin with a culture of 36 hours of the LMG-152 strain.
  • Rhizobium rhizogenes in a YMB medium containing 50 ppm of Kanamycin.
  • the infected plant pieces are grown on Mushashino medium for 48 hours at 28 ° C. After 48h, the infected plant pieces are subcultured into Mushashino liquid media containing 1000 ppm Cephalexin and 1000 ppm ampicillin every 4 days for 2 weeks. Then, the infected roots obtained are cultured in Mushashino liquid medium without antibiotic in the dark until hairy roots appear.
  • Example 8 Ginseng Root Cultivation We used the equipment and culture conditions of Example 4 by modifying only the culture medium (MS 1 ⁇ 2) and the type of cultivated hair roots (Panax Ginseng obtained in Example 7) and the speed of movement of the carpet (1 m every 2 days) and therefore harvest (1 bag every 2 days).

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  • Life Sciences & Earth Sciences (AREA)
  • Environmental Sciences (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
EP15735834.2A 2014-04-01 2015-03-20 Pfropfenströmungsherstellungsverfahren für pflanzenwurzeln Withdrawn EP3125675A2 (de)

Applications Claiming Priority (2)

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BE2014/0220A BE1021922B1 (fr) 2014-04-01 2014-04-01 Procede de production continu a ecoulement piston de racines de plantes hors sols par defilement d'un support de culture
PCT/BE2015/000009 WO2015149136A2 (fr) 2014-04-01 2015-03-20 Procédé de production continu à écoulement piston de racines de plantes hors sol par défilement d'un support de culture

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FR3072025B1 (fr) * 2017-10-06 2019-11-01 Plant Advanced Technologies Pat Extraits racinaires de plantes du genre morus et leurs utilisations
US11299700B1 (en) 2021-02-19 2022-04-12 Acequia Biotechnology, Llc Bioreactor containers and methods of growing hairy roots using the same

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US4292760A (en) * 1979-07-19 1981-10-06 Krave Carl A Method, apparatus and package for sprouting seeds
US4291499A (en) * 1979-08-14 1981-09-29 Prewer John R Propagation of plants
FR2590761B1 (fr) * 1985-11-29 1989-01-06 Beghin Say Sa Support de culture hors-sol comportant des particules superabsorbantes et utilisation de ce support pour la culture hors-sol
US4857464A (en) 1986-02-21 1989-08-15 Bio-Rational Technologies, Inc. Mist cultivation of cells
DE10016554A1 (de) 2000-04-03 2001-10-18 Rootec Ges Fuer Bioaktive Wirk Vorrichtung zum Kultivieren von pflanzlichen oder tierischen Gewebekulturen
US7531350B2 (en) 2005-04-20 2009-05-12 Agricultural Research Institute Bioreactor for growing fungus, plant cell, tissue, organ, hairy roots and plantlet
KR100842420B1 (ko) 2007-02-14 2008-07-01 주식회사 씨비엔바이오텍 생물반응기를 이용한 에키네시아 속 식물의 부정근대량생산법
US8114664B2 (en) 2007-07-12 2012-02-14 Worcester Polytechnic Institute Scalable wall bioreactor for culture of plant and animal tissues
CN102783414A (zh) 2011-05-19 2012-11-21 河南省海乐森药用细胞工程技术有限公司 一种适合于规模化培养植物毛状根的外循环云雾式生物反应器
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BE1021922B1 (fr) 2016-01-26
WO2015149136A3 (fr) 2015-11-19

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