WO2015149136A2

WO2015149136A2 - Continuous plug flow production method for plant roots outside soil by movement of a growing medium

Info

Publication number: WO2015149136A2
Application number: PCT/BE2015/000009
Authority: WO
Inventors: Pierre-Antoine Mariage; Thierry MARIQUE; Mario GODOY
Original assignee: Green2Chem S.A.
Priority date: 2014-04-01
Filing date: 2015-03-20
Publication date: 2015-10-08
Also published as: EP3125675A2; BE1021922B1; WO2015149136A3

Abstract

The invention relates to a continuous plug flow production method for plant roots outside soil, by movement of a growing medium, said medium acting as a confinement enclosure. According to the invention, such a method comprises the following consecutive steps for deployment of the growing medium for production of plant roots: said medium being provided with at least one first isolation means for compartmentalizing the medium and isolating the various compartments thus created from one another (1); seeding within said medium of a root inoculum through at least one first opening located, in the movement direction of the medium, downstream of said first isolation means (2); opening of at least one second isolation means allowing the medium to be compartmentalized and isolating the various compartments thus created from one another, situated, in the movement direction of the medium, downstream from said at least one first opening (3); supply by spraying of a growing environment through the at least one second opening, said at least one second opening being situated between said first and second isolation means (4); the isolation of the medium containing the roots for the purposes of growth by the at least one third isolation means allowing compartmentalization of the support and isolation of the various compartments thus created from one another (5); and harvesting of the roots (6).

Description

Process for the continuous production with piston flow of roots of plants above ground by scrolling a culture support

1. Field of the invention

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.

2. Solutions of the prior art

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.

The production of 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. simple. 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.

Some soil bacteria, such as Agrobacterium Rhizogenes, contain plasmids that can infect plant roots and cause them to grow abnormally. 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".

Currently, the main constraint for the industrial production of adventitious or hairy roots is the development and scaling of appropriate containers (bioreactors). The growth rate of plant biomass is indeed lower than that of yeast and bacteria. The doubling time of the observed biomass varies from 20 minutes in E. Coli, 60 minutes in Saccharomyces Cerevisiae, 100 minutes in Lactobacillus Plantarum. The doubling time of the plant cells present in the roots cultivated in-vitro varies from a few days to several weeks depending on the culture conditions. Existing bio-reactors therefore do not make it possible to produce industrially and economically sufficient quantities of biomasses and active molecules except for applications with very high added values.

Bioreactors in liquid media very quickly lead to difficulties in oxygenation of biomass and few accurate results of continuous culture have been published so far.

To eliminate the problem of limiting the transfer of oxygen in a liquid medium while maintaining a sufficient supply of nutrients, the use of a misted bioreactor as described by DiLorio et al (Appl Microbiol Biotechnol., Vol 37, pp 457-462) is an interesting alternative.

Existing nebulization processes are all carried out in batch culture and implement complex and expensive culture bioreactors or culture vessels of such processes are disclosed in WO200174992, US2006240544, EP0234868A2, CN102783414, US2009017529, Kim et al (Plant Cell Tiss Cooked Organ, Vol 98, pp 25-33, 2009), Huang et al (Enzyme and Microbial Technology, Vol 35 (1), pp 22-32, 2004), Wyslouzil et al (Biotechnol and Bioeng. Vol 70 (2), pp 143-150, 2000), Sivakumar et al (Biotechnol Bioeng Vol 107 (5), pp 802-813), they allow some applications in specific fields such as cosmetics and cosmetics. pharmaceuticals.

There are also disposable stirred bioreactors based on flexible plastics for the production of plant cells as described by Ducos and Al. (Adv Biochem Eng Biotechnol 2010; 115: 89-115; doi: 10.1007 / 10_2008_28) but these bioreactors are in the liquid phase, in batch and limited in size to a volume of 100. Examples of use of this type of reactor are also given by Eibl et al (Phytochem Rev. Vol 7, pp 593-598, 2008) and Stiles et al (Adv Biochem Eng Biotechnol, Vol 134, pp 91-114, 2013).

In view of this state of the art, there are no methods of continuous cultivation of adventitious and / or hairy roots allowing industrial production at the tonal scale. he There is therefore a need for this type of method to overcome the disadvantages of the existing processes mentioned above.

3. Objectives of the invention

The invention particularly aims to overcome these disadvantages of the prior art. More specifically, 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.

4. DESCRIPTION OF THE INVENTION In accordance with a particular embodiment, 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.

According to the invention, 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 ,

Feeding by nebulization of a culture medium through at least a second opening, said at least one second opening being situated between said first and second isolation means,

^• isolation of the medium containing the fully grown roots with at least a third insulation means to compartmentalize the support and isolate the various compartments thus created each other,

^• 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. Thus, when the second isolation means is opened, 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. powered by the nebulizing chamber. 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.

Thus, 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. Preferably, 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.

The term "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.

By the term "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 By the terms "scrolling a "support" or "support parade" is meant to indicate that the support is moving relative to where the seeding is performed.

By the terms "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.

By the term "insulation means" means a mechanical insulation means such as clip, clip.

The term "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. By the terms "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.

According to a preferred embodiment, 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. In other words, said at least one first opening and at least one second opening are merged. Thus, such an implementation allows the use of structure support and simplified manufacturing.

According to a preferred implementation, the at least first opening and / or the at least second opening are located on the upper part of the support.

Thus, the opening will not be encumbered by the roots and / or the liquid formed by the condensed mist. In addition 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.

According to a preferred implementation or according to the invention, 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.

Advantageously, 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.

By the term "solidifying means" is meant 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.

Thus, 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.

According to a preferred embodiment of the preceding embodiment, 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.

Thus, 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.

According to a preferred implementation or according to the invention, the method according to the invention is such that the step of deploying the culture medium corresponds to a running of said support.

Thus, 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.

According to one advantageous embodiment, 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 Thus, 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.

According to one advantageous embodiment, the process according to the invention is such that the roots are of the hair type.

Thus, the roots grown in the process do not require the addition of growth hormone in the culture medium.

According to an advantageous implementation, 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 μχη and preferably less than or equal to 2 μ m.

Thus, 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. In addition, 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 μτη. Example 3 below shows that the growth of Salvia miltiorrhiza roots is directly affected by the average size of the aerosol particles.

According to an advantageous implementation, the process according to the invention is such that the seeding of said support is carried out in a white chamber.

Thus, 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. According to an advantageous implementation, 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. Thus, 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 plaintiff company surprisingly discovered that the production of tanshinone could be increased by 200% by applying to the hair roots of Salvia miltiorrhiza a luminous treatment with a wavelength of 440 nm. Similarly and preferably, the roots can be illuminated at a photon intensity of 150 μ mole / sec / m ² at wavelengths of 600 nm +/- 20 nm to stimulate their growth.

According to an advantageous implementation, 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. Thus, the combination of different growth stimulation and / or production of active compounds either simultaneously, or successively, or continuously, or discontinuously allows the roots to be exposed to the conditions for optimal growth and growth. productivity and / or concentration of optimal active compounds.

According to an advantageous implementation of at least one of the two previous implementations, the method according to the invention is such that the stimulation and / or elicitation steps are performed on all or part of the support.

Thus, it is possible to stimulate growth and / or elicit the production of active compounds only when the roots are in their growth phase requiring it. It is by A possible example of eliciting the production of bioactive compounds only when root growth is complete, some of these compounds being inhibitors for growth proper.

According to an advantageous implementation, 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 muticus, Hypericum perforatum, Ipomea aquatica, Leontopodium alpinum, Lepidium meyenii and peruvianum, Lippia dulcis, Lithospermum erythrorhizon, Morinda citrifolia, Morus alba and nigra, Ophiorrhiza pumila, Panax ginseng and quinquifolium, Perilla fruitescnens, Piper methysticium, Polygonum cuspidatum, Ptychopetal olacoides, Pueraria lobata and phaseoloides, Rhodiola rosea, Salvia milthiorrhiza and prevalzkii, Sanguinaria canadensis, Sophora flavescens, Stizolobium hassjoo, Taxus brevifolia, Trigonella foenumgraceum, Whitania somnifera, Yucca shidigera, Wasabia japonica, preferentially in the root of the species Salvia milthiorrhiza and Panax ginseng. According to an advantageous implementation of the three preceding embodiments, 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. According to one advantageous embodiment, 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.

The term "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:

• a support feeding unit, · a support scrolling mechanism,

A nebulization chamber fed with a carrier gas,

^• a connection between the nebulizing chamber and the support, 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. Preferably, 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.

By the term "media feed unit" is meant 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. By the term "support scrolling mechanism" is meant 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. By the term "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 μτη, preferably less than or equal to 10 μχη, more preferably less than or equal to 5 μιη, most preferably less than or equal to 4 μνη and most preferably less than or equal to 2 μτη . 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.

By the term "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.

By the terms "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.

According to an advantageous embodiment or according to the invention, the device according to the invention is such that it comprises a white room for seeding. By "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. Preferably, 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.

5. List of figures

Other characteristics and advantages of the invention will appear more clearly on reading the following description of an embodiment of the method according to the invention, the presentation of a device capable of implementing a method according to the invention. the invention but also examples of root productions by implementing a method according to the invention. These examples are given as simple illustrative and non-limiting examples, and the appended drawings, among which:

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.

6. Description of an embodiment of 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:

1. Deployment of the culture support for production of plant roots, said support being provided with at least one first isolation means for compartmentalizing the support and isolating the different compartments thus created from each other (1),

2. 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 (2),

3. Opening at least a second isolation means for compartmentalizing the support and isolating the different compartments thus created from each other, located, in the direction of the scrolling of the support, downstream of said at least a first opening (3),

4. fogging a culture medium through at least a second opening, said at least one second opening being located between said first and second isolation means and pressurizing the support (4),

5. isolating the support containing the roots at the end of growth by at least a third isolation means for compartmentalizing the support and isolating the different compartments thus created from each other (5),

6. harvesting roots (6).

Advantageously, 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 particular example of a process according to the invention is described below. The method comprises the steps of:

1. unwinding of 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,

2. 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,

3. opening of at least a second insulating means in the form of a clip or closure clip for a bag, said second means being located, in the direction of the scrolling of the support, downstream of said at least one first opening, said second corresponding means, by moving the support, to the first isolation means of step 1 of the preceding step cycle,

4. fogging a culture medium through at least a second opening, said at least one second opening being located between said first and second insulating means and pressurizing the support, said support thereby forming a reactor or containment in the form of a tunnel,

5. isolation of the support containing the roots at the end of growth by at least a third means of isolation,

6. harvesting roots, during the whole process, 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. In addition, 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 μιη, preferably less than or equal to 10 μιη, more preferably less than or equal to 5 μm, most preferably less than or equal to 4 μνη and most preferably less than or equal to 2 μm 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. The support closed at both ends, has one or more openings for eliminating air or excess liquid and preferably maintained under pressure preferably using mist-laden air supplied by the nebulization chamber.

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.

To increase the active matter content of the roots, it is possible to elicit the production of these bioactive compounds by induced induced light stresses at wavelengths visible by conventional horticultural lighting, or at specific wavelengths. with lighting with 380-500 nm LEDs and preferably between 400-450 nm, or even illumination at UV wavelengths and preferably between 270-290 nm. An advantage of the method according to the invention is that the illumination can be arranged at any time of root growth. 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. As described in Example 4 below, the Applicant Company surprisingly discovered that the production of tanshinone could be increased by 200% by applying to the hair roots of Salvia Miltiorrhiza a light treatment with a wavelength of 430 nm.

Similarly and preferentially, as described in Example 5 below, 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.

It is possible to elicit the growth of roots and / or the production of bioactive molecules by using chemical compounds to add to the medium such as sodium acetate, jasmonic acid, methyl jasmonate, yeast extract, salicylic acid, fatty acids, acids C 12 fatty acids or gaseous compounds such as ethylene, carbon dioxide and / or nitrous oxide. Example 6 below shows the increased production of Tanshinone with an elicitation of 100 ppm of yeast extract. It is also possible to elicit the production of bioactive compounds by exposing the roots to ultrasound.

Other species can also be produced in the process described in this invention such as 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, Hypericum perforatum, Ipomea aquatica, Leontopodium alpinum, Lepidium meyenii and peruvianum, Lippia dulcis, Lithospermum erythrorhizon, Morinda citrifolia, Morus alba and nigra, Ophiorrhiza pumila, Panax ginseng and quinquifolium, Perilla fruitescnens, Piper methysticium, Polygonum cuspidatum, Ptychopetalum olacoides, Pueraria lobata and phaseoloides, Rhodiola rosea, Salvia milthiorrhiza and prevalzkii, Sanguinaria canadensis, Sophora flavescens, Stizolobium hassj oo, Taxus brevifolia, Trigonella foenumgraceum, Whitania somnifera, Yucca shidigera, Wasabia japonica. Fig 2 illustrates a device adapted to implement a method according to the invention. The reactor consists of the following parts described in Figure 2

1. a support in the form of a flexible plastic tunnel and sealed PVC (20) closed at both ends and deposited on a conveyor belt 15 m long and 1 m wide (21) fed from a roll of PVC wound and sterilized by irradiation. 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,

2. 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.

3. 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.

4. 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 μπ \ 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.

5. one or more air purges (25) equipped with Sartorius air filter and preceded by a Sulzer brand demister to prevent clogging. 6. one or more outlets to drain excess liquid.

Example 1 Transformation of Salvia Milhiorriza into Hairy Roots

The leaves of a healthy 2-year-old Salvia Miltiorriza are first disinfected with the following protocol:

1. Simple rinsing with tap water 2. Passage for 20 seconds in a 70% ethanol solution

3. Passage for 6 minutes in a solution of 6% calcium hypochlorite

4. 3 successive rinses with ultra pure water (step carried out in a laminar flow hood).

Then, 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.

After 4-5 days, the leaves are put back on a solid "MS" medium at 24 ° C and the roots appear after 2-3 weeks at the injury sites. Once sufficiently developed, these roots are recovered and placed in a liquid "MS" medium with stirring to promote growth. They are transplanted every two weeks in the same medium until a biomass quantity of 10 kg required for the inoculation of the reactor.

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.

Ingredients Ppm

Glucose 50000

Glycine 2

Myo-inositol 100

Nicotinic acid 0-5

Pyridoxyne HCL 0.5

Thiamine 0,1

CaCl ₂ 332

KH ₂ P0 ₄ 170

KN0 ₃ 1900

MgSO ₄ 180

NH ₄ N0 ₃ 1650

0.025 CoCl ₂

CuS0 ₄ 0.025

Under the laminar flow hood, 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.

After 15 days and then every 24 hours, the compartment n + 15 is isolated from the tunnel with the aid of 2 adjacent separation clamps and the plastic film is cut between the two clamps. The bags obtained are weighed to determine the root mass by deducting the weight of the bag. 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 3 Size of Mist Drops versus Reactor

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 2μπι to 4μπι and 10 / im.

We can see in Figure 3 that when the size of the fog particles is increased, nutrient transfer to the roots is significantly reduced with a significant decrease in root productivity of over 40% for 1.6 MHz and 75% for the frequency at 1 MHz compared to a frequency of nebulization at 2 MHz after 15 days of culture.

Example 4 Elicitation in light of the production of Tanshinone on roots Salvia Miltiorrhiza

In order to increase the active matter content of the roots, it is possible to elicit the production of these bioactive compounds by induced induced stress at the wavelengths visible by illumination, or at specific wavelengths with illumination. LEDs.

In the context of this example, we modified example 2 by adding a ramp of LED lamps (VEGELED 430 nm 9W from Colasse SA) to a 1 m above compartments 1 to 10 (10 spots per m). . These lamps are lit 10 hours a day providing a brightness of about 150 / * mole / s / m2.

We analyzed the concentration of Tanshinone A in roots exposed to 430 nm light and unexposed. The results clearly show a 250% increase in the concentration of Cryptanshinone and Tanshinone 2A and 233% of Tanshinone 1. Tanshinone A was analyzed according to the protocol described in Gupta et al (2011) on a Waters C18 column (5 μηι, 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 In the context of this example, we modified Example 2 by adding a ramp of LED lamps (VEGELED 600 nm 9W from Colasse SA) at 1 m above compartments 1 to 10 (10 spots per m). These lamps are lit 16 hours a day providing a brightness of about 150 μmol / s / m2.

We can see that after 15 days of operation, we see an increase of 34 to 38% in the amount of fresh biomass produced.

EXAMPLE 6 Chemical elicitation using yeast extract from the production of Tanshinone by Salvia Miltiorrhiza roots

In the context of this example, we modified example 2 by adding a ramp of LED lamps (VEGELED 600 nm 9W from Colasse SA) to a 1 m above compartments 1 to 10 (10 spots per m). . These lamps are lit 16 hours a day providing a brightness of about 150 // mole / s / m2. Tanshinone A was analyzed according to the protocol described in Gupta et al (2011) on a Waters C18 column (5 μηι, 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 7 Preparation of Hairy Roots of Panax Ginseng To obtain hairy roots of Panax Ginseng, laminated seeds purchased from Seines Baumaux (France) were soaked in water overnight and then treated with a 1% volumic solution. Cetrimide for 5 minutes.

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 ½) 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).

We observe in the following graph that the process described in the invention allows to produce after drying from 5.5 to 8.8 kg dry matter of Panax Ginseng roots every 2 days for a fairly stable Ginsenoside content of 4, 7 to 5.4% by weight.

Claims

1. A continuous piston-flow process for producing roots of plants above ground by scrolling a culture support, said support serving as confinement chamber (20), comprising the following successive steps:

Deployment of the culture support for production of plant roots, said support being provided with at least one first isolation means for compartmentalizing the support and isolating the different compartments thus created from each other (1),

Sowing within said support of a root inoculum through at least a first opening situated, in the direction of travel of the support, downstream of said at least first isolation means (2),

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 (3),

Feeding by nebulization of a culture medium through at least a second opening, said at least one second opening being situated between said first and second isolation means (4),

^• isolation of the carrier containing the end of growth the roots with at least a third insulation means to compartmentalize the support and isolate the various compartments thus created each other (5),

• harvesting roots (6), 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.

2. Method according to claim 1, such that the culture support (20) is in the form of a strip comprising at least one lower sheet and at least one sheet connected together by at least one securing means,

3. Method according to claim 2, such that the culture support (20) comprises at least one permeable sheet secured to at least a first lower sheet and at least a first upper sheet and located between them, said support being provided with at least one vent, at least one drain and at least one opening allowing the seeding and / or allowing the nebulization feed of a culture medium.

4. Method according to any one of the preceding claims, such that the step of deploying the culture support (1) corresponds to a running of said support,

The method of any of the preceding claims, such that the root inoculum comprises root pieces having a size of at least 50 mm and at most

100 mm,

6. Method according to any one of the preceding claims, such that the roots are of hairy type,

7. Method according to any one of the preceding claims, such that said at least one first opening and at least one second opening are combined,

8. Process according to any one of the preceding claims, such that the aerosol particles constituting the nebulization feed have an average diameter of less than or equal to 4 μηϊ.

9. Process according to any one of the preceding claims, such that the seeding of said support is carried out in a white chamber (23),

10. Method according to any one of the preceding claims, such that it comprises a step of stimulating root growth by illumination and / or modification of the composition of the culture medium,

11. Method according to any one of the preceding claims, such that it comprises a root elicitation step by illumination and / or modification of the composition of the culture medium and / or acoustic stimulation.

12. Method according to any one of claims 10 to 11, such that the stimulation and / or elicitation steps are performed on all or part of the support.

A method according to any root, such that the cultivated root is selected from the group consisting of Arabidopsis thaliana root, 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, Hypericum perforatum, Ipomea aquatica, Leontopodium alpinum, Lepidium meyenii and peruvianum, Lippia dulcis, Lithospermum erythrorhizon, Morinda citrifolia, Morus alba and nigra, Ophiorrhiza pumila, Panax ginseng and quinquifolium, Perilla fruitescnens, Piper methysticium, Polygonum cuspidatum, Ptychopetalum olacoides, Pueraria lobata e t phaseoloides, Rhodiola rosea, Salvia milthiorrhiza and prevalzkii, Sanguinaria canadensis, Sophora flavescens, Stizolobium hassjoo, Taxus brevifolia, Trigonella foenumgraceum, Whitania somnifera, Yucca shidigera, Wasabia japonica

14. Apparatus for semi-continuous production of roots of plants above ground by scrolling a culture support, said support serving as containment chamber (20), comprising: · a support supply unit,

A mechanism for scrolling the support (21),

A nebulization chamber fed with a carrier gas (24),

A connection between the nebulization chamber (24) and the support (20),

The support (20) being provided with isolation means making it possible to compartmentalize the support and to isolate the different compartments thus created from each other, at least one vent and at least one drain and at least one an opening allowing seeding and / or allowing the nebulization feed of a culture medium located between two successive isolation means.

15. Apparatus for semi-continuous production of roots of plants above ground by scrolling a culture support according to claim 14, such that it comprises a white chamber (22) for seeding.