WO2021099399A1 - Device for in vitro plant culture by temporary immersion in a nutritive liquid - Google Patents

Device for in vitro plant culture by temporary immersion in a nutritive liquid Download PDF

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Publication number
WO2021099399A1
WO2021099399A1 PCT/EP2020/082555 EP2020082555W WO2021099399A1 WO 2021099399 A1 WO2021099399 A1 WO 2021099399A1 EP 2020082555 W EP2020082555 W EP 2020082555W WO 2021099399 A1 WO2021099399 A1 WO 2021099399A1
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WO
WIPO (PCT)
Prior art keywords
culture
chamber
nutrient liquid
plant material
culture chamber
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PCT/EP2020/082555
Other languages
French (fr)
Inventor
Guillaume BONNAFOUX
Morgan GARCIA
Original Assignee
Cid Plastiques
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 Cid Plastiques filed Critical Cid Plastiques
Publication of WO2021099399A1 publication Critical patent/WO2021099399A1/en

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    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001—Culture apparatus for tissue culture
    • 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 present invention falls within the field of in vitro culture of plant material, by temporary immersion, in sterile condition, in particular intended for micro-propagation techniques by somatic organogenesis and embryogenesis.
  • Such a device will find particular application in the fields of horticulture and agriculture, where the multiplication of plants by in vitro culture in sterile conditions is common practice.
  • the technique of plant culture in vitro by temporary immersion consists in temporarily immersing in a liquid nutrient medium, a few minutes per day, a plant material in culture. Immersion can be total or partial.
  • an active phase of immersion in a liquid nutrient medium is alternated with a rest phase where the plant material is not immersed in the liquid nutrient medium.
  • the plant material in culture is emerged and without movement.
  • a film of nutrient medium is retained on the plant material. Said film of nutrient medium keeping moistened and supplied with nutrients, necessary for its growth, said plant material.
  • Plant culture in vitro by temporary immersion is a process which makes it possible to avoid the problems of asphyxiation of the culture and / or of the frictional force that are found with plant culture techniques where the plant material is permanently submerged and under agitation.
  • in vitro plant culture by temporary immersion also makes it possible to avoid the accumulation of culture costs linked to the multiplication of manipulations which, for example, in vitro culture in solid medium requires.
  • document WO 2012/146872 discloses a container for the in vitro culture of plant material by temporary immersion.
  • This container comprises two superimposed chambers, closed in the upper part by a cover.
  • the upper chamber is intended to contain the plant material to be cultivated.
  • the lower chamber is intended to contain the nutrient liquid.
  • This container also comprises means for the temporary transfer of at least part of the nutrient liquid from the lower chamber to the upper chamber.
  • the container consists of a one-piece outer part consisting of an upper part of large diameter forming the upper chamber and a lower part, of smaller diameter than the upper part, forming the lower chamber.
  • This container also comprises, housed in the upper chamber, a culture basket containing the plant material.
  • This basket has a central through orifice which allows to receive part of the nutrient liquid transfer means between the two chambers.
  • the basket comprises a filtration member which consists of a sieve which allows the distribution, from the lower chamber to the upper chamber, of the nutrient liquid and the overpressure gas.
  • the transfer means comprise a central connection duct, ensuring the water connection between the two chambers, and a lateral duct for the introduction of gas at the level of the lower chamber.
  • this container is closed by a removable cover.
  • This cover is provided with re-entrant hooking fins complementary to the ribs present at the upper peripheral edge of the upper chamber.
  • the tightness of the cover, assembled on the edge of the upper chamber, is ensured by its locking by screwing on the peripheral edge of the upper chamber, in combination with a seal.
  • a rotational movement of the cover on the container makes it possible to hermetically close the one-piece outer part by crushing the seal. In the locked position, the cover therefore exerts a support force directed towards the lower chamber and which crushes the seal.
  • said cover comprises a vent covered with a sterilizing filter to ensure the sterility of the interior volume of the container once the cover is locked.
  • the vent allows gas to pass between the interior and exterior of the container.
  • the technique of in vitro culture by temporary immersion, using this container consists in applying an overpressure in the lower chamber, containing the nutrient liquid.
  • the overpressure is applied by introducing a gas, sterilized through a sterilizing filter, through the side duct.
  • the introduction of gas is controlled and carried out using a pump delivering compressed air.
  • the overpressure generated pushes the nutrient medium from the lower chamber to the upper chamber.
  • the nutrient liquid from the lower chamber rises through the connection duct, then through the filtration member, to end up in the upper chamber.
  • the overpressure generates a risk of lifting of the cover, that is to say an air inlet or outlet near the seal which can no longer fulfill its role.
  • the mode of operation by overpressure of the container of document WO 2012/146872 results in the upper chamber being subjected to a permanent pressure due to the very slow evacuation of the air flow through the vent of the cover. This phenomenon of overpressure in the upper chamber tends to constrain and deform the container to the point of creating localized leaks.
  • the container disclosed in document WO2012 / 146872, requires a compressed air pump connected to each lower chamber of each container.
  • document WO2012 / 146872 envisages as many pumps as there are receptacles, which generates high costs and complex management in the case of large-scale cultivation requiring several receptacles.
  • the aim is to be able to carry out the technique of plant culture in vitro by temporary immersion in a nutritious liquid medium, on an industrial scale, without risk of contamination of the plant material, which is low production costs and uses little energy.
  • the solution provided for in document WO2012 / 146872 for the technique of plant culture in vitro by temporary immersion in a liquid nutrient medium should be optimized by making it more reliable by in particular limiting leaks.
  • the object of the present invention is to overcome the drawbacks of the state of the art.
  • a first aspect of the invention relates to a device for in vitro culture of plant material by temporary immersion in a nutrient liquid.
  • This device comprises: at least one culture vessel, each comprising: a / a culture chamber, which has an internal volume, and which is intended to contain the plant material to be cultivated, b / a nutrient liquid chamber which is intended to contain the nutrient liquid, c / transfer means for the temporary transfer of at least part of the nutrient liquid from said nutrient liquid chamber to the culture chamber, d / a removable cover to close said culture chamber as well as sealing means interposed between this cover and this culture chamber; e / means of communication, which the removable cover comprises, and which are intended to ensure communication between the internal volume of the culture chamber and the outside of the culture vessel.
  • said culture device also comprises, on the one hand, vacuum means for establishing, in the culture chamber, a pressure which is lower than the pressure in the nutrient liquid chamber and, on the other hand, connection means for connecting the vacuum means to said communication means of said at least one culture vessel.
  • said vacuum means comprise at least one suction means, preferably each suction means is constituted by a vacuum pump, said vacuum means further comprise at least one buffer tank interposed between said at least one suction means and said at least one culture vessel, said vacuum means also comprise at least one valve interposed between said at least a suction means and said at least one buffer tank.
  • said device of the invention comprises at least one valve interposed between said vacuum means and said at least one container.
  • said communication means consist of a duct which passes through the removable cover.
  • said communication means consist of an end piece which is fitted to said cover and which comprises an orifice passing through and communicating with an orifice which passes through said cover.
  • said device comprises at least two culture vessels each comprising communication means while the connection means comprise, on the one hand, a ramp connected to the vacuum means and, on the other hand, connection members. to connect said ramp to the means of communication that each of the culture vessels comprise.
  • said ramp adopts a linear shape and has one end connected to the vacuum means, while the connecting members are distributed over at least part of the length of the ramp.
  • said ramp takes the form of a loop, while the connecting members are distributed over at least part of the length of the loop and, on the other hand, the means of connection further comprise at least one connection duct for connecting said ramp to the vacuum means.
  • said device comprises a plurality of connecting conduits which extend from the ramp adopting the shape of a loop and which converge at a point connected to the vacuum means.
  • said at least one culture vessel comprises a vent to ensure communication between the internal volume of the nutrient liquid chamber and the exterior of such a culture vessel.
  • a second aspect of the invention relates to a method for in vitro culture of plant material by temporary immersion in a nutrient liquid, within at least one culture vessel comprising: f / a culture chamber (3 ), which has an internal volume, and which is intended to contain the plant material (M) to be cultivated, g / a nutrient liquid chamber (4) which is intended to contain the nutrient liquid (L), h / transfer means (5) for the temporary transfer of at least part of the nutrient liquid
  • Said method of in vitro culture of plant material by temporary immersion in a nutrient liquid comprises the following steps:
  • said method of in vitro culture of plant material by temporary immersion in a nutrient liquid is implemented by the device for in vitro culture of a plant material by temporary immersion in a nutrient liquid.
  • Figure 1 schematically shows an exploded side view of an embodiment of a culture vessel of the culture device of the invention
  • FIG.2 schematically shows a culture device of the invention comprising the culture container closed by its cover of Figure 1, vacuum means and connection means;
  • FIG.3 Figure 3 schematically shows a culture device of the invention multi-containers according to a first embodiment
  • FIG.4 Figure 4 schematically shows a top view of the connections of the culture device of the invention multi-container according to the first embodiment of Figure 3;
  • Figure 5 schematically shows a culture device of the invention multi-containers according to a second embodiment;
  • Figure 6 schematically shows a top view of the connections of the culture device of the invention multi-container according to the second embodiment of Figure 5;
  • Figure 7 schematically shows a top view of the culture device of the invention multi-container according to the second embodiment in the presence of a buffer tank.
  • the present invention relates to an in vitro culture device 1 of plant material M by temporary immersion in a nutrient liquid L.
  • Said plant material M to be cultivated consists of all plant tissues cultivated in vitro, such as micro-stems, micro-cuttings, callus, somatic embryos, or even plant material in the form of explants or cells. .
  • the nutrient liquid L consists of all known media allowing the growth and multiplication in vitro of said plant material M, such as for example a liquid medium containing the mineral salts of Murashige and Skoog (1962) or formulations derived from sugar , vitamins or even plant hormones.
  • the device 1 of the invention comprises at least one culture vessel 2.
  • Said container 2 comprises a culture chamber 3, a nutrient liquid chamber 4, means 5 for transferring the nutrient liquid L from one chamber to another, a removable cover 6 associated with sealing means 7 for its closure.
  • said container has a single removable cover 6 to ensure its closure and its sealing.
  • Said container 2 also comprises communication means 8 ensuring communication between its exterior and the internal volume of its culture chamber 3.
  • said container 2 comprises a one-piece outer part 21 covered by said removable cover 6. Consequently, said one-piece outer part 21 can be hermetically closed by said removable cover 6. According to the present invention, said outer part monobloc 21 is divided into a first and a second part.
  • Said first part defines the nutrient liquid chamber 4.
  • Said first part is intended to contain, within its internal volume, said nutrient liquid L.
  • said nutrient liquid L is contained directly within the internal volume of the first part of the one-piece outer part 21 of the container 2, that is to say within the internal volume of the nutrient liquid chamber 4.
  • Said second part defines the culture chamber 3.
  • Said second part is intended to contain, within its internal volume, said plant material M.
  • said second part defining the culture chamber 3, internalized in its internal volume, an interior part 31 in which is located said plant material M.
  • the internal volume of the culture chamber 3, intended to contain the plant material M corresponds to the volume occupied by the interior part 31 in which is finds the plant material M.
  • Said inner part 31 separates the nutrient liquid chamber 4 of plant material M of culture chamber 3.
  • the one-piece outer part 21, which said container 2 comprises has an arrangement along a horizontal axis.
  • the first part and the second part, dividing the one-piece outer part are arranged adjacent to one another, along a horizontal axis.
  • the culture chamber 3 and the nutrient liquid chamber 4 are positioned adjacent to each other in an adjacent manner, and they constitute the one-piece outer part 21, which the said container 2 comprises, covered by said removable cover 6.
  • said removable cover 6 covers both said culture chamber 3 adjacent to said nutrient liquid chamber 4, so as to hermetically close the entire one-piece outer part 21, that is to say say to hermetically close each of the two culture chambers 3 and nutrient liquid 4.
  • At least part of the wall of the culture chamber 3 communicates with at least part of the wall of the nutrient liquid chamber 4 L, through the transfer means 5.
  • said transfer means 5 allow the temporary transfer of at least part of the nutrient liquid L from the liquid chamber 4 nutrient L to the culture chamber 3.
  • said first part is superimposed on said second part as visible in FIG. 1.
  • the container 2 comprises a one-piece outer part 21 which consists of two superimposed annular parts, of different diameters.
  • the one-piece outer part 21 consists of a first annular upper part surmounting the second annular lower part.
  • the first and the second annular part respectively representing said culture chamber 3 and said nutrient liquid chamber 4.
  • Said culture chamber 3 having a diameter greater than that of said nutrient liquid chamber L.
  • Said chambers 3 and 4 are joined together. by a junction constituting an annular shoulder in the form of a crown on which the said inner part 31 can rest.
  • the advantage of a vertical configuration of the container 2, in the form of a single-piece outer part 21 comprising the culture chamber 3 superimposed on the nutrient liquid chamber 4, is that the in vitro culture device 1 is less bulky, compared to an arrangement along a horizontal axis of the culture 3 and nutrient 4 chambers.
  • this vertical configuration is economical in terms of the number of parts necessary to ensure the hermetic closure of both the culture chamber 3 and the nutrient liquid chamber 4 of the container 2.
  • the establishment of a single cover to hermetically close the two chambers 3 and 4 is sufficient.
  • said removable cover 6 covers and can hermetically close the entire one-piece outer part 21, so as to hermetically close the chamber. culture 3 and the nutrient liquid chamber 4.
  • the reduction in the number of parts making it possible to ensure the sealing also makes it possible to drastically reduce the possible zones of exit or entry of the flows within the chambers 3 and 4 and of the container culture 2.
  • the reduction in the number of covers necessary, ensuring the tightness of the chambers 3 and 4 makes it possible to reduce the risk of contamination of the plant material and to maintain the tightness of the entire culture vessel 2, while reducing its manufacturing cost.
  • the container 2 also comprises transfer means 5 for the temporary transfer of at least part of the nutrient liquid L, preferably all of the liquid L, from said chamber of nutrient liquid 4 to the culture chamber 3.
  • the transfer means 5 for the temporary transfer of at least part of the nutrient liquid L comprises at least one fluid passage duct connecting the nutrient liquid chamber 4 and culture chamber 3.
  • said at least one fluid passage duct extends, on the one hand, vertically and, on the other hand, inside the chamber of nutrient liquid 4 (in particular towards the bottom of this nutrient liquid chamber 4 and / or up to the proximity of this bottom) as well as inside the culture chamber 3, more particularly inside the inner part 31.
  • Said passage duct has one end positioned near the bottom of the nutrient liquid chamber 4, said end is intended to immerse inside the nutrient liquid L, at least in the rest phase.
  • said container 2 also comprises a removable cover 6.
  • Said removable cover 6 allows to close said culture chamber 3, more specifically, said removable cover 6 allows close the entirety of said one-piece outer part 21 that said container 2 comprises.
  • said removable cover 6 makes it possible to hermetically close said one-piece outer part 21 divided into a first part defining the nutrient liquid chamber 4 and into a second part defining said culture chamber 3.
  • said removable cover 6 makes it possible to close hermetically and simultaneously, both said nutrient liquid chamber 4 and said culture chamber 3.
  • sealing means 7, for example of the seal type, interposed between said cover 6 and said culture chamber 3 have the role of ensuring the hermetic closure by the cover of the one-piece outer part 21 which has, at the level of the culture chamber 3, an opening.
  • the cover 6 is manoeuvrable in rotation on the peripheral part of the opening of the culture chamber 3.
  • the culture chamber 3 has, on the periphery of its opening, flanges 62 defining between them peripheral openings 63.
  • Said cover 6 is provided, at the periphery, preferably regularly distributed, several re-entrant hooking fins 61 intended to be housed by a vertical translational movement through the peripheral openings 63 of the culture chamber 3, then by a clockwise rotation movement, below one of the flanges 62.
  • each of the fins 61 of the cover 6 is positioned below one of the corresponding flanges 62 of the opening of the culture chamber 3, so as to crush the seal present on the periphery of the culture chamber 3 and hermetically close said one-piece outer part 21 that said container 2 comprises.
  • each of the fins 61 of the cover 6 is dissociated from its corresponding flange 62 and is positioned through the peripheral openings 63 of the culture chamber 3.
  • said container 2 further comprises communication means 8 at its cover 6.
  • Said communication means 8 are intended to ensure communication between the internal volume of the culture chamber 3, and the outside of culture vessel 2.
  • said communication means 8 consist of at least one vertical duct that comprises said cover 6.
  • said communication means 8 consist of two vertical ducts which said removable cover 6 comprises.
  • the first vertical duct allows the suction of the internal gas flow, it allows within the device, in particular within the container 2, a return to atmospheric pressure.
  • the suction of the internal gas flow contained in said container 2 comprising said one-piece outer part 21 allows, if necessary, a more rapid descent of the nutrient liquid from the culture chamber 3 towards the nutrient liquid chamber 4.
  • the second vertical duct can for example allow the injection of a gas, in particular C02.
  • said culture device 1 comprises vacuum means 9, for example as shown in Figure 2.
  • Said vacuum means 9 have the role of reducing the pressure in the culture chamber 3 so that it becomes lower than the pressure contained in the nutrient liquid chamber 4.
  • said vacuum means 9 comprise at least one suction means 91 of gas, making it possible to reduce the pressure of the internal volume of the culture chamber 3.
  • Said suction means 91 being at the origin of the phenomenon of depression applied within said culture chamber 3.
  • said suction means 91 is constituted by a vacuum pump.
  • said vacuum means 9 comprise, in addition, at least one buffer tank 92 visible in Figure 2.
  • Said buffer tank 92 is interposed between said at least one suction means 91 and said at least one culture container 2. More specifically, said buffer tank 92 is interposed between said vacuum pump and said at least one container 2.
  • said buffer tank 92 is positioned:
  • Said buffer tank 92 can serve as a vacuum lock, so as not to directly apply a vacuum in the culture chamber 3 of the container 2.
  • the presence of the buffer tank 92 has the advantage of allowing the creation of a uniform vacuum before the connection with said at least one suction means 91 of each of said at least one receptacle 2 of the device 1.
  • the presence of a buffer tank 92 makes it possible to work with a suction means 91, in particular a vacuum pump, which has a reduced suction flow capacity compared to the minimum flow rate necessary for the operation of the device 1 not having a buffer tank 92.
  • the presence of a buffer tank 92 makes it possible to reduce the overall price of device 1 by integrating a less expensive vacuum pump with low flow capacity.
  • said vacuum means 9 comprise, again, at least one valve 93 interposed between said at least one suction means 91 and said at least one buffer tank 92.
  • said valve 93 consists of a manual valve or else of a solenoid valve, in particular a solenoid valve having an exhaust allowing return to atmospheric pressure.
  • Said valve 93 makes it possible to control the application of a pressure vacuum within said buffer tank 92, again with the aim of obtaining a vacuum within the buffer tank 92.
  • said device 1 also comprises connection means 10 for connecting the vacuum means 9 to said communication means 8 of said at least one culture vessel 2.
  • connection means 10 consist of pipes, conduits for the transfer of gas, in particular air.
  • the vacuum means 9 will allow the application of a pressure vacuum within the culture chamber 3.
  • the operation of the vacuum means 9 will allow aspiration of the gases contained in the culture chamber 3 of the container 2.
  • the gases are sucked using said communication means 8 which each container 2 comprises.
  • said device 1 comprises at least one valve 11 between said vacuum means and said at least one container 2, so as to control the application of a pressure vacuum in the culture chamber 3.
  • said communication means 8 consist of a conduit 81 which passes through the removable cover 6. Said conduit 81 makes it possible to connect the outside of the container 2, that is to say the connection means 10 with the internal volume of the culture chamber 3. Said communication means 8, that is to say the duct 81 allows the passage of gas between the internal volume of the culture chamber 3 and the exterior of the container 2. It is through the means of communication 8 and its duct 81 that the gases contained in the culture chamber 3 are sucked by the vacuum means 9 in operation.
  • said communication means 8 consist of a tip which is fitted to said cover (6).
  • This end piece has a through orifice which communicates with another orifice which passes through said cover (6).
  • Said nozzle allows the passage of gas between the internal volume of the culture chamber 3 and the outside of the container 2.
  • said device 1 comprises at least two culture vessels 2.
  • the multiplication of the number of receptacles 2 within the device 1 has the advantage of allowing d '' apply the technique of in vitro culture by temporary immersion in a nutrient liquid on an industrial scale.
  • said device 1 comprises six containers 2, or as visible in Figure 5, said device comprises 7 containers 2.
  • said device 1 comprises ten containers 2, the first container 2 being named M1, the second M2 and so on.
  • each container 2 comprises communication means 8 connected to the connection means 10.
  • said connection means 10 are configured to connect said vacuum means 9 to each of said communication means 8 which each of said at least two receptacles 2 comprises.
  • connection means 10 comprise, on the one hand, a ramp 100 and, on the other hand, connection members 101 as shown in Figures 3 to 7 .
  • Said ramp 100 is connected to the vacuum means 9.
  • Said connecting members 101 make it possible to connect the ramp 100 with each of the communication means 8 which each receptacle 2 of the multi-receptacle device 2 comprises.
  • said ramp 100 adopts a linear shape.
  • One of the ends of the linear ramp 100 is connected to the vacuum means 9.
  • one of the ends of the linear ramp 100 is connected for example directly to a suction means 91 of the vacuum pump type.
  • the connecting members 101 are distributed over at least part of the length of the linear ramp 100, preferably over its entire length.
  • said receptacles 2 are distributed on either side of the linear ramp 100.
  • said containers 2, called M1 to M10 are advantageously spaced regularly over the entire length of the linear ramp 100.
  • said receptacles 2 of the multi-receptacle device 1 2 are spaced apart and distributed regularly over the entire length of the linear ramp 100.
  • said ramp 100 adopts a loop shape, that is to say it takes any shape which is a closed shape, for example round, ovoid, rectangle type.
  • said connecting members 101 are distributed over at least part of the length of the loop.
  • said connecting members 101 are spaced regularly on the ramp 100 in a loop. More preferably, said connecting members 101 are distributed over the entire loop.
  • connection means 10 further comprises at least one connection duct 102.
  • Said connection duct 102 connects the ramp 100 in vacuum means loop 9.
  • said device 1 having a ramp 100 in a loop comprises a plurality of connecting conduits 102 which extend from the ramp 100 in the form of a loop and which converge in a point. This point is connected to the vacuum means 9.
  • said point is at the center of the ramp 100 in the form of a loop.
  • This has the effect of balancing the flow rates of the flows passing through the various conduits, so as to synchronize the movement of the nutrient liquid L of each of the culture vessels 2 connected to one another to the same ramp 100.
  • the operation of the different vessels 2 of the same device 1 is synchronized and allows use on an industrial scale.
  • said at least one culture vessel 2 comprises a vent 12.
  • the vent 12 is, of a on the one hand, present on the wall of the nutrient liquid chamber 4 and, on the other hand, positioned above the maximum level that the nutrient liquid L that the chamber includes nutrient liquid 4.
  • the vent 12 provides communication between the internal volume of the nutrient liquid chamber 4 and the exterior of the culture vessel 2.
  • said communication means 8 and said vent 12 comprise means for sterilizing the flows, for example of the sterilizing filter type.
  • the device 1 comprises control means making it possible to control the opening and closing of the valves 93 and / or 11, and to operate said vacuum means 9 or not. , in particular the operation of the suction means 91.
  • said vacuum means 9 of the device 1 make it possible to apply a vacuum to the culture chamber 3. Indeed, the vacuum means 9 will suck the air flow from the culture chamber 3, so that the pressure of the culture chamber 3 is lower than that of the nutrient liquid chamber 4. Following the difference in pressure between the culture chamber 3 and the nutrient liquid chamber 4, while respecting the mechanics of the fluids, the culture chamber 3 will seek to rebalance the pressures of its internal volume with respect to the pressures of the nutrient liquid chamber 4. The culture chamber seeks to make up for its loss of volume of air sucked in by sucking the volume contained in the nutrient liquid chamber 4. Thus, following this phenomenon and in order to rebalance the fluids present, there will be a rise in nutrient liquid L within the culture chamber 3.
  • the application of a vacuum within the culture chamber 3 allows the transfer of the nutrient liquid L from the nutrient liquid chamber 4 to the culture chamber 3.
  • the ace stops piration there will be a new pressure imbalance between the culture chamber 3 and the nutrient liquid chamber 4 which will generate a descent of the nutrient liquid L from the culture chamber 3 to the nutrient liquid chamber 4.
  • the operation of the vacuum means 9 makes it possible to lower the pressure of the culture chamber 3 below atmospheric pressure , while the nutrient liquid chamber 4 remains at atmospheric pressure.
  • the vacuum means 9 the nutrient liquid L will rise in the culture chamber 3.
  • the medium descends through gravity towards the nutrient liquid chamber 4.
  • the pressure values contained in the culture chamber 3 and the nutrient liquid chamber 4 never exceed the value of the atmospheric pressure, thus the return to the pressure equilibrium. between the internal volumes of the two chambers 3 and 4 is not sudden in the form of an overpressure.
  • the device 1 makes it possible to carry out an in vitro culture by temporary immersion in a nutrient liquid (L) which avoids, during the transfer of nutrient liquid from the nutrient liquid chamber 4 to the culture chamber 3 and vice versa, the formation of localized leaks resulting from a deformation of the internal volume of the container under the effect of the application of an overpressure in its internal volume.
  • a nutrient liquid L
  • the application of an internal vacuum and a return to atmospheric pressure within the culture chamber 3 prevents the phenomenon of deformation of the container 2, therefore prevents leaks and contamination of the plant material M.
  • the application of an internal vacuum and a return to atmospheric pressure within the culture chamber 3 prevents the phenomenon of deformation of the removable cover 2 hermetically closing said one-piece outer part 21 which said container 2 comprises.
  • the application of a vacuum in the culture chamber 3 of the container 2 by the vacuum means 9 of the culture device 1, allows the removable cover 6 to be pressed against the one-piece outer part 21 that said container 2 comprises with a greater pressure than that existing at atmospheric pressure.
  • the vacuum suction strengthens the tightness of the container 2 generated by the positioning of the removable cover 6 against the one-piece part 21.
  • the present invention also relates to a method of in vitro culture (1) of plant material (M) by temporary immersion in a nutrient liquid, within at least one culture vessel (2) comprising: a / a chamber culture (3), which has an internal volume, and which is intended to contain the plant material (M) to be cultivated, b / a nutrient liquid chamber (4) which is intended to contain the nutrient liquid (L), c / transfer means (5) for the temporary transfer of at least part of the nutrient liquid (M) by temporary immersion in a nutrient liquid, within at least one culture vessel (2) comprising: a / a chamber culture (3), which has an internal volume, and which is intended to contain the plant material (M) to be cultivated, b / a nutrient liquid chamber (4) which is intended to contain the nutrient liquid (L), c / transfer means (5) for the temporary transfer of at least part of the nutrient liquid (M) by temporary immersion in a nutrient liquid, within at least one culture vessel (2) comprising:
  • the gas suction step to create a vacuum in said culture chamber 3 has the advantage of increasing the pressure and the support that the cover may have on said chamber and therefore of strengthening the tightness of the container. 2.
  • the constraints of leakage or overpressure limit no longer exist with the in vitro culture method by temporary immersion of the invention.
  • said method of the invention is implemented using the device 1 of the invention.
  • the present invention has the advantage of overcoming the constraints of limiting the applicable overpressure, leaks and contamination of plants found with the in vitro culture vessel of plant material by temporary immersion in a nutrient medium disclosed in document WO2012 / 146872.

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
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  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention relates to a device for in vitro culture (1) of a plant material (M) by temporary immersion in a nutritive liquid (L), this device (1) comprising: at least one culture container (2) each comprising: •a. a culture chamber (3), which has an internal volume, and which is intended to contain the plant material (M) to be cultured; •b. a nutritive liquid chamber (4) which is intended to contain the nutritive liquid (L); •c. transfer means (5) for the temporary transfer of at least one portion of the nutritive liquid (L) from said nutritive liquid chamber (4) to the culture chamber (3); •d. a removable lid (6) for closing said culture chamber (3) and also sealing means (7) inserted between this lid (6) and this culture chamber (3); •e. communication means (8), comprised by the removable lid (6), and which are intended to ensure communication between the internal volume of the culture chamber (3) and the exterior of the culture container (2). The culture device (1) further comprises both pressure-reduction means (9) for establishing, in the culture chamber (3), a pressure which is lower than the pressure in the nutritive liquid chamber (4), and connecting means (10) for connecting the pressure-reduction means (9) to said communication means (8) of said at least one culture container (2). The invention also relates to a method for in vitro culture (1) of plant material (M) by temporary immersion in a nutritive liquid.

Description

Description Description
Titre de l'invention : Dispositif de culture végétale in vitro par immersion temporaire dans un liquide nutritif Title of the invention: Device for plant culture in vitro by temporary immersion in a nutrient liquid
[0001] [La présente invention entre dans le domaine de la culture in vitro de matériel végétal, par immersion temporaire, en condition stérile, notamment destiné aux techniques de micro propagation par organogénèse somatique et embryogénèse. [0001] The present invention falls within the field of in vitro culture of plant material, by temporary immersion, in sterile condition, in particular intended for micro-propagation techniques by somatic organogenesis and embryogenesis.
[0002] Elle concerne plus particulièrement un dispositif de culture végétale in vitro par immersion temporaire dans un liquide nutritif et son procédé de mise en oeuvre. [0002] It relates more particularly to a device for plant culture in vitro by temporary immersion in a nutrient liquid and its method of implementation.
[0003] Un tel dispositif trouvera une application particulière dans les domaines de l'horticulture et de l'agriculture, où la multiplication des végétaux par culture in vitro en condition stérile est une pratique courante. [0003] Such a device will find particular application in the fields of horticulture and agriculture, where the multiplication of plants by in vitro culture in sterile conditions is common practice.
[0004] La technique de culture végétale in vitro par immersion temporaire consiste à immerger temporairement dans un milieu nutritif liquide, quelques minutes par jour, un matériel végétal en culture. L'immersion peut être totale ou partielle. Pour permettre la croissance du matériel végétal, on alterne, une phase active d'immersion dans un milieu nutritif liquide, avec, une phase de repos où le matériel végétal n'est pas immergé dans le milieu nutritif liquide. Pendant la phase de repos, le matériel végétal en culture est émergé et sans mouvement. Toutefois, à cause de la phase d'immersion précédente, un film de milieu nutritif est retenu sur le matériel végétal. Ledit film de milieu nutritif maintien humidifié et alimenté en nutriments, nécessaires à sa croissance, ledit matériel végétal. The technique of plant culture in vitro by temporary immersion consists in temporarily immersing in a liquid nutrient medium, a few minutes per day, a plant material in culture. Immersion can be total or partial. To allow the growth of the plant material, an active phase of immersion in a liquid nutrient medium is alternated with a rest phase where the plant material is not immersed in the liquid nutrient medium. During the resting phase, the plant material in culture is emerged and without movement. However, because of the previous immersion phase, a film of nutrient medium is retained on the plant material. Said film of nutrient medium keeping moistened and supplied with nutrients, necessary for its growth, said plant material.
[0005] La culture végétale in vitro par immersion temporaire est un procédé qui permet d'éviter les problèmes d'asphyxie de la culture et/ou de force de friction que l'on retrouve avec des techniques de culture végétale où le matériel végétal est en permanence immergé et sous agitation. De plus, la culture végétale in vitro par immersion temporaire permet d'éviter également l'accumulation des coûts de culture liés à la multiplication des manipulations que demande par exemple la culture in vitro en milieu solide. [0005] Plant culture in vitro by temporary immersion is a process which makes it possible to avoid the problems of asphyxiation of the culture and / or of the frictional force that are found with plant culture techniques where the plant material is permanently submerged and under agitation. In addition, in vitro plant culture by temporary immersion also makes it possible to avoid the accumulation of culture costs linked to the multiplication of manipulations which, for example, in vitro culture in solid medium requires.
[0006] De manière connue, pour la mise en oeuvre de la technique de culture végétale in vitro par immersion temporaire dans un liquide nutritif, on utilise un récipient spécifique et adapté tel que celui divulgué dans le document WO2012/146872. In known manner, for the implementation of the technique of plant culture in vitro by temporary immersion in a nutrient liquid, a specific and suitable container such as that disclosed in document WO2012 / 146872 is used.
[0007] Plus spécifiquement, le document WO 2012/146872 divulgue un récipient pour la culture in vitro de matériel végétal par immersion temporaire. Ce récipient comprend deux chambres superposées, refermées en partie supérieure par un couvercle. [0007] More specifically, document WO 2012/146872 discloses a container for the in vitro culture of plant material by temporary immersion. This container comprises two superimposed chambers, closed in the upper part by a cover.
[0008] La chambre supérieure est destinée à contenir le matériel végétal à cultiver. La chambre inferieure est destinée à contenir le liquide nutritif. [0009] Ce récipient comporte également des moyens pour le transfert temporaire d'au moins une partie du liquide nutritif depuis la chambre inférieure jusqu'à la chambre supérieure. The upper chamber is intended to contain the plant material to be cultivated. The lower chamber is intended to contain the nutrient liquid. This container also comprises means for the temporary transfer of at least part of the nutrient liquid from the lower chamber to the upper chamber.
[0010] Plus précisément, le récipient consiste en une pièce extérieure monobloc constituée d'une partie supérieure de grand diamètre formant la chambre supérieure et d'une partie inférieure, de diamètre inférieur à la partie supérieure, formant la chambre inférieure. More specifically, the container consists of a one-piece outer part consisting of an upper part of large diameter forming the upper chamber and a lower part, of smaller diameter than the upper part, forming the lower chamber.
[0011] Ce récipient comporte également, logé dans la chambre supérieure, un panier de culture contenant le matériel végétal. This container also comprises, housed in the upper chamber, a culture basket containing the plant material.
[0012] Ce panier présente un orifice central traversant qui permet de recevoir une partie des moyens de transfert du liquide nutritif entre les deux chambres. This basket has a central through orifice which allows to receive part of the nutrient liquid transfer means between the two chambers.
[0013] Le panier comporte un organe de filtration qui consiste en un tamis qui permet la répartition, de la chambre inférieure vers la chambre supérieure, du liquide nutritif et du gaz de surpression. [0014] Les moyens de transfert comportent un conduit de raccordement central, assurant la liaison hydrique entre les deux chambres, et, un conduit latéral pour l'introduction de gaz au niveau de la chambre inférieure. The basket comprises a filtration member which consists of a sieve which allows the distribution, from the lower chamber to the upper chamber, of the nutrient liquid and the overpressure gas. The transfer means comprise a central connection duct, ensuring the water connection between the two chambers, and a lateral duct for the introduction of gas at the level of the lower chamber.
[0015] En outre, la partie supérieure de ce récipient est fermée par un couvercle amovible. Ce couvercle est muni d'ailettes d'accrochages rentrantes complémentaires à des nervures présentes en bordure périphérique supérieure de la chambre supérieure. L'étanchéité du couvercle, assemblé sur la bordure de la chambre supérieure, est assurée par son verrouillage par vissage sur la bordure périphérique de la chambre supérieure, en combinaison avec un joint d'étanchéité. Un mouvement de rotation du couvercle sur le récipient permet de fermer hermétiquement la pièce extérieure monobloc par écrasement du joint d'étanchéité. En position verrouillée, le couvercle exerce donc une force d'appui dirigée vers la chambre inférieure et qui écrase le joint. In addition, the upper part of this container is closed by a removable cover. This cover is provided with re-entrant hooking fins complementary to the ribs present at the upper peripheral edge of the upper chamber. The tightness of the cover, assembled on the edge of the upper chamber, is ensured by its locking by screwing on the peripheral edge of the upper chamber, in combination with a seal. A rotational movement of the cover on the container makes it possible to hermetically close the one-piece outer part by crushing the seal. In the locked position, the cover therefore exerts a support force directed towards the lower chamber and which crushes the seal.
[0016] De plus, ledit couvercle comporte un évent recouvert d'un filtre stérilisant pour garantir la stérilité du volume intérieur du récipient une fois le couvercle verrouillé. L'évent permet le passage de gaz entre l'intérieur et l'extérieur du récipient. [0016] In addition, said cover comprises a vent covered with a sterilizing filter to ensure the sterility of the interior volume of the container once the cover is locked. The vent allows gas to pass between the interior and exterior of the container.
[0017] En phase active d'immersion, la technique de culture in vitro par immersion temporaire, utilisant ce récipient, consiste à appliquer une surpression dans la chambre inférieure, contenant le liquide nutritif. La surpression est appliquée en introduisant un gaz, stérilisé via un filtre stérilisant, au travers du conduit latéral. L'introduction du gaz est contrôlée et réalisée à l'aide d'une pompe délivrant de l'air comprimé. In the active phase of immersion, the technique of in vitro culture by temporary immersion, using this container, consists in applying an overpressure in the lower chamber, containing the nutrient liquid. The overpressure is applied by introducing a gas, sterilized through a sterilizing filter, through the side duct. The introduction of gas is controlled and carried out using a pump delivering compressed air.
[0018] Le fonctionnement de la pompe rythme la durée des immersions du matériel végétal. [0018] The operation of the pump regulates the duration of the immersions of the plant material.
[0019] En effet, la surpression générée pousse le milieu nutritif depuis la chambre inférieure jusqu'à la chambre supérieure. En d'autres termes, sous l'effet de la surpression, le liquide nutritif de la chambre inférieure remonte au travers du conduit de raccordement, puis au travers de l'organe de filtration, pour se retrouver dans la chambre supérieure. [0020] Ainsi, le maintien de l'alimentation de la surpression, au niveau de la chambre inférieure, permet de maintenir le milieu nutritif dans la chambre supérieure pour une immersion du matériel végétal. Ceci constitue en la phase active d'immersion. Pendant cette phase active d'immersion, lors de la remonté du liquide nutritif, l'évent du couvercle permet d'évacuer le gaz contenu dans la chambre supérieure et qui est poussé par le liquide nutritif. In fact, the overpressure generated pushes the nutrient medium from the lower chamber to the upper chamber. In other words, under the effect of the overpressure, the nutrient liquid from the lower chamber rises through the connection duct, then through the filtration member, to end up in the upper chamber. [0020] Thus, maintaining the supply of the overpressure, at the level of the lower chamber, makes it possible to maintain the nutrient medium in the upper chamber for immersion of the plant material. This constitutes the active phase of immersion. During this active immersion phase, when the nutrient liquid is raised, the vent in the cover allows the gas contained in the upper chamber to be evacuated and which is pushed by the nutrient liquid.
[0021] Puis, on retourne vers une phase de repos, en stoppant l'alimentation de la surpression. L'arrêt de l'entrée de gaz, donc de la surpression, entraîne le retour par gravité du milieu nutritif de la chambre supérieure vers la chambre inférieure. L'évent du couvercle permet également, en phase de repos, d'évacuer la surpression continuant d'être alimentée par la descente du milieu nutritif [0022] Cependant en pratique, dans une configuration à la verticale, où le récipient est une pièce extérieure monobloc se composant d'une chambre de culture superposée à une chambre de liquide nutritif, la demanderesse a constaté, de manière désavantageuse, que le raccordement à un réseau d'alimentation d'air comprimé, pour générer le phénomène de surpression, provoque des fuites localisées au niveau du couvercle. Ces fuites au niveau du couvercle interviennent dès la mise en surpression de l'appareil, ainsi le couvercle recouvrant la partie supérieure du récipient ne permet plus de fermer hermétiquement la pièce extérieure monobloc constituant ledit récipient. Then, we return to a rest phase, stopping the supply of the overpressure. Stopping the gas inlet, and therefore the overpressure, causes the nutrient medium to return by gravity from the upper chamber to the lower chamber. The vent in the lid also makes it possible, in the rest phase, to evacuate the overpressure continuing to be supplied by the descent of the nutrient medium. However, in practice, in a vertical configuration, where the container is an external part. monobloc consisting of a culture chamber superimposed on a nutrient liquid chamber, the applicant has observed, disadvantageously, that the connection to a compressed air supply network, to generate the overpressure phenomenon, causes leaks located at the level of the cover. These leaks at the level of the cover occur as soon as the device is put under overpressure, thus the cover covering the upper part of the container no longer makes it possible to hermetically close the one-piece outer part constituting said container.
[0023] Désavantageusement, avec le récipient du document WO 2012/146872, la surpression génère un risque de soulèvement du couvercle, c'est-à-dire une entrée ou sortie d'air à proximité du joint d'étanchéité qui ne peut plus assurer son rôle. Le mode de fonctionnement par surpression du récipient du document WO 2012/146872 entraîne le fait que la chambre supérieure est soumise à une pression permanente due à l'évacuation très lente du flux d'air par l'évent du couvercle. Ce phénomène de surpression de la chambre supérieure tend à contraindre et déformer le récipient jusqu'à créer des fuites localisées. Disadvantageously, with the container of document WO 2012/146872, the overpressure generates a risk of lifting of the cover, that is to say an air inlet or outlet near the seal which can no longer fulfill its role. The mode of operation by overpressure of the container of document WO 2012/146872 results in the upper chamber being subjected to a permanent pressure due to the very slow evacuation of the air flow through the vent of the cover. This phenomenon of overpressure in the upper chamber tends to constrain and deform the container to the point of creating localized leaks.
[0024] Or, ces fuites localisées peuvent être à l'origine de la contamination du matériel végétal en croissance et de la perte de l'asepsie de la chambre supérieure de culture. However, these localized leaks can be the cause of the contamination of the growing plant material and the loss of asepsis of the upper culture chamber.
[0025] La présence des filtres stérilisants, au niveau de l'évent du couvercle et du conduit latéral de la chambre inférieure, est insuffisant pour garantir la stérilité de l'environnement autour du matériel végétal contenu dans le récipient divulgué dans le document WO2012/146872. L'utilisation du récipient divulgué possède donc ses limites en termes de surpression à appliquer, afin de conserver une pression admissible au sein de la chambre supérieure pour éviter qu'elle ne se déforme localement. Or dans un protocole de culture in vitro par immersion, où le milieu nutritif doit remonter vite vers la chambre de culture, il peut s'avérer nécessaire de dépasser cette limite de pression en risquant toutefois de déformer la chambre de culture, notamment son couvercle, et de créer des fuites donc des contaminations du matériel végétal. Or pour un usage industriel et une conservation de la stérilité du matériel végétal, ceci est impensable. [0026] De plus, le récipient, divulgué dans le document WO2012/146872, nécessite une pompe à air comprimé reliée à chaque chambre inférieure de chaque récipient. En effet, le document WO2012/146872 envisage autant de pompe que de récipient, ce qui génère des coûts élevés et une gestion complexe dans le cas d'une culture à grande échelle nécessitant plusieurs récipients. The presence of sterilizing filters, at the level of the vent of the cover and of the lateral duct of the lower chamber, is insufficient to guarantee the sterility of the environment around the plant material contained in the container disclosed in document WO2012 / 146872. The use of the disclosed container therefore has its limits in terms of overpressure to be applied, in order to maintain an admissible pressure within the upper chamber to prevent it from being deformed locally. However, in an in vitro culture protocol by immersion, where the nutrient medium must quickly rise to the culture chamber, it may prove necessary to exceed this pressure limit while at the risk of deforming the culture chamber, in particular its cover, and create leaks and therefore contamination of the plant material. However, for industrial use and preservation of the sterility of plant material, this is unthinkable. In addition, the container, disclosed in document WO2012 / 146872, requires a compressed air pump connected to each lower chamber of each container. Indeed, document WO2012 / 146872 envisages as many pumps as there are receptacles, which generates high costs and complex management in the case of large-scale cultivation requiring several receptacles.
[0027] C'est pourquoi et compte tenue de ces observations, il convient de trouver une solution alternative à celle mise en place avec le récipient existant. Le but est de pouvoir réaliser la technique de culture végétale in vitro par immersion temporaire dans un milieu liquide nutritif, à échelle industrielle, sans risque de contamination du matériel végétal, qui soit de faibles coûts de production et peu énergivore. Il convient d'optimiser la solution prévue par le document WO2012/146872 pour la technique de culture végétale in vitro par immersion temporaire dans un milieu liquide nutritif en la rendant plus fiable en limitant notamment les fuites. This is why and taking into account these observations, it is necessary to find an alternative solution to that implemented with the existing container. The aim is to be able to carry out the technique of plant culture in vitro by temporary immersion in a nutritious liquid medium, on an industrial scale, without risk of contamination of the plant material, which is low production costs and uses little energy. The solution provided for in document WO2012 / 146872 for the technique of plant culture in vitro by temporary immersion in a liquid nutrient medium should be optimized by making it more reliable by in particular limiting leaks.
[0028] La présente invention a pour but de pallier les inconvénients de l'état de la technique. The object of the present invention is to overcome the drawbacks of the state of the art.
[0029] Un premier aspect de l'invention a trait à un dispositif de culture in vitro d'un matériel végétal par immersion temporaire dans un liquide nutritif. Ce dispositif comprend: au moins un récipient de culture , chacun comportant : a/ une chambre de culture , qui présente un volume interne, et qui est destinée à contenir le matériel végétal à cultiver, b/ une chambre de liquide nutritif qui est destinée à contenir le liquide nutritif , c/ des moyens de transfert pour le transfert temporaire d'au moins une partie du liquide nutritif depuis ladite chambre de liquide nutritif vers la chambre de culture , d/ un couvercle amovible pour refermer ladite chambre de culture ainsi que des moyens d'étanchéité interposés entre ce couvercle et cette chambre de culture; e/ des moyens de communication, que comporte le couvercle amovible, et qui sont destinés à assurer une communication entre le volume interne de la chambre de culture et l'extérieur du récipient de culture. A first aspect of the invention relates to a device for in vitro culture of plant material by temporary immersion in a nutrient liquid. This device comprises: at least one culture vessel, each comprising: a / a culture chamber, which has an internal volume, and which is intended to contain the plant material to be cultivated, b / a nutrient liquid chamber which is intended to contain the nutrient liquid, c / transfer means for the temporary transfer of at least part of the nutrient liquid from said nutrient liquid chamber to the culture chamber, d / a removable cover to close said culture chamber as well as sealing means interposed between this cover and this culture chamber; e / means of communication, which the removable cover comprises, and which are intended to ensure communication between the internal volume of the culture chamber and the outside of the culture vessel.
Selon une spécificité de l'invention, ledit dispositif de culture comporte, encore, d'une part, des moyens de dépression pour établir, dans la chambre de culture, une pression qui est inférieure à la pression dans la chambre de liquide nutritif et, d'autre part, des moyens de raccordement pour raccorder les moyens de dépression auxdits moyens de communication dudit au moins un récipient de culture. According to a specific feature of the invention, said culture device also comprises, on the one hand, vacuum means for establishing, in the culture chamber, a pressure which is lower than the pressure in the nutrient liquid chamber and, on the other hand, connection means for connecting the vacuum means to said communication means of said at least one culture vessel.
Selon un mode préféré de réalisation du dispositif de l'invention : lesdits moyens de dépression comportent au moins un moyen d'aspiration , de préférence chaque moyen d'aspiration est constitué par une pompe à vide, lesdits moyens de dépression comportent, en outre, au moins une cuve tampon interposée entre ledit au moins un moyen d'aspiration et ledit au moins un récipient de culture, lesdits moyens de dépression comportent, encore, au moins une vanne interposée entre ledit au moins un moyen d'aspiration et ladite au moins une cuve tampon. According to a preferred embodiment of the device of the invention: said vacuum means comprise at least one suction means, preferably each suction means is constituted by a vacuum pump, said vacuum means further comprise at least one buffer tank interposed between said at least one suction means and said at least one culture vessel, said vacuum means also comprise at least one valve interposed between said at least a suction means and said at least one buffer tank.
De préférence, ledit dispositif de l'invention comporte au moins une vanne interposée entre lesdits moyens de dépression et ledit au moins un récipient. Preferably, said device of the invention comprises at least one valve interposed between said vacuum means and said at least one container.
Selon un autre mode de réalisation du dispositif de l'invention, lesdits moyens de communication consistent en un conduit qui traverse le couvercle amovible. According to another embodiment of the device of the invention, said communication means consist of a duct which passes through the removable cover.
Selon un autre mode de réalisation préféré du dispositif de l'invention, lesdits moyens de communication consistent en un embout qui équipe ledit couvercle et qui comporte un orifice traversant et communiquant avec un orifice qui traverse ledit couvercle. According to another preferred embodiment of the device of the invention, said communication means consist of an end piece which is fitted to said cover and which comprises an orifice passing through and communicating with an orifice which passes through said cover.
Avantageusement, ledit dispositif comporte au moins deux récipients de culture comportant, chacun, des moyens de communication tandis que les moyens de raccordement comportent, d'une part, une rampe raccordée aux moyens de dépression et, d'autre part, des organes de raccordement pour raccorder ladite rampe aux moyens de communication que comportent chacun des récipients de culture. Advantageously, said device comprises at least two culture vessels each comprising communication means while the connection means comprise, on the one hand, a ramp connected to the vacuum means and, on the other hand, connection members. to connect said ramp to the means of communication that each of the culture vessels comprise.
Selon un premier mode de réalisation du dispositif de l'invention, ladite rampe adopte une forme linéaire et présente une extrémité raccordée aux moyens de dépression, tandis que les organes de raccordement sont répartis sur une partie au moins de la longueur de la rampe. According to a first embodiment of the device of the invention, said ramp adopts a linear shape and has one end connected to the vacuum means, while the connecting members are distributed over at least part of the length of the ramp.
Selon un second mode de réalisation du dispositif de l'invention, ladite rampe adopte une forme de boucle, tandis que les organes de raccordement sont répartis sur au moins une partie de la longueur de la boucle et, d'autre part, les moyens de raccordement comportent en outre au moins un conduit de raccordement pour raccorder ladite rampe aux moyens de dépression. Avantageusement pour ce second mode de réalisation, ledit dispositif comporte une pluralité de conduits de raccordement qui s'étendent à partir de la rampe adoptant une forme de boucle et qui convergent en un point raccordé aux moyens de dépression. According to a second embodiment of the device of the invention, said ramp takes the form of a loop, while the connecting members are distributed over at least part of the length of the loop and, on the other hand, the means of connection further comprise at least one connection duct for connecting said ramp to the vacuum means. Advantageously for this second embodiment, said device comprises a plurality of connecting conduits which extend from the ramp adopting the shape of a loop and which converge at a point connected to the vacuum means.
Selon une autre caractéristique du dispositif de l'invention, ledit au moins un récipient de culture comporte un évent pour assurer la communication entre le volume interne de la chambre de liquide nutritif et l'extérieur d'un tel récipient de culture. According to another characteristic of the device of the invention, said at least one culture vessel comprises a vent to ensure communication between the internal volume of the nutrient liquid chamber and the exterior of such a culture vessel.
[0030] Un deuxième aspect de l'invention a trait à un procédé de culture in vitro de matériel végétal par immersion temporaire dans un liquide nutritif, au sein d'au moins un récipient de culture comportant : f/ une chambre de culture (3), qui présente un volume interne, et qui est destinée à contenir le matériel végétal (M) à cultiver, g/ une chambre de liquide nutritif (4) qui est destinée à contenir le liquide nutritif (L) , h/ des moyens de transfert (5) pour le transfert temporaire d'au moins une partie du liquide nutritifA second aspect of the invention relates to a method for in vitro culture of plant material by temporary immersion in a nutrient liquid, within at least one culture vessel comprising: f / a culture chamber (3 ), which has an internal volume, and which is intended to contain the plant material (M) to be cultivated, g / a nutrient liquid chamber (4) which is intended to contain the nutrient liquid (L), h / transfer means (5) for the temporary transfer of at least part of the nutrient liquid
(L) depuis ladite chambre de liquide nutritif (4) vers la chambre de culture (3) , i/ un couvercle amovible (6) pour refermer ladite chambre de culture (3) ainsi que des moyens d'étanchéité (7) interposés entre ce couvercle (6) et cette chambre de culture (3) ; j/ des moyens de communication (8), que comporte le couvercle amovible (6), et qui sont destinés à assurer une communication entre le volume interne de la chambre de culture (3)et l'extérieur du récipient de culture (2). (L) from said nutrient liquid chamber (4) to the culture chamber (3), i / a removable cover (6) to close said culture chamber (3) as well as sealing means (7) interposed between this cover (6) and this culture chamber (3); j / communication means (8), which the removable cover (6) comprises, and which are intended to ensure communication between the internal volume of the culture chamber (3) and the outside of the culture vessel (2) .
Ledit procédé de culture in vitro de matériel végétal par immersion temporaire dans un liquide nutritif comporte les étapes suivantes : Said method of in vitro culture of plant material by temporary immersion in a nutrient liquid comprises the following steps:
- on cultive ledit matériel végétal M au sein de ladite chambre de culture, - said plant material M is cultivated within said culture chamber,
- on transfère, de manière temporaire, lors de la phase active d'immersion, ledit liquide nutritif de la chambre de liquide nutritif vers la chambre de culture en générant une différence de pression entre lesdites chambres et, et en particulier les étapes suivantes : - Is transferred, temporarily, during the active immersion phase, said nutrient liquid from the nutrient liquid chamber to the culture chamber by generating a pressure difference between said chambers and, and in particular the following steps:
- on aspire les gaz contenus dans ladite chambre de culture jusqu'à ce que la pression de ladite chambre de culture soit inférieure à la pression de ladite chambre de liquide nutritif de sorte à générer ladite différence de pression, entre ladite chambre de culture et ladite chambre de liquide nutritif par dépression de ladite chambre de culture. - The gases contained in said culture chamber are sucked in until the pressure of said culture chamber is lower than the pressure of said nutrient liquid chamber so as to generate said pressure difference, between said culture chamber and said nutrient liquid chamber by depression of said culture chamber.
Selon une autre caractéristique ledit procédé de culture in vitro de matériel végétal par immersion temporaire dans un liquide nutritif est mis en oeuvre par le dispositif de culture in vitro d'un matériel végétal par immersion temporaire dans un liquide nutritif. According to another characteristic, said method of in vitro culture of plant material by temporary immersion in a nutrient liquid is implemented by the device for in vitro culture of a plant material by temporary immersion in a nutrient liquid.
[0031] D'autres caractéristiques et avantages de l'invention ressortiront de la description détaillée qui va suivre des modes de réalisation non limitatifs de l'invention, en référence aux figures annexées, dans lesquelles : Other characteristics and advantages of the invention will emerge from the detailed description which will follow of the non-limiting embodiments of the invention, with reference to the appended figures, in which:
[0032] [Fig.l] la figure 1 représente schématiquement une vue de profil éclaté d'un mode de réalisation d'un récipient de culture du dispositif de culture de l'invention ; [0032] [Fig.l] Figure 1 schematically shows an exploded side view of an embodiment of a culture vessel of the culture device of the invention;
[0033] [Fig.2] la figure 2 représente schématiquement un dispositif de culture de l'invention comprenant le récipient de culture refermer par son couvercle de la figure 1, des moyens de dépression et des moyens de raccordement ; [0033] [Fig.2] Figure 2 schematically shows a culture device of the invention comprising the culture container closed by its cover of Figure 1, vacuum means and connection means;
[0034] [Fig.3] la figure 3 représente schématiquement un dispositif de culture de l'invention multi- récipients selon un premier mode de réalisation, [0034] [Fig.3] Figure 3 schematically shows a culture device of the invention multi-containers according to a first embodiment,
[0035] [Fig.4] la figure 4 représente schématiquement une vue du dessus des raccordements du dispositif de culture de l'invention multi-récipient selon le premier mode de réalisation de la figure 3; [0036] [Fig.5] la figure 5 représente schématiquement un dispositif de culture de l'invention multi- récipients selon un second mode de réalisation ; [0037] [Fig.6] la figure 6 représente schématiquement une vue du dessus des raccordements du dispositif de culture de l'invention multi-récipient selon le second mode de réalisation de la figure 5; [0038] [Fig.7] la figure 7 représente schématiquement une vue du dessus du dispositif de culture de l'invention multi-récipient selon le second mode de réalisation en présence d'une cuve tampon. [0039] En référence à ces dessins, la présente invention concerne un dispositif de culture in vitro 1 d'un matériel végétal M par immersion temporaire dans un liquide nutritif L. [0035] [Fig.4] Figure 4 schematically shows a top view of the connections of the culture device of the invention multi-container according to the first embodiment of Figure 3; [0036] [Fig.5] Figure 5 schematically shows a culture device of the invention multi-containers according to a second embodiment; [0037] [Fig.6] Figure 6 schematically shows a top view of the connections of the culture device of the invention multi-container according to the second embodiment of Figure 5; [0038] [Fig.7] Figure 7 schematically shows a top view of the culture device of the invention multi-container according to the second embodiment in the presence of a buffer tank. With reference to these drawings, the present invention relates to an in vitro culture device 1 of plant material M by temporary immersion in a nutrient liquid L.
[0040] Ledit matériel végétal M à cultiver consiste en tous tissus végétaux cultivés in vitro, tel que des micro-tiges, des micro- boutures, du cal, des embryons somatiques, ou encore du matériel végétal sous forme d'explants ou de cellules. Said plant material M to be cultivated consists of all plant tissues cultivated in vitro, such as micro-stems, micro-cuttings, callus, somatic embryos, or even plant material in the form of explants or cells. .
[0041] Le liquide nutritif L consiste en tous milieux connus permettant la croissance et la multiplication in vitro dudit matériel végétal M, tel que par exemple un milieu liquide contenant les sels minéraux de Murashige et Skoog (1962) ou des formulations dérivées, du sucre, des vitamines ou encore des hormones végétales. The nutrient liquid L consists of all known media allowing the growth and multiplication in vitro of said plant material M, such as for example a liquid medium containing the mineral salts of Murashige and Skoog (1962) or formulations derived from sugar , vitamins or even plant hormones.
[0042] Le dispositif 1 de l'invention comprend au moins un récipient de culture 2. The device 1 of the invention comprises at least one culture vessel 2.
[0043] Ledit récipient 2 comporte une chambre de culture 3, une chambre de liquide nutritif 4, des moyens de transfert 5 du liquide nutritif L d'une chambre à l'autre, un couvercle amovible 6 associé à des moyens d'étanchéités 7 pour sa fermeture. En d'autres termes, ledit récipient comporte un seul couvercle amovible 6 pour assurer sa fermeture et son étanchéité. Said container 2 comprises a culture chamber 3, a nutrient liquid chamber 4, means 5 for transferring the nutrient liquid L from one chamber to another, a removable cover 6 associated with sealing means 7 for its closure. In other words, said container has a single removable cover 6 to ensure its closure and its sealing.
[0044] Ledit récipient 2 comporte également des moyens de communication 8 assurant une communication entre son extérieur et le volume interne de sa chambre de culture 3. Said container 2 also comprises communication means 8 ensuring communication between its exterior and the internal volume of its culture chamber 3.
[0045] Selon l'invention, ledit récipient 2 comporte une pièce extérieure monobloc 21 recouverte par ledit couvercle amovible 6. En conséquence, ladite pièce extérieure monobloc 21 peut être fermée hermétiquement par ledit couvercle amovible 6. Selon la présente invention, ladite pièce extérieure monobloc 21 est divisée en une première et une seconde partie. According to the invention, said container 2 comprises a one-piece outer part 21 covered by said removable cover 6. Consequently, said one-piece outer part 21 can be hermetically closed by said removable cover 6. According to the present invention, said outer part monobloc 21 is divided into a first and a second part.
[0046] Ladite première partie définit la chambre de liquide nutritif 4. Ladite première partie est destinée à contenir, au sein de son volume interne, ledit liquide nutritif L. De préférence, ledit liquide nutritif L est contenu directement au sein du volume interne de la première partie de la pièce extérieur monobloc 21 du récipient 2, c'est-à-dire au sein du volume interne de la chambre de liquide nutritif 4. Said first part defines the nutrient liquid chamber 4. Said first part is intended to contain, within its internal volume, said nutrient liquid L. Preferably, said nutrient liquid L is contained directly within the internal volume of the first part of the one-piece outer part 21 of the container 2, that is to say within the internal volume of the nutrient liquid chamber 4.
[0047] Ladite seconde partie définit la chambre de culture 3. Ladite seconde partie est destinée à contenir, au sein de son volume interne, ledit matériel végétal M. Selon l'invention, ladite seconde partie définissant la chambre de culture 3, internalise dans son volume interne, une pièce intérieure 31 dans laquelle se trouve ledit matériel végétal M. Dans ce cas, le volume interne de la chambre de culture 3, destinée à contenir le matériel végétal M correspond au volume occupé par la pièce intérieure 31 dans laquelle se trouve le matériel végétal M. Ladite pièce intérieure 31 sépare la chambre de liquide nutritif 4 du matériel végétal M de la chambre de culture 3. Said second part defines the culture chamber 3. Said second part is intended to contain, within its internal volume, said plant material M. According to the invention, said second part defining the culture chamber 3, internalized in its internal volume, an interior part 31 in which is located said plant material M. In this case, the internal volume of the culture chamber 3, intended to contain the plant material M corresponds to the volume occupied by the interior part 31 in which is finds the plant material M. Said inner part 31 separates the nutrient liquid chamber 4 of plant material M of culture chamber 3.
[0048] Selon un premier mode de réalisation, la pièce extérieure monobloc 21, que comporte ledit récipient 2, présente un agencement selon un axe horizontal. Selon cet agencement, la première partie et la seconde partie, divisant la pièce extérieure monobloc, sont agencées de manière adjacente l'une de l'autre, selon un axe horizontal. En d'autres termes, la chambre de culture 3 et la chambre de liquide nutritif 4 sont positionnées l'une à côté de l'autre de manière adjacente, et elles constituent la pièce extérieure monobloc 21, que comporte ledit récipient 2, recouvert par ledit couvercle amovible 6. Ainsi, ledit couvercle amovible 6 recouvre à la fois ladite chambre de culture 3 adjacente à ladite chambre de liquide nutritif 4, de sorte à fermer hermétiquement l'intégralité de la pièce extérieure monobloc 21, c'est-à-dire à fermer hermétiquement chacune des deux chambres de culture 3 et de liquide nutritif 4. Selon ce mode particulier de configuration, selon un axe horizontal, au moins une partie de la paroi de la chambre de culture 3 communique avec au moins une partie de la paroi de la chambre 4 de liquide nutritif L, au travers des moyens de transfert 5. En d'autres termes, lesdits moyens de transfert 5 permettent le transfert temporaire d'au moins une partie du liquide nutritif L depuis la chambre 4 de liquide nutritif L vers la chambre de culture 3. [0049] Selon un second mode de réalisation privilégié, ladite première partie est superposée sur ladite seconde partie tel que visible sur la figure 1. According to a first embodiment, the one-piece outer part 21, which said container 2 comprises, has an arrangement along a horizontal axis. According to this arrangement, the first part and the second part, dividing the one-piece outer part, are arranged adjacent to one another, along a horizontal axis. In other words, the culture chamber 3 and the nutrient liquid chamber 4 are positioned adjacent to each other in an adjacent manner, and they constitute the one-piece outer part 21, which the said container 2 comprises, covered by said removable cover 6. Thus, said removable cover 6 covers both said culture chamber 3 adjacent to said nutrient liquid chamber 4, so as to hermetically close the entire one-piece outer part 21, that is to say say to hermetically close each of the two culture chambers 3 and nutrient liquid 4. According to this particular mode of configuration, along a horizontal axis, at least part of the wall of the culture chamber 3 communicates with at least part of the wall of the nutrient liquid chamber 4 L, through the transfer means 5. In other words, said transfer means 5 allow the temporary transfer of at least part of the nutrient liquid L from the liquid chamber 4 nutrient L to the culture chamber 3. According to a second preferred embodiment, said first part is superimposed on said second part as visible in FIG. 1.
[0050] Dans ce second mode de réalisation, le récipient 2 comporte une pièce extérieure monobloc 21 qui se compose de deux parties annulaires superposées, de diamètres différents. La pièce extérieure monobloc 21 se compose d'une première partie supérieure annulaire surmontant la seconde partie inferieure annulaire. La première et la seconde partie annulaire représentant respectivement ladite chambre de culture 3 et ladite chambre de liquide nutritif 4. Ladite chambre de culture 3 ayant un diamètre supérieur à celui de ladite chambre de liquide nutritif L. Lesdites chambres 3 et 4 sont jointes entre elles par une jonction constituant un épaulement annulaire en forme de couronne sur lequel peut reposer ladite pièce intérieure 31. L'avantage d'une configuration à la verticale du récipient 2, sous forme de pièce extérieure monobloc 21 comportant la chambre de culture 3 superposée sur la chambre de liquide nutritif 4, est que le dispositif de culture in vitro 1 est moins volumineux, par rapport à une disposition selon un axe horizontal des chambres de culture 3 et nutritif 4. In this second embodiment, the container 2 comprises a one-piece outer part 21 which consists of two superimposed annular parts, of different diameters. The one-piece outer part 21 consists of a first annular upper part surmounting the second annular lower part. The first and the second annular part respectively representing said culture chamber 3 and said nutrient liquid chamber 4. Said culture chamber 3 having a diameter greater than that of said nutrient liquid chamber L. Said chambers 3 and 4 are joined together. by a junction constituting an annular shoulder in the form of a crown on which the said inner part 31 can rest. The advantage of a vertical configuration of the container 2, in the form of a single-piece outer part 21 comprising the culture chamber 3 superimposed on the nutrient liquid chamber 4, is that the in vitro culture device 1 is less bulky, compared to an arrangement along a horizontal axis of the culture 3 and nutrient 4 chambers.
En outre, cette configuration à la verticale est économique en termes de nombre de pièce nécessaire pour assurer la fermeture hermétique à la fois de la chambre de culture 3 et de la chambre de liquide nutritif 4 du récipient 2. En effet dans la configuration à la verticale du récipient 2, la mise en place d'un seul couvercle pour fermer hermétiquement les deux chambres 3 et 4 est suffisante. En effet, dans cette configuration, ledit couvercle amovible 6 recouvre et peut fermer hermétiquement l'intégralité de la pièce extérieure monobloc 21, de sorte à fermer hermétiquement la chambre de culture 3 et la chambre de liquide nutritif 4. La diminution du nombre des pièces permettant d'assurer l'étanchéité, permet également de réduire drastiquement les zones possibles de sortie ou d'entrée des flux au sein des chambres 3 et 4 et du récipient de culture 2. Ainsi, par rapport à l'état de la technique, la diminution du nombre de couvercle nécessaire, assurant l'étanchéité des chambres 3 et 4, permet de diminuer le risque de contamination du matériel végétal et de conserver l'étanchéité de tout le récipient de culture 2, tout en diminuant son coût de fabrication. In addition, this vertical configuration is economical in terms of the number of parts necessary to ensure the hermetic closure of both the culture chamber 3 and the nutrient liquid chamber 4 of the container 2. Indeed in the configuration at the bottom. vertical of the container 2, the establishment of a single cover to hermetically close the two chambers 3 and 4 is sufficient. In fact, in this configuration, said removable cover 6 covers and can hermetically close the entire one-piece outer part 21, so as to hermetically close the chamber. culture 3 and the nutrient liquid chamber 4. The reduction in the number of parts making it possible to ensure the sealing, also makes it possible to drastically reduce the possible zones of exit or entry of the flows within the chambers 3 and 4 and of the container culture 2. Thus, compared to the state of the art, the reduction in the number of covers necessary, ensuring the tightness of the chambers 3 and 4, makes it possible to reduce the risk of contamination of the plant material and to maintain the tightness of the entire culture vessel 2, while reducing its manufacturing cost.
[0051] Selon une autre caractéristique de l'invention, le récipient 2 comporte également des moyens de transfert 5 pour le transfert temporaire d'au moins une partie du liquide nutritif L, de préférence l'intégralité du liquide L, depuis ladite chambre de liquide nutritif 4 vers la chambre de culture 3. [0052] Selon un mode de réalisation préférentiel, les moyens de transfert 5 pour le transfert temporaire d'au moins une partie du liquide nutritif L comporte au moins un conduit de passage des fluides raccordant la chambre de liquide nutritif 4 et de la chambre de culture 3. According to another characteristic of the invention, the container 2 also comprises transfer means 5 for the temporary transfer of at least part of the nutrient liquid L, preferably all of the liquid L, from said chamber of nutrient liquid 4 to the culture chamber 3. According to a preferred embodiment, the transfer means 5 for the temporary transfer of at least part of the nutrient liquid L comprises at least one fluid passage duct connecting the nutrient liquid chamber 4 and culture chamber 3.
[0053] Selon le second mode de réalisation visible sur la figure 1, ledit au moins un conduit de passage des fluides s'étend, d'une part, de manière verticale et, d'autre part, à l'intérieur de la chambre de liquide nutritif 4 (notamment en direction du fond de cette chambre de liquide nutritif 4 et/ou jusqu'à proximité de ce fond) ainsi qu'à l'intérieur de la chambre de culture 3, plus particulièrement à l'intérieur de la pièce intérieur 31. Ledit conduit de passage présente une extrémité positionnée à proximité du fond de la chambre de liquide nutritif 4, ladite extrémité est destinée à plonger à l'intérieur du liquide nutritif L, au moins en phase de repos. According to the second embodiment visible in Figure 1, said at least one fluid passage duct extends, on the one hand, vertically and, on the other hand, inside the chamber of nutrient liquid 4 (in particular towards the bottom of this nutrient liquid chamber 4 and / or up to the proximity of this bottom) as well as inside the culture chamber 3, more particularly inside the inner part 31. Said passage duct has one end positioned near the bottom of the nutrient liquid chamber 4, said end is intended to immerse inside the nutrient liquid L, at least in the rest phase.
[0054] De plus, tel que visible sur la figure 1 et la figure 2, ledit récipient 2 comporte également un couvercle amovible 6. Ledit couvercle amovible 6 permet de refermer ladite chambre de culture 3, plus spécifiquement, ledit couvercle amovible 6 permet de refermer l'intégralité de ladite pièce extérieure monobloc 21 que comporte ledit récipient 2. Ainsi, ledit couvercle amovible 6 permet de fermer hermétiquement ladite pièce extérieure monobloc 21 divisée en une première partie définissant la chambre de liquide nutritif 4 et en une seconde partie définissant ladite chambre de culture 3. En d'autres termes, ledit couvercle amovible 6 permet de fermer hermétiquement et en simultanée, à la fois ladite chambre de liquide nutritif 4 et ladite chambre de culture 3. In addition, as visible in Figure 1 and Figure 2, said container 2 also comprises a removable cover 6. Said removable cover 6 allows to close said culture chamber 3, more specifically, said removable cover 6 allows close the entirety of said one-piece outer part 21 that said container 2 comprises. Thus, said removable cover 6 makes it possible to hermetically close said one-piece outer part 21 divided into a first part defining the nutrient liquid chamber 4 and into a second part defining said culture chamber 3. In other words, said removable cover 6 makes it possible to close hermetically and simultaneously, both said nutrient liquid chamber 4 and said culture chamber 3.
[0055] En outre, des moyens d'étanchéités 7, par exemple de type joint d'étanchéité, interposés entre ledit couvercle 6 et ladite chambre de culture 3 ont pour rôle d'assurer la fermeture hermétique par le couvercle de la pièce extérieure monobloc 21 qui présente, au niveau de la chambre de culture 3, une ouverture. In addition, sealing means 7, for example of the seal type, interposed between said cover 6 and said culture chamber 3 have the role of ensuring the hermetic closure by the cover of the one-piece outer part 21 which has, at the level of the culture chamber 3, an opening.
[0056] Selon un mode de réalisation privilégié visible sur la figure 1, le couvercle 6 est manoeuvrable en rotation sur la partie périphérique de l'ouverture de la chambre de culture 3. La chambre de culture 3 présente, en périphérie de son ouverture, des collerettes 62 définissant entre elles des ouvertures périphériques 63. Ledit couvercle 6 est muni, en périphérie, de préférence régulièrement répartis, de plusieurs ailettes d'accrochages rentrantes 61 destinées à venir se loger par un mouvement de translation verticale au travers des ouvertures périphériques 63 de la chambre de culture 3, puis par un mouvement de rotation horaire, en dessous de l'une des collerettes 62. Ainsi, dans la position verrouillée, chacune des ailettes 61 du couvercle 6 se positionne en dessous de l'une des collerette 62 correspondantes de l'ouverture de la chambre de culture 3, de sorte à écraser le joint d'étanchéité présent sur la périphérie de la chambre de culture 3 et fermer hermétiquement ladite pièce extérieure monobloc 21 que comporte ledit récipient 2. Au contraire, en position déverrouillée, chacune des ailettes 61 du couvercle 6 est dissociée de sa collerette 62 correspondante et vient se positionner au travers des ouvertures périphériques 63 de la chambre de culture 3. According to a preferred embodiment visible in Figure 1, the cover 6 is manoeuvrable in rotation on the peripheral part of the opening of the culture chamber 3. The culture chamber 3 has, on the periphery of its opening, flanges 62 defining between them peripheral openings 63. Said cover 6 is provided, at the periphery, preferably regularly distributed, several re-entrant hooking fins 61 intended to be housed by a vertical translational movement through the peripheral openings 63 of the culture chamber 3, then by a clockwise rotation movement, below one of the flanges 62. Thus, in the locked position, each of the fins 61 of the cover 6 is positioned below one of the corresponding flanges 62 of the opening of the culture chamber 3, so as to crush the seal present on the periphery of the culture chamber 3 and hermetically close said one-piece outer part 21 that said container 2 comprises. On the contrary, in the unlocked position, each of the fins 61 of the cover 6 is dissociated from its corresponding flange 62 and is positioned through the peripheral openings 63 of the culture chamber 3.
[0057] Selon l'invention, ledit récipient 2 comporte encore des moyens de communication 8 au niveau de son couvercle 6. Lesdits moyens de communication 8 sont destinés à assurer une communication entre le volume interne de la chambre de culture 3, et l'extérieur du récipient de culture 2. According to the invention, said container 2 further comprises communication means 8 at its cover 6. Said communication means 8 are intended to ensure communication between the internal volume of the culture chamber 3, and the outside of culture vessel 2.
[0058] Selon un mode de réalisation particulier visible sur la figure 1, lesdits moyens de communication 8 consistent en au moins un conduit vertical que comporte ledit couvercle 6. According to a particular embodiment visible in Figure 1, said communication means 8 consist of at least one vertical duct that comprises said cover 6.
Selon un autre mode de réalisation, lesdits moyens de communication 8 consistent en deux conduits verticaux que comporte ledit couvercle amovible 6. Le premier conduit vertical permet l'aspiration du flux gazeux interne, il permet au sein du dispositif, en particulier au sein du récipient 2, un retour à la pression atmosphérique. L'aspiration du flux gazeux interne contenu dans ledit récipient 2 comportant ladite pièce extérieur monobloc 21 permet le cas échant une descente plus rapide du liquide nutritif de la chambre de culture 3 vers la chambre de liquide nutritif 4. Le second conduit vertical peut par exemple permettre l'injection d'un gaz, notamment du C02. According to another embodiment, said communication means 8 consist of two vertical ducts which said removable cover 6 comprises. The first vertical duct allows the suction of the internal gas flow, it allows within the device, in particular within the container 2, a return to atmospheric pressure. The suction of the internal gas flow contained in said container 2 comprising said one-piece outer part 21 allows, if necessary, a more rapid descent of the nutrient liquid from the culture chamber 3 towards the nutrient liquid chamber 4. The second vertical duct can for example allow the injection of a gas, in particular C02.
[0059] Selon une spécificité de l'invention, ledit dispositif de culture 1 comporte des moyens de dépression 9, par exemple tel que visible sur la figure 2. Lesdits moyens de dépression 9 ont pour rôle de diminuer la pression dans la chambre de culture 3 de sorte qu'elle devienne inférieure à la pression contenue dans la chambre de liquide nutritif 4. According to a specific feature of the invention, said culture device 1 comprises vacuum means 9, for example as shown in Figure 2. Said vacuum means 9 have the role of reducing the pressure in the culture chamber 3 so that it becomes lower than the pressure contained in the nutrient liquid chamber 4.
[0060] Tel que visible sur la figure 2, lesdits moyens de dépression 9 comportent au moins un moyen d'aspiration 91 de gaz, permettant de diminuer la pression du volume interne de la chambre de culture 3. Ledit moyen d'aspiration 91 étant à l'origine du phénomène de dépression appliquée au sein de ladite chambre de culture 3. As seen in Figure 2, said vacuum means 9 comprise at least one suction means 91 of gas, making it possible to reduce the pressure of the internal volume of the culture chamber 3. Said suction means 91 being at the origin of the phenomenon of depression applied within said culture chamber 3.
[0061] De préférence, ledit moyen d'aspiration 91 est constitué par une pompe à vide. Preferably, said suction means 91 is constituted by a vacuum pump.
[0062] Selon un autre mode de réalisation préféré, lesdits moyens de dépression 9 comportent, en plus, au moins une cuve tampon 92 visible sur la figure 2. Ladite cuve tampon 92 est interposée entre ledit au moins un moyen d'aspiration 91 et ledit au moins un récipient de culture 2. Plus spécifiquement, ladite cuve tampon 92 est interposée entre ladite pompe à vide et ledit au moins un récipient 2. According to another preferred embodiment, said vacuum means 9 comprise, in addition, at least one buffer tank 92 visible in Figure 2. Said buffer tank 92 is interposed between said at least one suction means 91 and said at least one culture container 2. More specifically, said buffer tank 92 is interposed between said vacuum pump and said at least one container 2.
[0063] Tel que visible avec le dispositif 1 de la figure 2, ladite cuve tampon 92 est positionnée :As visible with the device 1 of Figure 2, said buffer tank 92 is positioned:
- en amont dudit récipient 2, avant son moyen de communication 8, - upstream of said container 2, before its means of communication 8,
- en aval de ladite pompe à vide. - downstream of said vacuum pump.
[0064] Ladite cuve tampon 92 permet de servir de sas de dépression, afin de ne pas appliquer directement un vide dans la chambre de culture 3 du récipient 2. La présence de la cuve tampon 92 a pour avantage de permettre la création d'un vide uniforme avant la connexion avec ledit au moins un moyen d'aspiration 91 de chacun desdits au moins un récipient 2 du dispositif 1. De plus, la présence d'une cuve tampon 92 permet de travailler avec un moyen d'aspiration 91, notamment une pompe à vide, qui présente une capacité de débit d'aspiration réduite par rapport au débit minimum nécessaire au fonctionnement du dispositif 1 ne présentant pas de cuve tampon 92. Ainsi, la présence d'une cuve tampon 92 permet de diminuer le prix global du dispositif 1 en intégrant une pompe à vide moins coûteuse à faible capacité de débit. Said buffer tank 92 can serve as a vacuum lock, so as not to directly apply a vacuum in the culture chamber 3 of the container 2. The presence of the buffer tank 92 has the advantage of allowing the creation of a uniform vacuum before the connection with said at least one suction means 91 of each of said at least one receptacle 2 of the device 1. In addition, the presence of a buffer tank 92 makes it possible to work with a suction means 91, in particular a vacuum pump, which has a reduced suction flow capacity compared to the minimum flow rate necessary for the operation of the device 1 not having a buffer tank 92. Thus, the presence of a buffer tank 92 makes it possible to reduce the overall price of device 1 by integrating a less expensive vacuum pump with low flow capacity.
[0065] Selon un mode de réalisation avantageux, lesdits moyens de dépression 9 comportent, encore, au moins une vanne 93 interposée entre ledit au moins un moyen d'aspiration 91 et ladite au moins une cuve tampon 92. Par exemple, ladite vanne 93 consiste en une vanne manuelle ou encore en une électrovanne, notamment une électrovanne disposant d'un échappement permettant le retour à la pression atmosphérique. According to an advantageous embodiment, said vacuum means 9 comprise, again, at least one valve 93 interposed between said at least one suction means 91 and said at least one buffer tank 92. For example, said valve 93 consists of a manual valve or else of a solenoid valve, in particular a solenoid valve having an exhaust allowing return to atmospheric pressure.
[0066] Ladite vanne 93 permet de contrôler l'application d'un vide de pression au sein de ladite cuve tampon 92, toujours dans le but d'obtenir au sein de la cuve tampon 92 un vide. Said valve 93 makes it possible to control the application of a pressure vacuum within said buffer tank 92, again with the aim of obtaining a vacuum within the buffer tank 92.
[0067] Selon l'invention, ledit dispositif 1 comporte également des moyens de raccordement 10 pour raccorder les moyens de dépression 9 auxdits moyens de communication 8 dudit au moins un récipient de culture 2. Par exemple, lesdits moyens de raccordement 10 consistent en des tuyaux, conduits de transfert de gaz, notamment d'air. According to the invention, said device 1 also comprises connection means 10 for connecting the vacuum means 9 to said communication means 8 of said at least one culture vessel 2. For example, said connection means 10 consist of pipes, conduits for the transfer of gas, in particular air.
[0068] Ainsi, au travers des moyens de raccordement 10, les moyens de dépression 9 vont permettre l'application d'un vide de pression au sein de la chambre de culture 3. En d'autres termes, le fonctionnement des moyens de dépression 9 va permettre une aspiration des gaz contenues dans la chambre de culture 3 du récipient 2. L'aspiration des gaz se fait à l'aide desdits moyens de communication 8 que comporte chaque récipient 2. Thus, through the connection means 10, the vacuum means 9 will allow the application of a pressure vacuum within the culture chamber 3. In other words, the operation of the vacuum means 9 will allow aspiration of the gases contained in the culture chamber 3 of the container 2. The gases are sucked using said communication means 8 which each container 2 comprises.
[0069] Selon un mode de réalisation particulier, ledit dispositif 1 comporte au moins une vanne 11 entre lesdits moyens de dépression et ledit au moins un récipient 2, de sorte à contrôler l'application d'un vide de pression dans la chambre de culture 3. [0070] Selon un mode de réalisation de l'invention, lesdits moyens de communication 8 consistent en un conduit 81 qui traverse le couvercle amovible 6. Ledit conduit 81 permet de relier l'extérieur du récipient 2, c'est-à-dire les moyens de raccordement 10 avec le volume interne de la chambre de culture 3. Lesdits moyens de communication 8, c'est-à-dire le conduit 81 permet le passage du gaz entre le volume interne de la chambre de culture 3 et l'extérieur du récipient 2. C'est au travers des moyens de communication 8 et de son conduit 81 que les gaz contenus dans la chambre de culture 3 sont aspirés par les moyens de dépression 9 en fonctionnement. According to a particular embodiment, said device 1 comprises at least one valve 11 between said vacuum means and said at least one container 2, so as to control the application of a pressure vacuum in the culture chamber 3. According to one embodiment of the invention, said communication means 8 consist of a conduit 81 which passes through the removable cover 6. Said conduit 81 makes it possible to connect the outside of the container 2, that is to say the connection means 10 with the internal volume of the culture chamber 3. Said communication means 8, that is to say the duct 81 allows the passage of gas between the internal volume of the culture chamber 3 and the exterior of the container 2. It is through the means of communication 8 and its duct 81 that the gases contained in the culture chamber 3 are sucked by the vacuum means 9 in operation.
Selon un autre mode de réalisation de l'invention, lesdits moyens de communication 8 consistent en un embout qui équipe ledit couvercle (6). Cet embout comporte un orifice traversant qui communique avec un autre orifice qui traverse ledit couvercle (6). Ledit embout permet le passage du gaz entre le volume interne de la chambre de culture 3 et l'extérieur du récipient 2. According to another embodiment of the invention, said communication means 8 consist of a tip which is fitted to said cover (6). This end piece has a through orifice which communicates with another orifice which passes through said cover (6). Said nozzle allows the passage of gas between the internal volume of the culture chamber 3 and the outside of the container 2.
[0071] Selon un mode de réalisation préféré, tel que visible sur les figures 3 à 6, ledit dispositif 1 comporte au moins deux récipients de culture 2. La multiplication du nombre de récipient 2 au sein du dispositif 1 a pour avantage de permettre d'appliquer la technique de culture in vitro par immersion temporaire dans un liquide nutritif à échelle industrielle. According to a preferred embodiment, as seen in Figures 3 to 6, said device 1 comprises at least two culture vessels 2. The multiplication of the number of receptacles 2 within the device 1 has the advantage of allowing d '' apply the technique of in vitro culture by temporary immersion in a nutrient liquid on an industrial scale.
[0072] Par exemple, tel que visible sur la figure 3, ledit dispositif 1 comporte six récipients 2, ou encore comme visible sur la figure 5, ledit dispositif comporte 7 récipients 2. For example, as visible in Figure 3, said device 1 comprises six containers 2, or as visible in Figure 5, said device comprises 7 containers 2.
[0073] Sur les figures 4, 6 et 7, ledit dispositif 1 comporte dix récipients 2, le premier récipient 2 étant nommé Ml, le second M2 et ainsi de suite. In Figures 4, 6 and 7, said device 1 comprises ten containers 2, the first container 2 being named M1, the second M2 and so on.
[0074] Selon l'invention, quand le dispositif 1 est multi-récipient 2, chaque récipient 2 comporte des moyens de communication 8 raccordés aux moyens de raccordement 10. En d'autres termes, lesdits moyens de raccordements 10 sont configurés pour raccorder lesdits moyens de dépression 9 à chacun desdits moyens de communication 8 que comporte chacun desdits aux moins deux récipients 2. According to the invention, when the device 1 is a multi-container 2, each container 2 comprises communication means 8 connected to the connection means 10. In other words, said connection means 10 are configured to connect said vacuum means 9 to each of said communication means 8 which each of said at least two receptacles 2 comprises.
[0075] Dans le cas des dispositif 1 multi-récipients 2, les moyens de raccordement 10 comportent, d'une part, une rampe 100 et, d'autre part, des organes de raccordement 101 tel que visible sur les figures 3 à 7. In the case of multi-receptacle device 1 2, the connection means 10 comprise, on the one hand, a ramp 100 and, on the other hand, connection members 101 as shown in Figures 3 to 7 .
[0076] Ladite rampe 100 est raccordée aux moyens de dépression 9. Said ramp 100 is connected to the vacuum means 9.
[0077] Lesdits organes de raccordement 101 permettent de raccorder la rampe 100 avec chacun des moyens de communication 8 que comporte chaque récipient 2 du dispositif 1 multi-récipients 2. [0078] Selon un premier mode de réalisation visible sur les figures 3 et 4, ladite rampe 100 adopte une forme linéaire. Une des extrémités de la rampe 100 linéaire est raccordée aux moyens de dépression 9. Tel que visible sur la figure 4, une des extrémités de la rampe 100 linéaire est raccordée par exemple directement à un moyen d'aspiration 91 de type pompe à vide. [0079] Selon ce premier mode de réalisation visible sur les figures 3 et 4, les organes de raccordement 101 sont répartis sur une partie au moins de la longueur de la rampe 100 linéaire, de préférence sur l'intégralité de sa longueur. Said connecting members 101 make it possible to connect the ramp 100 with each of the communication means 8 which each receptacle 2 of the multi-receptacle device 2 comprises. [0078] According to a first embodiment visible in FIGS. 3 and 4 , said ramp 100 adopts a linear shape. One of the ends of the linear ramp 100 is connected to the vacuum means 9. As can be seen in FIG. 4, one of the ends of the linear ramp 100 is connected for example directly to a suction means 91 of the vacuum pump type. According to this first embodiment visible in Figures 3 and 4, the connecting members 101 are distributed over at least part of the length of the linear ramp 100, preferably over its entire length.
[0080] Selon un mode de réalisation visible sur la figure 3, lesdits récipients 2 sont répartis de part et d'autre de la rampe 100 linéaire. According to one embodiment visible in Figure 3, said receptacles 2 are distributed on either side of the linear ramp 100.
[0081] Selon un mode de réalisation visible sur la figure 4, lesdits récipients 2, dénommés Ml à M10 sont avantageusement espacés régulièrement sur l'intégralité de la longueur de la rampe 100 linéaire. According to one embodiment visible in Figure 4, said containers 2, called M1 to M10 are advantageously spaced regularly over the entire length of the linear ramp 100.
[0082] Selon un mode de réalisation préféré, lesdits récipients 2 du dispositif 1 multi récipients 2 sont espacés et répartis régulièrement sur l'intégralités de la longueur de la rampe 100 linéaire. According to a preferred embodiment, said receptacles 2 of the multi-receptacle device 1 2 are spaced apart and distributed regularly over the entire length of the linear ramp 100.
[0083] Selon un second mode de réalisation visible sur les figures 5 à 7, ladite rampe 100 adopte une forme de boucle, c'est-à-dire qu'elle prend n'importe quelle forme qui est une forme fermée, par exemple de type ronde, ovoïde, rectangle. According to a second embodiment visible in Figures 5 to 7, said ramp 100 adopts a loop shape, that is to say it takes any shape which is a closed shape, for example round, ovoid, rectangle type.
[0084] Avantageusement, lesdits organes de raccordement 101 sont répartis sur au moins une partie de la longueur de la boucle. De préférence, lesdits organes de raccordement 101 sont espacés régulièrement sur la rampe 100 en boucle. De préférence encore, lesdits organes de raccordement 101 sont réparties sur l'intégralité de la boucle. Advantageously, said connecting members 101 are distributed over at least part of the length of the loop. Preferably, said connecting members 101 are spaced regularly on the ramp 100 in a loop. More preferably, said connecting members 101 are distributed over the entire loop.
[0085] Selon le second mode de réalisation visible sur les figures 5 et 7, avec une rampe 100 en boucle, les moyens de raccordement 10 comporte en plus au moins un conduit de raccordement 102. Ledit conduit de raccordement 102 raccorde la rampe 100 en boucle aux moyens de dépression 9. According to the second embodiment visible in Figures 5 and 7, with a ramp 100 in a loop, the connection means 10 further comprises at least one connection duct 102. Said connection duct 102 connects the ramp 100 in vacuum means loop 9.
[0086] De préférence et comme visible sur les figures 5 à 7, ledit dispositif 1 présentant une rampe 100 en boucle comporte une pluralité de conduits de raccordement 102 qui s'étendent à partir de la rampe 100 de forme de boucle et qui convergent en un point. Ce point est raccordé aux moyens de dépression 9. Preferably and as visible in Figures 5 to 7, said device 1 having a ramp 100 in a loop comprises a plurality of connecting conduits 102 which extend from the ramp 100 in the form of a loop and which converge in a point. This point is connected to the vacuum means 9.
[0087] Selon un mode privilégié, ledit point est au centre de la rampe 100 de forme de boucle. Ceci a pour effet d'équilibrer les débits des flux traversant les différents conduits, de sorte à synchroniser le mouvement du liquide nutritif L de chacun des récipients de culture 2 connectées entre eux à une même rampe 100. Ainsi, le fonctionnement des différents récipients 2 d'un même dispositif 1 est synchronisée et permet un usage à échelle industrielle. According to a privileged embodiment, said point is at the center of the ramp 100 in the form of a loop. This has the effect of balancing the flow rates of the flows passing through the various conduits, so as to synchronize the movement of the nutrient liquid L of each of the culture vessels 2 connected to one another to the same ramp 100. Thus, the operation of the different vessels 2 of the same device 1 is synchronized and allows use on an industrial scale.
[0088] Selon une autre caractéristique du dispositif 1 de l'invention, ledit au moins un récipient de culture 2 comporte un évent 12. De préférence et tel que visible sur les figures 1 et 2, l'évent 12 est, d'une part, présent sur la paroi de la chambre de liquide nutritif 4 et, d'autre part, positionné au- dessus du niveau maximum que peut atteindre le liquide nutritif L que comporte la chambre de liquide nutritif 4. L'évent 12 assure la communication entre le volume interne de la chambre de liquide nutritif 4 et l'extérieur du récipient de culture 2. According to another characteristic of the device 1 of the invention, said at least one culture vessel 2 comprises a vent 12. Preferably and as shown in Figures 1 and 2, the vent 12 is, of a on the one hand, present on the wall of the nutrient liquid chamber 4 and, on the other hand, positioned above the maximum level that the nutrient liquid L that the chamber includes nutrient liquid 4. The vent 12 provides communication between the internal volume of the nutrient liquid chamber 4 and the exterior of the culture vessel 2.
[0089] Selon un mode de réalisation privilégié et afin d'éviter une contamination du milieu végétal M, lesdits moyens de communication 8 et ledit évent 12 comprennent des moyens de stérilisation des flux, par exemple de type filtre stérilisant. According to a preferred embodiment and in order to avoid contamination of the plant medium M, said communication means 8 and said vent 12 comprise means for sterilizing the flows, for example of the sterilizing filter type.
[0090] Selon un mode privilégié de réalisation de l'invention, le dispositif 1 comporte des moyens de contrôle permettant de piloter l'ouverture et la fermeture des vannes 93 et/ou 11, et de faire fonctionner ou non lesdits moyens de dépression 9, notamment le fonctionnement du moyen d'aspiration 91. According to a preferred embodiment of the invention, the device 1 comprises control means making it possible to control the opening and closing of the valves 93 and / or 11, and to operate said vacuum means 9 or not. , in particular the operation of the suction means 91.
[0091] Ainsi, lesdits moyens de dépression 9 du dispositif 1 permettent d'appliquer dans la chambre de culture 3 une dépression. En effet, les moyens de dépression 9 vont aspirer le flux d'air de la chambre de culture 3, de sorte que la pression de la chambre de culture 3 soit inférieure à celle de la chambre de liquide nutritif 4. Suite à la différence de pression entre la chambre de culture 3 et la chambre de liquide nutritif 4, en respectant la mécanique des fluides, la chambre de culture 3 va chercher à rééquilibrer les pressions de son volume interne par rapport aux pressions de la chambre de liquide nutritif 4. La chambre de culture cherche à combler sa perte de volume d'air aspiré en aspirant le volume contenu dans la chambre de liquide nutritif 4. Ainsi, à la suite de ce phénomène et afin de rééquilibrer les fluides en présences, il y aura une montée du liquide nutritif L au sein de la chambre de culture 3. Ainsi, l'application d'une dépression au sein de la chambre de culture 3 permet le transfert du liquide nutritif L de la chambre de liquide nutritif 4 vers la chambre de culture 3. Dès l'arrêt de l'aspiration, il y aura un nouveau déséquilibre de pression entre la chambre de culture 3 et la chambre de liquide nutritif 4 ce qui va générer une descente du liquide nutritif L de la chambre de culture 3 vers la chambre de liquide nutritif 4. Thus, said vacuum means 9 of the device 1 make it possible to apply a vacuum to the culture chamber 3. Indeed, the vacuum means 9 will suck the air flow from the culture chamber 3, so that the pressure of the culture chamber 3 is lower than that of the nutrient liquid chamber 4. Following the difference in pressure between the culture chamber 3 and the nutrient liquid chamber 4, while respecting the mechanics of the fluids, the culture chamber 3 will seek to rebalance the pressures of its internal volume with respect to the pressures of the nutrient liquid chamber 4. The culture chamber seeks to make up for its loss of volume of air sucked in by sucking the volume contained in the nutrient liquid chamber 4. Thus, following this phenomenon and in order to rebalance the fluids present, there will be a rise in nutrient liquid L within the culture chamber 3. Thus, the application of a vacuum within the culture chamber 3 allows the transfer of the nutrient liquid L from the nutrient liquid chamber 4 to the culture chamber 3. As soon as the ace stops piration, there will be a new pressure imbalance between the culture chamber 3 and the nutrient liquid chamber 4 which will generate a descent of the nutrient liquid L from the culture chamber 3 to the nutrient liquid chamber 4.
[0092] Dans le cas, où la chambre de culture 3 et la chambre de liquide nutritif 4 sont à pression atmosphérique, le fonctionnement des moyens de dépression 9 permet d'abaisser la pression de la chambre de culture 3 en dessous de la pression atmosphérique, alors que la chambre de liquide nutritif 4 reste à pression atmosphérique. Ainsi, sous l'effet des moyens de dépression 9, le liquide nutritif L va monter dans la chambre de culture 3. Puis dès l'arrêt de l'application de la dépression au sein de la chambre de culture 3, le milieu redescend par gravité vers la chambre de liquide nutritif 4. Dans ce cas, les valeurs de pressions contenues dans la chambre de culture 3 et la chambre de liquide nutritif 4 ne dépassent jamais la valeur de la pression atmosphérique, ainsi le retour à l'équilibre des pressions entre les volumes internes des deux chambres 3 et 4 n'est pas brutal sous la forme d'une surpression. En conséquence, le couvercle amovible 6, lors du retour à l'équilibre des pressions ne subit pas de contraintes mécaniques générant des fuites de flux. Au contraire, lors du retour à l'équilibre des pressions entre les deux chambres 3 et 4, ledit couvercle amovible 6 sera juste plaqué avec moins de force contre ladite pièce extérieure monobloc 21 que comporte ledit récipient 2 In the case where the culture chamber 3 and the nutrient liquid chamber 4 are at atmospheric pressure, the operation of the vacuum means 9 makes it possible to lower the pressure of the culture chamber 3 below atmospheric pressure , while the nutrient liquid chamber 4 remains at atmospheric pressure. Thus, under the effect of the vacuum means 9, the nutrient liquid L will rise in the culture chamber 3. Then as soon as the application of the negative pressure within the culture chamber 3 is stopped, the medium descends through gravity towards the nutrient liquid chamber 4. In this case, the pressure values contained in the culture chamber 3 and the nutrient liquid chamber 4 never exceed the value of the atmospheric pressure, thus the return to the pressure equilibrium. between the internal volumes of the two chambers 3 and 4 is not sudden in the form of an overpressure. Consequently, the removable cover 6, when the pressure returns to equilibrium, is not subjected to mechanical stresses generating flow leaks. On the contrary, when the pressure returns to equilibrium between the two chambers 3 and 4, said removable cover 6 will be just pressed with less force against said one-piece outer part 21 that said container 2 comprises
[0093] Ainsi, lors de son fonctionnement, le dispositif 1 permet de réaliser une culture in vitro par immersion temporaire dans un liquide nutritif (L) qui évite, lors du transfert de liquide nutritif de la chambre de liquide nutritif 4 vers la chambre culture 3 et vice versa, la formation de fuites localisées résultant d'une déformation du volume interne du récipient sous l'effet de l'application d'une surpression dans son volume interne. Au contraire, l'application d'une dépression interne et un retour à une pression atmosphérique au sein de la chambre de culture 3 empêche le phénomène de déformation du récipient 2donc évite les fuites et la contamination du matériel végétal M. Plus précisément, l'application d'une dépression interne et un retour à une pression atmosphérique au sein de la chambre de culture 3 empêche le phénomène de déformation du couvercle amovible 2 fermant hermétiquement ladite pièce extérieure monobloc 21 que comporte ledit récipient 2. Avantageusement, l'application d'une dépression dans la chambre de culture 3 du récipient 2, par les moyens de dépression 9 du dispositif de culture 1, permet de plaquer par aspiration le couvercle amovible 6 contre la pièce extérieure monobloc 21 que comporte ledit récipient 2 avec une plus grande pression que celle existante à pression atmosphérique. L'aspiration par dépression renforce l'étanchéité du récipient 2 générée par le positionnement du couvercle amovible 6 contre la pièce monobloc 21. Thus, during its operation, the device 1 makes it possible to carry out an in vitro culture by temporary immersion in a nutrient liquid (L) which avoids, during the transfer of nutrient liquid from the nutrient liquid chamber 4 to the culture chamber 3 and vice versa, the formation of localized leaks resulting from a deformation of the internal volume of the container under the effect of the application of an overpressure in its internal volume. On the contrary, the application of an internal vacuum and a return to atmospheric pressure within the culture chamber 3 prevents the phenomenon of deformation of the container 2, therefore prevents leaks and contamination of the plant material M. More precisely, the application of an internal vacuum and a return to atmospheric pressure within the culture chamber 3 prevents the phenomenon of deformation of the removable cover 2 hermetically closing said one-piece outer part 21 which said container 2 comprises. Advantageously, the application of a vacuum in the culture chamber 3 of the container 2, by the vacuum means 9 of the culture device 1, allows the removable cover 6 to be pressed against the one-piece outer part 21 that said container 2 comprises with a greater pressure than that existing at atmospheric pressure. The vacuum suction strengthens the tightness of the container 2 generated by the positioning of the removable cover 6 against the one-piece part 21.
[0094] La présente invention concerne également un procédé de culture in vitro (1) de matériel végétal (M) par immersion temporaire dans un liquide nutritif, au sein d'au moins un récipient de culture (2) comportant : a/ une chambre de culture (3), qui présente un volume interne, et qui est destinée à contenir le matériel végétal (M) à cultiver, b/ une chambre de liquide nutritif (4) qui est destinée à contenir le liquide nutritif (L) , c/ des moyens de transfert (5) pour le transfert temporaire d'au moins une partie du liquide nutritif (The present invention also relates to a method of in vitro culture (1) of plant material (M) by temporary immersion in a nutrient liquid, within at least one culture vessel (2) comprising: a / a chamber culture (3), which has an internal volume, and which is intended to contain the plant material (M) to be cultivated, b / a nutrient liquid chamber (4) which is intended to contain the nutrient liquid (L), c / transfer means (5) for the temporary transfer of at least part of the nutrient liquid (
L) depuis ladite chambre de liquide nutritif ( 4) vers la chambre de culture (3) , d/ un couvercle amovible (6) pour refermer ladite chambre de culture (3) ainsi que des moyens d'étanchéité (7) interposés entre ce couvercle (6) et cette chambre de culture (3) ; e/ des moyens de communication (8), que comporte le couvercle amovible (6), et qui sont destinés à assurer une communication entre le volume interne de la chambre de culture (3)et l'extérieur du récipient de culture (2) , dans lequel : L) from said nutrient liquid chamber (4) to the culture chamber (3), d / a removable cover (6) to close said culture chamber (3) as well as sealing means (7) interposed between this cover (6) and this culture chamber (3); e / communication means (8), which the removable cover (6) comprises, and which are intended to ensure communication between the internal volume of the culture chamber (3) and the outside of the culture vessel (2) , in which :
- on cultive ledit matériel végétal M au sein de ladite chambre de culture (3), - said plant material M is cultivated within said culture chamber (3),
- on transfère, de manière temporaire, lors de la phase active d'immersion, ledit liquide nutritif (L) de la chambre de liquide nutritif (4) vers la chambre de culture (3) en générant une différence de pression entre lesdites chambres (3) et (4), caractérisé en ce que : - on aspire les gaz contenus dans ladite chambre de culture (3) jusqu'à ce que la pression de ladite chambre de culture (3) soit inférieure à la pression de ladite chambre de liquide nutritif (4) de sorte à générer ladite différence de pression, entre ladite chambre de culture (3) et ladite chambre de liquide nutritif (4) par dépression de ladite chambre de culture (3). - Is transferred, temporarily, during the active immersion phase, said nutrient liquid (L) from the nutrient liquid chamber (4) to the culture chamber (3) by generating a pressure difference between said chambers ( 3) and (4), characterized in that: - the gases contained in said culture chamber (3) are sucked in until the pressure of said culture chamber (3) is lower than the pressure of said nutrient liquid chamber (4) so as to generate said difference in pressure, between said culture chamber (3) and said nutrient liquid chamber (4) by depression of said culture chamber (3).
[0095] L'étape d'aspiration de gaz pour créer une dépression dans ladite chambre de culture 3 a pour avantage d'accentuer la pression et l'appui que peut avoir le couvercle sur ladite chambre et donc de renforcer l'étanchéité du récipient 2. Les contraintes de fuites ou de limite de surpression n'existe plus avec Le procédé de culture in vitro par immersion temporaire de l'invention. The gas suction step to create a vacuum in said culture chamber 3 has the advantage of increasing the pressure and the support that the cover may have on said chamber and therefore of strengthening the tightness of the container. 2. The constraints of leakage or overpressure limit no longer exist with the in vitro culture method by temporary immersion of the invention.
[0096] Avantageusement, ledit procédé de l'invention est mis en oeuvre à l'aide du dispositif 1 de l'invention. Advantageously, said method of the invention is implemented using the device 1 of the invention.
[0097] Ainsi, la présente invention a pour avantage de s'affranchir des contraintes de limitation de surpression applicable, de fuites et de contamination des végétaux retrouvées avec le récipient de culture in vitro de matériel végétal par immersion temporaire dans un milieu nutritif divulgué dans le document WO2012/146872. Thus, the present invention has the advantage of overcoming the constraints of limiting the applicable overpressure, leaks and contamination of plants found with the in vitro culture vessel of plant material by temporary immersion in a nutrient medium disclosed in document WO2012 / 146872.

Claims

Revendications Claims
[Revendication 1] [Dispositif de culture in vitro (1) d'un matériel végétal (M) par immersion temporaire dans un liquide nutritif (L), ce dispositif (1) comprenant : au moins un récipient de culture (2), chacun comportant : a. une chambre de culture (3), qui présente un volume interne, et qui est destinée à contenir le matériel végétal (M) à cultiver, b. une chambre de liquide nutritif (4) qui est destinée à contenir le liquide nutritif (L), c. des moyens de transfert (5) pour le transfert temporaire d'au moins une partie du liquide nutritif (L) depuis ladite chambre de liquide nutritif (4) vers la chambre de culture (3) , d. un couvercle amovible (6) pour refermer ladite chambre de culture (3) ainsi que des moyens d'étanchéité (7) interposés entre ce couvercle (6) et cette chambre de culture (3) ; e. des moyens de communication (8), que comporte le couvercle amovible (6), et qui sont destinés à assurer une communication entre le volume interne de la chambre de culture (3)et l'extérieur du récipient de culture (2) , caractérisé en ce que ledit dispositif de culture (1) comporte, encore, d'une part, des moyens de dépression (9) pour établir, dans la chambre de culture (3), une pression qui est inférieure à la pression dans la chambre de liquide nutritif (4) et, d'autre part, des moyens de raccordement (10) pour raccorder les moyens de dépression (9) auxdits moyens de communication (8) dudit au moins un récipient de culture (2). [Claim 1] [Device for in vitro culture (1) of a plant material (M) by temporary immersion in a nutrient liquid (L), this device (1) comprising: at least one culture vessel (2), each comprising: a. a culture chamber (3), which has an internal volume, and which is intended to contain the plant material (M) to be cultivated, b. a nutrient liquid chamber (4) which is intended to contain the nutrient liquid (L), c. transfer means (5) for the temporary transfer of at least part of the nutrient liquid (L) from said nutrient liquid chamber (4) to the culture chamber (3), d. a removable cover (6) for closing said culture chamber (3) as well as sealing means (7) interposed between this cover (6) and this culture chamber (3); e. communication means (8), which the removable cover (6) comprises, and which are intended to ensure communication between the internal volume of the culture chamber (3) and the outside of the culture vessel (2), characterized in that said culture device (1) also comprises, on the one hand, vacuum means (9) for establishing, in the culture chamber (3), a pressure which is lower than the pressure in the chamber of nutrient liquid (4) and, on the other hand, connection means (10) for connecting the vacuum means (9) to said communication means (8) of said at least one culture vessel (2).
[Revendication 2] Dispositif de culture in vitro (1) d'un matériel végétal (M) par immersion temporaire dans un liquide nutritif, selon la revendication précédente, caractérisé en ce que lesdits moyens de dépression (9) comportent au moins un moyen d'aspiration (91), de préférence chaque moyen d'aspiration est constitué par une pompe à vide. [Claim 2] Device for in vitro culture (1) of a plant material (M) by temporary immersion in a nutrient liquid, according to the preceding claim, characterized in that said vacuum means (9) comprise at least one means of suction (91), preferably each suction means is constituted by a vacuum pump.
[Revendication 3] Dispositif de culture in vitro (1) d'un matériel végétal (M) par immersion temporaire dans un liquide nutritif, selon la revendication précédente, caractérisé en ce que lesdits moyens de dépression (9) comportent, en outre, au moins une cuve tampon (92) interposée entre ledit au moins un moyen d'aspiration (91) et ledit au moins un récipient de culture (2). [Claim 3] Device for in vitro culture (1) of a plant material (M) by temporary immersion in a nutrient liquid, according to the preceding claim, characterized in that said vacuum means (9) further comprise at at least one buffer tank (92) interposed between said at least one suction means (91) and said at least one culture vessel (2).
[Revendication 4] Dispositif de culture in vitro (1) d'un matériel végétal (M) par immersion temporaire dans un liquide nutritif, selon la revendication précédente, caractérisé en ce que lesdits moyens de dépression (9) comportent, encore, au moins une vanne (93) interposée entre ledit au moins un moyen d'aspiration (91) et ladite au moins une cuve tampon (92). [Claim 4] Device for in vitro culture (1) of a plant material (M) by temporary immersion in a nutrient liquid, according to the preceding claim, characterized in that said vacuum means (9) comprise, again, at least a valve (93) interposed between said at least one suction means (91) and said at least one buffer tank (92).
[Revendication 5] Dispositif de culture in vitro (1) d'un matériel végétal (M) par immersion temporaire dans un liquide nutritif, selon l'une quelconque des revendications précédentes, caractérisé en ce qu'il comporte au moins une vanne (11) interposée entre lesdits moyens de dépression (9) et ledit au moins un récipient (2). [Revendication 6] Dispositif de culture in vitro (1) d'un matériel végétal (M) par immersion temporaire dans un liquide nutritif, selon l'une quelconque des revendications précédentes, caractérisé en ce que lesdits moyens de communication (8) consistent en un conduit (81) qui traverse le couvercle amovible (6) ou consistent en un embout qui équipe ledit couvercle (6) et qui comporte un orifice traversant et communiquant avec un orifice qui traverse ledit couvercle [Claim 5] Device for in vitro culture (1) of a plant material (M) by temporary immersion in a nutrient liquid, according to any one of the preceding claims, characterized in that it comprises at least one valve (11 ) interposed between said vacuum means (9) and said at least one container (2). [Claim 6] Device for in vitro culture (1) of a plant material (M) by temporary immersion in a nutrient liquid, according to any one of the preceding claims, characterized in that said communication means (8) consist of a duct (81) which passes through the removable cover (6) or consist of a nozzle which equips said cover (6) and which has a through orifice communicating with an orifice which passes through said cover
(6). (6).
[Revendication 7] Dispositif de culture in vitro (1) d'un matériel végétal (M) par immersion temporaire dans un liquide nutritif, selon l'une quelconque des revendications précédentes, caractérisé en ce qu'il comporte au moins deux récipients de culture (2) comportant, chacun, des moyens de communication (8) tandis que les moyens de raccordement (10) comportent, d'une part, une rampe (100) raccordée aux moyens de dépression (9) et, d'autre part, des organes de raccordement (101) pour raccorder ladite rampe (100) aux moyens de communication (8) que comportent chacun des récipient de culture (2). [Claim 7] Device for in vitro culture (1) of a plant material (M) by temporary immersion in a nutrient liquid, according to any one of the preceding claims, characterized in that it comprises at least two culture vessels (2) each comprising communication means (8) while the connection means (10) comprise, on the one hand, a ramp (100) connected to the vacuum means (9) and, on the other hand, connecting members (101) for connecting said ramp (100) to the communication means (8) which each of the culture vessels (2) comprise.
[Revendication 8] Dispositif de culture in vitro (1) d'un matériel végétal (M) par immersion temporaire dans un liquide nutritif, selon la revendication précédente, caractérisé en ce que ladite rampe (100) adopte une forme linéaire et présente une extrémité raccordée aux moyens de dépression (9), tandis que les organes de raccordement (101) sont répartis sur une partie au moins de la longueur de la rampe (100). [Claim 8] Device for in vitro culture (1) of a plant material (M) by temporary immersion in a nutrient liquid, according to the preceding claim, characterized in that said ramp (100) adopts a linear shape and has an end connected to the vacuum means (9), while the connecting members (101) are distributed over at least part of the length of the ramp (100).
[Revendication 9] Dispositif de culture in vitro (1) d'un matériel végétal (M) par immersion temporaire dans un liquide nutritif, selon la revendication 7, caractérisé en ce que, d'une part, ladite rampe (100) adopte une forme de boucle, tandis que les organes de raccordement (101) sont répartis sur au moins une partie de la longueur de la boucle et, d'autre part, les moyens de raccordement (10) comportent en outre au moins un conduit de raccordement (102) pour raccorder ladite rampe (100) aux moyens de dépression (9). [Claim 9] Device for in vitro culture (1) of a plant material (M) by temporary immersion in a nutrient liquid, according to claim 7, characterized in that, on the one hand, said ramp (100) adopts a loop shape, while the connection members (101) are distributed over at least part of the length of the loop and, on the other hand, the connection means (10) further comprise at least one connection duct ( 102) to connect said ramp (100) to the vacuum means (9).
[Revendication 10] Dispositif de culture in vitro (1) d'un matériel végétal (M) par immersion temporaire dans un liquide nutritif, selon la revendication précédente, caractérisé en ce qu'il comporte une pluralité de conduits de raccordement (102) qui s'étendent à partir de la rampe (100) adoptant une forme de boucle et qui convergent en un point raccordé aux moyens de dépression (9). [Claim 10] Device for in vitro culture (1) of a plant material (M) by temporary immersion in a nutrient liquid, according to the preceding claim, characterized in that it comprises a plurality of connecting conduits (102) which extend from the ramp (100) adopting a loop shape and which converge at a point connected to the vacuum means (9).
[Revendication 11] Dispositif de culture in vitro (1) de matériel végétal (M) par immersion temporaire dans un liquide nutritif, selon l'une quelconque des revendications précédentes, caractérisé en ce que ledit au moins un récipient de culture (2) comporte un évent (12) pour assurer la communication entre le volume interne de la chambre de liquide nutritif (4) et l'extérieur d'un tel récipient de culture (2). [Claim 11] Device for in vitro culture (1) of plant material (M) by temporary immersion in a nutrient liquid, according to any one of the preceding claims, characterized in that said at least one culture vessel (2) comprises a vent (12) to ensure communication between the internal volume of the nutrient liquid chamber (4) and the exterior of such a culture vessel (2).
[Revendication 12] Procédé de culture in vitro (1) de matériel végétal (M) par immersion temporaire dans un liquide nutritif, au sein d'au moins un récipient de culture (2) comportant : f. une chambre de culture (3), qui présente un volume interne, et qui est destinée à contenir le matériel végétal (M) à cultiver, g. une chambre de liquide nutritif (4) qui est destinée à contenir le liquide nutritif (L), h. des moyens de transfert (5) pour le transfert temporaire d'au moins une partie du liquide nutritif (L) depuis ladite chambre de liquide nutritif (4) vers la chambre de culture (3) , i. un couvercle amovible (6) pour refermer ladite chambre de culture (3) ainsi que des moyens d'étanchéité (7) interposés entre ce couvercle (6) et cette chambre de culture (3) ; j. des moyens de communication (8), que comporte le couvercle amovible (6), et qui sont destinés à assurer une communication entre le volume interne de la chambre de culture (3) et l'extérieur du récipient de culture (2), dans lequel : [Claim 12] A method of in vitro culture (1) of plant material (M) by temporary immersion in a nutrient liquid, in at least one culture vessel (2) comprising: f. a culture chamber (3), which has an internal volume, and which is intended to contain the plant material (M) to be cultivated, g. a nutrient liquid chamber (4) which is intended to contain the nutrient liquid (L), h. transfer means (5) for the temporary transfer of at least part of the nutrient liquid (L) from said nutrient liquid chamber (4) to the culture chamber (3), i. a removable cover (6) for closing said culture chamber (3) as well as sealing means (7) interposed between this cover (6) and this culture chamber (3); j. communication means (8), which the removable cover (6) comprises, and which are intended to ensure communication between the internal volume of the culture chamber (3) and the outside of the culture vessel (2), in which :
- on cultive ledit matériel végétal M au sein de ladite chambre de culture (3), - said plant material M is cultivated within said culture chamber (3),
- on transfère, de manière temporaire, lors de la phase active d'immersion, ledit liquide nutritif (L) de la chambre de liquide nutritif (4) vers la chambre de culture (3) en générant une différence de pression entre lesdites chambres (3) et (4), caractérisé en ce que : - Is transferred, temporarily, during the active immersion phase, said nutrient liquid (L) from the nutrient liquid chamber (4) to the culture chamber (3) by generating a pressure difference between said chambers ( 3) and (4), characterized in that:
- on aspire les gaz contenus dans ladite chambre de culture (3) jusqu'à ce que la pression de ladite chambre de culture (3) soit inférieure à la pression de ladite chambre de liquide nutritif (4) de sorte à générer ladite différence de pression, entre ladite chambre de culture (3) et ladite chambre de liquide nutritif (4) par dépression de ladite chambre de culture (3). - the gases contained in said culture chamber (3) are sucked in until the pressure of said culture chamber (3) is lower than the pressure of said nutrient liquid chamber (4) so as to generate said difference in pressure, between said culture chamber (3) and said nutrient liquid chamber (4) by depression of said culture chamber (3).
[Revendication 13] Procédé de culture in vitro (1) de matériel végétal (M) par immersion temporaire dans un liquide nutritif, selon la revendication précédente, caractérisé en ce qu'il est mis en oeuvre par le dispositif de culture in vitro (1) d'un matériel végétal (M) par immersion temporaire dans un liquide nutritif selon l'une quelconque des revendications 1 à 11.] [Claim 13] A method of in vitro culture (1) of plant material (M) by temporary immersion in a nutrient liquid, according to the preceding claim, characterized in that it is implemented by the in vitro culture device (1 ) of a plant material (M) by temporary immersion in a nutrient liquid according to any one of claims 1 to 11.]
PCT/EP2020/082555 2019-11-18 2020-11-18 Device for in vitro plant culture by temporary immersion in a nutritive liquid WO2021099399A1 (en)

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FR1912854 2019-11-18
FR1912854A FR3103078A1 (en) 2019-11-18 2019-11-18 Device for plant culture in vitro by temporary immersion in a nutrient liquid
FR2001991 2020-02-28
FR2001991A FR3103079B1 (en) 2019-11-18 2020-02-28 Device for invitro plant culture by temporary immersion in a nutrient liquid

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012146872A1 (en) 2011-04-28 2012-11-01 Centre De Cooperation Internationale En Recherche Agronomique Pour Le Developpement - C.I.R.A.D. Container for in vitro cultivation of plant material, by temporary immersion
WO2012156440A1 (en) * 2011-05-18 2012-11-22 Sopet Nv Container system for immersion growth regime
BRPI0004185B1 (en) * 2000-08-28 2016-01-12 Embrapa Empresa Brasileira De Pesquisa Agropecuária bioreactor systems for the cultivation of plant or animal cells, tissues or organs or cells of microorganisms by temporary or continuous immersion using a positive or negative pressure source
EP3069591A1 (en) * 2013-11-11 2016-09-21 Fibria Celulose S.A. Bioreactor for in vitro plant culture

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BRPI0004185B1 (en) * 2000-08-28 2016-01-12 Embrapa Empresa Brasileira De Pesquisa Agropecuária bioreactor systems for the cultivation of plant or animal cells, tissues or organs or cells of microorganisms by temporary or continuous immersion using a positive or negative pressure source
WO2012146872A1 (en) 2011-04-28 2012-11-01 Centre De Cooperation Internationale En Recherche Agronomique Pour Le Developpement - C.I.R.A.D. Container for in vitro cultivation of plant material, by temporary immersion
WO2012156440A1 (en) * 2011-05-18 2012-11-22 Sopet Nv Container system for immersion growth regime
EP3069591A1 (en) * 2013-11-11 2016-09-21 Fibria Celulose S.A. Bioreactor for in vitro plant culture

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AR120489A1 (en) 2022-02-16
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FR3103079B1 (en) 2021-10-29

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