BR112013027846B1 - container for in vitro culture of plant material, by temporary immersion - Google Patents

Abstract

container for the in vitro culture of plant material, by temporary immersion the present invention relates to a container for the in vitro culture of plant material, by temporary immersion, container (1) which comprises, on the one hand, an upper chamber ( 3) m designed to contain this plant material (m) to be cultivated, and an lower chamber (4) m designed to contain a nutritive liquid (l), as well as a means (5) to temporarily transfer at least a part of that nutrient liquid (l) from that lower chamber (4) to that upper chamber (3). according to the invention, the lower face (3c) of the upper chamber (3) m, intended to support this plant material, has a horizontal surface (s) between 0.02 and 0.07 m2, and the lateral face (3a ) of the upper chamber (3) has a height (h) between 40 and 130 mm. this combination of structural characteristics allows to improve the access to light and the photosynthetic activity of biomass, as well as to avoid the phenomena of compaction and stratification during the cultivation of plant material, to optimize the development of plant material.

Description

"CONTAINER FOR THE IN VITRO CULTURE OF VEGETABLE MATERIAL, BY TEMPORARY IMMERSION" [0001] The invention relates to the culture of plant material, and particularly to a container for the in vitro culture of plant material by temporary immersion, under sterile conditions.

[0002] The multiplication of plants by in vitro culture, under sterile conditions, is a common practice in the field of horticulture and agriculture.

[0003] This practice offers numerous advantages, but it generates a relatively high labor cost which is linked to the frequency of manipulations necessary for the transplantation of plant tissues on gelled nutritious media, but also to the quantity of containers and their cleaning.

[0004] In vitro culture techniques in liquid nutrient medium allow to limit this load. This type of culture has numerous advantages, unfortunately counterbalanced by important inconveniences, essentially linked to problems of asphyxiation of the culture and / or friction forces, when the plant material is permanently immersed and under agitation.

[0005] In order to prevent these inconveniences, a technique known as “temporary immersion” was developed, which consists, as its name suggests, of temporarily immersing (classically a few minutes a day) the plant material in culture (explants, cells, etc.) in a nutritious medium. Considering fluctuation phenomena or growth phenomena, this immersion in the nutrient medium can be partial or total. During the resting phase, which is the longest period, the cultivated material is immersed and without movement; but a film of nutrient medium, retained on the plant material by surface tensions at the end of the preceding immersion phase, keeps this material humidified and fed.

[0006] This process thus allows you to benefit from the advantages of in vitro culture in a liquid medium, avoiding choking and friction problems. It also allows to obtain a quality of culture development (cells, meristems, embryos, seedlings, etc.) that is particularly interesting in comparison to that obtained by other in vitro culture processes.

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2/19 [0007] A container adapted for this culture technique by temporary immersion is, for example, described in document FR - 2 370 743, marketed until today under the name "RITA".

[0008] The RITA container comprises two overlapping chambers, namely: an upper chamber designed to receive the plant material, and a lower chamber designed to contain the nutrient medium in a liquid form.

[0009] The dimensions of the upper chamber give it a vertically elongated shape, with a height and diameter of the order of 60 mm and 108 mm, respectively; the horizontal surface (in m2) / height (in m) ratio is thus around 0.15.

[0010] This container comprises a unitary body that forms an external part closed by a lid, and a basket for the separation of the two chambers. An inlet located on the cover allows the supply of an overpressure conducted in the lower chamber by a tube that flows into a compartment.

[0011] Overpressure causes a part of the nutrient medium to rise, from the lower chamber to the upper chamber, ensuring immersion and a mixture of plant material; stopping the overpressure allows the nutrient medium to return to the lower chamber by gravity.

[0012] The immersion of the plant material is thus carried out by displacing the nutrient medium, driven by an overpressure, and not by the mechanical displacement of the support basket. This absence of mechanical movements increases the reliability of the device.

[0013] This container allows an in vitro culture in sterile conditions, limiting the risk of infection, ensuring a good reliability of the culture during an important number of immersion cycles. This container is also interesting in terms of maintenance, disassembly and washing; above all, it is easily sterilizable between two cycles of use.

[0014] However, in practice, the inventors observed that, in the upper chamber, the plant material is present in a relatively compact form, which is unfavorable to mixing, to good exposure to light, and finally to development

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3/19 harmonious plant material.

[0015] This biomass also has a tendency to develop in a heterogeneous way over the height of the upper chamber, causing a stratification phenomenon.

[0016] In particular, the upper part of the biomass, more exposed to light and less physically disturbing, develops more than the central part of this biomass, physically compressed and disturbed by its access to light.

[0017] For these reasons, plant material evolves differently according to its position in biomass, which creates stratification with an increasing development gradient from bottom to top.

[0018] Now, the inventors even determined that, surprisingly, these phenomena of compaction and stratification are linked to the unsuitable shape and volume of the upper chamber of the container.

[0019] To prevent these inconveniences, the inventors have developed a container whose upper chamber is significantly wider than the upper chamber that equips the prior art container described in document FR-2 760 743 above, maintaining a similar height.

[0020] In this context, the applicant proposes a new structure for a container of in vitro culture of plant material, by temporary immersion, functioning in a similar way to that described in document FR-2 730 743.

[0021] The corresponding container comprises two overlapping chambers, namely - an upper chamber, intended to contain those plant tissues to be cultivated, and a lower chamber, intended to contain a nutritive liquid, these chambers being each delimited by a side face, on an upper side and on a lower side, a container which also includes means for the temporary transfer of at least a part (advantageously of almost or all) of that nutrient liquid, from that lower chamber to that upper chamber.

[0022] And, according to the present invention, the lower face of the upper chamber, intended to support the plant material, has a horizontal surface comprised between 0.02 and 0.07 m2; in addition, the side face of the upper chamber

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4/19 has a height between 40 and 130 mm.

[0023] The horizontal surface (in m2) / height (in m) ratio of the upper container chamber, according to the invention, is thus comprised between approximately 0.50 and 1.75.

[0024] By "horizontal surface" of the lower face of the upper chamber, we understand the surface bounded by the outline of that lower face, that is, in a horizontal plane that passes through that outline.

[0025] This horizontal surface also corresponds to the horizontal volume of that lower face of the upper chamber.

[0026] This horizontal surface is preferably between 0.03 and 0.04 m2, and preferably still on the order of 0.037 m2.

[0027] The inventors found that, unexpectedly, this particular dimensional relationship of the upper chamber, between the horizontal surface of its lower face and the height of its lateral face, confers several advantages to this in vitro culture vessel.

[0028] This container notably allows an optimum measurement of biomass in the upper chamber, with less competition for access to light.

[0029] This container also allows a homogeneous and synchronous development of plant material, thus suppressing the usual stratification phenomena.

[0030] According to a preferred embodiment, the upper chamber has a general cylindrical shape, of which:

- the bottom face has a radius between 75 and 150 mm, preferably on the order of 100 to 120 mm, and preferably also on the order of 110 mm (that is, a diameter between 150 mm and 300 mm, of preferably on the order of 200 to 240 mm, and preferably still on the order of 220 mm); and

- the side face has a height between 50 and 90 mm, preferably still on the order of 60 to 70 mm.

[0031] In the case of the lower face with a circular contour, the diameter / height ratio of the upper chamber can also be used to define the enlargement of the chamber

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Top 5/19 of the container.

[0032] In this case, this ratio is advantageously well above 1, preferably between 1.6 (150/90) and 6 (300/50), preferably still between 2.2 (200/90) and 4 , 8 (240/50) and still between 3.14 (220/70) and 3.66 (220/60).

[0033] According to an embodiment, the surface of the lower face of the upper chamber is greater than the surface of the lower face of the lower chamber and the surface of the upper face of the lower chamber.

[0034] According to an advantageous embodiment, the container comprises an external envelope delimited by a lower contour to which a bottom element is connected, and by an upper contour, delimiting an opening hermetically closed by a removable lid; the outer envelope comprises two parts, one, lower and the other, upper, separated by an internal interlayer, these lower and upper parts forming, respectively, the lateral faces of the lower chamber and the upper chamber, and this internal interlayer forming the upper face of the lower chamber and at least a part, preferably almost all or even all, of the lower face of the upper chamber.

[0035] In this case, the internal interlayer wall advantageously belongs to a complementary internal part, cooperating, in an airtight manner with the associated external envelope.

[0036] This complementary inner part advantageously comprises a side wall that rises from the contour of the interim inner wall to form a basket-shaped structure together, that side wall taking the shape of the outer envelope over at least a part, preferably , almost all or even the totality, from the height of its upper part.

[0037] The upper and lower parts of the outer wrap are advantageously connected by a part that forms an edge; and a peripheral band of the inner wall interspersed with a part that forms the rim; and a peripheral brace of the interim inner wall of the complementary intermediate piece is supported on that rim part by means of sealing means.

[0038] Still according to an advantageous realization feature, the means

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6/19 for the temporary transfer of the nutritive liquid include - at least one passage for water connection of the upper and lower chambers; and - means for introducing a gas into this lower chamber, in order to generate, on the one hand, a routing of at least a part (preferably almost or all) of the nutrient liquid, from that lower chamber to that upper chamber by a gas overpressure in that lower chamber and, on the other hand, a return by gravity of that nutrient liquid in that lower chamber, when that gas overpressure stops.

[0039] In this case, the connection passage between the chambers advantageously consists of a duct that extends through the height of the lower chamber, that duct being equipped with at least one lower orifice that opens in the vicinity of the lower face of that lower chamber and at least minus an upper orifice that opens at the level of the lower face of the upper chamber.

[0040] A part of at least the lower face of the upper chamber is then advantageously constituted by a filter element, obtaining a reserve, into which the upper orifice (s) of the upper conduit (s) empties (s). Link; this filtering element advantageously comprises a plurality of orifices for the distribution of the nutritive liquid, and, if applicable, the overpressure gas, moving towards the upper chamber; and the interim inner wall advantageously comprises a central concave part, open on the side of the upper chamber and at the level of which the filter element is taken to form the reserve.

[0041] This connection conduit advantageously ends with at least two upper holes, regularly distributed on the periphery of that conduit.

[0042] Still according to this advantageous characteristic, the means of introducing gas into the lower chamber consist of a duct that flows through its lateral face and disposed on the side of its upper face.

[0043] According to another advantageous embodiment, the container comprises (I) a lid provided with at least two recessed fitting fins, distributed over its periphery, and (II) an upper opening provided over its periphery of ferrule parts .

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7/19 [0044] This cover taken over this opening is maneuverable in rotation between a locked position, in which each of these locking fins is positioned under one of these ferrule parts, and - an unlocked position, in which these locking fins fitting are dissociated from these ferrules.

[0045] The present invention is further illustrated, without in any way being limited, by the following description of a particular embodiment, in connection with the attached drawings, in which:

figure 1 shows a general and perspective view of the container, according to the invention;

figure 2 schematically represents the container of figure 1, according to a vertical section plane;

figure 3 represents the container of figure 1, in an exploded manner;

figure 4 shows, in an enlarged manner, the detail IV of figure 2, corresponding to the upper end of the water connection duct located between the upper and lower chambers;

figure 5 illustrates, in an enlarged manner, the detail V of figure 2, corresponding to the means of being arranged between the external wrap and the complementary basket;

- figure 6 represents, in an enlarged manner, the detail VI of figure 2, corresponding to the sealing means arranged between the external wrap and the removable cover;

- figures 7 and 8 illustrate the operation of the container, according to the invention, with a resting phase, in which the nutritive liquid is located at the level of the lower chamber (figure 7) and a temporary immersion phase, in which the nutritive liquid is moved in the upper chamber to submerge / submerge the plant material (figure 8).

[0046] Container 1, represented in general in figures 1 to 3, constitutes a device that is adapted for the in vitro culture of plant material by temporary immersion, under sterile conditions.

[0047] This container 1 consists of a unitary device, in this case in the form

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8/19 general body, which has a general axis 1 [0048] As illustrated by figure 2, this container 1 comprises two chambers 3 and 4 superimposed, in this case, each in the general form of a straight circular cylinder, namely:

- an upper chamber 3, intended to contain the plant material M to be cultivated (figures 7 and 8), and

- a lower chamber 4, designed to contain a nutritive liquid L (figures 7 and 8).

[0049] These chambers 3 and 4 are each bounded by an annular lateral face 3a and 4a, by an upper face in the general form of disc 3b and 4b, and by a lower face also in general form of disc 3c and 4c .

[0050] This container 1 also includes means 5 for the temporary transfer of at least a part (preferably almost or all) of the nutrient liquid L, from the lower chamber 4 to the upper chamber 3, which will be described in sequence in relation to figures 2 and 3.

[0051] As developed below in relation to figures 7 and 8, the means 5 for the temporary transfer of the nutritive liquid L, are structured to generate, on the one hand, a routing of this nutritive liquid L, from the lower chamber 4 to the upper chamber 3 by a gas overpressure in that lower chamber 4 and, on the other hand, a return by gravity of that nutrient liquid in that lower chamber 4, when the gas overpressure is stopped.

[0052] According to the invention, the upper chamber 3 is designed to limit the phenomena of stratification and compaction of plant material M, and also to optimize access to the opening of this material.

[0053] For this, the circular lower face 3c of the upper chamber 3, intended to support the plant tissues M, in this case has a circular outline 3c1, whose radius R is of the order of 110 mm. In general, this radius R is advantageously between 75 and 150 mm, and preferably between 100 and 120 mm.

[0054] This contour 3c1 extends in a horizontal plane P, perpendicular to the general axis 1 'of the container 1, on which it delimits a surface or an area

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9/19 horizontal S (figure 2) of the order of 0.037 m2. In general, this horizontal surface S is between 0.02 m2 and 0.07 m2.

[0055] The lateral face 3a of that upper chamber 3 has a height H of the order of 60 to 70 mm. In general, this height H is advantageously between 40 and 130 mm, preferably still between 50 and 90 mm.

[0056] This upper chamber 3 thus presents a generally cylindrical shape relatively flat.

[0057] The lower chamber 4 has a radius of the order of 78 to 87 mm, preferably of the order of 82 mm (corresponding to the radius of its lower faces 4c and upper 4b) and a height of the order of 75 mm.

[0058] The surface of the lower face 3c of the upper chamber 3 is thus, in this case, greater than the surface of the lower face 4c of the lower chamber 4 and the surface of the upper face 4b of the lower chamber 4.

[0059] In order to constitute these chambers 3 and 4, the container 1 consists of a connection of different parts that are described in detail below in relation to figures 2 and 3.

[0060] The container 1 initially comprises a monobloc external part 6, comprising a lateral external envelope 7 which is delimited by:

- a lower contour 8, to which a disk-shaped bottom element 9 is attached; and

- an upper contour 10, delimiting an opening 11, intended to be hermetically sealed by a removable cover 12.

[0061] The outer casing 7 comprises, in this case, three annular parts distributed over its height, namely a tubular upper part 7a, a tubular lower part 7b and a junction part 7c that constitutes an annular ring in the shape of a crown.

[0062] These different parts 7a, 7b and 7c all extend coaxially to the same vertical axis 7 ', forming the axis of the outer envelope 7.

[0063] The upper parts 7a and lower 7b of the outer casing 7 each have a constant diameter, or at least approximately constant, at their respective heights.

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10/19 [0064] The upper part 7a of the outer casing 7 has, in addition, an upper diameter in relation to the diameter of the lower part 7b; the upper parts 7a and lower 7b of the outer casing 7 are thus connected, at the level of their frontal contours, by the part that forms the rim 7c.

[0065] The upper parts 7a and lower 7b of the outer casing 7 have, in addition to an identical height, or at least approximately identical, in relation to the upper chamber 3 and the lower chamber 4, respectively.

[0066] In particular, the upper part 7a of the outer casing 7 advantageously has a height between 50 and 90 mm, preferably still in the order of 60 to 70 mm.

[0067] The lower part 7b of the outer casing 7 and the bottom element 9 form, respectively, the side face 4a and the bottom face 4c of the lower chamber 4.

[0068] This lower part 7b of the casing 7 is equipped with a lat4eral duct 15, for the introduction of gas (for example air) directly into the lower Chamber 4.

[0069] A hydrophobic air filter F1, shown very schematically in figure 1, is advantageously attached to that side duct 15, in order to ensure sterility inside the container 1.

[0070] The lateral duct 15 flows through the lateral face 4a of the lower chamber 4 and is arranged on the side of its upper face 4b.

[0071] The longitudinal axis 15 'of this lateral duct 15 is oriented according to an upward slope towards the periphery, to limit the risk of the nutrient liquid L flowing through this lateral duct 15 to the outside and, thus, preventing the associated filter F1 is wet and rendered inoperative.

[0072] The rim part 7c supports a complementary inner part 16, in the form of a basket, which is intended to separate the two chambers 3 and 4 from the container 1 and which is intended to form at least part of the faces upper chamber 3.

[0073] This complementary internal part 16 comprises:

- a disk-shaped bottom wall 16a, intended to separate the two chambers 3 and 4 from the container 1; and

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11/19

- a tubular side wall 16b, rising from the outline of that bottom wall 16a.

[0074] The tubular side wall 16b internally folds the upper part 7a of the peripheral wrap 7, to form together the side face 3a of the upper chamber 3.

[0075] This tubular side wall 16b takes the shape of the outer casing 7 over the height of its upper part 7a; the diameter of the outer surface of the side wall 16b thus approximately corresponds to the diameter of the inner surface of the upper part 7a of the outer casing 7.

[0076] The bottom wall 16a constitutes an internal interlayer forming the upper face 4b of the lower chamber 4 and a part of the lower face 3c of the upper chamber 3.

[0077] A peripheral band 16a1 of this bottom wall 16a rests on the rim part 7c of the outer casing 7, by means of watertightness 17 (figure 5).

[0078] These sealing means 17 consist, for example, of a O-ring or square joint, made of a silicone-like material.

[0079] This back wall 16a presents a disc shape, very slightly tapered, in order to define a downward inclination of the periphery towards the center.

[0080] This form is useful to ensure an optimal collection of nutrient liquid L by gravity, at the end of the temporary immersion phase described below.

[0081] This bottom wall 16a also comprises a central concave part 16a2, open on the side of the upper chamber 3 and provided with a central through hole 16a3, to receive a part of the means 5 for the treatment of the nutritive liquid between the chambers 3 and 4.

[0082] The transfer means 5 comprise a filter element 18, in the form of a disc, which is brought to the level of this central concave part 16a2, in order to delimit together a reserve 19 for the flow of fluids (nutrient liquid and gas) .

[0083] The filter element 18 forms the central part of the bottom face 3c of the

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12/19 upper chamber 3. This filter element 18 consists of a kind of sieve that holds a screen 18a to be glued to a piece of plastic frame 18b.

[0084] The plastic frame part 18b comprises radial arms 18b1 in each of which holes 18b2 (in the case of three holes one centimeter apart) are opened for the distribution of the nutrient liquid L, and, if applicable, the overpressure gas, forwarding towards the upper chamber 3 (figures 2 and 3).

[0085] These means of transfer 5 also include:

- a complementary centerpiece 20 comprising a lower section 20a that constitutes a connection conduit, ensuring the water connection between the two chambers 3 and 4; and

- the aforementioned side flue 15, for introducing gas directly into the lower chamber 4.

[0086] The central part 20 is washed through the central hole 16a3 of the bottom wall 16a of the complementary basket 16. It comprises a longitudinal axis 20 'which extends coaxially in relation to the vertical axis 7' of the outer envelope 7.

[0087] Its connection duct 20a allows an immediate and total rise of the nutrient medium L.

[0088] For this purpose, this connection duct 20a extends the entire height of the lower chamber 4.

[0089] The ends of this connection duct 20a are provided with holes, namely:

- a lower axial orifice 20a1, opening in the vicinity of the lower face 4c of the lower chamber 4 (or in other words the upper face of the bottom floor 9); and

- two upper lateral holes 20a2, opening into the middle of the reserve 19 of the upper chamber 3.

[0090] The upper holes 20a2 of the connection duct 20 are diametrically opposed; they extend along a radial axis 20a2 ', perpendicular to the longitudinal axis 20'of this centerpiece 20 (figures 2 and 4).

[0091] This last structural feature, in combination with the

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13/19 the plurality of holes 18b2 of the filter element 18 and with the particular dimensional characteristics of the upper chamber 3, allows for an optimal distribution of the nutrient liquid L, when rising, from the lower chamber 4 to the upper chamber 3 and participates in improving the mixing of plant material M.

[0092] To be complete, that centerpiece 20 comprises an axial extension 20b, following the connection duct 20a on the side of the upper holes 20b. This axial extension 20b is intended to extend over the height of the upper chamber 3 and to be pushed axially towards the bottom element 9 by the complementary cover 12.

[0093] To complement the lid 12, the upper contour 10 of the outer casing 7 is provided, on its periphery, with four ferrule parts 10a, defining between them four peripheral openings 10b (figure 3).

[0094] For its connection, the cover 12 is provided with four recessed insertion fins 12a, regularly distributed on its periphery, designed to each come to be lodged (I) by a vertical translation, through one of the peripheral openings 10b, then (II) by a clockwise rotation movement, under one of the ferrule parts 10a (figures 2 and 6).

[0095] The watertightness of the cap 12 connected on the outer casing 7 is ensured by complementary annular structures of the rib / groove 21 type arranged on the surfaces in support of this cap 12 and the upper contour 10 of the peripheral casing 7, in combination with a joint tightness rating 22 (figure 6).

[0096] This cover 12 is also equipped with projecting elements 12b, for the manual operation in rotation over the upper contour 10 of the outer envelope 7 (figures 2 and 3). These projecting elements 12b also together form a receiving surface on which the bottom element 9 of a superimposed container 1 can be placed for storage purposes.

[0097] An outlet 12c, located on the lid 12, allows the gas to pass between the inside and the outside of the container 1, in one direction as in the other. This outlet 12c is advantageously connected to a hydrophobic sterilizing filter F2,

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14/19 represented very schematically in figure 1.

[0098] This exit 12c allows, when the nutrient liquid rises, in a first stage the evacuation of the gas contained in the upper chamber 3, pushed by the nutritive liquid, then in a second stage, evacuate the overpressure while continuing to be fed.

[0099] The cover 12 taken over the opening 11 is maneuverable by rotation over an angular sector (in the case of an eighth turn), according to two inverse directions to obtain two limit positions:

- a locked position (figures 1 and 2), in which, each of the locking fins 12a is positioned under one of the ferrule parts 10a, thus ensuring four clamping zones; and

- an unlocked position (figure 3), in which the locking fins 12a are dissociated from the ferrules 10a and come to be positioned through the peripheral openings 10b.

[0100] In the locked position, the cap 12 exerts a supporting force towards the bottom element 9 on, on the one hand, the axial extension 20b of the center piece 20 (figure 2) and, on the other hand, the upper contour free 16b1 from the tubular side wall 16b of the inner basket 16 (figure 6). The lid 12 in this case constitutes the upper face 3b of the upper chamber 3 of the container 1.

[0101] The outer part 6, the inner basket 16, the central part 20 and the lid 12, constituting the container 1, are advantageously manufactured in a transparent autoclavable plastic material, for example, in polycarbonate for clinical use.

[0102] The transparency of this material will allow to optimize the luminosity provided to the cultivated vegetable matter.

[0103] In addition, these different parts 6, 16, 20 and 12 are connected by simple fitting of complementary shapes, without additional means of connection, which facilitates, on the one hand, the operations of renewing the nutrient medium between two subcultures, and, on the other hand, the assembly and disassembly operations, when cleaning, but also sterilization operations intervening between two culture operations.

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15/19 [0104] The operation of this container 1 is developed below in relation to figures 7 and 8.

[0105] Initially, the different parts 6, 16, 18, 20 and 12 constituting container 1 are conveniently sterilized.

[0106] The plant material to be cultivated M is deposited on the bottom wall 16a of the inner basket 16 which is then introduced in the outer part 6, in order to rest on the junction part 7c of its outer envelope 7.

[0107] This plant material M consists, for example, of plant tissues grown in vitro, such as micro-stems and micro-cuttings, lime or somatic embryos. [0108] The positioning of the lid 12 on the outer casing 7 then allows to close the outer part 6 hermetically.

[0109] In addition, this cap 12 exerts an impulse on the free upper contour 16b1 of the side wall 16b of the basket 16, which causes the sealing means 17 to be compressed, of the junction part 7c of the outer casing 7 by the wall bottom 16a of the inner basket 16, thus defining the two chambers 3 and 4 of the container 1 (figure 7).

[0110] As mentioned above, cover 12 also exerts a support on the upper contiguous end of the axial extension 20b of the centerpiece

20.

[0111] The nutrient medium L, in liquid form, is, for example, taken in the lower chamber 4 of the container 1, before its sterilization, for its concomitant sterilization. This nutrient medium consists, for example, of a medium that contains the mineral salts of Murashige and Skoog (1962) or formulas derived from this formulation, of sugar, vitamins and vegetable hormones, in order to ensure multiplication and / or growth of plant material.

[0112] In the resting phase (figure 7), this nutrient medium L is thus located in the lower chamber 4.

[0113] In the active phase (figure 8), an overpressure is applied to the lower chamber 4, by introducing a gas (for example, air) through the lateral duct 15. This gas is, in this case, sterilized, when passing through the hydrophobic F1 filter attached

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16/19 in this lateral duct 15.

[0114] This overpressure pushes the nutrient medium L, thus causing it to rise through the connection duct 20a, then through the filter element 18, from the lower chamber 4 to the upper chamber 3.

[0115] When routing, the nutrient liquid L is effectively distributed through the upper outlet holes 20a2 of the connection duct 120, after the filter element 18, ensuring an effective mixture of plant material M.

[0116] Maintaining the overpressure feed makes it possible to maintain the nutrient medium L in the upper chamber 3 of the container 1, for an effective immersion of the plant material M [0117] Advantageously, the overpressure is applied for a period longer than that strictly necessary to raise the nutritious liquid L. The overpressure can, for example, be applied for a duration of one to two minutes, both or four hours. This causes a bubbling in that nutrient liquid L located in the upper chamber 3, improving its mixture and the immersion of the plant material M [0118] This maintenance of the overpressure also causes a renewal of the atmosphere contained inside the container 1. This aeration is particularly interesting, because it allows to avoid the harmful effect of an accumulation of gas inside the container 1 (notably ethylene and carbon dioxide).

[0119] This bubbling phenomenon is in this case optimized by the particular structure of the upper outlet holes 20a2 of the connection duct 20a, associated with the filter element 18, and the measured shape of the upper chamber 3.

[0120] The energetic bubbling thus obtained participates in an effective mixture of biomass, which is conducive to a homogeneous and synchronous development of plant material M.

[0121] Preferably, overpressure is achieved by introducing compressed air. This is provided by a pump that releases the oil-free compressed air. In order to protect the sterile internal environment of container 1, the air inlet is protected by the hydrophobic sterilizing filter F1;

[0122] Stopping the overpressure supply causes a return, for example

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17/19 gravity, of the nutrient medium L, from the upper chamber 3 to the lower chamber 4. [0123] The nutrient liquid L then undergoes an inverse route, passing successively through the filter element 18, the cavity 19 and the conduit connection 20a.

[0124] This return causes air to enter through outlet 12c of cover 12; in order to protect the sterile internal environment of container 1, this air inlet 12c is protected by the hydrophobic sterilizing filter F2.

[0125] The programming of the immersion cycles, and the operation of the container set 1, autonomously, can be carried out simply with the aid of a programmer or a programmable electrical outlet. This outlet allows you to control the operation of the pump, and therefore the rhythm of the immersions and their duration. [0126] To recover the plant material M, the operator just needs to open the container 1, by an appropriate maneuver of the lid 12.

[0127] With the cover 12 dissociated, the operator can access the upper chamber 3 through the upper opening 11 of the outer part 6, and eventually extract the inner part 16 in the form of a basket.

[0128] When a culture cycle is finished, the different parts 6, 12, 16, 18, 20, constituting container 1, can be easily separated for cleaning and sterilization, aiming at a later culture cycle.

[0129] Container 1, according to the invention, is adapted to be handled and transported by an operator. It is, moreover, adapted to be stored on shelves, superimposed one above the other; it can also be introduced in different laboratory devices (autoclave, laminar flow basket, etc.).

[0130] This container offers the following advantages:

- it allows an important measurement of the plant material, to improve access to light and, therefore, stimulate the photosynthetic capacity of seedlings and their behavior during the delicate stages of acclimation;

- it increases the mixture of plant material, to improve access to light and, therefore, stimulate the photosynthetic capacity of seedlings and their behavior, during the delicate stages of acclimatization;

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18/19

- it increases the mixture of plant material, during temporary immersions, notably due to a phenomenon of turbulence after propulsion of the liquid medium in the upper chamber, to improve the synchronization of plant development and, therefore, its homogeneity; this phenomenon allows to considerably reduce the manipulation work related to the selection and tripling of plant material; it also allows a random repositioning of the plants at the end of each immersion cycle and is thus opposed to the appearance of a stratification phenomenon;

- it is very simple to operate, robust, easy to transport and with a reduced number of parts to facilitate its use at the industrial level and reduce production costs;

- it facilitates the operations of marking plant material and changing the nutrient medium, due to its monoblock aspect, light and easily manipulated (easy opening of the lid and easy transfer of biomass, thanks to the internal basket);

- it has dimensions that give it a sufficient and interesting volume for industrial use, being limited so as not to generate complications when handling (transport, autoclaving, installation on the culture shelves, handling under laminar flow basket).

[0131] This container is, moreover, perfectly adapted to different plant materials (cell cultures, elements, seedlings).

[0132] It finds an application notably in the following domains:

- multiplication of plant species by micropropagation;

- multiplication of plant species by somatic embryogenesis;

- multiplication of “hairy roots” for the production of secondary metabolites or studies of functional genomics; and

- multiplication of plant cells for the production of secondary metabolites or for the amplification of embryogenic material.

[0133] As an example, tests allowed to observe a significant improvement in the plan of regeneration of plants from somatic embryos of coffee by

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19/19 application of the invention, going from 39%, for the previous RITA container to 90% for the container, according to the invention.

Claims (10)

1. Container for the in vitro culture of plant material, by temporary immersion, container (1) which has two overlapping chambers (3, 4), namely: an upper chamber (3), intended to contain this plant material (M ) to be cultivated and a lower chamber (4), designed to contain a nutritive liquid (L), chambers (3, 4) which are each bounded by a side face (3a, 4a), by an upper face ( 3b, 4b) and a lower face (3c, 4c), a container (1) which still has means (5) for the temporary transfer of at least a part of that nutrient liquid (L), from that lower chamber (4 ) up to that upper chamber (3), characterized by the fact that this lower face (3c) of the upper chamber (3), intended to support this plant material (M), has a horizontal surface (S) between 0.02 and 0 , 07 m ² , and because this side face (3a) of the upper chamber (3) has a height (H) is between 40 and 130 mm, in which the means (5) for the transfer temporary liquid nutrient (L) has -one or several passage (s) (20a) for the water connection of the upper (3) and lower (4) chambers and - means consisting of a side tube (15) for the introduction of a gas in that lower chamber (4), in order to generate, on the one hand, a passage of the nutrient liquid (L), from that lower chamber (4) to that upper chamber (3) by a gas overpressure in that lower chamber ( 4) and, on the other hand, a return by gravity of this nutrient liquid (L) in that lower chamber (4), when this gas overpressure is stopped, in which the connection passage (20a) between the chambers (3,4) consists of a conduit (20a) that extends vertically to the lower chamber (4), that conduit (20a) being equipped with one or more lower orifice (s) (20a1) opened in the lower face (4c) of that lower chamber (4) and one or more upper orifice (s) (20a2) opened in the lower face (3c) of the upper chamber (3). 2. Container according to claim 1, characterized by the fact that the upper chamber (3) has a general cylindrical shape, whose lower face (3c) has a radius (R) that is between 75 and 150 mm, and whose face lateral (3a) has a height (H) that is between 50 and 90 mm. Petition 870190135540, of 12/18/2019, p. 10/15 2/3 Container according to either claim 1 or claim 2, characterized in that the container (1) has an outer envelope (7) bounded by a lower contour (8) to which a bottom element (9) is attached and by an upper contour (10) that delimits an opening (11) hermetically closed by a removable cover (12), an external wrap (7) which comprises two parts, one, lower (7b) and the other, upper (7a) , separated by an interlayer inner wall (16a), these lower (7b) and upper (7a) parts forming, respectively, the lateral faces (4a, 3a) of the lower chamber (4) and the upper chamber (3), and this interlayer inner wall (16a) forming the upper face (4b) of the lower chamber (4) and part or all of the lower face (3c) of the upper chamber (3). 4. Container according to claim 3, characterized by the fact that the internal interlayer wall (16a) belongs to a complementary internal part (16), cooperating in an airtight manner as an associated external envelope (7). 5. Container according to claim 4, characterized by the fact that the complementary inner part (16) has a side wall (16b) that rises from the contour of the interim inner wall (16a) to form a structure together (16 ) in the form of a basket, that side wall (16b) taking the shape of the outer envelope (7) over a part or the entire height of its upper part (7a). 6. Container according to either of claims 4 or 5, characterized in that the upper (7a) and lower (7b) parts of the outer wrap (7) are connected by a joining part (7 c) that forms flange, and in which a peripheral strap (16a1) of the inner intermediate wall (16a) of the complementary intermediate piece (16) rests on that flange part (7c) by means of watertightness (17). 7. Container according to any one of claims 1 to 6, characterized in that part or all of the bottom face (3c) of the upper chamber (3) has a filter element (18) obtaining a reserve (19), into which the upper orifice (s) (20a2) of the connection conduit (20a) flows. 8. Container according to claim 7, characterized in that the filter element (18) has a plurality of holes (18b2) for the Petition 870190135540, of 12/18/2019, p. 11/15 3/3 distribution of the nutritive liquid (L) and, if it is the case of the overpressure gas, forwarding towards the upper chamber (3). 9. Container according to either of claims 7 or 8, considered in combination with any of claims 3 to 6, characterized in that the inner inner wall (16a) comprises a central concave part (16a2), open on the side of the upper chamber (3) and at the level from which the filter element (18) is taken to form the reserve (19). 10. Container according to any one of claims 1 to 9, characterized by the fact that the lateral duct (15) that flows through its lateral face (4a) and disposed on the side of its upper face (4b).