Classifications

• • 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
• • 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
  • A01G9/00—Cultivation in receptacles, forcing-frames or greenhouses; Edging for beds, lawn or the like
  • A01G9/02—Receptacles, e.g. flower-pots or boxes; Glasses for cultivating flowers
• • 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
  • A01H4/002—Culture media for tissue culture

Definitions

• the invention relates to the cultivation of plant material, and particularly a container for the in vitro culture of plant material, by temporary immersion, under sterile conditions.
• This practice has many advantages, but generates a relatively high labor cost which is related to the frequency of handling necessary to transplant the plant tissues on gelled nutrient media, but also to the amount of containers and their cleaning.
• temporary immersion a technique called "temporary immersion” has been developed, which consists, as its name suggests, in temporarily immersing (typically a few minutes per day) the plant material in culture (explants, cells , tissues, etc.) in a nutrient medium. Given flotation phenomena or growth phenomena, this immersion in the nutrient medium can be partial or total.
• This method thus makes it possible to benefit from the advantages of in vitro culture in a liquid medium, while avoiding problems of asphyxiation and friction. It also makes it possible to obtain a quality of development of the culture (cells, meristems, embryos, seedlings, etc.) which is particularly interesting in comparison with that obtained by other in vitro culture methods.
• the RITA container comprises two superimposed chambers, namely: an upper chamber for receiving the plant material, and a lower chamber for containing the nutrient medium in a liquid form.
• the dimensions of the upper chamber give the latter a vertically elongated shape, with a height and a diameter of about 60 mm and 108 mm, respectively ; the ratio of horizontal surface area (in m 2 ) to height (in m) is thus of the order of 0.15.
• This container comprises a unitary body forming an outer part closed by a lid, and a basket for separating the two chambers.
• An inlet on the lid allows the supply of a positive pressure conducted in the lower chamber by a tube opening into a bell.
• the overpressure causes a rise of a portion of the nutrient medium from the lower chamber to the upper chamber, ensuring immersion and mixing of the plant material; stopping the overpressure allows the return, by gravity, the nutrient medium in the lower chamber.
• the immersion of the plant material is thus achieved by displacement of the nutrient medium, pushed by an overpressure, and not by the mechanical movement of the support basket. This absence of mechanical movements increases the reliability of the device.
• This container allows in vitro culture in sterile conditions, limiting the risk of infection, while ensuring good reliability of the culture during a significant number of immersion cycles.
• Such a container is also interesting in terms of maintenance, disassembly and washing; it is particularly easily sterilizable between two cycles of use.
• the inventors have observed that, in the upper chamber, the plant material is present in a relatively compact form, which is unfavorable to stirring, to good exposure to light and finally to the harmonious development of the plant material.
• This biomass also tends to develop in a heterogeneous manner over the height of the upper chamber, resulting in a stratification phenomenon.
• the upper part of the biomass more exposed to light and little physical constraint, develops faster than the central part of this biomass, physically compressed and hindered for its access to light.
• the plant material evolves differently according to its position in the biomass, which creates a stratification with a gradient of development from bottom to top.
• the inventors have developed a container whose upper chamber is significantly wider than the upper chamber equipping the container of the prior art described in document FR-2,760,743 mentioned above, while maintaining a similar height.
• the Applicant proposes a new structure for an in vitro culture container of plant material, by temporary immersion, operating in a manner similar to that described in document FR-2,730,743.
• the corresponding container comprises two superimposed chambers, namely - an upper chamber, intended to contain said plant tissues to be cultivated, and - a lower chamber, intended to contain a nutritive liquid, which chambers are each delimited by a lateral face, by a face upper and lower side, which container further comprises means for the temporary transfer of at least a portion (preferably substantially all or all) of said nutrient liquid from said lower chamber to said upper chamber.
• the underside of the upper chamber intended to carry the plant material, has a horizontal surface between 0.02 and 0.07 m 2 ; in addition, the lateral face of the upper chamber has a height of between 40 and 130 mm.
• the ratio of the horizontal surface (in m 2 ) / height (in m) of the upper container chamber according to the invention is thus between about 0.50 and 1.75.
• horizontal surface of the lower face of the upper chamber is meant the surface delimited by the contour of this lower face, that on a horizontal plane passing through this contour.
• This horizontal surface still corresponds to the horizontal size of this lower face of the upper chamber.
• This horizontal surface is preferably between 0.03 and 0.04 m 2 , and more preferably of the order of 0.037 m 2 .
• Such a container allows in particular optimal spreading of the biomass in the upper chamber, with less competition for access to light.
• This container also allows a homogeneous and synchronous development of the plant material, thus eliminating the usual phenomena of stratification.
• the upper chamber has a generally cylindrical shape, of which:
• the lower face has a radius of between 75 and 150 mm, preferably of the order of 100 to 120 mm, and more preferably of the order of 1 10 mm (that is to say a diameter between 150 mm and 300 mm, preferably of the order of 200 to 240 mm, and more preferably of the order of 220 mm), and the lateral face has a height of between 50 and 90 mm, more preferably of the order of 60 to 70 mm.
• this ratio is advantageously much greater than 1, preferably between 1, 6 (150/90) and 6 (300/50), more preferably between 2.2 (200/90) and 4.8 (240/50), and again between 3.14 (220/70) and 3.66 (220/60).
• 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.
• the container comprises an outer envelope delimited by a lower edge to which is connected a bottom member and an upper edge defining an opening sealed by a removable cover;
• the outer casing has two parts, one lower and the other upper, separated by an intermediate inner wall, said lower and upper parts forming, respectively, the lateral faces of the lower chamber and the upper chamber, and said wall; inner insert forming the upper face of the lower chamber and at least a portion, preferably substantially all or all, of the lower face of the upper chamber.
• the intermediate inner wall advantageously belongs to an inner insert, cooperating hermetically with the associated outer casing.
• This inner insert advantageously comprises a side wall rising from the contour of the intermediate inner wall to form together a basket-shaped structure, said side wall conforming to the outer casing on at least a portion, preferably substantially all of or the whole of the height of its upper part.
• the upper and lower portions of the outer casing are advantageously connected by a shoulder portion; and a circumferential strip of the inner wall of the insert inserted resting on said shoulder portion by means of sealing means.
• the means for the temporary transfer of the nutritive liquid comprise - at least one passage for the water connection of the upper and lower chambers, and - means for introducing a gas into said lower chamber, so as to generate, on the one hand, a path of at least a portion (preferably almost all or all) of the nutrient liquid from said lower chamber to said upper chamber by a gas overpressure in said lower chamber and, secondly, a gravity return of said nutrient liquid in said lower chamber when stopping said gas overpressure.
• connection passage between the chambers advantageously consists of a duct extending over the height of the lower chamber, which duct is provided with at least one lower orifice opening near the lower face of said lower chamber and at least one upper opening opening at the lower face of the upper chamber.
• At least a portion of the lower face of the upper chamber is then advantageously constituted by a filter member closing a reservation in which opens or the upper openings of the connecting duct;
• this filtering member advantageously comprises a plurality of orifices, for the distribution of the nutritive liquid, and optionally the booster gas, moving towards the upper chamber;
• the intermediate inner wall advantageously comprises a concave central portion, open on the side of the upper chamber and at which the filter member is attached to form the reservation.
• connection duct is advantageously terminated by at least two upper orifices regularly distributed over the periphery of said duct.
• the gas introduction means in the lower chamber consist of a duct opening through its side face and formed on the side of its upper face.
• the container comprises (i) a lid provided with at least two reentrant gripping wings, distributed on its periphery, and
• This cover attached to said opening is operable in rotation between - a locked position, wherein each of said attachment fins is positioned below one of said flange portions, and - an unlocked position, wherein said fins of hooking are dissociated from said collars.
• FIG. 1 is a general perspective view of the container according to the invention.
• Figure 2 schematically shows the container of Figure 1, in a vertical sectional plane
• FIG. 3 shows the container of Figure 1, an exploded manner
• FIG. 4 shows an enlarged detail IV of Figure 2, corresponding to the upper end of the water connection conduit between the upper and lower chambers;
• FIG. 5 illustrates in an enlarged manner the detail V of Figure 2, corresponding to the sealing means formed between the outer casing and the basket insert;
• FIG. 6 shows an enlarged detail VI of Figure 2, corresponding to the sealing means formed between the outer casing and the removable cover;
• FIGS. 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 (FIG. 7) and a temporary immersion phase in which the nutritive liquid is moved to the upper chamber to submerge / drown the plant material (Figure 8).
• the container 1, shown generally in Figures 1 to 3, is a device that is suitable for in vitro culture of plant material by temporary immersion, under sterile conditions.
• This container 1 consists of a unitary device, here in the general shape of barrel, having a general axis 1 '.
• this container 1 comprises two superposed chambers 3 and 4, each here in the general shape of a right circular cylinder, namely:
• chambers 3 and 4 are each delimited by an annular lateral face 3a and 4a, by an upper face in the general shape of disk 3b and 4b, and by a lower face also in the general shape of disk 3c and 4c.
• This container 1 further comprises means 5 for the temporary transfer of at least a portion (preferably almost all or all) of the nutrient liquid L from the lower chamber 4 to the upper chamber 3, which will be described subsequently in connection with FIGS. 2 and 3.
• the means 5 for the temporary transfer of the nutritive liquid L are structured to generate, on the one hand, a path of this nutritive liquid L from the lower chamber 4 until the upper chamber 3 by a gas overpressure in said lower chamber 4 and, secondly, a gravity return of said nutrient liquid in said lower chamber 4 when stopping this gas overpressure.
• the upper chamber 3 is dimensioned to limit the phenomena of stratification and compaction of cultivated plant material M, and also to optimize access to light of this material.
• the circular underside 3c of the upper chamber 3, intended to carry the plant tissue M here has a circular contour 3cJ_ whose radius R is of the order of 1 10 mm.
• this radius R is advantageously between 75 and 150 mm, and preferably between 100 and 120 mm.
• This contour 3c_ extends in a horizontal plane P, perpendicular to the general axis 1 'of the container 1, on which it delimits a surface or a horizontal area S ( Figure 2) of the order of 0.037 m 2 .
• this horizontal surface S is between 0.02 m 2 and 0.07 m 2 .
• the lateral face 3a of this upper chamber 3 has a height H of the order of 60 to 70 mm.
• this height H is advantageously between 40 and 130 mm, more preferably between 50 and 90 mm.
• This upper chamber 3 thus has a generally cylindrical shape relatively flattened.
• 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 4b upper), and a height of the order 75 mm.
• the surface of the lower face 3c of the upper chamber 3 is thus here 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.
• the container 1 consists of an assembly of different parts which are described in detail below with reference to FIGS. 2 and 3.
• the container 1 comprises, first of all, an integral outer part 6, comprising a lateral outer envelope 7 which is delimited by:
• an upper rim 10 delimiting an opening 11 intended to be sealed hermetically by a removable cover 12.
• the outer casing 7 here comprises three annular parts distributed over its height, namely a tubular upper portion 7a, a tubular lower portion 7b and a joining portion 7c forming an annular shoulder in the form of a ring.
• the upper 7a and lower 7b portions of the outer shell 7 each have a constant diameter, or at least approximately constant, on their respective heights.
• the upper part 7a of the outer casing 7 has a larger diameter with respect to the diameter of the lower part 7b; the upper parts 7a and 7b of the outer casing 7 are thus connected, at their opposite edges, by the shoulder portion 7c_.
• the upper portions 7a and 7b of the outer casing 7 additionally have an identical height, or at least approximately the same, with respect to the upper chamber 3 and the lower chamber 4, respectively.
• the upper portion 7a of the outer casing 7 advantageously has a height of between 50 and 90 mm, more preferably of the order of 60 to 70 mm.
• the lower part 7b of the outer casing 7 and the bottom element 9 form, respectively, the lateral face 4a and the lower face 4c of the lower chamber 4.
• This lower part 7b of the casing 7 is provided with a lateral duct 15, for the introduction of gas (for example air) directly into the lower chamber 4.
• gas for example air
• a hydrophobic air filter F1_ shown very schematically in FIG. 1, is advantageously fixed to this lateral duct 15 in order to ensure sterility inside the container 1.
• the lateral duct 15 opens through the lateral face 4a of the lower chamber 4, and is formed on the side of its upper face 4b.
• the longitudinal axis 15 'of this lateral duct 15 is oriented along an upward slope towards the periphery, to limit the risk of flow of the nutrient liquid L through this lateral duct 15 to the outside and thus to prevent the associated filter ⁇ be wet and rendered inoperative.
• the shoulder portion 7c serves to support a basket-shaped insert 16, which is intended to separate the two chambers 3 and 4 of the container 1 and which is intended to form at least part of the faces of the chamber superior 3.
• This inner insert 16 includes:
• the tubular side wall 16b internally doubles the upper part 7a of the peripheral envelope 7, to form together the lateral face 3a of the upper chamber 3.
• This tubular side wall 16b matches the outer shell 7 over the height of its upper portion 7a; the diameter of the outer surface of the side wall 16b thus corresponds, with the clearance, to the diameter of the inner surface of the upper part 7a of the outer casing 7.
• the bottom wall 16a constitutes an intermediate inner wall which forms the upper face 4b of the lower chamber 4 and part of the lower face 3c of the upper chamber 3.
• sealing means 17 consist for example of an O-ring or square, made of a material of the silicone type.
• This bottom wall 16a has a disc shape, very slightly frustoconical, so as to define a downward slope from the periphery to the center.
• This form is useful for ensuring optimal collection of the nutrient liquid L by gravity, at the end of the temporary immersion phase described later.
• This bottom wall 16a further comprises a concave central portion 16a2, open on the side of the upper chamber 3 and provided with a through central orifice 16a3, to receive a portion of the means 5 for the transfer of the nutrient liquid between the chambers 3 and 4 .
• the transfer means 5 comprise a disk-shaped filter member 18 which is attached at the level of this concave central portion 16a2, so as to delimit together a reservation 19 for the flow of fluids (nutrient liquid and gas).
• the filtering member 18 constitutes the central part of the lower face 3c of the upper chamber 3.
• This filtering member 18 consists of a kind of screen comprising a cloth 18a bonded to a plastic reinforcement piece 18b.
• the plastic reinforcing member 18b has radial arms 18bJ_ in each of which are formed orifices 18b2 (here three orifices spaced one centimeter apart from each other), for the distribution of the nutrient liquid L, and where appropriate booster gas, traveling towards the upper chamber 3 ( Figures 2 and 3).
• These transfer means 5 further include:
• a central insert 20 comprising a lower section 20a constituting a connection duct, providing the water connection between the two chambers 3 and 4, and
• the central piece 20 is attached through the central opening 16a3 of the bottom wall 16a of the insert basket 16. It comprises a longitudinal axis 20 'which extends coaxially with respect to the vertical axis 7' of the outer casing 7.
• Its connecting duct 20a allows an immediate and total recovery of the nutrient medium L.
• this connecting duct 20a extends over the entire height of the lower chamber 4.
• this connecting duct 20a are provided with orifices, namely:
• the upper openings 20a2 of the connecting pipe 20 are diametrically opposed; they extend along a radial axis 20a2 ', perpendicular to the longitudinal axis 20' of this central piece 20 ( Figures 2 and 4).
• This last structural feature in combination with the plurality of orifices 18b2 of the filtering member 18 and with the particular dimensional characteristics of the upper chamber 3, allows in particular an optimal distribution of the nutrient liquid L during its ascent from the lower chamber 4 to the upper chamber 3 and participates in improving the brewing of plant material M.
• this central piece 20 comprises an axial extension 20b, continuing the connecting duct 20a on the side of the upper openings 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 cover 12 reported.
• the upper edge 10 of the outer casing 7 is provided on its periphery with four flange portions 10a defining between them four peripheral openings 10b (FIG. 3).
• the cover 12 is provided with four reentrant gripping fins 12a, regularly distributed on its periphery, intended to each be housed (i) by a vertical translation, through one of the peripheral openings 10b then (ii) by a clockwise rotational movement, below one of the flange portions 10a ( Figures 2 and 6).
• the tightness of the lid 12 assembled on the outer casing 7 is provided by complementary annular structures of the rib / groove type 21 formed on the bearing surfaces of this cover 12 and the upper edge 10 of the peripheral casing 7, combination with a seal 22 ( Figure 6).
• This cover 12 is further equipped with projecting members 12b, for the manual operation in rotation on the upper edge 10 of the outer casing 7 (FIGS. 2 and 3).
• projecting members 12b also together form a receiving surface on which the bottom element 9 of a superimposed container 1 can be placed for storage purposes.
• a vent 12c located on the cover 12, allows the passage of gas between the inside and the outside of the container 1, in one direction as in the other.
• This vent 12c is advantageously connected to a hydrophobic sterilizing filter 2, shown very schematically in FIG.
• This vent 12c thus enables the evacuation of the gas contained in the upper chamber 3, pushed by the nutritive liquid, during the rise of the nutrient liquid, and then, in a second step, to evacuate the continuous overpressure of to be fed.
• the lid 12 attached to the opening 1 1 is operable by rotation on an angular sector (here of an eighth of a turn), in two reverse directions to obtain two end-of-travel positions:
• the cover 12 comes to exert a bearing force directed towards the bottom element 9 on the one hand, the axial extension 20b of the central piece 20 ( Figure 2) and, on the other hand, the free upper edge 16bJ_ of the tubular side wall 16b of the inner basket 16 (FIG. 6).
• the lid 12 here constitutes the upper face 3b of the upper chamber 3 of the container 1.
• the outer part 6, the inner basket 16, the central piece 20 and the lid 12, constituting the container 1, are preferably each made of an autoclavable transparent plastic material, for example polycarbonate for clinical use.
• these different parts 6, 16, 20 and 12 are assembled by simply interlocking complementary shapes, without additional connecting means, which facilitates, on the one hand, nutrient medium renewal operations between two sub-cultures, and on the other hand, assembly and disassembly operations during cleaning operations, but also sterilization occurring between two culture operations.
• the various parts 6, 16, 18, 20 and 12 constituting the container 1 are suitably sterilized.
• the plant material to be cultivated M is deposited on the bottom wall 16a of the inner basket 16 which is then introduced into the outer part 6 so as to rest on the joining part 7c of its outer shell 7.
• This plant material M consists, for example, of plant tissues cultivated in vitro, such as micro-stems and microboutures, callus or somatic embryos.
• This cover 12 additionally exerts a push on the upper free edge 16bJ. of the side wall 16b of the basket 16, which causes the seals 17 to crush. the connecting portion 7c of the outer casing 7 by the bottom wall 16a of the inner basket 16, thereby defining the two chambers 3 and 4 of the container 1 ( Figure 7).
• the cover 12 also bears on the adjoining upper end of the axial extension 20b of the central piece 20.
• the nutrient medium L in liquid form, is for example reported in the lower chamber 4 of the container 1 before sterilization, for their concomitant sterilization.
• This nutrient medium consists for example of a medium containing the mineral salts of Murashige and Skoog (1962) or formulas derived from this formulation, sugar, vitamins and plant hormones, so as to ensure the multiplication and / or growth of plant material.
• this nutritive medium L is thus located in the lower chamber 4.
• an overpressure is applied in the lower chamber 4, by the introduction of a gas (for example air) through the lateral duct 15.
• a gas for example air
• This gas is here sterilized during the transition to through the hydrophobic filter H 2 attached to this lateral duct 15.
• This overpressure pushes the nutritive medium L, thus causing it to rise through the connecting duct 20a and then through the filtering member 18, from the lower chamber 4 to the upper chamber 3.
• the overpressure is applied for a period longer than that strictly necessary for the recovery of the nutritive liquid L.
• the overpressure may, for example, be applied for a period of one to two minutes, every two or four hours.
• This maintenance of the overpressure also causes a renewal of the atmosphere contained inside the container 1.
• This ventilation is particularly interesting because it avoids the detrimental effect of an accumulation of gas inside the container 1 (including ethylene and carbon dioxide).
• This bubbling phenomenon is here optimized by the particular structure of the upper through openings 20a2 of the connecting pipe 20a, associated with the filtering member 18, and the spreading shape of the upper chamber 3.
• the energetic bubbling thus obtained contributes to an efficient mixing of the biomass, which is conducive to a homogeneous and synchronous development of the plant material M.
• the overpressure is obtained by the introduction of compressed air.
• compressed air is supplied by a pump delivering oil-free compressed air.
• the air inlet is protected by the hydrophobic sterilizing filter PL
• Stopping the feed of the overpressure causes the return, by gravity, of the nutrient medium L from the upper chamber 3 to the lower chamber 4.
• the nutritive liquid L then undergoes a reverse path, passing successively through the filter member 18, the cavity 19 and the connecting pipe 20a.
• This return causes an air inlet through the vent 12c of the cover 12; in order to protect the sterile internal environment of the container 1, this air inlet 12c is protected by the hydrophobic sterilizing filter 2.
• the programming of the immersion cycles, and the operation of the entire container 1, autonomously, can be carried out in a simple manner using a programmer or a programmable electrical outlet.
• a plug makes it possible to control the operation of the pump, and therefore the rhythm of the immersions and their duration.
• the cover 12 dissociated the operator can access the upper chamber 3 through the upper opening 1 1 of the outer part 6, and optionally extract the inner part 16 in the form of basket.
• the various parts 6, 12, 16, 18, 20 constituting the container 1 can be easily separated for cleaning and sterilization for a subsequent crop cycle.
• the container 1 according to the invention is adapted to be handled and transported by an operator. It is also adapted to be stored on shelves, superimposed one above the other; it can also be introduced in different laboratory devices (autoclave, laminar flow hood, etc.).
• This container 1 offers the following advantages:
• This container is also perfectly adapted to different plant materials (cell cultures, organs, seedlings).
• tests have shown a significant improvement in the regeneration of plants from somatic embryos of coffee by the implementation of the invention, from 39% for the prior container RITA 90% for the container according to the invention.

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• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Developmental Biology & Embryology (AREA)
• Environmental Sciences (AREA)
• Cell Biology (AREA)
• Botany (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)
• Hydroponics (AREA)
• Cultivation Receptacles Or Flower-Pots, Or Pots For Seedlings (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

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Cited By (8)

Citations (3)

• 2011
  • 2011-04-28 FR FR1153642A patent/FR2974580B1/fr active Active
• 2012
  • 2012-04-25 WO PCT/FR2012/050919 patent/WO2012146872A1/fr active Application Filing
  • 2012-04-25 BR BR112013027846A patent/BR112013027846B8/pt active IP Right Grant
  • 2012-04-25 MX MX2013012473A patent/MX357396B/es active IP Right Grant
  • 2012-04-27 AR ARP120101483A patent/AR086142A1/es unknown

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