WO2012156440A1

WO2012156440A1 - Container system for immersion growth regime

Info

Publication number: WO2012156440A1
Application number: PCT/EP2012/059111
Authority: WO
Inventors: Juan Carlos PÉREZ GUERRA; Juan Nivaldo PÉREZ PONCE
Original assignee: Sopet Nv
Priority date: 2011-05-18
Filing date: 2012-05-16
Publication date: 2012-11-22
Also published as: BE1019588A5

Abstract

The present invention relates to an immersion growth system (28) and incubator (29) containing such a growth system (28) and a method for the in-vitro culture of biological material. The immersion-growing system (28) comprises two similar containers, in particular a growth container (1) and a medium container (2), wherein both containers comprise coupling means (7, 8, 13) for coupling the medium container (2) to the growth container (1), characterized by the fact that the growth container (1) is designed to be placed on top of the medium container (2),and that the coupling means (7, 8, 13) are provided at the front side of both containers.

Description

CONTAINER SYSTEM FOR IMMERSION GROWTH REGIME Technical field The present invention relates to an immersion growth system and process for the in-vitro culture of biological material, more in particularly plant cells, tissues, organs and embryos, seedlings or plants.

State of the art

Temporary immersion systems for in-vitro plant culture have been described and grouped into 4 categories according to operation : i) tilting and rocker machines, ii) complete immersion of plant material and renewal of nutrient medium, iii) partial immersion and a liquid nutrient renewal mechanism, iiii) complete immersion by pneumatic driven transfer of liquid medium and without nutrient medium renewal. The positive effects of temporary immersion of in-vitro plant culture are shown by proliferation of shoots and micro-cuttings, micro-tuberization and somatic embryogenesis. Immersion time, in particular the duration and/or frequency, is the most significant parameter for the efficiency of the system. Optimization of the volume of culture medium and volume of the culture container, also improves considerably the efficiency, in particular by shoots proliferation. Temporary immersion in general, improves the quality of the plant material (Etienne and Berthouly, 2002). FR 2730743 describes a container for the in-vitro culture via temporary immersion under sterile conditions, in a sealed container with a lid at the top, provided with a segment for plant tissue and a lower segment for the nutrient medium. One inlet in the lid makes possible to create a positive pressure in a bell-shaped chamber in the lower segment, wherein culture medium is pressed upwards from the lower to the upper segment, submerging then the plant material. The excess pressure is released by means of a valve, keeping the pressure balanced during the stationary periods.

MXPA04003837 discloses an invention, which relates to a system and process for the in-vitro culture of biological material, more in particular plant cells, tissues, organs and embryos, seedlings or plants. The system is integrated with a bioreactor suitable for the in-vitro culture of biological material, and an apparatus for delivering an upward/downward movement of the bioreactor. The bioreactor is formed by a chamber, having at least two containers, and a central coupling element. This final element is located on the platform of a device, suitable for providing a programmable upward/downward and cyclic movement of the bioreactor. The biological material and the liquid growth medium are stored in the containers, which are made of transparent material. The central coupling element forms a part that is suitable for the fastening of both containers, which are placed axially on either sides of each container. The coupling element contains a round flat piece provided with grooves and openings, suitable for better flow of liquids and gases, and further comprises a various gas and liquid outputs, where filters can be attached. The volume of the bioreactor can be increased by addition of one or a number of hollow profiles, which are attached between them, or to the container by means of coupling elements. The bioreactor perform immersion and aeration cycles to the biological material as a result of the upward/downward movement of the device, that provides a liquid flow from the first container to a second container, generated under the influence of gravity, while the plants remain present in one of the containers. In addition, achieves the bioreactor the culture via temporary immersion during all possible phases of the in-vitro culture: induction, multiplication, growth and adjustment.

The Twin Flask system (® BIT), contains two separated bottles next to each other, one for the growth medium and the other for plant material, made of glass or plastic, connected to each other by silicone tubing, through which, by application of over-pressure the growth medium is transferred from one bottle to the other (Escalona et al, 1998).

The existing systems have some disadvantages, using most of the systems round- shaped containers (beaker, bottles ...) for storage of plant material. By using round containers, the maximum useful volume is not optimally used for the culture to grow. In the systems where the containers (plant) material and growth medium are in the same chamber, the pressure cannot be separately regulated in both containers. In the systems where two separate containers are provided for (plant) material and growth medium, there are many tubes and connections present, making the assembly difficult, and leading to an increased risk of contamination. Additionally, the use of the light sources is suboptimal, as the used bottles do not have the appropriate shapes. In these systems, the ratio is between the total working surface area versus used surface for the culture, sub- optimal. Furthermore, the current systems are labor intensive in their setup and use.

The present invention addresses the above problems, for which a maximum amount of plant material is grown per unit of surface area, and on the other hand, the pressure-dependent parameters can be optimized in each separate container, resulting in an improved quality of culture. A maximal light entrance is created and the shape of the bottles is optimal for an homogeneous culture growth. The reduced labor required for the setup and ease use of the system are two further advantages, resulting in lower user costs.

Summary

In first place, the present invention relates to an immersion growth system comprising two related containers, in particular, a growth container, suitable to hold the plant material, and a medium container, suitable to hold the growth medium, wherein both containers comprise coupling means (7, 8, 13) for coupling the medium container (2) to the growth container (1), characterized by the fact that the growth container (1) is designed to be placed on top of the medium container (2), and that the coupling means(7, 8, 13) are provided at the front side of both containers..

One of the advantages of placing the growth container on top of the medium container as presented in this invention, is an optimum ratio between the necessary surface area for the complete immersion growth system, related to the available surface area for culturing plant material in the growth container.

Another advantage of the present invention, is the optimal light entrance in the growth container, which is made preferable of transparent material, and it is located at the upper part of the system, receiving all light originated at the light source, placed just above the system. The light incidence in the growth container is also not affected by the presence of connections ports or openings ports in the upper side of the growth container.

Furthermore, the present invention has the advantage that each container is independent and with separated accessibility, and thus the pressure is separately adjustable for each container. If for example, growth medium needs to be added or changed, then, this can be performed without interfering with the growth container.

Furthermore, the invention relates to containers, suitable to be used in the herein described immersion growth system. These containers are, among others, characterized by the correlation between their dimensions, in which the containers are wider than high, and also deeper than high. The containers are beam-shaped. In another aspect, the invention relates to an incubator, comprising a spatial repetition of immersion growth systems, according to the present invention. One advantage of the incubator herein described, is the optimized spatial efficiency, because first, all connections ports of the containers are placed at the same front side, and second, the containers are beam-shaped, with a minimum lost space between various growth systems. The incubator, as described in the present invention, also leads to reduce labor intensity by its simplified setup and increased user-friendliness.

The present invention also relates to a method for the in-vitro culture of plant material using the herein described immersion growth system and/or the herein described incubator.

Description of the figures

The following figures show preferred forms of the invention. Figure 1 is a perspective view of a preferred form of the invention, and contains the following elements: a growth container 1, placed on top of a medium container 2, both of which are connected to each other by means of coupling means 13 which is connected to the medium connectors 7 and 8 on the front side of each respective containers 1 and 2. Furthermore, both said front sides, said front sides of both containers also comprise a closeable supply opening supply opening(3, 4), suitable for supplying material and/or medium to and for removing material and/or medium from said containers. In the same front side of each container 1 and 2, in which the medium connectors and the closeable supply openings are located, is located an air connector 5 on the growth container, and 6 on the medium container. These air connectors allow air transfer in and/or out of the containers through air connecting elements 12 and 12', which further are connected to air filters 11 and 11' to allow air sterility. The medium container includes also in the same front side an injection point 9. At the bottom side of the medium container are two lateral handles 10 located. The upper side of the medium container includes a slope 19 towards the front side. Figure 2 illustrates the bottom view of a growth container as presented in this invention, where the stability elements 14 and 16 are present.

Figure 3 illustrates the lateral view of a growth container as presented in this invention, where the stability elements 14 and 16 are present at the bottom side.

Figure 4 shows the lateral view of a medium container according to the present invention, in which the stability element 15 is visible on the top side, and the handle 10 in the bottom side of the medium container. Figure 5 illustrates the top view of a medium container according to the present invention, in which the stability elements 15 and 17 are visible.

Figure 6 shows a view of an incubator according to the present invention, in which elements 18 are placed side by side in a shelf, in which each level is provided with a light source 20 and wherein further, two pressure air networks 22 and 23 are provided, which respectively, connect all medium containers and growth containers to an air pressure variation facility 24, which can use vacuum generated at 25 (vacuum pump) and/or compressed air generated at 26 (air compressor). The complete operation of the incubator is controlled with a computer-controlled system/software 27.

Each element 18 comprises two immersion systems that are placed against each other with both rear sides in contact.

Detailed description

The present invention relates to an immersion growth system comprising two related containers, in particular, a growth container, suitable to hold the plant material, and a medium container, suitable to hold the growth medium, wherein both containers possess connection ports for interconnect them, characterized by the facts that, the growth container is made to be placed on top of the medium container, and by the presence of connection ports solely located at the front side of both containers As described herein, the term "immersion growth system" refers to a system in which plant material is cultured by means of immersion of the mentioned material, in a growth medium. Preferably, the immersion is temporary. In such systems, the advantages of liquid growth medium are combined with the advantages of aeration/ventilation of the plant material.

As described herein, the term "coupling means" refers to the set of medium connectors and medium transfer elements which make possible to transfer liquid growth medium, from a medium container to a growth container, and vise versa.

In one form of the present invention, the medium connectors are preferably hollow tubular elements, which are located at the front side of each respective container, orientated with the longitudinal axe of such tubular elements perpendicular to the mentioned front side of the containers. The medium connectors have an opening in the front of the container, and an opening at the opposite end of the medium connector, communicating the outside and inside of each container. In these medium connectors the medium transfer elements can be connected. Preferably, the medium connectors are integrated in the container body. More preferably, the connectors can have different forms and shapes, influenced by the desired working conditions, kind of plant material or added functionality (eg. reduced or increased opening size to reduce or increase the drainage time, net-layer at the inside opening to avoid small plant material can go through the connectors, plug-in connection to speed up the connection action, etc.).

In a preferred form of the invention, these medium transfer elements are flexible plastic tubes, preferably made of silicone. The flexible plastic tubes have a preferable inside diameter between 3 mm and 10 mm, more preferably between 5 mm and 8 mm. In a preferred form according to the invention, have the flexible plastic tubes an outer diameter of between 6 mm and 12 mm, more preferably between 8 mm and 10 mm.

The medium connectors make the transfer of growth medium possible from a container to another container, where the two containers are placed one above the other. Preferably, in such setup one container is a growth container and the other container is a medium container. Most preferably, the upper container is the growth container and the lower container is the medium container and both are connected by means of a medium transfer element, which is connected to the medium connectors of the two containers. The preferred form of the invention does not contain any hoses, tubes or other elements inside the containers. This implies a low risk of contamination.

The term "closable supply opening" is an opening in the front side of each container which is suitable for transfer materials in/from the respective containers, which can be closed by means of a reusable closure. In a preferred form the closure is a cap, more preferably a screw cap. The closure is most preferably a screw cap with an inner silicone gasket. The inner silicone gasket ensures good sealing of the containers. The closable supply openings are suitable for both, solid and liquid matter, to be transported through it. Preferably, the closable supply openings, in accordance with the invention, are suitable for the placement and disposal through it of plant material and growth medium into or out of each container.

As here described, the term "pressure connection means" refers to the set of air connectors which make possible the transfer of a gas mixture under certain pressure in/from each container, which is further connected with the air pressure variation facilities by means of air connecting elements.

In one form of the present invention are the air connectors preferably hollow tubular elements, which are located on the front side of each container, orientated with the longitudinal axe of such tubular elements perpendicular to the mentioned front side of the containers. The air connectors have an opening in the front of the container and an opening at the opposite end of the air connector, communicating the outside and inside of each container. Preferably, the air connectors are integrated into the container body. At these air connectors the air connecting elements can be connected. In a preferred form of the invention, these air connecting elements are hollow tubes, preferably flexible hollow tubes manufactured from plastic, and more preferably from silicone. The flexible plastic tubes have a preferable inside diameter between 3 mm and 10 mm, more preferably between 5mm and 8mm. In a preferred form according to the invention, have the flexible plastic tubes an outer diameter of between 6 mm and 12 mm, more preferably between 8mm and 10mm. The air connectors can have different forms and shapes (eg. reduced or increased opening size to reduce or increase the air in/out speed, plug-in connection to speed up the connection action, double external opening as a "Y" to allow the use of different kind of air quality or different kind of gasses together and/or separated, etc.). The air connecting element is also connected to an air filter device, placed in such way that the gas mixture passing through the pressure connection means is previously filtrated. The air filter, in accordance with the present invention, is further characterized by its pores size for filtration, which are between 0.1 μιτι and 0.45 μιτι, more preferably between 0.15 μιτι and 0.3 μιτι. By preference, they are hydrophobic air filters. These filters have the function of sterilize all gas mixture transferred to the containers. The pressure connection means, according to the invention, are suitable for changing the pressure of each container separately. Most preferably, the pressure in each container is separately controlled via the pressure connection means, which are connected to the container in question and with an air pressure variation facility.

In a preferred embodiment, both medium connectors and air connectors can be foreseen by an external threat for receiving a closure cap thereby sealing all connectors during and/or after sterilization.

The "pressure variation facility" is referred to a set device in charge of generating and control the desired air pressure, which is further transferred via an air net to the pressure connection means, and further to each connected container. The pressure variation facility can generate and control both, vacuum (negative pressure) via a vacuum pomp and compressed air (positive pressure) via an air compressor.

The term "growth medium" refers to a medium that is preferably liquid, and contains all nutrients which are required for the culture, and that are essential for the growth of the plant material. The advantage of the liquid medium is that the chemical composition in the liquid, is far more uniform than in a solid medium. In addition, a liquid medium is easily transferred through the system than a solid medium. An additional advantage of the liquid medium is the improved nutrient uptake for the plant material, and moreover, economically more advantageous than solid growth medium. Liquid growth medium, suitable for use in the present invention, include for example Murashige and Skoog (MS) growth medium, without further limitations of choice. By "culture" means any biological material that is suitable for being cultivated by temporary immersion. Non-limiting examples are: potato, sugarcane, banana, orchids, eucalyptus, spatiphyllum sp., pineapple, and paulownia sp., among many others.

As here described, "container" refers to a container that is preferably made of a transparent plastic polymer that withstands the conditions of autoclave sterilization. More preferably, the container made from polycarbonate (PC). The form of the container, according to the present invention, has a rectangular shape, preferably beam-shaped, wherein the container possesses six sides: a front, a back, a left-hand, a right-hand, an upper and a bottom side. In addition, the dimensions of the container, according to the present invention, have the following properties:

- the height of the container is smaller than the depth of the container, with a correlation ratio comprised between 1 : 2,30 and 1 : 1,65

the height of the container is smaller than the width of the container, with a correlation ratio comprised between 1 : 1,75 and 1 : 1.25

the width of the container is smaller than the depth of the container, with a correlation ratio comprised between 1 : 1,36 and 1 : 1,30

The growth container, according to the present invention, is a container that is preferably made of a transparent plastic polymer that withstands the conditions of autoclave sterilization. More preferably, the container made from polycarbonate (PC). In another preferred form, is the growth container manufactured from a material that can be written or printed. The preferred form of a growth container, according to the present invention, has a rounded top side, most preferably the upper side of the growth container, manufactured from a highly transparent material, showing a convex shape as seen from outside the container. This means that the upper side from the inner container view sticks out (concave shape). This has the advantages that first, the light incidence is optimized in the growth container and secondly, that the accumulation of water condenses on the inner top side is prevented. In the growth container, according to the invention, the upper side is free of connectors, supply openings and/or elements. Another form of a growth container includes a flat upper side. The preferred form of a growth container, according to the invention, comprises a closable supply opening, with a sufficiently large diameter to allow a hand goes through. Therefore the plant material is easily accessible resulting in an increased user-friendliness. Preferably, the closeable supply opening provides a prominent neck from the front side of the growth container, so that the end of the closeable supply opening protrudes more than the container, making more accessible the content in the growth container. The preferred form of a growth container includes a medium connector for the growth medium, and an air connector, which are located on the same front side as the closeable supply opening. In one form of a growth container, the bottom side is similar to the top side of the medium container. This means that both surfaces have similar dimensions and form, allowing both containers to be placed on top of each other. In a preferred form, the outside surface of the bottom side of the growth container is complementary to the outside surface of the top side of the medium container, on which the growth container is positioned . Furthermore, this form of a growth container is characterized by the presence of stability elements at the bottom side. These stability elements are a combination of both, grooves and protuberances of the bottom or top sides of a container. Furthermore, these stability elements are suitable to be connected with the corresponding stability elements (15, 17) from the medium container (2). Preferably, one container is a growth container and the other is a medium container. Most preferably, the container with stability elements on the bottom side is a growth container and the container with stability elements on the top side is a medium container. In the form of the invention, contains the growth container four stability elements at the bottom side. Two stability elements are grooves in the surface of the bottom side, and two stability elements are protuberances in the surface of the bottom side. In other forms, comprises a container 5, 6, 7, 8, 9, 10 or more stability elements. Preferably, the grooves are placed in one line, parallel to the front or back side of the growth container, and the protuberances are placed in another line which is also parallel to the front or back side of the growth container. Most preferably, there is a line of protuberances close to the front side (16) of the growth container, and a line of grooves (14) close to the back side of the growing container. The form and orientation of the stability elements on the bottom of the growth container prevents the retention of growth medium during its removal from the growing container.

The growth container, according to a preferred form of the invention, is also characterized by the fact that the longitudinal direction of the medium connector, located on the front side, forms an angle with the bottom side of the growth container. This means that the join point of the medium connector with the growth container is higher than the end (tip) of the medium connector, where the medium transfer element is further connected.

The growth container, according to a preferred form of the invention, is also characterized by the fact that the ratio between the surface area of the bottom side of the growth container related to the height of the growth container is comprised between 20 cm ² / cm height and 40 cm ² / cm height.

The medium container, according to the present invention, is a container that is preferably made of a transparent plastic polymer that withstands the conditions of autoclave sterilization. More preferably, the container is made from polycarbonate (PC). In another preferred form, the medium container is manufactured from a material that can be written or printed. Preferably, the top and the bottom sides of the medium container are not parallel. Preferably, the top side of the medium container is inclined from the back side towards the front side, with a gradient between 2.5° and 5.0°, meaning that the back side is higher than the front side. Preferably, is this slope of 3.1°. This has the advantage that the bottom side of a growth container, once placed on top of the medium container, has a similar slope. This ensures that the front side of the growth container, once placed on top of the medium container, is lower than the back side of the same growth container. The preferred form of the growth container, is characterized by the fact that the medium connector, located on the front side, forms an angle with the bottom side of the growth container as herein described, allowing by this slope, that the growth medium which is located in the growth container, can be removed from the growth container, back to the medium container, only by using the gravity force. The preferred form of a medium container contains at the top side stability elements that are made to complement and stack with the stability elements of the growth container. These stability elements are a combination of both, grooves and protuberances of the bottom or top sides of a container. Furthermore, these stability elements are suitable for stacking with the complementary elements on the top or bottom of the other container. Preferably, one container is a growth container and the other is a medium container. Most preferably, the container with stability elements on the bottom side is a growth container and the container with stability elements on the top side is a medium container.

The connection between complementary stability elements is done by protuberances from one container, which stacks into complementary grooves from the other container. In one form, a protuberance is a block of material made of anti-slip material, such as rubber or soft plastic, which is attached to the top or bottom of a container. In a preferred form, according to the invention, a protuberance is a local increase outwards the surface of the container of the material from which the bottom or top of a container is manufactured. Such local increase can be differently shaped, and includes without limiting choice: a circular shape, a drop shape, an elliptical shape, a square, a rectangle or a polygon. The term "groove" correspond to a local increase of the container material, from the top or bottom of a container, with the condition that the material is increased towards the inside of the container. In the form according to the invention, a groove can have different shapes, and includes without limiting choice: a circular shape, a drop shape, an elliptical shape, a square, a rectangle or a polygon. In a preferred form of an immersion system, the grooves and protuberances of the bottom side of the growth container and the top side of the medium are complementary. This means that the protuberances at the bottom side of the growth container have a similar shape as the grooves at the top of the medium container, where the growth container is placed. Furthermore, are the grooves and protuberances, dimensioned in such way that they fit to each other. The grooves and protuberances are positioned in such way that a groove is always placed above or bellow a protuberance, from the complementary side of the other container and vice versa. This preferred form has the advantage that the containers placed can fit to each other, stable and secure, and that the top container does not slip off from the lower container. Preferably, has the growth container grooves at the bottom side and near the back side, and has protuberances near the front side. This has the advantage that, if such growth container is used in an immersion growth regime, according to the present invention, the growth medium can optimally flow back to the medium container under the unique influence of the gravity force during the drainage phase (see below).

The form of a medium container according to the present invention, contains also an injection point, which is located at the front side, suitable for the transfer of growth medium and/or chemicals to- and from- the medium container, without increasing the risk of contamination in the system. The term "injection point" refers to an opening, which can be closed and is suitable for the transfer of growth medium and/or chemicals to- and from- the medium container, preferable while the immersion growth system is in operation.

Preferably, the container bodies of the growth container and/or medium container may comprise one or more sensor attaching means suitable for attaching sensors under sterile conditions. Said sensor attaching means can comprise various kinds of ports, which can be joined such as glued to the container body, in order to attach under sterile conditions specific sensors and monitor the growth performance in real time.

The form of the medium container, according to the present invention, contains also a carrier system. The term "carrier system" means a system that makes possible to easily hold a medium container once assembled the immersion growth system, and to transport the whole immersion growth system with the hands. A possible form according to the invention is a handle placed on the left-hand and right-hand sides of the medium container. In a preferred form according to the present invention, the carrier system consists of two notches. One notch is located on the edge, between the bottom side and the left-hand side of the medium container. The second notch is located on the edge, between the bottom side and the right-hand side of the medium container. Both notches allow to grab the medium container with fingers and thereby to lift the entire immersion growth system. The notches are preferably centrally located, in the middle of the edge between the left-hand or right-hand sides and the bottom side. This form has the advantage that the left-hand and right-hand sides are free of protuberant elements, and thus two medium containers can be placed laterally against to each other, without loss of place.

In the present invention, the temporary immersion is achieved by transferring the growth medium, via the medium transfer element, out from the medium container to the growth container. In this way, the culture in the growth container is immersed in the growth medium. Preferably, the transfer of the growth medium out from the medium container is achieved by the influence of an external force. More preferably, is the transfer driven by pressure. Most preferably, the transfer of growth medium is driven by an external pressure, which is generated by the air pressure variation facility, which is connected to the medium container by the air connecting elements. Once the immersion phase is ended, the growth medium can be removed from the growth container. For this, the pressure generated in the medium container is released, so that the surrounding atmospheric pressure prevails inside the medium container, and the growth medium, under the unique action of gravity force, flows back from the growth container to the medium container via the medium transfer element. This is possible by the sloping top surface of the preferred form of a medium container, in combination with the medium connector, which is located on the front side of the growth container forming an angle with the bottom side of the growth container. This means that no additional energy is required during the stationary phase, being cost-effective. In a preferred form according to the invention, the front side of a container contains a medium connector and an air connector, which are located at opposite- transversal corner points of this container side. These corner points are localized by the sectioning of three planes, namely, the plane of the front side, related to the plane of the top or bottom sides, and with the left-hand or right-hand sides respectively, and vice versa.

The present invention relates also to an incubator, consisting of the spatial repetition of immersion growth systems as herein described, in which - the immersion growth systems are preferably grouped in one element, consisting of two immersion growth systems placed with the back side against each other

such an elements can be repeated in the horizontal plane to form a row of elements

- the incubator can consist of multiple rows of elements, which are repeated and distributed in the vertical plane

The form according to the invention, the incubator has a minimum of four elements, where one element is composed of two immersion growth systems, according to the invention, placed with the back side against each other. This has the advantage that both immersion growth systems have a free front side. Therefore, all elements required for the system operation and functioning are located at the front side, being possible to control and maintain both systems separately. The absence of internal connectors, tubes and other elements in the container prevents an increased risk of contamination. In the incubator, according to another form, there is a light source above the elements distributed horizontally, in such way that the light originated at the light source reaches completely the growth containers. A light source may be chosen from a set of fluorescent lamps, incandescent lamps, halogen lamps and LEDs. In another form, according to the invention, can the incubator and the elements placed on it, be lighted by indirect sunlight.

Another form comprises two elements side by side on a horizontal surface, with a light source above, and beneath, a second horizontal surface on which two other elements have been placed next to each other, with another light source on top. In a preferred form, an incubator is, according to the invention, a rack system comprising multiple rows on which a number of elements may be placed and wherein each row is illuminated over its entire length, and where multiple rows below each other are placed. The form according to the invention, comprises an incubator at least two elements in a row and at least 2 rows. Other forms of the invention contain 3, 4, 5, 6, 7, 8, 9, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more elements per row, and 3, 4, 5, 6, 7, 8, 9, 8, 9, 10 or more rows.

In a preferred form of the invention, all growth containers are connected to a pressure net via the pressure connection means, located at the front sides of the growth containers. All the medium containers are connected to a pressure net, different from the pressure net where the growth containers are connected.

The term "pressure net" means a pressure pipe with multiple lateral terminals, and is connected at one terminal with the air pressure variation facility. The terminals of the pressure net are suitable for being connected to the pressure connection means (5, 6, 11, 11', 12, 12'), according to the invention. As a result, the pressure in the growth containers can be regulated, and separately the pressure in the medium containers can be also regulated, where the pressure in all growth containers is the same but can differ from the pressure in all medium containers. The pressure in all medium containers is the same but can differ from the pressure in all growth containers.

Another aspect of the present invention relates to a method for the in-vitro growth of plant material by using the immersion growth system as described in this invention, or by using the incubator as also described in this invention, in which the growth medium can be transferred from the medium container to the growth container via the required connected elements and by means of applying a pressure variation in the medium container, and

the growth medium can flow out from the growth container to the medium container via the required connected elements and by means of the effects of the gravity force, and

- the stability elements from the growth container are connected to the complementary stability elements of the medium container. his process four phases can be defined :

The first phase is the stationary phase, when the growth medium is in the medium container and the culture in the growth container, and where no growth medium is transferred

The second phase is the immersion phase, wherein a generated pressure at the air pressure variation facility is introduced into the medium container via the pressure connection means. This pressure gives rise to a gas mixture under a pressure between 0.05 bar and 1.5 bar, more preferably between 0.2 bar and 0.8 bar. This pressure ensures that the growth medium is pumped/transferred through medium transfer element to the growth container. The culture will be, during this phase, immersed in growth medium. Once all growth medium is transferred from the medium container, then is air also transferred, avoiding as such that the growth medium returns back to the medium container. The frequency of immersion vary preferably between 1-10 times per day, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times a day, depending on the kind of culture to grow, and the selected growth regime. The immersion time vary between 1-20 minutes, preferably between 3-10 minutes.

The third phase is the drainage phase, which per definition starts after the immersion phase. At the start of this phase, the pressure is generated at the air pressure variation facility stopped. Thus, the growth medium flows, under the influence of gravity force back through the medium transfer element to the medium container. The preferred form of the medium container with a slope on the top side, makes it possible that the growth medium, without any external force from the growth container, which is located on top of the medium container, is transferred back into the medium container.

- The fourth phase is the ventilation phase. During this phase, air pressure is generated at the air pressure variation facility and this pressure is transferred to the growth container through the pressure connection means. The generated pressure is between 0,05 bar and 1,5 bar, more preferably between 0.2 bar and 0.8 bar. Due to this pressure, the gas mixture present in the growth container is replaced by a new gas mixture supplied by the air pressure variation facility. A similar effect can be obtained by using negative pressure (vacuum), which is also generated at the air pressure variation facility and brought to the medium container via the pressure connection means.

The invention is here bellow further explained, making reference to the following non-limiting examples.

Examples

The following examples are for illustrative purposes only and are preferred forms of the invention.

Example 1: An immersion growth system 28 as represented in figure 1. Here, the growth container 1 has the following dimensions: 14 cm height, 18 cm width and 24 cm depth. In this case there are 30cm² of base surface per cm of height. This growth container is connected through a silicone medium transfer element 13 in the left-bottom corner of the front side, with the medium connector 8 of the medium container 2. The medium transfer element 13 has an inner diameter of 6 mm and an outside diameter of 9mm. The growth container has a central closeable supply opening 3, located in the front side, and with an inner diameter of 8cm. The growth container has also a medium connector 7, which has an angle of inclination of 5°, measured from its longitudinal direction line and the surface of the bottom side where the medium transfer element is connected. At the top-right corner of the front side, is the air connector 5 located, on which the air connecting element 12' is connected, to further connect with the hydrophobic air filter 11', which finally connects the growth container with the air pressure variation facility. This filter has a pore size of 0.2 micrometers. The growth container has potato culture.

The coupled medium container 2 has the following dimensions: 11 cm height, 18 cm width and depth of 24cm. The growth container has been placed on top of the medium container and the stability is ensured by the stability elements 14 and 16, 15 and 17. The medium container has a central closeable supply opening 4 through which growth medium can be added or removed. The medium container contains Murashige and Skoog (MS) medium. The inner diameter of the closeable supply opening is 5 cm. At the top-right corner of the front side of the medium container, is the air connector 5 located, on which the air connecting element 12 is connected, to further connect with the hydrophobic air filter 11. This filter has a pore size of 0.2 micrometers. The carrier system 10 consists of notches of a width of 9 cm, and centrally located on the edge between the bottom side and either left-hand or right-hand sides. The depth of the notch is 3cm. Furthermore, has the medium container and injection point 9. This injection point has a diameter of 1.3 cm. The top side of the medium container exhibits a slope 19 with a gradient of 3.1°.

Example 2: An incubator as represented in figure 6. The incubator is a rack system placed in a growth chamber. The incubator has four shelves, on which seven elements 18 are placed . One element is composed of two immersion growth systems such as those in Example 1, with their back sides against each other. The left and right sides of the adjacent elements are also in complete contact with each other. There is a light source 20 above each shelf, in this example a fluorescent lamp. All growth containers from the immersion growth systems are, via the pressure connection means connected to the pressure net 23, which is also connected to the air pressure variation facility, consisting of electronically controlled pressure relief valves 24 which control the pressure supply of the vacuum pump 25 and the air compressor 26. The electronically controlled pressure valves 24, are controlled by the computer software, installed on a system 27. All medium containers of the immersion growth systems are, via their pressure connection means connected to a pressure net 22, that also is connected to the air pressure variation facility, consisting of electronically controlled pressure valves 24, which regulate the pressure supply of the vacuum pump 25 and the air compressor 26.

All immersion growth systems from the same incubator are at the same phase of the process: stationary phase, immersion phase, drainage phase and ventilation phase. During the stationary phase, there is no pressure or vacuum distributed through the pressure nets 22 and 23. At the start of the immersion phase, the pressure generated in the air compressor 26, it is distributed through the pressure net 22. This is regulated by the software of the system 27, which regulates the pressure valves 24, opening the compressed air to go through the pressure net 22. This pressure is equally distributed to all medium containers in the incubator. This phase lasts five minutes. During this phase, is the growth medium transferred from the medium containers to the growth containers, via the medium transfer element and allowing the culture to immerse. During the entire process, the immersion phase is repeated six times a day. After all the growth medium has transferred from the medium containers to the growth containers, then air start to pass through the medium transfer element, avoiding that the growth medium returns to the growth containers. Simultaneously, the pressure valves 24 regulate that no excess pressure reach the containers. Air drained out via the pressure connection means located in the growth containers, going through the pressure net 23 and being release at the air pressure variation facility 24. After the set time of immersion, is the pressure generated in the containers is released via the pressure nets 22 and 23, and regulated by the air pressure variation facility 24. Then, the air pressure falls in both containers and the growth medium flows back to the medium container. This phase is followed by the programmed (by system 27) ventilation phase, which is in this example performed by using vacuum pressure generated in the vacuum pump 25. The air pressure variation facility 24 allows the vacuum to expand through the pressure net 22, transferring this negative pressure to the medium containers. Because growth containers and medium containers are connected to each other via the medium transfer element, the vacuum generated in the medium container gives rise to the entry of fresh air into the growth container. This fresh air is also transferred through the medium connectors. After this phase, can the immersion growth systems return to stationary phase.

References

ETIENNE H . & BERTHOULY M. (2002). Temporary immersion systems in plant micropropagation. Plant Cell, Tissue and Organ Culture 69 : 215-231.

ESCALONA M, LORENZO JC, GONZALEZ B, DAQ UINTA M, FUNDORA Z,

BORROTO CG, ESPINOSA D, ARIAS E & ASPIOLEA ME (1998) New system f or invitro propagation of pineapple (Ananas comosus L. Merr). Pineapple News 5 : 5-7

Murashige T. and Skoog F. (1962). Physiol. Plant 15 :473.

Claims

1. An immersion growth system (28) comprising two complementary containers, in particular a growth container (1), suitable to hold the plant material, and a medium container (2), suitable to hold the growth medium, wherein both containers comprise coupling means (7, 8, 13) for coupling the medium container (2) to the growth container (1), characterized by the fact that the growth container (1) is designed to be placed on top of the medium container (2), and that the coupling means (7, 8, 13) are provided at the front side of both containers.

2. An immersion growth system (28) as is claim 1, where said front sides of both containers also comprise a closeable supply opening (3, 4), suitable for supplying material and/or medium to and for removing material and/or medium from said containers.

3. An immersion growth system (28) as in one of the preceding claims, whereby said front sides of the two containers also contain pressure connection means (5, 6, 11, 11 ', 12, 12'), suitable to be connected to an air pressure variation facility (24, 25, 26, 27), in order to separately regulate the pressure in both containers.

4. An immersion growth system (28) as in one of the preceding claims, whereby said pressure connection means (5, 6) and/or coupling means (7, 8) comprise an external threat for receiving a closure cap.

5. An immersion growth system (28) as in one of the preceding claims, wherein the surface of the bottom side of said growth container (1) is similar to the surface of the top side of the medium container (2).

6. An immersion growth system (28) as in the preceding claims, wherein the bottom side of said medium container (2) is not parallel to the top side of said same medium container (2).

7. A growth container (1) suitable for its use in an immersion growth system (28) as in one of the preceding claims, wherein said container comprises six sides, a top side, a bottom side, a left-hand side, a right-hand side, a front side and a back side, whereby said six sides form a beam-shaped structure.

8. A growth container as in claim 7, wherein

- the height of the container is smaller than the depth of the container, preferably in a ratio of height/depth of the container between 1 :2,30 and 1 : 1,65; and the height of the container is smaller than the width of the container, preferably in a ratio of height/width of the container between 1 : 1,75 and 1 : 1.25, and

the width of the container is smaller than the depth of the container, preferably in a ratio of width : depth of the container between 1 : 1,36 and 1 : 1,30.

9. A growth container (1) as in claims 7-8, suitable for its use in an immersion growth system (28) as in claim 1-6, wherein the closeable supply opening (3) is suitable for a hand to pass through.

10. A growth container (1) as in claims 7-9, wherein the ratio between the surface area of the bottom side and the height of the container is between 20 cm ² /cm and 40 cm ²/cm.

11. A growth container ( 1) as in claims 7-10, wherein the longitudinal direction of a medium connector (7), located at the front side of the growth container (1), forms an angle with the bottom side of the growth container

(1), in the sense that the join point of said medium connector (7) with the growth container (1) is higher than the end point of the same medium connector (7) on which coupling means (13) can be connected.

12. A growth container (1) as in claims 7-11, wherein the top side of the growth container (1) is made from a transparent material, and preferably convex and free of connectors, and/or openings.

13. A growth container (1) as in claims 7-12 in which stability elements (14, 16) are located in the plane of the bottom side of the container, suitable to be connected with the corresponding stability elements (15, 17) from the medium container (2).

14. A growth container (1) as in claims 7-13, characterized in that the growth container comprises one or more sensor attaching means, suitable for attaching sensors under sterile conditions.

15. A medium container (2) suitable for its use in an immersion growth system (28) as in claims 1-6, in which stability elements (15, 17) are located in the surface of the top side of the container, and are suitable to complement with the stability elements (14, 16) from the growth container (1).

16. A medium container (2) as in claim 15, suitable for its use in an immersion growth system (28) as in claims 1-6, wherein an injection point (9) is provided at a front side of the medium container (2), suitable for the transfer of growth medium and/or chemicals to and from the medium container (2) under sterile conditions.

17. A medium container (2) as in claims 15 and 16, in which a carrier system (10) is present.

18. A medium container (2) as in claims 15-17, characterized in that the medium container (2) comprises one or more sensor attaching means suitable for attaching sensors under sterile conditions.

19. An incubator (29), comprising a spatial repetition of immersion growth systems (28) as in any of the claims 1-6, wherein

the immersion growth systems are preferably grouped in a single element, comprising two immersion growth systems which are positioned with the back side against each other and

such element can be repeated in the horizontal plane to form a row of elements and

the incubator (29) can comprise several rows repeated in the vertical plane

wherein the incubator is further characterized by an air pressure variation facility (24, 25, 26, 27), in which all pressure connection means (5, 6, 11, 11 ', 12, 12') of the immersion growth systems are connected, by means of a pressure net (22, 23), and preferably the incubator comprises light sources (20), positioned in such way that all growth containers (1) from all rows, are illuminated.

20. A method for the in-vitro growth of plant material by the use of an immersion growth system (28) as in claims 1-6, or by the use of an incubator (29) as in claim 19, wherein

the growth medium can be transferred from the medium container (2) into the growth container (1) via the connected coupling means (7, 8,

13) and by a pressure variation (increase) in the medium container (2), and

the growth medium can be transferred from growth container (1) to the medium container (2) via the connected coupling means (7, 8, 13) by the effect of gravity force, and

the stability elements (14, 16) from the growth container are connected with the complementary stability elements (15, 17) from the medium container (2).