WO1996025484A1

WO1996025484A1 - In vitro culture container

Info

Publication number: WO1996025484A1
Application number: PCT/FR1996/000223
Authority: WO
Inventors: Claude Teisson
Original assignee: Centre De Cooperation Internationale En Recherche Agronomique Pour Le Developpement (Cirad)
Priority date: 1995-02-17
Filing date: 1996-02-12
Publication date: 1996-08-22
Also published as: FR2730743A1; FR2730743B1

Abstract

A container for in vitro culture using temporary immersion under sterile conditions in an integral body (1) sealed with a lid (2) and provided with a tray (3) defining an upper portion (4) for plant tissue and a lower portion (5) for a nutrient medium. An inlet (8) in the lid (2) enables a positive pressure to be provided in a bell-shaped chamber (13) in the lower portion via a tube (12) connected thereto, whereby the nutrient medium is forced upwards from the lower portion (5) to the upper portion until it covers the explants. The pressure is relieved through a vent (9) to ensure pressure equilibrium during rest periods.

Description

In vitro culture vessel

The invention is in the field of culture and more precisely of agriculture and horticulture. The present invention relates to in vitro culture, and more particularly relates to a container intended for in vitro culture, by temporary immersion, under sterile conditions.

The multiplication of plants by in vitro culture has become a common practice in the field of horticulture and agriculture. This practice offers many advantages, but nevertheless has several drawbacks, one of the most important of which is undoubtedly a high labor cost linked to the frequency and high technicality of the manipulations required.

In order to reduce this charge, in vitro culture in liquid medium has been developed. This type of culture has many advantageous, unfortunately counterbalanced by serious drawbacks, essentially linked to problems of asphyxiation of the culture, when the tissues are permanently submerged. To take advantage of the advantages of this technique, without suffering the disadvantages, processes have been developed which call for either the flotation of the explants on a raft or the use of a nutritive medium in the form of a mist. A process known as temporary or partial immersion of the explants has also been imagined. To date, this overly complex, unreliable, and expensive process has not experienced any real industrial development.

The temporary immersion process consists, as its name suggests, of temporarily immersing explants, tissues or plant cells, from the culture in vitro. Taking into account the floating phenomena, or the phenomena of growth, this immersion in the nutritive medium can be partial or total. During the rest phase, which constitutes the longest period, the explants are emerged, but the film of nutritive medium, retained by capillarity from the previous immersion phase, keeps them moist.

This process thus makes it possible to benefit from the advantages of in vitro culture in a liquid medium, while avoiding the problems of asphyxiation. This method also makes it possible to obtain a quality of development of the culture, which is particularly advantageous in comparison with that obtained by other in vitro culture methods, in particular the methods using a solid nutritive medium or in the form of a gel, or alternatively using floating on a raft, or using nutritious mist.

The present invention aims to overcome the drawbacks mentioned above by proposing a container intended for in vitro culture, by temporary immersion, under sterile conditions.

More particularly, an object of the invention is to have a container allowing in vitro culture under sterile conditions, limiting the risk of infection, while ensuring good reliability of the culture during a large number of immersion cycles. .

Another object of the invention is to have a maximum drainage surface, while avoiding pollution of the nutritive medium by plant tissues. Another object of the invention is also to propose a container for maintenance, disassembly and washing, which is easy and which can also be sterilized between two cycles of use. Another object of the invention is also to propose a container allowing the change of the nutritive medium without dismantling.

The invention relates to a container intended for in vitro culture, by temporary immersion, under sterile conditions, comprising, two compartments, an upper part intended to receive plant tissues and a lower part which can contain a nutritive medium in liquid form. According to the invention, the container comprises, a unitary body forming an outer envelope, closed by a cover, and a basket for separating said two parts, an inlet located on the cover allowing the supply of an overpressure conducted in the lower part by a tube opening into a bell, said overpressure causing a rise in part of the nutritive medium from the lower part in the upper part, the stopping of the overpressure allowing the return, by gravity, of the nutritive medium in the lower part.

This arrangement of the container provides many advantages. The immersion of the explants, or plant tissues, is carried out by displacement of the nutritive medium, pushed by an overpressure, and not by the mechanical displacement of a support basket. This absence of mechanical movements increases the reliability of the device. The air inlet from above makes it possible to group all the sealing problems between the inside of the container (sterile environment) and the outside on a single piece. This arrangement thus makes it possible to limit the phenomena of leakage, and to increase the reliability of the entire device.

The overpressure is applied for a time greater than that which is strictly necessary for the rise of the liquid. It follows a bubbling, production bubbles, or bubbling of the medium, improving the immersion of the plants, and a renewal of the internal atmosphere of the culture vessel. This aeration is particularly important because it avoids the harmful effect of an accumulation of gas inside the in-vitro culture vessel. The overpressure can, for example, be applied for a period of twenty minutes every two hours.

In different embodiments of the invention, each presenting their specific advantages, the following characteristics can optionally be combined:

- The bell forms with the side wall of the body an annular jacket serving as a lateral passage to the nutrient medium during transfers between the upper and lower parts.

- A vent located on the cover allows the passage of gas, between the inside and the outside of the container. This vent also allows pressure balancing during rest periods.

- The vent is connected to a hydrophobic sterilizing filter.

- The separation basket has openings for the passage of the nutrient medium. - A sieve with calibrated mesh is placed above the basket.

- The separation basket rests on a shoulder disposed inside the container, on the side wall of the body. - The tube extends inside the bell. This arrangement allows the transfer, in particular the replacement, of the nutrient medium without the need to dismantle or open the container. - A system of joints ensures a tightness of the plant tissues between the upper part and the lower part.

- The bell is fixed on the tube. Various other characteristics, objects and advantages of the invention will emerge from the following description, given by way of example and without limitation, with reference to the appended drawings in which: FIG. 1 is a longitudinal section of an embodiment of a device according to the invention. Figure 2 corresponds to the device of Figure 1, containing a nutritious medium in a resting situation. Figure 3 corresponds to the device of the

Figure 2, in an overpressure situation.

Figure 4 is an exploded view of another embodiment of the container according to the invention.

Figure 5 is an axial half-section of the container of Figure 4.

Figure 6 shows, front view with half section another embodiment of the container according to the invention.

As far as possible, the same reference numbers are used to designate similar elements in the various embodiments.

Referring more particularly to FIG. 1, the container intended for culture in vitro, by temporary immersion, under sterile conditions, comprises two compartments. An upper part 4 is intended to receive plant tissues, and a lower part 5 can contain a nutritive medium 23 in liquid form. A unitary body 1 forms an outer envelope. It is closed by a cover 2. A basket 3 of separation, separates the upper part 4 from the part low 5. An inlet 8, located on the cover 2, allows the supply of an overpressure. Advantageously, the inlet 8 is connected to a hydrophobic sterilizing filter. After filtration, the overpressure is conducted in the lower part 5 by means of a tube 12 opening into a bell 13.

The bell 13 forms with the side wall 6 of the body 1 an annular jacket 15. This annular jacket 15 serves as a lateral passage for the nutrient medium, during transfers between the upper 4 and lower 5 parts. This particular arrangement confers numerous advantages. It allows good mixing of the nutrient medium, and therefore improves its homogenization. It avoids too rapid movements during the transfer of the nutritive medium, and allows a good distribution of the arrival of the nutritive medium in the upper part. In this embodiment the bell 13 rests at the bottom of the container. In order to allow the passage of the nutritive medium, from the interior of the bell 13 in the annular jacket 15, at least one opening 20, or clearance, is arranged in the bottom of the bell 13.

A vent 9 located on the cover allows the passage of gas between the interior and the exterior of the container in one direction as in the other. Advantageously, this vent 9 is connected to a hydrophobic sterilizing filter. This vent 9 thus allows, during the ascent of the nutrient liquid 23, firstly the evacuation of the gas contained in the upper part 4 of the container, pushed by the nutrient liquid 23, then in a second time, to evacuate the overpressure continuing to be supplied.

Figures 2 and 3 show the container of Figure 1 in which the medium was placed nutrient 23. In the rest phase, corresponding to Figure 2, the nutrient medium 23 is in the lower part 5. In the active phase, corresponding to Figure 3, an overpressure is applied to the inlet 8, located on the cover 2. This overpressure is transmitted to the lower part 5 by a tube 12. In this embodiment, the seal between the tube 12 and the inlet 8 is ensured by a conical assembly. The tube 12 comprises in its lower part a lug 17. This lug 17 makes it possible to hold the bell 13 in the bottom of the container while preventing it from rising. In this embodiment, the bell 13 and the basket 3 of separation are produced in a single piece, in one piece. The upper part of the bell 13 is in this embodiment, common with the basket 3 of separation. At its periphery, the separation basket 3 rests on a shoulder 7, disposed inside the container, on the side wall 6 of the body 1. This arrangement makes it possible to avoid the transfer of part of the plant tissues located in the upper part 4, towards the lower part 5. It also ensures the centering of the bell 13 inside the container.

When the overpressure is applied, it is transmitted inside the bell 13. This overpressure pushes the nutrient medium 23, thereby causing it to rise from the bottom part 5 into the top part 4. In this embodiment, the bell 13 rests at the bottom of the container. Clearances 20, or passages are arranged in the lower edge of the bell in order to allow the passage, of the nutritive medium 23, between the interior of the bell and the annular jacket 15. The nutritive liquid rises laterally on the external walls of the Bell. The separation basket 3 has openings intended for the passage of the nutrient medium. Maintaining feeding the overpressure keeps the nutrient medium 23, in liquid form, in the upper part 4 of the container.

Advantageously, the overpressure is applied for a period greater than that strictly necessary for the ascent of the nutritive liquid 23. This causes the liquid, bubbling, located in the annular jacket 15 and in the upper part 4, improving, the ascent of the liquid, its stirring, and the immersion of the explants by creating a mist in the upper part of the upper part 4. This maintenance of the overpressure also causes a renewal of the atmosphere contained inside the container.

Preferably, the overpressure is obtained by the introduction of compressed air. This is supplied by a pump delivering oil-free compressed air. In order to protect the sterile medium contained inside the container, the air inlet is protected by a hydrophobic sterilizing filter. Stopping the supply of the overpressure causes the nutritive medium 23 from the top 4 to return to the bottom 5 by gravity.

The programming of the immersion cycles, and the operation of the entire device, independently, can be carried out in a simple manner using a programmer or a programmable electrical outlet. Such an outlet makes it possible to control the operation of the pump, and therefore the rate of immersion and their duration. In this embodiment, the tube 12 is extended inside the bell 13 by a lower part 22. This extension does not touch the bottom of the container and thus allows the passage of compressed air. The purpose of this extension is to allow extraction nutrient medium without the need to disassemble the container.

A suction pump connected to the inlet 8 makes it possible to remove the nutritive liquid 23. The inlet 8 then allowing the feeding of a new nutritive medium 23. This replacement is done without dismantling the container. Given the duration of culture, it is sometimes necessary to use successive nutrient media, of different compositions. Such changes which can be relatively frequent are easily carried out with the device of the invention.

Figures 4 and 5 show another embodiment of a container according to the invention. This container comprises a unitary external body 1, a cover 2, which is screwed onto the body 1 by means of a thread 11, a tube 12 opening into a bell 13, a basket 3 for separation, and a sieve 18. The fixing of the cover 2 on the body 1 is carried out by screwing but can also be carried out by equivalent means, in particular a clip type clip system. In this particular embodiment, the tube 12 comprises in its lower part a thread 14. This thread makes it possible to fix the basket 3 on the tube 12. The bottom of the basket 3 is striated to facilitate drainage, and has very fine openings allowing the passage of the nutrient medium 23.

Advantageously, the drainage surface is as large as possible, and corresponds substantially to a surface comparable to that of the horizontal section of the lower part 5. The use of the largest possible drainage surface gives several advantages , especially at the end of the submerged phase, a large area of drainage makes it possible to avoid a phenomenon of heaping or of too great compression of the biological material. The shape of the basket 3, and more particularly its rising side edges, allows easier handling when the explants come out.

For the in vitro culture of particularly fine plant tissues, a sieve 18 is added above the basket 3. This sieve 18 consists of a calibrated mesh fabric, welded to a tray having large openings. To facilitate the flow, a space is reserved between the basket 3 and the sieve 18. Preferably, the size of the mesh size is chosen between 100 and 250 μm. This sieve can constitute a renewable part, and thus be changed with each new use. In this embodiment, the screen 18 is fixed to the tube 12 by screwing.

The tube 12 and the bell 13 form a one-piece assembly in one piece. The arrangement of the opening 8 and the tube 12 allows the introduction of a cannula. This cannula allows the transfer of the nutritive medium, that is to say its replacement without having to dismantle the entire apparatus.

The internal sterile environment is protected by a seal 10a located on the cover 2. This seal makes it possible to ensure a good seal with the external environment.

Advantageously, a device for seals 16a, 16b seals the plant tissues between the upper part 4 and the lower part 5. A first seal 16a is located between the edge of the basket 3 and the shoulder 7, located in the part lateral 6 of body 1 unit. A second seal 16b is placed between the basket 3 and the bell 14. This device ensures the passage of the nutrient medium 23 by the openings in the bottom of the basket 3, thus ensuring good use of the drainage surface. This set of seals also makes it possible to avoid pollution of the nutrient medium 23 by plant tissues.

Figure 6 shows another preferred embodiment of the container according to the invention.

In this embodiment, the tube 12 is extended inside the bell by a lower part 22. This extension can come into contact with the bottom of the container. It is provided with lateral release 21. These clearances allow the passage of compressed air during immersion cycles. They also allow the passage of the nutrient medium, during its introduction or replacement.

This central tube 12 has a thread 14 allowing the bell 13 to be screwed. Above this thread 14, two lugs 17, 19 allow the screen 18 and the basket 3 to be held in place. The lower edge of the bell 13 does not come in contact with the bottom of the container, thus clearing an annular passage facilitating the movements of the nutritive medium.

A seal 10b located between the tube 12 and the cover 2 further improves the seal and the protection of the sterile environment.

Advantageously in different embodiments of the invention, the various constituent parts of the container are made of a material allowing their sterilization. This sterilization can be carried out by autoclaving. The different parts can be made of a transparent material. The drainage openings arranged in the bottom of the basket 3 are very fine slots with a length of approximately 1 mm and a width of approximately 3/10 of mm. The in vitro culture vessel has a capacity of approximately 1 liter. The lower part 5 can contain 250 ml of nutritive liquid. The container has a substantially cylindrical shape, a height between 14 and 16 cm, a diameter in the lower part of about 10 to 11 cm, and in the upper part of about 12 to 13 cm.

Claims

CLAIMS 1. Container intended for in vitro culture, by temporary immersion, under sterile conditions, comprising, two compartments, an upper part (4) intended to receive plant tissues and a lower part (5) which can contain a nutritive medium (23 ) in liquid form, characterized in that a unitary body (1) forming an outer envelope, closed by a cover (2), and a basket (3) for separating said two parts (4, 5), an inlet (8 ), located on the cover (2) allow the supply of an overpressure conducted in the lower part (5) by a tube (12) opening into a bell (13), said overpressure causing a rise in part of the medium nutrient (23) of the lower part (5) in the upper part (4), stopping the overpressure allowing the return, by gravity, of the nutritive medium in the lower part.

2. Container according to claim 1, characterized in that the bell (13) forms with the side wall (6) of the body (1) an annular jacket (15) serving as a lateral passage to the nutrient medium during transfers between the upper parts and bass (4, 5).

3. Container according to one of claims 1 and 2, characterized in that a vent (9) located on the cover allows the passage of gas, between the interior and the exterior of the container.

4. Container according to claim 3, characterized in that the vent (9) is connected to a hydrophobic sterilizing filter.

5. Container according to one of claims 1 to 4, characterized in that the basket (3) for separation has openings intended for the passage of the nutrient medium.

6. Container according to one of claims 1 to 5, characterized in that a sieve (18), with calibrated mesh, is arranged above the basket (3).

7. Container according to one of claims 1 to 6, characterized in that the basket (3) for separation rests on a shoulder (7) disposed inside the container, on the side wall (6) of the body (1 ).

8. Container according to one of claims 1 to

7, characterized in that the tube (12) extends inside the bell.

9. Container according to one of claims 1 to

8, characterized in that a seal device (16a, 16b) seals the plant tissues between the upper part (4) and the lower part (5).

10. Container according to one of claims 1 to 9, characterized in that the bell (13) is fixed on the tube (12).