System for transferring somatic plant embryos

Abstract

The present invention relates to a system and method for transferring clusters of plant cells. The present invention particularly relates to transferring somatic embryos. According to the present invention, a cluster of plant cells is transferred while it is held in a holding liquid, wherein during transfer, both the holding liquid and cluster of plant cells are arranged in a hollow transfer member.

Description

[0001] SYSTEM FOR TRANSFERRING SOMATIC PEANT EMBRYOS

[0002] The present invention relates to a system and method for transferring clusters of plant cells. The present invention particularly relates to transferring somatic embryos.

[0003] Within the context of the present invention, a cluster of plant cells may refer to a part of a plant during any stage of its growth. The present invention particularly relates to clusters of plant cells having a cluster size in a range between 0.01 and 125 mm3, more preferably between 1 and 27 mm3. Different clusters of plants cells may be arranged at substantially the same position in space. This situation is referred to as an agglomeration of different clusters of plant cells.

[0004] In somatic embryogenesis, a plant embryo is derived from a single somatic plant cell. Typically, multiple embryos are simultaneously generated within a given container. However, for the purpose of allowing the embryos to mature into seedlings, plants, or the like, a transfer is generally required from a container in which large quantities of embryos are held in a holding liquid to a different container in which a smaller number of embryos is held. In a particular embodiment, each embryo is transferred into a separate single container or into a separate single compartment of a multi-compartment container.

[0005] Several systems are known by which an embryo can be transferred from a first container to a second container. For example, systems are known in which embryos are picked up from the first container using tweezers that are mounted on a robotic arm. In other systems, a tubular member is used in which an embryo is picked up by applying a vacuum inside the tubular member. As a result of the vacuum, the embryo is pressed against an end part of the tubular member.

[0006] A drawback of the systems described above is that a relatively large risk exists that an embryo will adhere to the tweezers or the abovementioned tubular member, thereby preventing the release of this embryo into the second container. Consequently, additional steps are required for ensuring a proper release of the embryo into the second container.

[0007] An object of the present invention is to provide a system for transferring clusters of plant cells that are arranged in a holding liquid inside a first container, in which the abovementioned problems do not occur or at least to a lesser extent.

[0008] According to the present invention, this object is achieved using the system as defined in claim 1 , which comprises a hollow transfer member, a pressure unit for regulating a pressure in the hollow transfer member, and a controller for controlling the pressure unit. According to the present invention, the system is characterized in that the controller is configured to control the pressure unit to reduce a pressure inside the hollow transfer member allowing a singulated cluster of plant cells and a quantity of the holding liquid to be aspirated into the hollow transfer member, and for maintaining a pressure in the hollow transfer member such that the cluster of plant cells and at least a part of the quantity of holding liquid in which the cluster of plant cells is held are kept inside the hollow transfer member during transfer.

[0009] According to the present invention, the cluster of plant cells is held within the holding liquid during transfer, while the holding liquid itself is aspirated into the hollow transfer member. Put differently, a cluster of plant cells is transferred while it is held in a holding liquid, wherein during transfer, both the holding liquid and the cluster of plant cells are arranged in the hollow transfer member. In this manner, the risk of the cluster of plant cells being or becoming attached to the hollow transfer member is decreased.

[0010] The hollow transfer member may have a first opening through which the singulated cluster of plant cells and the quantity of holding liquid can be aspirated into the hollow transfer member and a second opening that is connected to the pressure unit. A size of the first opening may lie in a range between 1 and 80 mm2, preferably between 2 and 50 mm2. The first opening should be large enough so that the cluster of plant cells can be aspirated and / or sucked into the hollow transfer member. On the other hand, too large a first opening would result in an excessive amount of holding liquid being aspirated into the hollow transfer member for each transfer.

[0011] The pressure unit can be configured to reduce a pressure inside the hollow transfer member relative to a pressure outside of the hollow transfer member for the purpose of aspirating a singulated cluster of plant cells and a quantity of the holding liquid into the hollow transfer member while preventing the holding liquid and / or the singulated cluster of plant cells to leave the hollow transfer member through the second opening. When arranging the hollow transfer member in the first container and submerging the first opening in the holding liquid, some holding liquid will enter the hollow transfer member due to capillary action. By reducing the pressure on the opposite side of the interface between air and the holding liquid in the hollow transfer member, the level of holding liquid will rise even more and a cluster of plant cells can be aspirated into the hollow transfer member along with the holding liquid.

[0012] When removing the hollow transfer member from the holding liquid, a balance is preferably obtained so that the level of the holding liquid inside the hollow transfer member remains substantially constant. For example, the pressure unit can be configured to maintain a suction to the second opening and / or for maintaining a pressure in the hollow transfer member that is lower than a pressure outside of the hollow transfer member for the purpose of keeping the cluster of plant cells and at least a part of the quantity of the holding liquid in which the cluster of plant cells is held inside the hollow transfer member. Maintenance of the level of the holding liquid inside the hollow transfer member is particularly important during movement of the hollow transfer member. The under-pressure may compensate for the force of gravity acting on the holding liquid and the cluster of plant cells. By appropriately selecting the pressure inside the hollow transfer member it can be prevented that the holding liquid reaches the second opening. It should be noted that the abovementioned under-pressure can be actively created, for example by applying a suction force at the second opening. Alternatively, the under-pressure is created when aspirating the cluster of plant cells and the quantity of holding liquid into the hollow transfer member. After a predetermined amount of time, or after having detected that the cluster of plant cells is arranged inside the hollow transfer member, the second opening may be sealed to such an extent that air can no longer pass through the second opening. This can for instance be achieved using a valve arranged in between the second opening and the pressure unit. The valve can be operated by the controller.

[0013] The cluster of plant cells may comprise a plant embryo, such as a somatic embryo. Alternatively, the cluster of plant cells may comprise a callus, an anther, an ovary, or a clump.

[0014] The controller can further be configured for controlling the pressure unit to increase the pressure inside the hollow transfer member or to allow the pressure inside the hollow transfer member to increase for the purpose of releasing the holding liquid and the cluster of plant cells held therein from the hollow transfer member. The increase of pressure inside the hollow transfer member can be obtained by allowing air to enter the second opening to thereby establish a pressure inside the hollow transfer member above the interface between air and the holding liquid that is equal to the pressure outside the hollow transfer member. In this case, the cluster of plant cells and the holding liquid could leave the hollow transfer member due to gravity. Alternatively, an overpressure is generated by pumping air or another gaseous medium through the second opening into the hollow transfer member. This results in a flushing operation during which the holding liquid with the cluster of plant cells arranged therein is flushed out of the hollow transfer member.

[0015] The system may further comprise a transport unit for moving the hollow transfer member, wherein the controller is configured to control the transport unit for bringing the hollow transfer member in proximity to a singulated cluster of plant cells arranged in the first container prior to aspirating that singulated cluster of plant cells and holding liquid into the hollow transfer member. Additionally, or alternatively, the controller can further be configured to control the transport unit for bringing the hollow transfer member in proximity to a second container prior to releasing the holding liquid and the cluster of plant cells held therein from the hollow transfer member. The transport unit may comprise a robotic arm, an XYZ table, or a gantry system on which the hollow transfer member is mounted.

[0016] During each transfer, holding liquid is removed from the first container. When the level of the holding liquid is too low, the clusters of plant cells will stick to the walls of the first container. When the level of the holding liquid is too high, separate clusters of plant cells may be arranged on top of each other relative to a bottom wall of the first container thereby forming an agglomeration of clusters of plant cells. For that reason, it is advantageous to ensure that the level of the holding liquid is above a first threshold and below a second threshold. This can be obtained using a dispensing unit for dispensing, into the first container, holding liquid that is identical to and / or does not chemically react with the holding liquid already arranged in the first container. Hereinafter, the combination of the holding liquid that was already arranged in the first container from the start of the transfer process and the further holding liquid newly dispensed in the first container will be referred to as the holding liquid inside the first container.

[0017] The controller can be configured to maintain a substantially constant level of the holding liquid inside the first container during the transfer of a plurality of said clusters of plant cells by controlling the dispensing unit. For example, the controller can be configured to control the dispensing unit for releasing a predefined amount of the holding liquid after having performed a predefined number of transfers of clusters of plant cells. Alternatively, the system may further comprise a sensor for measuring a level of the holding liquid inside the first container. The controller may then be configured to control the dispensing unit based on output from the sensor, for example for maintaining a level of the holding liquid to remain between the abovementioned first and second thresholds.

[0018] The system may further comprise an imaging system, for example comprising one or more optical cameras, for identifying one or more singulated clusters of plant cells inside the first container. The controller can be configured to control the transport unit based on a position of the identified one or more singulated clusters of plant cells. The imaging system is not limited to optical cameras but may include other modalities by which an image can be constructed that allows the identification and positioning of singulated clusters of plant cells. For example, the imaging system may comprise an X-ray imaging system, an infrared imaging system, or a Chloroplast fluorescence imaging system. The imaging system may additionally or alternatively be configured for identifying an agglomeration of clusters of plant cells inside the first container.

[0019] The controller can be configured to control the pressure unit to reduce a pressure inside the hollow transfer member for the purpose of aspirating a quantity of the holding liquid through the first opening and into the hollow transfer member, to then control the transport unit to move the hollow transfer member to be above or near an identified agglomeration of clusters of plant cells, and to then control the pressure unit to increase the pressure inside the hollow transfer member or to allow the pressure inside the hollow transfer member to increase for the purpose of dispensing the holding liquid onto or in a near vicinity of the identified agglomeration of clusters of plant cells for singulating a cluster of plant cells from the agglomeration of clusters of plant cells. In this manner, the hollow transfer member is used for singulating a cluster of plant cells using the holding liquid in the first container.

[0020] It is generally desired to pick up a single cluster of plant cells at a time. To this end, the system may further comprise a movement system for mutually moving the dispensing unit and the first container. In this case, the controller can be configured to localize an agglomeration of clusters of plant cells in the first container using the imaging system, to mutually move the dispensing unit and first container to bring the dispensing unit at least near the agglomeration of clusters of plant cells, and to control the dispensing unit to dispense the holding liquid into the first container thereby singulating at least one cluster of plant cells from the agglomeration of clusters of plant cells.

[0021] Additionally, or alternatively, the controller can be configured for controlling the transport unit to cause the hollow transfer member to perform a movement, such as a vibration, a random movement or a stirring movement, while being at least partially submerged in the holding liquid thereby singulating at least one cluster of plant cells from an identified agglomeration of clusters of plant cells. The controller can in this case be configured to localize the agglomeration of clusters of plant cells in the first container using the imaging system, to control the transport unit to bring the hollow transfer member at least near the agglomeration of clusters of plant cells prior to causing the hollow transfer member to perform said movement.

[0022] The controller or a separate processing unit can be configured to evaluate, from a recorded image of a cluster of plant cells, one or more parameters of the cluster of plant cells. Then, the evaluated one or more parameters can be compared to respective target values. The controller or a separate processing unit may then determine whether or not to transfer the cluster of plant cells based on the comparison. The one or more parameters may comprise one or more out of the group of size, shape, colour, and an amount of free space around a cluster of plant cells.

[0023] Alternatively of additionally, the system may comprise a singulation unit for singulating clusters of plant cells inside the first container. As an example, the singulation unit may be a unit for imparting a mechanical vibration to the first container by directly engaging the first unit. Such unit may be arranged underneath the first container such that the first container is arranged in between the unit and the hollow transfer member.

[0024] Additionally, or alternatively, the singulation unit is a unit for imparting a mechanical vibration to the holding liquid inside the first container by directly engaging the holding liquid in the first container. For example, the singulation unit can be a vibration member configured to be inserted into the holding liquid.

[0025] The controller can be configured to initiate a next transfer of a cluster of plant cells if a distance between said cluster of plant cells and other clusters of plant cells inside the first container exceeds a predefined first threshold. If a cluster of plant cells to be transferred next cannot be identified, the controller may control the singulation unit for singulating clusters of plant cells inside the first container. Alternatively, as described above, the controller may control the movement system and dispensing unit, or the transport unit for singulating a cluster of plant cells. If no clusters of plant cells are detected, a signal may be generated triggering replacement of the first container with another first container that is filled with holding liquid and clusters of plant cells.

[0026] The imaging system can be configured to image the hollow transfer member or to image contents of the first container after the controller has controlled the pressure unit for aspirating a cluster of plant cells and holding liquid into the hollow transfer member for determining whether a singulated cluster of plant cells has been aspirated into the hollow transfer member. For example, the imaging system can determine whether a cluster of plant cells has been aspirated into the hollow transfer member by comparing an image before and after trying to aspire of a cluster of plant cells. The controller can be configured to control the transport unit and pressure unit to initiate a new transfer of a cluster of plant cells if it is determined that a singulated cluster of plant cells was not aspirated into the hollow transfer member.

[0027] The imaging system may be configured to image the hollow transfer member or to image contents of the second container after the controller has controlled the pressure unit to increase the pressure inside the hollow transfer member or to allow the pressure inside the hollow transfer member to increase for the purpose of releasing and / or flushing the holding liquid and the cluster of plant cells held therein from the hollow transfer member. Furthermore, the controller can be configured to control the pressure unit to provide another increase of the pressure inside the hollow transfer member or to allow the pressure inside the hollow transfer member to again increase for the purpose of releasing and / or flushing the holding liquid and the cluster of plant cells held therein from the hollow transfer member.

[0028] The system may comprise a plurality of second containers, wherein each second container is configured for receiving one or more clusters of plant cells. Alternatively, the second container may comprise a plurality of sub-compartments, wherein each sub-compartment is configured for receiving one or more clusters of plant cells.

[0029] The hollow transfer member may comprise a tubular member, such as an aspiration pipette.

[0030] According to a further aspect, the present invention provides a method for transferring clusters of plant cells that are arranged in a holding liquid inside a first container. This method comprises the steps of using a pressure unit for reducing pressure inside a hollow transfer member allowing a singulated cluster of plant cells and a quantity of the holding liquid to be aspirated into the hollow transfer member. The method additionally comprises maintaining a pressure in the hollow transfer member such that the cluster of plant cells and at least a part of the quantity of holding liquid in which the cluster of plant cells is held are kept inside the hollow transfer member during transfer.

[0031] The method may additionally comprise bringing the hollow transfer member in proximity to a singulated cluster of plant cells arranged in the first container prior to aspirating that singulated cluster of plant cells and holding liquid into the hollow transfer member and bringing the hollow transfer member in proximity to a second container prior to releasing or flushing the holding liquid and the cluster of plant cells held therein from the hollow transfer member. Additionally, or alternatively, an imaging system may be used that may include one or more optical cameras for identifying a singulated cluster of plant cells. A robotic arm, XYZ table, or gantry system on which the hollow transfer member is mounted may be automatically controlled to bring the hollow transfer member in proximity to the identified singulated cluster of plant cells. A pressure unit for reducing the pressure inside the hollow transfer member can automatically be controlled for allowing a singulated cluster of plant cells and a quantity of the holding liquid to be aspirated into the hollow transfer member. For the automated control, a controller may be used.

[0032] The method may further comprise after not being able to identify a singulated cluster of plant cells, using the hollow transfer member to perform a movement, such as a vibration, a random movement, or a stirring movement, while being at least partially submerged in the holding liquid, and / or imparting a vibration, a random movement, or a stirring movement to the first container, for singulating at least one cluster of plant cells from an identified agglomeration of clusters of plant cells.

[0033] The method may further comprise the following steps after not being able to identify a singulated cluster of plant cell. Firstly, the pressure unit is used to reduce a pressure inside the hollow transfer member for the purpose of aspirating a quantity of the holding liquid through the first opening and into the hollow transfer member. Then, the hollow transfer member is moved to be above or near an identified agglomeration of clusters of plant cells. Thereafter, the pressure inside the hollow transfer member is increased or the pressure inside the hollow transfer member is allowed to increase for the purpose of dispensing the holding liquid onto or in a near vicinity of the identified agglomeration of clusters of plant cells for singulating a cluster of plant cells from the agglomeration of clusters of plant cells.

[0034] Next, the present invention will be explained in more detail referring to the appended drawings, wherein:

[0035] Figures 1A-1F illustrate a general concept for transferring a cluster of plant cells in accordance with the present invention;

[0036] Figure 2 illustrates a system for transferring a cluster of plant cells in accordance with the present invention; and

[0037] Figure 3 illustrates a method for transferring a cluster of plant cells in accordance with the present invention.

[0038] A general concept for transferring a cluster of plant cells such as a somatic embryo is shown in figures 1A-1F. This concept will be explained by referring to system 1 shown in figure 2. Now referring to figure 1 A, first container 10 comprises a plurality of embryos 11A-11C that are held in a holding liquid 12. The embryos comprise a singulated embryo 11A that is separated by a distance dl from an agglomeration 1 IB of embryos, and a singulated embryo 11C that is separated by a distance d2 from agglomeration 1 IB. These embryos can be detected using an imaging system, which in figure 2 comprises an optical camera 61. More in particular, optical camera 61 may record an image of first container 10 with embryos 11 A-l 1C arranged therein. Using image analysis performed on the recorded image, a suitable embryo to be transferred next can be determined. This determination depends on whether an embryo is sufficiently far removed from its neighboring embryos. For example, for each embryo identified in the recorded image, a distance can be determined to its nearest neighboring embryo, and this distance can be compared to a predefined threshold. For example, referring to figure 1 A, it may be determined that embryo 11 A should be transferred next as dl is larger than the predefined threshold and d2 is smaller. Other criteria may be taken into account for determining which embryo to transfer next. For example, using analysis of the recorded image, one or more parameters of the embryo could be determined. Examples of such parameters are size, shape, color, an amount of space around the embryo, and the like. These parameters could alternatively or additionally be used for determining which embryo to transfer next. For example, in an embodiment, an embryo is only selected to be transferred next when it is sufficiently far removed from other embryos and if that embryo meets certain predefined targets for the abovementioned one or more parameters.

[0039] Determining an embryo to be transferred next based on a recorded image can be performed by controller 70 or it can be performed by a controller comprised in the imaging system. In the latter case, the imaging system sends information regarding the embryo to be transferred next to controller 70. This information may comprise position information on the embryo to be transferred next. In the former case, this position information is determined by controller 70 itself.

[0040] After having determined an embryo to be transferred next, controller 70 controls robotic arm 80 on which a hollow transfer member is mounted by which the embryo will be aspirated. Instead of using a robotic arm 80 other transport systems, such as a gantry system or XYZ table, can be used for enabling movement, more in particular 3D movement. Here, hollow transfer member comprises a tubular member 20. For this purpose, tubular member 20 has one of its ends 22 connected to a pressure unit 90. In figure 2, pressure unit 90 is mounted on robotic arm 80. Alternatively, pressure unit 90 is installed remote from robotic arm 80 using a conduit to tubular member 20.

[0041] On another end 21, tubular member 20 has an opening through which embryo 11A and a quantity of the holding liquid can be aspirated. A size of this opening lies in a range between 1 and 80 mm2, preferably between 2 and 50 mm2. In any event, according to the present invention, the opening is larger than the embryo to be transferred next. Accordingly, when tubular member 20 is evacuated, i.e. the pressure in tubular member 20 is lowered using pressure unit 90, the embryo and a quantity of holding liquid 12A will be aspirated into tubular member 20. This is shown in figures IB and 1C. According to the present invention, the pressure inside tubular member 20, more in particular at position Pl, is such that embryo 11A and the quantity of holding liquid 12A do not exit tubular member 20 towards pressure unit 90. A balance therefore is established between a capillary force acting on holding liquid 12A and a force acting on holding liquid 12A caused by a pressure difference between positions Pl and P0 on one hand, and the force of gravity acting on holding liquid 12A on the other hand. This balance will cause holding liquid 12A to remain arranged at one end of tubular member 20. Embryo 11 A may move within the quantity of holding liquid 12A and may move downward due to the force of gravity acting thereon.

[0042] For transferring embryo 11 A it is sufficient that at least part of the quantity of holding liquid 12A and embryo 11A remain inside tubular member 20 during transfer. To that end, a pressure difference is required between positions Pl and P0. However, the pressure at position Pl at the time of aspirating embryo 11 A need not be identical to the pressure at position Pl during transfer. For example, after aspirating embryo 11 A, end 22 of tubular member 20 connected to pressure unit 90 can be closed off, for example using a valve arranged in between tubular member 20 and pressure unit 90. This will cause some of holding liquid 12A to escape from tubular member 20. This will cause a decrease of pressure inside tubular member 20 at position Pl. A new balance can in this case be established in which embryo 11 A is held in a quantity of holding liquid that is smaller than the quantity of holding liquid that was aspirated.

[0043] In other embodiments, pressure unit 90 actively controls the pressure at position Pl during transfer. The pressure during transfer should be sufficient to keep embryo 11 A inside holding liquid 12A and inside tubular member 20.

[0044] Controller 70 will control robotic arm 80 based on the determined position of embryo 11 A. More in particularly, robotic arm 80 will first position tubular member 20 above embryo 11 A. Thereafter, as shown in figure 1A, tubular member 20 can make a movement such that end 21 thereof is below the interface between air and holding liquid 12. As shown in figure IB, pressure unit 90 can be controlled by controller 70 to reduce the pressure inside tubular member 20 after or during this movement to allow embryo 11A and a quantity of holding liquid 12A to be aspirated through end 21 into tubular member 20.

[0045] As shown in figure 1C, a predetermined amount of time after reducing the pressure, robotic arm 80 makes an upward movement to cause previously submerged end 21 of tubular member 20 to be positioned above the interface between air and holding liquid 12.

[0046] Optical camera 61 or another part of the imaging system may be used for verifying that embryo 11 A has been aspirated. For example, an image of first container 10 before and after the abovementioned steps may be compared to each other. Alternatively, optical camera 61 or another part of the imaging system may be used for recording an image of tubular member 20 after having made the abovementioned upward movement.

[0047] If it is determined, for example by controller 70 using image information from optical camera 61 or another part of a imaging system, that an embryo 11 A has not been aspirated into tubular member 20, the previous steps of determining an embryo to be transferred next and controlling robotic arm 80 and pressure unit 90 can be repeated.

[0048] Now referring to figure 2, if it is determined, for example by controller 70 and / or optical camera 61, that first container 10 does not comprise embryos that are sufficiently singulated, controller 70 may control singulation unit 50 to impart a mechanical vibration, a stirring motion, or other random motion to first container 10. This process may be repeated until an embryo has been sufficiently singulated. It should be noted that other means could be used for singulating embryos inside container 10. For example, controller 70 may control robotic arm 80 to make a stirring, vibrating, or stirring motion inside holding liquid 12 using tubular member 20. This is shown in figure ID.

[0049] Again referring to figure 2, system 1 may comprise a sensor 63 for detecting a level of holding liquid 12 inside first container 10. Preferably, the level of holding liquid 12 inside first container 10 is such that it accommodates a single layer of embryos. To maintain a sufficiently constant level of holding liquid 12 inside first container 10, controller 70 may control a dispensing unit 30 for dispensing holding liquid 12 into first container 10. For example, controller 70 can be configured to control dispensing unit 30 for repeatedly releasing an amount of holding liquid 12 during the process of transferring embryos 11A-11C. As an example, controller 70 may determine, using sensor 63, a decrease in a level of holding liquid 12 inside first container 10 and may control dispensing unit 30 for releasing an amount of holding liquid 12 for compensating this decrease. Additionally or alternatively, controller 70 may be configured to release a predefined amount of holding liquid after having performed a predetermined amount of actions for aspirating an embryo. For example, assuming that a quantity L ml of holding liquid is aspirated each time controller 70 controls pressure unit 90, controller 70 may control dispensing unit 30 to release N x L ml of holding liquid 12 after having performed N actions for aspirating an embryo. Preferably, the holding liquid dispensed by dispensing unit 30 is identical to holding liquid 12 that is already arranged in first container 10. Alternatively, a different holding liquid could be dispensed provided that this holding liquid does not chemically react with holding liquid 12 already arranged in first container 10.

[0050] As shown in figure IE, after having aspirated embryo 11 A, controller 70 controls robotic arm 80 to move towards a second container 40. In figure 2, second container 40 comprises a plurality of compartments 41 in which a respective embryo should be arranged. The positions of compartments 41 are preferably known to controller 70. Furthermore, controller 70 can be configured to arrange embryos 11A-11C inside compartments 41 according to a predefined schedule or pattern.

[0051] To arrange embryo 11A in compartment 41, controller 70 will control robotic arm 80 based on a position of compartment 41. When tubular member 20 is positioned above compartment 41, pressure unit 90 is controlled by controller 70 to increase a pressure at position Pl inside tubular member 20 causing the quantity of holding liquid 12A and embryo 11 A to be flushed out of tubular member 20.

[0052] An optical camera 62 or another part of the imaging system can be used for determining if an embryo has been successfully arranged in compartment 41. For example, controller 70 can perform image analysis on an image acquired using optical camera 62 for determining the presence and / or appropriate positioning of embryo 11 A inside compartment 41. If it is determined that an embryo has not been successfully arranged in compartment 41, controller 70 may control pressure unit 90, and optionally robotic arm 80, to perform another flushing operation.

[0053] Tubular member 20 can be embodied as an aspiration micropipette.

[0054] In the above, a controller 70 was described that is configured for performing various controlling and computational tasks. The present invention is not limited to controller 70 being a single unit. Alternatively, the various tasks can be performed by separated controlling units that together form controller 70.

[0055] It should be furthermore noted that the present invention is not limited to vertical positioning of tubular member 20. When the positioning of tubular member 20 deviates from a vertical positioning, the abovementioned balance of forces will be different. Still, regardless of the positioning of tubular member 20, according to the present invention, quantity of holding liquid 12A held in tubular member 20 is used as a carrier for embryo 11 A during transfer.

[0056] As shown in figure IF, after having released or flushed embryo 11 A from tubular member 20, controller 70 controls robotic arm 80 to move towards an embryo to be transferred next. If such an embryo cannot be identified, controller 70 controls singulation unit 50 for singulating one or more embryos.

[0057] The abovementioned process can be repeated until all embryos have been transferred.

[0058] Figure 3 illustrates a method for transferring a cluster of plant cells in accordance with the present invention. This method comprises step SI of using a pressure unit for reducing pressure inside a hollow transfer member thereby allowing a singulated cluster of plant cells and a quantity of the holding liquid to be aspirated into the hollow transfer member. Next, in step S2, a pressure in the hollow transfer member is maintained such that the cluster of plant cells and at least a part of the quantity of holding liquid in which the cluster of plant cells is held are kept inside the hollow transfer member during transfer of the hollow transfer member from the first container to a second container. In step S3, a pressure inside the hollow transfer member is increased or allowed to increase thereby releasing or flushing the cluster of plant cells and the quantity of holding liquid in which this cluster is held from the hollow transfer member and into the second container.

[0059] In the above, the present invention has been described using detailed embodiments thereof. However, the present invention is not limited to these embodiments. Rather, various modifications to these embodiments are possible without deviating from the scope of the present invention, which is defined by the appended claims and their equivalents.

Claims

CLAIMS1. A system (1) for transferring clusters (11A, 1 IB, 11C) of plant cells that are arranged in a holding liquid (12) inside a first container (10), the system comprising: a hollow transfer member (20) comprising a tubular member; a pressure unit (90) for regulating a pressure in the hollow transfer member, wherein the hollow transfer member has a first opening arranged at an end (21) of the tubular member, and a second opening that is arranged at another end (22) of the tubular member and that is connected to the pressure unit; and a controller (70) for controlling the pressure unit; characterized in that the system further comprises a valve arranged in between the second opening and the pressure unit, and in that the controller is configured to control the pressure unit to reduce a pressure inside the hollow transfer member relative to a pressure outside of the hollow transfer member allowing a singulated cluster of plant cells and a quantity of the holding liquid to be aspirated through the first opening into the hollow transfer member while preventing the holding liquid and / or the singulated cluster of plant cells to leave the hollow transfer member through the second opening, and for maintaining a pressure in the hollow transfer member by closing off the second opening of the hollow transfer member using the valve such that the cluster of plant cells and at least a part of the quantity of holding liquid in which the cluster of plant cells is held are kept inside the hollow transfer member during transfer thereof.

2. The system according to claim 1, wherein a size of the first opening lies in a range between 1 and 80 mm2, preferably between 2 and 50 mm2.

3. The system according to claim 1 or 2, wherein the second opening is configured to be closed off: a predetermined amount of time after the pressure unit has reduced the pressure inside the hollow transfer member; or after having detected that the singulated cluster of plant cells is arranged inside the hollow transfer member.

4. The system according to any of the previous claims, further comprising a dispensing unit (30) for dispensing, into the first container, holding liquid that is identical to and / or does not chemically react with the holding liquid already arranged in the first container, wherein the controller is configured to maintain a substantially constant level of the holding liquid inside the first container during the transfer of a plurality of said clusters of plant cells by controlling the dispensing unit.

5. The system according to claim 4, wherein the controller is configured to control the dispensing unit for releasing a predefined amount of the holding liquid after having performed a predefined number of transfers of clusters of plant cells.

6. The system according to claim 4, further comprising a sensor (63) for measuring a level of the holding liquid inside the first container, wherein the controller is configured to control the dispensing unit based on output from the sensor.

7. The system according to any of the previous claims, further comprising a transport unit for moving the hollow transfer member, wherein the controller is configured to: control the transport unit for bringing the hollow transfer member in proximity to a singulated cluster of plant cells arranged in the first container prior to aspirating that singulated cluster of plant cells and holding liquid into the hollow transfer member; and / or control the transport unit for bringing the hollow transfer member in proximity to a second container (40) prior to releasing the holding liquid and the cluster of plant cells held therein from the hollow transfer member.

8. The system according to claim 7, wherein the transport unit comprises a robotic arm (80), an XYZ table, or gantry system on which the hollow transfer member is mounted.

9. The system according to claim 7 or 8, further comprising an imaging system, for example comprising one or more optical cameras (61), for identifying one or more singulated clusters of plant cells inside the first container, wherein the controller is configured to control the transport unit based on a position of the identified one or more singulated clusters of plant cells; and / or for identifying an agglomeration of clusters of plant cells inside the first container.

10. The system according to claim 9, wherein the controller is configured to control the pressure unit to reduce a pressure inside the hollow transfer member for the purpose of aspirating a quantity of the holding liquid through the first opening and into the hollow transfermember, to then control the transport unit to move the hollow transfer member to be above or near an identified agglomeration of clusters of plant cells, and to then control the pressure unit to increase the pressure inside the hollow transfer member or to allow the pressure inside the hollow transfer member to increase for the purpose of dispensing the holding liquid onto or in a near vicinity of the identified agglomeration of clusters of plant cells for singulating a cluster of plant cells from the agglomeration of clusters of plant cells.

11. The system according to claim 4, 5 or 6, and claim 9, further comprising a movement system for mutually moving the dispensing unit and the first container, wherein the controller is configured to: localize an agglomeration of clusters of plant cells in the first container using the imaging system; mutually move the dispensing unit and first container to bring the dispensing unit at least near the agglomeration of clusters of plant cells; control the dispensing unit to dispense the holding liquid into the first container thereby singulating at least one cluster of plant cells from the agglomeration of clusters of plant cells.

12. The system according to claim 9, wherein the controller is configured for controlling the transport unit to cause the hollow transfer member to perform a movement, such as a vibration, a random movement or a stirring movement, while being at least partially submerged in the holding liquid thereby singulating at least one cluster of plant cells from an identified agglomeration of clusters of plant cells.

13. The system according to claim 12, wherein the controller is configured to: localize the agglomeration of clusters of plant cells in the first container using the imaging system; control the transport unit to bring the hollow transfer member at least near the agglomeration of clusters of plant cells prior to causing the hollow transfer to perform said movement.

14. The system according to any of the claims 9-13, wherein the controller or a separate processing unit is configured to: evaluate, from a recorded image of a cluster of plant cells, one or more parameters of the cluster of plant cells; compare the evaluated one or more parameters to respective target values; determine whether or not to transfer the cluster of plant cells based on the comparison;wherein the one or more parameters comprise one or more out of the group of size, shape, colour, and an amount of space around a cluster of plant cells.

15. The system according to any of the previous claims, wherein the clusters of plant cells comprise plant embryos, such as somatic embryos.

16. The system according to any of the previous claims, wherein the controller is further configured for controlling the pressure unit to increase the pressure inside the hollow transfer member or to allow the pressure inside the hollow transfer member to increase for the purpose of releasing the holding liquid and the cluster of plant cells held therein from the hollow transfer member.

17. The system according to any of the previous claims, further comprising a singulation unit for singulating clusters of plant cells inside the first container.

18. The system according to claim 17, wherein the singulation unit is a unit (50) for imparting a mechanical vibration to the first container by directly engaging the first container.

19. The system according to claim 17, wherein the singulation unit is a unit for imparting a mechanical vibration to the holding liquid inside the first container by directly engaging the holding liquid in the first container.

20. The system according to claim 19, wherein the singulation unit is a vibration member configured to be inserted into the holding liquid.

21. The system according to any of the previous claims, wherein the controller is configured to initiate a next transfer of a cluster of plant cells if a distance between said cluster of plant cells and other clusters of plant cells inside the first container exceeds a predefined first threshold.

22. The system according to claim 21, wherein the controller is configured to control: in so far as depending on claim 11, the movement system and the dispensing unit; or in so far as depending on claim 13, the transport unit; or in so far as depending on claim 18, the vibration unit; for singulating clusters of plant cells inside the first container if a cluster of plant cells to be transferred next cannot be identified using the imaging system.

23. The system according to claim 9 or any of the previous claims dependent thereon, wherein the imaging system is configured to image the hollow transfer member or to image contents of the first container after the controller has controlled the pressure unit for aspirating a cluster of plant cells and holding liquid into the hollow transfer member for determining whether a singulated cluster of plant cells has been aspirated into the hollow transfer member, wherein the controller is configured to control the transport unit and pressure unit to initiate a new transfer of a cluster of plant cells if it is determined that a singulated cluster of plant cells was not aspirated into the hollow transfer member.

24. The system according to claim 9 or any of the previous claims dependent thereon, wherein the imaging system is configured to image the hollow transfer member or to image contents of the second container after the controller has controlled the pressure unit to increase the pressure inside the hollow transfer member or to allow the pressure inside the hollow transfer member to increase for the purpose of releasing and / or flushing the holding liquid and the cluster of plant cells held therein from the hollow transfer member, wherein the controller is configured to control the pressure unit to provide another increase of the pressure inside the hollow transfer member or to allow the pressure inside the hollow transfer member to again increase for the purpose of releasing and / or flushing the holding liquid and the cluster of plant cells held therein from the hollow transfer member.

25. The system according to any of the previous claims, comprising a plurality of second containers each configured for receiving one or more clusters of plant material, or wherein the second container comprises a plurality of sub-compartments, each for sub-compartment being configured for receiving one or more clusters of plant material.

26. The system according to any of the previous claims, wherein the hollow transfer member comprises an aspiration pipette.

27. A method for transferring clusters of plant material that are arranged in a holding liquid inside a first container, comprising: providing a hollow transfer member that comprises a tubular member, wherein the hollow transfer member has a first opening arranged at an end of the tubular member, and a second opening that is arranged at another end of the tubular member and that is connected to a pressure unit;using the pressure unit for reducing pressure inside a hollow transfer member relative to a pressure outside of the hollow transfer member allowing a singulated cluster of plant cells and a quantity of the holding liquid to be aspirated through the first opening into the hollow transfer member while preventing the holding liquid and / or the singulated cluster of plant cells to leave the hollow transfer member through the second opening; maintaining a pressure in the hollow transfer member by closing off the second opening of the hollow transfer member using the valve such that the cluster of plant cells and at least a part of the quantity of holding liquid in which the cluster of plant cells is held are kept inside the hollow transfer member during transfer.

28. The method according to claim 27, comprising bringing the hollow transfer member in proximity to a singulated cluster of plant cells arranged in the first container prior to aspirating that singulated cluster of plant cells and holding liquid into the hollow transfer member; and bringing the hollow transfer member in proximity to a second container prior to releasing or flushing the holding liquid and the cluster of plant cells held therein from the hollow transfer member.

29. The method according to claim 27 or 28, further comprising: using an imaging system that for example includes one or more optical cameras for identifying a singulated cluster of plant cells; automatically controlling a robotic arm, XYZ table, or gantry system on which the hollow transfer member is mounted to bring the hollow transfer member in proximity to the identified singulated cluster of plant cells; automatically controlling a pressure unit for said reducing the pressure inside the hollow transfer member allowing a singulated cluster of plant cells and a quantity of the holding liquid to be aspirated into the hollow transfer member.

30. The method according to claim 29, further comprising after not being able to identify a singulated cluster of plant cells, using the hollow transfer member to perform a movement, such as a vibration, a random movement, or a stirring movement, while being at least partially submerged in the holding liquid, and / or imparting a vibration, a random movement, or a stirring movement to the first container, for singulating at least one cluster of plant cells from an identified agglomeration of clusters of plant cells.

31. The method according to claim 29, further comprising after not being able to identify a singulated cluster of plant cell: using the pressure unit to reduce a pressure inside the hollow transfer member for the purpose of aspirating a quantity of the holding liquid through the first opening and into the hollow transfer member; moving the hollow transfer member to be above or near an identified agglomeration of clusters of plant cells; increasing the pressure inside the hollow transfer member or to allow the pressure inside the hollow transfer member to increase for the purpose of dispensing the holding liquid onto or in a near vicinity of the identified agglomeration of clusters of plant cells for singulating a cluster of plant cells from the agglomeration of clusters of plant cells.

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