Systems and methods of separating and singulating embryos

Separating and singulating embryos employs a spray module configured to spray a plurality of embryos which are loaded on a porous substrate so as to separate and singulate the plurality of embryos, and a drying module configured to dry the plurality of separated and singulated embryos retained on the porous substrate while the porous substrate is moved across the drying module. A robotic arm operates to transfer the porous substrate from module to module, and a control device controls the operation of the system of separating and singulating embryos.

BACKGROUND

Modern silviculture often requires the planting of large numbers of genetically identical plants that have been selected to have advantageous properties. Production of new plants by sexual reproduction, which yields botanic seeds, is usually not feasible. Asexual propagation, via the culturing of somatic or zygotic embryos, has been shown for some species to yield large numbers of genetically identical embryos, each having the capacity to develop into a normal plant.

Somatic cloning is the process of creating genetically identical plants from plant somatic tissue. Plant somatic tissue is plant tissue other than the male and female gametes. In one approach to somatic cloning, plant somatic tissue is cultured in an initiation medium which includes hormones, such as auxins and/or cytokinins that initiate formation of embryogenic cells that are capable of developing into somatic embryos. The embryogenic cells are then further cultured in a maintenance medium that promotes multiplication of the embryogenic cells to form pre-cotyledonary embryos (i.e., embryos that do not possess cotyledons). The multiplied embryogenic cells are then cultured in a development medium that promotes development of cotyledonary somatic embryos.

At the end of development phase, the embryos may be present in a number of stages of maturity and development, and are typically attached to or imbedded in embryogenic suspensor mass (ESM). Separation and singulation are processing steps that typically occur at the end of development and maturation in which plant embryos are physically separated from each other and the underlying ESM before further processing, such as placement onto germination or pre-germination medium for further treatment prior to insertion into manufactured seeds.

SUMMARY

The present disclosure is directed to providing systems and methods of separating and singulating embryos for research purposes.

A system of separating and singulating embryos comprises a spray module configured to spray a plurality of embryos loaded on a porous substrate so as to separate and singulate the plurality of embryos, a drying module configured to dry the porous substrate upon which the plurality of separated and singulated embryos are retained, and a robotic arm operable of transferring the porous substrate from module to module in a predetermined sequence. The system of separating and singulating embryos further comprises a loading module, a dispensing module configured to dispense a blank porous substrate mounted in a frame which is transferred to a predetermined loading position at the loading module for loading embryos, and a bioreactor loading module for loading the porous substrate upon which the plurality of separated and singulated embryos are retained into a bioreactor, and a control device controlling the dispensing module, the spray module, the drying module, the conditioning module, and the robotic arm.

A method of separating and singulating embryos comprises the steps of spraying a plurality of embryos which are loaded on a porous substrate so as to separate and singulate the plurality of embryos at a spray module, drying the porous substrate upon which the plurality of separated and singulated embryos are retained, and transferring the porous substrate from module to module in a predetermined sequence by a robotic arm. The method of separating and singulating embryos further comprises the steps of dispensing a blank porous substrate from a dispensing module, loading a plurality of embryos onto the blank porous substrate while it is transferred to a predetermined loading position, loading the porous substrate upon which the plurality of separated and singulated embryos are retained into a bioreactor at a bioreactor loading module, and controlling the dispensing module, the spray module, the drying module, the bioreactor loading module, and the robotic arm by a control device.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As used herein, the term “embryogenic suspensor mass” (ESM) refers to early stage embryos in the process of multiplication by budding and cleavage.

As used herein, the term “embryogenic tissue” refers to an aggregate of tens to hundreds of embryogenic cells that form embryogenic suspensor mass.

As used herein, the term “plant embryo” refers to a somatic plant embryo. Somatic plant embryos may be produced by culturing embryogenic tissue by standard methods under laboratory conditions in which the cells comprising the tissue are separated from one another and urged to develop into minute complete embryos. As used herein, “plant embryo” includes embryos at various stages of development.

As used herein, the terms “cotyledonary embryo” refers to an embryo that possesses one or more cotyledons. Cotyledonary embryos have a well-defined elongated bipolar structure with latent meristem with cotyledonary primordial at one end and a potential radicle at the opposite end. The cotyledonary structure frequently appears as a small “crown” at one end of the embryo.

As used herein, the terms “separate”, “separation” refer to the process of separating mature embryos from attached ESM and immature embryos.

As used herein, the terms “singulate”, “singulation” refer to the process of acquiring individual, discrete embryos.

As used herein, the term “SSR” refers to the processes of the separation and singulation.

As used herein, the term “module” refers to a processing area or station.

As used herein, the term “COW” refers to the process of conditioning over water.

As used herein, the term “normal singulation” refers to the process of separating and singulating developed embryos contained in cultures after the Development and Stratification (a cold, late maturation treatment) processes.

As used herein, the term “early singulation” refers to the process of separating and singulating immature embryos part way through Development (majority embryos present are immature).

The somatic embryogenesis process is a process to develop plant embryos in vitro. Methods for producing plant somatic embryos are known in the art and have been previously described. Generally, the somatic embryogenesis process includes the steps of: (1) initiation or induction, to initiate formation of embryogenic tissue, such as embryogenic suspensor mass (ESM), which is a white mucilaginous mass that includes early stage embryos having a long, thin-walled suspensor associated with a small head with dense cytoplasm and large nuclei; (2) multiplication, sometimes referred to as maintenance, to multiply and mass produce embryogenic tissue; (3) development, to develop and form mature cotyledonary somatic embryos; and (4) post development steps such as separation, singulation, stratification, germination, growing into plants, such as through placement into manufactured seeds.

At the end of the development period, the embryos are to various degrees attached to and embedded in suspensor tissues and residual underdeveloped ESM, together with incompletely developed embryos, abnormally formed embryos, undersized or oversized embryos, and other pieces of non-embryo plant material, and to other embryos. It is important for subsequent normal germination to separate the embryos from the suspensor mass and from other embryos and to singulate the embryos into individual, discrete embryos.

The present application is directed to a system and method for separating and singulating embryos (referred to herein as the SSR system and method). The SSR system and method are designed to process a large number of small units of culture, e.g., culture on Petri plates. Thus, the SSR system and method are well suited for research focused activities such as genotype screening, e.g., as part of a clonal field test, and for research experiments where a high degree of replication on a small scale (e.g., a large number of Petri plates) are required.

As shown inFIG. 1, one embodiment of the separation and singulation system (referred to as the SSR system)10comprises five major stations or modules: (1) dispensing100; (2) loading200; (3) spray300; (4) drying400; and (5) bioreactor loading500. Blank s-frames or s-frames with disposed embryos may be transferred from module to module by use of a robotic arm600(as shown inFIGS. 2 and 3). At the same time, there may be a need of manually transferring the s-frames with disposed embryos from the drying module400to the bioreactor loading module500. The dispensing module100, the loading module200, the spray module300, the drying module400, the bioreactor loading module500, and the robotic arm600are contained in a sterile enclosure. The dispensing module100, the loading module200, the spray module300, and the drying module400may be arranged consecutively in a line from left to right, or vice versa. The SSR system10may implement an automated process of separating, singulating embryos disposed on the s-frames in a single spray step.

As shown inFIGS. 2 and 3, the loading module200, the spray module300, and the drying module400may include a loading platform210, a spray platform310, and a drying platform410which is divided into two sections, respectively. The loading platform310, the spray platform310, and the drying platform410may be installed on a support frame (not shown) which is fixed on an operation table (not shown). A dispensing slit110of the dispensing module100, the platforms210,310, and410may be located at the same height above the operation table by fixing on the support frame. In this way, the s-frames can flow from one end to another end of the processing line of the SSR system. The dispensing module100, the platforms210,310, and410may be designed to easily remove, insert, and/or sterilize.

The platforms210,310,410may be one integrated platform or separate ones. In some embodiments, the loading platform210and the spray platform310may be formed as one integrated platform with two sections; the drying platform410may be a separate one which is divided into two sections. As shown inFIGS. 2 and 3, the modules100,200,300, and400are arranged from left to right, which allows the s-frames dispensed from the module100to flow from left to right on the platform210,310, and410. In another embodiment, they can be arranged from right to left, whereby the s-frames flow from right to left.

In a further embodiment, two processing lines, a left line and a right line, may be built on the operation table. The handedness of the left line and the right line may be arranged opposite of each other. In the left line, the dispensing module100, the loading module200, the spray module300, the drying400, and the bioreactor loading module500may be arranged consecutively in a line from left to right, whereby s-frames flow from left to right. In the right line, the modules100,200,300,400, and500may be arranged in a line from right to left, whereby s-frames flow from right to left. The left and right processing lines flow towards each other so that one operator can load dried s-frames into either of two conditioning containers in the left line and the right line from one operating position. Another advantage of arranging two processing lines is that the operator may prefer one handedness over the other. Furthermore, the operator may switch back and forth between the two processing lines so as to relieve ergonomic strain.

As shown inFIGS. 2 and 3, an s-frame120includes a porous substrate122and a frame124. The porous substrate122may be mounted in the frame124. The porous substrate122includes a top surface and a bottom surface. The top surface of the porous substrate122may be used to receive a culture containing embryos. The porous substrate122may be any desired shape and dimension. While the porous substrate122as shown inFIGS. 2 and 3is rectangular, it can be any other desired shape, such as square shape. Exemplary dimensions may be from a surface area of about 10 square inches to 30 square inches or greater, such as 40 or 50 square inches. The fabric material of the porous substrate122should not be hydrophobic because it may lead to water pooling on the top surface of the porous substrate122while in the spray process at the spray module300. The fabric material must also be able to survive multiple autoclaving (15 psig steam—251 F). Examples of suitable material for the porous substrate122include polyester, polyamide, nylon, and so on. While fully developed embryos have typically been processed, various trials have shown that it is possible to process embryos at an earlier stage, thereby improving embryo quality. By way of example and without limitation, Saatifil Monofilament Polyester PES 810/55 may be used for normal singulation; Saatifil Monofilament Polyester PES 700/68 may be used for early singulation. For the purpose of imaging embryos which requires good color contrast between embryos and the porous substrates, the fabric material for making the porous substrate122may be preferably selected to have black or any other color which contrasts highly with the color of the embryos.

The porous substrate of an s-frame has a plurality of pores. The diameters of the pores may be referred to the opening width. The selection of opening width is generally related to dependent on the size of the embryos that users want to capture. Opening sizes between 425 microns and 2000 microns have been tried for loblolly pine and Douglas Fir embryos. By way of example and without limitation, the porous substrate may have an opening width in the range of from about 500 microns to about 2000 microns, such as about 810 micron for normal singulation and about 700 micron for early singulation.

The ratio of open area formed by all pores to the total area of the porous substrate may be referred to as open area percentage. The open area percentage may affect how easily water passes through the porous substrate when it is in the spray process at the spray module300. The opening width may also affect it too. When the open area percentage is below 50%, water may start to pool on the porous substrate in the spray process. The pooled water tends to float embryos out to the edges of the s-frame and tends to aggregate them. By way of example and without limitation, the open area percentage may be equal to or higher than 50%, preferably equal to or higher than 55%. By way of example and without limitation, the open area percentage of porous substrates with 810 micron opening width may be 55%; the open area percentage of porous substrates with 700 micron opening width may be 68.

As mentioned above, a blank s-frame can be ejected from the dispensing slit110of the s-frame dispensing module100. Then, the robotic arm600can move the blank s-frame to a predetermined position of the loading module200. When the s-frame120is moved to the predetermined position of the loading module200as shown inFIGS. 2 and 3, an operator may manually load a culture containing embryos from a Petri plate250to the approximate center of the s-frame120. By way of example and without limitation, the culture can be transferred from the single Petri plate250to the s-frame120by using a 2 inch×2 inch square mesh260on which the culture is originally plated. Typically, the culture does not fully cover the square mesh260. Thus, preferably less than 4 square inches of culture may be loaded to the s-frame120.

FIG. 4illustrates an embodiment of the loading platform210which is formed as one integrated platform with the spray platform310. A pair of steps212aand212bmay be formed longitudinally adjacent to the two opposite sides of the platform210,310, and510respectively. The steps212aand212bmay be used to support the two longitudinal sides of the frame of an s-frame so that the s-frame can be smoothly longitudinally moved by the robotic arm600from module to module. A rectangular recess214may be formed on the top surface of the loading platform210. A loading target216may be provided at the approximate center of the loading platform210. The loading target216may be used to show an operator a predetermined position for loading a culture. A pair of movable pins218aand218bmay be formed symmetrically at the two sides of the loading target216. The pair of pins218aand218bmay move up and down. When the robotic arm600moves an s-frame to and from the loading module200, the pins218aand218bmay move down. When the s-frame120is moved to the predetermined loading position, the pins218aand218bmay move up so as to capture the s-frame, whereby the operator cannot accidently move the s-frame while loading the culture from the square mesh260in the Petri plate250onto the s-frame120.

As shown inFIG. 4, a sink312may be formed in the spray platform310. The sink312may be connected to a drain314. The sink312and the drain314may be used for removing liquid and waste, such as embryogenic suspensor mass removed from the embryos, and embryos of undesired size, resulting from the separation and singulation process.

As shown inFIG. 5, the spray module300further comprises a spray nozzle320and a spray hood350. The spray nozzle320may be mounted to the approximate center of the internal top surface of the spray hood350by a first nut330and a second nut332. The height of the spray nozzle320relative to the spraying platform310may be adjusted by rotating a rotatable member336. The selection of the spray nozzle320may depend on the size, shape, and pressure of a desired spray. By way of example and without limitation, the spray nozzle320may be a narrow angle spray, such as 1/8GG-3007 FullJet Nozzle manufactured by Spraying System Co. The spay hood350may surround the spray nozzle320and the liquid spray produced by the nozzle320.

The spray hood350may be movably connected to a lift mechanism360(as shown inFIGS. 2 and 3) by a mounting frame352. The spray hood350and the mounting frame352may be designed to easily remove, insert, and/or sterilize. The lift mechanism360may raise and lower the spray hood350to engage the s-frame located at the predetermined spray position. The lift mechanism360may also raise the spray hood350to such a height that the robotic arm600can freely move below the spray hood350. The lift mechanism360may be of various constructions, such as using linear actuators. The spray hood350may be of a shape and size such that it engages around the frame of an s-frame at the predetermined spray position to form a seal, thus creating an isolated spray system. The isolated system contains the aerosols generated from the liquid spray emanating from the spray nozzle320, thereby reducing the possible spread of any contamination that may be present in the spray aerosols.

As shown inFIGS. 2 and 3, a pair of beam detectors312(one as a transmitter and another as a receiver) may be provided closely against the two opposite longitudinal sides of the spray platform310. The beam detectors312may be positioned in such way that a pair of beams314produced by the beam detector312are above and a certain distance away from the spray platform310. The beams314may be used for locating the s-frame under the spray hood350. The beam detectors312may be any suitable ones, such as laser beam detectors and infrared beam detectors. There may be proximity sensors (not shown) mounted on the lift mechanism360. The proximity sensors may determine when the spray hood is fully lowered or fully raised. When the spray hood350is fully lowered by the lift mechanism360to engage over the s-frame located at the predetermined spray position, the spray can be started.

The standard spray liquid is water; however, water with added osmotic agent, such as sugar or sugar alcohol, may be beneficial for early stage embryos. The spray nozzle320may produce a uniform, round, full spray pattern with medium to large sized drops (not atomized). By way of example and without limitation, the spray pressure at the spray nozzle320may range from 20 to 50 psi, preferably approximately 30 psi. If the spray pressure at the spray nozzle420is below 20 psi, the spray nozzle420may not develop a uniform spray pattern and may have “holes” in it.

The spray produced by the spray nozzle320may provide a downward force on the embryos disposed on the s-frame located at the spraying position. The downward force may push waste, such as suspensor tissues, residual underdeveloped ESM, and embryos of undesired size, through the pores of the porous substrate at the spraying position so as to separate desired embryos from the underlying suspensor mass and undersized embryos. Meanwhile, the downward force may, to some degree, singulate the embryos retained on the s-frame. The spray produced by the spray nozzle320may provide a tangential force on the plant embryos disposed on the s-frame. The tangential force may roll embryos out towards the edges of the s-frame and may cause the plant embryos to wiggle on the s-frame. The tangential force may, to some extent, singulate embryos separated by the downward force from ESM and undesired embryos by the downward force. However, the tangential force needs to be limited so that it will not have adverse effect on the separation process implemented by the downward force.

The downward force may be changed by various spray pressure. By the way of example, the spray pressure range of 20-50 psi may result in a downward force ranging from 0.0083 to 0.01441 lbs per in2. In addition to the spray pressure, the downward force may also be affected by other factors, such as nozzle type and spray distance. The downward force may be as high as 0.022 lbs per in2without damaging embryos. By the way of example, the downward force may be approximately 0.011 lbs per in2. The required downward force for separating embryos depends on ESM properties because ESM is a gel-like mat that has embryos embedded in it. Embryo development stage and culture temperature may affect how gelled ESM is. The downward force being approximately 0.022 lbs per in2works well for the majority of cultures following development and stratification (i.e., what it is referred above as “normal singulation”). A downward force being significantly less than 0.011 lbs per in2would be required for early singulation because the ESM mat is softer and less gelled at an early stage.

Spray time required for separation depends on the toughness of the ESM. Typically, tougher ESM need longer spray time. By the way of example without limitation, the spray time may range from 2 to 30 second, such as 20 seconds for normal separation and 4 seconds for early separation. The spray time may be up to a minute for very tough ESM which tends to result when cultures are stored for extended periods in stratification (a cold treatment).

FIG. 6is a cross section view of a full cone spray pattern. As shown in the figure, the top angle between two sides is referred to as spray angle370. The distance between the spray nozzle320and the surface of the s-frame at the spray position is referred to as spray distance372. The area covered by the spray produced by the spray nozzle320is referred to as coverage area. A theoretical coverage area376is shown inFIG. 6. A practical coverage area (not known) may diverge from the theoretical coverage area376.

By way of example and without limitation, the spray angle370may range from about 25 degrees to about 35 degrees, such as from about 26 degrees to about 32 degrees. Higher spray angles might be problematic because higher spray angles may lead to strong tangential force so that the tangential force rolls embryos out to the edges of the s-frame faster than a speed that the downward force pushes the ESM or undesired embryos through the s-frame. In that case, the embryos cannot be separated from the underlying suspensor mass and undersized embryos. A more narrow spray angle may be acceptable; however, in that case an increased spray distance may be needed to achieve the same coverage area.

As shown inFIG. 6, since the spray fans outward from the spray nozzle320, increasing the spray distance372may increase the coverage area376. At the same time, increasing the spray distance372may reduce downward impact force per unit area because of the increase of the coverage area while the amount of impacting water droplets remains the same. As mentioned above, the height of the spray nozzle320may be adjustable, which enables the spray nozzle320to be mechanically raised or lowered relative to the top of the spray hood350. This arrangement allows the users to adjust the spray distance372without fabricating a new spray hood and spray nozzle. The spray distance372(for a given spray angle) is a function of the desired spray coverage area and the downward impact force. By way of example and without limitation, if the spray distance is selected as being 7.25 inches, it will produce a spray pattern with a diameter of 3.5 inches at the s-frame surface. A desired spray coverage area resulting from the selection of spray distance may leave a certain size of outer area on the s-frame where little water is impacting the porous substrate of the s-frame. The unsprayed outer area of the s-frame may be useful for draining water and leaving an area for embryos to collect so that they are not up against the frame of the s-frame. By way of example and without limitation, the unsprayed outer area may range from 0.7 inch to 1.5 inch wide perimeter, such as ⅔ inch.

After separating and singulating at the spray module300, an s-frame130with embryos may be transferred by the robotic arm600to the drying module400. The drying module400is used to remove excess water or other spray liquid from the s-frame130upon which the separated and singulated embryos are deposited. A variety of methods may be used to remove the water or other spray liquid from the s-frame130. As shown inFIG. 7, one embodiment of the drying module400includes the drying platform410divided into two sections412and414, a vacuum housing420which is located between the two sections of the drying platform410, a narrow elongated opening422which is provided on the vacuum housing420. The elongated opening422is in communication with a vacuum source (not shown). The elongated opening422may be configured to ensure that the vacuum can be even across the length of the elongated opening422. Typically, the width of the elongated opening422may range from about 0.001 inches to 1.0 inch or greater, such as from 0.001 inches to about 0.1 inches, such as from 0.04 inches to 0.06 inches, such as about 0.02 inches. Other widths of the opening422may be suitable, depending on the dimensions of the s-frame.

During operation, the s-frame130may be transferred to the first section412of the drying platform410. The robotic arm600may move the s-frame across the opening422of the vacuum housing420to the second section414of the drying platform410. As the s-frame130is moved across the opening422, the bottom surface of the s-frame130is in contact with the opening422, which is in communication with the vacuum source, resulting in water or other spray liquid being removed from the s-frame130and air being drawn over and around the embryos disposed on the top surface of the s-frame130and through the s-frame130.

In some embodiments, the negative pressure generated by the vacuum source may range from about −0.5 psi to about 15 psi, such as from about −5 psi to about −12 psi. By the way of example without limitation, in one embodiment, the negative pressure is about −4.35 psi; the flow rate is about 170 liters per minute of air. In some embodiments, the negative pressure generated by the vacuum source may be constant as the elongated opening422is in communication with the vacuum source. In other embodiments, the negative pressure generated by the vacuum source may vary as the elongated opening422is in communication with the vacuum source.

In some embodiments, the s-frame130and the elongated opening422may move relative to each other at a speed in the range from about 1 millimeter per second to about 45 millimeters per second, preferably from about 1 millimeter per second to about 10 millimeters per second. In one embodiment, the s-frame130and the elongated opening422may move relative to each other at a speed of about 4 millimeters per second. By setting a certain speed of an s-frame with a given size relative to the elongated opening422, the s-frame drying time may be determined. For instance, when the inside width of the s-frame is about 4.72 inch and the speed of the s-frame relative to the elongated opening422is 4 millimeters per second, the s-frame drying time is about 30 seconds.

In one embodiment, the vacuum housing420with the elongated opening422is rectangular in shape (e.g., bar-shaped) and is sized, depending on the size of the porous substrate of the s-frame, such that the vacuum housing420with elongated opening422will be in contact with substantially all of an area of a cross section of the porous substrate, but preferably will not be in contact with the entire area of the porous substrate at any one time. In one embodiment, the vacuum housing420with the elongated opening422is sized and shaped such that the vacuum housing420with the elongated opening422in use will be in contact with less than 1% of the entire area of the porous substrate of the s-frame at any one time, such as from about 0.01% to about 0.1%, such as about 0.02% to about 0.05%, such as about 0.04%.

FIG. 8illustrates an overhead vision mechanism450in accordance with one embodiment. The overhead vision mechanism450can be used to count or at least estimate the number of embryos retained on the s-frame130after the separation and singulation process. The overhead vision mechanism450may be centered over the s-frame130after it has been dried and positioned on the second section414of the drying platform410. The location may be referred to the dry end of the drying module. As shown in theFIG. 8, the overhead vision mechanism450comprises an image generator452and a light source454. Both the image generator452and the light source454may be mounted in an arm460. The image generator452may be positioned to generate an image of embryos retained on the dried s-frame130. The light source454may be positioned to illuminate the dried s-frame130with separated and singulated embryos. The image generator452and the light source454may be mounted to face the dried s-frame130at approximately a right angle. The light source454may be positioned such that a beam of light produced by the light source454is approximately centered about the center of the dried s-frame130. Any of a variety of image generators452and light sources454may be used. By way of example and without limitation, the image generator452may be a high resolution camera.

After drying at the drying module400, the s-frame with separated and singulated embryos may be manually transferred to the fifth module of the SSR system, referred to herein as the “bioreactor loading module”500. The bioreactor loading module500may be used for loading the s-frame upon which the separated and singulated embryos are retained into a bioreactor where further bioprocessing may occur. The further bioprocessing may be further development, maturation, or conditioning depending on the stage of embryos. The further bioprocessing may also depend on what is in the bioreactor, such as development media, water, or a salt solution. For instance, after early singulation, the s-frames with the separated and singulated immature embryos may be loaded into a bioreactor in the presence of a development medium for further development and maturation of the separated and singulated immature embryos. In another embodiment, after normal singulation, the s-frames with the separated and singulated mature embryos may be loaded into a bioreactor in the presence of water or a salt solution for conditioning the separated and singulated mature embryos.

As described above, the SSR system comprises a number of modules or processing stations. During operation of the SSR system, the s-frames move sequentially through the processing line from the dispensing module100, the loading module200, the spray module300, and the drying module400. The s-frames may be transferred from module to module through the system by use of the robotic arm600.

As shown inFIGS. 2 and 3, the robotic arm600includes an arm610, a connecting member620, a base630, and a motor (not shown) for driving the robotic arm600. A downward opening612may be provided at the front end of the arm610; the opening612may be used to hold the left or right side of the frame of an s-frame so as to move the s-frame from module to module. The arm610may be connected to the connecting member620which may be fixed on the base630. The connecting member620may include a pneumatic cylinder (not shown) which can lower or raise the arm610. The pneumatic cylinder allows the arm610to engage or disengage the s-frame. When the arm610is lowered, the opening612may engage the s-frame so as to move it to any predetermined positions. When the arm610is raised, the opening may disengage the s-frame so that the robotic arm600can travel freely up and down the processing line. The base630may be movably attached to a linear slide rail650to provide the range of motion necessary to cover the processing area encompassed by the modules100,200,300, and400of the SSR system. The slide rail650may be positioned behind the processing line of the SSR system and fixed on the operation table. The maximum height of the robotic arm600needs to be a certain amount less than the height of the spray hood350raised by the lift mechanism360so that the robotic arm can freely move below the spay hood350when the spay hood350is raised by the lift mechanism360.

By way of example and without limitation, during operation of the SSR system10, the robotic arm600may be operable to transfer s-frames from module to module in a predetermined sequence. At any one time, a first s-frame may be positioned on the spray platform310, a second s-frame may be positioned on the loading platform410, and a third blank s-frame may be partially ejected from the dispensing module100. The robotic arm600may be initially located at any suitable position between the loading module200and the spray module300. Once the SSR system10is started by an operator, the robotic arm600may move to the leftmost end of the processing platform of the SSR system10and hold the right side of the third blank s-frame to pull it out from the s-frame canister102to the predetermined loading position at the loading module200. Meanwhile, the second s-frame with loaded embryos and ESM may be pushed towards the predetermined spray position by the third blank s-frame pulled out by the robotic arm600. The first s-frame with separated and singulated embryos after the spray process may be pushed towards the drying module400by the second s-frame. Then, the robotic arm600may adjust the second s-frame to the precise spray position. Next, the robotic arm600may hold the left side of a first s-frame with separated and singulated embryos and move it across the elongated opening422of the drying module400at a certain speed for drying the s-frame with separated and singulated embryos. The dried first s-frame may be manually transferred to the COW box520for conditioning developed embryos after normal singulation or transferred to any other suitable container for storing immature embryos after early singulation so as to clear the drying platform410for being prepared for next operation. When the first s-frame is entirely moved across the elongated opening422, the robotic arm600may return to its initial position and be prepared for the next motion circle based on a control signal from a control device700as shown inFIG. 9.

In an embodiment, the robotic arm may be controlled to move given distances so as to transfer an s-frame to the predetermined loading position or the predetermined spray position, or move an s-frame across the drying module400at a given speed. Alternatively, there may be provided a plurality of position sensors along the loading platform210, the spray platform310, and the drying platform410. The plurality of position sensors may be coupled to the control device700. The control device700may control the motion of the robotic arm600based on signals from the plurality of position sensors.

FIG. 9illustrates one embodiment of the control device700. As shown inFIG. 10, the control device700may be coupled to the dispensing module100, the spray module300, the drying module400, the bioreactor loading module500, and the robotic arm600. The control device700may be used to control the ejection of s-frames at the dispensing module100, control the lift mechanism360and spray time at the spray module300, control the drying time at the drying module400, and control the motion of the robotic arm600along the slide rail650.

As shown inFIG. 9, the control device700includes a processing unit706, a system memory708, and a system bus710that couples various system components including the system memory708and the processing unit706. The processing unit706may be any logic processing unit, such as one or more central processing units (CPUs), programmable logic controllers (PLC), distributed control system (DCS), and digital signal processors (DPS), etc. The system bus710can employ any known bus structures or architectures, including a memory bus with memory controller, a peripheral bus, and a local bus. The system memory708includes read-only memory (“ROM”)712and random access memory (“RAM”)714. A basic input/output system (“BIOS”)716, which can form part of the ROM712, contains basic routines that help transfer information between elements within the control device700, such as during start-up.

The control device700may include a storage device, such as a hard disk drive718for reading from and writing to a hard disk720, and an optical disk drive722and a magnetic disk drive724for reading from and writing to removable optical disks726and magnetic disks728, respectively. The optical disks726can be a CD or a DVD, while the magnetic disk728can be a magnetic floppy disk or diskette. The hard disk drive718, optical disk drive722, and magnetic disk drive724may communicate with the processing unit706via the system bus710. The drives718,722,724, and their associated computer-readable media720,726,728, may provide nonvolatile storage of computer-readable instructions, data structures, program modules and other data for the control device700. Although the depicted control device700employs hard disk720, optical disk726, and magnetic disk728, those skilled in the relevant art will appreciate that other types of computer-readable media that can store data and instruction accessible by a computer may be employed, such as magnetic cassettes, flash memory cards, Bernoulli cartridges, RAMs, ROMs, smart cards, etc. In other embodiments, the control device700need not include the drives718,722,724, and their associated computer-readable media720,726,728.

Program modules can be stored in the system memory708, such as an operating system730, one or more application programs732, other programs or modules734, drivers736, and program data738. While shown inFIG. 9as being stored in the system memory708, the operating system730, one or more application programs732, other programs or modules734, drivers736, and program data738can be stored on the hard disk720of the hard disk drive218, the optical disk726of the optical disk drive722and/or the magnetic disk728of the magnetic disk drive724. A user can enter commands and information into the control device700through input devices such as a keyboard742and/or a pointing device such as a mouse744. These or other input devices may be connected to the processing unit706through an interface746such as a universal serial bus “USB” interface that couples to the system bus710, although other interfaces such as a parallel port, a game port or a wireless interface or a serial port may be used. A touchscreen748or other display device may be coupled to the system bus710via a video interface750, such as a video adapter. Although not shown, the control device700can include other output devices, such as speakers, printers, etc. In other embodiment, the control device700needs not include any display device and any output devices, such as speakers, printers, etc. Optionally, the control device700may include a start button760and a stop button762for starting and stopping the application programs stored in the system memory708so as to implement necessary automated control of the SSR system.

The SSR system of the disclosure can significantly improve germination of plant embryos. Compared to existing standard protocol (e.g., hand transfers of embryos), the SSR system may improve germination of plant embryos by about 5% to about 7%.

FIG. 10shows a method800of separating and singulating embryos by using the SSR system.

At802, an s-frame may be at least partially ejected to a processing platform from an s-frame canister at a dispensing module of a SSR system. The s-frame may comprise a frame and a porous substrate which may be mounted in the frame and comprise a plurality of pores. The diameters of the pores may be referred to the opening width. The pore opening width may be in the range of from about 500 microns to about 2000 microns, such as about 810 micron for normal singulation and about 700 micron for early singulation. The ratio of open area formed by all pores to the total area of the porous substrate may be referred to as open area percentage. By way of example and without limitation, the open area percentage may be equal to or greater than 50%, preferably equal to or greater than 55%.

At804, the porous substrate may be transferred by a robotic arm to a predetermined loading position at a loading module

At806, a plurality of embryos may be loaded onto the top surface of the porous substrate at the predetermined loading position.

At808, the porous substrate with loaded embryos may be transferred by the robotic arm to a predetermined spray position at a spray module.

At810, when the porous substrate with disposed embryos is located at the predetermined spray position, a spray hood may be lowered to engage around the frame of the s-frame to form a seal, thereby creating a closed spray environment. A spray nozzle adjustably mounted at the approximate center of the internal top surface of the spray hood may be configured to discharge a spray pattern designed to separate desired embryos from the underlying ESM and undersized embryos by pushing ESM and undersized embryos through the pores of the porous substrate and singulate desired embryos retained on the s-frame by scattering them on the top surface of the porous substrate of the s-frame. The spray distance between the spray nozzle and the top surface of the porous substrate may be adjustable by raising or lowering the spray nozzle. The spray angle of the spray module may range from about 25 degrees to about 35 degrees, such as from about 26 degrees to about 32 degrees. The spray pressure at the spray nozzle320may range from 20 to 50 psi, preferably approximately 30 psi. The spray time may range from 2 to 30 second, such as 20 seconds for normal separation and 4 seconds for early separation. When the spray process is finished, the spray hood may be lifted by a lift mechanism. The s-frame with separated and singulated embryos may be transferred by the robotic arm to a drying module

At812, after spray separation and singulation at the spray module, the porous substrate with separated and singulated embryos may be transferred by the robotic arm to a drying module. The porous substrate with separated and singulated embryos may be moved by the robotic arm at a certain speed across an elongated opening of a vacuum housing located between the first section and the second section of a drying platform, whereby the bottom surface of the porous substrate of the s-frame being in contact with the elongated opening which is in communication with a vacuum source, results in spray liquid being removed from the s-frame and air being drawn over and around the embryos disposed on the top surface of the porous substrate and through the porous substrate. The dispensing module, the loading module, the spray module, the drying module may be arranged consecutively in a line from left to right or from right to left so that the porous substrate can be linearly transferred from one end to the other end of a processing line of the SSR system. The porous substrate may be transferred by the robotic arm from module to module in a predetermined sequence. Optionally, the SSR system may include two processing lines which are arranged opposite to each other.

At814, after drying at the drying module, the s-frame with separated and singulated embryos may be transferred to a bioreactor loading module for loading the s-frame upon which the separated and singulated embryos are retained into a bioreactor where further bioprocessing may occur. The further bioprocessing may be further development, maturation, or conditioning depending on the stage of embryos. The further bioprocessing may also depend on what is in the bioreactor, such as development media, water, or a salt solution. For instance, after early singulation, the s-frames with the separated and singulated immature embryos may be loaded into a bioreactor in the presence of a development medium for further development and maturation of the separated and singulated immature embryos. In another embodiment, after normal singulation, the s-frames with the separated and singulated mature embryos may be loaded into a bioreactor in the presence of water or a salt solution for conditioning the separated and singulated mature embryos.

Optionally, the method800of separating and singulating embryos by using the SSR system may comprise a step of counting or at least estimating the number of separated and singulated embryos by an overhead vision mechanism. The overhead vision mechanism may be centered over the porous substrate after it has been dried at the drying module. The overhead vision mechanism may comprises an image generator for capturing the image of the separated and singulated embryos retained on the porous substrate and a light source for illuminating the separated and singulated embryos retained on the porous substrate. The light source may be positioned such that a beam of light produced by the light source is approximately centered about the center of the porous substrate after it has been dried at the drying module.