Abstract:
A method of singulating embryos is provided. The method includes providing a plurality of embryos ( 40 ) within a system ( 20 ) and sensing ( 34 ) at least one of the plurality of embryos in a fluid. The method also includes dispensing ( 26 ) at least one of the plurality of embryos on a surface ( 28 ).

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application is entitled to and claims the benefit of priority under 35 U.S.C. §119 from U.S. Provisional Patent Application Ser. No. 61/247,047 filed Sep. 30, 2009, and titled “Method of Singulating Embryos,” the contents of which are incorporated herein by reference. 
    
    
     BACKGROUND 
     A sexual propagation for plants has been shown for some species to yield large numbers of genetically identical embryos, each having the capacity to develop into a normal plant. Such embryos must usually be further cultured under laboratory conditions until they reach an autotrophic “seedling” state characterized by an ability to produce their own food via photosynthesis, resist desiccation, produce roots able to penetrate soil, and fend off soil microorganisms. Some researchers have experimented with the production of artificial seeds, known as manufactured seeds, in which individual plant somatic or zygotic embryos are encapsulated in a seed coat. Examples of such manufactured seeds are disclosed in U.S. Pat. No. 5,701,699, issued to Carlson et al., the disclosure of which is hereby expressly incorporated by reference. 
     Typical manufactured seeds include a seed shell, synthetic gametophyte and a plant embryo. A manufactured seed that does not include the plant embryo is known in the art as a “seed blank.” Such a seed blank typically is a cylindrical capsule having a closed end and an open end. Synthetic gametophyte is placed within the seed shell to substantially fill the interior of the seed shell. A longitudinally extending hard porous insert, commonly known as a cotyledon restraint, may be centrally located within the synthetic gametophyte and includes a centrally located cavity extending partially through the length of the cotyledon restraint. The cavity is sized to receive the plant embryo therein. The well-known plant embryo includes a radicle end and a cotyledon end. The plant embryo is deposited within the cavity of the cotyledon restraint cotyledon end first and is sealed within the seed blank by at least one end seal. There is a weakened spot in the end seal to allow the radicle end of the embryo to penetrate the end seal. 
     There are automated processes available to mass produce manufactured seeds of the type described above. One such automated process is described in U.S. patent application Ser. No. 10/982,951, entitled System and Method of Embryo Delivery for Manufactured Seeds, and assigned to Weyerhaeuser Company of Federal Way, Washington, the disclosure of which is hereby expressly incorporated by reference. 
     Currently, embryos are manually plucked from a growing medium and are physically placed on the plate for retrieval and insertion into a seed blank. Although such manual processes are effective, they are not without their limitations. As a non-limiting example, such manual operations are both labor and time intensive and, therefore, expensive. As part of the process to produce large numbers of somatic embryos available for insertion in manufactured seeds, it is desirable to minimize the manual labor element from the process. 
     SUMMARY 
     This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. 
     A method of singulating embryos is provided. The method includes providing a plurality of embryos within a system and sensing at least one of the plurality of embryos in a fluid. The method also includes dispensing at least one of the plurality of embryos on a surface. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
       The foregoing aspects and many of the attendant advantages of this invention will become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein: 
         FIG. 1  is a diagrammatical view of one example of a system using a method of singulating embryos in accordance with one embodiment of the present disclosure; 
         FIG. 2A  is a flow diagram of a method of singulating embryos in accordance with one embodiment of the present disclosure; 
         FIG. 2B  is a continuation of the flow diagram of  FIG. 2A ; 
         FIG. 3A  is a flow diagram of a method of singulating embryos in accordance with another embodiment of the present disclosure; and 
         FIG. 3B  is a continuation of the flow diagram of  FIG. 3A . 
     
    
    
     DETAILED DESCRIPTION 
       FIG. 1  diagrammatically depicts an automated system  20  for implementing a method of singulating embryos in accordance with one embodiment of the present disclosure. The system  20  is suitably mounted in conjunction with an assembly for assembling manufactured seeds (not shown) or is remotely located from such an assembly. 
     The system  20  includes an embryo storage assembly  22 , a programmable logic controller (PLC)  24 , a placement mechanism  26 , and an embryo deposit assembly  28 . The embryo storage assembly  22  includes a singulation vessel  30 , a lift mechanism  32 , and a sensor  34 . The singulation vessel  30  is suitably a container having a plurality of embryos  40  suspended in a fluid, such as a sterile, Nanopure water. Preferably, the fluid is agitated to a sufficient degree to suspend all embryos  40 . The singulation vessel  30  is mounted on the lift mechanism  32 . 
     The lift mechanism  32  includes a base plate  50  coupled to a well-known lift  52 , such as a screw drive or a scissor lift, to assist in maintaining a substantially constant head at the outlet of the singulation vessel  30 . Within the meaning of this disclosure and used in this context, the term “substantially” is intended to include engineering acceptable variations resulting in a nearly constant fluid flow rate. 
     Although the use of a lift  52  to assist in maintaining a substantially constant head, other devices known to maintain a substantially head are also acceptable. As a non-limiting example, a pump (not shown) may be placed in fluid communication with the singulation vessel  30  to maintain the substantially constant flow rate. Thus, such devices are acceptable equivalents and are within the scope of the present disclosure. Further, while maintaining a substantially constant head is preferred, a variable head is also within the scope of the present disclosure as described in greater detail below. 
     Embryos  40  are transported between the singulation vessel  30  and the placement mechanism  26  by fluid flowing through tubing  60 . The tubing  60  extends between the singulation vessel  30  and the placement mechanism  26  and the sensor  34  is suitably positioned adjacent the tubing  60  to sense and/or detect embryos  40  within the tubing  60 , as described in greater detail below. 
     In the illustrated and exemplary embodiment, the flow rate of embryos  40  through the tubing  60  is controlled by the lift  52 . Specifically, and as is well-known, the flow rate within the tubing  60  is proportional to the square root of the vertical distance between the outlet of the tubing  60  at the placement mechanism  26  and the liquid level in the singulation vessel  30 . As the fluid in the singulation vessel  30  is decreased, the height of the singulation vessel  30  is raised by the lift mechanism  32 . The lift  52  raises the singulation vessel  30  at a fixed rate proportional to the flow rate of fluid inside of the tubing  60  to maintain a substantially constant flow rate. In other embodiments, the lift  52  may be raised or lower to increase or decrease, respectively, the flow rate. 
     The tubing  60  includes an inner diameter sufficiently large to permit entry of a single embryo  40  to enter the tubing  60  at any given time. Although multiple embryos  40  may be positioned longitudinally within the tubing  60 , it is desirable that only a single embryo may enter the tubing  60  at any given time. It is also preferred that the tubing  60  be of a material, such as silicone, that is transparent or semi-transparent to permit detection of an embryo within the tubing  60  by the sensor  34 . 
     The sensor  34  is a well-known, laser-based visual sensor used to detect when an embryo  40  exits the singulation vessel  30 . One such sensor  34  is model No. LV-H300/100 Series, manufactured and sold by Keyence Corporation of Osaka, Japan. The sensor  34  is suitably mounted to the base plate  50  with the tubing  60  operatively disposed between components of the sensor  34 . The sensor  34 , in turn, is in communication with the PLC  24 . 
     The system  20  may include a second, well-known sensor (not shown) in communication with the singulation vessel  30 . This second sensor is used to measure the hydrostatic head of the fluid in the singulation vessel  30 . One such sensor is model No. FW-H07, manufactured and sold by Keyence Corp. of Osaka, Japan. Such a sensor uses ultrasonic sound waves to measure distance. Although an ultrasonic sensor is preferred, other types of sensors, including laser and radar based, are within the scope of the present disclosure. The second sensor is in communication with the PLC  24 . 
     The well-known PLC  24  suitably has an operator interface to control the singulation process and the raising and lowering of the lift mechanism  32 . One such PLC  24  is a DirectLOGIC 205 Modular Programmable Logic Controller (DL205 PLC), manufactured and sold by Koyo Electronics Industries Co., Ltd. of Tokyo, Japan. 
     The PLC  24  is programmable to interface with the lift mechanism  32 , the sensor  34 , the second sensor, and the placement mechanism  26  during operation of the system  20 , as well as to permit the operator to adjust operational parameters. Operational parameters, such as the number of embryos  40  placed on the embryo deposit assembly  28 , the spacing between the embryos  40 , and the location of embryos  40  on the embryo deposit assembly  28  may all be programmed as desired. 
     The PLC  24  may be programmed to control the spacing and placement of embryos  40  on the embryo deposit assembly  28  by tracking the embryo as it flows through the tubing  60 . In such an embodiment, the PLC  24  includes a clock or timer and a registry. One such registry is an embryo location registry (“ELR”). The ELR includes binary registers that represent locations along the length of the tubing  60 . As an example, the ELR may segregate the tubing  60  into fifty registers, which represent fifty sequential locations in the tubing  60 . The first register location is suitably located closest to the sensor  34  and the last register is located at the end of the tubing  60  where it connects to the placement mechanism  26 . The ELR tracks and logs as a function of time the path of embryos within the tubing  60 , as described in greater detail below. 
     The placement mechanism  26  includes a robotic arm  80 . Motion of the robotic arm  80  is controllable relative to the embryo deposit assembly  28  to position the outlet of the tubing  60  over an open location on the embryo deposit assembly  28 . One suitable robotic arm  80  is an Ultramotion robotic arm, model No. DA25-HT17-8 NO-B/4, manufactured and sold by Ultramotion of Mattituck, N.Y. To achieve the desired motion of the robotic arm  80 , the placement mechanism  26  also includes a well-known stepping motor (not shown), such as model No. PK266-E2.0A, manufactured and sold by Oriental Motor U.S.A. Corp. of Torrance, Calif. 
     The robotic arm  80  has two degrees of freedom to provide precise placement of embryos  40  on the embryo deposit assembly  28 . In that regard, it is preferred that the robotic arm  80  translates longitudinally along an axis indicated by the arrow  70 . Further, the robotic arm  80  moves along the axis perpendicular to arrow  70 , i.e., in and out of the page. The outlet of the tubing  60  on the robotic arm  80  is suitably oriented at an angle relative to a vertical axis so that, as the fluid exits from the tubing  60 , it is not perpendicular to the embryo deposit assembly  28 . 
     It is also desirable that the robotic arm  80  is controlled by the PLC  24 , in combination with the ELR, sensor  34 , and/or the second sensor. As a non-limiting example, if an embryo  40  is detected by the sensor  34 , it sends a signal to the PLC  24  indicating the presence of the embryo. This signal is entered in the ELR as a “true.” If an embryo  40  is not detected by the sensor  34 , then the register is “false.” A “true” registry is noted as a “1,” while a “false” registry is noted as a “0.” 
     The number of registries in the ELR is a function of the length of the tubing  60 . For example, if the tubing  60  is 20 inches long and there are fifty registers, each register represents 0.4 inches of tubing  60 . Further, in this example, the travel time of an embryo from the sensor  34  to the placement mechanism  26  is approximately one second. As a result, each registry of the ELR represents approximately 20 ms of time. The clock updates the registry every 20 ms, such that the registers are shifted forward and each register is updated with a “1” or a “0.” Further, the speed of the robotic arm  80  is also updated every 20 ms and is programmed to match the spacing between the embryos, as desired by the operator to control the spacing of the embryos deposited onto the embryo deposit assembly  28 . 
     The embryo deposit assembly  28  includes a singulation frame  82  and a drainage vessel  84 . The singulation frame  82  suitably includes a supporting material that allows fluid to pass through while retaining embryos. The supporting material also preferably provides a color contrast between the supporting material and the embryo such that there is contrast between the embryos and the supporting material. One such supporting material suitable for use with the system  20  is Nitex® nylon, model No. 03-125/45. The drainage vessel  84  suitably supports a vacuum (not shown) for fluid removal and to aid in holding the embryos in a fixed location. 
     Operational aspects of the system  20  constructed in accordance with one embodiment of the present disclosure may be best understood by referring to  FIGS. 2A-2B . The beginning of the operational sequence is represented by the start block  100  by initiating the system  20  to zero the ELR, indicated by the block  102 . Also, fluid flow through the system  20  is initiated and the lift mechanism  32  raises the singulation vessel  30  at a rate to maintain a substantially constant liquid head throughout the system  20 . This is illustrated by the block  104 . 
     The timer is enabled, indicated by block  110 , and the sensor  34  determines whether an embryo  40  is detected in the tubing  60  and indicated by the decision block  106 . If an embryo  40  is detected by the sensor  34 , a “1” is placed in the first registry location of the ELR, indicated by the block  108 . Thereafter, the timer is evaluated to determine whether or not a predetermined period of time, such as 20 ms, has expired, and as indicated by the decision block  112 . If an embryo is not detected by the sensor  34 , the PLC will advance ahead to the block  112  and evaluate whether the timer has timed out. 
     If the timer has not timed out, the ELR returns to block  106  to evaluate whether an embryo has been detected. If the timer has timed out, then the ELR shifts the registry by one position forward, indicated by the block  114 . Also, as indicated by the block  116 , the timer is reset. 
     As indicated by the block  118 , the PLC evaluates whether there is a “1” in the last ELR registry, indicating the presence of an embryo  40  at the very end of the tube  60 . If there is a “0” in the last registry, indicating that there is no embryo in the last registry, the PLC determines whether every registry of the ELR is a “0,” indicated by the block  120 . If every registry is empty, the robotic arm  80  is turned off, as indicated by the block  122 , and the PLC returns back to block  110  to enable the increment timer and to evaluate whether an embryo is again detected by the sensor  34 , as indicated by block  106 . 
     Referring back to the block  118 , if the last registry in the ELR contains a “1,” then the PLC evaluates whether any other registry in the ELR contains a “1,” thereby indicating the presence of another embryo in the tubing  60 . This is indicated by the block  124 . As represented by the block  126 , if no other registry in the ELR contains a “1,” then the speed of the robotic arm  80  is set to a minimum speed. This may be accomplished by an inclusion of a lookup table containing predetermined robotic arm speeds as a function of the number of embryos in the tubing  60 . Such a lookup table is well-known to one of ordinary skill in the art. 
     If there is a “1” in any one or more other registry of the ELR, then the PLC sets the robotic arm speed based on the last and next to the last registry positions in the ELR by referring to the lookup table, as noted above. This is indicated by the block  128 . Thereafter, as indicated by the block  130 , the output speed is transmitted to the robotic arm  80 . 
     Before depositing the embryo onto the singulation frame  82 , the “X” position of the robotic arm  80  relative to the width of the singulation frame  82  is evaluated. Specifically, as indicated by the block  132 , the “X” position of the robotic arm  80  is evaluated to determine whether it has reached the maximum width of the singulation frame  82 . If yes, then the robotic arm  80  is advanced one position forward in the longitudinal direction, or “Y” direction, of the singulation frame  82  and the direction of the robotic arm  80  in the “X” direction is reversed, as indicated by the block  134 . 
     After the “X” position of the robotic arm  80  is reversed, the PLC zeroes out the “X” position, indicated by the block  136 . Thereafter, the embryo is deposited on the singulation frame  82 , as indicated by the block  138 . Returning to block  132 , if the “X” position is not reached, the blocks  134  and  136  are bypassed and the embryo is deposited on the singulation frame  82 , as noted in block  138 . 
     It is desired that the PLC  24  be programmed to control the robotic arm  80  such that it deposits embryos in a predetermined position on the singulation frame  82 . As a non-limiting example, the PLC  24  may be programmed such that the robotic arm  80  deposits embryos on the singulation frame  82  on their sides. In such a position, both the cotyledon and radical ends contact the supporting material of the singulation frame  82 , or only the cotyledon or radical end contacts the supporting material of the singulation frame  82 . As another non-limiting example, the robotic arm  82  may deposit embryos on the supporting material such that succeeding embryos are spaced from preceding embryos. Accordingly, such predetermined positions, as well as equivalents thereof, are within the scope of the disclosure. 
     After the embryo is deposited on the singulation frame  82 , and as indicated by the block  140 , the ELR determines whether a desired number of embryos deposited on the singulation frame  82  have been reached. If “no,” the ELR is returned to block  110  and the evaluation is repeated. If the maximum number of embryos has been deposited on the singulation frame  82 , the process is now complete, as indicated by the block  142 . 
     Operation of an alternate method of singulating embryos may be best understood by referring to  FIGS. 3A and 3B . It should be noted that components of this alternate embodiment that are the same as those described with respect to the first embodiment of  FIGS. 3A and 3B  have the same reference number. 
     The beginning of the operational sequence is represented by the start block  100  by initiating the system  20  to “0” the ELR, indicated by the block  102 . Simultaneously, fluid flow through the system  20  is initiated and indicated by the block  204 . An increment timer  1  is enabled, indicated by the block  206 , and the singulation rate, or data point, is calculated, as indicated in the block  208 . 
     The embryo singulation rate is compared to the set point to determine whether or not the embryo singulation rate is equal to the set point, as indicated by the decision block  210 . The singulation rate is defined as the number of detected embryos per unit time. To calculate it, the number of embryos detected in a moving window of time is divided by the size (in time) of the window, e.g., 50 detections in the last 60 seconds. The window is “moving” forward in time, as the most recent window is always used. If the embryo singulation rate does not equal that set point, the hydrostatic head setpoint is adjusted. If the singulation rate needs to be decreased, the hydrostatic head setpoint is lowered. This is indicated by the block  212 . Then the hydrostatic head of the liquid within the singulation vessel  30  is measured by the second sensor. One such ultrasonic sensor is described above. This is indicated by the block  214 . 
     Still referring to  FIG. 3A , a comparison of the liquid hydrostatic head is made relative to the set point to determine whether or not the hydrostatic head is equal to the set point, as indicated by the block  216 . If the hydrostatic head is not at the set point, the raise rate of the singulation vessel  30  by the lift mechanism  32  is adjusted, as indicated by the block  218 . In summary, the singulation rate controller adjusts the hydrostatic head setpoint (i.e., the target flow rate of fluid/embryos) and the hydrostatic head controller adjusts the rise rate of the singulation kettle in an attempt to drive the hydrostatic head to its target (aka setpoint). Following adjustment of the hydrostatic head, calculate the length (i.e., number of registers) of the ELR, as indicated by the block  220 . The length of the ELR is calculated based on the distance between the sensor ( 34 ) and the outlet of tubing ( 60 ) and the flow rate of the fluid (i.e., hydrostatic head). As the flow rate (head) increases the velocity of the fluid/embryos increases in tubing ( 60 ), which is turn reduces the time between detection and placement on s-frame ( 82 ). The number of registers required is this time divided by the time of timer  2  in block  220 . Following block  220 , a second increment timer is enabled, as shown in the block  221 . 
     The sensor  34  determines whether an embryo  40  is detected in the tubing  60  and indicated by the decision block  106 . If an embryo  40  is detected by the sensor  34 , a “1” is placed in the first registry location of the ELR, indicated by the block  108 . Thereafter, the second increment timer is evaluated to determine whether or not a predetermined period of time, such as 20 milliseconds, has expired, and as indicated by the decision block  222 . If an embryo is not detected by the sensor  34 , the PLC will advance ahead to block  222  to determine whether the second increment timer has timed out. 
     If the second increment timer has not timed out, the ELR returns to block  106  to evaluate whether an embryo has been detected. If the second increment timer has timed out, then the ELR shifts the registry by one position forward and places a “1” in the next registry location, indicated by the block  114 . Also, as indicated by the block  116 , the second increment timer is reset. 
     As indicated by the decision block  118 , the PLC evaluates whether there is a “1” in the last or “trigger” ELR registry, indicating the presence of an embryo  40  at the very end of the tube  60 . If there is a “0” in the last registry, indicating that there is no embryo in the last or trigger registry, the PLC determines whether the first incremental timer has timed out, indicated by the decision block  224 . If the first incremental timer has not timed out, then the PLC will advance back to enable the second increment timer, indicated by the block  221 . If, however, the first increment timer has timed out, the PLC returns back to enable Timer 1 , as indicated by the block  206 . 
     Returning to the decision block  118 , if the last or trigger registry in the ELR contains a “1,” then the PLC deposits an embryo on the singulation frame  82 , as noted in the block  138 . After depositing the embryo onto the singulation frame  82 , the “X” position of the robotic arm relative to the width of the singulation frame  82  is evaluated. Specifically, as indicated by the block  132 , the “X” position of the robotic arm  80  is evaluated to determine whether it has reached the maximum width of the singulation frame  82 . If it has reached the maximum width of the singulation frame  82 , then the robotic arm  80  is advanced one position forward in the longitudinal direction, or “Y” direction, of the singulation frame  82 , and the direction of the robotic arm  80  in the “X” direction is reversed, as indicated by the block  134 . After the “X” position of the robotic arm is reversed, the PLC zeroes out the “X” position, indicated by the block  136 . 
     If the “X” position is not reached in block  132 , the robotic arm  80  is moved one position in the “X” axis, as indicated by the block  226 . Doing so moves the robotic arm  80  to the next open position on the singulation frame  82 . Thus, removal of at least one of the plurality of embryos may be synchronized with the data point, such as the hydrostatic head, and the flow rate. 
     Thereafter, the PLC determines whether a desired number of embryos deposited on the singulation frame  82  have been reached, as indicated by the block  140 . If the desired number of embryo counts has not been reached, the program returns to block  204  and the process is repeated. If the maximum number of embryos has been deposited on the singulation frame  82 , the process is now complete, as indicated by the block  142 . 
     While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention. As a non-limiting example, the sensor  34  may be positioned at any point along the tubing  60 . In one alternate embodiment, the sensor  34  may be positioned adjacent the robotic arm  80 . In such an alternate embodiment, the PLC  24  may be programmed to actuate the robotic arm  80  to deposit the sensed embryo as soon as it receives an input signal from the sensor  34 . Positioning the sensor  34  adjacent the robotic arm  80  works in a system  20  that has either constant or non-constant fluid flow. Also, the method of the present disclosure may be implemented in a variety of systems and, therefore, the described system for implementing the method is provided for illustration purposes only and is not intended to be limiting.