Abstract:
A method of delivering cultivated plant embryos including the step of orientating a plurality of plant embryos in a predetermined orientation. Analyzing each of the plurality of embryos according to a predetermined quality criteria to identify qualified embryos. Determining positional measurements of the qualified embryos and positioning a first seed coat relative to the qualified embryos. The method also includes the step of inserting one of the qualified embryos in the seed coat according to the positional measurements of the qualified embryos to minimize damage to and contamination of the qualified embryos.

Description:
RELATED APPLICATION 
     The present invention claims the benefit of U.S. provisional patent application serial No. 60/150,292, filed Aug. 23, 1999. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates generally to manufactured seeds and, more particularly, to a system for the delivery of plant embryos to various growing platforms. 
     BACKGROUND OF THE INVENTION 
     Modern agriculture, including silviculture, often requires the planting of large numbers of substantially identical plants genetically tailored to grow optimally in a particular locale or to possess certain other desirable traits. Production of new plants by sexual reproduction can be slow and is often subject to genetic recombinational events resulting in variable traits in its progeny. As a result, asexual propagation 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, such as those 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 coat, a synthetic gametophyte and a plant embryo. The seed coat is usually a capsule having a closed end and an open end. The synthetic gametophyte is placed within the seed coat, such that it substantially fills the seed coat. A cotyledon restraint may be centrally located within the synthetic gametophyte. The cotyledon restraint includes a centrally located cavity extending partially through its the length and 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. The plant embryo is typically sealed within the seed coat by at least one end seal. 
     In the past, delivery of the plant embryo within the seed coat has utilized a liquid-based transport system to move the plant embryo through the manufactured seed production line. In such a liquid-based transport system, plant embryos are placed in a container of liquid to orient them in a like direction. The plant embryos are caused to float to the top of the container, such that each embryo floats upwardly within the container cotyledon end first. From the top of the container, additional liquid is used to propel the plant embryos out of the container while maintaining their cotyledon end first orientation. Liquid is then used to transport the plant embryos through the remaining manufactured seed production line steps. Although such liquid-based plant embryo delivery systems are effective at transporting plant embryos, they are not without their problems. 
     First, both system response and plant embryo movements through the system are slow because electromechanical actuators are required for controlling the liquid flow. Second, handling of the plant embryo is not precise. Often it is difficult to manipulate a plant embryo suspended in liquid, as it is difficult to manipulate any objects suspended in liquid. Third, it is difficult to reliably detect plant embryos because of their small size, the requirement for a large diameter transport tube, and cavitation in the liquid. Additionally, it is difficult to analyze each plant embryo for quality when it is suspended in liquid. Further, removing all of the liquid after the plant embryo is placed in the cavity of the cotyledon restraint is difficult. Removing all of the liquid from the embryo is desirable because liquid may cause early germination or rot. Slow throughput of the liquid system requires multiple liquid systems to meet the overall production quantity goals. Finally, the large numbers of components in a liquid delivery system present reliability problems, as well as difficulties in maintaining the system. 
     Thus, there exists a need for a plant embryo delivery system that is capable of reliably producing a large number of manufactured seeds at a relatively low cost, and minimizing the risk of damaging or contaminating the plant embryo. 
     SUMMARY OF THE INVENTION 
     In accordance with one embodiment of the present invention, a method of delivering cultivated plant embryos is provided. The method includes the step of orientating a plurality of embryos in a predetermined orientation. The method also includes analyzing each of the plurality of embryos according to a predetermined quality criteria to identify qualified plant embryos. Further, the steps of determining the positional measurements of the qualified embryos, and positioning a first seed coat relative to the qualified embryos are also included in the method of the present invention. The method further includes the step of inserting one of the qualified embryos in the seed coat according to the positional measurements of the qualified embryos to minimize damage to and contamination of the qualified embryos. 
     The method of delivering a plant embryo of the present invention has several advantages over currently available plant embryo delivery systems. The delivery system of the present invention uses mini-robotic pick and place systems with motion control to increase the speed and accuracy of the embryo delivery system. Embryo manipulation is transformed from a non-precise environment to a precise environment at the front end of the embryo processing on the manufacturing line. In a robotics system, precise information about an object and the ability to move that object with precision allows the opportunity to move the object faster. The overall system is simpler because it utilizes computerized electronics and machine control equipment. Using less components and, therefore, less equipment results in a more reliable system. Further, liquid is removed from around the embryo as one of the first process steps, thereby eliminating the potential for liquid contamination of the cotyledon restraint. Finally, electronically viewing the embryo is simpler without liquid in the path of viewing. 
     Thus, a method of delivering plant embryos in a manufactured seed formed in accordance with the present invention has a high degree of reliability, and is able to mass produce manufactured seeds or deliver embryos in a given orientation in a plate, greenhouse container or other seed designs. Further such a method for delivering plant embryos also minimizes the risk of damaging or contaminating the plant embryo during the process of manufacturing the seed. 
    
    
     BRIEF 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 partial schematic view of an embryo delivery system formed in accordance with one embodiment of the present invention; 
     FIG. 2 is a partial side planar view showing a first robotic arm and conveyer belt for an embryo delivery system formed in accordance with one embodiment of the present invention; 
     FIG. 3 is a partial side view of a measurement assembly for an embryo delivery system formed in accordance with one embodiment of the present invention shown in non-measuring position; 
     FIG. 4 is a partial side view of a measurement assembly for an embryo delivery system formed in accordance with one embodiment of the present invention shown in a measuring position; 
     FIG. 5 is a partial top planar view of the measurement assembly shown in FIGS. 3 and 4 with the measuring assembly shown in a measuring position; 
     FIG. 6 is a partial top planar view of the measurement assembly shown in FIGS. 3 and 4 with the measurement assembly shown in both a measuring position and a transfer position; 
     FIG. 7 is a partial top view of a second robotic arm for an embryo delivery system formed in accordance with one embodiment of the present invention showing measurements of a plant embryo; 
     FIG. 8 is an enlarged view of a plant embryo received within a tip of the second robotic arm shown in FIG. 7; 
     FIG. 9 is a partial side planar view of the second robotic arm for an embryo delivery system formed in accordance with one embodiment of the present invention showing rotation of the robotic arm to deposit the plant embryo within a seed coat; 
     FIG. 10 is a top planar view of a tray receptacle for an embryo delivery system formed in accordance with one embodiment of the present invention; and 
     FIG. 11 is an enlarged view of a portion of the receptacle tray for an embryo delivery system formed in accordance with one embodiment of the present invention. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     FIGS. 1-6 illustrate a preferred embodiment of an embryo delivery system (EDS)  20  constructed in accordance with the present invention. For ease of illustration and clarity, various components of the EDS  20  are broken into FIGS. 1-6. One embodiment of the EDS  20  includes four major stages of delivery. The first stage includes an embryo orientation and imaging system  22  (FIG.  1 ). The second stage includes a first transfer assembly  24  (FIG.  2 ). The third stage includes an embryo measurement assembly  26  (FIGS.  3  and  4 ). The fourth stage includes an embryo placement assembly  28  (FIGS. 5-8) and a two-dimensional positioning table  30  (FIG.  2 ). 
     As may be best seen by referring to FIG. 1, the embryo orientation and imaging system  22  includes an embryo orientation assembly  40 , a controller assembly  42 , a vacuum system  44 , a conveyor system  46 , and an imaging system  48 . The embryo orientation assembly  40  may be a well known assembly, such as that disclosed in U.S. Pat. No. 5,284,765, issued to Bryan et al., the disclosure of which is hereby incorporated by reference. The embryo orientation assembly  40  includes a containment vessel  60  and a control valve  62  in communication with the controller assembly  42  to selectively regulate the output of plant embryos from the containment vessel  60 . The containment vessel  60  is filled with a liquid and has a plurality of plant embryos  64  contained therein. Suitably, the plant embryos  64  placed in the containment vessel  60  are caused to float by adjusting the specific gravity of the liquid within the containment vessel  60  to be higher than the specific gravity of the embryos  64  by a predetermined amount. Floating embryos have been found to sustain a higher percentage of acceptable or qualified embryos for implantation in a manufactured seed coat, as is described in greater detail below. 
     The controller assembly  42  includes detectors  70   a - 70   c  and a controller  72 . The first detector  70   a  is suitably a well known photoelectric sensor. Other sensors, such as optical or infrared, are also within the scope of the invention. The first detector  70   a  is disposed adjacent the top of the containment vessel  60 . The controller  72  polls the first detector  70   a  to determine when an embryo or embryos  64  have floated to the top of the containment vessel  60 . When the controller  72  determines that the first detector  70   a  has detected an embryo  64 , the controller  72  activates a solenoid (not shown). The solenoid in turn actuates a pump  61 , connected to a reservoir  63 , and a valve  62  that permits liquid to flow in at the top of the containment vessel  60  to direct the embryo  64  into the tube which will transport the embryo out of the containment vessel  60  and onto the conveyor system  46 . This stream of liquid forces the embryo  64  into the tube toward the conveyor system  46 . 
     The second detector  70   b  is located adjacent the end of the tube of the containment vessel  60 . When the controller  72  determines that the second detector  70   b  has detected a passing embryo  64 , it activates a well known conveyor drive motor  86  of the conveyor system  46 , such that an embryo  64  is transferred to the conveyor system  46  without disturbing the orientation as it is ejected from the containment vessel  60 . The second detector  70   b  is in communication with the controller  72  and may be adjusted to control the number and frequency in which plant embryos  64  are released from the containment vessel  60 . 
     Still referring to FIG. 1, plant embryos  64  are ejected from the containment vessel  60  in a predetermined orientation. Suitably, each plant embryo  64  is emitted from the containment vessel  60 , such that the embryos  64  come out of the containment vessel  60  cotyledon end first. Although orientating plant embryos such that they are emitted cotyledon end first is preferred, other orientations, such as emitting plant embryos  64  root end first, are also within the scope of the present invention. The plant embryos  64  are ejected onto the conveyor system  46  and transported to the imaging system  48 . 
     The conveyor system  46  includes a well known continuous and liquid porous conveyor belt  80  and is driven by a motor  86 . The vacuum system  44  is suitably disposed near the outlet of the containment vessel  60 , such that when the plant embryos  64  are emitted from the containment vessel  60 , they are vacuumed to remove additional or excess liquid on the plant embryos  64 . The vacuum system  44  vacuums excess liquid from the plant embryos  64  through the porous conveyor belt  80 . Although it is preferred that the vacuum process occur at a single location, additional locations, such as continuously vacuuming the plant embryo as it is being transferred to the imaging system, are also within the scope of the present invention. 
     After the plant embryos  64  have been subjected to the vacuum system  44 , the conveyor system  46  is activated to transfer the plant embryos  64  to the imaging system  48 . A third detector  70   c  is disposed near the imaging system  48 . When the controller  72  determines from polling that the third detector  70   c  has detected an embryo  64 , it signals the conveyor drive motor  86  to turn off, thereby positioning the embryo  64  in a suitable location for imaging by the imaging system  48 . 
     The imaging system  48  includes an imaging camera  82 , such as a digital camera, and a well known detector sensor (not shown). As the plant embryo  64  is transferred into the range of the detector sensor, the detector sensor sends a signal to the main computer  84 . The main computer  84 , in turn, sends a signal to the controller  72  to stop the conveyor belt  80 , thereby positioning the plant embryo  64  beneath the digital camera  82 . The camera  82  acquires and digitally stores images that will be used to determine whether an embryo is considered qualified to be placed in a manufactured seed. 
     Information from the imaging camera  82  is sent to the main computer  84  and is processed by a software program, such as that disclosed in PCT Application Serial No. PCT/US99/12128, entitled: Method for Classification of Somatic Embryos, filed Jun. 1, 1999, the disclosure of which is hereby expressly incorporated by reference. The software program makes a qualitative determination of the plant embryo  64  and, based on predetermined parameters, defines and stores which plant embryos are considered to be qualified and which are considered to be unqualified embryos. 
     Referring to FIG. 2, the first transfer assembly  24  will now be described in greater detail. The first transfer assembly  24  includes a robotic arm assembly  90  movably attached to a rail  92 . The robotic arm assembly  90  includes a housing  94  and an arm  96 . The lower end of the arm  96  includes a vacuum tip end adapted to selectively seize a plant embryo  64 . As a non-limiting example, if a plant embryo  64  is deemed to be qualified by the software program to be placed into a manufactured seed, it is plucked off the conveyor belt  80  by the vacuum tip end of the robotic arm  96 . The vacuum tip seizes the middle section of the plant embryo  64  and transfers the qualified plant embryo to the embryo measurement assembly  26 . Unqualified plant embryos are rejected off the end of the conveyor into a trash receptacle  81 . Although the preferred actuation for the robotic arm assemblies has movement in two axes, movement in more than two axes, such as a three axes system, is also within the scope of the present invention. 
     Referring to FIGS. 3 through 5, the embryo measurement assembly  26  includes a precision robotic arm embryo holder assembly  100  and a first laser micrometer  102 . Preferably, the precision robotic arm embryo holder assembly  100  has motion in two axes, wherein the first axis is into a laser micrometer measurement plane  108 , and as indicated by the Z-direction of FIGS. 3 and 4. The second axis of motion is horizontally perpendicular to the measurement plane  108 , and as indicated by the X-direction of FIG.  5 . 
     The precision robotic arm embryo holder assembly  100  includes a vacuum activated embryo holder assembly  104  and is adapted to releasably receive the plant embryo  64  from the first robotic arm  96  (FIG.  2 ). During operation, after receiving the plant embryo  64  from the first robotic arm  96 , the embryo holder assembly  104  slides along the housing  106  coupled to a frame  107  to move the tip of the root end of the plant embryo  64  into the well known two-dimensional laser micrometer measurement plane  108  emitted from the laser micrometer  102 . A set of XYZ positional measurements is collected about the tip of the root end of the plant embryo  64 . The set of XY positional information is recovered from the laser micrometer and the Z position is recovered from the known distance of the embryo measurement assembly  26  relative to the laser micrometer measurement plane  108 . The XY positional measurement of the tip of the root end of the plant embryo  64  permits the plant embryo  64  to be precisely transferred to the embryo placement assembly  28 . 
     Referring now to FIGS. 5-9, the embryo placement assembly  28  will now be described in greater detail. As may be best seen by referring to FIG. 9, the embryo placement assembly  28  includes a third robotic arm embryo holder  120 , a housing  122 , and a rail  124 . The housing  122  is pivotally attached to the rail  124  by a pivot and slide assembly  126 . Referring back to FIG. 5, after the XYZ positional measurements of the tip end of the plant embryo  64  are determined, the plant embryo  64  is transferred from the embryo measuring assembly  26 , held in place by the embryo holder assembly  104 , and precisely into the third robotic arm embryo holder  120 . In this position, the plant embryo  64  is held in a predetermined position by the embryo holder  104 . 
     The third robotic arm embryo holder  120 , attached to the housing  122  by the rail  124 , is moved, using information received about the position of the tip of the root end of the plant embryo  64  into a position where the cavity  130  of the third robotic arm embryo holder  120  is placed over the tip of the root end of the plant embryo  64 . The vacuum is activated to pick up the embryo and deactivated to the embryo holder, thereby transferring holding control of the plant embryo  64  from the embryo measurement assembly  26  to the embryo placement assembly  28 . In this position, the precision robotic arm embryo holder assembly  100  translates away from the laser micrometer  102  to a known stop position and in the direction indicated by the arrow  128  (FIG.  6 ). In this precise stop position, the plant embryo  64  is transferred from the embryo holder assembly  104  to the third robotic arm embryo holder  120  of the embryo placement assembly  28 . 
     As may be best seen by referring to FIG. 8, the end of the third robotic arm embryo holder  120  includes a conical cavity  130  in communication with a vacuum tube  132 . When the plant embryo  64  is transferred from the embryo measuring assembly  26  to the embryo placement assembly  28 , the root end of the plant embryo  64  is received within the conical tip cavity  130  and is held therein by the vacuum tube  132 . In this position, the third robotic arm embryo holder  120 , attached to the housing  122  and slide assembly  126 , is moved away from the laser micrometer measurement plane  10  until the plant embryo  64  is moved totally out of the laser micrometer measurement plane  108 . In this position, the cotyledon end of the plant embryo  64  protrudes out of the assembly  120 . 
     As received within the third robotic arm embryo holder  120 , the embryo placement assembly  28  translates back towards the laser micrometer  102 . The precision measurement of the center of the cotyledon end of the plant embryo  64  is calculated and the length of the protrusion, indicated by the distance X, of the cotyledon end from the end of the third robotic arm embryo holder  120  is also calculated. The circumference of the cotyledon end is a standard measurement obtained from the well known laser micrometer. The center of the cotyledon end of the plant embryo  64  can be precisely calculated from that measurement. 
     As may be best seen by referring to FIG. 9, after the center and length of the cotyledon end of the plant embryo  64  is determined, the housing  122  and third robotic arm embryo holder  120  pivot downwardly towards the two-dimensional positioning table  30 . The two-dimensional positioning table  30  selectively translates in two dimensions. In particular, the table  30  is permitted to move fore and aft, as well as in the lateral direction. Although a two-dimensional table is preferred, a table capable of movement in other directions, such as a three-dimensional table, is also within the scope of the present invention. 
     Located on top of the table  30  is a receptacle tray  134 . The receptacle tray  134  includes a plurality of cavities  136  extending vertically therethrough. Suitably, there may be a total of  96  cavities located in the receptacle tray  134 . However, a receptacle tray  134  having more or less cavities is also within the scope of the present invention. 
     Received within each cavity  136  is a well known manufactured seed  38 , such as that disclosed in U.S. Pat. No. 5,701,699, issued to Carlson et al., the disclosure of which is hereby incorporated by reference. The two-dimensional positioning table  30  includes an imaging camera (not shown) to precisely locate and store the center of the opening of the cotyledon restraint in the manufactured seed. Having the positional information of the cotyledon restraint opening of the manufactured seed and the position information of the cotyledon end of the embryo  64  held by the vacuum tip of the third robotic arm embryo holder  120 , the third robotic arm embryo holder  120  positions the embryo  64  above the cotyledon restraint opening of the manufactured seed. The third robotic arm embryo holder  120  positions the embryo  64  above the opening of the cotyledon restraint and lowers the embryo  64  therein to a predetermined depth within the opening and above the bottom of the opening. At this point, the vacuum tip is turned off and a short burst of air gently releases the embryo  64  from the vacuum tip  120  and into the cotyledon restraint of the manufactured seed. 
     Operation of the EDS may be best understood by referring to FIGS. 1-11. After the embryo  64  is delivered from the manufactured seed production line, the embryo  64  is placed in the containment vessel  60  of the embryo orientation assembly  40 . As noted above, the embryos are placed within the containment vessel  60  to segregate the floating from non-floating embryos  64 . The plant embryos are caused to float to the top of the container, such that the plant embryo floats upwardly within the container cotyledon end first. From the top of the container, additional liquid is used to propel the plant embryos out of the container while maintaining their cotyledon end first orientation. 
     As the embryos are detected exiting the delivery tube, the detector  70  causes the controller  72  to start the porous conveyor belt  80  moving such that the embryos  64  will be placed on the conveyor belt  80  at close or at the same speed at which they are exiting the delivery tube. This ensures that the embryos  64  will be placed on the conveyor belt  80  and maintain their orientation, rather than dropped on the belt  80  and randomly lose their orientation as they bounce to settle on the belt  80 . Simultaneously, the vacuum  44  starts and the vacuum nozzle located beneath the conveyor belt  80  vacuums off any excess liquid around the plant embryo that has drained on the porous belt  80  and seeped to below the belt  80 . 
     Moving on the conveyor belt  80 , the embryo  64  is again detected by a well known photoelectric detector and the conveyor belt  80  is stopped by the controller  42  in the correct position for the imaging camera  82 . The imaging camera  82  acquires and digitally stores the necessary images that will be used to determine whether the embryo  64  can be considered qualified to be placed in a manufactured seed. 
     If the embryo  64  is qualified to be placed in a manufactured seed, it is plucked off the conveyor belt  80  by the vacuum tip located at the end of the first arm  96 . The vacuum tip picks up the embryo  64  from the middle section of the embryo  64 , places the embryo  64  on a second vacuum tip of the embryo placement measurement  26 . The embryo holder assembly  104  holds the lower surface of the embryo  64 , with the root end protruding sideways from the vacuum tip. The vacuum tip is fastened to a two-axes motion control table that will move the tip of the embryo  64  into a two-dimensional laser micrometer field  108 , thereby calculating a set of XYZ positional measurements about the root end of the embryo  64 . The set of XY position information is recovered from the laser micrometer  102  and the Z position is recovered from the precision motion of the controlled table controller. 
     Having the three-dimension position information for the tip of the root end of the embryo  64 , the precision motion control table controller moves the tip to a position that will allow the root end of the embryo  64  to be placed precisely into the opening of another vacuum tip of the embryo placement assembly  28 . The embryo  64  held by the third robotic arm embryo holder  120  then moves back into the laser micrometer  102 , where the position measurement of the center of the cotyledon end of the embryo  64  is calculated and the length of the protrusion of the cotyledon end from the end of the vacuum tip is also calculated. 
     As noted above, simultaneous with or prior to the acquisition of the precision information for the embryo, a second imaging system such as OMRON Vision Systems Model F350, F300 or F200, locates the position of the opening of the cotyledon restraint in the manufactured seed secured to the two-dimensional positioning table  30 . As a result, having both the positional information of the cotyledon restraint opening of the manufactured seed and the position information of the cotyledon end of the embryo, the third robotic arm embryo holder  120  positions the embryo above the cotyledon restraint opening and precisely lowers the embryo  64  within the cotyledon restraint. 
     The previously described version of the present invention provides several advantages over currently available embryo delivery systems. First, the overall system is simpler and more reliable than the liquid-based systems currently available by using a combination of robotics, computers, vision systems, motion controlled components, laser micrometers and other basic electronics. Further, the embryos may be accurately placed into the cotyledon restraint without damaging or contaminating the embryos. Thus, a method and apparatus of delivering plant embryos in a manufactured seed formed in accordance with the present invention has a high degree of reliability, is able to mass produce manufactured seeds and minimize the risk of damaging or contaminating the plant embryo during the process of manufacturing the seed. 
     From the foregoing description, it can be seen that an embryo delivery system formed in accordance with the present invention incorporates many novel features and offers significant advantages over currently available systems. While the presently preferred embodiments of the invention have been illustrated and described, it is to be understood that within the scope of the appended claims, various changes can be made therein without departing from the spirit of the invention.