Abstract:
The present invention provides apparatus and methods useful, for example, for introducing a desired spacing between or classifying and sorting objects, e.g. plant embryos. Objects carried serially in a fluid stream enter the apparatus via an upstream conduit. A sensor associated with the conduit provides information regarding an object at a particular location in the upstream conduit and produces a signal. A switch coupled to the upstream conduit directs the fluid stream to an appropriate downstream conduit by applying a force to a conduit, e.g., by aligning the upstream conduit with a downstream conduit to create a fluid-tight path. Apparatus according to the present invention are particularly useful for manipulating fragile multicellular biological objects such as plant embryos.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application is a divisional of application Ser. No. 09/619,773 filed on Jul. 20, 2000, now U.S. Pat. No. 6,354,770, as a divisional application of application Ser. No. 08/883,757, now U.S. Pat. No. 6,145,247, filed on Jun. 27, 1997, and claiming the benefit of Provisional Patent Application No. 60/022,001 filed on Jun. 27, 1996. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates generally to apparatus and methods for automated handling of objects carried in a fluid stream, in particular, to apparatus and methods for sorting living multicellular biological objects such as plant embryos. 
     BACKGROUND OF THE INVENTION 
     Modern agriculture often requires the planting of large numbers of substantially identical plants selected to grow optimally in a particular locale or to possess certain other desirable traits. Production of new plants by sexual reproduction, which yields botanic seeds, is a lengthy, labor-intensive process that is often subject to genetic recombinational events resulting in variable traits in the progeny. Furthermore, inbred strains used to perform such crosses often lack vigor, resulting in low seed productivity. 
     Botanic seeds, such as those produced by conventional plant breeding, have food-storage organs and protective structures that shelter plant embryos from the harsh soil environment, nurture the embryo during sowing and germination, and enable the seed to survive until conditions are favorable for germination. 
     In view of the disadvantages of producing large numbers of identical progeny plants by sexual means, propagation of commercially valuable plants via culturing of somatic or zygotic plant embryos has been intensively studied. For some species such “asexual” propagation has been shown to yield large numbers of genetically identical embryos, each having the capacity to develop into a normal plant. Unfortunately the resulting embryos lack the protective and nutritive structures found in natural botanic seeds. As a result, the embryos are usually 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. 
     Much effort has been directed to the development of techniques for embryogenesis of agronomically important plant species, including conifer species. See, e.g., U.S. Pat. Nos. 4,957,866, 5,034,326, and 5,036,007. Totipotent plant tissue is developed in culture to a stage similar to the natural zygotic embryos occurring in mature seeds. For conifers, these are very small, commonly ranging from about 2-4 mm in length. Embryos have a bipolar form which anticipates the ultimate plant. One end has a latent radicle or root, and the other end has a latent cotyledon and appears similar to a tiny crown. 
     Somatic embryos lack the endosperm of the natural seed. In order to provide nutrients to the embryo at the time of germination, somatic embryos may be placed on a solid germination medium that contains the necessary carbohydrate and other nutrients, on a growing medium, or on synthetic soil that is saturated with an appropriate nutrient solution. Sterility must be maintained until after the resulting plantlet is well established. Somatic embryos also lack a seed coat and thus are more susceptible than botanic seed to mechanical damage, desiccation, and attack by pathogens and pests. 
     A preferred method of germinating a unit of totipotent plant tissue, e.g., a plant somatic embryo, is to incorporate it into a manufactured seed (i.e., “artificial seed” or “seed analog”). A number of versions of manufactured seed have been described in the patent literature, including U.S. Pat. Nos. 4,562,663; 4,583,320; 4,615,141; 4,715,143; 4,777,762; 4,779,376; and 4,780,987 and Canadian Patent No. 1,241,552. More advanced versions of manufactured seed that display an improved germination rate are disclosed in U.S. Pat. Nos. 5,427,593 and 5,236,469, incorporated herein by reference. 
     Methods and apparatus are needed for producing manufactured seed on a commercial scale. If an economical production rate is to be obtained, this process must be automated as much as possible. 
     One step in this production of manufactured seed is the selection of totipotent plant tissue, e.g., somatic embryos, that are mature enough to incorporate into manufactured seed. There is typically significant variation in morphological normalcy and embryo maturity in somatic embryos produced by conventional tissue culture methods. Manufactured seed containing morphologically abnormal or immature embryos seldom germinate into normal plantlets. Tedious manual selection has been the standard solution to this problem. 
     Various apparatus have been described for sorting microscopic biological objects such as single cells. See, U.S. Pat. Nos. 3,560,754, 3,710,933, 3,791,517, 3,987,307, and 4,175,662. These apparatus are generally not useful for sorting larger, multicellular biological objects, particularly macroscopic objects such as plant embryos. 
     A method has been described for separating loblolly pine zygotic embryos and celery somatic embryos according to maturity criteria using sucrose density gradients (Velho et al.;  HortScience , Programs and Abstracts (suppl.), p. 137, 1989 [Abstract, 87th Annual Meeting of the American Society of Horticultural Science, Tucson, Ariz., Nov. 4-8, 1990]). 
     U.S. Pat. No. 5,284,765 describes a method of directionally orienting plant embryos in a liquid flotation medium. 
     Published International Application WO 91/00781 describes the use of a scanner to identify and determine the location of plant embryos and a pipetting mechanism to remove the plant embryos from the liquid culture medium. 
     Harrell et al.,  Computers and Electronics in Agriculture  9:13-23, 1993, describes a system for classifying plant embryos. Mature embryos arc fixed, manually introduced into the system under non-sterile conditions, and optically imaged. Images of the objects are analyzed using a neural network. Objects identified as mature embryos are deflected out of a gap in a conduit in a medium-filled harvest chamber by an injection of culture medium from a control nozzle and collected. Rejected structures pass through the gap and enter a settlement chamber. 
     There remains a need for automated apparatus and methods for rapidly and efficiently handling multicellular biological objects such as plant embryos under aseptic conditions without subjecting the objects to mechanical forces that would cause substantial damage. In particular, there is a need for apparatus and methods for rapidly separating embryos that are acceptable for producing manufactured seed from unacceptable embryos and delivering the acceptable embryos in an aseptic fluid stream to a location for incorporation into manufactured seed. 
     SUMMARY OF THE INVENTION 
     It is an object of the present invention to provide apparatus and methods for introducing a fixed spacing between objects carried in a fluid stream. 
     It is another object to provide apparatus and methods for classifying objects carried in a fluid stream. 
     It is another object of the invention to provide apparatus and methods for sorting objects belonging to various classes. 
     It is another object of the invention to provide apparatus and methods for performing these functions without subjecting fragile biological objects such as plant embryos to mechanical forces, e.g., shear forces, that would damage and reduce the viability of the objects. 
     It is a further object of the invention to provide such apparatus and methods that maintain an aseptic environment for the biological objects to prevent contamination. 
     The foregoing objects have been achieved by providing apparatus and methods for directing objects carried serially by a fluid (e.g., a liquid such as water or a culture medium, air, etc.) to a desired destination. The apparatus includes an upstream, or source, fluid conduit and two or more downstream, or destination, fluid conduits. A sensor, e.g., a fiber-optic sensor, is associated with the upstream conduit and provides information regarding objects in the upstream conduit, e.g., the presence of the object at a particular location in the upstream conduit or an image of the object. A switch coupled to the upstream fluid conduit is selectively operable to deliver the fluid stream, and objects carried by the fluid stream, from the upstream conduit to the appropriate downstream conduit. 
     For example, according to one embodiment of the invention, the switch is selectively operable to apply a force to at least one of the upstream or downstream fluid conduits, e.g., to align an end of an upstream conduit with an end of a downstream conduit according to the information provided by the sensor to produce a single fluid-tight path for the fluid stream and for objects carried therein. Alternatively, the switch comprises a fluid chamber that is selectively operable to be aligned with an upstream conduit to receive the object and then to be moved into alignment with a downstream conduit in order to direct the object thereto. 
     According to one embodiment of the invention, the upstream conduit is normally connected to a first downstream conduit. A sensor associated with the upstream conduit produces a signal upon detecting the presence of an object at a particular location in the upstream conduit. The switch responds to the signal by delivering the fluid stream to a second downstream conduit. Then, after a predetermined delay, the switch reconnects the upstream conduit with the first downstream conduit. This permits the detected object and a unit volume of the fluid in which the object is carried to enter the second downstream conduit. This embodiment is useful, for example, for automatically achieving a desired spacing between objects that are randomly spaced as they enter the upstream conduit. 
     According to another embodiment of the invention, the apparatus includes an optical sensor associated with the upstream conduit that produces an image of an object at a particular location in the upstream conduit and transmits the image to a signal processor, which processes the image for display on a monitor for viewing and classification by a human operator, who transmits a signal corresponding with the classification of the object. Alternatively, in an automated apparatus, the signal processor transmits the processed image to a computerized image recognition system that classifies the object and produces a signal corresponding to the classification of the object that causes the switch to direct the object to the appropriate downstream conduit. Apparatus according to the invention are particularly useful for spacing and sorting living biological objects, especially fragile multicellular, macroscopic objects such as plant embryos. The apparatus are designed such that fluid flow is substantially nonturbulent (i.e., laminar) to reduce or eliminate mechanical damage to fragile objects resulting from shear forces. Moreover, the apparatus can be maintained and operated under conditions that maintain asepsis of the medium in which the objects are suspended and prevent contamination of the objects themselves. 
     The apparatus can be fully automated. Two or more apparatus according to the invention can be arranged in series or in parallel for spacing, orienting, or sorting objects according to multiple criteria. For example, in a commercial process for producing manufactured seed, embryos carried in a fluid stream in a randomly spaced fashion can be directed to one embodiment of the invention to achieve a regular spacing, then directed to another embodiment of the invention in series with the spacing apparatus to classify and sort the embryos and direct embryos that are acceptable for manufactured seed to a location for manufactured seed assembly. 
     The present invention also provides related methods for directing objects to a desired location, producing a substantially regular spacing between objects, classifying and sorting objects, and producing manufactured seed that include plant embryos sorted by such methods, as well as manufactured seed produced by such methods. 
     Those skilled in the art will appreciate the utility of this invention which is not limited to the specific experimental modes and materials described herein. 
     The foregoing and other features and advantages of the invention will become more apparent from the following detailed description and accompanying drawings. 
    
    
     BRIEF DESCRIPTION OF DRAWINGS 
     FIG. 1 is a perspective view of a two-position fluid switch according to one embodiment of the invention. 
     FIG. 2 is a top sectional view of the fluid switch of FIG. 1 in the second position, wherein dashed lines indicate the first position. 
     FIG. 3 is a sectional side view of the fluid switch of FIG.  1 . 
     FIG. 4 is a top view of a three-position fluid switch according to another embodiment of the invention. The switch is in the third position for viewing purposes, wherein dashed lines indicate the first and second positions. 
     FIG. 5 is a sectional side view of the fluid switch of FIG.  4 . 
     FIG. 6 is a sectional end view of the fluid switch of FIG.  4 . 
     FIG. 7 is a top view of another embodiment of a three-position fluid switch with a shuttle  172  that is adapted to receive an optical cell  174 . 
     FIG. 8 is a sectional side view of the fluid switch of FIG.  7 . 
     FIG. 9 is a sectional end view of the fluid switch of FIG.  7 . 
     FIG. 10A is an enlarged perspective view of an optical cell. 
     FIG. 10B is a cross-sectional view of the optical cell of FIG.  10 A. The dimensions of the optical cell bore are shown (“a”). 
     FIG. 11 is a top view of a three-position fluid switch with a rotary shuttle according to another embodiment of the invention. 
     FIG. 12 is a side view of the fluid switch of FIG.  11 . 
     FIG. 13 is an end view of the fluid switch of FIG. 11 including a servo motor for selectively rotating the rotary shuttle. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The following description of various embodiments of apparatus according to the invention discusses use of the apparatus in a process for the production of manufactured seed comprising plant embryos, e.g., for achieving a desired spacing between randomly spaced embryos that are entrained in an aseptic liquid stream (e.g., water or an aqueous plant cell culture medium) or for sorting and separating viable, mature, morphologically normal plant embryos from other objects such as nonviable embryos or non-embryo structures. The invention is not considered limited thereto, however, but would be useful for a variety of purposes, for example, for classifying and separating a wide variety of microscopic or macroscopic living or non-living objects, particularly fragile macroscopic objects. 
     FIGS. 1-3 show a two-position fluid switch  20  according to the invention. The fluid switch  20  comprises a body  22  that includes an upstream body portion  24  having an upstream end  26  and a downstream end  28  and an adjacent downstream body portion  30  having an upstream end  32  and a downstream end  34 . The downstream body portion  30  is preferably mounted to the upstream body portion  24  with screws  36 . An upstream bore  38  is defined by and extends into upstream body portion  24 . 
     A shuttle cavity  42  is also defined by the upstream body portion  24  adjacent the downstream body portion  30 . A slide, or shuttle,  44  is slidably disposed in the shuttle cavity  42 . The shuttle  44  dimensionally conforms to the shuttle cavity  42  to allow the shuttle  44  to be moved back and forth in the shuttle cavity  42  by an actuator  46 , such as a standard two-way pneumatic positioning cylinder that is attached to one end of the shuttle  44 . A wide variety of conventional actuators can be employed. A calibration stop post  48  threadably extending through the upstream body portion  24  and into the shuttle cavity  42  provides an adjustable stop for the shuttle  44 . A cylindrical shuttle bore  50 , preferably having substantially the same diameter as the upstream bore  38 , is defined in the shuttle  44 . The shuttle bore  50  can be axially aligned with the upstream bore  38  or can be displaced a distance to either side of the upstream bore  38  by side-to-side movement of the shuttle  44  in the shuttle cavity  42 . 
     As shown in FIGS. 1-3, the downstream body portion  30  defines spaced-apart nonintersecting first and second downstream bores  54 ,  56 , respectively, preferably having substantially the same diameter as the upstream bore  38 . The downstream bores  54 ,  56  each have a corresponding open upstream end  58 ,  60 , respectively. Surrounding each upstream end  58 ,  60  is a respective gland adapted to receive a silicone O-ring  62  to provide a liquid-tight seal between the upstream ends  58 ,  60  and the shuttle  44 . The O-rings are preferably made of silicone rubber or other suitable material. 
     The downstream bores  54 ,  56  are equilaterally spaced apart from each other on opposite sides of an axis defined by the upstream bore  38  and are substantially coplanar with the upstream bore  38  and the shuttle bore  50 . The length of the shuttle  44  and the positions of the downstream bores  54 ,  56  are such that the shuttle bore  50  can be aligned with either of the downstream bores  54 ,  56 . 
     An aseptic upstream conduit  64 A, e.g., a length of a flexible transparent tubing (e.g., Tygon® tubing, Norton Co.), is coaxially connected to the upstream bore  38  to deliver objects entrained in a fluid stream, e.g., biological objects such as plant embryos, to the shuttle  44 . An aseptic flexible conduit  64 B is also axially connected to the upstream bore  38  and the shuttle bore  50  to deliver fluid-entrained objects through the shuttle  44 . For plant embryos, the fluid is preferably water or an aqueous culture medium, although for other objects, air or another fluid can be employed. 
     The upstream conduit  64  preferably has an inner diameter that is greater than that of the diameter of objects entrained in the liquid stream but small enough to ensure that the objects pass through the fluid switch  20  serially (e.g., about one-eighth inch in diameter for conifer embryos). The upstream conduit  64  has an open downstream end  66  (FIG. 2) that is flush with the surface of the shuttle  44  adjacent the upstream end of the downstream body portion  32 . 
     First and second downstream conduits  68 ,  70  (which may be either flexible or inflexible, and which may be transparent or non-transparent) are disposed within the first and second downstream bores  54 ,  56 , respectively, in the downstream body portion  30 . The upstream opening  58 ,  60  of each of the first and second downstream bores preferably has a diameter substantially equal to the inside diameter of the upstream and downstream conduits. 
     As it moves reciprocatively in the shuttle cavity  42 , the shuttle  44  carries the upstream conduit  64 B with it. Free lateral movement of the upstream conduit  64 B is facilitated by a V-shaped void  52  defined by the upstream body portion  24 . In this way, the downstream end  66  of the upstream conduit  64 B can be aligned with either the upstream end  58  of the first downstream bore (“first position”) or the upstream end  60  of the second downstream bore (“second position”), respectively, to provide a single, continuous pathway for the movement of the fluid stream and objects carried by the fluid stream through the fluid switch  20 . The O-rings  62  provide a fluid-tight seal between the downstream end of the upstream conduit  66  and an upstream opening  58  or  60  of a downstream bore or between the downstream end  66  of the upstream conduit and the upstream end  32  of the downstream body portion when the shuttle is moving between the first and second positions. Thus, the fluid path through the switch is fluid-tight, i.e., both fluid leakage and contamination of the fluid and objects carried therein is prevented. 
     The upstream sensor bores  80 ,  82  are aligned on opposite sides of and horizontally intersect the upstream bore  38 . Sensors  84 ,  86  that are suitable for detecting the presence of an object in the upstream conduit (e.g., a light-path or ultrasonic sensor, such as a FS2 series color mark fiber sensor, model FU-75, Keyence Corp., Osaka, Japan) are disposed in the upstream sensor bores  80 ,  82  (one sensor serving as a light or ultrasonic transmitter, the other sensor serving as a receiver). 
     Upon detection of an object in the upstream conduit  64 A, the upstream sensors  84 ,  86  transmit a signal to a signal processor  87 . The signal processor processes the signal and transmits a processed signal to a programmable logic controller  88  (PLC, e.g., model KX-10R(T), Keyence Corp., Osaka, Japan) that includes the appropriate switching logic (preferably electrical or pneumatic) and drive circuitry to control the actuator  46 , which moves the shuttle  44  to a desired position. 
     The fluid switch  20  can be used, for example, to achieve a substantially regular spacing between embryos entrained in a liquid stream. The shuttle  44  is maintained in the second position until an embryo is detected by upstream sensors  84 ,  86 , which transmit a signal to the signal processor  87 , which processes the signal and transmits the processed signal to the controller  88 . After an appropriate delay, the controller  88  causes the actuator  46  to move the shuttle  44  to the first position. After a preset delay, controller  88  causes actuator  46  to move shuttle  44  back to the second position, thereby establishing a predetermined gap or spacing between embryos in the first downstream conduit  68 . Water is discharged through the second downstream conduit  70  to be recycled until another embryo is sensed by the upstream sensors  84 ,  86 . 
     Such a two-position fluid switch  20  can also be used to classify and separate normal embryos from other objects (e.g., immature embryos, morphologically abnormal embryo structures, debris, etc.) that are spaced apart in a liquid stream. After an object enters the fluid switch  20  via the upstream conduit  64 A, the upstream sensors  84 ,  86  (e.g., standard fiber optics borescopes, such as models A8-260-F45 or A8-260-R45, Genesys Instruments, Inc.) generate images of the object and transmit the images to a signal processor  87 , which in turn transmits a processed signal to a monitor  90   a . A human operator views the monitor and classifies the object as a normal embryo (“accept”) or other object (“reject”). Based on the classification, the operator sends a signal to the controller  88 , which causes the actuator  46  to move the shuttle  44  to the appropriate position. If the object is classified as a normal embryo, the shuttle  44  is moved to the first (“accept”) position to permit the embryo to continue to a location for incorporation into manufactured seed. If an object is classified as a non-embryo, the shuttle  44  is moved to the second (“reject”) position to permit the non-embryo to continue to a waste receptacle or other desired destination (or vice versa). A delay can be introduced between classification of the object and movement of the shuttle. In an alternate embodiment, the monitor  90   a  (and human operator) is replaced with a computerized image processor  90   b , which automatically analyzes and classifies the object on the basis of the processed signal received from the signal processor  87  and transmits a signal according to the classification to the controller  88 . 
     As shown, the upstream conduit  38  is coupled to only one of the downstream conduits  54  or  56  at a time to create a single enclosed fluid path for the object. Moreover, neither of the downstream conduits need intersect with the upstream bore, permitting a connection to be formed between a single upstream fluid conduit and three or more downstream fluid conduits, if desired, as is exemplified below. 
     Three-Position Fluid Switch 
     One embodiment of a three-position fluid switch  100  according to the present invention is shown in FIGS. 4-6. The body  102  of the fluid switch (shown for the sake of simplicity as a one-piece body in FIGS. 4-6, although a two-piece body as shown in FIGS. 1-3 can be used) defines a shuttle cavity  104 . A shuttle  106  defining a shuttle bore  108  is disposed in the shuttle cavity  104 . The body  102  also defines an upstream bore  110 . A flexible, transparent upstream conduit  112  is disposed within and coaxially connected to the upstream bore  110  and has an open downstream end  114  inserted in the shuttle bore  108 . The body  102  also defines upstream sensor bores  116 ,  118  corresponding to upstream optical sensors  120 ,  122  (shown in FIG. 4 as “eye” symbols) to detect the presence of an object in the upstream conduit  112 . The body  102  also defines a V-shaped void  124  for unhindered movement of the upstream conduit  112  by the shuttle  106 . These elements are similar in design and function to analogous elements of the two-position fluid switch described above. 
     Spaced apart, parallel first, second, and third downstream bores  126 ,  128 , and  130 , respectively, having corresponding open upstream ends  132 ,  134 ,  136  are defined by the body  102 . First and second downstream conduits  138 ,  140  (which need not be flexible or transparent), are disposed in the corresponding first and second downstream bores  126 ,  128 . 
     The shuttle  106  is reciprocally slidable to align the open downstream end  114  of the upstream conduit  112  with the open end  132  of the first downstream bore (“first position”), the open end of the second downstream bore  134  (“second position”), or open end  136  of the third downstream bore (“third” or “viewing position”). O-rings  142  provide a liquid seal between the downstream end  114  of the upstream conduit  112  and any of the upstream openings  132 ,  134 ,  136  of a downstream bore when the shuttle  106  is so aligned or between the downstream end  114  of the upstream conduit and the shuttle cavity  104  to prevent leakage when the shuttle  106  is moving between these positions. 
     As shown in FIGS. 4-6, the fluid switch  100  includes a vision system that includes three downstream optical sensors (e.g., borescopes)  144 ,  146 ,  148  (shown in FIGS. 4-6 as eye symbols), that are oriented to produce images of an object located in the upstream conduit  112  in the shuttle bore  108  from three views. The sensor  144  provides a horizontal view in an upstream direction along an axis defined by the third downstream bore  130 . The sensor  144  is disposed in and closes the third downstream bore  130  downstream of a fluid bleed channel  150 , by means of which fluid can exit the system or be recycled. The sensor  144  is stationary during operation of the fluid switch  100 . The sensor  146  is positioned in the sensor bore  152  to provide a second horizontal view along the long axis of the shuttle  106  and can be either stationary during operation of the fluid switch  100  or attached to and move with the shuttle  106 . The sensor  148  is positioned in the sensor bore  154  to provide a third view vertically downward at substantially a right angle to the shuttle bore  108  when the switch  100  is in the third position. The sensor  148 , like the sensor  146 , can be either stationary during operation of the switch  100  or be attached to and move back and forth with the shuttle  106 . The sensors  144 ,  146 , and  148  are positioned such that the viewing end of each sensor is proximate the shuttle bore  108  when the switch  100  is in the third position, so as to provide images of an object in the shuttle bore with a minimum of distortion. 
     A programmable logic controller  158  controls the positioning of the shuttle  106  by a first actuator  160 , e.g., a two-way pneumatic cylinder that is attached to the shuttle  106  by a threaded rod  162 , and a second, opposing, two-way pneumatic cylinder  164  that limits the travel of the shuttle  106  by means of rod  165 . 
     In use, a normal embryo or other object entrained in a liquid stream enters fluid switch  100  via the upstream conduit  112  and moves past upstream sensors  120 ,  122 . Upon detection of an object at a particular location in the upstream conduit, the upstream sensors  120 ,  122  send a signal (such as an object image) to a signal processor  155 , which processes the signal and transmits the processed signal to the controller  158 . After a preset delay, the controller  158  signals actuators  160 ,  164  to move the shuttle  106  to the third position. The object enters the shuttle bore  108 , displacing fluid through the fluid bleed channel  150 . The controller  158  then signals a fluid flow control means (e.g., a pump or valve upstream of the fluid switch  100  or a valve downstream of the fluid bleed channel  150 ) to stop fluid flow, thereby maintaining the position of the object in the shuttle bore  108  at a position suitable for the downstream sensors  144 ,  146 ,  148  to generate images of the object. The object images are processed by a conventional signal processor  156  and transmitted to a conventional monitor  157   a  for viewing and classification by a human operator, or, in an alternate embodiment, transmitted to a computerized image processing system  157   b  for analysis and classification. The human operator or image processing system  157   b  transmits a signal corresponding to the classification to the controller  158 , which causes the actuators  160 ,  164  to move the shuttle  106  to the corresponding first or second position, permitting the object to continue into the first or second downstream conduit  138 ,  140 , respectively. After a preset delay to permit the object to move a distance downstream of the shuttle  106 , the controller  106  causes the actuators  160  and  164  to return the shuttle  106  to the third position. 
     As shown in FIGS. 4-6, the downstream sensors  144 ,  146 ,  148  provide orthogonal views of the object. However, the downstream sensors  146  and  148  can be disposed at various angles, e.g., downstream sensor  144  can be oriented as shown to provide an end-on view of the object, with downstream sensors  146  and  148  oriented at a right angle with respect to downstream sensor  144  and at a 60-degree angle with respect to each other. 
     Another embodiment of a three-position switch  170  is shown in FIGS. 7-10. The shuttle  172  is adapted to receive an optical cell  174  (shown in enlarged views in FIGS.  10 A and  10 B). The bore  176  of the optical cell  174  has a square cross-section with interior dimensions (“a” in FIG. 10B) that are substantially the same as the interior diameters of the upstream conduit  178  and the first and second downstream conduits  180  and  182 , respectively. The sensor  184  is disposed in the third downstream bore  190  and is stationary during operation of the switch  170 . The sensor  186  is disposed in the sensor bore  192  and can be either stationary during operation of the switch  170  or attached to and move with the shuttle  172 . The sensor  188  is in a vertical orientation so as to view an object through the sensor bore  194  and, like the sensor  186 , can be either stationary during operation of the switch  170  or be attached to and move with the shuttle  172 . The sensors  184 ,  186  and  188  are positioned such that the viewing end of each sensor is proximate the optical cell  174  when the switch  170  is in the third position so as to provide images of an object in the optical cell bore  176  with a minimum of distortion. Gaps between the surface of the optical cell  174  and the sensors  186 ,  188  can be reduced by disposing a lens or optical flat of an optically clear material (not shown) in contact with a surface of the optical cell  174  at the end of sensor bores  192  and  194  (in sensor bore  194 , such a lens or optical flat is preferably coplanar with the surface of the shuttle  172 ) to reduce light reflection and distortion of object images. The switch  170  is otherwise similar in construction and operation to the three-position switch  100  shown in FIGS. 4-6. 
     The optical cell  174  is preferably made of an optically clear material (e.g., an optical-grade plastic or glass) to reduce optical distortion of object images produced by the downstream sensors  184 ,  186  and  188 . The flat interior surfaces of the optical cell bore  176  are also intended to reduce optical distortion of object images in the optical cell bore  176  that are produced by the downstream sensors  186  and  188 . (Alternatively, if the optical cell  174  has a cylindrical bore, images of an object in the optical cell  174  that are obtained by downstream sensors  186  and  188  through the curved surface of the optical cell bore can be corrected by an appropriate cylindrical lens.) 
     Three-Position Fluid Switch with Rotary Shuttle 
     An alternative embodiment including a rotary shuttle is shown in FIGS. 11-13. The fluid switch  200  has a body  202  (shown as a one-piece body) defining a shuttle cavity  204 , in which is disposed a disk-shaped rotary shuttle  206  having a thickness slightly less than the width of the shuttle cavity  204 . 
     The body  202  defines first, second, and third upstream bores  208 ,  210 ,  212 , respectively, upstream of the shuttle cavity  202 . The upstream bores have corresponding open downstream ends  214 ,  216 ,  218 . In the upstream bores are disposed corresponding first, second and third upstream conduits  220 ,  222 ,  224 . 
     The body  202  also defines first, second, and third downstream bores  226 ,  228 ,  230 , respectively, downstream of the shuttle cavity  204 . The downstream bores  226 ,  228 ,  230  have corresponding open upstream ends  232 ,  234 ,  236 . The downstream conduits  238 ,  240  are disposed in the first downstream bore  226  and the third downstream bore  230 , respectively. No conduit need be disposed in the second downstream bore  228 , which may therefore have a different diameter than the first and third downstream bores,  226 ,  230 , respectively. A fluid bleed channel  242  is connected to the second downstream bore  228  close to the upstream end  234 . An O-ring  243  is disposed in a gland around each of the open ends  232 ,  234 ,  236  to provide a liquid seal between the open end and the shuttle  206 . 
     The shuttle  206  defines first, second, and third shuttle bores  244 ,  246 ,  248 , respectively, each having a diameter substantially the same as the inside diameter of the upstream and downstream conduits. 
     The upstream, downstream, and shuttle bores are spaced apart and lie on an arcuate plane such that corresponding upstream, downstream, and shuttle bores can be aligned as shown in FIGS. 7-9. The shuttle can be rotated such that the second shuttle bore  246  is selectively aligned with any one of the downstream bores. 
     Adjacent the second upstream bore  210  are upstream sensor bores  250 ,  252  in which are disposed upstream optical sensors  254 ,  256 , respectively (shown in FIGS. 11-13 as eye symbols). Upon detection of an object in the second upstream conduit  222 , the upstream sensors  254 ,  256  transmit a signal to a signal processor  257 , which in turns transmits a signal to controller  258 . The controller  258  controls rotation of the shuttle  206  via a servo stepper motor  260  or analogous actuator. 
     Unlike the two-position fluid switch  20  and three-position fluid switches  100  and  170  described above, the shuttle  206  in the three-position switch  200  does not carry the end of an upstream conduit. As a result, the upstream and downstream conduits need not be flexible. At least the second upstream conduit  222  is preferably transparent. 
     The fluid switch  200  also includes an imaging system that includes three optical sensors  264 ,  266 ,  268  (shown in FIGS. 11-13 as eye symbols). The first optical sensor  264  is disposed in a sensor bore  270  that horizontally intersects the second upstream bore  210  at about a right angle. The second optical sensor  266  is disposed in a sensor bore  272  that vertically intersects the second upstream bore  210 . All three optical sensors  264 ,  266 ,  268  are stationary during operation of the fluid switch  200 . As shown in FIGS. 11-13, sensor bores  270 ,  272  and respective sensors  264 ,  266  disposed therein are oriented at approximately right angles to each other to provide orthogonal views of an object in the upstream bore  210 . The third optical sensor  268  is disposed in and closes the second downstream bore  228  to provide a view of an object upstream along the axis defined by the second upstream bore  210  and the second downstream bore  228 . Thus, the three optical sensors  264 ,  266 ,  268  provide views of an object along three intersecting axes. As discussed above, the angle along which the object is viewed by the downstream sensors can be varied. 
     The sensors  264 ,  266 ,  268  generate images of an object and transmit the images to a conventional signal processor  273  to convert the object images into a form suitable for a conventional monitor  274   a  for viewing and classification by a human operator, or, in an alternate embodiment, for analysis and classification by a computerized image processing system  274   b . The human operator viewing monitor  274   a  or the image processing system  274   b  then sends a signal corresponding to the classification to the controller  258 . The controller  258  controls rotation of shuttle  206  via a servo stepper motor  260  (the support bracket for the motor  260  is not shown). 
     In use, the upstream sensors  254 ,  256  detect an object in the second upstream conduit  222  and transmit a signal to the signal processor  257 , which processes the signal and transmits the processed signal to the controller  258 . After a preset delay, the controller  258  stops fluid flow in the second upstream conduit  222 , and thus the movement of the object carried by the liquid in the upstream conduit  222 , to permit viewing of the object by the sensors  264 ,  266  (i.e., at the “first viewing position”). The sensors  264 ,  266  generate images of the object, which are processed by signal processor  273  and transmitted to the monitor  274   a  to be viewed and analyzed by a human operator, who classifies the object (accept/reject) and sends a signal to the controller  258  corresponding to the classification. Alternatively, a computerized image processing system  274   b  analyzes and classifies the object on the basis of the processed image received from the signal processor  273  and sends an appropriate signal based on the object classification to the controller  258 . The controller  258  stops fluid flow in the second upstream conduit  222  by signalling the motor  260  to rotate the shuttle  206  to a position at which the shuttle  206  blocks the flow of liquid and entrained objects. Alternatively, the controller  258  can stop fluid flow in the second upstream conduit  222  by signalling a fluid flow control means (e.g., a pump or valve) upstream of the fluid switch  200  or, when the shuttle  206  is in the second position, by signalling a fluid flow control means such as a valve downstream of the fluid bleed channel  242 . 
     If a reject decision is made, an appropriate signal is sent to the controller  258 , which causes the motor  260  to rotate the shuttle  206  until the downstream end  216  of the second upstream bore  210 , the second shuttle bore  246 , and the upstream end  234  of the second downstream bore are aligned (“second position”). The object enters the second shuttle bore  246  and liquid is pushed into the fluid bleed channel  242 . Then, after a preset delay to permit the object to enter the second shuttle bore  246 , the shuttle  206  is rotated until the second shuttle bore  246  is aligned with the downstream end  214  of the first upstream bore  208  and the upstream end  232  of the first downstream bore  226  (“first” or “reject position”). (If desired, after a preset delay to allow the embryo to enter the second shuttle bore  246  and before the shuttle  206  is rotated to the first position, fluid flow in the second upstream conduit  222  can be stopped as described above). After a preset delay to allow the rejected object to be pushed by purge liquid from the first upstream conduit  220  into the downstream conduit  238 , the controller  258  signals the motor  260  to rotate the shuttle  206  back to the second position to begin the next cycle (if necessary, the controller  258  also signals the fluid flow control means to resume fluid flow.) The rejected object eventually continues to an appropriate destination, e.g., a waste receptacle. The second shuttle bore  246  thus serves: first, as a portion of a continuous, enclosed fluid-tight path that includes the second upstream conduit  222 , the second shuttle bore  246 , and the second downstream bore  228 ; second, as a fluid chamber to receive the embryo (and a volume of fluid) and deliver the embryo to an appropriate downstream fluid conduit, in this instance, the downstream conduit  238 ; and third, as part of a second fluid-tight path, in this case including the first upstream conduit  220 , the second shuttle bore  246 , and the downstream conduit  238 . 
     If an accept decision is made, the shuttle  206  is rotated to the second position. After a preset delay to permit the object to enter the second shuttle bore  246 , the controller  258  stops fluid flow in the second upstream conduit  222  to maintain the position of the embryo in the second shuttle bore  246  (“second viewing position”). The third sensor  268  then generates additional images of the object that are transmitted to the monitor  274  and analyzed by the human operator, who again classifies the object (accept/reject). 
     Once this second classification is complete, an appropriate accept or reject signal is sent to the controller  258 . If the object is rejected, the shuttle  206  is rotated to the reject position and the object is pushed into the downstream conduit  238 , as discussed above. If the object is accepted as a normal, mature embryo, the shuttle  206  is rotated until the second shuttle bore  246  is aligned with the downstream end  218  of the third upstream bore  212  and the upstream end  236  of the third downstream bore  230  (“third” or “accept position”). In this position, purge liquid from the third upstream conduit  224  pushes the embryo into the downstream conduit  240 . Eventually the embryo continues to a location for incorporation into manufactured seed. Thus, the fluid switch  200  employs a two-stage process of classification and sorting instead of the one-stage classification and sorting process described above for the three-position fluid switch  100 . 
     The above-described fluid switches  20 ,  100 , and  200 , including the bodies and shuttles thereof, can be made of a variety of materials, e.g., plastic materials (including opaque plastics such as Delrin® or clear plastics such as acrylics, including plexiglass), metals (e.g., food-grade stainless steel), ceramics, etc., preferably non-phytotoxic, food grade materials that can be sterilized by standard techniques. For production of high quality images of objects, it is preferable that at least the shuttle be made of an opaque, non-reflective material, such as black Delrin®. 
     It will be readily appreciated by those of ordinary skill in the art that the above-described embodiments of the present invention can be modified, for example, to couple multiple upstream conduits to one downstream conduit or to multiple downstream conduits. 
     Although embodiments of the invention are described above in terms of a shuttle moving the downstream end of the upstream conduit into alignment with the upstream end of a downstream fluid conduit, in alternative embodiments the upstream end of a downstream fluid conduit can be moved into alignment with the downstream end of an upstream fluid conduit. In such an embodiment of the invention, the upstream conduit could be inflexible and stationary. 
     Classification of Objects 
     Features for distinguishing morphologically normal, mature embryos from “non-embryos” (including immature embryos, morphologically abnormal embryos, and non-embryo structures such as debris) include but are not limited to: size characteristics, e.g., length and diameter; shape characteristics, e.g., circularity, symmetry, and elongation; surface characteristics, e.g., roughness, etc.; the presence, size, and normalcy of anatomical features, e.g. cotyledons (see, e.g., Buchholz and Stimert,  Ill. Acad. Sci. Trans.  38:27-50, 1945); and so on. Images of objects generated by optical imaging systems (including borescopes as discussed above), for example, can be readily and automatically analyzed by object-recognition software. Currently available software can be used or readily adapted for use in classifying objects such as plant embryos on the basis of various characteristics, including, but not limited to, those listed above. 
     Methods of Producing Manufactured Seed 
     A number of versions of manufactured seed and methods for their production have been described in the patent literature, including U.S. Pat. Nos. 4,562,663; 4,583,320; 4,615,141; 4,715,143; 4,777,762; 4,779,376; and 4,780,987 and Canadian Patent No. 1,241,552. More advanced versions of manufactured seed that display an improved germination rate are disclosed in U.S. Pat. Nos. 5,427,593 and 5,236,469, incorporated herein by reference. 
     Fluid switches according to the present invention are particularly well suited for automated methods useful in a commercial process of producing manufactured seed. Plant embryos are first directionally oriented and introduced into a flowing liquid stream into a conduit, preferably without the need for human manipulation, e.g., as described in U.S. Pat. No. 5,284,765, incorporated herein by reference. A substantially regular spacing between the embryos is achieved employing a fluid switch according to the present invention, as described above. Next, normal, mature embryos having a high probability of germinating and developing into normal plants are separated from other objects such as immature or morphologically abnormal embryos and non-embryo structures by means of a fluid switch according to an embodiment of the invention and delivered to an assembly location for incorporation into a manufactured seed. 
     Briefly stated, to assemble one embodiment of a manufactured seed, a unit of totipotent plant tissue is disposed relative to a hydrated gel so as to permit liquid transfer from the gel to the embryo. At least the shoot (or cotyledon) end of the plant tissue is enclosed by a shoot restraint, which is adapted to resist penetration by the shoot upon germination, to permit access of the plant tissue to gases and liquids, and to be shed distally off the shoot during germination. The plant tissue, gel, and shoot restraint are enclosed within a substantially rigid capsule. The capsule (or manufactured seed coat) protects the plant tissue from mechanical damage, desiccation, and pathogens and pests when the manufactured seed is placed on or in soil. 
     All publications and published patent documents cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. 
     Having illustrated and described the principles of the present invention, it should be apparent to persons skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. We claim all modifications that are within the spirit and scope of the appended claims.