Patent Abstract:
A process of mechanical separation of embryos from seeds for genetic transplantation employs counter-rotating cylinders together with one or more culling, hydration, separation, and viability testing steps to provide high-throughput of viable, transplantable tissue.

Full Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
   This application is a division of U.S. application Ser. No. 10/710,067, filed Jun. 16, 2004 now U.S. Pat. No. 7,402,734, which claims the benefit of U.S. Provisional application 60/320,278 filed on Jun. 16, 2003, each of the disclosure of which are hereby incorporated by reference in their entirety. 

   BACKGROUND OF INVENTION 
   The present invention relates to plant cell transformation in which genetic material is inserted into plant cells to modify resulting plants, and in particular, the invention relates to an apparatus for collecting embryonic tissue from seeds that may be used for such transformation. 
   The genetic transformation of plants may be used to develop crops with improved yield, insect and disease resistance, herbicide tolerance, and increased nutritional value. In such transformation, new genes are introduced into the chromosomal material of existing plant cells. Various methods have been developed for transferring genes into plant tissue including high velocity microprojection, microinjection, electroporation, direct DNA uptake and, Agrobacterium-mediated gene transformation. 
   Once the gene is successfully introduced into the chromosomal material of the plant cells, new inheritable germ line tissue must be developed (e.g., seeds) so that the new plant may be propagated. One way this may be done is by selecting only cells that have accepted the new gene and culturing the callus of these cells into a new viable plant. The time required to develop a plant from a single cell is lengthy. 
   Shortened development times may be obtained by directly treating meristematic tissue of a preformed plant embryo. The meristematic tissue is formative plant tissue of cells that will differentiate to produce different plant structures including the seeds or germ line tissue. A number of plant embryos may be treated and selection or screening techniques used later to determine which of those plants have incorporated the new genetic information into their germ line tissue. 
   U.S. Pat. No. 6,384,301 assigned to the assignee of the present invention and hereby incorporated by reference describes a method of genetically transforming soybeans ( Glycine max ) using Agrobacterium mediated gene transfer directly on the meristematic cells of soybean embryos. In this procedure, the seeds are soaked to initiate germination. After germination has begun, the embryo is excised from the seed and the primary leaf tissue removed to expose the meristem of the soybean embryo. The meristem is formative plant tissue that will differentiate to give rise to different parts of the plant. 
   Although seeds are inexpensive, the considerable labor involved in excising the embryos, transferring the genetic material into the embryos, and cultivating the embryos makes it desirable to reduce damage to the embryo that could result in this effort being applied to tissue that is ultimately non-viable. For this reason, the excision of plant embryos is performed by hand. 
   In the manual process, surface sterilized seeds are aseptically handled one at a time with gloved hands. They are oriented in a manner as to eject the seed coat with applied force. Then the cotyledons are separated and removed leaving the seed embryo. The embryonic leaves are removed near the area of the primary meristem. Recovery of viable embryos for genetic transfer is less than 100% even with this hand method and may be as little as 70% with high quality seeds. 
   Bacterial contamination of the embryos after excision is a significant concern. Manual excision of the embryos allows early separation of the seed coat from the remainder of the seed to prevent contamination of the embryo with bacteria found on the seed coat, which normally protects the embryo. 
   Skilled personnel performing manual excision can often recognize abnormal embryos at the time of excision and discard them, substantially improving downstream yields. 
   Despite the advantages of manual excision, individual separation of each plant embryo from its seed is extremely labor intensive and stands as a barrier to a scaling up of the transformation process in which, typically, many plants must be treated to yield a successful few transformations. 
   What is needed is a process that can significantly increase the availability of transformable embryos without unacceptably increasing total costs of transformation, the latter which will rise if damage to embryos or bacterial contamination of the embryos causes fruitless cultivation of large numbers of non-viable embryos. 
   SUMMARY OF INVENTION 
   The present inventors have developed an automated technique for excision of transformable tissue from seeds that sufficiently reduces embryo damage and bacterial contamination such as might render mechanical separation impractical. A mechanical excision machine is combined with optional seed culling, improved hydration of the seeds, and automated separation of the embryos to make automatic excision practical. Additional techniques to reduce bacterial contamination incident to such automation, particularly between the seed coat and the embryo, are provided. 
   Specifically then, the present invention provides for automated preparation of transformable plant tissue by hydrating plant seeds to soften the seed tissue and then passing the hydrated seeds through a mechanical separator that divides the seeds into separate cotyledon, seed coat and embryo. Genetic material is then introduced into the cells of the separated embryo. 
   It is one object of the invention to provide for the high volume automated excision of transformable plant tissue. 
   The mechanical separator may provide opposed moving surfaces applying a shear force to the hydrated seeds. 
   It is another object of the invention to provide for a simple mechanical separator that separates the seed components without undue damage to the embryo. The shear force on the hydrated seeds coaxes the seeds apart along their natural separation points. 
   The opposed moving surfaces may be rollers having different rolling speeds. 
   Thus it is another object of the invention provide for shear surfaces that are easily manufactured. 
   The rollers may be co-rotating. 
   It is another object of the invention to provide a mechanism that is adaptable to a continuous or semi-continuous batch process. 
   The rollers may have serpentine roller faces. 
   It is another object of the invention to provide a surface that envelops the outer surface of the seeds to separate them and distribute the shearing force evenly to reduce damage to the embryos. 
   The rollers may have an outer elastomeric surface. 
   Thus, it is another object of the invention to provide for improved grip and reduced pressure on the seed coat. 
   The moving surfaces may comprise at least two successive sets of opposed rollers. 
   Thus, it is another object of the invention to provide for a series of graduated separations of the seed coats to increase yield. 
   The separation of the moving surfaces may be adjusted according to the type of seeds. The amount of shear between the moving surfaces may also be adjusted according to the type of seed. 
   Thus, it is another object of the invention to provide a machine suitable for the processing of a variety of different seed types. 
   The seeds may be sprayed with liquid as they pass through the mechanical separator. 
   It is another object of the invention to reduce bacterial contamination incident to such mechanical separations by a constant dilution or disinfecting of such contamination with sterile liquid or a disinfectant solution. 
   Liquid may be sprayed against the rollers to strike the rollers in a direction opposite rotation of the rollers. 
   It is another object of the invention to provide for a cleaning of the rollers that minimizes damage to attached embryos. 
   The volume or mass flow of seeds into the mechanical separator may be controlled to a predetermined constant value. 
   It is thus another object of the invention to minimize damage to the embryos that may be caused by an excessive number of seeds entering the rollers. 
   The seeds may be culled based on predetermined seed characteristics such as color, size, moisture, germplasm or density prior to their mechanical separation. 
   Thus it is another object of the invention to compensate for the lack of human visual inspection in mechanical excision by a tight control of seed type at a stage where rejection of seeds is relatively inexpensive. 
   The step of hydrating the seeds may include rinsing the seeds and then holding them for at least one hour followed by a soaking of the seeds. 
   It is thus another object of the invention to provide for a hydration in a manner that reduces cracking of the cotyledons such as may promote damage to the embryo. 
   The rinsing, holding, and soaking may be performed in a container in which seeds are introduced, the container having a drain and an inlet, the inlet communicating with the first rinse liquid reservoir, and a second soak liquid reservoir different from the rinse liquid reservoir and including a valve position between the inlet and the rinse liquid reservoir and the inlet and the soak liquid reservoir and the drain, the valve communicating with an electronic timer for controlling the rinse, holding, and soaking automatically. 
   Thus it is another object of the invention to allow more complex schedules for hydrating the seeds without undue seed handling. It is another object of the invention to allow the use of reservoirs into which different additives may be introduced permitting different rinse and soak materials to be used in hydrating the seeds. 
   The rinse may include an antimicrobial such as a bleach or other disinfecting solution. 
   Thus it is another object of the invention to reduce the bacterial load upstream of their mechanical excision, the latter which may cause contamination of the embryos. 
   After the mechanical separation, the cotyledons, seed coats, and embryos may be passed into a separating machine to separate the embryos from the seed coats and the cotyledons. 
   Thus it is another object of the invention to eliminate the need to manually sort through separated seed material such as would reduce the benefit of mechanical excision. 
   The separating machine may include a weir allowing the seed coats to wash over the top of the weir and the embryos and cotyledons to pass to the bottom of the weir. 
   Thus it is another object of the invention to provide a separation system that works naturally with the mixture of liquid and seed parts exiting the separation machine. It is another object of the invention to separate the dirty seed coats from the embryos early in the separation process to reduce the risk of contamination. 
   The separating machine may include a screen separating the cotyledons from the embryos. 
   Thus it is another object of the invention to reduce manual effort necessary to extract the embryos from the cotyledons. 
   The method may include, after the mechanical separation, a step of culturing the embryos for a predetermined period in a liquid medium to cull nonviable embryos. 
   It is thus another object of the invention to provide a mechanism that may, if necessary, accommodate a higher rate of nonviable embryos in mechanical separation without incurring excessive cultivation costs. 
   These particular objects and advantages may apply to only some embodiments falling within the claims and thus do not define the scope of the invention. 

   
     BRIEF DESCRIPTION OF DRAWINGS 
       FIG. 1  is a flow chart showing principal steps of the present invention such as may include: culling, hydration, excision, separation, and a viability test; 
       FIG. 2  is a schematic diagram of an apparatus used in the hydration step of  FIG. 1  allowing automatic control of seed hydration; 
       FIG. 3  is a simplified representation of an apparatus used in the excision step of  FIG. 1  providing a series of opposed rollers which separate the seed parts by a sheering action; 
       FIG. 4  is a perspective view of one roller of the device on  FIG. 3 ; 
       FIG. 5  is a cross-section through a pair of rollers of  FIG. 3  taken along line  5 - 5  of  FIG. 4  showing a setting of the separation of the rollers using a gauge; 
       FIG. 6  is a fragmentary enlarged view of one pair of opposed rollers of  FIG. 3  showing liquid sprays directed to prevent the rollers from clogging and to direct process flow; 
       FIG. 7  is an elevational cross-sectional view of a weir in a collection vessel after the final rollers of  FIG. 3  such as separates the seed coats from the cotyledons and embryos; 
       FIG. 8  is an elevational cross-section through a separation device that may follow the weir of  FIG. 7  employing a screen to separate the cotyledons and remaining seed coats from the embryos; 
       FIG. 9  is a figure similar to  FIG. 8  of an alternative embodiment of the separation device using a reciprocating sifting platform; 
       FIG. 10  is a figure similar to that of  FIGS. 8 and 9  showing an alternative separation device employing a rotating drum having an outer peripheral screen; 
       FIG. 11  is an elevational cross-section of a sucrose separation system in which a predetermined density of sucrose solution separates embryos from the remaining portions of the seed; 
       FIG. 12  is a flow diagram of an inoculation step in which the embryos are treated with Agrobacterium and processed in a viability test in a liquid media prior to culturing; 
       FIGS. 13   a  and  13   b  are simplified elevational views of the path of seeds from an auger feeder into the apparatus of  FIG. 3 , the elevational views superimposed on plots of seed distribution with and without a spreader bar used to provide a more uniform seed distribution; 
       FIG. 14  is an alternative embodiment of the separation devices of  FIGS. 8-10  using air agitation; 
       FIG. 15  is a first embodiment of a nozzle assembly for the air agitation of the device of  FIG. 14 ; and 
       FIG. 16  is a second embodiment of a nozzle assembly for the air agitation of the device of  FIG. 14 . 
   

   DETAILED DESCRIPTION 
   Referring now to  FIG. 1 , generally the mechanized method  10  of the present invention receives harvested soybeans or other seeds  12  from which transformable plant tissue will be extracted. The seeds  12  are ideally harvested at a predetermined internal moisture suitable for isolating transformable material therefrom, e.g., 8-14% internal moisture for soybeans, and held in stable storage conditions prior to use. 
   The seeds  12  may be subject to an optional culling step  14  intended to remove seeds  12   a  with a high degree of bacterial or fungal contamination and also seeds  12   a  that may for any reason statistically fail to produce viable embryonic tissue with the present invention. These latter reasons may include parameters such as the size of the seed or other physical characteristics that in other contexts would be unobjectionable and may be adjusted empirically by variation of the parameters and measurement of ultimate yields of the viable tissue. 
   Preferably, the culling step  14  is performed mechanically and may include a size culling using standard seed sorting techniques eliminating the seeds  12  above and below a predetermined size, optical sorting using high speed sorting equipment readily available on the market such as employs a camera and vision system to reject seeds  12  that are selected from one or more of the following criteria, color, size, shape or density. Examples of culling methods may include the use of an automatic scale after size sorting, or an optical sorter suitable for this purpose is the Satake Scan Master II manufactured by Satake USA Inc., of Houston, Tex. Other culling techniques may also be employed including culling by moisture content. Culling may also occur after hydration, as it has been determined that seeds with seed coats that have been damaged become imbibed faster than seeds with intact seed coats. 
   The culling step  14  is intended in part to replace the unconscious selecting of seeds by technicians performing the manual excision of the prior art, and to reduce bacterial and fungal load on the seeds  12  that may, in the mechanical process, create greater potential for contamination of the embryos. The optional culling step  14  may be quite aggressive because the seeds  12  prior to the excision are inexpensive. 
   Referring now to  FIG. 2 , the seeds  12   b  that pass the optional culling step  14  move to an optional hydration step  16  in which liquid may be introduced into the seeds  12  to soften the cotyledons and the seed coats reducing the possibility of damage of the embryo during the following excision step  18 . The hydration step  16  is preferably performed automatically, but may be performed manually. Referring again to  FIG. 2 , in a preferred embodiment hydration is performed through the use of a sterilized hydration container  20  having a four-liter capacity and a false bottom  22  perforated by a series of holes  24  smaller than the size of the seeds  12   b . The holes  24  lead to a drain chamber  26  communicating via an outlet hose  28  and valve  30  to a drain  32 . 
   The seeds  12  are placed on top of the false bottom  22  and a retainer plate  34  having holes  36 , also smaller than the average seed  12   b , is placed to rest lightly on top of the seeds  12   b  to prevent them from floating. An upper, removable lid  38  of the container  20  provides two inlets  40  and  42 . The first inlet  40  communicates via valve  44  to a rinse reservoir  46  containing a solution of sterile liquid and 200 ppm of Clorox. The second inlet  42  communicates via valve  48  to a tissue culture solution reservoir  50  containing a suitable plant tissue culture medium, such as bean germination medium (BGM) as described in U.S. Pat. No. 6,384,301. The tissue culture medium may also contain antimicrobials such as cefotaxime, Bravo, Benlate, Captan, and Carbenicillin. Other fungicides, disinfectants, plant hormones, antibiotics, and hydrogen peroxide may optionally be used in the tissue culture solution reservoir  50 . The liquid in both reservoirs  46  and  50  is held at room temperature. 
   An electronic timer  52  communicates with each of the valves  44 ,  30 , and  48  and is programmed so to initially, at a predetermined time before the excision process, to close valve  30  and open valve  44  for a predetermined time to fill the container  20  with the rinse solution from the rinse reservoir  46  after which valve  44  is closed. The rinse solution is held in place for three to ten minutes as valve  30  is opened to drain the container  20  through outlet hose  28 . 
   This first rinsing of the seeds  12   b  allows them to begin to absorb moisture but is not so pronounced as to cause cracking of the cotyledons such as might be caused by uneven expansion of the cotyledon material in the presence of excessive liquid. Rinsing also serves to further reduce surface contaminants. Other ways to prevent cracking include pre-incubation in a humid atmosphere or seed priming. 
   At least one hour later and preferably two hours later, the timer  52  operates to close valve  30  and open valve  48  for a predetermined time to fill the container  20  with the tissue culture media from the tissue culture solution reservoir  50 . The tissue culture media is held within the chamber for 8-13 hours after which the tissue culture media is drained by the timer  52  opening valve  30 . The container  20  is then refilled (via valve  44  operated by timer  52 ) with rinse solution from the rinse reservoir  46  for 15-30 minutes without draining (timer  52  holding valve  30  closed), the excess solution being used as a carrier for the excision step or drained (i.e., for use with an auger) as will now be described. When the seeds  12  are contained in a tissue culture medium without circulation, an ethylene inhibitor may be used. 
   Other methods of hydration are also contemplated in the present invention including an aerobic method in which the liquid is sprayed on the seeds without accumulating or where a gas is bubbled through the growth medium using an aerator or the like or media may be recirculated. It is also envisioned that other sizes and shapes of containers with different combinations of inlets and outlets, different methods of separating liquid from seeds, different solutions for different times, and the like may also serve the purpose of hydration. 
   Referring now to  FIGS. 1 and 3 , after hydration, the seeds  12   b  are poured together with the rinse liquid into a hopper  54  of an auger feed  56  such as provides a controlled feeding of the seeds  12   b  and rinse liquid into a first hopper  58  of an automated excision machine  60 . Such auger feeds  56  are well known in the art. The speed of the feeding of the seeds  12   b  is determined initially by inspection to reduce clumping of the seeds  12   b  at the rollers and to minimize visual damage to the embryos. Ultimately this feed speed may be determined empirically by using varying speeds and observing embryo viability. The auger feed  56  may be an Accu-Rate Feeder, manufactured in Whitewater, Wis. Other feed systems may be used in place of the auger feed  56  including, for example, pumps (with the seeds held in a slurry), conveyor belts, or vibrating conveyor systems such as are well known in the art. In addition, the rinse liquid could be separated from the seeds prior to input into the feeder. This step may also be performed manually without the use of a feeder. 
   Referring now to  FIGS. 3 and 13   a , the auger feed  56  provides a discharge tube  57 , ejecting seeds  12  along a horizontal axis perpendicular to the axis of rotation of rollers  62 ,  66  and  70  as will be described below. The seeds  12  fall from the discharge tube  57  through hopper  58  into a gap between the rollers  62 , concentrated along a centerline  160  by the limited size and circular aperture of the discharge tube  57 . 
   This spatial concentration of seeds  12 , shown by a seed distribution curve  162  peaking near the centerline  160 , can cause a crushing of seeds  12  when multiple seeds  12  pass through the rollers  62  gapped to provide efficient separation of the seed coat embryos and cotyledons at the edges of the rollers  62 . 
   Accordingly, referring to  FIG. 13   b , a diverter bar  164  may be placed between the discharge tube  57  and the rollers  62  extending fully across the hopper  58  along the axis of discharge tube  57  at the centerline  160 . This diverter bar  164  reduces the peak of the new seed distribution  162 ′ providing a smaller seed distribution variance  170  than the seed distribution variance  170 ′ obtained without the diverter bar as shown in  FIG. 13   a.    
   Similar methods of mechanical redistribution to even the solid flows may be made prior to or between successive sets of rollers if more than one roller pair are utilized. 
   The rollers  62 ,  66  and  70  are part of an automated excision machine  60  performing the excision step  18  of the present invention to separate the seeds  12   b  into embryos  12   c , cotyledons  12   d , and seed coats  12   e . The excision operation may be conducted in a clean room to minimize contamination from bacteria and mold. 
   The first hopper  58  of the automated excision machine  60  directs the seeds  12   b  into a pair of horizontally opposed rollers  62 , each rotating about mutually parallel horizontal axes. The seeds  12  pass through these rollers  62  to be received by a second hopper  64  and a second pair of horizontally opposed rollers  66  with mutually parallel horizontal axes. The seeds  12  pass between these rollers  66  and are received by a third hopper  68  and a following third pair of horizontally opposed rollers  70  with mutually parallel horizontal axes. 
   From the last set of rollers  70 , the seeds  12  fall into a collection vessel  72  as will be described further below. The use of three separate stages of rollers ensures that the components of most seeds  12  are fully separated by the time they arrive in the collection vessel  72 . 
   The left rollers as depicted in  FIG. 3 , (i.e., rollers  62   a ,  66   a  and  70   a ) turn clockwise in unison as driven by overlapping timing belts  74   a  which is driven by a first motor  76  attached to a first motor controller  78 . The clockwise direction causes a downward progression of the seeds  12  between the roller pairs. 
   Similarly, the right rollers as depicted in  FIG. 3 , (i.e., rollers  62   b ,  66   b  and  70   b ) are interconnected by overlapping timing belts  74   b  and turned by a second motor  80  having an independent second motor controller  82 . Here, a counterclockwise direction causes a downward progression of the seeds  12  between the roller pairs. 
   A sprocket  84  on motor  80  and engaging with the teeth of the timing belt  74  is larger than the corresponding sprocket  86  on motor  76  so as to provide a different (faster) rotational rate to the rollers  62   b ,  66   b , and  70   b  on the right than the rollers  62   a ,  66   a , and  70   a  on the left. For example, the rollers on the right may turn at about 30 rpm and the rollers on the left may turn at about 90 rpm. The motor controllers  82  and  78  may be adjusted to further refine the speed difference. Seeds  12  contacting both rollers of a pair thus experience a shear force acting on their outer surfaces. 
   It will be understood that other methods of driving the rollers at controlled speeds may be used including gear drives, direct drive servo motors, and the like. It is also understood that different speeds of turning the rollers may be used. 
   Referring still to  FIG. 3 , a sterile liquid or disinfectant solution source may attach through liquid line  87  to a flow meter  88  to be metered via pressure regulator  90  into a manifold connected to a set of spray heads  92   a  through  92   g . The liquid may further contain additional ingredients to surface sterilize or condition the embryos including but not limited to disinfectants, ethylene inhibitors, antioxidants, and surfactants. Spray head  92   a  is directed down-ward through hopper  58  to provide a steady wash of sterile liquid or disinfectant solution to wash the seeds  12  through the excision machine  60  and to lubricate and orient the seeds  12  and to dilute any contamination that may be introduced from the seed coats  12   e . The rate of liquid flow and pressure may be controlled to empirically determined values. 
   Spray heads  92   e  through  92   g  spray the under surface of rollers  70   a ,  66   a , and  62   a , respectively, directed against the tangential direction of rotation of the rollers to help dislodge seed material stuck on the rollers and further urge the seed through the machine. Likewise, spray nozzles  92   c  through  92   f  spray the under surface of rollers  62   b ,  66   b , and  70   b , respectively, directed against the tangential direction of rotation of the rollers. 
   It is anticipated that other methods may be used to introduce liquids into this step. Examples include, but are not limited to, the use of a distribution manifold, overflow weir, pipe, etc. 
   A sterile air source from air filter  96  may be connected to the liquid manifold via a valve  98  to purge the water lines between use to prevent the accumulation of biofilm and bacterial contamination. The air further dries the lines and provides a positive pressure to the lines reducing the risk of contamination of the lines. 
   Referring now to  FIG. 4 , each roller  62 ,  66 , and  70  has a generally cylindrical central portion  100  presenting a serpentine longitudinal profile  108 . The cylindrical central portion  100  is mounted on a concentric longitudinal axle  102 . The axle  102  may be supported at either end by conventional ball bearings  104 , and includes at one end, a sprocket  106  such as receives toothed timing belts  74   a  or  74   b  as described with respect to  FIG. 3 . The cylindrical central portion  100  may be coated with an elastomeric material, such as neoprene, Buna-N, chlorobutyl, EPDM, Viton, etc., that is resistant to wear and provides a cleanable and sanitizable surface that nevertheless is soft so as to conform slightly to the seed  12   b  and to provide improved gripping of the seeds  12 . Referring momentarily to  FIG. 3 , the softness of the elastomeric material may be increased for lower roller pairs with the roller pair  62   a  and  62   b  providing the hardest outer surface and the roller pair  70   a  and  70   b  providing the softest outer surface. For example, the elastomeric material of the upper rollers may be durometer  35  of the next pair of rollers, durometer  25  and  35 , and the bottom pair, both durometer  25 . It is understood that different seeds may require a particular gap angle, geometry, configuration, outer profile, diameter, or durometer. 
   Referring now to  FIG. 5 , the serpentine profile  108  of each roller  62   a ,  66   a , or  70   a  may be aligned with a corresponding surface serpentine profile  108 ″ of the corresponding roller  66   b ,  62   b , and  70   b  to which it is opposed to create therebetween, a substantially constant width serpentine channel  110  whose cross-section encourages separation of the seeds  12   b  as they pass through the rollers and provides for multiple engaging surfaces that are curved to conform with the curved outer periphery of the seeds  12   b . Setting of the separation between pairs of the rollers may be accomplished by lateral movement  111  of bearing  104  and may be facilitated by the insertion of a feeler gauge  113  at either edge of the central portion to ensure the rollers are substantially parallel. 
   Referring to  FIG. 6 , the bearing  104  may be held on a pillow block  112  having ears, one of which is mounted pivotally to a frame (not shown) of the automated excision machine  60  and the other which is mounted to an elongated hole  114  in the frame so as to allow lateral motion  111 , as shown in  FIG. 5 . The roller separation or diameter may be changed to accommodate different types of seeds  12  and may be increased for lower roller pairs with the roller pair  62   a  and  62   b  providing the narrowest serpentine channel  110  and the roller pair  70   a  and  70   b  providing the widest serpentine channel. 
   Other methods of excising the seeds  12  other than rollers are contemplated including disks, rollers with pins and the like which may stab at the cotyledons and press them together. 
   Referring now to  FIG. 7 , in an initial stage of the separation process  117  (of  FIG. 1 ), collection vessel  72  fills with clean liquid or disinfectant solution  116  produced from the nozzles  92  and also, in part, from the rinse liquid used during the hydration step  16 . An opening  118  near the upper edge of the collection vessel  72  provides a weir  120  over which liquid  116  may flow near the surface of the collection vessel  72 . Although the inventors do not wish to be bound by a particular theory, it is believed that the seed coats  12   e  entrap air during the excision step  18  and thus float out over the weir  120  to be separated from the cotyledons  12   d  and embryos  12   c , the latter which settle to the bottom of the collection vessel  72 . This early separation of the seed coats  12   e  in a wash of sterile liquid or disinfectant is believed to significantly reduce bacterial or fungal contamination of the embryos  12   c  and prevents the seed coats  12   e  from trapping embryos  12   c  or clogging separation screens in later separation steps. 
   Referring now to  FIG. 8 , the embryos  12   c  may be separated from the cotyledons  12   d  by means of a hydroscreen  126  providing a sloped wire mesh  128  (Tyler number six screen) having square openings approximately one-quarter inch on a side. Other functionally similar materials may be used in place of the wire mesh including, for example, perforated sheets of metal or plastic, loosely woven and non woven fabrics, nets, grids, and the like. 
   The wire mesh  128  is sloped so that a mixture of cotyledons  12   d  and embryos  12   c  in a sterile liquid or disinfectant solution may be introduced at the upper edge of the sloped wire mesh  128  to wash generally down the slope, at which point embryos  12   c  pass through the wire mesh  128 , whereas cotyledons  12   d  follow the wire mesh  128  to its edge and are discharged through an ejection port  132 . A separate drain port  134  may be provided for the embryos  12   c.    
   In an alternative embodiment, the cotyledons  12   d  and embryos  12   c , as shown in  FIG. 9 , may be introduced into a tray submerged in sterile liquid or disinfectant solution and having a bottom wire mesh  128 . The tray may be reciprocated in a horizontal direction  140  so that the embryos  12   c  pass through the wire mesh  128  into an outer container. The tray  129  may be removed from the outer container  131  and the embryos  12   c  recovered. 
   Referring now to  FIG. 14 , in an alternative embodiment, the tray  129  of  FIG. 9  may be adapted to provide a cylindrical wall with an upper flange  174  allowing it to rest on top of the upper lip of a cylindrical tank  176 . As before, the bottom of the tray is fit with a wire mesh  128 . The wire mesh  128  is sized to block cotyledons and seed coats but to allow passage of the embryos. 
   The cylindrical tank  176  is filled with liquid to a liquid level  186  so that seeds placed within the tray  129  (when the tray  129  is in the tank  176 ) are submerged within the liquid at rest on the wire mesh  128 . A cap  188  may fit over the top of the tank  176  covering the tray  129  to pre-vent splashing. 
   Positioned beneath the tray  129 , when the tray is in position in the tank  176 , is an aerator assembly  190  having a central hub  192  from which horizontal and radially extending spokes  194  are attached. The hub  192  provides a connection to an air line  196  which receives a source of high-pressure air through valve  200  controlled by pulse timer  202 . 
   Referring to  FIG. 16 , the hub  192  may be a generally cylindrical inverted cup attached and sealed to a vertical air pipe  212  by a lower bearing  214  fit about the vertical air pipe  212 . The bearing  214  allows the hub  192  to rotate freely about a vertical axis. The spokes  194  attached to the hub are hollow tubes communicating with the interior of the hub  192  (and hence with the vertical air pipe  212 ) at one end and plugged at their opposite ends. The spokes  194  have a series of upwardly facing holes  216  allowing the escape of air bubbles  210  and at least one laterally opening hole  218 . This laterally opening hole  218  reinforced by other similarly oriented holes in other spokes  194  provides for rotative motion under the reactive force of escaping air bubbles  210  moving the spokes  194  in a circular motion to ensure even distribution of the air impinging on the bottom of the wire mesh  128 . 
   The pulse timer  202  receives a waveform  204  providing for an agitation time period  206  and a rest time period  208 . This duration of each of these time periods  206  and  208  may be freely adjusted so as to provide alternating periods of intense agitation of the liquid in the tray  129  as moved by the liquid roiled by the discharge of air bubbles  210  from the aerator assembly  190 . 
   The discharge of air during the agitation time period  206  is such as to lift the cotyledons, seed coats, and embryos (not shown in  FIG. 14 ) from the wire mesh  128 . During the rest time period  208 , the lifted material descends again through the liquid so that the embryos may pass through the wire mesh  128  unobstructed by seed coats and cotyledons which tend to fall through the liquid at a different rate. 
   The tank  176  has a funnel shaped bottom  180  terminating in an outlet for  182  having a control valve  184 . The embryos selectively passing through the wire mesh  128  are received by the funnel shaped bottom  180  and may be discharged through the outlet for  182  as controlled by valve  184 . 
   Referring to  FIG. 15 , the air jet assembly  190 ′ may alternatively be a stationary ring or other figuration so as to introduce air bubbles  210  of sufficient volume to provide the necessary agitation. Instead of bubbles, the liquid itself may be pumped using impellers or other pumping systems in place of the air jet assembly  190 ′. 
   Sufficient air to produce a vigorous boiling of the liquids within the tray  129  can provide not only improved separation of the seed coats, cotyledons and embryos, but may provide for some excision as well. 
   Referring to  FIG. 10 , in yet another alternative embodiment, a drum  135  may be partially immersed approximately one-third to one-half in liquid held in container  141 . The drum  135  has wire mesh  128  attached to its outer cylindrical periphery and may filled with cotyledons  12   d  and embryos  12   c  into solution and rotated as indicated by arrow  142 , causing the embryos  12   c  to pass out of the drum  135 , which retains the cotyledons  12   d.    
   It is envisioned that other methods of embryo separation may also be used. For example, manual or automated sieving may be performed. Manual sieving may be performed using sieve trays immersed in liquid and gently shaking the trays. 
   Referring to  FIG. 11 , in an alternative separation method, the cotyledons  12   d  and embryos  12   c  may be introduced into a sucrose solution  146  of predetermined density selected to cause flotation of the embryos  12   c  and the sinking of the cotyledons  12   d  and seed coats  12   e  which may then be separated by a skimming or pouring off the embryos  12   c . The sucrose solution should be approximately 30-40% with thirty-seven percent preferred; however, concentrations of 10-70% will also provide some separation. After a few minutes, the embryos  12   c  rise to the surface of the container. The sucrose may be substituted with other biologically neutral compounds such as propylene glycol or Ficol, for example. 
   For each of these processes, the removed embryos may not be perfect, however, experimentation has shown that embryos with obscured meristems are still transformable. This separation need not be perfect as transformable tissue includes the embryo  12   c  with the primary leaves removed or with the primary leaves intact or with a partial cotyledon  12   d.    
   Referring now to  FIGS. 1 and 12 , once the embryos  12   c  are collected, they may be rinsed in sterile liquid or other solutions and then may be inoculated in a gene transfer step  155  with the desired genes using one of a variety of techniques, for example in soybean, sonication, as described in U.S. Pat. No. 6,384,301 issued May 7, 2002, assigned to the assignee of the present invention and hereby incorporated by reference, or particle delivery as described in U.S. Pat. No. 5,914,451 issued Sep. 22, 1992, assigned to the assignee of the present invention and also hereby incorporated by reference. Monocotyledonous plants could be transformed using the methods described in U.S. Pat. No. 5,591,616 issued Jan. 7, 1997, or PCT application WO95/06722 published Mar. 9, 1995, herein incorporated by reference. Cotton could be transformed using the methods described in U.S. Pat. No. 5,846,797 issued Dec. 8, 1998, or U.S. Pat. No. 5,004,863 issued Apr. 2, 1991 all hereby incorporated by reference. 
   Optionally, as indicated in process block  156  in  FIG. 1 , after sonication or other gene transfer step  155 , the trans-planted embryos  150  may be placed in a liquid culture  152  for fifteen to thirty days to identify which embryos  12   c  are still viable. This culturing also allows easier identification of the root and stem tips of the embryos  12   c  for proper planting of the viable embryos in an agar block  154  or further culture in liquid medium for selection. Up to this viability test, the amount of hand labor may be negligible and therefore nonviable embryos may still be removed at relatively low cost. Viability may also be tested on solid or semi-solid medium as well as liquid medium. 
   The proven viable embryos  12   c  are then grown on an agar block  154  such as may be treated with compounds or environmental conditions to help identify those embryos that have successfully received the implanted gene according to methods described in above-referenced U.S. Pat. No. 6,384,301. 
   The above-described techniques may be suitable for any plant whose transformable tissue can be derived from seeds and is especially useful for seeds of oilseed plants, such as soybean, canola, rapeseed, safflower, and sunflower, as well as other plants of commercial interest, such as legumes, cotton, corn, rice and wheat. 
   Generally each of the steps of  FIG. 1  may be used independently of the others. It is specifically intended that the present invention not be limited to the embodiments and illustrations contained herein, but include modified forms of those embodiments including portions of the embodiments and combinations of elements of different embodiments as come within the scope of the following claims.

Technology Classification (CPC): 1