Abstract:
A method of separating an embryo suspension mass is provided. The method includes supplying an embryo suspension mass culture having a plurality of first particles of a first size and a plurality of second particles of a second size different at least in part from the first size of the first particles. The method also includes suspending the embryo suspension mass culture in a fluid to create a mixture and forcing the mixture through a filter while maintaining the mixture in the fluid to separate the first particles from the second particles.

Description:
BACKGROUND 
     Asexual propagation of plants has been shown for some species to yield large numbers of genetically identical embryos, each having a capacity to develop into a normal plant. Such embryos are usually further cultured under laboratory conditions until they reach an autotrophic “seedling” state characterized by an ability to produce their own food via photosynthesis, resist desiccation, produce roots able to penetrate soil, and fend off soil microorganisms. Some researchers have experimented with the production of artificial seeds, known as manufactured seeds, in which individual plant somatic or zygotic embryos are encapsulated in a seed coat. Examples of such manufactured seeds are disclosed in U.S. Pat. No. 5,701,699, issued to Carlson et al., the disclosure of which is hereby expressly incorporated by reference. 
     An embryo suspension mass (“ESM”) is used to cultivate embryos that are subsequently inserted into manufactured seeds. Current somatic embryogenesis involves the ongoing maintenance and planting of ESM having diverse mixtures of cell clump sizes and types. The inventors of the current disclosure have discovered that small, usually single cells from the ESM generally have a higher yield than larger cells. That is, small cells tend to have a higher rate of survival and ultimately germinate into healthy plants or trees. 
     Currently available methods for size separation include passively pouring cell culture onto a sieve or set of nested sieves. Another currently available method is density-based centrifugation where cell culture is disposed in a test tube with a separation media, such as Percoll or Ficoll. The test tubes are centrifuged to segregate the cells on the basis of density. 
     Although such methods for size separation are effective, they are not without their problems. As a non-limiting example, such methods often subject the cells to contamination. Additionally, when centrifugation is included as part of the density separation, the centrifuge often causes shearing and compaction damage to the cells. 
     Thus, there exists a need for a method of separating an ESM that is capable of reliably separating the ESM at a relatively low cost, and minimizing the risk of damaging or contaminating the ESM. 
     SUMMARY 
     This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. 
     A method of separating an embryo suspension mass is provided. The method includes supplying an embryo suspension mass culture having a plurality of first particles of a first size and a plurality of second particles of a second size different at least in part from the first size of the first particles. The method also includes suspending the embryo suspension mass culture in a fluid to create a mixture and forcing the mixture through a filter while maintaining the mixture in the fluid to separate the first particles from the second particles. 
    
    
     
       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 an isometric view of a filtering device constructed in accordance with one embodiment of the present disclosure; 
         FIG. 2  is a cross-sectional side planar view of a manufactured seed; 
         FIG. 3  is an exploded isometric view of the filtering device of  FIG. 1 ; and 
         FIG. 4  is a cross-sectional isometric view of the filtering device of  FIG. 1 . 
     
    
    
     DETAILED DESCRIPTION 
     A filtering device  120  constructed in accordance with one embodiment of the present disclosure may be best seen by referring to  FIG. 1 . The filtering device  120  may be suitably adapted for inclusion in an automated assembly used to cultivate embryos used in the construct of artificial seeds. Alternatively, such a filtering device  120  is suitably adapted to stand alone as a separate assembly to harvest either embryos or culture used to cultivate embryos for insertion into well-known manufactured seeds. 
     For clarity and background, the structure of a manufactured seed  20  is described with reference to  FIG. 2 . The manufactured seed  20  includes a cylcap  22 , a seed shell  24 , nutritive media  26 , such as a gametophyte, and a dead end seal  28 . The seed shell  24  is suitably formed from a section of tubular material. In one embodiment, the seed shell  24  is a sectioned straw of fibrous material, such as paper. The sections of straw may be pre-treated in a suitable coating material, such as wax. 
     The cylcap  22 , also known as a restraint, is suitably manufactured from a porous material having a hardness strong enough to resist puncture or fracture by a germinating embryo, such as a ceramic or porcelain material, and includes an end seal portion  30  and a cotyledon restraint portion  32 . The cotyledon restraint portion  32  is suitably integrally or unitarily formed with the end seal portion  30 . The cylcap  22  also includes a longitudinally extending cavity  34  extending through the end seal portion  30  and partially through one end of cotyledon restraint portion  32 . The cavity  34  is sized to receive a plant embryo  42  therein. 
     In certain embodiments, as the cylcap  22  is suitably manufactured from a porous material, it may be desirable to coat the cylcap  22  with a barrier material to reduce the rate of water loss and restrict or reduce microbial entry. Such barriers include wax, polyurethane, glaze, nail polish, and a coating sold by Airproducts Airflex 4514. 
     The embryo  42  is disposed within the cavity  34  and is suitably sealed therein by a live end seal  43 . The live end seal  43  includes a primary end seal  44  and a secondary end seal  21 . The primary end seal  44  is suitably formed from a PCL material described above and includes a centrally located opening  50 . The opening  50  is sized to correspond to diameter of the cavity  34  of the cylcap  22  to permit a germinating embryo  42  to pass therethrough. The primary end seal  44  is suitably attached to the end seal portion  30  by a variety of methods, including glue or heat bonding. Finally, the manufactured seed  20  includes a dead end seal  28  and may include a tertiary seal  60 . 
     As may be best seen by referring to  FIGS. 3 and 4 , the filtering device  120  includes a beaker  122 , a rotating assembly  124 , and a basket  126 . The beaker  122  is a well-known beaker adapted to hold a fluid (not shown). Attached to the beaker  122  is a well-known aerator  128 . Such an aerator  128  includes an aeration tube  130 , a cap  132 , and a clamp  134 . The aerator  128  is suitably disposed within the beaker  122  and, as is well known, aerates the contents of the beaker  122 . 
     The rotating assembly  124  includes a feed tube  140 , an arbor nut  142 , a magnet  144 , and a pin  146 . The feed tube  140  is “broken” for ease of illustration and includes a supply port  150  extending through one sidewall of the tube  140 . One end of the feed tube  140  defines a feed end  152 , while the other end defines an anchor end  154 . The anchor end  154  is sized to slidably receive the pin  146  therein. 
     The pin  146  is suitably a cylindrically-shaped member and includes a spherically-shaped tip  160  extending from one end of the pin  146 . The other end of the pin  146  includes a shoulder  162 . The tip  160  is preferably spherically-shaped to minimize damage to particulate matter, such as embryo suspension mass (“ESM”), supplied through the feed end  152  of the feed tube  140 , as described in greater detail below. 
     After the pin  146  is inserted into the anchor end  154  of the feed tube  140 , a screw  148  is inserted into a corresponding port (not shown) formed in the shoulder  162  of the pin  146 . Insertion of the screw  148  into the port of the pin  146  causes that end of the pin  146  to expand within the anchor end  154  of the feed tube  140 , thereby attaching the pin  146  within the feed tube  140  by compression fit. Thereafter, the magnet  144  is inserted through a channel  164  extending through one end of the arbor nut  142 . 
     The rotating assembly  124  is attached to the basket  126  by an impeller  170 . The impeller  170  includes an externally-threaded stem  172  sized to threadably engage corresponding internal threads within the arbor nut  142 . As attached, the impeller  170  rotates with the rotating assembly  124  by a well-known magnetic drive assembly (not shown) suitably disposed beneath the beaker  122 . The feed end  152  of the feed tube  140  extends through the basket  126  and through a beaker cap  180  and is sealed thereto by an end cap  182  and O-ring  184 . 
     As may be best seen by referring to  FIG. 4 , the basket  126  includes a plurality of pores  190  sized to filter the ESM. Specifically, the ESM includes a plurality of first particles of a first size and a plurality of second particles of a second size. As is known in the art, ESM is a population of variably sized clusters and associations of single embryonal cells with suspensor cells and individual suspensor cells and cellular debris. Thus, as used within this disclosure, the term “particle” is intended to include single embryonal cells, multiple embryonal cells, suspensor cells, cellular debris, embryos, etc., and any combination thereof. 
     During operation of the filtering device  120 , the ESM is filtered by the basket  126  to segregate the plurality of particles constituting the ESM. As an example, the pores  190  may range between a diameter of 0.25 mm-1.5 mm. As non-limiting examples, the pores  190  may be 0.5 mm, 0.75 mm, 1.00 mm, or 1.25 mm in diameter. In the non-limiting example of a basket  126  having pores  190  that measure 0.5 mm in diameter, ESM filtered through the basket  126  will permit particles having a diameter of approximately 0.5 mm and smaller to pass through the pore  190 . As such, the basket  126  filters at least particles having a diameter of approximately 0.5 mm from particles having a diameter greater than 0.5 mm. 
     Operation of the filtering device  120  may be best understood by referring to  FIGS. 1 and 4 . A supply of ESM is provided to the feed tube  140  through the feed end  152 . The ESM matter flows within the feed tube  140  where it exits into the basket  126  through the supply port  150 . The basket  126  is disposed within the beaker  122  filled with a suspension media, such as fluid. As noted above, the flow of ESM is cushioned to limit damage to the particulate matter of the ESM by the spherical end  160 . 
     The ESM suspended in the fluid of the beaker  122  creates a mixture that is forced through the filter or basket  126  by, in one example, centrifugal force. As the mixture is forced through the pores  190  of the basket  126 , it separates a plurality of first particles of a first size from a plurality of second particles of a second size different at least in part from the first size of the first particles. As an example, if the first particles are cells of larger, more mature embryos and the second particles are smaller in size than the more mature cells, the smaller cells pass through the pores  190  if they are smaller than the diameter of the ports  190  and into the base of the beaker  122 . In the example where the basket  126  is magnetically driven to induce a rotating force, the centrifugal force of the rotating basket  126  propels the second particles through the pores  190 . 
     While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention. As a non-limiting example, the filtering device  120  may include a second basket or filtering layer where the second basket includes pores of a different diameter from the first basket. In such an embodiment, the filtering devices provide two layers of filtration to segregate particulates from the ESM matter into separate stages. Although two baskets are described, it should be apparent that more baskets may be included to provide as many layers of filtering as desired.