# US 9,040,301 B2 · Separator device, deposition device and system for handling of somatic plant embryos > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,040,301 B2 - Document ID: US9040301B2 - Status: Granted - Kind: B2 - Application number: 13/500,886 - Filed: 2010-10-08 - Published: 2012-08-09 - Granted: 2015-05-26 - Earliest claimed: 2009-10-09 - Assignees: Georgia Tech Research Corporation - Applicants: Cyrus K. Aidun - Inventors: Cyrus K. Aidun - Web page: https://patentgrep.com/patent/US9040301B2 ## CPC - A01H 4/001: {Culture apparatus for tissue culture} - C12N 5/00: Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00) - A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract Methods and devices for separating fluid-suspended plant somatic embryos and embryogenic tissue based on differences in their fluid drag properties are disclosed. Deposition method and device for depositing plant somatic embryos into embryo receiver comprising growth substrate by means of a fluid jet is disclosed. An automated system for processing plant somatic embryos from the bioreactor to the growth substrate is also disclosed. ## Claims ### Claim 1 A method of separating fluid-suspended embryos from immature embryogenic tissue comprising the steps of: a) providing a suitable separator container, said container containing fluid having a density lower than of the embryos to be separated, being essentially cylindrical in shape, having an essentially flat bottom wall and an essentially vertical axis, and comprising a conduit in communication with the fluid in the container at an axial region of the bottom wall during operation, and providing a rotation object positioned inside the container for inducing an axisymmetric rotating flow; b) creating a sink vortex at the axial region of the bottom wall by draining fluid from said conduit, and inducing an axisymmetric rotating flow in the fluid relative to the bottom wall by rotating the rotation object, thus: i) creating a viscous boundary layer at the bottom wall; and ii) creating a radial pressure gradient in the separator container; c) introducing the fluid-suspended embryos and immature embryogenic tissue to be separated into the fluid present in the separator container at a location away from the bottom wall, thus: i) sedimenting the embryos faster than the immature embryogenic tissue; ii) allowing the embryos to enter the viscous boundary layer while not allowing the immature embryogenic tissue to enter the viscous boundary layer; iii) drawing the embryos entering the viscous boundary layer into the axial region of the bottom wall; and d) collecting embryos from said axial region of the bottom wall in the fluid drained from the conduit, whereby the embryos collected are essentially separated from immature embryogenic tissue. ### Claim 2 (depends on 1) The method according to claim 1, further comprising a) providing a suitable separator container further comprising a conduit in communication with the fluid in the container at the axial region of the bottom wall during operation, wherein the conduit is placed and dimensioned such that embryos drawn into the axial region of the bottom wall during operation enter into the conduit by gravitational settlement; and b) collecting embryos from said conduit. ### Claim 3 (depends on 2) The method according to claim 2, further comprising the step of modulating the sedimentation velocity of the embryos in the conduit by means of inducing fluid flow through the conduit into the separator container. ### Claim 4 (depends on 1) The method according to claim 1, wherein the method is adapted for batchwise operation and further comprises selectively collecting the embryos during a time period after the sedimentation of the embryos has occurred but before the immature embryogenic tissue has had time to sediment. ### Claim 5 (depends on 1) The method according to claim 1, wherein the method additionally comprises the step of replacing the fluid in the separator container after processing a batch of embryos with fresh fluid. ### Claim 6 (depends on 1) The method according to claim 1, wherein the method is adapted for continuous operation such that it comprises feeding fluid into the separator container at a rate exceeding the rate of fluid flow from the conduit. ### Claim 7 (depends on 2) The method according to claim 2, wherein the method is adapted for continuous operation such that it comprises feeding fluid into the separator container at a rate exceeding the rate of fluid flow from the conduit. ### Claim 8 A device for separating fluid-suspended embryos and immature embryogenic tissue from each other comprising: a) separator container, which during operation contains fluid having a density lower than the density of the embryos to be separated, said container being essentially cylindrical in shape, having an essentially flat bottom wall, an essentially vertical axis and comprising a fluid conduit in communication with the inside of the container, located at the axial region of the bottom wall; b) rotation object positioned inside the container for inducing an axisymmetric rotating flow in the fluid relative to the bottom wall, whereby during operation: i) a viscous boundary layer is created at the bottom wall; ii) a radial pressure gradient is created in the separator container; c) means of introducing the fluid-suspended embryos and immature embryogenic tissue to be separated into the fluid present in the separator container at a location away from the bottom wall, whereby during operation: i) the embryos sediment substantially faster than the immature embryogenic tissue; ii) the embryos enter the viscous boundary layer while the immature embryogenic tissue remains substantially outside the viscous boundary layer; iii) the embryos entering the viscous boundary layer are drawn into the axial region of the bottom wall and into the conduit; and d) means of collecting embryos from said conduit; whereby the embryos collected are essentially separated from immature embryogenic tissue. ### Claim 9 (depends on 8) The device according to claim 8, wherein i) the conduit is placed and dimensioned such that embryos drawn into the axial region of the bottom wall during operation enter into the conduit by gravitational sedimentation. ### Claim 10 (depends on 8) The device according to claim 8, wherein the means collecting embryos comprise means of collecting the embryos from the conduit without substantially altering the volume of fluid in the container. ### Claim 11 (depends on 10) The device according to claim 10, wherein the means of removing the embryos from the conduit without altering the volume of fluid in the container comprise a valve or a set of valves. ### Claim 12 (depends on 8) The device according to claim 8, wherein i) the device comprises means of draining fluid from said conduit, whereby during operation a sink vortex is created at the axial region of the bottom wall; and ii) the means of collecting embryos comprise means of collecting the embryos from the sink vortex in the fluid drained from the conduit. ### Claim 13 (depends on 8) The device according to claim 8, wherein the device is adapted for batchwise operation and further comprises means of collecting the embryos selectively during a time period after the sedimentation of the embryos has occurred but before the immature embryogenic tissue has had time to sediment. ### Claim 14 (depends on 8) The device according to claim 8, wherein the device is adapted for continuous operation and further comprises a separator container comprising a second outlet at the top of the separator container and means of feeding fluid into the separator container at a rate exceeding the rate of fluid flow from the conduit. ### Claim 15 (depends on 14) The device according to claim 14, wherein the second outlet is implemented by means of a separator container which is open at the top. ### Claim 16 (depends on 14) The device according to claim 14, wherein the means of collecting embryos from the sink vortex comprise means of collecting the fluid exiting the conduit. ### Claim 17 (depends on 8) The device according to claim 8, wherein the device additionally comprises means of replacing the fluid in the separator container. ### Claim 18 (depends on 8) The device according to claim 8, wherein the device comprises means of draining fluid from the axial region of bottom wall during operation, comprising a conduit extended from above or any other direction to the proximity of the axial region of the bottom wall. ### Claim 19 (depends on 8) The device according to claim 8, wherein the separator container has a diameter in the range of 3-30 cm. ### Claim 20 (depends on 8) The device according to claim 8, wherein the fluid conduit has an area of 0.01%-10% of the area of the bottom wall. ### Claim 21 (depends on 8) The device according to claim 8, wherein the means of inducing an axisymmetric rotating flow result in a rotational speed in the range of 5-1200 rpm in the fluid. ### Claim 22 (depends on 8) The device according to claim 8, wherein the means of inducing an axisymmetric rotating flow comprise a rotating disk- or cylinder-shaped object. ### Claim 23 (depends on 8) The device according to claim 8, wherein the means of introducing embryos and tissue is located at an axial location near the surface of the fluid. ### Claim 24 (depends on 8) The device according to claim 8, wherein the fluid height during operation is 0.8-1.2 times the diameter of the separator container. ### Claim 25 (depends on 13) A system for processing plant embryos suspended in a fluid, comprising a separator device according to claim 13, and one or more components selected from the group consisting of: a) disperser unit to disperse the embryos and the embryogenic tissue suspended in a fluid, located upstream of the separator device, and optionally a bioreactor, as embryo source, located upstream of the disperser unit; b) orientation and sorting unit for orienting and sorting the embryos suspended in a fluid, located downstream of the separator device; and c) a deposition device, located downstream of the orientation and sorting unit, if present. ### Claim 26 (depends on 25) The system according to claim 25, the system comprising the deposition device, wherein the deposition device is for depositing fluid-suspended plant embryos while maintaining the orientation of the embryo and the deposition device comprises: i) a flow channel dimensioned such that the embryos may travel with fluid flowing through the channel but are restricted to travelling either in a crown-first or crown-last orientation by dimensional constraints, said flow channel emanating to an outlet; ii) an embryo receiver; and iii) means of forming a free jet of fluid emanating from the outlet, wherein the free jet during operation is aligned with an embryo receiver thus allowing embryos suspended in the fluid to be deposited in the embryo receiver; wherein the means of forming a free jet comprise a flow channel section immediately upstream of the outlet having a straight section with length at least equal to the largest cross-sectional inside dimension of the flow channel. ### Claim 27 (depends on 26) The system according to claim 26, wherein the length of the straight section of the deposition device is at least 10 times the largest cross-sectional inside dimension of the flow channel. ### Claim 28 (depends on 26) The system according to claim 26, wherein a tip of the outlet of the deposition device is configured to be positioned during operation at one to three flow channel diameters away from the embryo receiver. ### Claim 29 (depends on 26) The system according to claim 26, wherein the embryo receiver of the deposition device has an opening for depositing the embryo having a smallest dimension of at least 10% larger than the largest cross-sectional diameter of the embryo to be deposited. ### Claim 30 (depends on 26) The system according to claim 26, wherein the deposition device further comprises means for stabilising the jet delivering the embryos comprising means of encapsulating the jet delivering the embryos in another fluid jet with substantially larger diameter.