NZ622105B2 - Automated system and methods for separating and singulating plant embryos - Google Patents

Abstract

automated system and methods for separating and grading plant embryos is disclosed. The process of separating and grading plant embryos is expedited by use of this system. A series of stations are disclosed for the separation (200), grading (300, 70, 74), drying (400) and storage (500) of plant embryos grown in embryogenic suspensor mass. The embryos can be moved between each station in any order by a programmable robotic arm (114, 120). Its use in silviculture is specifically disclosed. mbryos grown in embryogenic suspensor mass. The embryos can be moved between each station in any order by a programmable robotic arm (114, 120). Its use in silviculture is specifically disclosed.

Description

WO 2013/101536 PCT/US2012/070267 AUTOMATED SYSTEM AND METHODS FOR SEPARATING AND SINGULATING PLANT EMBRYOS CROSS-REFERENCE TO RELATED APPLICATION 5 This application is entitled to and claims the benefit of priority from US.

Nonprovisional Patent ation Ser. No. ,478 filed December 29, 2011, and titled “AUTOMATED SYSTEM AND METHODS FOR SEPARATING AND SINGULATING PLANT EMBRYOS,” the contents of which are orated herein by reference. 10 BACKGROUND Modern ulture often requires the planting of large numbers of genetically identical plants that have been selected to have advantageous properties. Production of new plants by sexual reproduction, which yields botanic seeds, is usually not feasible.

Asexual propagation, via the culturing of somatic or zygotic embryos, has been shown for 15 some species to yield large s of genetically identical embryos, each having the capacity to develop into a normal plant. c cloning is the process of creating genetically cal plants from plant tissue other than male and female gametes. In one approach to somatic cloning, plant tissue is cultured in an initiation medium that includes es, such as auXins and/or 20 cytokinins, to te formation of embryogenic tissue, such as embryogenic suspensor masses, that are capable of developing into somatic embryos. The embryogenic tissue is then further cultured in a multiplication medium that promotes multiplication and mass production of the embryogenic tissue. The embryogenic tissue is then cultured in a development medium that promotes pment and maturation of cotyledonary somatic 25 embryos that may, for example, be placed on germination medium to produce 2 germinants, and subsequently transferred to soil for further growth, or alternatively, placed within manufactured seeds and sown in soil where they ate to yield ngs.

Manufactured seeds are described, for example, in U.S. Patent Nos. 5,564,224; 5,687,504; 5,701,699; and 6,119,395. 5 The somatic embryogenesis process typically is laborious and inefficient. For example, a labor intensive step in the embryogenesis process is the selective harvesting from development medium of individual embryos suitable for germination. s have been made to te the harvesting of cotyledonary embryos. At the end of the development phase, the s may be present in a number of stages of maturity 10 and development, and are typically attached to or imbedded in embryogenic suspensor mass.

Separation and singulation are processing steps that occur at the end of development and maturation in which plant embryos are physically separated from each other and the underlying embryogenic suspensor mass (ESM) before further processing such as, for example, insertion into manufactured seed, or placement onto germination or pre-germination 15 medium for further treatment prior to insertion into manufactured seed.

The present invention is directed to an automated system and methods for separating and singulating plant embryos in a sterile environment and on a commercial scale.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present ion. It 20 is not suggested or represented that any or all of these matters formed part of the prior art base or were common l knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

SUMMARY 25 Throughout the description and claims of this ication, the word “comprise” and ions of the word, such as “comprising” and “comprises”, is not intended to exclude other additives, ents, integers or steps.

This summary is provided to introduce a ion of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to 30 identify key features of the claimed subject , nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In one aspect, the present invention is directed to an automated system for separating and ating plant embryos comprising: a tion module constructed and arranged to 3 separate a plurality of plant embryos from attached embryogenic suspensor mass, and sort the plant embryos according to size; a ation module constructed and arranged to singulate the ted and sorted plant embryos into dual, discrete embryos, and to deposit the singulated embryos onto a porous substrate; a drying module constructed and ed to dry 5 the porous substrate upon which the singulated plant embryos are disposed; and a robotic arm, said robotic arm operable to transport the plant embryos from module to module in a predetermined sequence, wherein the robotic arm is programmable such that the robotic arm is not limited to move from module to module in the predetermined ce, and n the robotic arm may move among the modules in response to one or more signals, and perform 10 functions associated with ting and singulating plant embryos in addition to transporting the plant embryos from module to module, y optimizing the transport of plant embryos through the system and maximizing the use of each module to separate and ate plant s. The automated system for separating and singulating plant embryos may further comprise a storage module for g the plant embryos before the plant embryos are 15 transported to the separation module. The automated system for separating and ating plant embryos may further comprise a docking module for receiving plant embryos from the drying module, and for storing the plant embryos in containers that provide an environment suitable for further maturation of the plant embryos.

In one aspect, the present invention is directed to automated methods of separating and 20 ating plant embryos, comprising the steps of: (a) transferring a plurality of plant embryos to a separation module constructed and arranged to separate a ity of plant embryos from attached embryogenic suspensor mass, and sort the plant embryos according to size; (b) transferring the separated and singulated plant embryos from step (a) to a 25 singulation module constructed and arranged to singulate the plant embryos into individual, discrete embryos, and to deposit the singulated embryos onto a porous ate; (c) transferring the singulated embryos from step (b) to a drying module constructed and arranged to dry the porous substrate upon which the singulated plant embryos are ed; and 30 (d) wherein the plant embryos are transported from module to module by a robotic arm, said robotic arm operable to transport the plant embryos from module to module in a predetermined sequence, wherein the robotic arm is programmable such that the robotic arm is not limited to move from module to module in the predetermined sequence, and wherein the 3a robotic arm may move among the modules in response to one or more signals, and perform functions ated with separating and ating plant s, in addition to transporting the plant embryos from module to module, thereby zing the transport of plant embryos through the system and maximizing the use of each module to separate and singulate plant 5 embryos.

. DESCRIPTION OF THE DRAWINGS The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the 10 following detailed description, when taken in conjunction with the accompanying drawings, wherein: FIGURE 1 is a flow diagram illustrating the process of separating and singulating plant embryos utilizing a representative embodiment of the system of the invention.

FIGURE 2 rates a perspective view of a storage module in ance with one 15 embodiment of the system of the invention.

FIGURE 3 illustrates a perspective view of a vision system in accordance with one embodiment of the system of the invention.

FIGURE 4 illustrates a perspective view of a separation module in accordance with one embodiment of the system of the invention. 20 FIGURE 5 illustrates a ctive view of a spray hood and spray apparatus in accordance with one embodiment of a separation module of the system of the invention.

WO 2013/101536 PCT/US2012/070267 FIGURE 5A illustrates a view axial to the rotation of the spray apparatus shown in FIGURE 5 indicating the direction of rotation of the spray apparatus and spray emanating from the nozzles.

FIGURE 6 illustrates a perspective view of a singulation module in accordance with one ment of the system of the invention.

FIGURE 7 illustrates a perspective view of an embryo dispensing assembly in accordance with one ment of a singulation module of the system of the invention.

FIGURE 8 rates a perspective view of an embryo deposit assembly in accordance with one embodiment of the ation module of the system of the 10 invention.

FIGURE 9A illustrates a perspective view of a drying module in accordance with one embodiment of the system of the invention.

FIGURE 9B illustrates a perspective view of a drying module in ance with one embodiment of the system of the invention. 15 FIGURE 10 illustrates a perspective view of a docking module in accordance with one embodiment of the system of the invention.

FIGURE ll illustrates a top planar view in accordance with one embodiment of the system of the invention.

FIGURE 12 rates a perspective view of a robotic arm in accordance with one 20 embodiment of the system of the invention.

DETAILED DESCRIPTION As used herein, the term "embryogenic suspensor mass" (ESM) refers to early stage embryos in the process of multiplication by budding and cleavage.

As used herein, the term ogenic tissue" refers to an aggregate of tens to 25 hundreds of genic cells that form an embryogenic suspensor mass.

As used herein, the term "plant embryo" refers to a somatic plant embryo.

Somatic plant embryos may be produced by culturing embryogenic tissue by standard methods under laboratory conditions in which the cells comprising the tissue are separated from one another and urged to develop into minute complete embryos. As used 30 herein, "plant " includes embryos at s stages of development.

As used herein, the term "cotyledonary embryo" refers to an embryo that possesses one or more cotyledons. Cotyledonary embryos have a well defined elongated WO 2013/101536 PCT/US2012/070267 bipolar structure with latent meristem with cotyledonary primordia at one end and a ial radicle at the opposite end. The cotyledonary structure frequently appears as a small "crown" at one end of the .

As used herein, the term "module" refers to a processing area or station.

As used herein, the terms "separate" "separation" refers to the process of separating cotyledonary embryos from attached embryogenic suspensor mass and sorting the embryos according to size.

As used herein, the terms "singulate" or lation" refers to the process of sing embryos on a substrate as individual, discrete embryos. 10 As used herein, the term "SAS" refers to the separation and singulation processes.

The somatic embryogenesis process is a process to develop plant embryos in vitro. Methods for producing plant somatic embryos are known in the art and have been previously bed (see, e.g., US. Patent Nos. 4,957,866; 5,034,326; 5,036,007; 5,041,382; 5,236,841; 549; 5,482,857; 5,563,061; and 5,821,126). Generally, the 15 somatic genesis process includes the steps of: (l) initiation or induction, to initiate formation of embryogenic , such as embryogenic suspensor mass (ESM), which is a white mucilaginous mass that es early stage embryos having a long, thin—walled suspensor associated with a small head with dense cytoplasm and large nuclei; (2) multiplication, sometimes referred to as maintenance, to multiply and mass produce 20 embryogenic tissue; (3) development, to develop and form mature cotyledonary somatic embryos; and (4) post development steps such as separation, singulation, stratification, germination, growing into plants, such as through placement into manufactured seeds.

The somatic embryogenesis process is labor intensive. Efforts have been made to automate and scale—up the s to facilitate the production of tens of thousands of 25 plant embryos. For example, the multiplication step may be d out in a commercial—scale liquid bioreactor. At the end of the multiplication step, embryogenic tissue may be transferred to development medium for a period of time to develop into cotyledonary s. At the end of the pment period, the cotyledonary embryos are to various degrees attached to and embedded in suspensor s and residual 30 underdeveloped ESM, together with incompletely developed embryos, ally formed embryos, undersized or oversized embryos, and other pieces of non—embryo plant material, and to other embryos. It is important for subsequent normal germination to WO 2013/101536 2012/070267 separate the embryos from the suspensor mass and from other embryos and to ate the embryos into individual, te s.

Automating the separation and singulation steps is important for commercial scale—up of the embryogenesis process, as well as for productivity and worker well—being.

In one aspect, according to the present ion, an automated system for separating and singulating cotyledonary plant embryos is provided. A flow diagram of the process of separating and singulating plant embryos utilizing a entative embodiment of the system of the invention is illustrated in FIGURE 1. Referring to FIGURE 1, one embodiment of the separation and singulation system of the invention 10 (referred to herein as the "SAS system") comprises five major stations or modules: (i) staging 100; (ii) separation 200; (iii) singulation 300; (iv) drying 400; and (v) docking 500. The embryos are transferred from module to module by use of a c arm. The modules and robotic arm are contained in a sterile enclosure. In one embodiment, the sterile enclosure is a HEPA—filtered laminar flow chamber. 15 A first module of the SAS system is referred to herein as the "staging module." The staging module 100 is used to store embryos developed en masse and attached to ESM until processing begins. In one embodiment, the developed s are stored on a porous membrane mounted in a frame, referred to herein as a development frame or "d— frame." 20 A entative embodiment of the staging module 100 is shown in FIGURE 2.

Referring to FIGURE 2, the staging module 100 includes an insulated compartment 10 having a door 11 and one or more shelves 12 for storing one or more d—frames 20 containing disposed embryos. The d—frames 20 with disposed embryos may be transferred to the staging module from an area in which the embryos were developed 25 and/or matured into cotyledonary embryos. The staging module 100 may store d—frames with or without development medium. The d—frames with disposed embryos may be stored in the ted compartment 10 in ners 14 with lids 15. An elevator and slide system (not shown) may be used to transport a container 14 holding a d—frame 20 from the insulated compartment 10 to a presentation area 150 (Shown in figure 3) where 30 the lid 15 is removed from the container 14 and the robotic arm picks up a d—frame 20 and moves it to the separation module 200.

WO 2013/101536 PCT/US2012/070267 As shown in FIGURE 3, in one embodiment, an overhead vision mechanism 16, having a camera 18 mounted in an arm 19, is used to locate the d—frame 20 inside the container 14 to assist the robotic arm in picking up the d—frame 20. Alternatively, the container 14 may be designed such that the d—frame 20 is precisely located within the container 14 and the robotic arm can pick up the d—frame 20 without the aid of a vision mechanism.

A second module of the SAS system is referred to herein as the "separation module." At the end of the development period, the cotyledonary embryos are to various degrees attached to and embedded in sor tissues and residual underdeveloped 10 ESM, together with incompletely developed embryos, abnormally formed embryos, undersized or oversized embryos, and other pieces of non—embryo plant material, and to other embryos. It is important for uent normal ation to separate the embryos from the suspensor mass and from other embryos to yield individual embryos.

The separation module 200 is used to separate developed embryos from the underlying 15 suspensor mass and from each other, and to sort the embryos according to size.

A representative embodiment of the separation module 200 is shown in FIGURES 4 and 5. Referring to FIGURE 4, the separation module 200 is shown as ing a circular separation spray hood 30, one or more sieves 40 and 42, a separation vessel 44, and a lift mechanism 48. The separation vessel 44 is used to collect liquid and 20 waste, such as embryogenic suspensor mass removed from the embryos, and s of undesired size or shape, resulting from the separation process, as further described below.

The separation vessel 44 may be connected to an outlet 46 for removing the waste and liquid from the separation vessel 44. In one ment, the separation vessel 44 is disposed on top of the lift mechanism 48, and the one or more sieves 40 and 42 are in 25 t with, and stacked in a series on top of, the tion vessel 44. Referring to FIGURE 5, a spray tus 32 is mounted to the underside of the separation spray hood 30. A spray apparatus suitable for use in the automated system of the invention is described in US. Application No. incorporated herein by reference.

At the separation module 200, the robotic arm (not shown) inverts a d—frame 20, 30 transferred from the g module 100, places it on the top sieve 40, and thereafter the spray apparatus 32 sprays liquid on the inverted d—frame 20 to remove the ed embryos and attached ESM from the d—frame 20.

WO 2013/101536 PCT/US2012/070267 More than one sieve may be used to separate the embryos from the ESM and to sort the embryos according to size. The mesh opening sizes of the sieve(s) 40 and 42 are selected so as to capture the desired sized embryos. The sieves 40 and 42 may be arranged in a stack such that a first sieve 40 with a first mesh opening size is placed on top of a second sieve 42 with a second mesh opening size that is smaller than the first mesh opening size. By way of e, the first sieve 40 may be of a mesh opening size such that s of the desired size, undersized embryos, and the embryonal suspensor mass pass through the first sieve 40, and embryos that are larger than the desired size are captured on the surface of the first sieve 40. The second sieve 42 may be of a mesh 10 opening size such that embryos of the desired size are captured on the second sieve 42, and undersized embryos and the embryonal suspensor mass pass through the second sieve 42 into the separation vessel 44 and may be discarded through the outlet 46.

During the separation process, in one embodiment, the first sieve 40 is removed from the stack of sieves by the robotic arm and the oversized embryos are ded. The 15 second sieve 42 may be further sprayed by the spray apparatus 32 to facilitate the removal of undersized s and residual ESM. The second sieve 42, containing embryos of the desired size, is subsequently transferred by the robotic arm to the singulation module 300.

In some embodiments, more than two sieves may be used, each sieve having a 20 ent mesh opening size from the mesh g size of each of the other sieves to further sort the s according to size/shape. For example, three sieves may be used, or even four sieves may be used. The mesh opening sizes may vary in the range from about 500 microns to about 2400 microns. For example, mesh opening sizes of 500, 850, 1000, 1180, 1400, 1700, 2000, and 2400 microns may be used. In one embodiment, the 25 first sieve may have a mesh opening size of about 2400 microns. In one embodiment, the second sieve may have a mesh opening size of about 1400 microns.

By adjusting the mesh g size/shape of the one or more sieves 40 and 42, only those embryos within a desirable size/shape range are selected, resulting in a population sing mostly embryos separated from each other and substantially free 30 of suspensor tissues.

Again referring to FIGURE 5, a spray apparatus 32 is mounted to the underside of the separation spray hood 30. In one ment, the separation module utilizes a spray WO 2013/101536 PCT/US2012/070267 apparatus 32 comprising a plurality of spray nozzles that are configured to discharge spray ns designed to push the plant embryos through the porous substrate and also move the s across the surface of the porous substrate. In one embodiment, the spray nozzles are selected from the group consisting of nozzles that discharge a cone shaped—spray pattern, a fan—shaped spray pattern, an oval—shaped spray pattern, and combinations thereof. In one embodiment, a ation of spray nozzles 34 and 36 are d on the rotatable arm 38 of the spray apparatus 32, wherein a first spray nozzle 34 discharges liquid in the form of a cone—shaped spray pattern; and a second spray nozzle 36 discharges liquid in the form of a fan—shaped spray pattern. In one 10 embodiment, only nozzles 34 that discharge liquid in a cone—shaped pattern may be used.

In one embodiment, only nozzles 36 that rge liquid in a fan—shaped pattern may be used. Of course, other spray nozzles may be used, for example s that discharge liquid in an oval—shaped spray pattern.

The spray nozzles 34 and 36 are shown as ed on the spray apparatus arm 38 15 such that the spray nozzles 34 that discharge a cone—shaped spray pattern are alternated with the spray nozzles 36 that discharge a fan—shaped spray pattern.

The spray nozzles 34 and 36 perform different functions during the separation process. Spray nozzles 34, which discharge liquid in the form of a cone—shaped spray pattern, produce a gentle spray and cover a wide area. Spray nozzles 36, which discharge 20 liquid in the form of a fan—shaped spray pattern, are particularly effective in removing the ESM from the embryos.

The spray tus is powered to rotate around a rotational aXis 39. The spray nozzles are either configured or oned along the spray arm 38 to cooperatively provide ntially uniform spray coverage during rotation of the spray arm 38. The 25 spray nozzles 34 and 36 are canted relative to the onal aXis 39 of the spray apparatus. The spray nozzles 34 and 36 may be canted relative to the rotational aXis 39 of the spray apparatus at an angle in the range from about 220 to about 25", shown in FIGURE 5 as "or".

As shown in FIGURE 5A, the spray apparatus 32 rotates in a direction opposite to 30 the direction that the spray nozzles 34 and 36 are canted such that the spray nozzles provide both a downward and tangential force on the plant embryos disposed on a sieve.

The downward force pushes the embryos h the openings of the sieves 40 and 42.

WO 2013/101536 PCT/US2012/070267 10 The tangential force moves the ESM and the embryos across the sieves 40 and 42. As the mesh of the sieve is uneven, the tangential force causes the plant embryos to wiggle on the sieve, and when they ly orient in a vertical position, they present their slender profile to the mesh opening, thus allowing them to pass through the sieve if they are of the proper diameter. Without random wiggles, many of the embryos remain flat, and thus only expose their long aXis to the mesh openings, and are less likely to pass through.

As shown in FIGURE 5, the separation spray hood 30 surrounds the spray apparatus 32. During operation of the separation module 200, the lift mechanism 48 raises the separation vessel 44/sieves 40 and 42 assembly to engage the separation spray 10 hood 30. The lift mechanism 48 can be of s constructions, ing using linear actuators to effectuate the lifting of the sieves 40 and 42.

The spray hood 30 is of a shape and size such that it engages around the separation vessel 54 to form a seal, thus creating a closed spray system. The closed system contains the aerosols generated from the liquid spray emanating from the spray 15 nozzles 34 and 36 of the spray apparatus 32, thereby ng the spread of any contamination that may be present in the spray aerosols.

The separation spray hood 30 may further include a vent (not shown) h which air displaced by the spray emanating from the spray nozzles 34 and 36 is directed outside of the closed system. 20 After processing at the separation module 200, the robotic arm may move the sieve 42 containing embryos of the desired size to a singulation module 300, which is the third module of the SAS system. The ation module is shown in FIGURES 6—8. The singulation module 300 is used to deposit individual, te embryos onto a substrate in such a manner that the embryos are not touching each other or any object. Singulating 25 the embryos into dual embryos allows the embryos to be subsequently imaged and robotically picked up for insertion into a manufactured seed.

During operation of the singulation module 300, embryos are deposited as individual discrete embryos onto a substrate. The substrate upon which singulated embryos are deposited may be a porous ate d in a frame. A framed porous 30 substrate suitable for use in the present invention is referred to herein as a ation frame or "s—frame" 72. The porous substrate allows fluid to pass through while retaining embryos. The porous substrate may be of a color different than the color of the embryos WO 2013/101536 2012/070267 11 so as to provide a contrast between the porous substrate and the embryo. One such porous substrate suitable for use in the singulation module 300 is NiteX® nylon, model No. 03—125/45 black color.

Describing the singulation module in more detail as shown in FIGURE 6, in one embodiment, the singulation module 300 includes an embryo dispensing assembly 50, a programmable logic controller (PLC) 64, a singulation mechanism 70, which is used to deposit individual embryos on an e 72, a sensor 66, an embryo dispensing tubing 58, and an embryo deposit assembly 74. Also, an s—frame 72 is shown in FIGURE 6 disposed on the embryo deposit assembly 74. 10 Referring to FIGURE 7, the embryo dispensing ly 50 includes a singulation spray hood 52, to which is mounted a spray apparatus (not shown), a singulation vessel 54, a stir plate 56, a mass balance 60, a lift mechanism 62, and a sensor 66 (as shown in FIGURE 6). An embryo dispensing tubing 58 extends from the singulation vessel 54. The singulation vessel 54 is in communication with the stir plate 15 56, which is disposed on the mass balance 60, which in turn is disposed on the lift mechanism 62, which is used to linearly raise or lower the singulation vessel 54. In one embodiment, the lift mechanism 62 may be an elevator, During operation, embryos are received at the embryo dispensing assembly 50 disposed on a sieve 42. The sieve 42 is inverted over, and placed on top of, the 20 ation vessel 54. Liquid emanating from the spray apparatus mounted to the ation spray hood 52 is sprayed onto the inverted sieve 42 to ge the embryos from the inverted sieve 42 and into the ation vessel 54. The spray apparatus is also used to supply the singulation vessel 54 with a suitable fluid, e. g. sterile water.

The lift mechanism 62 raises the ation vessel/sieve ly to engage the 25 singulation spray hood 52. The spray hood 52 is of a shape and size such that it engages around the sieve 42 to form a seal, thus creating a closed spray system. The closed system contains the aerosols generated from the liquid spray emanating from the spray tus, thereby reducing the spread of any contamination that may be present in the spray aerosols. 30 The singulation vessel 54 is in communication with a stir plate 56 to stir the fluid in the singulation vessel 54 to a sufficient degree to maintain the embryos in suspension in the fluid. The stir plate 56 is in communication with the PLC 64 (shown in figure 6) to WO 01536 PCT/US2012/070267 12 automatically adjust the amount of stirring that occurs. The PLC 64 is programmed so that the stirring speed linearly, or at another rate, decreases as the fluid level in the singulation vessel 54 decreases as embryos are dispensed from the singulation vessel 54 through the embryo dispensing tubing 5 8.

The embryo dispensing tube 58 extends between the singulation vessel 54 and the singulation mechanism 70. s are orted from the singulation vessel 54 to the singulation mechanism 70 by fluid flowing through the embryo sing tubing 58.

The flow rate of embryos through the tubing 58 is controlled by the lift mechanism 62, as r described below. 10 The fluid volume in the singulation vessel 54 is determined using a mass balance 60. The mass balance 60 is used to measure the mass of the fluid inside the singulation vessel 54. Mass is used to ly measure the flow rate of fluid exiting the singulation vessel 54, which is directly related to the time rate of mass decrease of the fluid in the singulation vessel 54; i.e., fluid exits the embryo dispensing tubing 58 at the 15 outlet 59 of the singulation mechanism 70 at the same rate that mass decreases in the singulation vessel 54. Alternatively, mass may be used as an indirect measurement of "total head." As used herein, the term "total head" refers to the total hydrostatic pressure at the outlet 59 of the embryo dispensing tubing 58 (as shown in FIGURES 6 and 8) from the ation mechanism 70, and it ines the flow rate of the fluid out of the 20 singulation vessel 54.

The flow rate determines the velocity of the fluid in the embryo dispensing tubing 5 8. The velocity of the fluid in turn fixes the time of flight of the embryos through the embryo dispensing tubing 58, and thus controls when an embryo will exit the embryo dispensing tubing 58 at the outlet 59 of the singulation mechanism 70, and be ted 25 on the s—frame 72. The flight time is used to synchronize the motion of the singulation mechanism 70 with the exiting of the embryos from the ation mechanism 70.

The flow rate of embryos flowing through the embryo dispensing tubing 58 is controlled by the lift mechanism 62. The lift mechanism 62 is used to raise or lower the singulation vessel 54, as required, to maintain the mass of fluid exiting the singulation 30 vessel 54 per unit time fixed. Alternatively, when mass is used as an indirect measurement of total head, the lift mechanism 62 is used to raise or lower the singulation vessel 54, as required, to maintain a constant total head; i.e. a constant height between the WO 2013/101536 PCT/US2012/070267 13 outlet 59 of the embryo dispensing tubing 58 at the singulation mechanism 70 and the fluid level inside the singulation vessel 54.

The PLC 64 is in ication with the lift mechanism 62 to l raising and ng of the lift mechanism 62. The lift mechanism 62 raises the singulation vessel 54 at a fixed rate proportional to, or otherwise related to, the flow rate of fluid exiting the singulation vessel 54 to maintain a substantially constant flow rate. The singulation vessel 54 may be raised or lowered to increase or decrease, respectively, the flow rate.

In one embodiment, the embryo dispensing tubing 58 es an inner diameter of a size to permit only a few embryos, for example one to three embryos, to enter the 10 tubing 58 at any given time. Although only a few embryos enter the tubing 58 at a given time, multiple embryos may be positioned longitudinally within the tubing 58 over time.

Depending on the size of the opening, the embryos may be positioned within the tubing 58 side by side, end to end, or a ation of positions. The number of embryos entering the tubing at any given time is generally controlled by creating sed 15 turbulence at the outlet from the singulation vessel 54. This can be accomplished, for example, by placing a stir bar inside the singulation vessel 54 near the outlet from the singulation vessel 54, and by placing the stir bar in a well at the bottom of the singulation vessel 54 near the outlet. Additionally, controlling the density of embryos per unit volume of fluid in the singulation vessel 54 may assist in controlling the number of 20 embryos entering the tubing at any given time. A suitable density for controlling the number of embryos entering the tubing at any given time may be, for example, around one embryo per 5 ml of fluid. zing the length of the embryo dispensing tubing 58 between the singulation vessel 54 and outlet 59 of the singulation mechanism 70, as well as 25 minimizing obstructions in the flow path, are helpful to maintain a consistent and free flow of embryos h the embryo dispensing tubing 58.

In one embodiment, the embryo sing tubing 58 is composed of a material, such as silicone, that is transparent or ransparent to permit visual detection of an embryo within the tubing 58 by the sensor 66. 30 As shown in FIGURE 6, a sensor 66 is suitably positioned on top of the singulation mechanism 70 just in front of the rotational axis 71 to sense and/or detect embryos within the tubing 58, and to detect when an embryo exits the singulation WO 2013/101536 PCT/US2012/070267 14 vessel 54. The sensor 66 is in communication with the PLC 64. The PLC 64 may be programmed to track the path of embryos within the tubing 58 and control the spacing and placement of embryos on the embryo deposit assembly 74.

The sensor 66 may be a based visual . One such sensor 66 is model No. LV—H300/100 Series, ctured and sold by Keyence Corporation of Osaka, Japan.

The singulation ism 70 is used to deposit individual embryos on an s— frame 72. Again referring to FIGURE 6, the singulation mechanism 70 is shown as being in the form of a robotic arm. The singulation mechanism 70 includes two drives to 10 provide precise placement of embryos on an s—frame 72. A first drive causes movement of the singulation mechanism 70 in a first axis, for example, linearly back and forth in a direction along its length. A second drive causes movement of the singulation mechanism 70 in as second axis, for example rotating clockwise and counterclockwise.

In one embodiment, the singulation mechanism rotates about vertical axis 71 over a very 15 small angle, for example +/— ll degrees, depending on the size of the s—frame 72.

The axis of rotation 71 of the singulation mechanism 70 is located distal to the outlet 59 of the embryo dispensing tubing 58 such that the outlet of the embryo dispensing tubing 58 moves in an arc about axis 71.

In one ment, the ation ism 70 is mounted on a rotary drive, 20 which in turn is mounted on a linear drive. A suitable rotary drive is sold by Techno, Inc. of New Hyde Park, New York, part number HL36SOM26030. A suitable linear drive is sold by Parker Hannifin Corp. of Cleveland, Ohio, part number ER032BLTRA000DO0FSRN0l50A.

The combination of movement of the singulation mechanism in the first and 25 second axes results in the t of the plant embryos onto the porous substrate in a two— dimensional array.

The singulation ism 70 is in communication with the PLC 64. The PLC 64 is programmable so as to permit an operator to adjust operational ters.

Operational parameters, such as the number of embryos placed on an s—frame 72, the 30 spacing between the embryos, and the location of embryos on the s—frame 72 may all be programmed as desired.

WO 2013/101536 PCT/US2012/070267 15 The singulation mechanism 70 deposits embryos on an s—frame disposed on the embryo deposit assembly 74. Referring to FIGURE 8, the embryo deposit assembly 74 includes a first singulation frame holding area 76 and a second singulation frame holding area 78, located side—by—side the first holding area 76, a retention mechanism 80, and a rotating mechanism 82. These ents are mounted on an underlying frame structure 86.

The first singulation frame holding area 76 further comprises a vacuum source (not shown) that is in communication with an aperture 84 located in the center of holding area 76. During operation, the vacuum source is in communication with the s— 10 frame 72 to rapidly remove fluid from the s—frame 72 and to aid in holding the deposited embryos in a fixed location on the s—frame 72.

The second singulation frame holding area 78 is used to hold an empty s—frame 72 awaiting transfer to the first singulation frame g area 76, or alternatively, to hold an s—frame 72 with singulated embryos awaiting transfer to the drying module 400. The 15 ng mechanism 82 is used to support and to rotate s—frame(s) 72 about a central aXis 88 between the first singulation frame g area 76 and the second singulation frame holding area 78.

The ion mechanism 80 is pivotably attached to the first singulation frame holding area 76 via pivot rod 79, and is used to hold an s—frame 72 in place during 20 singulation. To this end, the retention mechanism includes a pair of arms 81 that can be rotated about rod 79 to overlie the side margins of an s—frame 72. Pins 83 extend laterally from the distal end of arms 81 to bear against the side margins of the s—frame 72 at a location about midway along the length of the e 72 side margins.

During er of s—frame(s) between the first singulation frame holding area 76 25 and the second singulation frame holding area 78, the retaining mechanism 80 pivots upward about rod 79 to relieve the s—frame 72, and the ng mechanism 82 is raised and rotated 180 s about aXis 88, thereby moving an empty s—frame 72 into the first singulation frame holding area 76, and moving an s—frame 72 with singulated embryos into the second singulation frame holding area 78. The rotating ism 82 is then 30 d back into position, and the retaining mechanism 80 is ed to locking position over an empty s—frame 72.

WO 2013/101536 PCT/US2012/070267 16 During ion of the singulation module 300, an empty s—frame 72 is transferred from the second singulation frame holding area 78 to the first ation holding area 76 by the rotating mechanism 82 and is positioned within the first singulation frame holding area 76 to receive singulated embryos from the singulation mechanism 70.

The SAS system of the invention may further include a wetting n (not shown) in which an empty s—frame 72 is immersed in water before being transferred to the second singulation frame holding area 78. Wetting the s—frame 72 is beneficial in aiding removal of fluid by the vacuum source 84 from the e 72 during the 10 singulation process.

The SAS system of the invention may include more than one singulation module 300, for example two singulation s 300, to accommodate multiple sieves transferred from the tion module 200, thereby increasing efficiency and productivity. 15 After embryos are deposited on an s—frame 72 at the embryo deposit assembly 74, the s—frame 72 with disposed embryos may be transferred by the robotic arm to the fourth module of the SAS system, referred to herein as the "drying module." The drying module 400 is used to remove excess water from the substrate upon which the embryos are deposited at the singulation module 300, e.g., an s—frame 72. A y of methods 20 may be used to remove the water from the s—frames 72.

In one embodiment, the drying module 400 includes a mechanism to remove excess water by blowing dry air over and/or through the es.

One embodiment of the drying module 400 includes a drying assembly 90, shown in Figure 9A. The drying assembly 90 includes a porous substrate 92. The porous 25 substrate 92 may be, for example, fritted glass or a porous metal sheet. The porous substrate 92 is ed in a frame structure 91, which supports the porous substrate 92 over a vacuum source (not . During operation, an s—frame 72, with disposed embryos, is placed on the porous substrate 92. The porous substrate is brought into communication with a vacuum source through a pipe 93 and a vacuum is applied to 30 remove water from the s—frame 72. The vacuum may be applied for a period of time for additional drying as the vacuum source moves air through the s—frame 72.

WO 2013/101536 PCT/US2012/070267 17 One ment of the drying module 400 includes a drying mechanism 94, shown in FIGURE 9B. The drying mechanism 94 includes a platform 95 divided into two sections; a mechanical slide arm 96, driven by a motor (not shown); and a housing 97, which is located between the two sections of the platform 95, having a narrow ted opening 98. The elongated opening 98 is in communication with a vacuum source (not shown).

During ion, an s—frame 72 with disposed embryos is placed onto a section of the platform 95. The mechanical slide arm 96 pushes the s—frame 72 across the opening 98 of the housing 97. As the s—frame 72 moves across the opening 98, the 10 bottom surface of the s—frame 72 is in contact with the opening 98, which is in communication with the vacuum , resulting in liquid being removed from the s— frame 72 and air being drawn over and around the embryos disposed on the top surface of the s—frame 72 and through the s—frame 72.

The SAS system of the invention may include more than one drying module 400, 15 for example two drying modules 400, to accommodate multiple s—frames 72 transferred from the singulation module 300, thereby increasing efficiency and productivity.

After drying at the drying module 400, the s—frame 72 with disposed embryos may be transferred by the robotic arm to the fifth module of the SAS system, referred to herein as the "docking module" 500. As shown in FIGURE 10, in one embodiment, the docking 20 module 500 includes one or more containers 110, each having a door 112 and one or more shelves 113 for storing the s—frames 72 with disposed singulated embryos. In one embodiment the containers provide an environment suitable for further maturation of the plant embryos, for example conditioning over water. The docking module may be ured such that it can communicate with ent used in processing the embryos 25 after singulation and , for e inserting the embryos into manufactured seed or placing the embryos onto germination medium.

As described above, the SAS system of the invention ses a number of modules or processing stations for ting and singulating embryos. During operation of the SAS , embryos moves sequentially through the process from the staging 30 module 100, to the separation module 200, to the singulation module 300, to the drying module 400, and finally to the docking module 500.

WO 2013/101536 PCT/US2012/070267 18 The embryos, disposed on porous substrates (e.g. d—frames 20, sieves 42, or s— frames 72) are transferred from module to module through the system by use of a programmable robotic arm 114 shown in FIGURE 11. In one embodiment, the robotic arm 114 is in the form of a 6—axis articulating arm, as shown in FIGURE 12, in communication with an external motion control device (not shown). Referring to FIGURE 12, the robotic arm includes a base 116, a lower arm 117, an upper arm 118, and a wrist 119. The first axis, located at the base 116, allows the robot to rotate from left to right up to 180 degrees from the center point. The second axis allows the lower arm 117 to extend d and backward. The third axis allows the upper arm 118 to raise and 10 lower. The fourth axis s the upper arm 118 in a circular motion. The fifth axis allows the wrist 119 to tilt up and down. The sixth axis allows the wrist 119 to rotate in a circular motion. A le robotic arm and control device are sold by Denso Robotics of Long Beach, California, Model VS—6577G and RC7M, respectively.

The robotic arm 114 performs all al handling tasks n each processing 15 station or module. For example, the robotic arm 114 may perform tasks such as, but not limited to, (i) picking—up a d—frame 20 from the storage module 100, transferring it to and from the separation module 200, and inverting it over the stack of sieves 40 and 42; (ii) picking—up a sieve 42 with disposed embryos and transferring it to the singulation module 300; (iii) picking—up an empty s—frame 72, erring it to the wetting station, 20 and then transferring it to the second singulation frame holding area 78; (iv) transferring an e 72 with disposed embryos to the drying module 400; and (v) transferring an s— frame with disposed embryos from the drying module 400 to the docking module 500.

The robotic arm 114 performs other tasks, including, but not limited to, sieve stacking & parking as well as set—up and tear—down of components of the singulation 25 module 300. The robotic arm 114 is not limited to performing tasks in a predetermined sequence. The robotic arm 114 performs task in variable sequences, as needed, thereby increasing efficiency and productivity. For e, the c arm can transfer a d— frame 20 to the separation module 200 and invert it over the sieve 40; pick up an s—frame 72 from the drying module 400 and transfer it to the docking module 500; then return to 30 the tion module 200 to pick—up a sieve 42 and transfer it to the singulation module 300; etc.

WO 01536 PCT/US2012/070267 19 Referring to FIGURE 11, the robotic arm 114 is movably attached to a programmable linear slide structure 120 to provide the range of motion necessary to cover the large area encompassed by all the process modules and stations of the SAS system. In one embodiment, the slide 120 provides about 2.5 meters of travel along the length of the SAS system and is positioned by a seventh axis auxiliary control that is embedded into the robotic arm 114 external l device. The amount of travel may vary depending on the size of the SAS system. The linear slide 120 motion is fully integrated into the robotic arm 114 motion to precisely control the positioning of the robotic arm 114. The c arm 114 movements, as it performs the task associated with 10 each module, and the motion of the robotic arm 114 along the liner slide 120, are controlled to minimize the robotic arm 114 passing over exposed embryos, thereby zing the probability of biological ination erring from the c arm 114 and its tools, described below, to the embryos.

In some embodiments, more than one robotic arm may be used. When multiple 15 robotic arms are used, each robotic arm may be designated to perform one or more specific tasks. In some embodiments, the robotic arm(s) may be mounted to the ceiling.

In order to facilitate handling of d—frames 20, s—frames 72, and sieves 40 and 42 by the robotic arm 114, three specific end—of—arm tools are utilized. The tools are automatically exchanged by the robotic arm 114 as needed. The robotic arm 114 further 20 comprises an automatic quick—change coupling device 115, shown in FIGURE 11, to enable tool changes by the robotic arm 114. Each tool has a ic parking holder and location within the SAS system. The robotic arm 114 moves to the appropriate location to ge tools, as needed. Each coupling device 115 r comprises multiple electrical circuits in communication with the robotic arm 114 controller to identify each 25 tool with a unique t signal. After a tool change, the robotic arm 114 controller verifies that the robotic arm has picked up the intended tool.

The SAS system of the invention significantly ses productivity and rate of processing of embryos. In operation, the SAS system of the invention has achieved a production rate of about 100 singulated embryos deposited per s—frame, and about 35 s— 30 frames processed per hour, which may result in about 3500 singulated embryos ed per hour, and about 28,000 singulated embryos produced in an eight hour period. By including more than one singulation module 300 and more than one drying module 400 in WO 2013/101536 PCT/US2012/070267 20 the SAS system, s can be processed through singulation and drying continuously, thereby greatly increasing the tion rate.

Additionally, the components of the modules of the SAS system are designed to allow for rapid change—out for sterilization, and to change components, as needed, to s multiple clonal lines and avoid cross contamination between embryonic cell lines.

In one aspect, the present ion es automated methods of separating and singulating plant embryos. The methods of the invention e the steps of: (a) depositing a plurality of plant embryos attached to embryogenic suspensor mass onto a first porous substrate; (b) spraying the plant embryos and embryogenic suspensor mass 10 with a liquid discharged from a spray apparatus sing a plurality of spray nozzles, wherein the spray nozzles are configured to discharge spray patterns designed to push the plant embryos through the porous substrate and also move the embryos across the surface of the porous substrate; and (c) transferring the plant embryos from step (b) to a singulation mechanism and depositing the embryos on a second porous substrate as 15 individual, discrete embryos to produce singulated embryos.

In one embodiment, the first porous substrate is located on top of a stack of one or more porous substrates, each having a pore opening size. The porous ates may be arranged in the stack according to pore opening size, in sing order, such that the substrate having the largest pore opening size is on top of the stack, and the substrate 20 having the smallest pore opening size is on the bottom of the stack, thereby sorting the plant embryos according to size.

In one embodiment, the pore opening size of the porous substrates ranges from about 500 microns to about 2400 microns. Substrates of pore opening sizes of, for example, 500, 850, 1000, 1180, 1400, 1700, 2000, and 2400 microns may be used. In one 25 embodiment, the porous substrate on the top of the stack may have a pore opening size of about 2400 s, and the porous substrate on the bottom of the stack may have a pore size g of about 1400 microns.

In one embodiment, the automated methods for separating and singulation plant embryos comprises the steps of: (a) erring a plurality of plant embryos to a 30 separation module ucted and arranged to separate a plurality of plant embryos from attached embryogenic suspensor mass, and sort the plant embryos according to size; (b) transferring the separated and singulated plant embryos from step (a) to a singulation WO 2013/101536 PCT/US2012/070267 21 module constructed and arranged to ate the plant embryos into individual, discrete s, and to deposit the singulated embryos onto a porous substrate; (c) transferring the singulated embryos from step (b) to a drying module constructed and arranged to dry the porous substrate upon which the singulated plant embryos are disposed; and (d) wherein the plant embryos are transported from module to module by a robotic arm, said c arm operable to transport the plant embryos from module to module in a predetermined ce, wherein the robotic arm is programmable such that the robotic arm is not limited to move from module to module in the predetermined sequence, and wherein the robotic arm may move among the modules in response to one or more 10 signals, and perform functions associated with separating and singulating plant embryos, in addition to transporting the plant embryos from module to module, thereby optimizing the transport of plant embryos h the system and maximizing the use of each module to separate and singulate plant embryos.

The automated system and methods of the invention can be used to separate and 15 singulate plant embryos from any plant species, such as dicotyledonous or monocotyledonous plants, gymnosperms, etc. Conifer embryos are suitable for use in the SAS system of the ion and may be from any conifer species including, but not d to, species within the genera Pinus, Picea, Tsuga, tsuga, Thuja, Juniperis, LariX, and Sequoia. 20 While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made n without departing from the spirit and scope of the invention.

Claims (21)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An automated system for separating and singulating plant embryos comprising: a separation module constructed and arranged to separate a plurality of plant embryos from attached embryogenic suspensor mass, and sort the plant embryos according to size; 5 a singulation module constructed and ed to singulate the separated and sorted plant embryos into individual, te embryos, and to deposit the singulated embryos onto a porous ate; a drying module constructed and arranged to dry the porous substrate upon which the singulated plant embryos are disposed; and 10 a robotic arm, said robotic arm operable to transport the plant embryos from module to module in a predetermined sequence, wherein the robotic arm is programmable such that the robotic arm is not limited to move from module to module in the predetermined sequence, and wherein the robotic arm may move among the modules in response to one or more signals, and perform functions ated with separating and singulating plant s, in addition to 15 transporting the plant embryos from module to , thereby optimizing the transport of plant embryos through the system and maximizing the use of each module to te and singulate plant embryos.

2. The automated system for separating and singulating plant embryos of Claim 1, further comprising a staging module for g the plant embryos before the plant embryos 20 are transported to the separation .

3. The automated system for separating and singulating plant embryos of Claim 1 or Claim 2, wherein the separation module further comprises: one or more sieves for receiving plant embryos and ed embryogenic suspensor mass, for separating the plant embryos from the embryogenic suspensor mass, and for sorting 25 the plant embryos according to one or more desired sizes; a liquid spray source for forcing the embryogenic suspensor mass and plant embryos of undesired size through the sieves; a vessel for collecting liquid from the spray source, embryogenic suspensor mass, and plant embryos of undesired size. 23

4. The automated system for separating and singulating plant embryos of Claim 3, wherein the liquid spray source comprises a spray apparatus, said spray apparatus comprising a plurality of spray nozzles, wherein the spray nozzles are configured to discharge spray patterns designed to push the plant embryos through the porous substrate and also move the 5 embryos across the e of the porous substrate, and wherein the spray nozzles are selected from the group consisting of nozzles that discharge a cone-shaped spray pattern, a fan-shaped spray pattern, an haped pattern, and combinations thereof.

5. The automated system for separating and ating plant embryos of any one of Claims 1 to 4, wherein the singulation module further comprises: 10 an embryo dispensing assembly for dispensing plant embryos in a fluid flow to a singulation mechanism; a singulation mechanism for receiving plant embryos from the embryo dispensing assembly, and for ting the plant embryos onto a porous substrate in discrete embryos; an embryo deposit assembly for holding the porous substrate in place to receive plant 15 embryos from the singulation mechanism; and a ller programmable to control the flow of plant embryos from the embryo dispensing assembly to the singulation mechanism, and to control the deposit of plant embryos by the singulation mechanism onto the porous ate.

6. The automated system for separating and singulating plant s of Claim 5, 20 wherein the embryo dispensing assembly further comprises: a vessel for g plant embryos ded in a fluid; a mass balance for measuring the volume of the fluid in the vessel; a lift mechanism for raising and lowering the vessel to in a desired flow rate of fluid exiting the vessel; and 25 a controller programmable to control the raising and ng of the vessel by the lift mechanism, depending on the volume of the fluid in the vessel.

7. The automated system for separating and singulating plant embryos of Claim 5 or Claim 6, wherein the embryo deposit assembly r comprises: a first holding area for holding a porous substrate to receive plant embryos ejected 30 from the singulation mechanism; and 24 a second holding area for (a) holding a porous substrate until it can be erred to the first holding area; or (b) holding a porous substrate with disposed embryos, which has been transferred from the first holding area to the second holding area.

8. The automated system for separating and singulating plant embryos of Claim 7, 5 further sing a rotating mechanism for transferring a porous substrate between the first g area and the second holding area.

9. The ted system for separating and singulating plant embryos of Claim 7 or Claim 8, wherein the first holding area further comprises a vacuum source in communication with the porous substrate to remove water from the porous substrate.

10 10. The automated system for ting and singulating plant embryos of any one of Claims 5 to 9, wherein the singulation mechanism further comprises: a first drive that causes movement of the singulation mechanism in a first axis; and a second drive that causes movement of the singulation mechanism in a second 15 axis, wherein movement of the singulation mechanism in the first and second axes results in the deposit of the plant embryos onto the porous substrate in a two-dimensional array.

11. The ted system for separating and singulating plant embryos of any one of Claims 5 to 10, wherein the controller is programmable to control the number of plant 20 embryos deposited on the porous substrate by the singulation mechanism, the spacing n the embryos on the porous substrate, and the location of embryos on the porous substrate.

12. The automated system for separating and singulating plant embryos of any one of Claims 1 to 11, wherein the drying module further comprises: a drying mechanism for removing liquid from the porous substrate upon which the 25 plant embryos are disposed; and a vacuum source to tate the removing of liquid from the porous substrate by the drying mechanism.

13. The automated system for separating and singulating plant embryos of any one of Claims 1 to 12, further sing a docking module for receiving plant embryos from the 25 drying module, and for storing the plant embryos in ners that provide an environment le for further maturation of the plant embryos.

14. The automated system for separating and singulating plant embryos of Claim 13, wherein the g module is configured to icate with ent used in 5 processing the plant embryos after ation and drying.

15. The automated system for separating and singulating plant embryos of any one of Claims 1 to 14, further comprising one or more tools suitable to be coupled to the robotic arm through a coupling device, wherein each tool has a unique electrical signal, and n the ng device further comprises multiple electrical circuits in communication with a 10 controller of the robotic arm to identify the unique electrical signal of each tool.

16. The automated system for separating and singulating plant embryos of Claim 1, further comprising a sterile enclosure for enclosing the modules of the automated system.

17. An automated method for separating and singulating plant s comprising the steps of: 15 (a) transferring a plurality of plant embryos to a separation module constructed and arranged to separate a ity of plant embryos from attached embryogenic suspensor mass, and sort the plant embryos according to size; (b) transferring the separated and singulated plant embryos from step (a) to a singulation module constructed and arranged to singulate the plant embryos into individual, 20 discrete embryos, and to deposit the singulated embryos onto a porous ate; (c) transferring the singulated embryos from step (b) to a drying module ucted and arranged to dry the porous substrate upon which the singulated plant embryos are disposed; and (d) wherein the plant embryos are transported from module to module by a robotic 25 arm, said robotic arm operable to transport the plant embryos from module to module in a predetermined sequence, wherein the robotic arm is programmable such that the robotic arm is not limited to move from module to module in the predetermined sequence, and wherein the robotic arm may move among the modules in response to one or more signals, and perform functions ated with separating and singulating plant embryos, in addition to transporting 30 the plant embryos from module to module, thereby zing the transport of plant embryos 26 through the system and maximizing the use of each module to separate and ate plant embryos.

18. The automated method of claim 17, wherein the robotic arm is in a form of a 6- 5 axis articulating arm.

19. The automated method of claim 18, wherein the robotic arm further comprises an end-of-arm tool, the end-of-arm tool being changeable. 10

20. The automated method of claim 19, wherein the robotic arm further comprises a coupling device for changing the end-of-arm tool.

21. An automated system according to claim 1, substantially as herein described with reference to any one or more of the