BR122017021419B1 - METHODS AND APPARATUS FOR PREPARING AND OBTAINING EMBRYOS SUITABLE FOR TISSUE CULTURE AND/OR GENETIC TRANSFORMATION - Google Patents

Abstract

The present invention relates to methods and apparatus for rapid isolation of monocotyledonous plant embryos suitable for transformation or tissue culture. The invention includes mechanical devices for substantially isolating plant embryos for use as transformable explants. Suitable media for isolating plant embryos and methods for their preparation are also provided.

Description

[001] Split from PI0815980-7, deposited on 08/29/2008.

BACKGROUND OF THE INVENTION

[002] This application claims priority to U.S. Provisional Application Serial No. 60/969,287, filed on August 31, 2007, the complete description of which is incorporated herein by reference.

1. Field of Invention

[003] The present invention generally relates to substantially isolating target plant tissues, such as embryos, which are suitable for genetic transformation or tissue culture.

2. Description of Related Art

[004] The preparation of tissues for plant propagation, regeneration and transformation is exhausting and very laborious, especially when it generally involves the manual excision of transformable or cultivable plant tissues. For example, in maize (Zea mays), individual immature embryos are typically removed manually to provide genetically transformable explants. Manual excision of embryonic tissues is laborious and poses a risk of ergonomic injury to the worker. Furthermore, when large quantities of transformable plant tissue are required for high-yield transformation and plant production, more workers must be employed and trained to meet the increased demand. Additionally, there can be significant variability in the quality of the plant tissues obtained, depending on the level of experience, care, attention and fatigue of individual workers.

[005] Tissue variability and lack of adaptability to automation in previous techniques for isolating transformable plant tissues is problematic, because low tissue quality negatively affects tissue culture, genetic transformation and propagation of the subsequent plants. Nevertheless, to produce even a single transgenic plant suitable for commercial development and use in agriculture, it may be necessary to produce tens of thousands of individual transformation events in a single species. Therefore, there is a great need in the art for improved methods of preparing target plant tissues that are more efficient, reduce the overall ergonomic burden on workers, reduce the amount of labor required to process the plant materials, and/or that provide plant fabrics that are of higher quality and more consistent than handcrafted fabrics.

SUMMARY OF THE INVENTION

[006] In one aspect, the invention provides a method for obtaining embryos suitable for tissue culture and/or genetic transformation, comprising at least partially removing an embryo from a plant seed in a liquid medium consisting essentially of water and/or an osmotic agent with an osmolality of about 0 mOsm/kg to about 600 mOsm/kg, wherein the embryo remains viable for tissue culture and/or genetic transformation after excision of the embryo from the seed of the plant. The osmotic agent may further be an inert osmotic agent. In a further embodiment, the method comprises removing, at least partially, a plurality of embryos from a population of plant seeds or ears in the liquid medium. The osmotic agent may be selected from the group consisting of mannitol, sorbitol, glucose or other osmotic agents. In particular embodiments, the medium consists essentially of water and mannitol at a concentration of between about 0.05 M to about 0.5 M, or sucrose at a concentration of between about 0.05 M to about 0.5 M. The method may further comprise the step(s) of genetically transforming the embryo and regenerating the transgenic plant from the transformed embryo.

[007] In certain embodiments of the method, the step of genetically transforming the embryo comprises the use of a coculture medium comprising a bactericide. In particular embodiments, the bactericide is carbenicillin. In other embodiments, the coculture medium comprises about 0.5 to 1.5 mg/l of 2,4-D.

[008] The embryo may be a member of the Poaceae, such as corn, rice, wheat or millet. In particular embodiments, the embryo is a corn embryo or a millet embryo. In other embodiments, the embryo may be a soybean embryo, a cotton embryo, or another dicot embryo.

[009] In another embodiment, the method comprises preparing a liquid medium in a media preparation system comprising: (a) a water inlet; (b) an inlet for the osmotic agent; and (c) a chamber for mixing the water and the osmotic agent to produce the liquid medium, wherein the water inlet and the osmotic agent inlet are coupled to the mixing chamber to allow release of the water and the osmotic agent into the chamber for mixing. In certain embodiments, the water inlet and/or osmotic agent inlet are coupled to a chamber or chambers for containing the water and/or the osmotic agent. The method may further comprise coupling the mixing chamber to a liquid jet apparatus. In other embodiments, the method further comprises sterilizing the liquid medium with a sterilizer selected from the group consisting of a filter, a UV or gamma radiation source, and a sterilizing heat source. The sterilizer can sterilize the water and/or osmotic agent before entering the mixing chamber. Alternatively, the sterilizer sterilizes the liquid medium concurrently with and/or after entering the mixing chamber. In another embodiment, the method comprises sterilizing the liquid medium after the liquid medium leaves the mixing chamber.

[0010] The method may further comprise measuring the complete level of one or more chambers for containing the water, the chamber for containing the osmotic agent or the chamber for mixing the water and the osmotic agent. In additional embodiments, the method comprises releasing the water and osmotic agent into the water and osmotic agent mixing chamber. In particular embodiments, the method comprises controlling the release by electronically detecting the release of water and/or the osmotic agent.

[0011] In another aspect, the method for preparing a plant embryo suitable for tissue culture and/or genetic transformation comprises: (a) placing a plant seed tissue comprising plant embryos and/or integuments of seed in an aqueous environment; (b) contacting the tissue with an agent that selectively couples to embryos or seed coats; and (c) isolating at least one first embryo based on selective coupling of the agent to the embryo or seed coat. In certain embodiments, the agent comprises gas bubbles. In particular embodiments, the gas bubbles have an average dimension wider than about 100 μm to about 1 mm. In certain embodiments, the gas bubbles comprise a gas selected from the group consisting of air, oxygen, nitrogen, and a combination thereof. Furthermore, in certain embodiments, step (c) comprises isolating the first embryo based on the buoyancy of the embryo.

[0012] The method may also comprise including in the aqueous environment a surfactant that reduces the coalescence of bubbles with each other. In certain embodiments, the surfactant is selected from the group consisting of a polyether, PPG (poly(propylene glycol)) and PEG (poly(ethylene glycol)). In particular embodiments, PPG has a molecular weight of about 340 to about 3500 Daltons and PEG has a molecular weight of about 100 Daltons to about 9000 Daltons.

[0013] In other embodiments, the agent comprises a second liquid that is immiscible with the aqueous environment. The second liquid may be selected from the group consisting of vegetable oil, such as canola oil, mineral oil or other hydrophobic liquid compatible with the survival and transformation of the embryos.

[0014] In certain embodiments, the plant seed tissue comprises embryos produced, at least partially, by excision of an embryo from a plant seed into a liquid medium (aqueous environment) consisting essentially of water and/or a osmotic agent with an osmolality of about 0 mOsm/kg to about 600 mOsm/kg, wherein the embryo remains viable for tissue culture and/or genetic transformation after excision of the embryo from the plant seed. In particular embodiments, the aqueous environment essentially consists of a medium comprising water and/or an osmotic agent having an osmolality of about 7 mOsm/kg to about 500 mOsm/kg. The method may comprise placing the plant seed tissue in an aqueous environment without first separating the embryo from the non-embryonic tissue. In a particular embodiment, the plant seed tissue is a corn plant seed tissue. In other embodiments, the plant seed tissue is soybean plant seed tissue or cotton plant seed tissue.

[0015] In another aspect, the invention provides an apparatus for preparing plant embryo tissue suitable for tissue culture and/or genetic transformation, comprising: (a) a container for containing plant seed tissue comprising a plurality of plant embryos and non-embryonic tissue, such as plant seed coats, in an aqueous environment; and (b) at least one first injector for releasing into the aqueous environment an agent that selectively couples to embryos or seed coats, wherein the injector produces gas bubbles having an average diameter of between about 0.1 mm to about 1mm. In certain embodiments, the invention provides an apparatus, wherein the container is filled with the medium and plant tissue comprising the embryo and non-embryonic tissue. In particular embodiments, the apparatus further comprises a collector for separating embryonic tissue based on the buoyancy of the embryos within the aqueous environment. In still other embodiments, the gas bubbles may comprise a gas selected from the group consisting of air, oxygen, nitrogen, and a combination thereof.

[0016] In another aspect, the invention provides a method for preparing plant embryos suitable for tissue culture and/or genetic transformation comprising: (a) directing a first jet of liquid medium onto a corn kernel or other tissue comprising a plant embryo to extract the endosperm from the grain or tissue; and (b) directing a second jet of liquid medium onto the grain or tissue to extract an embryo from the grain or tissue. In certain embodiments, the liquid medium consists essentially of water or an osmotic agent having an osmolality of about 7 mOsm/kg to about 500 mOsm/kg. In a particular embodiment, the grain may be comprised in an ear of corn. In certain embodiments, the method may further comprise moving the corn cob relative to the first and second jets to remove the endosperm and kernel embryo in succession. In certain embodiments, the first and/or second jet comprises a width smaller than that of the corn kernel. In particular embodiments, the first and/or second jet comprises a width of about 0.0762 mm (0.003") and a height of about 25.4 mm (1") and the first and/or second jet are produced in a pressure between about 206.8 kPa (30 psi) to about 517.1 kPa (75 psi).

[0017] In certain embodiments, the first and/or second jet may be directed from an injector that produces a stable laminar liquid flow at a distance of at least 63.5 mm (2.5") from the injector. In In other embodiments, the grain may be positioned approximately 44.45 - 50.8 mm (1% - 2") from the tip of the injector. In certain embodiments, the first and/or second jets contact each grain in a row of grains found on the cob with substantially the same force.

[0018] In another aspect, the invention provides an apparatus for obtaining embryos from corn or other plants suitable for tissue culture and/or genetic transformation comprising (a) at least a first jet of liquid for directing the medium onto a grain or other corn tissue comprising a plant embryo; and (b) an apparatus for containing the grain or other fabric in the middle path. The corn kernel can be contained on an ear of corn. In certain embodiments, the apparatus for containing the grain or other fabric comprises a blade or a cylindrical blade. In other embodiments, the apparatus for containing the grain or other fabric comprises a screen or a plurality of slits; or a cam or pressure screw that applies force to the tissue to be restrained. The seed or fruit tissue may be contained on the grain or other tissue containment apparatus by mechanical force, friction, centrifugal force or suction force. In particular embodiments, the support may comprise a pressure cam, thread or screw. In certain embodiments, the apparatus for containing the grain or other tissue is suspended in a gas phase, a liquid phase, or is partially suspended in both gas and liquid phases. In other embodiments, the apparatus for containing the grain or other fabric is fixed relative to a fluid force or is movable relative to a fluid force.

[0019] Another aspect of the invention comprises a method for preparing plant embryos suitable for tissue culture and/or genetic transformation, wherein the apparatus for containing the grain or other tissue is centrifuged in a container to apply force to the grain or other fabric.

[0020] The apparatus can further be defined as comprising a first and second liquid jets. Additionally, in certain embodiments the device for containing the grain comprises means for moving the corn cob relative to the first and second liquid jets to control the contact angle between the first and second liquid jets and the grain. In particular embodiments, the apparatus further comprises a detector for identifying endosperm tissue and removed embryos.

[0021] In certain embodiments, the apparatus further comprises at least one first separator for isolating embryos from non-embryonic tissue. In particular embodiments, the separator separates embryos suitable for tissue culture from non-embryonic tissue by a method selected from the group consisting of size exclusion, differential density, and differential hydrophobicity. The apparatus may be further defined as comprising a sieve for separating embryo from non-embryonic tissue based on size. In a particular embodiment, the grain containment apparatus comprises at least a first motor for moving the corn cob relative to the liquid jet or vice versa.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 describes an embodiment of an apparatus provided by the invention that uses positive mechanical pressure to substantially isolate embryos, as described in Example 4.

[0023] Figure 2 describes an embodiment of a device provided by the invention that uses positive pressure in the liquid jet to dislodge embryos from seeds as described in Example 7. Caption: (A) robot with movement in dimensions X, Y and Z , (B) motor to rotate the corn cob, (C) fastener, (D) handle to hold the corn cob, (E) baffle to prevent material from splashing upward, (F) flange to prevent material from splash upward, (G) opening to direct liquid, (H) transparent tube, (I) corn cob, (J) stirring screen, (K) cotton gauze or other porous material, and (L) waste container .

[0024] Figure 3 depicts an embodiment of an assembly mechanism that uses a magnetic "handle" by which the ear of corn can be attached to a robotic arm, as described in Example 7.

[0025] Figure 4 describes an embodiment of an injector useful in certain aspects of the invention, as described in Example 7. This injector generates a substantially uniform jet of liquid, like a flat sheet.

[0026] Figure 5 describes an embodiment of an apparatus useful in the methods of the invention, as described in Example 13. This apparatus includes an injector for generating a substantially laminar liquid jet and an optional suction head. Figure 5 (above) represents a cross-section of one embodiment of such an apparatus, showing how the injector, optional suction head, and corn cob can be positioned relative to one another. Figure 5 (below) schematically represents an ear of corn positioned in the device. Legend (A) base, (B) injector, (C) shaft coupled to the base of the cob, (D) suction head, (E) corn cob and (F) opening for liquid flow guidance.

[0027] Figure 6 represents an embodiment of a component useful for applying negative pressure useful in the methods of the invention, as described in Example 13. Legend: (A) one or more openings for guiding the liquid flow.

[0028] Figures 7A-C represent different aspects of an embodiment of an apparatus that uses a combination of forces and is useful in certain aspects of the invention, as described in detail in Example 13. This apparatus includes a pump with a leading edge capable of applying a pre-defined amount of mechanical pressure to the base of grains that previously had the pericarp open or truncated and a component to apply liquid with negative pressure. Such apparatus may further include a means for dispersing liquid or directing the flow of liquid.

[0029] Figure 8 represents an embodiment of the invention, in which the top of a cylinder is completely, partially or substantially covered with a membrane or sheet of flexible material that is discretely smaller than the diameter of the corn cob and the handle at the which the corn cob is attached to. Specifically, an anti-splash silicone rubber membrane (0.79375 mm (1/32") thickness) from McMaster-Carr (McMaster-Carr, Atlanta, GA; e.g., cat. no. 9010K11) is shown. 25.4 mm (1") diameter was made into the membrane. The corncob was able to move downward through the opening and also back up through it.

[0030] Figure 9 represents a 1 liter polymethylpentene graduated cylinder with the bottom cut out. Three jets of liquid are passed into holes cut in the cylinder wall. The complete device can be autoclaved before use. For operation, the top of the cylinder may be fitted with an anti-splash baffle and the corn cob lowered into the cylinder such that the liquid jets dislodge the contents of each kernel. The dislodged material then falls to the bottom of the cylinder for further processing.

[0031] Figure 10 represents an ear of corn showing the location of the embryo within an individual grain. Also shown is an embodiment of the invention in which a jet of liquid is directed to the basipetal side of the grain, as opposed to the acropetal side where the embryo is located.

[0032] Figure 11 illustrates an embodiment of a Media Preparation System provided by the invention.

[0033] Figure 12 illustrates a general view of the mixing chamber of a media preparation system provided by the invention.

[0034] Figure 13 illustrates a close-up view of the mixing chamber.

[0035] Figure 14 represents a zoom of the mixing chamber, with a positioning device and a support plate.

[0036] Figure 15 represents a zoom of the lower portion of the Media Preparation System in operating position.

[0037] Figure 16 represents a zoom of the upper portion of the Media Preparation System in operating position.

[0038] Figure 17 represents a cross-sectional view of an apparatus for separating maize embryos into phases.

[0039] Figure 18 illustrates a linden wood device for dispersing spiral-shaped bubbles.

[0040] Figure 19 illustrates embryos floating on top of the foam produced by a linden wood device for dispersing spiral-shaped bubbles.

[0041] Figure 20 illustrates the separation of embryos from endosperms using a flotation process.

[0042] Figure 21 depicts a vacuum filter device used to collect foam embryos on the surface of an air-liquid interface of an embryo separator.

[0043] Figure 22 comprises a schematic diagram of an embryo separation device as described in Example 19.

[0044] Figure 23 represents an alternative device for separating embryos by floating as described in Example 20.

[0045] Figure 24 illustrates a top view of an embryo extractor in a combination device for extracting and separating corn embryo tissue from culture tissue.

[0046] Figure 25 represents a bottom view of an extractor for rotating and extracting embryos from multiple ears.

[0047] Figure 26 illustrates a side view of an embryo separator.

[0048] Figure 27 illustrates an exemplary liquid level regulator.

[0049] Figure 28 illustrates a metal screen and slot fasteners for seed and/or fruit tissue.

[0050] Figure 29 represents a leaf and a cylindrical embodiment of a seed or fruit holder.

[0051] Figure 30 illustrates an embodiment of a seed or fruit fastener comprising a cam or pressure screw.

[0052] Figure 31 illustrates an explant separator comprising a linden wood bubble dispersion device used for separating cotton embryonic tissue.

[0053] Figure 32 represents cotton explants floating on the surface of the foam produced in the microbubble dispersion device shown in Figure 31.

[0054] Figure 33 represents a comparison of the purity of cotton explants produced either through excision and sieving (left) or excision, sieving and then further purification using micro-bubble technology (right).

DETAILED DESCRIPTION OF THE INVENTION

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs, when employed in the context of the present application. Where there are inconsistencies between the text of the order and the material incorporated by reference, the definitions and meanings provided in this order are proposed. The nomenclature used herein and the manufacturing or laboratory procedures described below are well known and commonly employed by those skilled in the art.

[0056] The phrases "substantially isolated" or "extracted" refer to the processing of a target tissue (e.g., an embryo or other tissue explant) that resides in or forms part of a major tissue complex (e.g., a seed), such that the target tissue is physically separated from at least half of the main complex. In some embodiments, a substantially isolated target tissue can be physically separated from at least about 20%, 30%, 40%, 50%, 60%, 70%, 85%, 90%, 95%, 97%, 98 % or 99% of the main complex or any fraction thereof. In other embodiments, the target tissue is physically separated from more than about 80% to about 100%, about 90% to about 100%, or about 99% to about 100% from the main complex or any intermediate fraction. In some embodiments, the target tissue can be physically separated from about 100% of the main complex.

[0057] Although a substantially isolated target tissue is physically separated from some percentage of the main complex, it does not necessarily need to be purified from that complex. In other words, the substantially isolated target tissue can remain in a batch with the main tissue complex, as long as the target tissue is physically separated from the complex (as described above). In some embodiments, however, it may be desirable to remove some or all of the separated complex from the substantially isolated target tissue. All such embodiments are within the scope of the present invention.

[0058] The phrase "target plant tissue" refers to a portion of a plant or seed tissue that is sought to substantially isolate. In the present invention, target plant tissue refers to any portions of a plant or plant seed that can be substantially isolated and used for genetic transformation or tissue culture. In some embodiments, the target plant tissue is an embryo, such as an immature embryo of a monocot such as corn. In other embodiments, the target plant tissue is from a dicotyledonous plant such as soybean (Glycine sp. including Glycine max) or cotton (Gossypium sp. including G. hirsutum).

[0059] The phrase "suitable for genetic transformation" and "suitable for tissue culture" refers to plant tissues that are competent for transformation or growth under conditions suitable for plant culture, respectively. One skilled in the art can quickly determine whether a particular target tissue is suitable for genetic transformation or tissue culture through the use of routine experimentation. For example, a sample from a batch of substantially isolated target tissues can be cultured on suitable plant media (also known to those skilled in the art) to determine whether the tissues are capable of development and regeneration. Similarly, substantially isolated target tissue samples can be subjected to transformation and selected for the presence of a heterologous nucleic acid molecule. Such techniques are routine and can quickly identify which tissues are competent for transformation or tissue culture and which, if any, are not.

SUBSTANTIALLY ISOLATING TARGET PLANT TISSUE

[0060] The present invention provides methods for substantially isolating target plant tissues for genetic transformation and/or tissue culture. In some embodiments, the target plant tissue is an embryo. In one embodiment, the embryos are monocot embryos, such as corn. In some embodiments, the substantially isolated target plant tissue may be isolated in whole or in part. For example, a batch of substantially isolated immature embryos may include intact embryos, partial embryos, or mixtures thereof. Preferably, the intact and/or partial tissues are suitable for genetic transformation, tissue propagation, plant regeneration or other tissue culture applications.

[0061] As tissues are isolated using, for example, flows of a selected medium, a collection receptacle must be provided. In some embodiments, it is useful to provide a cover for such collection receptacles in order to improve the efficiency of the apparatus, reduce clutter, prevent undesirable spatter from the jet stream, and/or limit leakage of extracted tissue during collection. Any suitable receptacle or receptacle cover may be employed. Examples are given elsewhere in this application and are also known to those skilled in the art.

[0062] Suitable covers for the receptacle may include those made of metals, wood, glass, screens, fabrics, plastics, rubbers, latex, acrylics and functionally equivalent materials. In some embodiments, the material is flexible in a manner that allows penetration and removal of an ear of corn, while maintaining a substantially watertight seal during the extraction process. The material may be provided with a suitable opening to permit entry and removal of an ear of corn. In some embodiments, the material is solid and contains a flexible hole to receive and contain the cob during extraction. In other embodiments, the material is flexible. Such flexible materials can be stretched over the receptacle to form a liquid-tight fitting, yet allowing insertion of a cob both by penetrating the material and providing an opening to receive the cob. In still other embodiments, the material is a net or screen that has a flexible opening.

[0063] The covers can be removable or semi-permanently attached. In some embodiments, the materials are secured by an elastic band or equivalent security means. In other embodiments, the cover is held in place by weights, friction rings, hooks, springs or other functionally equivalent security means.

[0064] The cover can be made of flexible material and have variable thicknesses. These factors can be varied in order to obtain the desired effects for insertion and extraction of corn cobs. The following table (Table 1) illustrates some hardness and density parameters of silicone coatings. The invention, however, is not limited to these few choices.Table 1. Hardness and density parameters of coatings.

[0065] In one embodiment, the collecting receptacle is covered with a membrane or sheet of flexible material that is discretely smaller than the diameter of the corn cob and the handle to which it is attached. In an alternative embodiment, small incisions are made in the membrane to provide greater flexibility. The membrane may be coupled to the cylinder by means such as an elastic band or a fitted friction ring or the like. Figure 8 illustrates a cover embodiment and coupling means described above.

[0066] In some embodiments, the material is autoclavable. Autoclavable materials are well known to those skilled in the art. For example, a flexible material such as a sheet of soft silicone rubber can be used. One skilled in the art is aware of the possible materials and physical arrangements that will permit extraction while providing a collecting vessel that reduces escape of the extracted tissue and prevents undesirable spatter.

[0067] Suitable procedures for culturing and regenerating plant tissue are well known in the art. See, for example, United States Patent Number 5,550,318 to Adams et al., United States Patent Number 5,780,708 to Lundquist et al., United States Patent Application Publication Number 2004/0210958 to Duncan et al. , United States Patent Application Publication Number 2004/0016030 to Lowe et al., and United States Patent Application Publication Number 2004/0244075 to Cai et al., which describe useful processing methods with corn and Application Publication United States Patent Number 2003/0024014 to Cheng et al. which describes useful processing methods with wheat, all of which are incorporated by reference herein in their entirety. Tissue culture applications may include at least one process selected from transformation, callus formation, formation of differentiated plant tissue, formation of at least one mature plant, formation of at least one fertile mature plant, and combinations of these processes. Plants regenerated from immature extracted embryos can be regenerated, for example, through differentiation of dedifferentiated tissue (callus). Regenerated plants can be grown to maturity to provide mature plants, including fertile mature plants. The extracted immature embryos and extracted non-embryonic tissues may also be used for other purposes, such as, but not limited to, genetic or biochemical analysis.

[0068] The methods and apparatus of the present invention can be applied to any monocot plants of interest. Preferred monocots include, but are not limited to, members of the Poaceae family, including grain crops such as corn, wheat, barley, oats, rye, sorghum, millet and rice. Particularly preferred monocots include Zea species including corn (Zea mays) and millet (e.g., Pennisetum glaucum, Pennisetum sp. Setaria sp, Panicum sp.) which have multiple grains (seeds) typically arranged in rows on an ear.

[0069] In general, monocot seeds from which target tissues are substantially isolated are provided in any suitable manner. For example, the seeds may be attached to the ear or handle on which the seeds develop; In some embodiments, the monocot seeds can be removed from the ear or stalk before substantially purifying the target tissue.

[0070] In some embodiments, an opening in the pericarp or seed coat of monocot seeds is provided. This may be achieved by any suitable technique, such as, but not limited to, making a hole, puncture or incision with a needle, awl, blade or other suitable implement. In some applications of the method, no pericarp tissue needs to be removed; In other embodiments, opening the pericarp may include removing at least part of the pericarp and possibly some non-embryonic tissue (e.g., endosperm). Preferably, the opening is sufficient to substantially separate the embryo from the seed. In some embodiments, it may only be necessary to weaken the pericarp sufficiently (e.g., by abrasion or other physical, chemical, or enzymatic treatment) such that the application of force to the seed results in substantial isolation of the target tissue, such as the embryo.

[0071] In such a method, force is generally applied to the seeds sufficient to substantially isolate the target tissue, such as an immature embryo, wherein the substantially isolated target tissue is suitable for genetic transformation and tissue culture. Force can be applied to multiple seeds consecutively or simultaneously. The applied force may be continuous or non-continuous (e.g., pulsatile or wavelike) and is generally applied mechanically, that is, through the use of a device or machine rather than the human hand. The amount of force applied is preferably sufficient to overcome the adhesion of the target (e.g., embryo) and the non-target (e.g., non-embryonic tissue such as endosperm) to each other, thus allowing separation of the target and non-target tissues. . Any force or forces can be employed to remove the target tissue from its seed and multiple forces can be used in combination, sequentially or simultaneously. Suitable forces include, but are not limited to, positive pressure fluid jet, positive pressure liquid jet, mechanical positive pressure, negative pressure, centrifugal force, linear acceleration, linear deceleration, shear fluid, turbulent fluid flow, and laminar fluid flow. . Fluid forces can be exerted by any fluid, for example, gases or liquids or a combination of both.

[0072] Because a corn embryo is located on the acropetal side of a kernel, it is possible to direct a jet of liquid to the basipetal side of the kernel, if desired, to successfully eject the embryo (See, for example, Figure 10 ). In such an arrangement, the full force of the jet generally does not directly impact the embryo. Particularly, the substantial amount of force is applied only indirectly to the embryo itself. Therefore, greater forces can be applied to the device to accelerate embryo removal without substantially increasing the damage to the embryos to be removed.

[0073] Large impact forces can be provided by forcing large quantities of liquid through the apparatus of the present invention. In some embodiments, however, large impact forces can be generated without using more liquid. For example, in some embodiments, the size of the jet opening is reduced such that the same volume of liquid can be used at greater speed. Since the energy of a moving object is proportional to the square of the speed, a jet with the same volume can have much greater energy. A simple equation for the kinetic energy of a moving object is equal to (1/2)(m)(v2). Calculation of the actual energy impact of a liquid jet may take into account other factors known to those skilled in the art. Additionally, some embodiments may use a combination of increased fluid and changes in the size of the jet openings to obtain the desired force or energy.

[0074] Injectors with gpm graduations of about 0.01 to about 0.25, for example, can be used in the present invention, or about 0.01 to about 0.2, or about 0.01 to about 0.1, or about 0.01 to about 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.015 or whatever whole number or fraction between these quantities. In some embodiments, injectors with low gpm ratings may be used, such as 0.033 or 0.021 gpm. When such injectors are used at high pressure but directed toward the opposite side of the grain from the embryo, accelerated embryo collection can be achieved while avoiding damage to the embryo.

[0075] In some embodiments, multiple jets are provided in an apparatus of the present invention. Such a device is useful for equalizing the force exerted by fluid jets while decreasing the time needed to collect embryos from a corn cob.

[0076] In some embodiments, the apparatus may have 2, 3, 4, 6, 8, 10 or more jet openings/injectors as desired to conduct fluid force. In one embodiment there are three openings. Such an apparatus is depicted in Figure 9. In some embodiments, the openings are provided as narrow-angle, horizontally oriented, laminar jet streams. However, other embodiments of the jet openings are provided elsewhere throughout this application. The stainless steel spray nozzle components that connect directly to the polymethylpentene graduated cylinder depicted in Figure 9 are shown in Table 2.Table 2. Spray nozzle components.

[0077] The remaining components are mainly piping and fittings with a quick-connect male adapter at the fluid source on the right of the photo (Figure 9).

[0078] A method and apparatus of the invention can further provide for the separation of substantially isolated target tissue, such as immature embryos, from non-embryonic tissue such as endosperm, glumes and seed coat or pericarp tissues. Separation may be achieved by one or more suitable techniques, including, but not limited to, size exclusion separation (e.g., by filtration in one or more filtration steps), separation based on hydrophobicity, hydrophilicity or other forces, and separation by mass or density differentials (e.g. centrifugal separation, settling and decantation). The separation step or steps may be optional, for example, where no additional isolation of intact or partial embryos is required for their use in tissue culture.

[0079] The invention is particularly suitable for applications where a large number of target tissues must be provided, for example, in high-throughput or selection processes or when processing a batch for genetic transformation or tissue culture. Automation of the method is possible, for example, by using robotics or mechanical handling of corn cobs or seeds, opening the pericarp, applying force to the seed or optional separation steps. Such automation may use optical or mechanical sensors to assist in positioning the corn cobs or seeds in relation to the force or forces applied or in the separation step. In one embodiment, the method provides substantially isolated embryos at a rate of between about 250 to 50,000 or more embryos per day, or between about 500 to about 100,000, or between about 250 to about 50,000, or between about 250 to about 20,000, or between about 250 to about 10,000, or between about 250 to about 5000, or between about 250 to about 3000, or between about 250 to about 1000 embryos per day; or between about 1000 to about 50,000 embryos per day and the like, including any fraction or whole number between any of the ranges mentioned above. As noted above, the present invention overcomes the significant yield limitations of manual excision of embryos.

APPARATUS FOR SUBSTANTIALLY ISOLATING TISSUE FROM TARGET PLANTS

[0080] The present invention also provides an apparatus for substantially isolating target tissues, such as corn embryos, which are suitable for genetic transformation and tissue culture. In one embodiment of separating corn embryos, such an apparatus may comprise at least one opening for directing a flow of fluid, wherein the flow of fluid contacts the kernels of a corn cob and substantially isolates the embryos from the kernels. Generally, it is preferred that the fluid flow contact as many grains in a given period of time as is convenient, in order to isolate the embryos more quickly. The opening may include a single opening or multiple openings, for example, single or multiple injectors, which may include injectors that produce planar, round, oval, fan-shaped or other patterned jets of fluid, and adjustable, movable or stationary, and which can generate a fluid flow of any suitable size and medium. The fluids may be gases, such as air, nitrogen or mixtures of gases, liquids, such as water, physiological saline or various culture media, media as described below, or combinations. Suitable fluid flows include, but are not limited to, fluid jets, such as single or multiple columnar jets; planar, cone-shaped or fan-shaped jets or sprays; and blade-like jets, laminar fluid flow, and turbulent fluid flow. Adequate fluid flows can result in a variety of forces to remove the embryo from its grain, including positive pressure or negative pressure or both; such forces may be uniform or non-uniform, continuous or non-continuous (such as pulsatile or wave force) or any combination thereof.

[0081] An apparatus of the invention may further include a means for moving the target tissue to be substantially purified and the fluid flow relative to each other. For example, either the corn cob containing the seeds or the fluid flow or both can move. Various embodiments of the apparatus can be used with single or multiple, intact or partial ears of corn. For example, the corn cob or cobs may be attached to a support or handle, which is moved relative to the fluid flow. In other embodiments, however, the ear or ears of corn need not be individually coupled to a fastener, but may be moved freely in a manner that allows multiple kernels to be contacted by the force used to remove embryos from the kernels. The means for moving at least one ear of corn relative to the fluid flow may rotate the ear of corn and the opening relative to each other, or may move the flow of fluid along the longitudinal axis of the ear of corn, or may provide any three-dimensional movement of the corn cob and slot relative to each other, such as a combination of rotation and longitudinal movement.

[0082] The invention further provides a separator for separating target tissues from non-target tissues. For example, embryos may be separated from non-embryonic tissues, wherein the separated embryos comprise at least some maize embryos suitable for genetic transformation or tissue culture. Separators may operate by a mechanism that includes, but is not limited to, size exclusion separation (e.g., using a screen, sieve, perforated surface, or other device capable of excluding objects of a certain size), hydrophobicity-based separation, or other attractive forces (e.g., using a solid or fluid material that can attract or repel embryos) and separation by mass or density differentials (e.g., using a centrifuge or using differential settling solutions). In certain embodiments, the separator may be optional, for example, where no additional isolation of intact or partial embryos is required for their use in genetic transformation or tissue culture.

[0083] Substantially isolated immature embryos include at least some embryos, such as intact or partial immature embryos, suitable for applications in tissue culture, callus formation, direct embryogenesis, formation of differentiated plant tissue, formation of at least one plant mature, formation of at least one fertile mature plant and combinations of these processes, as described above. Substantially isolated immature embryos and non-embryonic tissues may also be used for other purposes, such as, but not limited to, genetic or biochemical analysis.

[0084] The present invention further provides an apparatus for substantially isolating in a mechanical process multiple corn embryos suitable for genetic transformation or tissue culture from at least one immature corn cob. In one embodiment, the apparatus comprises at least one component selected from (a) at least one solid surface for applying positive mechanical pressure to the exterior of the kernels of an immature corn cob; (b) an opening for directing a flow of fluid. wherein the fluid flow contacts the kernels on the corn cob; and (c) an opening for applying negative pressure to the fluid contacting the grains on the cob; wherein the component applies sufficient force to substantially isolate embryos from the grains suitable for genetic transformation or tissue culture. Forces can be applied to multiple seeds consecutively or simultaneously, in a continuous or non-continuous manner, and are generally applied mechanically rather than manually. Multiple forces can be used in combination, sequentially or simultaneously. Suitable forces include, but are not limited to, positive pressure fluid jet, positive pressure liquid jet, positive mechanical pressure, negative pressure, centrifugal force, linear acceleration, linear deceleration, fluid shear, turbulent fluid flow, and fluid flow. of laminar fluid. Fluid forces can be exerted on any fluid, gases or liquids or combinations of both.

[0085] Combination apparatus of the invention may optionally include a means for moving at least one ear of corn relative to the source or sources of force for isolating embryos or parts thereof. The cob or cobs can be moved relative to the force source such that the force or forces contact as many grains in a given period of time as is convenient, in order to isolate the embryos more quickly.

[0086] Combination apparatus of the invention may further include at least one means for further separation of substantially isolated immature embryos suitable for genetic transformation or tissue culture. Separators may function by a mechanism that includes, but is not limited to, size exclusion separation, separation based on attractive forces, and separation by mass or density differentials.

LIQUID MEDIUM FOR EXCISION OF EMBRYOS FROM CORN OR OTHER PLANTS

[0087] The present invention provides in one embodiment, means and methods for substantially isolating target plant tissues suitable for genetic transformation or tissue culture. For example, a fluid jet device uses liquid to excise embryos. requiring substantial amounts of fluid for excision. Thus, for example, about 20 l of a liquid medium may be needed to separate embryos from an ear of corn. Therefore, the medium is preferably easily prepared (consisting essentially of one or two ingredients), sterilizable, for example, through an in-line filtration unit, and can flow through a fluid jet apparatus operating at a pressure of about 206.8 - 517.1 kPa (about 30-75 psi), for example, about 275.18 - 413.7 kPa (about 40-60 psi). Furthermore, preferably, it does not require pH adjustment before use. To save resources and costs, it is also preferably reusable.

[0088] Culture media such as Lynx #1013 and Lynx #1902 inoculation medium (half the concentration of Lynx #1013) have been used successfully in an automated method and in an apparatus for excising maize embryos for use in tissue culture. However, these media have multiple ingredients and require pH adjustment before use. Thus, the present inventors have developed means that can be used to optimize the efficiency of an automated embryo excision method for tissue culture. Surprisingly, the inventors have found, in particular, that media consisting essentially of water and/or an osmotic agent, with an osmolality of about 0 mOsm/kg to about 600 mOsm/kg, are useful.

[0089] In certain embodiments, an osmolality of about 0 mOsm/kg to about 600 mOsm/kg for the osmotic agent is preferred, including about 7 mOsm/kg to about 500 mOsm/kg, about 13 mOsm/kg kg to about 300 mOsm/kg, about 25 mOsm/kg to about 300 mOsm/kg, about 50 mOsm/kg to about 300 mOsm/kg, about 13 mOsm/kg to about 200 mOsm/kg , about 13 mOsm/kg to about 100 mOsm/kg or about 100 mOsm/kg to about 300 mOsm/kg. In specific embodiments, the media provided consists essentially of sterile distilled water, dilute calcium chloride (e.g., about 10 ppm), 0.05% MES, pH 5.4 to 5.8, about 5% sucrose +/- MS salts, or about 0.05 to 0.5 M mannitol, including 0.1 to 0.2 M mannitol solutions. Although the transformation frequency (TF) of plant embryos separated by the use of some of such media liquids may sometimes be reduced relative to that found when using, for example, Lynx #1013 Inoculation Medium, the significant time and cost efficiencies that arise from the use of simpler media may outweigh any reduction in FT by allowing producing more explants using the same amount of resources.

[0090] In specific embodiments of the invention, a medium for excision comprises as an osmotic agent mannitol and/or sucrose, such means essentially consisting of 0.05 to 0.5M mannitol. Such media have been found to be capable of producing viable explants. In particular embodiments, an excision medium consists essentially of about 0.1 to 0.2 M mannitol. The osmolality of a 0.2 M mannitol solution in water is, for example, about 225 mOsm/kg. In other embodiments, sterile distilled water, as well as 5% (w/v) sucrose have also been found to be simple, yet effective, media for use with the invention.

MEDIA PREPARATION SYSTEM

[0091] A Media Preparation System (MPS) for use with a fluid jet apparatus is another embodiment of this invention. MPS, for example, can be used to provide a means of excision described above. The MPS may consist of a box (MPSH) and a mixing chamber (MC). The MPS can be made from any suitable material, such as aluminum (e.g. aircraft grade 7075 aluminum) or steel. The mixing chamber generally comprises a tank with an upper and lower end and may comprise a flange and a cover plate. The cover plate was generally provided with one or more sealing rings or other sealing means sealing the mixing chamber. The cover plate comprises openings for insertion and removal of liquid and other media ingredients. Liquid and other ingredients of the media may be added through one or more inlets, comprised in the mixing chamber, which may communicate with a chamber and/or a supply line that contains and/or delivers a supply of the ingredients. The cover plate may also comprise an electronic sensor for monitoring a specified volume of the medium being prepared. The mixing chamber can be oriented on a support plate and held in operating position by fixing means such as mounting pins and positioning brackets. The support plate can be slide-connected to the MPSH. In certain embodiments, a mixing chamber is connected to a fluid jet apparatus, for example, through steel or polycarbonate tubing.

[0092] A mixing assembly can be coupled to the cover plate or to another part of the mixing chamber. The assembly may comprise an impeller and a shaft mounted on a suspension means such as a high temperature bearing. The mixer can be autoclaved before use. The MPS may further comprise a Programmable Logic Controller (PLC) in communication with sensors to monitor the ingress of components, preparation of the media and subsequent removal of the media, for example, through a fluid outlet tube to the fluid extraction apparatus. fluid jet. One or more fluid inlet and outlet tubes may comprise an in-line filtration unit for sterilizing the prepared media.

DEVICES FOR EXCISION OF STAGE EMBRYOS

[0093] Methods and apparatus for preparing multiple embryos suitable for tissue culture are embodiments of the invention, which methods comprise using at least two fluid streams to substantially extract an embryo and endosperm from a seed, and the apparatus comprises at least one opening for directing a first fluid flow and a second fluid flow to substantially extract an embryo from a seed nucleus. In certain embodiments, the seed or seed kernel is a corn cob or corn kernel.

[0094] Fluid flows can be gas flows or liquid flows. In certain embodiments, the fluid streams comprise a liquid, and in particular embodiments, the liquid consists essentially of distilled water, about 5% sucrose, or about 0.05 M - 0.5 M mannitol, e.g., about from 0.1 to 0.2 M mannitol. The fluid flow may exert a force to substantially extract an embryo, comprising one or more forces selected from the group consisting of a positive pressure fluid jet, positive pressure liquid jet, mechanical positive pressure, negative pressure, centrifugal force, linear acceleration, linear deceleration, fluid shear, turbulent fluid flow and laminar fluid flow. Fluid flow can be continuous or pulsatile. In particular embodiments, the fluid flow is sterile.

[0095] An apparatus of the invention may further comprise a means for detecting removed endosperm and embryo tissue and a means for channeling endosperms or embryos for automation. The means for detection and the means for channeling may be linked, for example, electronically, to assist in automation. The apparatus may also comprise at least one separator for separating embryos from non-embryonic tissue, wherein the separated embryos are embryos suitable for tissue culture. The separator can separate embryos from non-embryonic tissues by differential hydrophobicity, by size exclusion or by density differential. The embryonic tissue can be cultured, transformed and/or regenerated to provide at least one fertile plant. In particular embodiments, the embryo is a corn embryo and the plant is a corn plant.

EMBRYO SEPARATION BY FLOATING

[0096] The invention further provides an apparatus and methods for separating embryos based on the different affinity of a selected agent in relation to non-embryonic seed tissue. In certain embodiments, embryos, such as corn embryos produced by a fluid jet excision process, are contacted and coupled to bubbles through hydrophobic interactions. In other embodiments, seed coats are contacted and coupled to bubbles. The bubbles may comprise one or more gases selected from the group consisting of an inorganic gas, such as argon, air, O2, N2; and a covalent organic gas such as methane, ethane or propane, which do not seriously affect the viability and transformability of the removed embryos.

[0097] Bubble size has been discovered to play an important role in the efficiency of the separation process. In practice, (gas) bubbles that have a range of sizes can be generated. However, any bubble size that is suitable for attaching to embryos or seed coats and elevating them to the surface of the fluid is within the scope of this invention.

[0098] The size distribution of the bubbles can vary depending on several factors such as:

[0099] Frit uniformity: the openings between the particles that make up a frit can be variable, allowing bubbles of different sizes to form.

[00100] Gas flow rate: The rate of gas flow through the frit that generates bubbles also affects the size of the bubble - for example, a rapid flow of gas results in larger bubbles.

[00101] Surfactants and their concentrations: additives to the media, such as mannitol and PEG, as well as proteins and other substances with surfactant properties released from broken corn kernels can affect the size of the bubble.

[00102] Bubble fusion: as the bubbles rise towards the top of the tank, some of them merge with other bubbles.

[00103] Hydrostatic pressure: bubbles near the bottom of a float tank will, on average, be slightly smaller because of the greater pressure at the bottom of the tank, although this effect may be small given the size of the float tank.

[00104] Furthermore, it is not necessary for a single bubble to carry a single embryo or seed coat all the way to the surface. Instead, an embryo or seed coat may be only partially transported to the surface by a single bubble, only to be transported later by a sequential coupling of other bubbles. An embryo or seed coat may also not reach the surface and become embedded in the foam the first time, but may circulate several times before reaching the air-liquid phase interface (e.g., the "foam").

[00105] Large bubbles, for example, about 1 mm in diameter (that is, if spherical; or with larger dimensions if non-spherical) or larger, do not achieve good separation for several reasons. For example, such bubbles move fast enough that they hydrodynamically push embryos as well as waste out of their way. Thus, such large bubbles tend not to adequately contact the embryos. Such bubbles also do not have sufficient contact time with the embryos or seed coats to allow efficient bubble coupling. Finally, such bubbles generate a high enough shear force while rising to the surface of the fluid that any embryos that are attached to the bubbles tend to be displaced before reaching the surface.

[00106] Small bubbles, for example, bubbles around 100 microns in diameter or less, attach to waste as well as embryos. This is because the bubbles move so slowly through the fluid that they do not push smaller materials (e.g., endosperm fragments) out of the way. Thus, such bubbles contact and attach to embryos as well as other smaller debris. Finally, such bubbles do not generate sufficient shear force to efficiently displace the coupled residues.

[00107] Medium-sized bubbles (between 100 μm and 1 mm in size) were comparatively more efficient in achieving embryo purification. Such bubbles apparently produce a hydrodynamic displacement small enough to allow the bubble to contact the embryos, thus allowing coupling to occur. Furthermore, such bubbles do not generate sufficient shear force to displace coupled embryos.

[00108] Another aspect of the separation process refers to the physical nature of the gas present in the bubbles, that is, the selectivity of the gas bubbles for the embryos or seed coats. Air is approximately 21% oxygen and 78% nitrogen. Oxygen and nitrogen are both covalently linked diatomic molecules that have a strong affinity for the similarly covalent (nonpolar) waxy portions of embryos, such as cotton embryos. On the contrary, such gases are less efficient in coupling with relatively polar endosperm residues, which remain floating in the medium. In summary, the bubbles compete with the aqueous fluid (e.g., water or mannitol/water) for coupling with the waxy surface of the embryo. As both oxygen and nitrogen molecules exceed water molecules in their covalent character, they preferentially couple to the surface of the embryo. Thus, the relatively lipophilic nature of oxygen and nitrogen and the relatively hydrophilic nature of the aqueous medium explain the connection of bubbles to lipophilic portions, for example, on the surface of the cotton embryo. Alternatively, bubbles were found to efficiently couple to soybean seed coats that rose to the "foam" at the liquid-air interface, while embryos, including soybean embryonic axes and cotyledons, were found to remain in the liquid phase. Therefore, agents that differentially couple to various seed components and direct them to distinct locations, phases or fractions can be used to enrich a given location, phase or fraction for a seed component, such as an embryo or a portion of an embryo.

[00109] In one embodiment, air is used to prepare bubbles. Other gases useful in the practice of the invention include O2 or N2, as well as covalent inorganic gases including noble gases, such as argon. Covalent organic gases such as methane, ethane and propane and other lipophilic gases or mixtures of gases that do not seriously affect the viability and transformability of corn embryos can also be used.

[00110] In the broadest sense, "bubbles" can be of any material that exhibits a preferential bond with embryos, including solids or liquids or mixtures thereof that selectively couple to embryo surfaces. Formats such as a movable flat surface, e.g. a mat or a disk to which embryos can selectively attach can be used. In one embodiment, embryos floating on the surface of the liquid medium were preferentially coupled to a sheet of hydrophobic filter paper (Whatman PS water-repellent phase separation paper, impregnated with silicone). In another embodiment, canola oil mixed with an embryo and waste suspension preferentially attached to the embryos and transported them to the surface as the oil rose to the surface.

[00111] Bubbles that come into contact with each other can merge within milliseconds. Stabilizing the bubbles as they rise preserves an appropriate bubble size distribution as well as promoting a stable foam on top of the buoyancy liquid. Several surfactants have been shown to be useful in preventing coalescence and stabilizing bubbles. Preferably, the surfactant is an ionic or nonionic surfactant, such as a polyether. The polyether can be PEG. PEG may have an average molecular weight of about 100, 300, 400, 600, 900, 1000, 1450, 3350, 4500, 8000 and 9000. Preferably, PEG has an average molecular weight of about 8000. PEG is used in a water-soluble concentration sufficient to prevent premature coalescence of bubbles. Another suitable polyether is PPG. PPG has a molecular weight of about 340 to about 3500. Preferably, PPG has a molecular weight of 340. PPG can be used in a water-soluble concentration sufficient to prevent bubble coalescence.

[00112] In one embodiment, bubbles may be produced by a fine-pore fritted glass dispersion tube (Chemglass, NJ, USA) connected to an aquarium pump to pump air through a sterile filter and into the tube. glass dispersion for creating bubbles. The dispersion tube can be placed in a container such as a graduated cylinder or beaker containing a mixture of embryos and suspended waste. However, the strong convection currents created by the large population of bubbles generated by this device produced sufficient shear force to reduce its effectiveness in separating embryos from waste. Alternatively, the bubbles may be produced by another method, such as a multi-point source bubble generator (e.g., a bubble dispersion device using linden wood fragments for or a porous ceramic surface).

[00113] In some embodiments, a surfactant can be used to stabilize the bubbles and prevent their coalescence. The surfactant can be an ionic or non-ionic surfactant.

[00114] The surfactant is used in a water-soluble concentration sufficient to prevent coalescence of the bubbles. In certain embodiments, the surfactant is a polyether. In particular embodiments, the surfactant may be PEG or PPG. The molecular weight of the PEG surfactant can be between about 100 to about 9000 Daltons, for example, about 100, 300, 400, 600, 900, 1000, 1450, 3350, 4500, 8000 and 9000 Daltons. Alternatively, the surfactant may be PPG and the molecular weight of the PPG may be between about 340 to 3500 Daltons, for example, about 340 Daltons.

[00115] Bubbles can be produced by a bubble creation and dispersion method. For example, a fine-pore glass dispersal tube or sprinkler can be connected to an aquarium pump and placed in a container containing a mixture of embryos and suspended waste. In certain embodiments, the bubble production and dispersion device comprises multiple bubble point sources, such as an individual linden tree fragment. The fragments can be inserted into a matrix, such as the length of silicone tubing, and optionally wound into a spiral and secured.

[00116] When embryos are carried to a liquid interface by bubbles, they can be removed from the surface of the foam by a suitable method, such as gravity drainage or a skimmer or vacuum device. The device can be automated.

COMBINATION DEVICE AND METHODS FOR EXTRACTION AND SEPARATION OF EMBRYOS

[00117] In additional embodiments, the invention provides a combination device for extracting and separating embryos suitable for tissue culture. The extractor can be in communication with the separator or they can work separately. The extractor comprises one or more jets of fluid for extracting an embryo, such as a corn embryo from a seed. The seed may be in a kernel on an ear of corn. More than one jet of fluid can be directed onto a single spike and more than one spike can be placed simultaneously in the extractor.

[00118] After extraction of embryos from grains, embryos and residues may fall or be conveyed to an embryo separator comprising, for example, a flotation chamber. The method for conduction to and from the flotation chamber may be a screened conductive mat or mats or a screen that can separate waste from the embryos through size exclusion. The mat or screen may be a braided or molded thermoplastic. After adding the embryos to the flotation chamber, the embryos are preferably separated from other waste, for example, via the bubble method described, and float to the surface where they can be removed through an opening or channel.

[00119] Therefore, the corn cobs can be harvested and the embryos can be removed using a jet of fluid. The apex of the grain may be removed to facilitate excision of the embryo by the fluid jet and the grain contents are substantially removed from the grain by the jet. The fluid jet may be a liquid jet comprising the described excision medium with an osmolality of about 0-600 mOsm/kg. In particular embodiments, the excision medium is a sterile solution, e.g., 0.1-0.2 M mannitol. Embryonic as well as non-embryonic tissue (e.g., endosperm) can be extracted from the ear and kernel by jet. Subsequently, the embryos can be substantially separated from the non-embryonic tissue, for example, by the described separation methods and apparatus, using shear forces including embryo flotation, by sieving or the like. However, even embryos or groups of embryos that are not separated from other tissue comprising a grain may be of use for subsequent tissue culture, including transformation and regeneration of transgenic plants.

[00120] If a flotation method is used for separation, a surfactant can be added to the medium in which the embryos are separated. The surfactant, such as PEG or PPG, reduces bubble coalescence, maintaining an effective average bubble size to promote efficient separation of embryos from non-embryonic tissue, and the deposition of embryos at the air-liquid interface where they can be collected by flow of liquid, through a skimmer, through a vacuum device or similar. By such methods, substantially removed and separated plant embryos can be obtained, suitable for the production of transformed tissue and transformed plants.

PROCESSED PLANTS AND METHODS FOR THEIR PRODUCTION

[00121] The present invention also provides a transformed plant, such as a corn plant, cotton plant or soybean plant, produced by steps including (a) providing at least one transformable target tissue using one or all of the methods or apparatus herein. described; (b) introducing a selected nucleic acid molecule into the transformable target tissue to produce a transformed explant; and (c) cultivating the transformed monocot plant from the transformed explant. Preferred plants of the invention include members of the Poaceae family, including grain crops such as corn, millet, wheat and rice; as well as transformed dicotyledonous plants such as cotton or soybean plants.

[00122] Particularly preferred monocotyledonous plants include Zea species, such as Zea mays. The transformed corn contains at least one heterologous nucleic acid molecule capable of imparting a desired characteristic to the transformed corn, such as herbicide resistance, pest resistance, cold germination tolerance, water deficiency tolerance, increased productivity, increased yield, and similar. Practical transformation methods and materials for making transgenic monocot plants of this invention (e.g., various media and receptor target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are described, for example, in United States Patent Number 6,194 636 to McElroy et al., United States Patent Number 6,232,526 to McElroy et al., United States Patent Application Publication Number 2004/0216189 to Houmard et al., United States Patent Application Publication Number 2004 /0244075 to Cai et al., who describe methods useful for corn, and United States Patent Application Publication Number 2003/0024014 to Cheng et al., who describe methods useful for wheat, all of which are incorporated herein by reference. Single or multiple heterologous nucleic acid molecules can be used for transforming monocot plants of the invention; for example, constructs for the coordinated decrease or increase of gene expression are described in United States Patent Application Publication Number 2004/0126845 to Van Eenennaam et al., which is incorporated herein by reference. Numerous methods for transforming other plants, including dicotyledonous plants, are well known in the art. For example, methods for transforming soybean and cotton plants are described in U.S. Patent Application Serial Number 12/045,502 or U.S. Patent 7,002,058, each of which is incorporated herein by reference.

[00123] In certain embodiments, for example, when a method of genetic transformation mediated by Agrobacterium or other bacteria is used, the culture response or frequency of transformation of embryogenic tissue isolated by the methods of the present invention can be enhanced by the use of a coculture medium comprising a bactericide such as carbenicillin and/or 2,4-D at a concentration of about 0.5-1.5 mg/l. In a particular embodiment, the coculture medium comprises the ingredients of Medium 1898 of Table 9.

[00124] The seeds of the fertile transgenic plants resulting from the invention can be collected and used to develop progeny generations, including hybrid generations, of transformed plants that include the heterologous nucleic acid molecule in their genome. Therefore, the present invention includes both primary transformed plants ("R0" plants, produced by the transforming embryos provided by a method of the invention) and their progeny carrying the heterologous nucleic acid molecule. Such progeny of transgenic plants can be prepared by crossing a transformed monocot plant of the invention that has a heterologous nucleic acid molecule with a second plant that does not have the construct. A transformed plant of the invention can also be crossed with a plant line that has other heterologous nucleic acid molecules that confer another characteristic, to produce a progeny of plants that have heterologous nucleic acid molecules that confer multiple characteristics.

[00125] In order to provide a clear and consistent understanding of the application and claims, including the scope given to such expressions, the following definitions are provided.

[00126] "Embryo" is that part of a seed that consists of precursor tissues (meristematic tissues) for leaves, stem and root, as well as one or more cotyledons. Once the embryo begins to develop (germinate), it becomes a seedling.

[00127] "Meristem" or "meristematic tissue" consists of undifferentiated cells, the meristematic cells, which differentiate to produce multiple plant structures including stem, roots, leaves, germinal tissue and seeds. Meristematic cells are the targets for transformation to obtain transgenic plants.

[00128] "Explant" is an expression used to refer to the target material for transformation. Therefore, it is used alternatively with "meristematic tissue" or "embryo" in the latter embodiments.

EXAMPLES

[00129] Those skilled in the art will appreciate the various advantages of the methods and compositions provided by the present invention. The following examples are included to demonstrate preferred embodiments of the invention. It should be considered by those skilled in the art that the methods described in the following examples represent techniques discovered by the inventors that work well in the practice of the invention and therefore may be considered to constitute the preferred modes for its practice. However, those skilled in the art should consider, in light of the present description, that various changes can be made to the specific embodiments that are described and still obtain the same or similar result without departing from the spirit and scope of the invention. All references cited herein are incorporated by reference to the extent that they complement, explain, provide support or teach a methodology, techniques or compositions employed herein.

Example 1: Method for extruding multiple corn embryos

[00130] This example describes a method that uses positive mechanical pressure from an extruder device to produce embryos suitable for tissue culture or genetic transformation.

[00131] The kernel tips were sterilely removed from an immature corn cob (Zea mays) with a vegetable peeler. The dehuller was pressed from the basal end of the corn cob to the apical end using a gentle sawing motion to obtain a quick, accurate truncation of the kernels. Although in this example the individual grains are truncated to expose the internal tissues, in other embodiments it may be necessary only to ensure that an opening (such as a hole, an incision or abrasion) is made in the pericarp without actually removing the pericarp material. Where intact embryos are desired (e.g., intact embryos for transformation), the size of any opening is preferably sufficient to permit removal without the embryo without damaging it. Opening of the pericarp can be achieved using any suitable device, including, but not limited to, blades and abrasive materials. For example, a vegetable peeler is considered to be relatively safe and quick to use; it has a regulated cutting depth and also requires less experience to use than a scalpel. Other tools with similar functions can be used. Devices for opening the pericarp are preferably sterilizable, for example, in an autoclave or by heating or chemical sterilization. These pericarp treatment processes can be automated; for example, a blade or blades or a scraper may be motorized.

[00132] A sterile extruder (in this case, a 4 mm diameter rod) was pushed against the base of the truncated grains. Other devices suitable for extrusion may be employed. Preferably, such devices should have a size and shape capable of applying a relatively localized force to the base of the truncated grains to eject the embryos and endosperms. Preferably, the applied force is of sufficient magnitude and is applied in a suitable direction such that the advance of the extruder does not "ride" the grains ahead. The back blade of the extruder preferably also provides a surface upon which the ejected embryos and endosperm accumulate; for example, a flat piece of stainless steel with a rounded front edge can be used. In this example, the embryos are gently squeezed out of the pericarp, followed by the endosperms. The extruded embryos and endosperms settled on top of the extruder rod and were not crushed during the process.

[00133] The mixture of embryos and endosperms was washed with an aqueous liquid medium (for example, water, liquid medium or saline) on a sterilized screen that has diamond-shaped openings (about 2 x 3 millimeters). The endosperms were observed to be largely retained and the smaller embryos and some minor endosperm residues were washed through the sieve into a collecting receptacle. The collected embryos were washed twice to remove small debris.

[00134] The washed embryos were then purified by a flotation process. In the first step of the flotation process, the aqueous fluid medium was completely removed from the collection receptacle, which was allowed to dry quickly (e.g., about one minute) such that the remaining aqueous medium came off the waxy surface of the embryos, exposing it. directly to the air. New aqueous medium was added and most of the embryos floated because their waxy surfaces were not re-wetted by the fluid. Non-embryonic tissues, such as endosperm residues, remained submerged in the medium and a clear separation of embryos and non-embryonic tissues was obtained. Flotation of extruded embryos can be improved by more rapid, complete, or reproducible removal of the aqueous medium, such as through the use of aspiration or capillary action (e.g., use of a sterile pad placed in the collection receptacle to absorb fluid). out of the extruded embryos).

[00135] A production of approximately 100 embryos was isolated in this preliminary experiment, in which only a portion of the embryo-endosperm material from the entire ear was processed. These results demonstrate that the methods of the present invention are practical and convenient for collecting a large number of immature embryos from corn cobs.

[00136] Embryos isolated by the method of the invention can later be used in tissue culture procedures, for example, regeneration methods to generate transgenic corn plants. The transfer of isolated embryos to the culture medium was easily done by placing forceps, with the ends closed together, under the floating embryos, lifting them free of the liquid with the forceps and placing them on a culture medium. Another technique may be to remove isolated embryos with an instrument that has a hydrophobic surface. An additional technique may be to transfer the embryos by hydrophobicity, for example, transferring them to the surface of a medium through a small puff of air or a sudden mechanical movement, such that their kinetic energies exceed the hydrophobic force that holds them. on the instrument.

Example 2: Visual confirmation of embryo size

[00137] This example describes an improvement to an embodiment of the method of the present invention, as described in Example 1. Using the approach described in Example 1, immature corn embryos need to be as close as possible to the truncated part of the kernel in order to to be ejected in large numbers. Variation in the size of the immature corn embryo is an important consideration in gauging the amount of kernel apex to be removed. Embryos tend to be widest in the middle section of the cob, with embryos sometimes smaller toward the ends. Smaller embryos, for example less than about 1.5 millimeters in length, are more difficult to remove unless they are close to truncation.

[00138] One way to ensure that the maximum amount of grain has been decapitated above embryos of varying sizes is to observe the cob during the decapitation process under minimum magnification. For example, glasses with minimal magnification (e.g., Donegan Opti-VISOR binocular magnifier equipped with No. 7 lenses, which provide 2.75X magnification) were used to aid visual confirmation of embryo size and adequate truncation of the pericarp. . If the first cut did not remove enough of the grain apex, a second cut can be made. Other magnifying devices using the same or similar magnifications may be used. For example, the lenses available for the Opti-VISOR provide magnification ranging from 1.5 to 3.5X.

Example 3: Extrusion of embryos and endosperms

[00139] This example describes an improvement to an embodiment of the method of the present invention, as described in Example 1. Motorized devices can be used to assist in the extrusion of embryos and endosperms. For example, an electric slitter such as a WeCheer 320 electric slitter (WeCheer Industrial Co., Taichung Hsien, Taiwan, R.O.C) adapted with a round extrusion device can be used to reduce the force a person needs to exert to eject the embryos. and endosperms. Other electrical devices are available and can be similarly used. Preferably, the "slitter" portion of such a tool (or any part of the tool that may come into contact with the embryos) can be conveniently sterilized, for example, by insertion into a sterilization sphere.

[00140] In one experiment, the blade of a stainless steel weighing spatula was curved back on itself to provide an extruder device that has a rounded front blade. After insertion into a WeCheer 320 electric slitter, a portion approximately 10 centimeters long protruded from the electric slitter mandrel. This set was used to eject the embryos and endosperms from the individual rows of decapitated grains. As the extruder device (modified spatula) was moved down a row of grains, a slight tendency for the spatula to slip to the left or right was observed; however, this tendency can be corrected by including a spatula extension similar to a small keel over each outer edge.

Example 4: Mechanized extrusion of the embryo

[00141] This example describes an improvement of an embodiment of the method of the present invention, as described in Example 1. Mechanization of the embryo extrusion process can be obtained by the use of a suitable device, such as, but not limited to, the device described here and represented schematically in Figure 1. This device includes two motors. The first motor (D) is a stepper motor that can rotate the corn cob (A) such that new rows of kernels are exposed to the two extrusion rods (G), which apply force to squeeze the embryos and endosperms out of the its pericarps.

[00142] The rods (G) are conveniently located on opposite sides of the cob in order to balance the pressure applied to the cob in relation to the longitudinal axis of the cob. However, a single rod may be used or more than two rods may be used; where multiple shanks are used, it is preferable that they are positioned so as to evenly distribute the resulting mechanical pressure around the tenon. The rod does not need to be a straight rod; In one embodiment of the device, a flexible "ring" surrounding the circumference of the tang is used instead of a rigid rod. In another embodiment, multiple short rods or bearings are arranged in a flexible, circular configuration that can slide along the longitudinal axis of the cob, applying mechanical pressure to several or all rows of grains simultaneously.

[00143] The second motor is connected to the pinion gear (E) which connects to a support (F) such that a linear movement up and down the cob occurs. The base of the tang is securely attached to a handle (B) by means of a screw extending from the handle to the base of the tang. The narrow middle portion of the handle is square such that it will not rotate unless the support (C) to which it is attached is rotated by the stepper motor (D).

[00144] Before insertion into the machine, the apices of the grains are decapitated as in Example 1 so that the embryos and endosperms can be squeezed. To begin the process, the tang is lowered until the two rods (G) are close to the base of the tang just below the handle (B). Then, the rods are pressed against both sides of the tenon and the support and pinion assembly pulls the tenon upwards. As this occurs, the embryos and endosperms are removed from a pair of rows, fall down into the collection plate (H) remaining at the base (I) and accumulate in a pile (J). When the shanks approach the apical end of the cob, the cob is retracted upward to its original starting position and slightly rotated by the stepper motor until new rows of kernels are in position.

[00145] Various degrees of automation of this machine are possible, including sensors to automatically adjust the vertical start and end positions as well as the initial and end rotational positions. A bracket and pinion is not the only method by which linear motion can be achieved. Pneumatic or hydraulic may be preferred for some applications. The rods (G) can be opened automatically by a suitable mechanism. When a new cob is loaded, it may be preferable to raise the cob to a position high enough to clear the shanks.

Example 5: Hydrophobic separation of embryos

[00146] This example describes a modification to an embodiment of the method of the present invention, as described in Example 1. In separation applications, the material of interest often appears at the interface of dissimilar phases (e.g., between aqueous and lipophilic solvents) . Removing the material of interest from such an interface can present problems and has, in the past, been a manual process involving intimate contact with the extractor and the material to be extracted. Often, the only way to successfully separate a component is to use a material of the same polarity or hydrophobicity/hydrophilicity. In the case of immature corn embryos extruded by a method of the invention, the embryos are found at the water/air interface. The surface of corn embryos is waxy, that is, lipophilic or hydrophobic, and when an embryo cuticle is contacted with a substance of similar hydrophobicity, the embryo tends to adhere to the hydrophobic surface. The hydrophobicity of the embryo reduces the surface tension of the water around it, which helps the embryo to "float" on the surface of the water/air interface.

[00147] An approach that takes advantage of these physical characteristics may be to touch the floating embryos with a hydrophobic material such as a hydrophobic filter paper, for example, Whatman No. 1 PS paper, which is a paper for separation of silicone-impregnated water-repellent phase (Whatman plc, Brentford, Middlesex, U.K.). And as an example, a piece of sterile hydrophobic filter paper can be lowered over an entire container of floating embryos and remove them all at once. In another example, a small piece of the hydrophobic paper can be used to successively remove several embryos and transfer them to the next container. In a third example, either a small piece of hydrophobic paper or the tip of a hydrophobic pipette can be used to contact and remove individual embryos and then dispense them with a puff of air from the pipettor. Common pipette tips can also be modified for such use by inserting the pipette tip into a short length of a hydrophobic tube (e.g., a silicone tube). The embryo can then be removed by hydrophobic attraction to the distal end of the hydrophobic tube and then released by blowing air through the pipette. The reduced surface tension around hydrophobic embryos helps them float on a water surface, and floating embryos can also be transported by moving them over the water surface (for example, by a jet of air directed at the embryos). Embryo removal and disposal can be automated using modifications of existing devices, such as machines designed for colony removal or for rescuing protein spots on stained two-dimensional protein gels.

Example 6: Additional methods of embryo ejection or extrusion

[00148] The method of the present invention encompasses the use of various types of force or combination of forces, to separate the embryo from its seed. This example describes additional embodiments. In a basic method as described in Example 1, positive mechanical pressure is applied to the base of the truncated seed (such as a kernel of corn) to eject the embryo through the truncated apex of the seed.

[00149] In another embodiment, centrifugal force can be used to eject the embryo. For example, an ear of corn (whose kernels have previously been truncated) can be rotated about its longitudinal axis at a speed sufficient to eject the embryos and/or endosperms in a radial trajectory. Rotation may be achieved by any suitable technique, such as, but not limited to, contacting the apical end of an ear of corn with a freely rotating cone, wherein the rotation of the ear is maintained within a limited longitudinal range, e.g. , by coupling the basal end of the tenon to a handle that is then inserted into a support within which it can rotate. In an exemplary embodiment using centrifugal force, about one-third of the apex of each kernel on an ear of corn is removed with a scalpel and the ear is rolled over a surface to release the embryo and endosperm within the kernels. The cob was broken into two pieces, each about 750 millimeters long. Each piece was placed in a 250 milliliter centrifuge bottle with about 100 milliliters of water. These were centrifuged for 15 minutes at 5000 rpm to eject the embryos. Examination of the ears after centrifugation showed that, in some portions of the ear, all embryos were removed by centrifugation, while in other areas, few or no embryos were removed. The ejected material was centrifuged and the supernatant removed to leave a slurry, which contained intact embryos (estimated to include about 20 percent of the total number of embryos). In another example, an immature ear of corn was harvested (typically between about 10 to about 14 days after pollination). The cob is disinfected and under sterile conditions the apex of each kernel is cut off. The tenon is mounted on a drill bit in an electric drill (or similar device) and the tenon is surrounded by a large sterile collecting vessel (e.g., a large glass beaker). The spike is rotated enough to eject the immature embryos and the ejected tissues are collected from the sterile container. Immature embryos are collected, for example, by manual collection, or by rinsing the container with sterile culture medium and recovering the enriched fraction containing the embryos (e.g., by sieving, by use of a liquid density gradient, or by other methods to separate embryos from non-embryonic tissue as described elsewhere in this specification). Immature embryos (or calluses derived from immature embryos) can subsequently be used for transformation. Improved results using these and other centrifugation methods can be obtained by determining preferred centrifugation times and speeds by routine testing.

[00150] Another modality employs the maceration of a mass of grains. An immature ear of corn is harvested (typically between about 10 to about 14 days after pollination). The cob is disinfected. The pericarp can be opened under sterile conditions or the kernels can be left intact. The kernels are removed from the cob by any suitable procedure, including, but not limited to, the use of a scalpel or other bladed tool. Once separated from the cob, the kernels are placed in tissue culture medium. The grain-medium mixture may be subjected to further tissue disruption using a suitable cutting device, such as, but not limited to, a mixer. Immature embryos are collected, for example, by manual collection or by rinsing the container with sterile tissue culture medium and recovering the enriched fraction containing the embryos (e.g., by sieving, by use of a liquid density gradient, or by other methods for separating embryos from non-embryonic tissue as described elsewhere in this specification). Immature embryos (or calluses derived from immature embryos) can subsequently be used for transformation.

[00151] In an additional embodiment, jets of fluids (gases or liquids or combinations thereof) can be used to dislodge the embryos. An example of this approach is to automatically rotate an ear of corn in a gradual or continuous (helical) manner beyond a stationary jet, collecting the ejected material containing the embryos, and then isolating the embryos if necessary, for example, by size separation in a screen or sieve or similar. Where the corn cob is oriented vertically (with respect to its longitudinal axis), it may be preferred to rotate the cob in an upward helical direction, or otherwise move the cob relative to the jet such that the extracted embryos tend to flow downward. .

[00152] In another embodiment, linear deceleration or linear acceleration can be employed to dislodge or eject the embryos. For example, an ear of corn can be shocked parallel to the longitudinal axis of the ear and with enough force to eject the embryos and endosperms. An ear of corn can be enclosed in a suitable sterile high-impact resistant support, which can be subjected to sudden acceleration or deceleration, for example, by a vigorous impact (e.g., such as that of a wooden hammer).

[00153] Another improvement of the method may be to facilitate the ejection or extrusion of the embryo from a truncated seed. For example, embryos can be detached or displaced from their native positions within the seed by applying a force to the apices of intact seeds(e.g., by applying pressure to the apices of rows of corn kernels on an intact ear or by rolling or pressing the ears themselves onto a surface before decapitating the kernel apices). Embryos can also be released within the seed by applying vibration, for example, ultrasound. Another approach may be to remove additional non-embryonic tissue, such as additional sidewall material (pericarp), prior to ejection or extrusion of the embryo. For example, a V-shaped knife or other instrument can be used to remove some of the sidewalls of corn kernels in rows on the cob.

Example 7: Automated embryo isolation using positive pressure fluid jet

[00154] This example describes an additional embodiment of the present invention. In this example, an automated device uses a positive-pressure jet of fluid to dislodge embryos from seeds. Referring to Figure 2, a robotic gripper C (preferably capable of moving in three dimensions by means of a robot A and a motor B or by an equivalent means) picks up an ear of corn I (by a handle D having a deflector E) in a position defined by a frame on the robot platform. The robot inserts the corn cob into the tube H (optionally made of transparent material to facilitate visual observation) at a starting position below the flange F. The positive pressure fluid jet is introduced through the opening G and the cob is simultaneously elevated (in the Y dimension) and rotated by robot A and motor B, respectively, preferably resulting in the corn cob being struck by the fluid jet, inducing the dislodgement of the embryo and endosperm. The fluid passing through opening G may be at least one gas, at least one liquid, or any combination thereof. The fluid jet may exert force continuously or non-continuously, for example, as in pulses. As embryos and endosperms are dislodged by the positive pressure fluid jet from opening G, they fall onto screen J, which retains the endosperms while allowing the embryos to fall to the collecting surface K (e.g., sterile gauze) below. Excess fluid can optionally be collected in a receptacle for waste or recycling L. After the embryo removal process for each ear of corn is completed, the inside of the tube can be washed manually or with an automated jet above or below the flange F.

Example 8: Methods of processing crude embryo preparations

[00155] Embryo preparations obtained by methods such as those described in Examples 1 to 7 may include both intact embryos and partial embryos, which may be accompanied by non-embryonic tissue, such as endosperm and glumes. Some applications may not need further treatment or separation steps, for example in a mass transformation of such a "raw" embryo preparation, where the embryos (intact or partial) do not need to be separated from the non-embryonic tissue. For example, callus derived from either intact or partial immature corn embryos can be used for transformation, regeneration and production of fertile transgenic plants. Therefore, both intact and partial embryos can serve as transformable explants and do not need to be separated from each other. However, in other cases it may be desirable to further purify the embryos from a crude embryo preparation.

[00156] Procedures in which some difficulties may be encountered in processing crude embryo preparations include: (1) removal of non-embryonic tissues (e.g., cellular debris, starch grains, undesirable proteins), (2) efficient removal of excess of liquid from embryos after extrusion or washing using liquid, and (3) adding liquid with minimal turbulence such that the embryos float and do not become submerged.

[00157] A porous material is useful for separating non-embryonic tissue from embryos. Any suitable porous material may be employed, preferably having a mesh or pore size small enough to retain embryos but allowing non-embryonic tissue or minor debris to pass through and capable of being sterilized (e.g., by autoclave, heat, irradiation or chemical sterilization). The suitability of materials is easily judged or tested by simple experimentation by one skilled in the art. Examples of suitable materials include cotton gauze or other textile material and other screens or sieves. In some embodiments, perforated solid materials may be used, including perforated ceramics, polymers, metals, or glasses (e.g., in the form of a Buchner funnel or similar separation funnel). Appropriately graded cotton gauze, for example, has a mesh size small enough to retain embryos but allows smaller debris to pass through and is autoclavable. The cotton gauze can be attached to a screen or ring (e.g., the embryo-containing screen of collection surface K in Figure 2 and described in Example 7) to enable the gauze and all retained embryos to be simultaneously submerged for easy rinsing. For example, cotton gauze can be easily attached to a screen via an elastic band or similar (e.g. silicone tubing); Such screens are easily manufactured, for example, from a beaker or graduated cylinder made of autoclavable material (e.g., polypropylene, polymethylpentene, polycarbonate, or autoclavable glass) cut into sections. Cotton gauze has strong capillarity, allowing liquid to be efficiently extracted from the embryos, thus exposing their waxy epidermis to air before floating. In the flotation stage, the cotton gauze is simply submerged in an aqueous liquid, allowing the embryos to float.

Example 9: Substantial isolation of embryos using a fluid jet

[00158] This example describes an additional embodiment of the present invention. In this example, multiple embryos were dislodged from the seeds by a positive pressure fluid jet.

[00159] In the simplest example, a 200 microliter pipette tip was attached to a sink tap with Parafilm®. When the water was turned on, a jet emerged from the pipette tip with considerable force. Tap water pressure was estimated to be about 413.7 kPa (about 60 pounds per square inch). This jet of fluid (liquid) was formed over an immature corn cob (contained in a beaker) where the kernels had been decapitated as described in Example 1. As the jet struck each kernel, the endosperm and embryo were ejected and collected in the beaker. As the endosperm at this stage is a relatively soft tissue, it was fragmented into many small pieces by the jet, while the embryos appeared to remain intact.

[00160] The endosperm and embryo tissue dislodged by the jet were decanted directly onto a No. 60 cotton gauze (other suitable porous materials, such as a hydrophilic screen of appropriate mesh size, can be substituted). Different "grades" of cotton gauze are available (e.g. grades 10, 20, 30, 40, 50, 60, 70, 80 and 90, where mesh openings decrease with higher grades) and the grade or size mesh sizes appropriate for the average size and shape of a given type of embryo are easily selected by simple experimentation. The embryos and larger fragments of the endosperm were retained on the upper surface of the cotton gauze. Before the next step, the cotton gauze was allowed to partially dry by draining excess liquid. This removed liquid from the tissues and exposed the embryo's surfaces to air. When the gauze was dipped in a watery liquid, the embryos floated because their waxy epidermis did not remoisten.

[00161] In a simple configuration, the cotton gauze (or other suitable porous material) can be manually stretched or held over a receptacle or waste container as the liquid containing the crude embryo preparations is poured onto the cotton gauze. For sterile work, cotton gauze can be tied to rigid screens, which can be autoclaved before use. Sieves attached with handles, such as those available at kitchen supply stores, can also be used in the method.

Example 10: Devices for embryo extraction using a fluid jet

[00162] This example describes various embodiments of an apparatus for mechanically preparing multiple corn embryos suitable for tissue culture.

[00163] One embodiment includes an apparatus for preparing multiple corn embryos using a jet of fluid, generally similar to the apparatus described in Figure 2. A transparent cylinder open at the bottom was made by cutting the ends of a graduated cylinder of 1 liter autoclavable polymethylpentene (PMP). A pipette tip (e.g., Gilson DistriTip®, gradually narrowed to avoid the formation of back pressure) was attached to the side of the cylinder and served as an opening to guide a stream of fluid as a jet through a hole drilled in the wall of the cylinder. cylinder. Fluid (in this case, water) was fed through the pipette tip from PharMed® high-pressure autoclavable peristaltic pump tubing. The water was released from a laboratory sink faucet, but it could be an aqueous fluid released by a pump or other source. Using a pump capable of delivering a sterile fluid is preferable when, for example, a sterile culture medium or a sterile saline solution is found to be superior to water as a liquid for substantial isolation of embryos. An example of a suitable pump is a Masterflex® pump with an L/S pressure pump (Cole-Parmer Instrument Co., Vernon Hills, IL) that can deliver sterile liquid up to 689.5 kPa (100 psi) when used with a high pressure piping.

[00164] An ear of corn with previously decapitated grains was manually positioned inside the cylinder. After the cob was properly positioned inside the cylinder, each grain was subjected to the positive pressure of the water jet. This resulted in the extrusion of embryos and non-embryonic tissues from the grains. Examination of the cob after treatment indicated efficient removal of embryos from the kernels. The removed material was washed from the inside of the cylinder walls into an embryo collector positioned underneath the cylinder. The embryo collector included: (1) a thick plastic screen (on which larger debris was captured), heat fused to the cut top of a Tri-Pour® plastic beaker and superimposed above (2) a thinner screen (gauze of Class 60 cotton, on which the extruded embryos were captured), secured with an elastic band from the cut top of a second Tri-Pour® plastic beaker and superimposed above (3) a waste collection beaker or other container (in which minor waste, non-embryonic tissues and liquid waste were collected).

[00165] Modifications to these and similar modalities are easily made by one skilled in the art. For example, with respect to positioning the corn cob or seed for application of the fluid jet, the cob may be held manually in place, or preferably mounted securely within the cylinder by a movable bearing capable of moving the cob in three dimensions. For example, the tenon may be mounted on a screw-down metal or polymer rod, such as a polypropylene rod, which may be used to move the tenon along its longitudinal axis as well as to rotate the tenon). Another example of a mounting mechanism is described in Figure 3, which illustrates a magnetic "handle" by which the spike can be attached to a robotic arm.

[00166] In other embodiments, however, the ear or ears of corn need not be individually secured to a fastener, but may be freely movable in a manner that allows multiple kernels to be contacted by the force used to remove embryos from the kernels. For example, at least one spike or multiple spikes may be carried or fixed between at least one support, such as, but not limited to, at least one flat surface, a screen, a grid, a wire mesh, a sieve, platform, bearing , rod or guide wire and a track, wheel or track on rollers. Such a support may be movable or induce the tenon or tenons to move, for example, by vibration, rolling, gravity or other mechanisms. Substantially isolated embryos may pass through the platform itself if the platform is porous (e.g., made of mesh). The spike or spikes may also float on a fluid in a manner that allows each spike to rotate or otherwise move freely while floating. The fluid, such as a liquid containing substantially isolated embryos, may be continuously drained, optionally through a filtration or sedimentation device or collected by centrifugation.

[00167] Devices for obtaining movement along the longitudinal axis of a corn cob include, but are not limited to, ball screw-directed blades or belt-directed blades, such as those commercially available from various manufacturers such as Techno. Inc. (New Hyde Park, NY; Technoisel.com). To obtain rotational motion to turn an ear of corn, a stepper motor can be used, for example, a stepper motor coupled to a sliding plate. The rotational movement may be provided by rolling devices, for example by parallel round or tubular rollers between which the corn cob is clamped and rotated.

[00168] The shape of the fluid jet can be advantageously modified according to the desired application. For example, a narrow column-shaped jet of uniform diameter is useful for removing embryos from a seed at once. Where it is desirable to increase the rate at which embryos are substantially isolated, multiple embryos may be simultaneously removed from their seeds by a jet of fluid; This can be achieved, for example, by using at least a single jet of fluid that covers a larger area, or by using multiple jets simultaneously. In one embodiment, multiple jets, such as multiple parallel, narrow, or column-shaped jets (e.g., produced by multiple injectors similar to those used in Example 9 and optionally connected to each other by a hose, are used to direct the jet of fluid with positive pressure over multiple seeds to substantially isolate their embryos substantially and simultaneously. Automation of these and other devices may further include optical or mass sensors to assist in positioning the spike and the fluid jet relative to each other.

[00169] In another embodiment, at least one fluid jet that covers a larger area (for example, in which the fluid jet simultaneously impacts multiple grains or multiple rows of grains on an ear of corn) can be used. The dimensions of such a jet preferably allow the jet to enter the grains and weaken the embryos. Typically, corn embryos used in genetic experiments are immature and generally range in size from about 1.8 to about 2.2 millimeters long; The grains that house these immature embryos are generally in the size range of about 4 to about 5 millimeters wide. For embryos of this size, a suitable fluid jet may be, for example, between about 0.5 to about 1 millimeter wide.

[00170] Any suitable means for producing such a wider fluid jet may be used, such as, but not limited to, injectors that generate non-columnar fluid jets. Examples of suitable injectors include, but are not limited to, injectors that generate a flat spray pattern and injectors that generate a fan-shaped or cone-shaped spray pattern. In one example, a commercially available flat spray nozzle (number 23990-1/4-04, Spraying Systems Co., Dillburg, PA) was used with a Masterflex® L/S pump (model 77250-62) to pump liquid to 1 liter per minute and 206.84 kPa (30 psi); embryos were removed from an ear of corn under these conditions. Another example of a preferred injector is an injector that generates a fluid jet in the form of a flat "sheet" of fluid, as illustrated in Figure 4. Such an injector preferably is capable of generating a uniform, flat fluid jet that maintains a coherent, uniform sheet-like flow over at least a sufficient distance to allow the flow to contact more than one seed (and preferably several seeds) at the same time. The new injector illustrated in Figure 4 is designed to generate a uniform laminar jet that is about 0.5 to 1 millimeter thick, greater than about 20 millimeters wide, and which maintains follicular flow over a distance of about 15 to approximately 30 millimeters from the injector opening. This latter distance allows the jet to be moved along the grain rows with minimal adjustments required for differences in the distance between the grain surface and the nozzle opening.

[00171] Without regard to the area or shape of the jet or the spray pattern generated by the injector or the opening through which the liquid flows, injectors or openings are preferably used with flow rates and pressures sufficient to generate sufficient fluid force to dislodge the embryo from its seed, without damaging the embryo. In some embodiments it is preferable to use a small flow rate and possibly a higher pressure to minimize consumption of fluid (such as a medium) as well as to minimize waste generated.

Example 11: Using a gas jet to substantially isolate embryos

[00172] This example describes additional embodiments of methods and devices for mechanically preparing multiple corn embryos suitable for genetic transformation and tissue culture. As described in Example 6, gas jets can also be used for substantial isolation of multiple embryos. An apparatus similar to that described in Example 10 was modified for use with gas. A 1-milliliter pipette tip (Marsh Bio Products-ABGene, Rochester, NY; catalog number TN1300RS) was attached to the side of a 1-liter polymethylpentene graduated cylinder and served as the opening to guide an air stream as a jet through from a hole drilled in the cylinder wall. Air was supplied by a compressor pressurized at between about 413.7 to about 689.5 kPa (60 to about 100 psi). An air valve, for convenience, was positioned in line between the compressor and the pipette tip. The jet of air emerging from this pipette tip was used to dislodge the embryos from a prepared ear of corn. Examination of the grains after they were air blasted showed that the thick pericarp remained in place and surrounded by paper-like glumes and the contents of the pericarp (embryo and endosperm) had been removed. Examination of the tissue retained by the class 60 cotton gauze showed that it included dislodged embryos as well as some glumes dislodged by the high-pressure air jet. Corn glumes have a waxy surface like embryos and also float after the flotation procedure. Using lower air pressures can reduce glume contamination.

Example 12: Substantial isolation of embryos using other fluid forces

[00173] This example describes additional embodiments of methods and devices for mechanically preparing multiple corn embryos suitable for genetic transformation and tissue culture. Forces exerted by fluids, other than the positive fluid pressure of a fluid jet, can be used to substantially isolate embryos. In one experiment, the tops of the kernels were removed from an ear of corn, which was placed inside a bottle containing sterile distilled water and shaken vigorously by hand. This resulted in the substantial isolation of 90 of the 200 ear embryos. Another experiment repeated the previous procedure, except that agitation was carried out in a mechanical vat mixer. In this experiment, 56 embryos were substantially isolated from the 190 ear embryos. In a third experiment, a similar procedure was performed, except that the corn cob had been pre-submerged in medium 211 (1 liter: Duchefa's N6 Basal Salt Mixture: 1 pkg (i.e., 3.952 g) (Gold Biotechnology Inc. , St. Louis, MO, USA); 2,4-D (1 mg/ml): 1 ml; ; L-Asparagine Monohydrate: 0.91g; Myo-inositol: 100 mg; pH 5.8), and mixing was carried out in a paint mixer. In this experiment, 109 embryos were substantially isolated from the 210 ear embryos. In these cases, the non-fluid jet force from the movement of the liquid around the corn cob resulted in substantial isolation of the embryos; fluid force may include fluid with turbulent flow, fluid with laminar flow, shear from the fluid flow, fluid with negative pressure (e.g., resulting in cavitation), or combinations thereof. Forces also include forces generated by acoustic techniques, such as by an acoustic wave or waves (pulsed or continuous) in both the gas and fluid phases.

[00174] The preceding examples (including Examples 9-11) describe the use of a jet of fluid to remove embryos from an immature cob. During these procedures, it was observed that the fluid jet generally also induced the release of at least part of the grain's endosperm. Endosperm tissue was observed to be softer and more friable than embryos and tended to disintegrate to varying degrees (in contrast to embryos which tend to remain intact). It is possible for endosperms to disintegrate after exposure to the shear caused by the fluid jet. This shear is considered to be non-uniform, resulting in the variability of the observed disintegration; Nevertheless, a large proportion of the endosperm material that was sufficiently disintegrated to pass through the cotton gauze left a retentate composed of a semi-pure embryo preparation.

[00175] When a low-pressure jet from a standard laboratory squirt bottle was directed at the retentate of the cotton gauze, more of the remaining endosperm tissue was further disintegrated and washed through the gauze, leaving behind a relatively small embryo preparation. purer. Therefore, it is reasonable to predict that if the retentate is uniformly exposed to a shear force of the correct intensity, all or substantially all of the remaining endosperm may disintegrate and pass through the gauze. Such shear force may be generated by any suitable means, such as, but not limited to, a single jet, multiple jets, a sheet-like jet or a curtain-like jet, rapidly moving jets, and acceleration and deceleration of endosperms. Furthermore, if the jet used to initially release the grain contents is intended to expose a greater proportion of the endosperms to shear during ejection, an initial embryo preparation of greater purity can be obtained.

[00176] The following is a non-limiting modality for applying shear to further purify the embryos. Once embryos and partially disintegrated endosperm are released from a cob, the remaining endosperm can be rapidly fragmented by liquid flow, for example from a spray gun, which strikes the endosperm evenly and simultaneously. A suitable type of injector is a total conical injector. Full conical nozzles generate a spray pattern completely filled with droplets. An internal blade inside the injector imparts controlled turbulence to the liquid before it exits the orifice, enabling the spray pattern to form. Commercially available injectors have spray patterns that are round, square, or oval. An example of a full cone nozzle is known as the "UniJet® Spray Nozzle, Standard Spray, Small Capacity" (Spraying Systems Co., Wheaton, IL; order number TG-SS0.3).

Example 13: Devices that use a combination of forces

[00177] This example describes several additional embodiments of the method of the invention, which uses a combination of forces to substantially isolate multiple seed embryos.

[00178] Figure 5 illustrates a device that uses a more voluminous fluid jet (as described in Example 10). Such a device includes an injector for generating fluid flow such as a more voluminous fluid jet (e.g., a flat fluid jet) and optionally a suction head or component for applying negative pressure fluid (e.g., by vacuum or suction), to dislodge embryos and/or to collect dislodged embryos. Figure 5A (top) illustrates a cross-section of an example of such a device, showing how the injector, optional suction head, and corn cob can be positioned relative to each other. The corncob, injector and optional suction head can be moved relative to each other; for example, the corncob may be stationary while the injector and optional suction head are moved, or the injector and suction head are stationary while the corncob is moved. Figure 5B (bottom) schematically illustrates a corn cob positioned in the device and shows the injector positioned to generate a flat fluid jet in which the jet impacts multiple grains in a row.

[00179] Figure 6 illustrates an embodiment of a suitable suction head or component for applying fluid with negative pressure (for example, by vacuum or suction), as is optionally used in the device of Figure 5 and which can also be used alone to substantially isolate the embryos. The suction head may include one or more openings through which negative pressure fluid may be applied. The suction head may also include a means for dispensing fluid (such as gas or liquid, e.g., water or media), for example, multiple openings in the suction head. For use with corn, the suction head is preferably shaped to follow the contours of a typical corn cob and is preferably capable of capturing embryos from multiple kernels or multiple rows of kernels. It is anticipated that the suction head may be manufactured from a rigid material (such as stainless steel or other metals) or a flexible material to allow easier conformation of the suction head to the contours of a corn cob or its combinations. Embryos can be substantially isolated by any combination of positive mechanical pressure (exerted, for example, by the leading edge of the suction head), negative fluid pressure (e.g., suction or vacuum), and fluid force (such as, but not limited to positive pressure from a jet of fluid, fluid with turbulent flow and fluid with laminar flow capturing the material inside the grain).

[00180] Devices for applying force to substantially isolate embryos, such as are described in Examples 1, 3, 4, 6, 7, 9, 10 and the present example (including, but not limited to, the devices illustrated in Figures 5 and 6 ) can be moved relative to the corncob. The tang can be stationary or the device can be stationary or both can be moved. Because corn seed typically occurs in relatively uniform rows arranged parallel to the longitudinal axis of the corn cob, the device typically is moved (relative to the cob) such that the device passes parallel to the longitudinal axis of the corn cob and following a row. or multiple rows of grain. However, the movement of such devices relative to the cob may follow the circumference of the cob or may be random or may be any combination of suitable movements.

[00181] Figures 7A to 7C illustrate different plans of a device embodiment that uses a combination of forces to substantially isolate multiple seed embryos (in this example, corn). This device includes a head with a leading edge capable of applying a predefined amount of mechanical pressure to the base of grains that have previously had the pericarp opened or truncated, such that embryos are expelled from the grain in a manner similar to those described in Examples 1, 3 and 4. The device further includes a component for applying negative fluid pressure (e.g., by vacuum or suction) to displace embryos and/or to collect the displaced embryos. The expelled embryos (and accompanying non-embryonic tissues) are thus separated from the corncob and can be collected by applying negative fluid pressure. The collected embryos and non-embryonic tissues can be further separated, if desired, by suitable methods such as size separation, hydrophobic separation or differential centrifugation. A variation of such a device may include a means for dispensing fluid (such as liquid, e.g., water or medium), for example, multiple openings in the suction head.

[00182] The embryo extraction devices illustrated in Figures 5, 6 and 7 are described as illustrative examples that are not intended to be limiting. These and other devices may include additional components, for example, means for separating embryos from non-embryonic tissue or fluids used in the substantial isolation process.

Example 14: Feasibility Data

[00183] The multiple monocot embryos provided by using the methods and devices of the present invention are most preferably embryos suitable for application in genetic transformation or tissue culture, such as plant transformation and regeneration. This example further illustrates the utility of methods of the invention in providing multiple monocot embryos that are viable and suitable for genetic transformation or tissue culture. In this example, the quality of immature corn embryos obtained by different excision methods was compared in their response to transformation by Agrobacterium tumefasciens.

[00184] Corn embryos excised using this apparatus and method were transformed using standard methods known in the art of corn transformation (e.g., Cai et al; Patent Application Publication 2004/0244075). Four experiments (designated A, B, C and D, respectively) were performed. Each experiment compared embryos obtained by manual excision with embryos obtained by a method of the present invention: excision by a liquid jet (experiments A, B and D) or excision by a gas jet (experiment C). The liquid jet in experiments A and B used ordinary tap water and an injector made from a pipette tip. Experiment C tested a gas jet using air from a compressed air pump and an injector made from a pipette tip. The liquid jet in experiment D used a concentrated MSPL % medium (see Table 1 of United States Patent Application Publication Number 2004/0244075 by Cai et al) as the liquid and a solid stream injector with an equivalent orifice diameter to 0.020 inch (catalog number TP000050-SS, Agricultural Division of Spraying Systems Co., Wheaton, IL).

[00185] The corn cobs were harvested twelve days after pollination and sterilized by immersion in a 1 liter bottle of 80% ethanol for 3 minutes. Embryos were excised manually by cutting the upper third of the grain with a scalpel and removing the embryo from the grain using a narrow spatula. The collected embryos were excised in 1 milliliter of % MSPL medium in a single microcentrifuge tube (Eppendorf). The medium was removed and replaced with 1 milliliter of Agrobacterium tumefasciens prepared as described below.

[00186] The embryos were substantially isolated using a jet of fluid (liquid or gas), following procedures similar to those described in Examples 10 and 11. The jet of fluid was used to excise the embryos remaining on the ears after removal of the third top of the grain with a scalpel. The spike was positioned such that the jet of fluid was aimed at individual cut kernels in succession to dislodge both the embryo and non-embryonic tissue (endosperm). The contents of the kernel removed from the cob were passed through a coarse screen to remove large pieces of endosperm and the embryos were collected on sterilized cotton gauze. Embryos were transferred using a small gauze spatula to a microcentrifuge tube containing 1 milliliter of %MSPL medium. After all embryos were collected, the %MSPL medium was removed and replaced with 1 milliliter of Agrobacterium tumefasciens inoculant.

[00187] The embryos prepared by the various excision methods were subjected to the same inoculation, selection and regeneration procedures. Suitable procedures, including descriptions of media and reagents for plant transformation using glyphosate selection and GFP as a reporter, have been described in United States Patent Application Publication Number 2004/0244075 to Cai et al.

[00188] The embryos were inoculated with 1.0 milliliter of Agrobacterium for 5 minutes. The contents of the microcentrifuge tube were poured onto a plate with coculture medium, excess Agrobacterium was removed with a pipette, and the contents were cocultured for 18 hours at 23 degrees Celsius. The embryos were then transferred to MS induction medium and cultured at 30 degrees Celsius for 13 days. Transformation-derived calli were cultured at 27 degrees Celsius for 11 days before regeneration. At this time, GFO-positive sectors were counted using a fluorescence microscope. For regeneration, calli derived from each embryo were individually transferred to MS/6 BA medium and grown in a lighted environment for 7 days, after which each matured callus was transferred to MSOD medium and returned to the lighted environment for 17 days. additional. The resulting seedlings were transferred to Phytatrays® containing regeneration medium (consisting of 2.165 g of MS Basal Salts, 5 milliliters of 100X MS Vitamins and 20 grams of sucrose made with 1 liter in water and autoclaved, pH adjusted with KOH to 5.8 , solidified by autoclaving with 3 g of Phytagel® and with 0.75 milliliter of 1 milligram per milliliter of indole-3-butyric acid, 0.5 milliliter of 1 milligram per milliliter of 1-naphthaleneacetic acid and 0.2 milliliter of glyphosate 0.5 molar). After 3 weeks, transgenic plants were consolidated by transplanting rooted seedlings into peat pots containing soil mixture and grown at 26 degrees Celsius.

[00189] The results of these experiments are summarized in Table 3. The number of embryos that were transformed is estimated from the number of GFP-positive embryos. Table 3. Viability and frequency of transformation of excised embryos. n/a: data not evaluated

[00190] The overall frequency of transformation and regeneration is given as the percentage of GFP-positive plants regenerated from the inoculated embryos. These results demonstrated that the various methods and devices of the present invention are useful for providing multiple monocot embryos suitable for genetic transformation or tissue culture.

Example 15: Development of liquid media to excise corn embryos

[00191] The fluid jet apparatus required about 20 l of liquid to excise the embryos from a cob, necessitating the development of an excision medium that is simple to prepare (i.e., containing only about one or two ingredients). , which can be readily prepared, which is sterilized by filtration preferably through an in-line filtration unit, which can flow through the fluid jet apparatus at a preferred operating pressure of 275.8 - 413.7 kPa (40-60 psi) and that did not require pH adjustment before use. Such media and its preparation should be low cost, reduce media preparation time, and allow for automation. This example provides several such means.

[00192] Culture media such as Lynx 1013 (inoculation medium) and Lynx 1902 (half-strength Lynx 1013) have been successfully used for the excision of corn embryos useful for transformation using a fluid jet apparatus. Lynx 1013 comprises (per liter): MS Basal Salts (Phytotech; PhytoTechnology Laboratories, Shawnee Mission, KS): 2.165g; MS Vitamins (100X; Phytotech): 10 ml; Glucose (Phytotech): 36 g; Sucrose (Phytotech): 68.5 g; Proline (Fisher): 0.115 g. The medium was adjusted to pH 5.4 with KOH and then sterilized by filtration. Although these media worked well, they contained numerous ingredients, some of which had to be sterilized by filtration. These media also require pH adjustment before use.

[00193] Various other liquid media (Table 4) have been tested for the excision of transformable corn embryos from corn cobs. The excised embryos were then used for transformation according to the methods described elsewhere in this description and the transformation frequency (FT) was determined for each excision medium tested. FT was defined as the number of unique transformation events regenerated in plants divided by the number of embryos inoculated with Agrobacterium.

[00194] All media tested, including water, were capable of producing embryos that were transformable (see Table 4). However, media comprising mannitol produced FT comparable to the Lynx 1902 control medium. Mannitol media is a simple media with only two ingredients (mannitol and water), does not require pH adjustment prior to use, and is sterilizable by filtration, making -Significantly cheaper and more convenient to use.

[00195] The concentration of mannitol in the medium is between about 0.05 M to about 0.5 M. Preferably the concentration of mannitol in the medium is about 0.1 M. More preferably, the concentration of mannitol in the medium is is about 0.2 M. A suitable concentration of mannitol for the excision medium, however, can be determined by one skilled in the art of plant tissue culture, simply by varying the concentration of mannitol.

[00196] Potential representative osmotic measurements of the selected media are found in Table 4. Readings were taken using a Wecor 5100C Vapor Pressure Osmometer (Wecor, Logan, UT, USA), with calibration using standardized solutions of 100, 290 and 1000 mOsm/ kg. A medium with an osmotic potential (i.e., molality) between about 0 mOsm/kg to about 500 mOsm/kg is suitable for the excision of maize embryos for tissue culture, including, for example, about 7 mOsm /kg to about 300 mOsm/kg. For example, a solution of 0.2 M mannitol in water has an osmolality of about 222-230 mOsm/kg, while a solution of 0.05% MES in water has an osmolality of about 7 mOsm/kg. . This osmotic potential range is preferably obtained by adding one or two compounds to the medium, such as Lynx #1937, #1932 and #1162 media. Preferably, such compounds do not have a significant adverse effect on the cultivability and transformability of tissue from excised embryos. Table 4. Osmotic potential measurements of exemplary solutions for use in embryo isolation n/a = not carried out

Example 16: Construction of a fluid jet excision media preparation system

[00197] This example describes a media preparation system (MPS) for use with a fluid jet apparatus. The fluid jet devices tested required 20 liters of medium, which required time and effort to make and use. The MPS of the present invention can prepare large quantities of medium quickly. It also allows preparation of a specific volume of medium for a particular size of corn cob.

[00198] The MPS comprised a Mixing Chamber (MC) and an MPS Box (MPSH). The MC was designed to be easily detachable from the MPSH for easy autoclaving sterilization. Both MC and MPSH can be made from any suitable material. Preferably they are made of a material such as steel or aluminum. As shown in Figures 11 and 12, MC comprises a tank that has an upper end and a lower end, a flange that is coupled to the upper end of the tank, and a cover plate to cover the upper end and to seal the chamber. mixing. Preferably, the tank is sealed with the cover plate by a sealing ring provided on the flange. The cover plate is preferably made of corrosion-resistant grade 7075 aircraft grade aluminum to reduce the weight of the mixing chamber. In one embodiment, the tank is provided with three pins at the lower end, each on three sides as shown in Figure 12, for orientation and positioning of the tank on the mixing chamber support plate. In a working position, the three pins engage with the three positioning brackets provided on the support plate as shown in Figures 12 and 14. A positioning plate (Figure 12 and 13) is also provided at the bottom of the tank at the fourth side, to hold the tank in a working position on the mixing chamber support plate. Preferably, the positioning plate has a 6.35 mm (1/4") NPT thread for mounting a quick connector to drive a positioning arm with a handle (Figure 14) to secure the mixing chamber onto the support plate (e.g., Figure 13).

[00199] The cover plate is provided with an opening for inserting a tube for bringing in a liquid such as water to make the medium, an opening for inserting a tube for taking the prepared medium which is connected with a jet apparatus of fluid, an opening for inserting a tube for adding medium ingredients, preferably stainless steel, into the mixing chamber (e.g. Figure 12). When working, separate sealing rings seal several pipes with openings. The cover plate is also provided with an opening for insertion of a box containing an ultrasonic sensor to monitor a specific volume of media that needs to be prepared. Preferably, the opening for the ultrasonic sensor housing is provided on one side of the cover plate. In the working position, several openings provided on the mixing chamber cover plate are connected to corresponding tubes provided on the lower surface of the upper box platform (Figure 11). These tubes are preferably made of steel. The various openings connect with the mixing chamber as shown in Figure 11, for example via connecting tubes. The piping is preferably made of polycarbonate.

[00200] A medium mixing assembly is coupled to the lower end of the cover plate, such that the assembly preferably hangs in the center of the tank. The assembly comprises an impeller with two folded blades, a stainless steel shaft sealed with a sealing ring in the cover plate, a high temperature ball bearing suitable for operation at high temperature, such as above 204°C (400° F), present during autoclaving and a spider-type coupling (for example, Figure 12) for coupling the assembly with a motor in the MPSH (Figure 16).

[00201] A threadable block is mounted on the lower side of the mixing chamber supporting the plate and is connectable with a ball screw mounted on a ball bearing provided on the upper surface of the lower box platform. The support plate is also slidable via four wheels onto the four legs of the MPS box as shown in Figure 15. The wheels roll along the t-slots of the four legs to facilitate movement up and down the mixing chamber. when the stepper motor provided on the bottom surface of the MPSH bottom platform is turned on.

[00202] After autoclaving, the mixing chamber is positioned on the support plate by the three pins and the positioning plate. The positioning arm with the handle is inserted into the quick connector. The arm is pushed down and rotated 90 degrees clockwise with the help of the clamp to position and secure the mixing chamber onto the support plate. The positioning arm is coupled to the cam and mounted on a spring that is welded to the support plate. The spring pushes the arm and cam upward. When placed in position, the rear pin of the mixing chamber pushes an electrical switch, which sends a signal to a Programmable Logic Controller (PLC). This signal informs the PLC that the mixing chamber is positioned on the mixing chamber support plate. The stepper motor provided on the lower platform of the box pushes the mixing chamber towards the upper platform of the box where, in the working position, the various openings provided on the cover plate of the mixing chamber become connected with the corresponding tubes. provided on the lower surface of the upper box platform (Figure 16).

[00203] After receiving an operation signal, the PLC opens an electromagnetic valve connected to a water system to fill the mixing chamber with water. The ultrasonic sensor monitors the water level in the mixing chamber. When the water level reaches the specified level in the mixing chamber, the PLC closes the electromagnetic water valve and waits for a signal from the operator indicating that the excision medium components are in the mixing chamber. When the PLC receives that signal, it drives a motor provided on the upper surface of the MPSH upper platform and operationally linked to the mixing assembly. Thus, the mixing assembly begins to mix the medium. Once the medium is ready, it is pumped using a gear pump through the medium outlet pipe to the fluid jet apparatus according to a PLC program.

[00204] In one embodiment, the prepared medium outlet/supply connected to the fluid jet apparatus is provided with an in-line filtration unit such as a 0.2 μm Absolute FiberFlo® Hollow Fiber Capsule Filter (Minntech Filtration Technologies Group, Minneapolis, MN) to sterilize the medium prior to its use in the fluid jet apparatus for embryo excision.

Example 17: Embryo isolation by gradual excision

[00205] This example describes an apparatus for preparing multiple corn embryos suitable for grain tissue culture on a corn cob, comprising at least one opening for directing a first flow of fluid at a first time to substantially extract the endosperms. from the grains and a second flow of fluid at a second time to substantially extract the embryos from the grains, the extracted embryos being suitable for tissue culture. The apparatus further comprises means for moving at least one ear of corn relative to the first and second fluid streams. The means for moving the at least one ear of corn relative to the first and second fluid streams moves the fluid flows along the longitudinal axis of the at least one ear of corn. In this particular embodiment of apparatus, each fluid flow is a liquid flow. The liquid stream may consist, for example, essentially of mannitol and water in a concentration of about 0.05 M to about 0.5 M mannitol. The fluid flow may alternatively be a gas flow. The apparatus may further comprise a means for detecting excised endosperm or embryos. The apparatus may also comprise a means for channeling excised endosperm or embryos so as to separate the endosperm from the embryo. The apparatus may also comprise a means for connecting the excised endosperm and embryo detection means to the excised endosperm and embryo channeling means electronically for automation. The apparatus may further comprise at least one separator for separating embryos from non-embryonic tissues, wherein the separated embryos comprise maize embryos suitable for tissue culture. The separator may comprise a size exclusion device. Alternatively, the separator may separate embryos from non-embryonic tissue by differential hydrophobicity or differential density.

[00206] The present example also describes a method for providing monocot embryos suitable for tissue culture comprising: (a) providing monocot seeds containing embryos that have an opening in the pericarp of the seeds; and (b) applying a first force at a first time point to the seeds to substantially extract the endosperms from the seeds and a second force at a second time point to substantially extract the embryos from the seeds, the extracted embryos being suitable for tissue culture . The forces comprise one or more forces selected from a positive pressure fluid jet, a positive pressure liquid jet, positive mechanical pressure, negative pressure, centrifugal force, linear acceleration, linear deceleration, fluid shear, turbulent fluid flow and laminar fluid flow. The method further comprises separating embryos from non-embryonic tissue by methods selected from the group consisting of size exclusion separation, hydrophobic separation and differential density. Monocot seeds are supplied on at least one ear. The monocot may be from the Poaceae family, such as Zea species (for example, Zea mays). The tissue culture step may include one or more transformation and regeneration steps, which result in at least one fertile transformed plant.

[00207] As shown in Figure 17, the apparatus is provided with a flat jet injector (1) to produce a laminar flat flow (6), two stepper motors controlled by a programmable logic controller (PLC) and a gear pump digital. A stepper motor rotates the cob (2), which has the corn kernels from which the crown has been removed to facilitate excision, mounted on a shaft in an airtight chamber (3) and the second motor advances the cob along. along the axis such that the flow acts on each grain for substantially the same amount of time. The digital gear pump forces the liquid medium through the injector as specified by PLC rate and pressure. The injector (1) produces a flow of liquid that is about 0.0762 mm (about 0.003") wide and about 25.4 mm (about 1") high. Generally the flow width is smaller than the grain width. In the tested pressure range of 206-517.1 kPa (3075 psi), laminar flow was stable up to a distance of up to 5 cm (2"), 12.7-7.62 cm (5-3") from the injector (1) and did not disperse before reaching the ear. The spike (2) can be positioned 3.2 - 5 cm (1%" - 2") from the injector (1) such that the liquid flow acts on each grain in a row with substantially the same force. The amount and duration of force can be manipulated by changing the liquid pressure in the injector, adjusting the distance from the spike to the injector and the space between the injector plates as well as other parameters such as the angle of entry of the liquid flow into the injector. grain and cob rotation speed.

[00208] In the first stage, a PLC according to a PLC program opens a channel (4) through a valve (5) to collect the endosperm and adjusts a pressure and a flow rate through the injector (1) by sending signals control unit for the digital gear pump and rotates and advances the spigot (2) by the two stepper motors. The liquid flow (6) controlled by the PLC first removes the endosperm (7) from the grains, leaving the embryos (9) attached to the grains (7). The amount of time and force required to remove the endosperm can be adjusted depending on the size of the endosperm, the extent of the cut in the kernel crown, and the differences between ear and kernel sizes, among other parameters. The mixture of liquid medium and endosperms collected in the channel (4) can be pumped through a filtration system. After filtration, the liquid medium can be reused in the excision process.

[00209] In the second phase, the PLC according to a PLC program closes the channel (4) and opens a channel (10) through the valve (5) for embryo collection, adjusting a pressure and a flow rate through the injector (1) and rotating and advancing the spigot. The liquid flow (6) removes the embryos (9) and the rest of the endosperms (8) from the grains (7). Further separation of embryos, if desired, can be done using separators described elsewhere. The amount of time and force required to remove the endosperm can be adjusted depending on the size of the endosperm, the extent of the cut in the kernel crown, and the differences between ear and kernel sizes, among other parameters. The mixture of liquid medium and embryos collected in the channel (10) is subjected to a separation device (e.g., Example 18) to isolate the embryos from the waste and liquid, and the liquid can be pumped through a filtration system to reuse in the excision process.

[00210] The chamber (3) can be equipped with an emitter (11) and a preferential photoelectric sensor (12) for detecting embryos in the liquid medium that emerges after excision. During the first phase, the signal confirming the appearance of embryos in the exiting medium instructs the PLC to complete phase one. During the second phase, the signal that confirms the disappearance of the embryos in the exiting liquid medium alerts the PLC to complete phase two and may also instruct the PLC to turn off the device.

[00211] Corn embryos excised using this apparatus and method were transformed using standard methods in the art of corn transformation (e.g., Cai et al; US Patent Application Publication 2004/0244075). All treatments used Lynx #1986 (0.2 M Mannitol, non-sterile) for embryo excision. Representative transformation frequencies (TF) are shown in Table 5, indicating that the embryos produced by this apparatus and method were transformable and provided transgenic plants. Table 5. Transformation of maize embryos excised by the fluid jet apparatus and method gradual "FJ (Fluid Jet) Phase with endosperm" refers to embryos that, after collection, were inoculated with endosperm and left on the same plate (embryos and endosperm together). "FJ Non-endosperm Stage" refers to embryos that, after collection, were removed to a plate with fresh medium leaving the endosperm behind.

Example 18: Embryo separation process

[00212] This example describes a flotation process for separating embryos from a mixture produced by a fluid jet excision process. The parameters for efficient separation are described here. In this process, gas bubbles were generated in a fluid and coupled to excised corn embryos present in the fluid. The bubbles floated to the surface (i.e., fluid-air interface), allowing embryos to be collected preferentially, while leaving behind endosperm material and other debris.

[00213] To reduce the number of bubbles per unit volume and generate a more uniform bubble field such that a lower shear force was generated, a device was constructed containing multiple bubble source points. The device was constructed by inserting individual linden tree fragments into pre-drilled holes in the length of silicone tubing (Masterflex® C-96400-16 silicone tubing, Cole-Parmer). After inserting linden chips into the silicone tubing, one end of the tubing was closed with a stainless steel bearing. The tubing is then wound into a spiral and secured with a metal wire as shown in Figure 18. The spiral-shaped air dispersion device combined with the use of PEG as a surfactant has been shown to purify 95% of embryos from almost all endosperm as shown in Figure 19. Figure 20 illustrates the amount of endosperm separated from the embryo fraction by bubble flotation using the spiral-shaped air dispersion device and PEG. Other linden-based bubblers can be built or purchased from, for example, aquarium suppliers. The Petri dish on the left of Figure 20 shows the large amount of endosperm interspersed with embryos when the flotation process is not used. In comparison, the Petri dish on the right shows the reduction in the amount of endosperm cofractionated with the embryos when the flotation process of the present invention was used. Bubbles can also be produced by ceramic materials that have various pore sizes.

[00214] In order to detach the bubbles from the source material that produces them, it is desirable that the source material be of an opposite nature to that of the bubbles, with respect to the degree of polarity (i.e., hydrophobicity). Since air bubbles are covalent in nature, they will tend to detach quickly from a material that is polar in nature. Conversely, bubbles that emerge from a surface that is also covalent in nature, such as a porous plastic sprinkler, will tend to adhere to the surface and coalesce and grow to a larger size before detaching. In this regard, Chemglass sprinkler, limewood, and ceramic are all desirable choices. The surface of the lime tree is composed of cellulose, hemicellulose and lignin which are polar and therefore hydrophilic, as are sintered glass sprinkler and ceramic materials.

[00215] In one example, once the embryos were carried to the surface by the bubbles and deposited in the foam, they were collected from the foam with a slotted spoon and then cleaned from the foam. In another experiment, a vacuum filtration device (e.g., Figure 21) was used to collect embryos from the foam surface.

[00216] In one experiment, embryos and endosperms were excised from donor ears using a fluid jet apparatus (FJ) and collected on cotton gauze (CC). The mixture of embryos and endosperm was then placed in a graduated cylinder filled with Lynx 1013 and 20 μl of 20% PPG (polypropylene glycol mono-butyl ether - Average Molecular Weight 340. Aldrich Cat. No. 438103; Sigma Chemical Co ., St. Louis, MO) and bubbles were produced by air pumped through a narrow-pore fritted glass dispersion tube (Chemglass) by an aquarium pump (bubbler). Separated embryos, substantially free of endosperm material and other debris, were collected as described above. The separated embryos were used for Agrobacterium-mediated maize transformation as noted above. Transformation frequencies are shown in Table 6.Table 6. Transformation of float-separated corn embryos using PPG-stabilized air bubbles.

[00217] In another experiment, a linden-based bubbler was used to separate embryos from endosperms excised by the fluid jet apparatus. In general, the linden-based bubbler was placed in a beaker in a sterile laminar flow hood. The bubbler tubing was connected to an air filter at one end. The other end of the air filter was connected to an aquarium pump. Separation medium (0.2 M Mannitol with 20 μl of 20% PEG (Average Molecular Weight 8000, Sigma No. P21390)) was added to the beaker over the bubbler. The embryo and endosperm from the fluid jet were collected and transferred to the beaker. Air was bubbled through the bubbler. The floating embryos were transferred to a small Petri dish and used for transformation using standard methods known in the art of corn transformation as noted above. Transformation frequencies are shown in Table 7.Table 7. Transformation of float-separated corn embryos using PEG-stabilized air bubbles.

Example 19: Apparatus for separating embryos by floating

[00218] Figure 22 shows an apparatus for separating embryos by flotation comprising a flotation column (E) connected to an inlet tube (A) at the upper end of the column for adding a mixture of embryos, endosperm and other waste , an air dispersion duct (G) at the lower end of the column for the production of air bubbles to elevate the embryos and an outlet tube (H) also at the lower end of the column for regulating the liquid level and removing residues from the bottom of the column. The bottom of the column is preferably inclined to facilitate the removal of non-floating residues. The buoyancy column is also provided with a drainage conduit (C) to collect the foam containing the embryos. The air dispersion duct is also provided with a valve (F) to prevent backflow of the liquid column into the air dispersion tube.

[00219] To operate this apparatus, the flotation column is filled with a liquid medium compatible with the subsequent transformation and tissue culture of the embryos until the liquid level reaches the drainage drain (D). The drain drain has a siphon tube (B) open to the air in the ropo. A surfactant is added to the liquid medium to prevent premature coalescence of bubbles and stabilize the foam for coupling and ascent of the embryos to the top of the flotation column.

[00220] The next step in the operation of the apparatus is to turn on the air supply to the air dispersion duct. The pores in the dispersal duct are small enough to produce bubbles that rise so slowly that their contact with the hydrophobic surface of the embryo is long enough to allow coupling of the bubbles to the embryos.

[00221] Then, a mixture of embryos, endosperm and waste, produced by the fluid jet apparatus containing the desired concentration of a surfactant (such as PEG at a concentration of 20 parts per million) is fed into the inlet duct. As the slurry enters the buoyancy column, the suspended particles encounter the bubble column of the air dispersion device. The bubbles first attach to the embryos and preferentially lift the embryos to the surface of the buoyancy column where they become part of the foam. As foam accumulates, it exits the buoyancy column through the drain conduit (C).

[00222] During operation, the column can be inclined at a discrete angle with the vertical towards the drainage conduit such that the rising bubbles are concentrated on the rear wall of the column and tend to push the foam towards and towards the conduit.

[00223] The endosperm-rich waste, which does not float, falls to the bottom of the flotation column and accumulates at the lowest point of the inclined bottom. These residues are removed from the column during automatic liquid leveling of the column, which occurs when the slurry of embryos and endosperm residues is initially fed into the feed hopper at the beginning of column operation or may be removed at the end of float column operation. .

[00224] In another embodiment, the liquid level in the float tank is a level maintained with an outlet drain and the foam that rises to the surface is continuously drawn into the drain. This action can be facilitated by the existence of more bubbles that arise on the side of the tank away from the drain such that there is a circulation produced that arises on the side away from the drain and dissipates on the drain side.

[00225] In another embodiment, the liquid level in the float tank may be maintained below the drain level until a sufficient amount of foam and embryos has accumulated and then the liquid level is raised such that the foam and embryos overflow to the drain.

Example 20: Alternative devices for separating embryos by flotation

[00226] This example describes another embodiment of apparatus designed to separate corn embryos from corn endosperm through a flotation process that uses bubbles and surfactants. The apparatus can be used with an embryo excision apparatus, such as a fluid jet apparatus as described previously in the application.

[00227] In the apparatus (Figure 23), the embryos and endosperms flow into a chamber equipped with a medium for producing bubbles. Bubbles can be generated by air forced through a sprinkler. Preferably, the size (diameter) of the bubble is about 100 μm to 500 μm. A bubble stabilizing agent such as polyethylene glycol (PEG) is also added at a concentration of about 1 to about 100 ppm, for example, 20 ppm. The embryos preferentially attach to bubbles through hydrophobic interaction and the bubbles elevate the coupled embryos to the surface of the chamber. The addition of PEG enables the bubbles to transport their "loads" of embryos to the surface. PEG also helps to create a foam layer on top of the surface and the embryos collected on top of the foam layer are carried into the drainage conduit. A motorized skimmer device can be used to facilitate the flow of the foam containing the embryos to the outlet, where they pass through an imaging station or counting device to count the number of embryos, and are collected into containers as needed. A switch operatively linked to an imaging station or counting device can be provided to dispense desired numbers of embryos into different containers. The containers can be connected to a vacuum collection device to remove the liquid leaving behind the embryos for later use.

[00228] The apparatus is further provided with a means for bringing the liquid into and out of the chamber. The liquid level in the chamber can be controlled by a liquid leveling device. The endosperms and other residues that are not selectively coupled to the bubbles are discarded through means to remove the liquid from the bottom of the container, such as a waste outlet drain. The same liquid medium that is used for excision of the embryos can be used to fill the embryo separation chamber. The medium that leaves the device can be recycled for later use during the excision and separation processes.

Example 21: Combination device for excision and separation of corn embryos for tissue culture

[00229] This example illustrates a combination device or an integrated device comprising an embryo extractor as shown in Figures 24-25 and an embryo separator as shown in Figure 26. Preferably, the embryo extractor is in communication with the separator . However, the embryo extractor or separator may be operated separately and the outlet of the embryo extractor may be the inlet of the embryo separator.

[00230] With reference to Figure 24 which is a top view, the embryo extractor is equipped with at least one jet of fluid (C) for extracting embryos from each ear (B). More than one jet of fluid can be directed at a single spike. Each tang in Figure 24 is held in position by spring-loaded retainers (A) at one end of the tangs and a mechanized retainer (D) at the other end that can be driven by a track mechanism (E). A side view of the apparatus is shown in Figure 25. Typically, the crowns of the kernels on the ear are partially or completely removed to facilitate embryo extraction.

[00231] Any liquid medium can be used to produce the fluid jet (C), for example, as described in Example 15. Sterile water can be used to excise the embryos as long as the period between the embryo's initial contact with the water and the immersion in the culture medium in the flotation tank is short. A filter can be used to sterilize the water. Alternatively, the liquid medium comprises mannitol or another solute of suitable osmotic strength as described above.

[00232] As shown in Figure 26, after extraction of the embryos from the grains, the embryos and waste optionally fall or are added to an embryo separator, which comprises a double screen conveyor belt system connected to a flotation chamber . The double screen conveyor belt has a movable coarse screen (A) that captures coarse material but allows embryos and other smaller material to pass through to a finer screen (H) that retains embryos and similar sized material but which allows very fine material and liquid to pass through to the liquid waste drain (W). Material collected on the coarse screen is removed in the coarse screen wash tank (F) and is assisted by the waste stream below the liquid waste drain (W) and drained through the coarse screen wash tank conduit (E) and the wash tank drain (G). The material collected on the fine screen (H) is washed freely in the fine screen washing tank (N) with the aid of a fresh medium and preferably a foaming medium through the inlet (M). If the embryos do not move from the fine screen (H) to the fine screen wash tank (N), agitation can be used. For example, embryos captured on the fine screen are dislodged in the flotation medium as they pass around a roller (K) and also by the impact of production of the medium added through (M). The release of fresh medium and frother is adjusted such that residues that accumulate in the flotation chamber are continuously removed through the liquid level regulator inlet (U) and outlet ducts (V).

[00233] Molded thermoplastic screens, which are not woven, may be preferred for use in this apparatus as they do not fall apart. Such screens are available (e.g., McMaster-Carr, Atlanta, GA) in various screen (woven) thicknesses, braid widths, and screen opening sizes. Screens made from polypropylene are also preferred because they can be autoclaved. The exact dimensions of the screen can be selected empirically by the operator depending on the expected sizes of the embryos and the waste that is introduced into the separator.

[00234] With reference to Figure 26, the thick screen (A) and the thin screen (H) can be supported by rollers. Preferably, the thick screen (A) is supported by a mechanized roller (B), a free roller (C) and a cleaning roller (D). The fine screen (H) is supported by a mechanized roller (I), a free roller (J), a cleaning roller (K) and an elevator roller (L). The rollers may be linear-cylindrical or other than linear-cylindrical, preferably concave-cylindrical because they allow trough-type conveyor belts to be used, which typically have less product loss along their boundaries than flat belts. Chute-style conveyor belts can allow for higher belt speeds and inclines. One or more of the rollers may be mechanized, for example, roller (B) and may have textured surfaces to grip the screen more firmly. It may be desirable to machine only one roll directly and have the other roll machined, for example roll (C) from the first by a belt or gear. Side or center supports or guides under the coarse and fine screens may be desirable to prevent submersion and loss of liquid along the edges of the screens. Additional support rollers may also be desirable.

[00235] After the material collected on the fine screen (H) moves to the fine screen wash tank (N), it falls past the bubble deflector (O) and enters the float chamber (P) where it finds rising air bubbles (S) produced, for example, by an air dispersion duct (T). At this point, the air bubbles preferentially attach to the hydrophobic surfaces of the embryos, carrying them to the surface where they are deposited in a foam through the side opening of the flotation chamber (Q) and the embryo release conduit (foam). (R). If embryos sinking in the flotation chamber (P) are not adequately exposed to the bubbles, a mixing device such as a magnetic stirrer can be provided in the lower portion of the chamber (P) to improve exposure.

[00236] The liquid level in the float tank is regulated by the inlet of fresh and foaming media from (M) and by the liquid leveling system (e.g., Figure 27) comprising a U-shaped inlet tube and a outlet tube (V) in which excess liquid along with waste enters the U-tube and exits through the tube (V). A U-shaped tube connects (U) and (V) with the highest level in the tube reaching the level at which the liquid is to be maintained. The top of the U-tube is also fitted with an anti-siphonage opening. Other liquid leveling devices such as those based on buoyancy, based on optics, based on conductivity and based on electricity can be provided for regulating the level of liquid in such a device.

Example 22: Apparatus for isolating transformable tissues from seeds and fruits

[00237] The compositions, methods and apparatus of the present invention can be used to isolate transformable tissue, for example, embryos from seeds and fruits of other monocotyledonous and dicotyledonous plants, including without limitation, corn, wheat, barley, soybeans, sunflower, cotton , canola, peppers, tomatoes, raspberries and strawberries among others.

[00238] A suitable fastener, for example, a blade having holes and/or grooves suitable for containing seeds or fruits of various shapes and sizes is provided (e.g., Figure 28). The fastener may be made of a suitable material such as plastic or metal. The fastener may be a flat sheet or rolled into a cylinder as shown in Figure 29 and suspended in a gas phase such as an air or liquid phase or may be partially suspended in the gas and liquid phases. Seeds and fruits can be secured onto the clamp by a suitable force, such as mechanical and/or suction force. The fastener may be fixed relative to a fluid force, for example, a liquid stream of the medium described in Example 15, or it may be movable relative to the fluid force.

[00239] In another embodiment shown in Figure 30, a Cam or Pressure Screw, for example, a screw, is inserted into one end of the cylinder to apply pressure to the seeds and fruits to facilitate isolation of the embryos. Isolated embryos can be separated from waste by the various methods described elsewhere in this application.

[00240] In another embodiment of the present invention, the sheet or cylinder can be centrifuged in a container to apply forces to the retained seeds or fruits to isolate the embryos.

[00241] Embryos can be separated from other tissues as described in Examples 18-20 and used for transformation and regeneration of various plant species. For example, soybean embryos can be transformed using the methods described in U.S. Patent 7,002,058. Transformation methods for other plants are known in the art.

Example 23: Methods for separating cotton and soybean embryos

[00242] This example illustrates the usefulness of the compositions, methods and apparatus of the present invention in separating cotton and soybean embryos from seed tissue, thus demonstrating broader utility. Cotton and soybean seeds were crushed by the method and apparatus described in U.S. Patent Application Serial No. 12/045,502, filed March 10, 2008, and U.S. Patent Application Publication 20050005321, and added to a mannitol solution. 0.2 M with 0.0012% PEG 8000 for the isolation of cotton embryos and about 0.003% PEG8000 for the isolation of soybean embryos, using the apparatus shown in Figure 31. Bubbles were produced, e.g. , as described in Example 18. Most of the cotton embryonic axes were found accumulated with the blisters as shown in Figures 32-33. Cotton embryos were prepared for transformation using the methods described in U.S. Patent Application Serial No. 12/045,502. In the case of soybeans, the broken embryos (embryonic axis and cotyledons) were found at the bottom of the container, while most of the seed coats floated to the top with the bubbles. The embryonic axes and cotyledons were then separated by differential density methods. For example, in a 20% solution of Ficoll in sucrose, the embryonic axes rise to the surface, while the cotyledons dive to the bottom of the container. Soybean embryos can be transformed using the method described in US Patent 7,002,058.

Example 24: Development of a co-culture environment to intensify the frequency of transformation

[00243] In some experiments, the use of coculture medium 1233 (U.S. Patent Application Publication 2004/00244075) in the corn transformation process, using embryos prepared by a fluid jet method and separated by a flotation method , resulted in lower transformation frequencies (FTs). In order to maintain or intensify the transformation frequency, a new coculture medium called "1898" was tested and unexpectedly found to increase TFs.

[00244] Several sets of experiments were conducted to compare the 1233 coculture medium and the 1898 coculture medium. Among other compositional differences (see Table 9), the 1898 coculture medium has a lower level of 2,4-D and has Carbenicillin. Immature corn cob embryos were excised and separated as described in the Examples above. The separated embryos were divided into two treatments. Embryos in both treatments were inoculated with the plant transformation vector pMON97367 comprising a gus and a CP4 expression cassette. Embryos from treatment 1 were cocultured on medium 1233 and embryos from treatment 2 were cocultured on medium 1898. After overnight coculture, the embryos were processed by the method described in U.S. Patent Application Publication 2004/00244075.

[00245] Table 8 shows that the use of the 1898 coculture medium resulted in improvements in all key indicators such as culture response, events created, events transferred to Phytatrays, events transferred to soil and % FT compared to the use of coculture medium 1233. Table 9 gives the compositions of the media used. Table 8. Increased transformation frequency with coculture medium 1898. Table 9. Compositions of the media used in the present invention. Means 1233, 1278, 1073, 1071, 1084 are from Cai et al.; US Patent Application Publication 2004/00244075. Medium 1898 is from US Patent Application Publication 2008/0124727. *Comprising 1250 mg/L nicotinic acid (Sigma), 250 mg/L pyridoxine HCl (Sigma), 250 mg/L thiamine HCl (Sigma), and 250 mg/L calcium pantothenate (Sigma).

[00246] All materials and methods described and claimed here can be made and used, as instructed by the above description, and without exhaustive experimentation, by a person skilled in the art. Although the materials and methods of this invention have been described in terms of preferred embodiments and illustrative examples, it will be clear to those skilled in the art that variations may be applied to the materials and methods described herein without departing from the concept, spirit and scope of the invention. All similar substituents and modifications clear to those skilled in the art are considered to be within the concept, spirit and scope of the invention as further defined by the appended claims.

REFERENCES

[00247] The following references, to the extent that they provide exemplary procedures or other details supplementary to those described here, are specifically incorporated herein by reference. U.S. Patent 5,550,318 U.S. Patent 5,780,708 U.S. Patent 6,194,636 U.S. Patent 6,232,526 U.S. Patent 7,002,058 U.S. Patent 7,150,993 U.S. Patent Application 10/710,067 U.S. Patent Application 11 /613,031 U.S. Patent Application 12/ 045,502 U.S. Patent Publication 2003/0024014 U.S. Patent Publication 2004/0016030 U.S. Patent Publication 2004/0126845 U.S. Patent Publication 2004/0210958 U.S. Patent Publication 2004/0216189 U.S. 2004/0244075 U.S. Patent Publication 2005/0246786 U.S. Patent Publication 2008/0124727 U.S. Provisional Patent Application 60/894,096 U.S. Provisional Patent Application 60/915,066

Claims (28)

1. Method for preparing corn embryos suitable for tissue culture and/or genetic transformation, characterized by the fact that it comprises (a) directing a first jet of liquid medium onto a corn grain to extract the endosperm from the grain; and (b) directing a second jet of liquid medium onto the grain to extract an embryo from the grain. 2. Method according to claim 1, characterized by the fact that the liquid medium consists of water or an osmotic agent with an osmolality of 0 mOsm/kg to 600 mOsm/kg. 3. Method according to claim 2, characterized in that the osmotic agent is selected from the group consisting of mannitol, sorbitol, glucose and sucrose. 4. Method, according to claim 3, characterized by the fact that the medium consists of water and mannitol in a concentration between 0.05 M and 0.5 M. 5. Method, according to claim 3, characterized by the fact that the medium consists of water and sucrose in a concentration between 0.05 M and 0.5 M. 6. Method according to claim 1, characterized by the fact that the grain is comprised on an ear of corn. 7. Method, according to claim 1, characterized by the fact that it comprises moving the corn cob in relation to the first and second jets to remove the endosperm and the grain embryo in succession. 8. Method, according to claim 1, characterized by the fact that the first and/or second jets comprise a width smaller than the width of the corn grain. 9. Method, according to claim 1, characterized by the fact that the first and/or second jets comprise a width of 0.0762 mm (0.003") and a height of 25.4 mm (1"). 10. Method, according to claim 1, characterized by the fact that the first and/or second jets are produced with a pressure between 206.8 kPa (30 psi) to 517.1 kPa (75 psi). 11. Method according to claim 1, characterized by the fact that the first and/or second jets are directed from an injector that produces a stable laminar fluid flow at a distance of at least 63.5 mm (2 .5") of the injector. 12. Method according to claim 1, characterized by the fact that the grain is positioned 44.45 - 50.8 mm (1% - 2") from the tip of the injector. 13. Method according to claim 1, characterized by the fact that the first and/or second jets contact each grain in a row of grains found on the cob with the same force. 14. Method for preparing plant embryos suitable for tissue culture and/or genetic transformation, characterized in that an apparatus for containing a grain or other tissue is centrifuged in a container to apply force to the grain or other tissue . 15. Apparatus for obtaining plant embryos suitable for tissue culture and/or genetic transformation, characterized in that it comprises (a) at least a first jet of fluid and a second jet of fluid for directing the medium onto a grain or other corn tissue comprising a plant embryo; (b) an apparatus for containing the grain or other fabric in the media path; and (c) at least one first separator for isolating embryos from non-embryonic tissue. 16. Apparatus, according to claim 15, characterized by the fact that the embryo is contained within a corn grain. 17. Apparatus, according to claim 16, characterized by the fact that the corn grain is comprised on an ear of corn. 18. Apparatus according to claim 17, characterized by the fact that the apparatus for containing the grain comprises means for moving the corn cob relative to the first and second jets of fluid to control the angle between the first and second jets of fluid and the grain. 19. Apparatus according to claim 15, characterized by the fact that it further comprises a detector for identifying endosperm tissue and excised embryos. 20. Apparatus according to claim 15, characterized by the fact that the apparatus for containing the grain or other fabric comprises a flat or cylindrical blade. 21. Apparatus according to claim 15, characterized by the fact that the apparatus for containing the grain or other fabric comprises a screen, a plurality of slits; or a cam or pressure screw that applies force to the fabric. 22. Apparatus, according to claim 15, characterized by the fact that the seed or fruit tissue is fixed on the apparatus by containing the grain or other tissue by a mechanical force, friction, centrifugal force or force suction. 23. Apparatus according to claim 15, characterized by the fact that the apparatus for containing the grain or other tissue is suspended in a gas phase, a liquid phase or is partially suspended in the gas and liquid phases. 24. Apparatus according to claim 15, characterized by the fact that the apparatus for containing the grain or other tissue is fixed in relation to a fluid force. 25. Apparatus according to claim 15, characterized by the fact that the apparatus for containing the grain or other fabric is movable with respect to a fluid force. 26. Apparatus according to claim 15, characterized by the fact that the separator separates embryos suitable for tissue culture from non-embryonic tissue by a method selected from the group consisting of size exclusion, differential density and differential hydrophobicity. 27. Apparatus according to claim 26, characterized by the fact that it further comprises a sieve for separating embryo from non-embryonic tissue based on size. 28. Apparatus according to claim 17, characterized by the fact that the apparatus for containing the grain comprises at least one first motor for moving the corn cob relative to the fluid jet.